Analysis of Occurrence of Elements in Tissues of the Knee Joint

*Wojciech Roczniak, Magdalena Babuśka Roczniak, Elżbieta Cipora and Barbara Brodziak Dopierała*

#### **Abstract**

The mineral structure of bones is never static, it is a living structure, reacting and adapting to load and having the ability to remodel. Skeletal cells work continuously to maintain the remodelling process therefore they are in a constant state of dynamic balance both in the sense of composition and structure, and they react to external mechanical forces. The remodelling processes that occur in the bone tissue allow for a proper functioning of this tissue, as well as for inclusion of additional elements, toxic ones included, in the remodelled bone, and they affect the metabolic processes occurring therein. This may result in disturbances in the osteoarticular system, manifested by changes in the bone tissue and within other organs. The influence of tobacco smoking on the content of strontium, lead, calcium, phosphorus, sodium and magnesium has not been confirmed. Non-smokers showed a high iron content in knee joint tissues compared to smokers. There were no statistically significant differences in the content of cadmium, nickel, copper and zinc in women and men in the studied knee joint components. With age, an increase in the content of chromium in knee joint tissues was observed, while gender, place of residence and occupational exposure had no effect.

**Keywords:** knee joint tissues, structural and trace elements, environmental hazards

#### **1. Introduction**

Many joints can be distinguished in the human body, but one of them stands out in terms of function and size. It is the knee joint [1]. It belongs to a group of complex joints and connects the femur and the tibia together. This largest joint, apart from the mentioned elements, is formed by the sesamoid bone in the form of the kneecap, and two pieces of meniscus, which allow to match the joint surfaces to each other during movement. The knee joint allows making straightening and flexion movements, but also rotational movements possible only in incomplete joint flexion [2]. The entire structure of the knee joint is strengthened by strong internal and external ligaments. The afore-mentioned joint is the second most strained joint in the human body, after the ankle joint. Due to the powerful force that the quadriceps exerts on the kneecap (max. 300 kg), the knee joint is exposed to overload. Taking into account the functions of the knee joint, it must be both mobile and flexible, as well as resistant to pressure [3, 4].

The mineral structure of bones is never static, it is a living structure, reacting and adapting to load and having the ability to remodel. Skeletal cells work continuously to maintain the remodelling process therefore they are in a constant state of dynamic balance both in the sense of composition and structure, and they react to external mechanical forces. The remodelling processes that occur in the bone tissue allow for a proper functioning of this tissue, as well as for inclusion of additional elements, toxic ones included, in the remodelled bone, and they affect the metabolic processes occurring therein. This may result in disturbances in the osteoarticular system, manifested by changes in the bone tissue and within other organs. Maintaining all the characteristics of the knee joint is possible thanks to the balance of many elements of the bone tissue that are responsible for individual properties of bones, which in turn create separate joints. Elements occurring in large quantities, e.g. calcium, magnesium, phosphorus, and those with low content - the so-called trace elements, e.g. Strontium, can be found in the bone tissue. Regardless of the amount of elements contained in the bone tissue, all of them are important and play significant roles. Mostly, calcium and phosphorus are part of bone hydroxyapatite. However, during the mineralisation process, metal ions present in the blood plasma may be built into the bone tissue, and their uptake will depend on the affinity of a given metal for mineral and extracellular matrix, as well as the concentration of metal ions in the blood plasma, and the degree of skeletal mineralisation. Strontium present in the knee joint is a trace element, although it plays a special role in the bone remodelling process of the human body [5, 6]. It is accumulated mainly in bones due to the high similarity to calcium [7] but unlike it, it is absorbed from food much less efficiently and in a larger percentage it is excreted [8]. Previous in vivo studies demonstrate the effect of strontium on improvement of mechanical characteristics of bones [7]. These studies also proved the effectiveness of treatment with small doses of strontium in the form of strontium chloride. Their conclusion is that 9–26 week Sr. therapy activates bone building and also stops bone resorption in humans [8].

Iron is a cofactor in many enzymes and cells in redox reactions. Low levels of iron ions can be detrimental to cells, while an excess of iron ions can lead to the production of reactive oxygen species through the Fenton reaction. The cellular iron content is strictly regulated by homeostatic mechanisms to maintain the right amount of iron in cells. Nickel ions and other divalent metals can compete with iron ions to enter the cell through DMT1 (divalent metal transporter 1) because they have similar ionic radii. Therefore, metal ions can affect many other processes dependent on the presence of iron in cells. As an enzymatic cofactor, iron is involved in bone matrix synthesis (activation of lysyl hydroxylase) and in 25-hydroxy-cholecalciferol hydroxylase synthesis. What is more, thanks to active vitamin D, iron ions stimulate the absorption of calcium ions in the intestine. Iron deficiencies in rats led to poor mineralisation of skeletons and pathological changes in the micro-architecture of the spongy substance. In turn, administration of estrogens' increases the accumulation of iron in hamsters and facilitates the uptake of iron ions by lymphocytes in culture. The deficiency of iron ions in young rats leads to a decrease in the mechanical strength of femurs and the cortical bone. In severe iron deficiency, both bone strength and mineral density decrease. Excessive iron ion content in mice leads to increased oxidative stress. Oxidative stress mediates in bone loss through changes in bone remodelling. In rats with severe anaemia due to iron deficiency, the concentration of the N-terminal pro-collagen type I was low, which reduced bone formation and mineralisation [9]. These parameters returned to normal values after a diet with a normal iron content. There is no data, given the importance of iron for bone health in humans. Whereas, osteopenia was observed in patients with genetically determined hemochromatosis, and a very high iron content in tissues. Thus, the

**35**

*Analysis of Occurrence of Elements in Tissues of the Knee Joint*

bone health at a time when peak bone mass is achieved [9, 10].

protective or destructive effects of iron on bones depend on its concentration. Iron deficiency related anaemia is still important for public health. Deficiencies of iron ions in women of childbearing age and in adolescents may also have an effect on

Chromium in living organisms occurs as a trace element, yet its presence is extremely important. According to research on osteoblasts, chromium suppresses the level of osteocalcin the too high levels of which may accompany the osteoporosis process. It does not inhibit collagen production [11]. Reduced bone resorption in postmenopausal women has also been observed, and thus the prevention of osteoporosis together with an adequate level of chromium replenishment [12]. Due to its properties, chrome has been used in the production of orthopaedic implants. There are still concerns about local toxicity of chromium contained in prostheses, many studies address this issue [13–15]. Trace elements have a significant effect on the growth, development and condition of bone tissues. Changes in the mineral composition of bone tissues may cause degenerative changes and fractures. According to recent epidemiological data, the incidence of osteoarthritis around the world varies between 2 and 15% of the population. In Poland, this disease affects approximately 7–8 million people; in 40% of cases, degenerative changes are located within the hip joint and in 25% in the knee joint [8]. Trace elements have a significant effect on the growth, development and condition of bone tissues. Changes in the mineral composition of bone tissues may cause degenerative changes and fractures. The deficiency of some trace elements such as zinc, selenium, copper may increase the risk of bone resorption, inhibiting bone growth. Environmental exposure to lead and cadmium is associated with the risk of a number of chronic diseases related to ageing, cardiovascular diseases, chronic renal failure and osteoporosis. The deficiency of some trace elements such as zinc, selenium, copper may increase the risk of bone resorption, inhibiting bone growth. Among metals that can affect the skeleton, there is no significant distinction between cellular effects and effects caused by the accumulation in the mineral or extracellular matrix. A given metal will undergo significant accumulation in the mineral and cause changes in its properties. Therefore, by studying specific mechanisms of the accumulation of metal ions, more information about bone mineralisation processes can be obtained. From a practical point of view, the influence of metal ions is often studied

The goal of the manuscript was to determine the content of trace elements (Cd, Ni, Fe, Cr, Sr., Cu, Zn, Pb) and structural elements (Ca, P, Mg) in knee joint tissues. A diversified content of elements was determined in particular elements of the knee joint: tibia, femur and meniscus. Differences in the content of selected elements in the studied tissues between particular groups: women and men, smokers and non-smokers, inhabitants of cities and villages, people at risk of exposure and not exposed, patients operated on due to degenerative changes, and depending on age were considered in the research. The next stage of the research was a correlation analysis in the occurrence of elements, taking into account antagonistic and synergistic changes. For this purpose, various statistical methods were used to determine the dependence between the content of elements in bone

Tissues that were examined were acquired intraoperatively during knee arthroplasty procedures based on the consent of the Bioethical Commission 2/2013 of 18.06.2013. The studied population consisted of women (n = 36) and men (n = 14)

tissues, e.g. main factors analysis and group similarity analysis.

*DOI: http://dx.doi.org/10.5772/intechopen.95418*

in relation to applied implants and prostheses.

**2. Research goal**

#### *Analysis of Occurrence of Elements in Tissues of the Knee Joint DOI: http://dx.doi.org/10.5772/intechopen.95418*

*Trace Elements and Their Effects on Human Health and Diseases*

building and also stops bone resorption in humans [8].

Iron is a cofactor in many enzymes and cells in redox reactions. Low levels of iron ions can be detrimental to cells, while an excess of iron ions can lead to the production of reactive oxygen species through the Fenton reaction. The cellular iron content is strictly regulated by homeostatic mechanisms to maintain the right amount of iron in cells. Nickel ions and other divalent metals can compete with iron ions to enter the cell through DMT1 (divalent metal transporter 1) because they have similar ionic radii. Therefore, metal ions can affect many other processes dependent on the presence of iron in cells. As an enzymatic cofactor, iron is involved in bone matrix synthesis (activation of lysyl hydroxylase) and in 25-hydroxy-cholecalciferol hydroxylase synthesis. What is more, thanks to active vitamin D, iron ions stimulate the absorption of calcium ions in the intestine. Iron deficiencies in rats led to poor mineralisation of skeletons and pathological changes in the micro-architecture of the spongy substance. In turn, administration of estrogens' increases the accumulation of iron in hamsters and facilitates the uptake of iron ions by lymphocytes in culture. The deficiency of iron ions in young rats leads to a decrease in the mechanical strength of femurs and the cortical bone. In severe iron deficiency, both bone strength and mineral density decrease. Excessive iron ion content in mice leads to increased oxidative stress. Oxidative stress mediates in bone loss through changes in bone remodelling. In rats with severe anaemia due to iron deficiency, the concentration of the N-terminal pro-collagen type I was low, which reduced bone formation and mineralisation [9]. These parameters returned to normal values after a diet with a normal iron content. There is no data, given the importance of iron for bone health in humans. Whereas, osteopenia was observed in patients with genetically determined hemochromatosis, and a very high iron content in tissues. Thus, the

The mineral structure of bones is never static, it is a living structure, reacting and adapting to load and having the ability to remodel. Skeletal cells work continuously to maintain the remodelling process therefore they are in a constant state of dynamic balance both in the sense of composition and structure, and they react to external mechanical forces. The remodelling processes that occur in the bone tissue allow for a proper functioning of this tissue, as well as for inclusion of additional elements, toxic ones included, in the remodelled bone, and they affect the metabolic processes occurring therein. This may result in disturbances in the osteoarticular system, manifested by changes in the bone tissue and within other organs. Maintaining all the characteristics of the knee joint is possible thanks to the balance of many elements of the bone tissue that are responsible for individual properties of bones, which in turn create separate joints. Elements occurring in large quantities, e.g. calcium, magnesium, phosphorus, and those with low content - the so-called trace elements, e.g. Strontium, can be found in the bone tissue. Regardless of the amount of elements contained in the bone tissue, all of them are important and play significant roles. Mostly, calcium and phosphorus are part of bone hydroxyapatite. However, during the mineralisation process, metal ions present in the blood plasma may be built into the bone tissue, and their uptake will depend on the affinity of a given metal for mineral and extracellular matrix, as well as the concentration of metal ions in the blood plasma, and the degree of skeletal mineralisation. Strontium present in the knee joint is a trace element, although it plays a special role in the bone remodelling process of the human body [5, 6]. It is accumulated mainly in bones due to the high similarity to calcium [7] but unlike it, it is absorbed from food much less efficiently and in a larger percentage it is excreted [8]. Previous in vivo studies demonstrate the effect of strontium on improvement of mechanical characteristics of bones [7]. These studies also proved the effectiveness of treatment with small doses of strontium in the form of strontium chloride. Their conclusion is that 9–26 week Sr. therapy activates bone

**34**

protective or destructive effects of iron on bones depend on its concentration. Iron deficiency related anaemia is still important for public health. Deficiencies of iron ions in women of childbearing age and in adolescents may also have an effect on bone health at a time when peak bone mass is achieved [9, 10].

Chromium in living organisms occurs as a trace element, yet its presence is extremely important. According to research on osteoblasts, chromium suppresses the level of osteocalcin the too high levels of which may accompany the osteoporosis process. It does not inhibit collagen production [11]. Reduced bone resorption in postmenopausal women has also been observed, and thus the prevention of osteoporosis together with an adequate level of chromium replenishment [12]. Due to its properties, chrome has been used in the production of orthopaedic implants. There are still concerns about local toxicity of chromium contained in prostheses, many studies address this issue [13–15]. Trace elements have a significant effect on the growth, development and condition of bone tissues. Changes in the mineral composition of bone tissues may cause degenerative changes and fractures. According to recent epidemiological data, the incidence of osteoarthritis around the world varies between 2 and 15% of the population. In Poland, this disease affects approximately 7–8 million people; in 40% of cases, degenerative changes are located within the hip joint and in 25% in the knee joint [8]. Trace elements have a significant effect on the growth, development and condition of bone tissues. Changes in the mineral composition of bone tissues may cause degenerative changes and fractures. The deficiency of some trace elements such as zinc, selenium, copper may increase the risk of bone resorption, inhibiting bone growth.

Environmental exposure to lead and cadmium is associated with the risk of a number of chronic diseases related to ageing, cardiovascular diseases, chronic renal failure and osteoporosis. The deficiency of some trace elements such as zinc, selenium, copper may increase the risk of bone resorption, inhibiting bone growth. Among metals that can affect the skeleton, there is no significant distinction between cellular effects and effects caused by the accumulation in the mineral or extracellular matrix. A given metal will undergo significant accumulation in the mineral and cause changes in its properties. Therefore, by studying specific mechanisms of the accumulation of metal ions, more information about bone mineralisation processes can be obtained. From a practical point of view, the influence of metal ions is often studied in relation to applied implants and prostheses.

#### **2. Research goal**

The goal of the manuscript was to determine the content of trace elements (Cd, Ni, Fe, Cr, Sr., Cu, Zn, Pb) and structural elements (Ca, P, Mg) in knee joint tissues. A diversified content of elements was determined in particular elements of the knee joint: tibia, femur and meniscus. Differences in the content of selected elements in the studied tissues between particular groups: women and men, smokers and non-smokers, inhabitants of cities and villages, people at risk of exposure and not exposed, patients operated on due to degenerative changes, and depending on age were considered in the research. The next stage of the research was a correlation analysis in the occurrence of elements, taking into account antagonistic and synergistic changes. For this purpose, various statistical methods were used to determine the dependence between the content of elements in bone tissues, e.g. main factors analysis and group similarity analysis.

Tissues that were examined were acquired intraoperatively during knee arthroplasty procedures based on the consent of the Bioethical Commission 2/2013 of 18.06.2013. The studied population consisted of women (n = 36) and men (n = 14) from 41 up to 82 years of age. Those people lived mainly in the areas of southern Poland, with the largest number of people coming from Upper Silesia.

#### **3. Discussion of research results**

Bone tissue has the ability to accumulate chemical elements and incorporate them into its structure, which is why it is often used to determine the impact of not only environmental but also occupational exposure.

Some metals such as zinc, iron and copper are closely related to human health because they are essential for maintaining normal physiological functions. However, heavy metal ions that are environmental pollutants show adverse health effects. Cadmium and lead can replace other elements that change the course of a number of biochemical reactions and can act as inhibitors, usually due to formation of complex compounds with sulphhydryl groups of proteins. Exposure to heavy metal compounds can affect genetic material and increase susceptibility to diseases. The World Health Organisation (WHO) classified some heavy metals such as cadmium, lead, mercury and arsenic as pollutants that need to be closely monitored [16]. The accumulation of an adequate amount of harmful heavy metal compounds in the human body changes the hormonal metabolism and narrows blood vessels. Metals are considered a risk factor for fractures and degenerative diseases in osteoporosis [17, 18].

As results from tests for the presence of elements, the average content of strontium in the entire knee joint reaches 17,50 mg/kg. There are no significant differences between Sr. depending on gender. The following strontium content can be distinguished in individual elements of the knee joint: meniscus - 1,44 mg/kg; femur - 24,60 mg/kg; while in tibia - 26,64 mg/kg. It is easy to see that the highest level of strontium in the examined knee joint is in the tibia, the lowest in the meniscus. The effect of smoking on the level of the element determined in the knee joint was also examined. The obtained results confirm a high level of Sr. in smokers compared to non-smokers, however the differences shown are not statistically significant.

Phosphorus present in the knee joint is the main component of all tissues of the human body. It plays a key role in mineralisation of the skeleton [19]. The content of phosphorus in individual bones is different. For example, in the femoral and tibial bones, the level of this element is 24 times higher than in the meniscus. The average content of phosphorus in the knee joint is 36,04 mg/kg. The obtained result is almost twice higher compared to strontium discussed above [20]. The level of phosphorus determined in the knee joint is slightly predominant among men, depending on gender groups. The differences generated in the study did not reach the statistically significant level.

In the case of Pb, Ca, P, Na, Mg content, significant statistical differences occurred in individual elements of the knee joint (Kruskal-Wallis ANOVA test, p < 0,001). The lead content in the meniscus was 0,32 mg/g, in the tibia 2,67 mg/g, and in the femur 2,64 mg/g. The highest calcium content was in the tibia – 122,57 g/kg, and in the femur – 112,45 g/kg, in the meniscus the content was about 23 times lower and was 5,08 g/kg. The phosphorus content was similar, the highest in the tibia – 55,34 g/kg and in the femur – 50,56 g/kg, and the lowest in the meniscus – 2,21 g/kg. In the case of sodium in the tibial and femoral bones, the content was 5,50 and 5,56, and in the meniscus – 2,11 g/kg. The magnesium content was as follows: tibia 1,55, femur 1,42, and meniscus 0,10 mg/kg. The highest content of Sr., Pb, Ca, P, Na, Mg was in the tibia and the smallest in the meniscus. Statistically significant differences between men and women occurred only in the tibia and related to lead content (U Mann–Whitney U test, p = 0,011).

**37**

in **Figure 1**.

*Analysis of Occurrence of Elements in Tissues of the Knee Joint*

individual elements of the knee joint and sex and smoking.

prosthesis implanted (32,81 μg/g < 36,96 μg/g).

The influence of tobacco smoking on the content of strontium, lead, calcium, phosphorus, sodium and magnesium did not cause statistically significant differences. Among those elements, only the content of strontium was greater in people who smoked tobacco. Whereas, the contents of lead, calcium, phosphorus, sodium and magnesium were higher in non-smokers. There were no differences between

Another element that is significant and present in the knee joint is iron. As an enzyme cofactor, it participates in the formation of bone matrix. Iron deficiency in the human body, especially a significant deficiency, leads to a decrease in both density and bone strength. Deficiency, as well as excess of Fe adversely affects the system. Too high value of the element increases oxidative stress [21]. According to research on rats, iron deficiency leads to diseased changes in the spongy substance of discs and weakened skeletal mineralisation. For this reason, the mechanical strength of the femoral bones decreases considerably [22]. The effect of iron content on the health of human bones has not yet been studied. There are works on Fe that suggest that maintaining high levels of iron may help in the prevention of bone fractures in older women [21]. Studies concerning iron content in selected knee joint tissues are slightly different when compared to other elements studied. In the case of iron, its highest content was found in the femur, in which the level is 41,91 μg/g. The second, extreme iron value was determined at 27,04 μg/g in the tibial bone. An intermediate level was determined in the meniscus - i.e. 38,68 μg/g. There were no statistically significant differences between the tibial and the femoral bones in gender groups, but the marked values among women predominated over those marked in the opposite sex. The marked Fe content in the meniscus was different in the group of men. Differences turned out to be statistically insignificant in this case as well. The studies also included iron levels in the knee joint tissues in relation to smokers and non-smokers. In non-smokers, high Fe levels (39,11 μg/g) were determined compared to smokers (25,47 μg/g). The last examined dependence related to the iron level in knee joint tissues was the operation of knee arthroplasty. The study of this correlation shows that Fe levels are lower in patients with a knee

Chromium is found in living organisms as a trace element. Its presence is extremely important, it inhibits the level of osteocalcin the high level of which accompanies the osteoporosis process, as results from studies on osteoblasts. Reduced bone resorption in postmenopausal women has also been observed, and thus the prevention of osteoporosis together with an adequate level of chromium replenishment [23]. Thanks to its properties, Cr has been used in the production of orthopaedic implants. Nevertheless, there are many concerns about possible local toxicity of prostheses with its content. This problem has been addressed in many manuscripts [13]. As in the case of iron, the highest chromium content in the knee joint occurs in the femur (1,64 μg/g). Slightly lower levels are

found in the tibial bone (1,27 μg/g), with the lowest in the meniscus (1,18 μg/g). A slightly higher level of Cr is recorded among men however without statistical significance. An interesting examined dependence is the increase in chromium levels in the knee joint along with age. Among the respondents, its highest level (1,78 μg/g) was recorded in people over 70 years of age. The Cr content of the knee joint in residents of cities is almost twice as high (2,30 μg/g) compared to inhabitants of villages (1,20 μg/g). In smokers, a higher level of some metals in the body can be seen which is due to their presence in tobacco smoke. In the case of chromium, this dependence was not confirmed. Its lower level was examined in people smoking cigarettes (1,00 μg/g) compared to non-smokers (1,47 μg/g). The content of elements in the knee joint tissues in female and male were showed

*DOI: http://dx.doi.org/10.5772/intechopen.95418*

*Trace Elements and Their Effects on Human Health and Diseases*

only environmental but also occupational exposure.

**3. Discussion of research results**

diseases in osteoporosis [17, 18].

the statistically significant level.

from 41 up to 82 years of age. Those people lived mainly in the areas of southern

Bone tissue has the ability to accumulate chemical elements and incorporate them into its structure, which is why it is often used to determine the impact of not

Some metals such as zinc, iron and copper are closely related to human health

As results from tests for the presence of elements, the average content of strontium in the entire knee joint reaches 17,50 mg/kg. There are no significant differences between Sr. depending on gender. The following strontium content can be distinguished in individual elements of the knee joint: meniscus - 1,44 mg/kg; femur - 24,60 mg/kg; while in tibia - 26,64 mg/kg. It is easy to see that the highest level of strontium in the examined knee joint is in the tibia, the lowest in the meniscus. The effect of smoking on the level of the element determined in the knee joint was also examined. The obtained results confirm a high level of Sr. in smokers compared to non-smokers, however the differences shown are not statistically significant.

Phosphorus present in the knee joint is the main component of all tissues of the human body. It plays a key role in mineralisation of the skeleton [19]. The content of phosphorus in individual bones is different. For example, in the femoral and tibial bones, the level of this element is 24 times higher than in the meniscus. The average content of phosphorus in the knee joint is 36,04 mg/kg. The obtained result is almost twice higher compared to strontium discussed above [20]. The level of phosphorus determined in the knee joint is slightly predominant among men, depending on gender groups. The differences generated in the study did not reach

In the case of Pb, Ca, P, Na, Mg content, significant statistical differences occurred in individual elements of the knee joint (Kruskal-Wallis ANOVA test, p < 0,001). The lead content in the meniscus was 0,32 mg/g, in the tibia 2,67 mg/g, and in the femur 2,64 mg/g. The highest calcium content was in the tibia – 122,57 g/kg, and in the femur – 112,45 g/kg, in the meniscus the content was about 23 times lower and was 5,08 g/kg. The phosphorus content was similar, the highest in the tibia – 55,34 g/kg and in the femur – 50,56 g/kg, and the lowest in the meniscus – 2,21 g/kg. In the case of sodium in the tibial and femoral bones, the content was 5,50 and 5,56, and in the meniscus – 2,11 g/kg. The magnesium content was as follows: tibia 1,55, femur 1,42, and meniscus 0,10 mg/kg. The highest content of Sr., Pb, Ca, P, Na, Mg was in the tibia and the smallest in the meniscus. Statistically significant differences between men and women occurred only in the tibia and related to lead content (U Mann–Whitney U

because they are essential for maintaining normal physiological functions. However, heavy metal ions that are environmental pollutants show adverse health effects. Cadmium and lead can replace other elements that change the course of a number of biochemical reactions and can act as inhibitors, usually due to formation of complex compounds with sulphhydryl groups of proteins. Exposure to heavy metal compounds can affect genetic material and increase susceptibility to diseases. The World Health Organisation (WHO) classified some heavy metals such as cadmium, lead, mercury and arsenic as pollutants that need to be closely monitored [16]. The accumulation of an adequate amount of harmful heavy metal compounds in the human body changes the hormonal metabolism and narrows blood vessels. Metals are considered a risk factor for fractures and degenerative

Poland, with the largest number of people coming from Upper Silesia.

**36**

test, p = 0,011).

The influence of tobacco smoking on the content of strontium, lead, calcium, phosphorus, sodium and magnesium did not cause statistically significant differences. Among those elements, only the content of strontium was greater in people who smoked tobacco. Whereas, the contents of lead, calcium, phosphorus, sodium and magnesium were higher in non-smokers. There were no differences between individual elements of the knee joint and sex and smoking.

Another element that is significant and present in the knee joint is iron. As an enzyme cofactor, it participates in the formation of bone matrix. Iron deficiency in the human body, especially a significant deficiency, leads to a decrease in both density and bone strength. Deficiency, as well as excess of Fe adversely affects the system. Too high value of the element increases oxidative stress [21]. According to research on rats, iron deficiency leads to diseased changes in the spongy substance of discs and weakened skeletal mineralisation. For this reason, the mechanical strength of the femoral bones decreases considerably [22]. The effect of iron content on the health of human bones has not yet been studied. There are works on Fe that suggest that maintaining high levels of iron may help in the prevention of bone fractures in older women [21]. Studies concerning iron content in selected knee joint tissues are slightly different when compared to other elements studied. In the case of iron, its highest content was found in the femur, in which the level is 41,91 μg/g. The second, extreme iron value was determined at 27,04 μg/g in the tibial bone. An intermediate level was determined in the meniscus - i.e. 38,68 μg/g. There were no statistically significant differences between the tibial and the femoral bones in gender groups, but the marked values among women predominated over those marked in the opposite sex. The marked Fe content in the meniscus was different in the group of men. Differences turned out to be statistically insignificant in this case as well. The studies also included iron levels in the knee joint tissues in relation to smokers and non-smokers. In non-smokers, high Fe levels (39,11 μg/g) were determined compared to smokers (25,47 μg/g). The last examined dependence related to the iron level in knee joint tissues was the operation of knee arthroplasty. The study of this correlation shows that Fe levels are lower in patients with a knee prosthesis implanted (32,81 μg/g < 36,96 μg/g).

Chromium is found in living organisms as a trace element. Its presence is extremely important, it inhibits the level of osteocalcin the high level of which accompanies the osteoporosis process, as results from studies on osteoblasts. Reduced bone resorption in postmenopausal women has also been observed, and thus the prevention of osteoporosis together with an adequate level of chromium replenishment [23]. Thanks to its properties, Cr has been used in the production of orthopaedic implants. Nevertheless, there are many concerns about possible local toxicity of prostheses with its content. This problem has been addressed in many manuscripts [13]. As in the case of iron, the highest chromium content in the knee joint occurs in the femur (1,64 μg/g). Slightly lower levels are found in the tibial bone (1,27 μg/g), with the lowest in the meniscus (1,18 μg/g). A slightly higher level of Cr is recorded among men however without statistical significance. An interesting examined dependence is the increase in chromium levels in the knee joint along with age. Among the respondents, its highest level (1,78 μg/g) was recorded in people over 70 years of age. The Cr content of the knee joint in residents of cities is almost twice as high (2,30 μg/g) compared to inhabitants of villages (1,20 μg/g). In smokers, a higher level of some metals in the body can be seen which is due to their presence in tobacco smoke. In the case of chromium, this dependence was not confirmed. Its lower level was examined in people smoking cigarettes (1,00 μg/g) compared to non-smokers (1,47 μg/g). The content of elements in the knee joint tissues in female and male were showed in **Figure 1**.

Apart from the elements discussed above, the presence of nickel, cadmium, zinc and copper in the knee joint can be distinguished. Many factors affect their level. These include: the type of tissue being examined, gender, place of residence, nicotinism, age, occupational exposure. The lowest content in knee joint tissues is shown by cadmium. Nickel is characterised by a higher level in women's knee joints (tibia – 0,29 μg/g, femur – 0,36 μg/g, meniscus – 0,69 μg/g) in relation to men (tibia – 0,22 μg/g, femur – 0,28 μg/g, meniscus – 0,42 μg/g). The lowest percentage of copper in the knee joint in women concerns the femur (0,36 μg/g), and in men

**39**

**Author details**

**4. Conclusions**

Wojciech Roczniak1

and Barbara Brodziak Dopierała2

provided the original work is properly cited.

between nickel and copper.

\*, Magdalena Babuśka Roczniak1

of Laboratory Medicine, Medical University of Silesia, Sosnowiec, Poland

\*Address all correspondence to: wojciech\_roczniak@interia.pl

women and men in the studied knee joint components.

1 The Jan Grodek Higher Vocational State School, Medical Institute, Sanok, Poland

2 Department of Toxicology and Bioanalysis, School of Pharmacy with the Division

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Elżbieta Cipora1

*Analysis of Occurrence of Elements in Tissues of the Knee Joint*

the tibial bone (0,31 μg/g). In the studied joint, the element that has an advantage over the others is zinc. In smokers, an increased level of cadmium in knee joint tissues is observed due to its content in tobacco smoke. Zinc affects the condition, growth and development of bone tissue. The deficiency of zinc or copper leads to an increased bone resorption and thus inhibits their growth. What is more, zinc is responsible for the activity of vitamin D. Its deficiency leads to osteoporosis.

Keeping the balance in the mineral composition of bone tissue is very important and any deviations from the norm may cause degenerative changes and fractures [24].

The results of the presented research results indicate that the bone tissue of the femur and the tibia of the knee joint can be used to determine the content of such elements as lead, cadmium, chromium, zinc, magnesium, potassium and calcium. There was 24 times more phosphorus, 23 times more calcium, 18 times more strontium, 15 times magnesium, 8 times lead, and 3 times sodium in the femur and the tibia compared to the meniscus. However, copper and nickel showed a high content in connective tissue (meniscus) compared to bone tissue (tibia and femur). High values of metals can affect the structure of bone tissue and cause a change in composition and its properties. One of the most common correlations described in the literature on the subject has been confirmed - it is a synergistic correlation

The influence of tobacco smoking on the content of strontium, lead, calcium, phosphorus, sodium and magnesium has not been confirmed. Non-smokers showed a high iron content in knee joint tissues compared to smokers. There were no statistically significant differences in the content of cadmium, nickel, copper and zinc in

With age, an increase in the content of chromium in knee joint tissues was observed, while gender, place of residence and occupational exposure had no effect.

*DOI: http://dx.doi.org/10.5772/intechopen.95418*

*Analysis of Occurrence of Elements in Tissues of the Knee Joint DOI: http://dx.doi.org/10.5772/intechopen.95418*

the tibial bone (0,31 μg/g). In the studied joint, the element that has an advantage over the others is zinc. In smokers, an increased level of cadmium in knee joint tissues is observed due to its content in tobacco smoke. Zinc affects the condition, growth and development of bone tissue. The deficiency of zinc or copper leads to an increased bone resorption and thus inhibits their growth. What is more, zinc is responsible for the activity of vitamin D. Its deficiency leads to osteoporosis.

Keeping the balance in the mineral composition of bone tissue is very important and any deviations from the norm may cause degenerative changes and fractures [24].

#### **4. Conclusions**

*Trace Elements and Their Effects on Human Health and Diseases*

Apart from the elements discussed above, the presence of nickel, cadmium, zinc and copper in the knee joint can be distinguished. Many factors affect their level. These include: the type of tissue being examined, gender, place of residence, nicotinism, age, occupational exposure. The lowest content in knee joint tissues is shown by cadmium. Nickel is characterised by a higher level in women's knee joints (tibia – 0,29 μg/g, femur – 0,36 μg/g, meniscus – 0,69 μg/g) in relation to men (tibia – 0,22 μg/g, femur – 0,28 μg/g, meniscus – 0,42 μg/g). The lowest percentage of copper in the knee joint in women concerns the femur (0,36 μg/g), and in men

*The content of elements in the knee joint tissues in female and male.*

**38**

**Figure 1.**

The results of the presented research results indicate that the bone tissue of the femur and the tibia of the knee joint can be used to determine the content of such elements as lead, cadmium, chromium, zinc, magnesium, potassium and calcium. There was 24 times more phosphorus, 23 times more calcium, 18 times more strontium, 15 times magnesium, 8 times lead, and 3 times sodium in the femur and the tibia compared to the meniscus. However, copper and nickel showed a high content in connective tissue (meniscus) compared to bone tissue (tibia and femur). High values of metals can affect the structure of bone tissue and cause a change in composition and its properties. One of the most common correlations described in the literature on the subject has been confirmed - it is a synergistic correlation between nickel and copper.

The influence of tobacco smoking on the content of strontium, lead, calcium, phosphorus, sodium and magnesium has not been confirmed. Non-smokers showed a high iron content in knee joint tissues compared to smokers. There were no statistically significant differences in the content of cadmium, nickel, copper and zinc in women and men in the studied knee joint components.

With age, an increase in the content of chromium in knee joint tissues was observed, while gender, place of residence and occupational exposure had no effect.

#### **Author details**

Wojciech Roczniak1 \*, Magdalena Babuśka Roczniak1 , Elżbieta Cipora1 and Barbara Brodziak Dopierała2

1 The Jan Grodek Higher Vocational State School, Medical Institute, Sanok, Poland

2 Department of Toxicology and Bioanalysis, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia, Sosnowiec, Poland

\*Address all correspondence to: wojciech\_roczniak@interia.pl

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Kapandji A.I.: Anatomia funkcjonalna stawów. Kończyna dolna TOM 2. Elsevier Urban & Partner, Wrocław 2013.

[2] Nordin M., Frankel V.H.: Basic biomechanics of the musculoskeletal system. 4th edition. Wolter Kluwer; Lippincott Wiliams & Wilkins 2012.

[3] Lemiesz G.I.: The effectiveness of rehabilitation procedure after the reconstruction of the anterior cruciate ligament according to the Norwegian protocol. Pol. Ann. Med. 2011; 18(1): 82-95.

[4] Trzaska T.: Aktualne metody rekonstrukcji więzadła krzyżowego przedniego. Medicina Sportiva 2012; 6: 19-22.

[5] Brodziak-Dopierała, B.; Kwapuliński, J.; Kusz, D.; Gajda, Z.; Sobczyk, K.: Interactions between concentrations of chemical elements in human femoral heads. Arch. Environ. Contam. Toxicol. 2009; 57: 203-210.

[6] Branca, F.; Vatuena, S.: Calcium, physical activity and bone health— Building bones for stronger future. Public Health Nutr. 2001; 4: 117-123.

[7] Nordberg, G.F.; Fowler, B.A.; Nordberg, M.; Friberg, L.T.: Handbook on the Toxicology of Metals. Elsevier: London, UK, 2008.

[8] Uenishi, K.: Nutrition and bone health. Conclus. Clin. Calcium 2010; 20: 940-943.

[9] Diaz-Castro, J.; Lopez-Frias, M.R.; Campos, M.S.; Lopez-Frias, M.; Alferez, M.J.M.; Nestares, T.; Ojeda, M.L.; Lopez-Aliaga, I.: Severe nutritional iron-deficiency anemia has a negative effect on some bone turnover biomarkers in rats. Eur. J. Nutr. 2012; 51: 241-247.

[10] Balogh, E.; Paragh, G.; Jeney V. Influence of iron on bone homeostasis. Pharmaceuticals 2018; 11(4): 107.

[11] Allen, M.J.; Myer, B.J.; Millett, P.J.; Rushton, N.: The effects of particulate cobalt, chromium and cobalt-chromium alloy on human osteoblast-like cells in vitro. J. Bone Jt. Surg. Br. 1997; 79: 475-482.

[12] McCarty, M.F.: Anabolic effects of insulin on bone suggest a role for chromium picolinate in preservation of bone density. Med. Hypotheses 1995; 45: 241-246.

[13] Scharf, B.; Clement, C.C.; Zolla, V.; Perino, G.; Yan, B.; Elci, S.G.; Purdue, E.; Goldring, S.;Macaluso, F.; Cobelli, N.; Vachet, R.W.; Santambrogio, L.: Molecular analysis of chromium and cobalt-related toxicity. Sci. Rep. 2014; 4: 5729.

[14] Wang, J.Y.; Wicklund, B.H.; Gustilo, R.B.; Tsukayama, D.T.: Titanium, chromium and cobalt ions modulate the release of bone-associated cytokines by human monocytes/ macrophages in vitro. Biomaterials 1996; 17: 2233-2240.

[15] Rakow, A.; Schoon, J.; Dienelt, A.; John, T.; Textor, M.; Duda, G.; Perka, C.; Schulze, F.; Ode, A.: Influence of particulate and dissociated metal-on-metal hip endoprosthesis wear on mesenchymal stromal cells in vivo and in vitro. Biomaterials 2016; 98: 31-40.

[16] Rebolledo, J.; Fierens, S.; Versporten, A.: Brits, E.;, De Plaen, P.; Nieuwenhuyse, A.V.: Human biomonitoring on heavy metals in Ath: methodological aspects. Arch. Public Health 2011; 69: 10.

[17] Kido, S.: Secondary osteoporosis or secondary contributors to bone loss in fracture. Bone metabolism and heavy

**41**

*Analysis of Occurrence of Elements in Tissues of the Knee Joint*

*DOI: http://dx.doi.org/10.5772/intechopen.95418*

metals (cadmium and iron). Clin. Calcium 2013; 23: 1299-1306.

2016; 23(4): 223-231.

2001; 28: 446-453.

1555-1561.

Press: Cambridge, UK, 2007.

[21] Zofková. I.; Nemcikova, P., Matucha, P.: Trace elements and bone health. Clin. Chem. Lab. Med. 2013; 51:

[22] Parelman, M.; Stoecker, B.; Baker, A, Medeiros *D. iron* restriction negatively affects bone in female rats and mineralization of hFOB osteoblast cells. Exp. Biol. Med. 2006; 231: 378-386.

[23] Brodziak-Dopierała B., Roczniak W., Jakóbik-Kolon A., Kluczka J., Koczy B., Kwapuliński J., Babuśka-Roczniak M.: Correlations between iron kontent in knee joint tissues and chosen indices of peripheral blond morphology. Adv. Clin. Exp. Med.

2017; 26 (7): 1077-1083.

[24] Brodziak-Dopierała, B.,

Kwapuliński, J., Sobczyk K., Wiechuła, D: Distribution of magnesium, calcium, sodium and potassium in tissues of the hip joint. Magnes. Res. 2013; 26: 125-131.

[18] Hee-Sook, L., Hae-Hyeog, L., Tae-Hee, K., Bo-Ra L.: Relationship between heavy metal exposure and bone mineral density in Korean adult. J. Bone Metab.

[19] Carter, D.R.; Beaupré, G.S.: Skeletal function and form: mechanobiology of skeletal development, aging and regeneration. Cambridge University

[20] Dahl, S.G.; Allain, P.; Marie, P.J.; Mauras, Y.; Boivin, G.; Ammann, P.; Tsouderos, Y.; Delmas, P.D.; Christiansen, C.: Incorporation and distribution of strontium in bone. Bone. *Analysis of Occurrence of Elements in Tissues of the Knee Joint DOI: http://dx.doi.org/10.5772/intechopen.95418*

metals (cadmium and iron). Clin. Calcium 2013; 23: 1299-1306.

[18] Hee-Sook, L., Hae-Hyeog, L., Tae-Hee, K., Bo-Ra L.: Relationship between heavy metal exposure and bone mineral density in Korean adult. J. Bone Metab. 2016; 23(4): 223-231.

[19] Carter, D.R.; Beaupré, G.S.: Skeletal function and form: mechanobiology of skeletal development, aging and regeneration. Cambridge University Press: Cambridge, UK, 2007.

[20] Dahl, S.G.; Allain, P.; Marie, P.J.; Mauras, Y.; Boivin, G.; Ammann, P.; Tsouderos, Y.; Delmas, P.D.; Christiansen, C.: Incorporation and distribution of strontium in bone. Bone. 2001; 28: 446-453.

[21] Zofková. I.; Nemcikova, P., Matucha, P.: Trace elements and bone health. Clin. Chem. Lab. Med. 2013; 51: 1555-1561.

[22] Parelman, M.; Stoecker, B.; Baker, A, Medeiros *D. iron* restriction negatively affects bone in female rats and mineralization of hFOB osteoblast cells. Exp. Biol. Med. 2006; 231: 378-386.

[23] Brodziak-Dopierała B., Roczniak W., Jakóbik-Kolon A., Kluczka J., Koczy B., Kwapuliński J., Babuśka-Roczniak M.: Correlations between iron kontent in knee joint tissues and chosen indices of peripheral blond morphology. Adv. Clin. Exp. Med. 2017; 26 (7): 1077-1083.

[24] Brodziak-Dopierała, B., Kwapuliński, J., Sobczyk K., Wiechuła, D: Distribution of magnesium, calcium, sodium and potassium in tissues of the hip joint. Magnes. Res. 2013; 26: 125-131.

**40**

241-247.

*Trace Elements and Their Effects on Human Health and Diseases*

[10] Balogh, E.; Paragh, G.; Jeney V. Influence of iron on bone homeostasis. Pharmaceuticals 2018; 11(4): 107.

[11] Allen, M.J.; Myer, B.J.; Millett, P.J.; Rushton, N.: The effects of particulate cobalt, chromium and cobalt-chromium alloy on human osteoblast-like cells in vitro. J. Bone Jt. Surg. Br. 1997; 79:

[12] McCarty, M.F.: Anabolic effects of insulin on bone suggest a role for chromium picolinate in preservation of bone density. Med. Hypotheses 1995; 45:

[13] Scharf, B.; Clement, C.C.; Zolla, V.; Perino, G.; Yan, B.; Elci, S.G.; Purdue, E.; Goldring, S.;Macaluso, F.; Cobelli, N.; Vachet, R.W.; Santambrogio, L.: Molecular analysis of chromium and cobalt-related toxicity. Sci. Rep. 2014; 4:

[14] Wang, J.Y.; Wicklund, B.H.; Gustilo, R.B.; Tsukayama, D.T.: Titanium, chromium and cobalt ions modulate the release of bone-associated

cytokines by human monocytes/ macrophages in vitro. Biomaterials

Dienelt, A.; John, T.; Textor, M.; Duda, G.; Perka, C.; Schulze, F.; Ode, A.: Influence of particulate and dissociated metal-on-metal hip endoprosthesis wear on mesenchymal stromal cells in vivo and in vitro. Biomaterials 2016; 98:

[17] Kido, S.: Secondary osteoporosis or secondary contributors to bone loss in fracture. Bone metabolism and heavy

1996; 17: 2233-2240.

[15] Rakow, A.; Schoon, J.;

[16] Rebolledo, J.; Fierens, S.; Versporten, A.: Brits, E.;, De Plaen, P.; Nieuwenhuyse, A.V.: Human biomonitoring on heavy metals in Ath: methodological aspects. Arch. Public

Health 2011; 69: 10.

475-482.

241-246.

5729.

31-40.

[1] Kapandji A.I.: Anatomia

Wrocław 2013.

**References**

82-95.

19-22.

2009; 57: 203-210.

London, UK, 2008.

940-943.

funkcjonalna stawów. Kończyna dolna TOM 2. Elsevier Urban & Partner,

[2] Nordin M., Frankel V.H.: Basic biomechanics of the musculoskeletal system. 4th edition. Wolter Kluwer; Lippincott Wiliams & Wilkins 2012.

[3] Lemiesz G.I.: The effectiveness of rehabilitation procedure after the reconstruction of the anterior cruciate ligament according to the Norwegian protocol. Pol. Ann. Med. 2011; 18(1):

[4] Trzaska T.: Aktualne metody rekonstrukcji więzadła krzyżowego przedniego. Medicina Sportiva 2012; 6:

[5] Brodziak-Dopierała, B.; Kwapuliński, J.; Kusz, D.; Gajda, Z.; Sobczyk, K.: Interactions between concentrations of chemical elements in human femoral heads. Arch. Environ. Contam. Toxicol.

[6] Branca, F.; Vatuena, S.: Calcium, physical activity and bone health— Building bones for stronger future. Public Health Nutr. 2001; 4: 117-123.

[7] Nordberg, G.F.; Fowler, B.A.; Nordberg, M.; Friberg, L.T.: Handbook on the Toxicology of Metals. Elsevier:

[8] Uenishi, K.: Nutrition and bone health. Conclus. Clin. Calcium 2010; 20:

[9] Diaz-Castro, J.; Lopez-Frias, M.R.; Campos, M.S.; Lopez-Frias, M.; Alferez, M.J.M.; Nestares, T.; Ojeda, M.L.; Lopez-Aliaga, I.: Severe nutritional iron-deficiency anemia has a negative effect on some bone turnover biomarkers in rats. Eur. J. Nutr. 2012; 51:

**43**

**1. Introduction**

**Chapter 4**

Diseases

**Abstract**

*and Alok S. Tripathi*

Natural Iron Chelators as

*Naheed Waseem A. Sheikh, Satish B. Kosalge,* 

Potential Therapeutic Agents for

the Treatment of Iron Overload

*Tusharbindu R. Desai, Anil P. Dewani, Deepak S. Mohale* 

Iron overload disease is a group of heterogeneous disease, which is caused either due to hereditary or acquired condition. Excess of iron participate in redox reactions that catalyzes the generation of reactive oxygen species (ROS) and increases oxidative stress, which causes cellular damage and encourage the cell injury and cell death. The electronic databases of Scopus, PubMed and Google Scholar have been intensively searched for the research as well as review articles published with the full text available and with the key words such as natural iron chelating agent, synthetic iron chelating agents, iron overload disease, oxidative stress and antioxidant which were appearing in the title, abstract or keywords. In light of the literature review presented in this artial, based on meta-analyses, we suggest that iron chelating agents were used for the management of iron overload disease. These agents were having wide spectrum of activity, they were not only used for the management of iron overload disease but also used as anticancer and antioxidant in various oxidative stress mediated diseases. Last from many years Desferoxamine (DFO) was used as standard iron chelator but currently two new synthetic iron chelators such as Deferiprone (DFP) and Deferasirox (DFS) are available clinically. These clinically available synthetic iron chelators were having serious side effects and certain limitations. Phytochemicals such as flavonoids and polyphenols compounds were having iron chelating as well as antioxidant property with no or minimal side effects. Hence, this review provides an updates on natural iron chelation therapy for the safe and efficacious management of iron overload diseases.

**Keywords:** Natural iron chelating agents, synthetic iron chelating agents,

Iron is an essential element for all living organism. It participates in various biochemical reactions like oxygen transport, electron transfer, energy metabolism and DNA synthesis. The biological action of iron is largely due to its chemical properties as a transition metal, although these properties make it potentially toxic. Total body iron

iron overload disease, oxidative stress, antioxidant

#### **Chapter 4**

## Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload Diseases

*Naheed Waseem A. Sheikh, Satish B. Kosalge, Tusharbindu R. Desai, Anil P. Dewani, Deepak S. Mohale and Alok S. Tripathi*

#### **Abstract**

Iron overload disease is a group of heterogeneous disease, which is caused either due to hereditary or acquired condition. Excess of iron participate in redox reactions that catalyzes the generation of reactive oxygen species (ROS) and increases oxidative stress, which causes cellular damage and encourage the cell injury and cell death. The electronic databases of Scopus, PubMed and Google Scholar have been intensively searched for the research as well as review articles published with the full text available and with the key words such as natural iron chelating agent, synthetic iron chelating agents, iron overload disease, oxidative stress and antioxidant which were appearing in the title, abstract or keywords. In light of the literature review presented in this artial, based on meta-analyses, we suggest that iron chelating agents were used for the management of iron overload disease. These agents were having wide spectrum of activity, they were not only used for the management of iron overload disease but also used as anticancer and antioxidant in various oxidative stress mediated diseases. Last from many years Desferoxamine (DFO) was used as standard iron chelator but currently two new synthetic iron chelators such as Deferiprone (DFP) and Deferasirox (DFS) are available clinically. These clinically available synthetic iron chelators were having serious side effects and certain limitations. Phytochemicals such as flavonoids and polyphenols compounds were having iron chelating as well as antioxidant property with no or minimal side effects. Hence, this review provides an updates on natural iron chelation therapy for the safe and efficacious management of iron overload diseases.

**Keywords:** Natural iron chelating agents, synthetic iron chelating agents, iron overload disease, oxidative stress, antioxidant

#### **1. Introduction**

Iron is an essential element for all living organism. It participates in various biochemical reactions like oxygen transport, electron transfer, energy metabolism and DNA synthesis. The biological action of iron is largely due to its chemical properties as a transition metal, although these properties make it potentially toxic. Total body iron

content of an adult is 3–5 g (~ 55 mg/kg for male and ~ 45 mg/kg female). Majority of body iron is incorporated within hemoglobin (Hb) (60–70%) as circulating RBC; while about 20–30% of iron is present in the form of ferritin and hemosiderin as a spare iron in hepatocytes and reticuloendothelial (RE) macrophages. Remaining body iron is primarily located in myoglobin and enzymes such as cytochromes, peroxidases, catalases, xanthine oxidase and some mitochondrial enzymes. A healthy adult absorbs near about 1–2 mg/day of iron from the diet, which reimburses the non-specific loss of iron by exfoliation of intestinal epithelial cells. Moreover menstruating women additionally losses iron during the menstrual cycle. Erythropoiesis daily requires about 30 mg of iron, which is largely provided by the recycling of iron through RE macrophages.

The dietary iron is absorbed from the small intestine and the normal level of iron is regulated by feedback mechanism between its requirement and absorption. The dietary iron is present either as haeme or as inorganic ferric iron (Fe3+). Absorption of haeme iron takes place directly without the aid of active carrier transport. However, haeme iron is a minor source of dietary iron. The primary source of dietary iron is Fe3+ which has to be reduced to ferrous (Fe2+) form by acid reducing agents for efficient uptake. Iron is transported across the membrane by two distinct transporters. The divalent metal transporter 1 (DMT1) present at luminal membrane carries Fe2+ into the intestinal epithelial cell. This Fe2+ and iron released from the haeme is transported across the basolateral membrane by another iron transporter ferroportin (FP). Gut has a mechanism to prevent the entry of excess iron in the body. After reaching to the intestinal epithelial cell, iron is either transported to plasma or oxidized to Fe3+ and complexed with apoferritin to form ferritin, the cytosolic protein in which iron is stored. Ferritin usually remains stored in intestinal epithelial cells for 2–4 days after that the cells were shed off. This process is called as exfoliation. Whenever the body iron is low, the ferritin is either not formed or dissociates quickly to release iron. This released iron is transported to the blood [1].

The free form of iron is extremely toxic. The Fe2+ on entering into plasma it is rapidly oxidized to Fe3+ and complexed with apotransferrin (Apo-Tf) a glycoprotein to form transferrin (Tf). Two Fe3+ residues bound to one Tf molecule. This complex bound to membrane bound transferrin receptor 1 (Tfr1), present on erythropoietic and other cells. The Tf–Tfr1 complex is engulfed by receptor mediated endocytosis. The endosomes of erythropoietic and other cells become acidified through engulfed proton complex, which leads to conformational changes, which dissociates iron from the complex. The released Fe3+ is reduced to Fe2+ and transported out of the endosomes by DMT1. This released Fe2+ is utilized for hemoglobin synthesis or other biochemical process; Apo-Tf and Tfr1 are return to the cell surface for further cycles. In iron deficiency and haemolytic anemia, Tfr1 receptors up regulation take place at erythropoietic cells, but not at other cells. Under physiological conditions, all circulating iron is bound to transferrin. Nontransferrin bound iron (NTBI) can increase in a pathological condition like iron overload disease (**Figure 1**) [2, 3, 14].

Once the iron enters the cell, the fraction that is not needed for immediate use is stored by ferritin and haemosiderin in RE cells of liver, spleen and bone marrow. Iron status of the body regulates the synthesis of apoferritin. When the iron status is low, the iron regulating element (IRE) at DNA is blocked and synthesis of apoferritin is not taken place, whereas more Tf is produced. Moreover excess of apoferritin is synthesized to trap iron when the iron store is high [4, 5].

The plasma iron obtained from three primary sources, firstly from constant degradation of older RBC (lifespan ~120 days), secondly from stored iron from RE cells in liver, spleen and bone marrow while thirdly from intestinal absorption. The conservation and recycling of iron are necessary to reload the iron contained within Hb. The recycling takes place at macrophage, which phagocytes the RBC and liberates iron in haeme form by haeme oxygenase-1 [6].

**45**

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload…*

Daily excretion of iron in an adult male is approximately 1–2 mg, mostly as exfoliated intestinal epithelial cells, some RBCs and in bile. The primary route of excretion of iron is feces, while skin, urine and sweat are minor routes. In menstruating women, monthly menstrual loss of iron is about 0.5–1 mg/day. Excess of iron is required during last two trimesters of pregnancy for expansion of RBC mass,

The iron homeostasis maintained by its controlled absorption, recycling and storage. The iron is regulating peptide hormone, hepcidin produced mainly by the liver, whereas a smaller amount is produced in other organs like lung and heart plays a vital role in this regards. Hepcidin act by degradation of FP, an iron efflux transporter, concerned about transportation of iron across the basolateral membrane in the intestine. Iron overload increases whereas anemia and hypoxia decrease the synthesis of hepcidin [8]. Hepcidin synthesis is regulated by bone morphogenetic protein (BMP)/SMAD pathway via activation of hepcidin transcription. The loss of hepatic SMAD4 gene results in iron overload due to the failure of hepcidin-mediated degradation of FP [9].

The biochemical estimation of body iron status depends on serum based indica-

transfer to the fetus and to compensate the loss during delivery [7].

**2. Determination of serum iron**

*Iron absorption pathways by the enterocyte.*

tors, as follows

**Figure 1.**

1.Serum iron (SI)

2.Serum ferritin (SF)

3.Transferrin saturation

4.Soluble transferrin receptor (sTfR)

5.Erythrocyte protoporphyrin

*DOI: http://dx.doi.org/10.5772/intechopen.98749*

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload… DOI: http://dx.doi.org/10.5772/intechopen.98749*

**Figure 1.**

*Trace Elements and Their Effects on Human Health and Diseases*

is synthesized to trap iron when the iron store is high [4, 5].

ates iron in haeme form by haeme oxygenase-1 [6].

The plasma iron obtained from three primary sources, firstly from constant degradation of older RBC (lifespan ~120 days), secondly from stored iron from RE cells in liver, spleen and bone marrow while thirdly from intestinal absorption. The conservation and recycling of iron are necessary to reload the iron contained within Hb. The recycling takes place at macrophage, which phagocytes the RBC and liber-

content of an adult is 3–5 g (~ 55 mg/kg for male and ~ 45 mg/kg female). Majority of body iron is incorporated within hemoglobin (Hb) (60–70%) as circulating RBC; while about 20–30% of iron is present in the form of ferritin and hemosiderin as a spare iron in hepatocytes and reticuloendothelial (RE) macrophages. Remaining body iron is primarily located in myoglobin and enzymes such as cytochromes, peroxidases, catalases, xanthine oxidase and some mitochondrial enzymes. A healthy adult absorbs near about 1–2 mg/day of iron from the diet, which reimburses the non-specific loss of iron by exfoliation of intestinal epithelial cells. Moreover menstruating women additionally losses iron during the menstrual cycle. Erythropoiesis daily requires about 30 mg of iron, which is largely provided by the recycling of iron through RE macrophages.

The dietary iron is absorbed from the small intestine and the normal level of iron is regulated by feedback mechanism between its requirement and absorption. The dietary iron is present either as haeme or as inorganic ferric iron (Fe3+). Absorption of haeme iron takes place directly without the aid of active carrier transport. However, haeme iron is a minor source of dietary iron. The primary source of dietary iron is Fe3+ which has to be reduced to ferrous (Fe2+) form by acid reducing agents for efficient uptake. Iron is transported across the membrane by two distinct transporters. The divalent metal transporter 1 (DMT1) present at luminal membrane carries Fe2+ into the intestinal epithelial cell. This Fe2+ and iron released from the haeme is transported across the basolateral membrane by another iron transporter ferroportin (FP). Gut has a mechanism to prevent the entry of excess iron in the body. After reaching to the intestinal epithelial cell, iron is either transported to plasma or oxidized to Fe3+ and complexed with apoferritin to form ferritin, the cytosolic protein in which iron is stored. Ferritin usually remains stored in intestinal epithelial cells for 2–4 days after that the cells were shed off. This process is called as exfoliation. Whenever the body iron is low, the ferritin is either not formed or dissociates quickly to release iron. This released iron is transported to the blood [1]. The free form of iron is extremely toxic. The Fe2+ on entering into plasma it is rapidly oxidized to Fe3+ and complexed with apotransferrin (Apo-Tf) a glycoprotein to form transferrin (Tf). Two Fe3+ residues bound to one Tf molecule. This complex bound to membrane bound transferrin receptor 1 (Tfr1), present on erythropoietic and other cells. The Tf–Tfr1 complex is engulfed by receptor mediated endocytosis. The endosomes of erythropoietic and other cells become acidified through engulfed proton complex, which leads to conformational changes, which dissociates iron from the complex. The released Fe3+ is reduced to Fe2+ and transported out of the endosomes by DMT1. This released Fe2+ is utilized for hemoglobin synthesis or other biochemical process; Apo-Tf and Tfr1 are return to the cell surface for further cycles. In iron deficiency and haemolytic anemia, Tfr1 receptors up regulation take place at erythropoietic cells, but not at other cells. Under physiological conditions, all circulating iron is bound to transferrin. Nontransferrin bound iron (NTBI) can increase in a pathological condition like iron overload disease (**Figure 1**) [2, 3, 14]. Once the iron enters the cell, the fraction that is not needed for immediate use is stored by ferritin and haemosiderin in RE cells of liver, spleen and bone marrow. Iron status of the body regulates the synthesis of apoferritin. When the iron status is low, the iron regulating element (IRE) at DNA is blocked and synthesis of apoferritin is not taken place, whereas more Tf is produced. Moreover excess of apoferritin

**44**

*Iron absorption pathways by the enterocyte.*

Daily excretion of iron in an adult male is approximately 1–2 mg, mostly as exfoliated intestinal epithelial cells, some RBCs and in bile. The primary route of excretion of iron is feces, while skin, urine and sweat are minor routes. In menstruating women, monthly menstrual loss of iron is about 0.5–1 mg/day. Excess of iron is required during last two trimesters of pregnancy for expansion of RBC mass, transfer to the fetus and to compensate the loss during delivery [7].

The iron homeostasis maintained by its controlled absorption, recycling and storage. The iron is regulating peptide hormone, hepcidin produced mainly by the liver, whereas a smaller amount is produced in other organs like lung and heart plays a vital role in this regards. Hepcidin act by degradation of FP, an iron efflux transporter, concerned about transportation of iron across the basolateral membrane in the intestine. Iron overload increases whereas anemia and hypoxia decrease the synthesis of hepcidin [8]. Hepcidin synthesis is regulated by bone morphogenetic protein (BMP)/SMAD pathway via activation of hepcidin transcription. The loss of hepatic SMAD4 gene results in iron overload due to the failure of hepcidin-mediated degradation of FP [9].

#### **2. Determination of serum iron**

The biochemical estimation of body iron status depends on serum based indicators, as follows


**Figure 2.** *Laboratory measurement of iron indicators needed to calculate transferrin saturation.*

These indicators present challenges for clinical practice and national nutrition surveys, and often iron status interpretation is based on the combination of several indicators (**Figure 2**). The diagnosis of iron deficiency through SF concentration, the most commonly used indicator, is complicated by concomitant inflammation. The sTfR concentration is an indicator of functional iron deficiency that is not an acute phase reactant, but challenges in its interpretation arise because of the lack of assay standardization, common reference ranges, and common cutoffs. However it is unclear which indicators are best suited to assess excess iron status. The value of hepcidin, non–transferrin-bound iron, and reticulocyte indexes is being explored in research settings. Serum based indicators are generally measured on fully automated clinical analyzers. Although international reference materials have been available for years, the standardization of immunoassays is complicated by the heterogeneity of antibodies used and the absence of physicochemical reference methods to establish "true" concentrations. The assessment of iron status in NHANES was based on the multi-indicator ferritin model. However, the model did not indicate the severity of iron deficiency and produced categorical estimates. Recently, iron status assessment in NHANES has used the total body iron stores (TBI) model, in which the log ratio of sTfR to SF is assessed. Together, sTfR and SF concentrations cover the full range of iron status. The TBI model better predicts the absence of bone marrow iron than SF concentration alone, and TBI can be analyzed as a continuous variable. Additional consideration of methodologies, interpretation of indicators and analytic standardization is important for further improvements in iron status assessment [10].

#### **3. Iron overload disease**

Iron overload disease is also known as haemochromatosis is a group of heterogeneous disease which is caused either due to hereditary or acquired condition. Iron overload disease is characterized by the accumulation of iron in the body with or without organ dysfunction [11, 12]. Iron overload is unavoidable since there is no physiologically regulated mechanism for excretion of excess iron. During iron overload, the low molecular weight iron can play an essential catalytic role in the initiation of free radical reactions. These free radicals have the potential to damage cellular macromolecules like lipids, proteins, carbohydrates and nucleic acids resulting in cell injury, impaired cell function and integrity or cell death. The rate of free radical generation determines the intensity of cell injury [13].

**47**

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload…*

A. Familial or hereditary haemochromatosis (Primary iron overload).

g.Autosomal dominant haemochromatosis (Solomon Islands).

B. Acquired haemochromatosis (Secondary iron overload).

*3.1.1 Familial or hereditary haemochromatosis (primary iron overload)*

Hereditary haemochromatosis (HH, HFE1) is the most prevalent form of primary iron overload disease. HFE1 is an autosomal recessive disorder caused due to a mutation in HFE gene on chromosome 6, that resulting in iron overload and variable multiorgan dysfunction. More than 20 mutations of HFE gene were identified, but the most clinically significant mutations, however, are the C282Y and H63D. The C282Y mutation is a missense mutation that causes the tyrosine to replace cysteine at position 282, whereas H63D mutation is characterized by a histidine to aspartic acid substitution at position 63 in the HFE protein [15]. The H63D mutation may add to minor increases in serum iron levels, but in the absence of C282Y, there was no clinical significance of the H63D mutation. Approximately 85–90% of HFE patients were C282Y homozygotes while 3–5% of subjects with HFE may be C282Y/H63D compound heterozygous [16]. Another mutation of HFE gene is S65C

The haemochromatosis is classified into two main categories, namely primary

*DOI: http://dx.doi.org/10.5772/intechopen.98749*

and secondary haemochromatosis [14].

i.C282Y homozygosity.

ii.C282Y, H63D heterozygosity.

iii.Other HFE gene mutations.

c.Ferroportin mutation (HFE4).

d.Aceruloplasminemia.

f. Neonatal iron overload.

a.Iron loading anemia's.

c.African iron overload.

b.Transfusional iron overload.

d.Iron overload in chronic liver disease.

e.Atransferrinaemia.

a. JuVenile haemochromatosis (HFE2).

b.Transferrin receptor 2 mutation (HFE3).

**3.1 Types of iron overload disease (haemochromatosis)**

a.Hereditary haemochromatosis (HH, HFE1).

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload… DOI: http://dx.doi.org/10.5772/intechopen.98749*

#### **3.1 Types of iron overload disease (haemochromatosis)**

The haemochromatosis is classified into two main categories, namely primary and secondary haemochromatosis [14].

A. Familial or hereditary haemochromatosis (Primary iron overload).

	- i.C282Y homozygosity.

*Trace Elements and Their Effects on Human Health and Diseases*

These indicators present challenges for clinical practice and national nutrition surveys, and often iron status interpretation is based on the combination of several indicators (**Figure 2**). The diagnosis of iron deficiency through SF concentration, the most commonly used indicator, is complicated by concomitant inflammation. The sTfR concentration is an indicator of functional iron deficiency that is not an acute phase reactant, but challenges in its interpretation arise because of the lack of assay standardization, common reference ranges, and common cutoffs. However it is unclear which indicators are best suited to assess excess iron status. The value of hepcidin, non–transferrin-bound iron, and reticulocyte indexes is being explored in research settings. Serum based indicators are generally measured on fully automated clinical analyzers. Although international reference materials have been available for years, the standardization of immunoassays is complicated by the heterogeneity of antibodies used and the absence of physicochemical reference methods to establish "true" concentrations. The assessment of iron status in NHANES was based on the multi-indicator ferritin model. However, the model did not indicate the severity of iron deficiency and produced categorical estimates. Recently, iron status assessment in NHANES has used the total body iron stores (TBI) model, in which the log ratio of sTfR to SF is assessed. Together, sTfR and SF concentrations cover the full range of iron status. The TBI model better predicts the absence of bone marrow iron than SF concentration alone, and TBI can be analyzed as a continuous variable. Additional consideration of methodologies, interpretation of indicators and analytic standardization is important for further improvements in iron status assessment [10].

*Laboratory measurement of iron indicators needed to calculate transferrin saturation.*

Iron overload disease is also known as haemochromatosis is a group of heterogeneous disease which is caused either due to hereditary or acquired condition. Iron overload disease is characterized by the accumulation of iron in the body with or without organ dysfunction [11, 12]. Iron overload is unavoidable since there is no physiologically regulated mechanism for excretion of excess iron. During iron overload, the low molecular weight iron can play an essential catalytic role in the initiation of free radical reactions. These free radicals have the potential to damage cellular macromolecules like lipids, proteins, carbohydrates and nucleic acids resulting in cell injury, impaired cell function and integrity or cell death. The rate of

free radical generation determines the intensity of cell injury [13].

**46**

**3. Iron overload disease**

**Figure 2.**

	- a.Iron loading anemia's.
	- b.Transfusional iron overload.
	- c.African iron overload.
	- d.Iron overload in chronic liver disease.

#### *3.1.1 Familial or hereditary haemochromatosis (primary iron overload)*

Hereditary haemochromatosis (HH, HFE1) is the most prevalent form of primary iron overload disease. HFE1 is an autosomal recessive disorder caused due to a mutation in HFE gene on chromosome 6, that resulting in iron overload and variable multiorgan dysfunction. More than 20 mutations of HFE gene were identified, but the most clinically significant mutations, however, are the C282Y and H63D. The C282Y mutation is a missense mutation that causes the tyrosine to replace cysteine at position 282, whereas H63D mutation is characterized by a histidine to aspartic acid substitution at position 63 in the HFE protein [15]. The H63D mutation may add to minor increases in serum iron levels, but in the absence of C282Y, there was no clinical significance of the H63D mutation. Approximately 85–90% of HFE patients were C282Y homozygotes while 3–5% of subjects with HFE may be C282Y/H63D compound heterozygous [16]. Another mutation of HFE gene is S65C

#### *Trace Elements and Their Effects on Human Health and Diseases*

that leads to substitution of serine for cysteine, this kind of mutation also results into mild iron overload in the compound to heterozygous type HFE1 [17].

The C282Y mutation leads to disruption of a disulfide bridge that decreases the affinity of HFE gene towards β2 microglobulin and TfR1. This type of HFE gene mutation is retained in the Golgi complex [18]. This mutation resulted in decreased uptake of plasma transferrin-bound iron (TBI) by the duodenal crypt cells. This decreased uptake of iron would result into false iron deficiency even increasing total body iron stores that results into upregulation of iron regulating protein (IRP), DMT1 and ferroportin 1 (Fpn1) expression and increased iron absorption from the intestine [19, 20].

Mutation of a gene other than HFE gene is a rare genetic disorder of iron metabolism, also responsible for the development of the iron overloaded disease. Juvenile haemochromatosis, a type 2 haemochromatosis (HFE2) is a rare autosomal recessive disorder resulting in iron overload during the second and third decades of life. Some patients with HFE2 have mutations in hepcidin or hemojuvelin genes [21, 22]. Mutations of transferrin receptor-2 gene situated on chromosome 7q22 responsible for the development of transferrin receptor 2 mutations (HFE3) type of haemochromatosis. HFE3 is inherited in an autosomal recessive fashion [23]. HFE4 is caused due to mutations in ferroportin gene (IREG1, MTP1 or SLC11A3). It is inherited in an autosomal dominant fashion. These mutations are rare and should not routinely be screened for diagnosis purpose, but should be considered if HH cannot be diagnosed by conventional HFE gene mutations [24]. Other forms of hereditary haemochromatosis like aceruloplasminemia, atransferrinaemia, neonatal iron overload and autosomal dominant haemochromatosis (Solomon Islands) are rare conditions and they comprise of the very negligible proportion of inherited haemochromatosis [25].

#### *3.1.2 Acquired haemochromatosis (secondary iron overload)*

The ineffective erythropoiesis like thalassaemia, hereditary sideroblastic anemia and certain myelodysplastic syndromes, the hyperplastic erythroid marrow stimulate the iron absorption to a level that leads to the clinical iron overloaded condition. There is a direct relationship between erythropoiesis and iron absorption [26]. Each unit of blood contains 200–250 mg of iron. Chronic blood transfusion therapy is required in a condition like β thalassaemia, bone marrow failure and sickle cell anemia. Excess of transfusion, the iron load, initially accumulates in the RE macrophages but iron may deposit later in the parenchymal cells of the liver, heart, pancreas and endocrine tissue. Phlebotomy is not a treatment alternative because of the underlying anemia. Iron chelation therapy with Desferoxamine (DFO) administered continuous infusion is often the only option. Another oral iron chelator, Deferiprone (DFP) and Deferasirox (DFS) has been studied but has limitations in their efficacy [27].

Iron overload in sub-Saharan Africa was believed initially due to ingestion of large amounts of iron obtained from traditional home brewed beer fermented in non-galvanized steel drums. Moreover, only a few numbers of these beer drinkers acquire iron overload, suggesting that a genetic predisposition may be involved in the development of iron overload. This indicates that the gene locus is not related to the HFE gene, but maybe specific putative locus has not been identified [28]. Whereas heterozygosity for a common polymorphism (Q248H) in Fpn1 gene was identified in African and African-American person with iron overload [29].

Liver cirrhosis may increase the hepatic iron deposition. It seen in non-biliary cirrhosis likes alcoholic liver disease, chronic viral hepatitis and non-alcoholic steatohepatitis [30]. Very few cases were reported to have iron overload due to liver cirrhosis. The mechanism is not known, heterozygosity for the C282Y mutation in the HFE gene may play an important role [31]. In cirrhosis, there is a decrease in the

**49**

**4.2 Current iron chelator**

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload…*

synthesis of Tf whereas an increase in the plasma NTBI levels, which may contribute to hepatic iron overload [32]. Reduced incorporation of iron into the RBC as well as reduced RBC lifespan in liver cirrhosis related hypersplenism, combined with

Iron overload may lead to produce organ complications such as liver failure (fibrosis and rarely carcinoma), Cardiac abnormalities (cardiomyopathy, arrhythmias and heart failure), and hepatosplenomegaly. The other symptoms include chronic abdominal pain, weakness, fatigue, joint pain, arthritis, osteoporosis, arthralgia, loss of libido, impotence, infertility, hyperpigmentation of the skin, cutaneous atrophy, flattening of nails and loss of hair [12]. Endocrine abnormalities like diabetes mellitus, hypothyroidism, hypoparathyroidism, hypocortisolism, adrenal insufficiency hypothalamic–pituitary dysfunction, pancreatic dysfunction, amenorrhoea, delayed puberty and hypogonadism seen with iron overloaded patients [34]. Iron overload also leads to kidney damage [35]. Accumulation of iron in the brain may lead to neurodegenerative diseases like Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, progressive supranuclear palsy, corticobasal degeneration and superficial siderosis [36].

Iron chelators typically contain donor atoms like oxygen, nitrogen or sulfur which form coordinate bonds with the bound iron. The donor atoms determine the preference of the chelator for either the Fe2+ or Fe3+ oxidation states [37]. Chelators that contain nitrogen and sulfur as donor atoms can prefer not only Fe2+ but also other divalent metals such as Cu2+ and Zn2+ [38]. Iron chelators may be classified by their binding structures. Bidentate iron chelator such as DFP requires three molecules each with two iron binding sites Fe3+ (3:1 ratio). A tridentate iron chelator DFS requires two molecules for Fe3+ (2:1 ratio); whereas hexadentate iron chelator, DFO binds Fe3+ in a 1:1 ratio [39]. Iron can coordinate six ligands in an octahedral arrangement. Hence DFO has the highest affinity for iron. The effectiveness of iron chelator determine by how wholly and efficiently it form the complex; thus affinity and stoichiometry of iron chelator play an essential role for its therapeutic effectiveness [40]. Iron chelators were mainly focused on the management of iron overload conditions due to multiple blood transfusions as the supportive treatment of disease like β-thalassaemia, sickle cell disease and myelodysplasia [41]. An iron chelator is having a broad spectrum of activity, they were not only used for the management of iron overload disease, but also as in the treatment of cancer due to their ability to sequester metals essential to tumor growth [42]. Other than the iron chelation, they also play an essential role as antioxidant in various oxidative stress mediated diseases like liver disease [43], ischemic reperfusion injury [44], atherosclerosis [45], diabetes mellitus

[46], inflammation [47], infectious disease [48] and neurologic disease [49].

used for the treatment of iron overloaded conditions.

In current medicine, iron chelators include natural compounds derived from microorganisms such as siderophores and synthetic iron chelators were clinically

portocaval shunting may also contribute to the liver iron overload [33].

*DOI: http://dx.doi.org/10.5772/intechopen.98749*

**3.2 Complications of iron overload disease**

**4. Iron chelator: pharmacology and toxicology**

**4.1 Concept and chemistry of iron chelator**

synthesis of Tf whereas an increase in the plasma NTBI levels, which may contribute to hepatic iron overload [32]. Reduced incorporation of iron into the RBC as well as reduced RBC lifespan in liver cirrhosis related hypersplenism, combined with portocaval shunting may also contribute to the liver iron overload [33].

#### **3.2 Complications of iron overload disease**

*Trace Elements and Their Effects on Human Health and Diseases*

that leads to substitution of serine for cysteine, this kind of mutation also results into mild iron overload in the compound to heterozygous type HFE1 [17].

The C282Y mutation leads to disruption of a disulfide bridge that decreases the affinity of HFE gene towards β2 microglobulin and TfR1. This type of HFE gene mutation is retained in the Golgi complex [18]. This mutation resulted in decreased uptake of plasma transferrin-bound iron (TBI) by the duodenal crypt cells. This decreased uptake of iron would result into false iron deficiency even increasing total body iron stores that results into upregulation of iron regulating protein (IRP), DMT1 and ferroportin 1 (Fpn1) expression and increased iron absorption from the intestine [19, 20]. Mutation of a gene other than HFE gene is a rare genetic disorder of iron metabolism, also responsible for the development of the iron overloaded disease. Juvenile haemochromatosis, a type 2 haemochromatosis (HFE2) is a rare autosomal recessive disorder resulting in iron overload during the second and third decades of life. Some patients with HFE2 have mutations in hepcidin or hemojuvelin genes [21, 22]. Mutations of transferrin receptor-2 gene situated on chromosome 7q22 responsible for the development of transferrin receptor 2 mutations (HFE3) type of haemochromatosis. HFE3 is inherited in an autosomal recessive fashion [23]. HFE4 is caused due to mutations in ferroportin gene (IREG1, MTP1 or SLC11A3). It is inherited in an autosomal dominant fashion. These mutations are rare and should not routinely be screened for diagnosis purpose, but should be considered if HH cannot be diagnosed by conventional HFE gene mutations [24]. Other forms of hereditary haemochromatosis like aceruloplasminemia, atransferrinaemia, neonatal iron overload and autosomal dominant haemochromatosis (Solomon Islands) are rare conditions and they comprise of the very negligible proportion of inherited

**48**

haemochromatosis [25].

*3.1.2 Acquired haemochromatosis (secondary iron overload)*

(DFS) has been studied but has limitations in their efficacy [27].

The ineffective erythropoiesis like thalassaemia, hereditary sideroblastic anemia and certain myelodysplastic syndromes, the hyperplastic erythroid marrow stimulate the iron absorption to a level that leads to the clinical iron overloaded condition. There is a direct relationship between erythropoiesis and iron absorption [26]. Each unit of blood contains 200–250 mg of iron. Chronic blood transfusion therapy is required in a condition like β thalassaemia, bone marrow failure and sickle cell anemia. Excess of transfusion, the iron load, initially accumulates in the RE macrophages but iron may deposit later in the parenchymal cells of the liver, heart, pancreas and endocrine tissue. Phlebotomy is not a treatment alternative because of the underlying anemia. Iron chelation therapy with Desferoxamine (DFO) administered continuous infusion is often the only option. Another oral iron chelator, Deferiprone (DFP) and Deferasirox

Iron overload in sub-Saharan Africa was believed initially due to ingestion of large amounts of iron obtained from traditional home brewed beer fermented in non-galvanized steel drums. Moreover, only a few numbers of these beer drinkers acquire iron overload, suggesting that a genetic predisposition may be involved in the development of iron overload. This indicates that the gene locus is not related to the HFE gene, but maybe specific putative locus has not been identified [28]. Whereas heterozygosity for a common polymorphism (Q248H) in Fpn1 gene was identified in African and African-American person with iron overload [29].

Liver cirrhosis may increase the hepatic iron deposition. It seen in non-biliary cirrhosis likes alcoholic liver disease, chronic viral hepatitis and non-alcoholic steatohepatitis [30]. Very few cases were reported to have iron overload due to liver cirrhosis. The mechanism is not known, heterozygosity for the C282Y mutation in the HFE gene may play an important role [31]. In cirrhosis, there is a decrease in the

Iron overload may lead to produce organ complications such as liver failure (fibrosis and rarely carcinoma), Cardiac abnormalities (cardiomyopathy, arrhythmias and heart failure), and hepatosplenomegaly. The other symptoms include chronic abdominal pain, weakness, fatigue, joint pain, arthritis, osteoporosis, arthralgia, loss of libido, impotence, infertility, hyperpigmentation of the skin, cutaneous atrophy, flattening of nails and loss of hair [12]. Endocrine abnormalities like diabetes mellitus, hypothyroidism, hypoparathyroidism, hypocortisolism, adrenal insufficiency hypothalamic–pituitary dysfunction, pancreatic dysfunction, amenorrhoea, delayed puberty and hypogonadism seen with iron overloaded patients [34]. Iron overload also leads to kidney damage [35]. Accumulation of iron in the brain may lead to neurodegenerative diseases like Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, progressive supranuclear palsy, corticobasal degeneration and superficial siderosis [36].

#### **4. Iron chelator: pharmacology and toxicology**

#### **4.1 Concept and chemistry of iron chelator**

Iron chelators typically contain donor atoms like oxygen, nitrogen or sulfur which form coordinate bonds with the bound iron. The donor atoms determine the preference of the chelator for either the Fe2+ or Fe3+ oxidation states [37]. Chelators that contain nitrogen and sulfur as donor atoms can prefer not only Fe2+ but also other divalent metals such as Cu2+ and Zn2+ [38]. Iron chelators may be classified by their binding structures. Bidentate iron chelator such as DFP requires three molecules each with two iron binding sites Fe3+ (3:1 ratio). A tridentate iron chelator DFS requires two molecules for Fe3+ (2:1 ratio); whereas hexadentate iron chelator, DFO binds Fe3+ in a 1:1 ratio [39]. Iron can coordinate six ligands in an octahedral arrangement. Hence DFO has the highest affinity for iron. The effectiveness of iron chelator determine by how wholly and efficiently it form the complex; thus affinity and stoichiometry of iron chelator play an essential role for its therapeutic effectiveness [40].

Iron chelators were mainly focused on the management of iron overload conditions due to multiple blood transfusions as the supportive treatment of disease like β-thalassaemia, sickle cell disease and myelodysplasia [41]. An iron chelator is having a broad spectrum of activity, they were not only used for the management of iron overload disease, but also as in the treatment of cancer due to their ability to sequester metals essential to tumor growth [42]. Other than the iron chelation, they also play an essential role as antioxidant in various oxidative stress mediated diseases like liver disease [43], ischemic reperfusion injury [44], atherosclerosis [45], diabetes mellitus [46], inflammation [47], infectious disease [48] and neurologic disease [49].

#### **4.2 Current iron chelator**

In current medicine, iron chelators include natural compounds derived from microorganisms such as siderophores and synthetic iron chelators were clinically used for the treatment of iron overloaded conditions.

#### *4.2.1 Siderophores*

Siderophores are the low molecular mass with high affinity iron chelating compounds that are secreted by the iron dependent microorganisms such as bacteria and fungi. They serve primarily as iron transport across the cell membrane. Wide range of siderophores is available such as Ferrichrome, DFO, Fusarinine, Ornibactin, Enterobactin, Bacillibactin, vibriobactin and Azotobactin. The commonly used siderophore is DFO [50].

#### *4.2.1.1 Desferoxamine (DFO)*

DFO is the most common clinically used siderophore. It has been used for the treatment of iron overloaded diseases last for decades and it remains the current standard for the iron chelation therapy [51]. The toxic effect of an excess of iron in iron overloaded disease is majorly due to NTBI; iron has both redox activity as well as ability to concentrate in highly vascular tissues such as hepatic, cardiac and endocrine tissue [52]. DFO is a hexadentate iron chelator, which can bind Fe3+ in a 1:1 ratio as shown in **Figure 3**. DFO is a multifunctional therapeutic agent, which can detoxify NTBI by its chelation property and heme proteins by ferryl reduction as well as free radical scavenging action. DFO also acts as a reducing agent, which prevent the oxidation of membrane lipids by removing high-oxidation states of heme iron, like ferryl myoglobin (Mb) or Hb [53].

Because of its high molecular weight (656.79), DFO is not orally bioavailable. Hence it is administered via subcutaneous injection at a dose of 50 mg/kg/day as a 10% solution in sterile water (0.50 or 2.0 g vials). Additionally, DFO has a short half-life of about 5–10 min, therefore to improve its efficacy; the required dose is injected over a period of 4–12 hrs via a small portable peristaltic pump. DFO is poorly metabolized by transamination, β -oxidation, decarboxylation and N-hydroxylation. DFO is excreted as its 1:1 complex with iron mostly in the urine and a small amount in feces [54].

#### *4.2.2 Synthetic iron chelator*

#### *4.2.2.1 Deferiprone (DFP)*

DFP is a synthetic oral iron chelator that has shown comparable efficacy to DFO and is more effective than DFO in the removal of excess iron from the heart. An advantage of DFP is that Fe3+ chelate of DFP carries no net charge and therefore, DFO-iron complex can easily penetrate the membrane. Additionally, the combination of DFO and DFP is widely used now a day without any new toxic effects [51].

**51**

*4.2.2.2 Deferasirox (DFS)*

*Chelation of iron with Deferasirox.*

**Figure 4.**

**Figure 5.**

*Chelation of iron with Deferiprone.*

for Fe3+ (2:1 ratio) as shown in **Figure 5**.

**4.3 Limitations of current iron chelation therapy**

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload…*

The clinical DFP is used limited in thalassemia major; however, its use in agranulocytosis and arthropathy [55]. Bidentate iron chelator such as DFP requires three molecules each with two iron binding sites Fe3+ (3:1 ratio) as shown in **Figure 4**.

DFS is another synthetic oral chelator that has recently approved by US-FDA for the use in the treatment of iron overload diseases. DFS is effective in removing excess of iron from the liver. DFS has good tolerance, though monitoring is required for renal function [56]. DFS is as effective as DFO in maintaining the iron balance. The combinations of DFO and DFS or the combination of two orally active iron chelator DFP and DFS have been suggested as a treatment option for transfusional iron overload. DFS is used in the treatment of uncommon anemia like aplastic anemia, Diamond–Blackfan anemia and Fanconi's anemia, which are associated with iron overload [57]. DFS is tridentate iron chelator DFS requires two molecules

All these iron chelators are having severe side effects and certain limitations. The side effects and limitations of DFO include irritation at the infusion site, growth retardation, skeletal changes, ocular and auditory disturbances, hypersensitivity reactions and systemic allergic reaction such as rash, urticaria, anaphylactic

*DOI: http://dx.doi.org/10.5772/intechopen.98749*

**Figure 3.** *Chelation of iron with Desferoxamine.*

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload… DOI: http://dx.doi.org/10.5772/intechopen.98749*

**Figure 4.** *Chelation of iron with Deferiprone.*

*Trace Elements and Their Effects on Human Health and Diseases*

Siderophores are the low molecular mass with high affinity iron chelating compounds that are secreted by the iron dependent microorganisms such as

bacteria and fungi. They serve primarily as iron transport across the cell membrane. Wide range of siderophores is available such as Ferrichrome, DFO, Fusarinine, Ornibactin, Enterobactin, Bacillibactin, vibriobactin and Azotobactin. The com-

DFO is the most common clinically used siderophore. It has been used for the treatment of iron overloaded diseases last for decades and it remains the current standard for the iron chelation therapy [51]. The toxic effect of an excess of iron in iron overloaded disease is majorly due to NTBI; iron has both redox activity as well as ability to concentrate in highly vascular tissues such as hepatic, cardiac and endocrine tissue [52]. DFO is a hexadentate iron chelator, which can bind Fe3+ in a 1:1 ratio as shown in **Figure 3**. DFO is a multifunctional therapeutic agent, which can detoxify NTBI by its chelation property and heme proteins by ferryl reduction as well as free radical scavenging action. DFO also acts as a reducing agent, which prevent the oxidation of membrane lipids by removing high-oxidation states of

Because of its high molecular weight (656.79), DFO is not orally bioavailable. Hence it is administered via subcutaneous injection at a dose of 50 mg/kg/day as a 10% solution in sterile water (0.50 or 2.0 g vials). Additionally, DFO has a short half-life of about 5–10 min, therefore to improve its efficacy; the required dose is injected over a period of 4–12 hrs via a small portable peristaltic pump. DFO is poorly metabolized by transamination, β -oxidation, decarboxylation and N-hydroxylation. DFO is excreted as its 1:1 complex with iron mostly in the urine

DFP is a synthetic oral iron chelator that has shown comparable efficacy to DFO and is more effective than DFO in the removal of excess iron from the heart. An advantage of DFP is that Fe3+ chelate of DFP carries no net charge and therefore, DFO-iron complex can easily penetrate the membrane. Additionally, the combination of DFO and DFP is widely used now a day without any new toxic effects [51].

*4.2.1 Siderophores*

monly used siderophore is DFO [50].

heme iron, like ferryl myoglobin (Mb) or Hb [53].

and a small amount in feces [54].

*4.2.2 Synthetic iron chelator*

*4.2.2.1 Deferiprone (DFP)*

*4.2.1.1 Desferoxamine (DFO)*

**50**

**Figure 3.**

*Chelation of iron with Desferoxamine.*

**Figure 5.** *Chelation of iron with Deferasirox.*

The clinical DFP is used limited in thalassemia major; however, its use in agranulocytosis and arthropathy [55]. Bidentate iron chelator such as DFP requires three molecules each with two iron binding sites Fe3+ (3:1 ratio) as shown in **Figure 4**.

#### *4.2.2.2 Deferasirox (DFS)*

DFS is another synthetic oral chelator that has recently approved by US-FDA for the use in the treatment of iron overload diseases. DFS is effective in removing excess of iron from the liver. DFS has good tolerance, though monitoring is required for renal function [56]. DFS is as effective as DFO in maintaining the iron balance. The combinations of DFO and DFS or the combination of two orally active iron chelator DFP and DFS have been suggested as a treatment option for transfusional iron overload. DFS is used in the treatment of uncommon anemia like aplastic anemia, Diamond–Blackfan anemia and Fanconi's anemia, which are associated with iron overload [57]. DFS is tridentate iron chelator DFS requires two molecules for Fe3+ (2:1 ratio) as shown in **Figure 5**.

#### **4.3 Limitations of current iron chelation therapy**

All these iron chelators are having severe side effects and certain limitations. The side effects and limitations of DFO include irritation at the infusion site, growth retardation, skeletal changes, ocular and auditory disturbances, hypersensitivity reactions and systemic allergic reaction such as rash, urticaria, anaphylactic reaction, with or without shock and angioedema. DFO is an expensive drug and cannot be afforded by the majority of patients. The primary in a challenge with DFO therapy is patient's adherence. DFO is having poor oral bioavailability and short t1/2, therefore it is administered by slow subcutaneous infusion over a period of 8–12 hrs for 5–7 days/week. This leads to lower patient compliance. The slow subcutaneous DFO infusion affect quality of life as the slow infusion can produce troublesome, time-consuming and painful. Patient's poor compliance resulted in gaps during chelation therapy, which leads to increase the plasma iron level, which causes further damage.

The major side effect of DFP is that it produces agranulocytosis, which can be reversed by discontinuation of therapy. The other side effects of DFP include gastrointestinal discomfort, arthropathy, increased liver-enzyme levels, low plasma zinc level, the progression of hepatic fibrosis associated with an increase in iron overload or hepatitis C and joint pain [58].

DFS produces various side effects such as agranulocytosis, gastrointestinal discomfort, skin rash, loss of hearing and visual impairment. Especially in geriatric patients and other patients with high risk of myelodysplastic syndrome, hepatic or renal impairment and thrombocytopenia are prone to develop hepatic failure, kidney failure and gastrointestinal hemorrhage with the use of DFS. Also, these agents are not suitable for use during pregnancy [59].

#### **5. Herbal iron chelators**

Due to these side effects and limitations, the use of synthetic iron chelators is suboptimal. Taking into account the paucity of iron chelating agents, scientists are putting their efforts towards the finding of therapeutically potential iron chelator to get maximum possible benefits with fewer harmful effects. Plants containing flavonoids and polyphenolic compounds possess iron chelating and antioxidant property [60, 61]. Due to the specific chemical structure of flavonoids, they can chelate iron and forms the soluble as well as stable iron-flavonoids complex.

**53**

**Plant** *Caesalpinia sappan*

*Curcuma longa* *Triticum aestivum*

*Tetracarpidium conophorum*

Enhydra fluctuans

*Terminalia chebula*

Terminalia belerica

Emblica officinalis

*Caesalpinia crista*

*Cajanus cajan* Tinospora cordifolia

*Medicago sativa* *Allium porrum* *Silybum marianum*

Nerium indicum Clerodendrum colebrookianum

*Melilotus officinalis*

*Salvia virgata* *Epilobium hirsutum*

*Caulerpa racemosa*

*Mangifera foetida*

*Coriandrum sativum*

**Table 1.**

*Natural Iron chelating agents.*

Lamiaceae

Fabaceae Lamiaceae Onagraceae Caulerpaceae Anacardiaaceae

Apiaceae

Horse mango

Coriander

sea grapes

great willowherb

Leaves Whole plant

Leaves Whole plant

In-Vivo

In-Vivo

In-Vivo

In-vitro and In-vivo

wand sage

East Indian glory bowe

Yellow sweet clover

Arial Shoot

In-Vivo

In-vitro and In-vivo

Leaves

In-Vivo

Euphorbiaceae

Asteraceae Combretaceae Combretaceae Euphorbeaceae

Caesalpiniaceae

Fabaceae Menispermaceae

Fabaceae Alliaceae Asteraceae Apocynaceae

Kaner

Milk thistle

Leek

Alfalfa

Heart-leaved moonseed

Stem Arial Arial Seeds Leaves

In-Vivo

In-Vivo

In-Vitro and In-Vivo

In-Vitro and In-Vivo

In-vitro

Pigeon pea

Indian gooseberry

Crested fever nut

Leaves Leaves

In-vitro

In-vitro

Fruit

In-vitro

Bahera

Myrobalan

Helencha

**Family** Caesalpiniaceae

Zingiberaceae

Poaceae

Turmeric Wheat Grass African Walnut

Nut Leaves

Fruit Fruit

In-vitro

In-vitro

In-vitro

In-vitro

Sappan wood

**Common name**

**Part used**

Wood rhizomes Whole grass

In-vitro, In-vivo and clinical study

Hydroalcoholic

Aqueous Aqueous Hydroalcoholic

Hydroalcoholic

Hydroalcoholic

Hydroalcoholic

Hydroalcoholic

Hydroalcoholic

Aqueous and Methanol

Hydroalcoholic

Aqueous and methanolic

Methanol Aqueous and Methanol

Aqueous and methanol fraction

Dichloromethane

Aqueous and Methanol fraction

Aqueous and Ethanolic

Aqueous Hydroalcoholic

[81]

[82]

[83]

[80]

[79]

[76]

[78]

[75, 76]

[77]

[72–74]

[73, 74]

In-Vivo

In-vivo

**Type of study**

**Extract** Hydroalcoholic

Aqueous

**References**

[64] [65] [66–68]

[69] [70] [71] [71] [71] [71] [71] [71]

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload…*

*DOI: http://dx.doi.org/10.5772/intechopen.98749*

**Figure 6.** *Typical metal chelation sites of flavonoid.*


*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload… DOI: http://dx.doi.org/10.5772/intechopen.98749*

> **Table 1.** *Natural Iron chelating agents.*

*Trace Elements and Their Effects on Human Health and Diseases*

causes further damage.

**5. Herbal iron chelators**

overload or hepatitis C and joint pain [58].

agents are not suitable for use during pregnancy [59].

reaction, with or without shock and angioedema. DFO is an expensive drug and cannot be afforded by the majority of patients. The primary in a challenge with DFO therapy is patient's adherence. DFO is having poor oral bioavailability and short t1/2, therefore it is administered by slow subcutaneous infusion over a period of 8–12 hrs for 5–7 days/week. This leads to lower patient compliance. The slow subcutaneous DFO infusion affect quality of life as the slow infusion can produce troublesome, time-consuming and painful. Patient's poor compliance resulted in gaps during chelation therapy, which leads to increase the plasma iron level, which

The major side effect of DFP is that it produces agranulocytosis, which can be reversed by discontinuation of therapy. The other side effects of DFP include gastrointestinal discomfort, arthropathy, increased liver-enzyme levels, low plasma zinc level, the progression of hepatic fibrosis associated with an increase in iron

DFS produces various side effects such as agranulocytosis, gastrointestinal discomfort, skin rash, loss of hearing and visual impairment. Especially in geriatric patients and other patients with high risk of myelodysplastic syndrome, hepatic or renal impairment and thrombocytopenia are prone to develop hepatic failure, kidney failure and gastrointestinal hemorrhage with the use of DFS. Also, these

Due to these side effects and limitations, the use of synthetic iron chelators is suboptimal. Taking into account the paucity of iron chelating agents, scientists are putting their efforts towards the finding of therapeutically potential iron chelator to get maximum possible benefits with fewer harmful effects. Plants containing flavonoids and polyphenolic compounds possess iron chelating and antioxidant property [60, 61]. Due to the specific chemical structure of flavonoids, they can chelate iron and forms the soluble as well as stable iron-flavonoids complex.

**52**

**Figure 6.**

*Typical metal chelation sites of flavonoid.*

Flavonoids possess three possible sites for metal chelating that can bind metal ions as follows (**Figure 6**).

i. 3-hydroxy-4-ketone groups in the C-ring

ii. 5-hydroxy group in the A-ring and 4-carbonyl group in the C–ring

iii. 3′,4′-dihydroxy groups, located on the B-ring.

The complex is then excreted in urine and feces [62]. Flavonoids and polyphenolic compounds illustrate their antioxidant activity through various mechanisms, such as free radicals scavenging, transition metals chelation and inhibition of various enzymes [63].

Some medicinal plants were reported to have In-vitro iron chelating potential, while other medicinal plants have been screened for In-vivo iron chelation activity, whereas some plants were clinically evaluated for the treatment of iron overload in β thalassaemia patients as showed in **Table 1**.

#### **6. Conclusion**

Chelation therapy is the preferred medical treatment for reducing the toxic effects of metals. Chelating agents are capable of binding to toxic metal ions to form complex structures which are easily excreted from the body removing them from intracellular or extracellular spaces.

Presently, siderophore like DFO and synthetic iron chelators such as DFP and DFS were used for the treatment of iron overload diseases. These iron chelators have severe side effects and certain limitations. As compared to siderophores and synthetic iron chelators, natural iron chelators are usually less toxic and have minimum side effects. Additionally, these medicines possess antioxidant property, which plays an essential role in the treatment of iron overload disease and its complications associated with oxidative stress. Therefore, need to search for more safe and effective treatment of iron overloaded disease has become an area of current research interest.

**55**

**Author details**

Anil P. Dewani3

Naheed Waseem A. Sheikh1

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload…*

\*, Satish B. Kosalge1

1 Hi-Tech College of Pharmacy, Chandrapur, Maharashtra, India

3 P. Wadhwani College of Pharmacy, Yavatmal, Maharashtra, India

4 Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh, India

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 School of Pharmacy, RK University, Rajkot, Gujarat, India

\*Address all correspondence to: wsheikh2@gmail.com

provided the original work is properly cited.

and Alok S. Tripathi4

, Deepak S. Mohale3

, Tusharbindu R. Desai<sup>2</sup>

,

*DOI: http://dx.doi.org/10.5772/intechopen.98749*

*Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload… DOI: http://dx.doi.org/10.5772/intechopen.98749*

## **Author details**

*Trace Elements and Their Effects on Human Health and Diseases*

i. 3-hydroxy-4-ketone groups in the C-ring

iii. 3′,4′-dihydroxy groups, located on the B-ring.

β thalassaemia patients as showed in **Table 1**.

intracellular or extracellular spaces.

as follows (**Figure 6**).

various enzymes [63].

**6. Conclusion**

Flavonoids possess three possible sites for metal chelating that can bind metal ions

The complex is then excreted in urine and feces [62]. Flavonoids and polyphenolic compounds illustrate their antioxidant activity through various mechanisms, such as free radicals scavenging, transition metals chelation and inhibition of

Some medicinal plants were reported to have In-vitro iron chelating potential, while other medicinal plants have been screened for In-vivo iron chelation activity, whereas some plants were clinically evaluated for the treatment of iron overload in

Chelation therapy is the preferred medical treatment for reducing the toxic effects of metals. Chelating agents are capable of binding to toxic metal ions to form complex structures which are easily excreted from the body removing them from

Presently, siderophore like DFO and synthetic iron chelators such as DFP and DFS were used for the treatment of iron overload diseases. These iron chelators have severe side effects and certain limitations. As compared to siderophores and synthetic iron chelators, natural iron chelators are usually less toxic and have minimum side effects. Additionally, these medicines possess antioxidant property, which plays an essential role in the treatment of iron overload disease and its complications associated with oxidative stress. Therefore, need to search for more safe and effective treatment of iron overloaded disease has become an area of current research interest.

ii. 5-hydroxy group in the A-ring and 4-carbonyl group in the C–ring

**54**

Naheed Waseem A. Sheikh1 \*, Satish B. Kosalge1 , Tusharbindu R. Desai2 , Anil P. Dewani3 , Deepak S. Mohale3 and Alok S. Tripathi4

1 Hi-Tech College of Pharmacy, Chandrapur, Maharashtra, India

2 School of Pharmacy, RK University, Rajkot, Gujarat, India


\*Address all correspondence to: wsheikh2@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[21] A. Roetto, G. Papanikolaou, M. Politou, F. Alberti, D. Girelli, J. Christakis, D. Loukopoulos, C. Camaschella, Mutant

antimicrobial peptide hepcidin is associated with severe juvenile

Ludwig, M.L.E. MacDonald, P.L. Franchini, M. Dube, L. Andres, J. MacFarlane, N. Sakellaropoulos, M. Politou, E. Nemeth, J. Thompson, J.K. *Natural Iron Chelators as Potential Therapeutic Agents for the Treatment of Iron Overload… DOI: http://dx.doi.org/10.5772/intechopen.98749*

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[17] C. Mura, O. Raguenes, C. Ferec, HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis, Blood 93 (1999) 2502-2505, https://doi.org/10.1182/ blood.V93.8.2502.

[18] J.N. Feder, Z. Tsuchihashi, A. Irrinki, V.K. Lee, F.A. Mapa, E. Morikang, C.E. Prass, S.M. Starnes, R.K. Wolff, S. Parkkila, W.S. Sly, R.C. Schatzman, The hemochromatosis founder mutation in HLA-H disrupts β2-microglobulin interaction and cell surface expression, J. Biol. Chem. 272 (1997) 14025-14028, https://doi. org/10.1074/jbc.272.22.14025.

[19] H. Zoller, R.O. Koch, I. Theurl, R.O. Koch, W. Vogel, P. Obrist, A. Pietrangelo, G. Montosi, D.J. Haile, Expression of duodenal iron transporters divalentmetal transporter 1 and ferroportin 1 in iron deficiency and iron overload, Gastroenterology 120 (2001) 1412-1419, https://doi.org/10.1053/gast.2001.24033.

[20] K.A. Stuart, G.J. Anderson, D.M. Frazer, L.W. Powell, M. McCullen, L.M. Fletcher, D.H.G. Crawford, Duodenal expression of iron transport molecules in untreated haemochromatosis subjects, Gut 52 (2003) 953-959, https:// doi.org/10.1136/gut.52.7.953.

[21] A. Roetto, G. Papanikolaou, M. Politou, F. Alberti, D. Girelli, J. Christakis, D. Loukopoulos, C. Camaschella, Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis, Nat. Genet. 33 (2003) 21-22, https://doi.org/10.1038/ng1053.

[22] G. Papanikolaou, M.E. Samuels, E.H. Ludwig, M.L.E. MacDonald, P.L. Franchini, M. Dube, L. Andres, J. MacFarlane, N. Sakellaropoulos, M. Politou, E. Nemeth, J. Thompson, J.K.

Risler, C. Zaborowska, R. Babakaiff, C.C. Radomski, T.D. Pape, O. Davidas, J. Christakis, P. Brissot, G. Lockitch, T. Ganz, M.R. Hayden, Y.P. Goldberg, Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis, Nat. Genet. 36 (2004) 77-82, https://doi.org/10.1038/ng1274.

[23] C. Camaschella, A. Roetto, A. Cali, M.D. Gobbi, G. Garozzo, M. Carella, N. Majorano, A. Totaro, P. Gasparini, The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22, Nat. Genet. 25 (2000) 14-15, https://doi. org/10.1038/75534.

[24] G. Montosi, A. Donovan, A. Totaro, C. Garuti, E. Pignatti, S. Cassanelli, C.C. Trenor, P. Gasparini, N.C. Andrews, A. Pietrangelo, Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene, J. Clin. Invest. 108 (2001) 619-623, https://doi.org/10.1172/JCI13468.

[25] A. Hayashi, Y. Wada, T. Suzuki, A. Shimizu, Studies on familial hypotransferrinemia: unique clinical course and molecular pathology, Am. J. Hum. Genet. 53 (1993) 201-213.

[26] P. Pootrakul, K. Kitcharoen, P. Yansukon, P. Wasi, S. Fucharoen, P. Charoenlarp, G. Brittenham, M.J. Pippard, C.A. Finch, The effect of erythroid hyperplasia on iron balance, Blood 71 (1988) 1124-1129, https://doi. org/10.1182/blood.V71.4.1124.1124.

[27] A.V. Hoffbrand, F. Al-Refaie, B. Davis, N. Siritanakatkul, B.F.A. Jackson, J. Cochrane, E. Prescott, B. Wonke, Long term trial of deferiprone in 51 transfusion-dependent iron overloaded patients, Blood 91 (1998) 295-300, https://doi.org/10.1182/blood.V91.1.295.

[28] V. Gordeuk, J. Mukiibi, S.J. Hasstedt, W. Samowitz, C.Q. Edwards, G. West, S. Ndambire, J. Emmanual, N. Nkanza, Z. Chapanduka, M. Randall, P. Boone, P. Romano, R.W. Martell, T. Yamashita, P.

**56**

jn/134.1.1.

*Trace Elements and Their Effects on Human Health and Diseases*

regulation of hepcidin expression, Cell Metab. 2 (2005) 399-409, https://doi. org/10.1016/j.cmet.2005.10.010.

[10] M, P, Christine and C, L, Anne, Laboratory methodologies for indicators of iron status: strengths, limitations, and analytical challenges, Am. J. Clin. Nutr. 106 (2017) 1606S-1614S https:// doi.org/10.3945/ajcn.117.155887.

[11] A, Pietrangelo, Haemochromatosis, Gut 52 (2003) ii23–ii30, https://doi. org/10.1136/gut.52.suppl\_2.ii23.

[12] A. Siddique, K.V. Kowdley, Review article: The iron overload syndromes, Aliment Pharmacol. Ther. 35 (2012) 876-893, https://doi.org/10.1111/ j.1365-2036.2012.05051.x.

[13] G. Papanikolaou, K. Pantopoulos, Iron metabolism and toxicity, Toxicol. Appl. Pharm. 202 (2005) 199-211,

[14] D. Trinder, C. Fox, G. Vautier, J. K. Olynyk, Molecular pathogenesis of iron overload, Gut 51 (2002) 290-295, http://

[15] J.N. Feder, A. Gnirke, W. Thomas, Z. Tsuchihashi, D.A. Ruddy, A. Basava, F. Dormishian, R.Jr. Domingo, M.C. Ellis, A. Fullan, L.M. Hinton, N.L. Jones, B.E. Kimmel, G.S. Kronmal, P. Lauer, V.K. Lee, D.B. Loeb, F.A. Mapa, E. McClelland, N.C. Meyer, G.A. Mintier, N. Moeller, T. Moore, E. Morikang, C.E. Prass, L. Quintana, S.M. Starnes, R.C. Schatzman, K.J. Brunke, D.T. Drayna, N.J. Risch, B.R. Bacon, R.K. Wolff, A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis, Nat. Genet. 13 (1996) 399-408, https://doi.

dx.doi.org/10.1136/gut.51.2.290.

https://doi.org/10.1016/j. taap.2004.06.021.

org/10.1038/ng0896-399.

[16] B.R. Bacon, L.W. Powell, P.C. Adams, T.F. Krisena, J.H. Hoofnagle, Molecular medicine and hemochromatosis: at the

[1] N. Abbaspour, R. Hurrell, R. Kelishadi, Review on iron and its importance for human health, J. Res.

[2] N.C. Andrews, Forging a field: the golden age of iron biology, Blood 112 (2008) 219-230, https://doi.org/10.1182/

[3] M.W. Hentze, M.U. Muckenthaler, N.C. Andrews, Balancing acts: molecular control of mammalian iron metabolism, Cell 117 (2004) 285-297, https://doi. org/10.1016/s0092-8674(04)00343-5.

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treatment for the patients with Thalassemia, Ori. Pharm. Exp. Med. 7 (2008) 466-476, https://doi.org/ 10.3742/OPEM.2008.7.5.466.

[68] P. Das, A. Mukhopadhyay, S. Mandal, B.C. Pal, R. Mishra, D. Mukherjee, S. Mukhopadhyay, J. Basak, M. Kar, In vitro studies of iron chelation activity of purified active ingredients extracted from Triticum aestivum Linn. (Wheat Grass), Eur. J.

Med. Plants 2 (2012) 113-124.

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2628-2643.

[70] L.N. Patralekh, G. Mukherjee, In vitro studies on antioxidant and ironchelating activity of Enhydra fluctuans Lour, Sci. Cult. 76 (2010) 537-539.

[71] R. Sarkar, N. Mandal, Study of iron chelating and DNA protective activities in hydroalcoholic extract of Indian medicinal plants, Int. J. Pharm. Bio. Sci.

[72] R. Patel, P. Tirgar, Evaluation of beneficial effects of Medicago sativa (Alfalfa) in iron-overload conditions, J. Chem. Bio. Phy. Sci. Sec. B. 3 (2013)

[73] A. Mirzaei, M. Abbasi, S. Sepehri, M. Mirzaei, The effects of Allium

[69] B.M. Olabinri, O.O. Eniyansoro, C.O. Okoronkwo, P.F. Olabinri, M.T. Olaleye, Evaluation of chelating ability of aqueous extract of Tetracarpidium conophorum (African walnut) in vitro, Int. J. Appl.

[66] T.R. Desai, J.K. Solanki, P. Buch, R.K. Goyal, Triticum aestivum

(wheatgrass) formulation: An alternate

[67] P.R. Tirgar, T.R. Desai, Investigation into iron chelating activity of Triticum aestivum (wheat grass) in iron-dextran induce iron overload model of thalassaemia, J. Pharm. Res. 4 (2011)

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3066-3069.

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[58] A.N. Saliba, A.R. Harb, A.T. Taher, Iron chelation therapy in transfusiondependent thalassemia patients: current strategies and future directions, J. Blood Med. 6 (2015) 197-209, https://doi.

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[61] M.A. Ebrahimzadeh, F. Pourmorad, A.R. Bekhradnia, Iron chelating activity, phenol and flavonoid content of some medicinal plants from Iran, Afr. J. Biotechnol. 7 (2008) 3188-3192, https://

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cbpa.2007.04.025.

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blood.V51.3.415.415.

[62] C. Hershko, Determinants of fecal and urinary iron excretion in desferrioxamine treated rats, Blood 51 (1978) 415-423, https://doi.org/10.1182/

[63] P. Cos, T.D. Bruyne, N. Hermans, S. Apers, D.V. Berghe, A.J. Vlietinck, Proanthocyanidins in health care: current and new trends, Curr. Med. Chem. 11 (2004) 1345-1359, https://doi.

[64] R. Safitri, A.M. Maskoen, M.R.A.A. Syamsunarno, M. Ghozali, R. Panigoro, Iron chelating activity of Caesalpinia sappan L. extract on iron status in iron overload rats (*Rattus norvegicus* L.), AIP Conf. Proc. 2002, 020050-1-020050-6, https://doi.org/10.1063/1.5050146.

[65] Y. Jiao, J. Wilkinson IV, E.C. Pietsch, J.L. Buss, W. Wang, R. Planalp, F.M. Torti, S.V. Torti, Iron chelation in the biological activity of curcumin, Free Radic. Biol. Med. 40 (2006) 1152-1160,

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[75] M. Nematbakhsh, Z. Pezeshki, B.A. Moaeidi, F. Eshraghi-Jazi, A. Talebi, H. Nasri, S. Baradaran, M. Gharagozloo, T. Safari, M. Haghighi, Protective role of Silymarin and Deferoxamine against iron dextran-induced renal iron deposition in male rats, Int. J. Prev. Med. 4 (2013) 286-292.

[76] M.A. Ebrahimzadeh, M. Khalili, M. Azadbakht, M. Azadbakh, Salvia virgata Jacq. and Silibum marianum L. gaertn display significant iron-chelating activity, Inter. J. Pharm. Sci. Res. 7 (2016) 3756-3763, https://doi.org/10.13040/ IJPSR.0975-8232.7(9).3756-63.

[77] N.B. Ghate, D. Chaudhuri, S. Panja, N. Mandal, Nerium indicum leaf alleviates iron-induced oxidative stress and hepatic injury in mice, Pharm. Biol. 53 (2015) 1066-1074, https://doi.org/10. 3109/13880209.2014.959612.

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[82] P. Fajri, A. Estuningtyas, M. Louisa, H. Freisleben, The preventive effect of Mangifera foetida L. leaf extract administered simultaneously to excess iron on markers of iron overload in sprague dawley rats, Med. J. Indones. 26 (2017) 246-252, http://dx.doi. org/10.13181/mji.v26i4.1829.

[83] R. Talaei, A. Kheirollah, H.B. Rezaei, E. Mansouri, G. Mohammadzadeh, Protective effects of hydro-alcoholic extract of Coriandrum sativum in rats with experimental iron-overload condition, Jundishapur J. Nat. Pharm. Prod. 13 (2018) 1-9, https://doi.org/ 10.5812/jjnpp.65028.

**Chapter 5**

Disease

**Abstract**

**1. Introduction**

**63**

*and Biju Prava Sahariah*

Chromium Genotoxicity

*Jyoti Kant Choudhari, Jyotsna Choubey,*

regulating the DEGs and their gene ontology.

Associated with Respiratory

*Mukesh Kumar Verma, Anand Kumar Jayapal*

Chromium existing in the biosphere in prominent two forms Cr (III) and Cr (VI) is a well-studied heavy metal. Cr (III) is considered as non-harmful and necessary element in diet whereas Cr(VI) is extremely toxic exerting various negative health impacts on human and other organisms. Mining activity is must for extracting economic minerals and a large number of people are related to these sites as worker or habitants and a major source of chromium exposure. Present chapter discusses genotoxic nature of chromium considering respiratory disease resulted from chromium exposure. The genotoxicity is illustrated in terms of chromium induced differential expressed genes (DEGs), transcription factors and microRNA

**Keywords:** Mine tailing, Chromium Toxicity, Genotoxicity, Gene expressions

For the growth of economy of a country and improving living status of population, industrial functioning is mandatory which is in other hand associated activities including supply of power, raw materials, processing and discharge of waste. For a major section of industries, power supply is from coal or electricity generated from coal, and the raw materials are various form of ores received from mining. Mine tailing is the fine residual mine dump after completion of mining left with dug out soil, scattered residuals and disturbed ecosystem. The major source of chromium in the mine tailings is the residual ores present in traces not extracted with economic point of view and mineral processing chemicals that are left unattended. Chromium (Cr), a valuable element often finds its utility in metallurgical, chemical, and refractory industries due to its pigment property, hardness and persistence. From environment point of view, chromium exists in three oxidative states, elemental chromium (0) that does not exist naturally, whereas trivalent chromium (Cr III) is rather stable followed by hexavalent chromium (Cr VI) based on the different number of electrons and therefore varied properties [1]. Hexavalent chromium is extremely toxic even in low concentration and listed as carcinogenic, hematotoxic and altering genetic material whereas, Cr (III) is regarded as micronutrient in human diet. When Cr is left unattended in mine tailings, it can be transported by

#### **Chapter 5**

## Chromium Genotoxicity Associated with Respiratory Disease

*Jyoti Kant Choudhari, Jyotsna Choubey, Mukesh Kumar Verma, Anand Kumar Jayapal and Biju Prava Sahariah*

#### **Abstract**

Chromium existing in the biosphere in prominent two forms Cr (III) and Cr (VI) is a well-studied heavy metal. Cr (III) is considered as non-harmful and necessary element in diet whereas Cr(VI) is extremely toxic exerting various negative health impacts on human and other organisms. Mining activity is must for extracting economic minerals and a large number of people are related to these sites as worker or habitants and a major source of chromium exposure. Present chapter discusses genotoxic nature of chromium considering respiratory disease resulted from chromium exposure. The genotoxicity is illustrated in terms of chromium induced differential expressed genes (DEGs), transcription factors and microRNA regulating the DEGs and their gene ontology.

**Keywords:** Mine tailing, Chromium Toxicity, Genotoxicity, Gene expressions

#### **1. Introduction**

For the growth of economy of a country and improving living status of population, industrial functioning is mandatory which is in other hand associated activities including supply of power, raw materials, processing and discharge of waste. For a major section of industries, power supply is from coal or electricity generated from coal, and the raw materials are various form of ores received from mining. Mine tailing is the fine residual mine dump after completion of mining left with dug out soil, scattered residuals and disturbed ecosystem. The major source of chromium in the mine tailings is the residual ores present in traces not extracted with economic point of view and mineral processing chemicals that are left unattended. Chromium (Cr), a valuable element often finds its utility in metallurgical, chemical, and refractory industries due to its pigment property, hardness and persistence. From environment point of view, chromium exists in three oxidative states, elemental chromium (0) that does not exist naturally, whereas trivalent chromium (Cr III) is rather stable followed by hexavalent chromium (Cr VI) based on the different number of electrons and therefore varied properties [1]. Hexavalent chromium is extremely toxic even in low concentration and listed as carcinogenic, hematotoxic and altering genetic material whereas, Cr (III) is regarded as micronutrient in human diet. When Cr is left unattended in mine tailings, it can be transported by

natural means to nearby waterbody, added with acid mine drainage, and surrounding ecosystem expanding the circumference of toxicity exposure [2]. This chapter emphasises on toxicity of hexavalent chromium in genetic level that influence expression of genes, the transcript factors controlling the differentially expressed genes and finally to find out the major indicating and influenced genetic factors with functional analysis of gene ontology for respiratory units of human.

Persons developed coughing, wheezing, and decreased forced volume after an inhalation exposure to a sample of Cr(III) sulfate [10]. Combine effect of Cr(III)

among 60 ferrochromium workers squeezed out subjective symptoms of coughing, wheezing, and dyspnea whereas control remained neutral [11]. These symptoms might get puzzled with smoking issue to clarify the accurate problem of the diseases [11]. While considering respiratory issue, animals are also often exposed to chromium similar to the human. Henderson et al. [12] in histological examination with exposure of 0.9–25 mg Cr(III) trichloride for 30 min observed alterations in lung

) investigated

and Cr(VI) as total chromium (0.02–0.19 mg total chromium/m<sup>3</sup>

*Chromium Genotoxicity Associated with Respiratory Disease*

*DOI: http://dx.doi.org/10.5772/intechopen.97336*

**2. Chromium-gene interactions in respiratory disease**

Comparative toxico-genomics database (CTD, http://ctdbase.org) is a recognised well informed/updated, openly accessible database. It purposes to provide detail knowledge and information about the impacts of exposure of environ-

The core block of the database basically manually curated contains updated information regarding interaction and relationships among chemicals, genes, proteins and their resulted specific disease in terms of functional and pathways to incorporate new hypotheses expressing underlying mechanisms of disease and

In this work, all Chromium- gene /protein interactions for respiratory disease are downloaded from CTD, in which Chromium- gene /protein interactions associ-

KEGG (http://www.genome.jp/) is a knowledge base for systematic analysis of gene functions, linking genomic information with higher-order functional information [15]. For the analysis of Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/) is a great option. DAVID provides various functional annotation tools for researchers to understand biological meaning behind large list of genes. [16] Gene ontology (GO) analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis can be performed for analysing differentially expressed genes (DEGs) at the functional level based on DAVID Bioinformatics Resources 6.8. P < 0.05 as the cut-off criterion. Researchers can upload all DEGs to the online software DAVID to identify overrepresented GO categories and KEGG pathways. The curated genes in CTD for each respiratory disease can be uploaded to DAVID 6.8 Beta

ated to the following 04 respiratory disease are selected for further analysis according to MESH ID used in CTD— Lung Neoplasma, Pulmonary Fibrosis and Lung disease. Chromium- gene/protein interactions associated to this respiratory disease are collected for further analysis. According to the reference score on relationships between chemicals-genes, genes-diseases and chemicals-diseases [14], lung neoplasms is recognised as most likely having the maximum connectivity with chromium. (**Table 1**). From the identified 168 chromium gene with in respiratory

tissues associated with mild inflammation.

mental elements (pollutants) on human health.

environmental contamination [13].

**3. Results and discussion**

disease, 131 genes are unique.

**65**

**3.1 Gene function enrichment analysis**

#### **1.1 Source and toxicity of chromium**

Toxicity of chromium is directly influenced by the chromium species with valence with number of electrons and thus their properties. The Cr(VI), is a powerful oxidizing agent and plainly toxic to human and other organisms causing adverse effect to blood cells, renal cells, allergic conditions and organs of most part of body failure. Chromium can significantly find its route of exposures through dermal chromium contact in waste sites, inhalation of chromium emissions and ingestion of contaminated water or food grown in chromium contaminated soil. Also, erosion products and emissions from road and cement dust, leather, paints and or any Cr used materials contribute to inhalation of chromium. Dermal ulcers, irritation and sensitization of respiratory/lungs are consecutive result of chromium contact. In the plasma and cells, Cr(VI) readily get reduced to Cr(III), and thereafter excreted in the urine. Trivalent chromium is the form of chromium that is essential to human health and counted as an essential trace mineral in the human diet. Hexavalent chromium is recognised as genotoxic as it can damage genetic information in living cells, causes DNA mutations, and possibly the formation of cancerous tumours. Chromates (chromium salts) formed from hexavalent chromium also finds utilization in manufacture leather products, paints, cement, mortar, anti-corrosives, and other things. They are carcinogenic and allergenic.

#### **1.2 Physiologic effects of chromium exposure in respiratory disease**

Occupational exposures often include mixed exposure to both Cr(III) and Cr (VI) [3]. Chromium compounds, when inhaled, causes respiratory tract irritants, resulting in airway irritation, airway obstruction, and lung, nasal, or sinus cancer. Radiographic analysis from several reports revealed enlargement of the hilar region and lymph nodes [4, 5]. Consistent associations have been found between employment in the chromium industries and significant risk for respiratory cancer. Moller et al. [6] reported systemic reactions characterised with anaphylactoid reaction in a young welder having chromium (VI) vapor fume exposures. Following an experiment with sodium chromate inhalation at a concentration of 29 μg/m<sup>3</sup> , formation of static urticaria, angioedema and severe bronchospasm simultaneously with plasma histamine rising in threefold was documented and suggested direct positive leukocyte inhibitory factor of sodium chromate.

A number of nasal mucosa injury cases in Cr (VI) exposed workers at concertation of nearly 20 μg/m<sup>3</sup> (against US permissible standard 5 μg/m<sup>3</sup> ) for 5 months to 10 years characterised with inflamed mucosa and ulcerated/perforated septum was recorded in a study with 43 chrome-plating plants and tanneries in Sweden [7, 8]. Huge number of complaints for nasal irritations was documented in a detail epidemiological study with Tokyo (Japan) housewives residing near chromium slag contaminated construction site [7]. U.S has recommended chromate and chromic acid at workplace to be 5 μg/m<sup>3</sup> as permissible standard. Gibb et al. [9] observed that with less than 30 days median time for nasal ulceration diagnosis from first exposure, median Cr (VI) concentration matched the Sweden report. Occupational exposure to Cr(III) has also been associated with respiratory effects.

*Chromium Genotoxicity Associated with Respiratory Disease DOI: http://dx.doi.org/10.5772/intechopen.97336*

natural means to nearby waterbody, added with acid mine drainage, and surrounding ecosystem expanding the circumference of toxicity exposure [2]. This chapter emphasises on toxicity of hexavalent chromium in genetic level that influence expression of genes, the transcript factors controlling the differentially expressed genes and finally to find out the major indicating and influenced genetic factors with functional analysis of gene ontology for respiratory units of human.

Toxicity of chromium is directly influenced by the chromium species with valence with number of electrons and thus their properties. The Cr(VI), is a powerful oxidizing agent and plainly toxic to human and other organisms causing adverse effect to blood cells, renal cells, allergic conditions and organs of most part of body failure. Chromium can significantly find its route of exposures through dermal chromium contact in waste sites, inhalation of chromium emissions and ingestion of contaminated water or food grown in chromium contaminated soil. Also, erosion products and emissions from road and cement dust, leather, paints and or any Cr used materials contribute to inhalation of chromium. Dermal ulcers, irritation and sensitization of respiratory/lungs are consecutive result of chromium contact. In the plasma and cells, Cr(VI) readily get reduced to Cr(III), and thereafter excreted in the urine. Trivalent chromium is the form of chromium that is essential to human health and counted as an essential trace mineral in the human diet. Hexavalent chromium is recognised as genotoxic as it can damage genetic information in living cells, causes DNA mutations, and possibly the formation of cancerous tumours. Chromates (chromium salts) formed from hexavalent chromium also finds utilization in manufacture leather products, paints, cement, mortar, anti-corrosives, and other things. They are carcinogenic and allergenic.

**1.2 Physiologic effects of chromium exposure in respiratory disease**

ment with sodium chromate inhalation at a concentration of 29 μg/m<sup>3</sup>

leukocyte inhibitory factor of sodium chromate.

**64**

Occupational exposures often include mixed exposure to both Cr(III) and Cr (VI) [3]. Chromium compounds, when inhaled, causes respiratory tract irritants, resulting in airway irritation, airway obstruction, and lung, nasal, or sinus cancer. Radiographic analysis from several reports revealed enlargement of the hilar region and lymph nodes [4, 5]. Consistent associations have been found between employment in the chromium industries and significant risk for respiratory cancer. Moller et al. [6] reported systemic reactions characterised with anaphylactoid reaction in a young welder having chromium (VI) vapor fume exposures. Following an experi-

static urticaria, angioedema and severe bronchospasm simultaneously with plasma histamine rising in threefold was documented and suggested direct positive

5 months to 10 years characterised with inflamed mucosa and ulcerated/perforated septum was recorded in a study with 43 chrome-plating plants and tanneries in Sweden [7, 8]. Huge number of complaints for nasal irritations was documented in a detail epidemiological study with Tokyo (Japan) housewives residing near chromium slag contaminated construction site [7]. U.S has recommended chromate and chromic acid at workplace to be 5 μg/m<sup>3</sup> as permissible standard. Gibb et al. [9] observed that with less than 30 days median time for nasal ulceration diagnosis from first exposure, median Cr (VI) concentration matched the Sweden report. Occupational exposure to Cr(III) has also been associated with respiratory effects.

A number of nasal mucosa injury cases in Cr (VI) exposed workers at concertation of nearly 20 μg/m<sup>3</sup> (against US permissible standard 5 μg/m<sup>3</sup>

, formation of

) for

**1.1 Source and toxicity of chromium**

*Trace Elements and Their Effects on Human Health and Diseases*

Persons developed coughing, wheezing, and decreased forced volume after an inhalation exposure to a sample of Cr(III) sulfate [10]. Combine effect of Cr(III) and Cr(VI) as total chromium (0.02–0.19 mg total chromium/m<sup>3</sup> ) investigated among 60 ferrochromium workers squeezed out subjective symptoms of coughing, wheezing, and dyspnea whereas control remained neutral [11]. These symptoms might get puzzled with smoking issue to clarify the accurate problem of the diseases [11]. While considering respiratory issue, animals are also often exposed to chromium similar to the human. Henderson et al. [12] in histological examination with exposure of 0.9–25 mg Cr(III) trichloride for 30 min observed alterations in lung tissues associated with mild inflammation.

#### **2. Chromium-gene interactions in respiratory disease**

Comparative toxico-genomics database (CTD, http://ctdbase.org) is a recognised well informed/updated, openly accessible database. It purposes to provide detail knowledge and information about the impacts of exposure of environmental elements (pollutants) on human health.

The core block of the database basically manually curated contains updated information regarding interaction and relationships among chemicals, genes, proteins and their resulted specific disease in terms of functional and pathways to incorporate new hypotheses expressing underlying mechanisms of disease and environmental contamination [13].

#### **3. Results and discussion**

In this work, all Chromium- gene /protein interactions for respiratory disease are downloaded from CTD, in which Chromium- gene /protein interactions associated to the following 04 respiratory disease are selected for further analysis according to MESH ID used in CTD— Lung Neoplasma, Pulmonary Fibrosis and Lung disease. Chromium- gene/protein interactions associated to this respiratory disease are collected for further analysis. According to the reference score on relationships between chemicals-genes, genes-diseases and chemicals-diseases [14], lung neoplasms is recognised as most likely having the maximum connectivity with chromium. (**Table 1**). From the identified 168 chromium gene with in respiratory disease, 131 genes are unique.

#### **3.1 Gene function enrichment analysis**

KEGG (http://www.genome.jp/) is a knowledge base for systematic analysis of gene functions, linking genomic information with higher-order functional information [15]. For the analysis of Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/) is a great option. DAVID provides various functional annotation tools for researchers to understand biological meaning behind large list of genes. [16] Gene ontology (GO) analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis can be performed for analysing differentially expressed genes (DEGs) at the functional level based on DAVID Bioinformatics Resources 6.8. P < 0.05 as the cut-off criterion. Researchers can upload all DEGs to the online software DAVID to identify overrepresented GO categories and KEGG pathways. The curated genes in CTD for each respiratory disease can be uploaded to DAVID 6.8 Beta


**Term Count P value FDR Genes**

*Chromium Genotoxicity Associated with Respiratory Disease*

*DOI: http://dx.doi.org/10.5772/intechopen.97336*

26 1.17E-21 2.49E-18 CRP, PDGFB, HIF1A, TNF, GJA1, FGF9,

14 1.39E-18 1.49E-15 EDN1, INSR, PTGS2, SOD2, ESR1, TNF, EGFR,

26 1.23E-15 8.80E-13 CDKN1B, HILPDA, PDGFB, EGFR, IGF1R,

13 7.81E-15 2.78E-12 JUN, EDN1, PDGFB, PTGS2, TNF, EGFR,

16 1.07E-14 3.27E-12 TGFB1, ANXA2, INSR, TNF, MMP9, EGFR,

Aging 18 1.68E-15 8.89E-13 JUN, TGFB1, OGG1, STAT3, FOS, TYMS,

Response to drug 22 2.08E-15 8.89E-13 CDKN1A, JUN, TGFB1, CDKN1B, OGG1,

Extracellular space 49 1.61E-24 3.37E-22 SERPINA1, CXCL8, TFRC, HILPDA, LECT2,

Protein complex 21 1.48E-12 1.55E-10 PDGFRA, CDKN1A, FEN1, CDKN1B, PARP1,

Extracellular region 37 9.09E-12 6.36E-10 CRP, SERPINA1, CCL11, CXCL8, TFRC,

Cytosol 49 4.84E-09 2.54E-07 CDKN1A, CDKN1B, FAM13A, SMC2, SOX2,

Term Count P value FDR Genes

ERBB3, MYC, ERBB2, HRAS, TGFB1, CAV1, STAT3, FN1, MAPK14, MTOR, VEGFA, ACTA2, AR, IL6, IFNG, IL1B, KIT, TP53, TLR4, NFE2L2

MTOR, IL6, IFNG, IL1B, AKT1, PTX3, TLR4

EFNB2, FGF9, MYC, MAPK1, SOX9, TIMP1, HRAS, PDGFRA, EDN1, TGFB1, INSR, STAT3, FN1, IL2, TGFBR2, VEGFA, AR, IL6, IFNG, KIT, BCL2L1

EEF2, TGFBR2, SOD1, GCLC, IL6, CAT, CYP1A1, SERPING1, CCL2, AKT1, TIMP1, NFE2L2

STAT3, APOA1, FOS, TYMS, PTGS2, SOD2, TGFBR2, SOD1, IL4, IL6, IFNG, CCND1, MYC, CAT, CYP1A1, CTNNB1, FYN

MTOR, TGFBR2, IL6, MYC, CCL5, AKT1, HMOX1

MTOR, VEGFA, CCND1, CHEK2, IL1B, ERBB2, AKT1, SOX9, HRAS, MAPK3

HMGB1, TNF, FGF9, TIMP1, SFTPB, EDN1, ANXA2, GPX3, MMP2, WNT5A, APOA1, MMP9, MMP10, ACTA2, ACE2, AZGP1, IFNG, IL1B, CAT, KIT, SERPING1, CRP, CCL11, GSTP1, PDGFB, EGFR, ERBB3, PRDX1, CCL5, CCL2, HMOX1, TGFB1, ACE, FN1, APOC3, LAMB1, PRDX6, IL2, SOD1, VEGFA, IL4, IL6, CPE, PTX3

CDKN2A, OGG1, CAV1, BRCA2, PTGS2, SOD1, ACTA2, AR, MYC, COL6A1, AKT1, MAPK1, CTNNB1, SOX9, TP53, MAPK3

PDGFB, HMGB1, TNF, FGF9, CCL5, CCL2, TIMP1, SFTPB, EDN1, TGFB1, ACE, GPX3, MMP2, WNT5A, FN1, APOA1, APOC3, LAMB1, MMP9, MMP10, IL2, SOD1, VEGFA, IL4, ACE2, IL6, AZGP1, IFNG, IL1B, COL6A1, SERPING1, PTX3

GJA1, CASP8, CCND1, MYC, AKT1, HRAS, GPX1, ANXA2, APOA1, FOS, TGFBR2,

**Biological Process** Positive regulation of gene expression

Positive regulation of nitric oxide biosynthetic process

Positive regulation of cell proliferation

Positive regulation of smooth muscle cell proliferation

Positive regulation of protein phosphorylation

**Cellular Component**

**67**

#### **Table 1.**

*Selected Respiratory diseases and related chromium-interacted gene.*

(https://david-d.ncif-crf.gov/tools.jsp) with *Homo sapiens* as the background population [17] for GO analysis.

GO analysis results for Cr toxicity in respiratory organs shows that that chromium interacted genes in respiratory disease are involved in the biological processes (BP) such as positive regulation of gene expression, positive regulation of cell proliferation, response to drug positive regulation of protein phosphorylation. (**Table 2**) For molecular function (MF), genes are enriched in identical protein binding, enzyme binding, transcription factor binding and protein phosphatase binding (**Table 2**). In addition, GO cell component (CC) analysis also displayed that the gene are significantly enriched in the extracellular space, protein complex, extracellular region and extracellular exosome (**Table 2**).

**Table 3** contains the most significantly enriched pathways of the chromium interacting genes by KEGG analysis. The interacting genes are enriched in Pathways in cancer, Proteoglycans in cancer, HIF-1 signalling pathway and TNF signalling pathways.

#### **3.2 Gene-TFs-miRNAs regulation**

The transcription factors (TFs) as well as microRNAs (miRNAs), are recognised for their huge share in transacting and gene regulations with various common logics and regulatory factors for gene regulation in multicellular genomes [18, 19]. The library of ENCODE and ChEA Consensus TFs from ChIP-X in EnrichR (http://amp.pharm.mssm.edu/Enrichr/ [20, 21]) can be used for the possible TFs


(https://david-d.ncif-crf.gov/tools.jsp) with *Homo sapiens* as the background

extracellular region and extracellular exosome (**Table 2**).

*Selected Respiratory diseases and related chromium-interacted gene.*

GO analysis results for Cr toxicity in respiratory organs shows that that chromium interacted genes in respiratory disease are involved in the biological processes (BP) such as positive regulation of gene expression, positive regulation of cell proliferation, response to drug positive regulation of protein phosphorylation. (**Table 2**) For molecular function (MF), genes are enriched in identical protein binding, enzyme binding, transcription factor binding and protein phosphatase binding (**Table 2**). In addition, GO cell component (CC) analysis also displayed that the gene are significantly enriched in the extracellular space, protein complex,

**Inference chromium-interacted genes (n) Gene**

ACE,AKT1,APOA1,APOC3,AR,AVPI1,BCL2L1,BRCA2,CASP8, CCND1,CDKN1A,CDKN1B,CDKN2A,CEACAM1,CHEK2, COL6A1,COX17,CRP,CTNNB1,CWH43,CYP1B1,DPYD,EEF2, EFNB2,EGFR,EGR1,ERBB2,ERBB3,ESR1,FAS,FEN1,FGF9,FOS, GCLC,GPX1,GPX3,GSTM1,GSTP1,HILPDA,HMOX1,HRAS, IDS,IER2,IFNG,IL1B,IL2,IL6,JUN,JUNB,LECT2,MAP2K7, MAPK1,MAPK14,MAPK3,MIR21,MIR494,MMP10,MYC,NOS2, OGG1,PCNA,PDCD4,PRDX1,PRDX6,PRKN,PTMA,SERPINA1, SERPING1,SFTPB,SIDT2,SMC2,SOX2,SOX9,TERT,TFRC, TGFBR2,TNF,TP53,TRP53,USP18,WNT5A

*Trace Elements and Their Effects on Human Health and Diseases*

ACE,AKT1,ANXA2,APOA1,AZGP1,CASP8,CAV1,CDKN1A, CDKN2A,CPE,CYP1B1,FOS,GCLC,GJA1,GPX1,GSTM1, HMOX1,IER2,IFNG,IL10,IL1B,IL6,JUN,MAPK1,MAPK3,MMP1, SFTPB,TGFB1,TLR4,TNF,TP53,TYMS

ACE2, ACTA2, CAT, CCL11, CCL2, CCL5, CXCL8, EDN1, FAM13A, FN1, FYN, HMGB1, HMOX1, IL1B, IL4, IL6, LAMB1, MMP2, MMP9, MTOR, NFE2L2, PARP1, PDGFB, PTX3, SERPINA1, SOD1,STAT3,TIMP1,TNF

SERPINA1,SFTPB,SOD2,TNF,VEGFA

Lung Injury ACE, ACE2, CCL2, CYP1A1, HMOX1, IL6, PARP1, SIRT1, TNF 09 3.12

TGFB1, TNF 02 5.86

MMP2 01 2.55

Lung Diseases ACE, BST1, HARS, HIF1A, IGF1R, INSR, KIT, PDGFRA,PTGS2,

**count**

**Inference Score**

81 74.7

32 58.22

29 25.25

14 7.62

**Table 3** contains the most significantly enriched pathways of the chromium interacting genes by KEGG analysis. The interacting genes are enriched in Pathways in cancer, Proteoglycans in cancer, HIF-1 signalling pathway and TNF signalling

The transcription factors (TFs) as well as microRNAs (miRNAs), are recognised for their huge share in transacting and gene regulations with various common logics and regulatory factors for gene regulation in multicellular genomes [18, 19]. The library of ENCODE and ChEA Consensus TFs from ChIP-X in EnrichR (http://amp.pharm.mssm.edu/Enrichr/ [20, 21]) can be used for the possible TFs

population [17] for GO analysis.

**3.2 Gene-TFs-miRNAs regulation**

pathways.

**66**

**Disease Name**

Lung Neoplasms (Cr VI)

Lung Neoplasms(Cr

III)

Pulmonary Fibrosis

Asthma, Occupational

Nose Neoplasms

**Table 1.**


and related networks. The TargetScan library in EnrichR can be used for the possible miRNA interaction. TFs are identified to be significantly associated with the genes involved in the respiratory disease. TRIM28, NFE2L2, EGR1 GATA2, PPARG,

*Pathway analysis for the chromium interacting genes related to Respiratory Disease.*

**Term Count P value FDR Genes**

*Chromium Genotoxicity Associated with Respiratory Disease*

*DOI: http://dx.doi.org/10.5772/intechopen.97336*

**Term Count P value FDR Genes**

21 1.14E-07 6.65E-06 PDGFRA, JUN, TGFB1, GSTM1, NOS2, TFRC,

41 5.78E-24 5.60E-22 CDKN1A, CDKN1B, CXCL8, GSTP1, PDGFB,

30 1.79E-21 8.69E-20 CDKN1A, HIF1A, TNF, EGFR, IGF1R, ERBB3,

21 3.91E-18 1.26E-16 CDKN1A, EDN1, CDKN1B, NOS2, TFRC, INSR,

21 2.09E-17 5.07E-16 JUN, TGFB1, ACE, CXCL8, NOS2, FOS, MAPK14,

17 2.75E-12 3.34E-11 JUN, EDN1, FOS, PTGS2, MAPK14, TNF, MMP9,

14 7.57E-12 8.16E-11 TGFB1, CDKN2A, STAT3, BRCA2, EGFR,

23 1.29E-11 1.25E-10 PDGFRA, EGR1, CDKN1A, JUN, TGFB1, PCNA,

Bladder cancer 14 1.10E-14 2.13E-13 CDKN1A, CXCL8, CDKN2A, MMP2, MMP9,

Hepatitis B 21 1.87E-14 3.02E-13 CDKN1A, JUN, TGFB1, CDKN1B, PCNA, CXCL8,

Prostate cancer 16 1.94E-12 2.69E-11 PDGFRA, CDKN1A, CDKN1B, PDGFB, EGFR,

PDGFB, TYMS, PTGS2, SOD1, VEGFA, CEACAM1, TERT, ERBB3, CHEK2, CCL5, KIT, DPYD, CAT, HMOX1, BCL2L1

BRCA2, PTGS2, HIF1A, EGFR, IGF1R, CASP8, FGF9, CCND1, MYC, ERBB2, AKT1, MAPK1, HRAS, MAPK3, PDGFRA, JUN, TGFB1, NOS2, CDKN2A, MMP2, WNT5A, STAT3, FN1, LAMB1, FOS, MMP9, MTOR, TGFBR2, VEGFA, AR, IL6, KIT, CTNNB1, FAS, TP53, BCL2L1

CCND1, MYC, ERBB2, AKT1, MAPK1, HRAS, MAPK3, TGFB1, CAV1, MMP2, WNT5A, STAT3, FN1, MIR21, MAPK14, MMP9, ESR1, MTOR, VEGFA, PDCD4, CTNNB1, FAS, TP53, TLR4

STAT3, HIF1A, EGFR, MTOR, IGF1R, VEGFA, IL6, IFNG, ERBB2, AKT1, HMOX1, MAPK1, TIMP1, TLR4, MAPK3

TNF, IL2, TGFBR2, IL6, CASP8, IFNG, IL1B, CCL5, FAS, CCL2, AKT1, MAPK1, TLR4, MAPK3

EGFR, VEGFA, CCND1, MYC, ERBB2, MAPK1, HRAS, TP53, MAPK3

STAT3, FOS, TNF, MMP9, IL6, CASP8, CCND1, MYC, FAS, AKT1, MAPK1, HRAS, TP53, TLR4, MAPK3

MTOR, IGF1R, AR, CCND1, ERBB2, AKT1, MAPK1, CTNNB1, HRAS, TP53, MAPK3

IL6, CASP8, IL1B, CCL5, FAS, CCL2, AKT1, MAPK1, MAP2K7, MAPK3

TGFBR2, VEGFA, CCND1, ERBB2, AKT1, MAPK1, TP53, BCL2L1, MAPK3

CDKN2A, WNT5A, PDGFB, FOS, TNF, IL2, TGFBR2, IL6, TERT, CCND1, CHEK2, MYC, AKT1, CTNNB1, HRAS, TP53, BCL2L1

Protein

activity

Pathways in cancer

Proteoglycans in cancer

HIF-1 signaling pathway

Chagas disease (American trypanosomiasis)

TNF signaling pathway

Pancreatic cancer

HTLV-I infection

**Table 3.**

**69**

**Table 2.**

homodimerization

*Gene ontology analysis of Cr interacted genes.*


#### **Table 2.**

**Term Count P value FDR Genes**

*Trace Elements and Their Effects on Human Health and Diseases*

Extracellular exosome

**Molecular Function** Identical protein binding

Transcription factor

Protein phosphatase

binding

binding

**68**

Mitochondrion 26 7.77E-07 2.62E-05 FEN1, GSTP1, OGG1, COX17, TYMS, GJA1,

Membrane raft 11 7.89E-07 2.62E-05 ACE2, GJA1, CASP8, ANXA2, CAV1, FAS,

Enzyme binding 22 9.84E-15 2.02E-12 JUN, TGFB1, GSTM1, PCNA, PARP1, CAV1,

Protein binding 90 3.45E-08 2.41E-06 CDKN1A, FEN1, CDKN1B, SERPINA1,

Cytokine activity 12 3.53E-08 2.41E-06 IL4, IL6, EDN1, TGFB1, IFNG, IL1B, WNT5A,

40 8.72E-07 2.62E-05 CRP, SERPINA1, PCNA, TFRC, GSTP1, SMC2,

31 2.03E-15 8.34E-13 SERPINA1, PCNA, TFRC, LECT2, PDGFB,

15 9.22E-09 1.13E-06 JUN, PARP1, CDKN2A, GPX3, STAT3,

9 1.10E-08 1.13E-06 CEACAM1, CDKN1B, ERBB2, STAT3,

ACTA2, AR, IL1B, DPYD, CAT, TP53, GSTP1, TYMS, USP18, HIF1A, PRDX1, HMOX1, MAPK1, FYN, HARS, MAP2K7, MAPK3, JUN, GSTM1, NOS2, CDKN2A, STAT3, EEF2, MAPK14, PRDX6, MTOR, SOD1, GCLC, PDCD4, CTNNB1, FAS, NFE2L2, BCL2L1

CASP8, MYC, PRDX1, CYP1B1, AKT1, MAPK1, FYN, HARS, MAPK3, GPX1, PARP1, CDKN2A, MMP2, MAPK14, SOD2, SOD1, CAT, CYP1A1, TP53, BCL2L1

FYN, EEF2, TNF, EGFR, TGFBR2

GJA1, FGF9, PRDX1, MAPK1, TIMP1, MAPK3, ACE, GPX1, ANXA2, GPX3, INSR, WNT5A, FN1, APOA1, APOC3, LAMB1, EEF2, MAPK14, SOD2, MMP9, PRDX6, SOD1, ACTA2, ACE2, BST1, AZGP1, CEACAM1, IL1B, COL6A1, CAT, SERPING1, CPE, CTNNB1, FAS

TNF, EGFR, IGF1R, CASP8, ERBB3, CHEK2, PRDX1, ERBB2, AKT1, MAPK1, FYN, JUN, PARP1, CAV1, STAT3, FN1, APOA1, SOD2, MMP9, ESR1, SOD1, VEGFA, FAS, PTX3, TP53, BCL2L1

APOA1, PTGS2, MAPK14, HIF1A, ESR1, EGFR, AR, CCND1, CAT, CYP1A1, AKT1, HMOX1, CTNNB1, FYN, MAP2K7, TP53

HMGB1, FOS, HIF1A, ESR1, AR, CCND1, MYC, MAPK1, CTNNB1, TP53

CTNNB1, MAPK14, MAP2K7, TP53, EGFR

CXCL8, TFRC, OGG1, HILPDA, HMGB1, BRCA2, TNF, IGF1R, SMC2, SOX2, GJA1, CASP8, CCND1, MYC, CHEK2, AKT1, SOX9, TIMP1, HRAS, PDGFRA, EDN1, PARP1, ANXA2, GPX3, MMP2, WNT5A, APOA1, FOS, MMP9, TGFBR2, ACE2, AR, AZGP1, CEACAM1, DPYD, KIT, SERPING1, TLR4, TP53, AVPI1, CRP, CCL11, PCNA, GSTP1, COX17, PDGFB, PTGS2, USP18, HIF1A, EGFR, EFNB2, ERBB3, TERT, PRDX1, CCL5, ERBB2, HMOX1, MAPK1, FYN, MAP2K7, MAPK3, EGR1, JUN, TGFB1, NOS2, CDKN2A, INSR, CAV1, STAT3, FN1, EEF2, MAPK14, ESR1, PRDX6, MTOR, SOD1, VEGFA, IL4, IL6, CYP1A1, PDCD4, CTNNB1, FAS, PTX3, NFE2L2, BCL2L1

TIMP1, HMGB1, TNF, IL2, VEGFA

*Gene ontology analysis of Cr interacted genes.*


**Table 3.**

*Pathway analysis for the chromium interacting genes related to Respiratory Disease.*

and related networks. The TargetScan library in EnrichR can be used for the possible miRNA interaction. TFs are identified to be significantly associated with the genes involved in the respiratory disease. TRIM28, NFE2L2, EGR1 GATA2, PPARG, ZMIZ1 and ESR1 are significant for respiratory disease influencing DEGs. The regulated genes for each of these TFs for chromium toxicity are shown in **Table 4** followed by the miRNAs identified for chromium interacting genes involved in the Respiratory diseases in **Figure 1**.

#### **3.3 Comparable chemicals**

Information about biological effects of a chemical at genetic level can be extensively extracted from CTD to create new hypotheses with a lot of interaction pathways and networks among genes-contaminants and diseases [22].

This highly contributes in identifying similar contaminants responsible for specific diseases. Comparable chemicals extracted from CTD for the possible sharing with many of the networks common to chromium in respiratory disease are given in **Table 5**. Mercury, SB 203580, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4 one, 2,3-dimethoxy-1,4-naphthoquinone, were found interacting with 102, 81,77and 61 chromium-iInteracting genes in Respiratory disease.


**Figure 1.**

*Gene-TFs-miRNA Interaction Network.*

4-(4-fluorophenyl)-2-(4 hydroxyphenyl)-5-(4-pyridyl)

imidazole

**71**

**Chemical CAS RN Similarity**

*Chromium Genotoxicity Associated with Respiratory Disease*

*DOI: http://dx.doi.org/10.5772/intechopen.97336*

2,3-dimethoxy-1,4-naphthoquinone 6956-96-3 0.174285714 61 Niacin 0.150997151 53 Antimony 7440-36-0 0.143333333 43 Antimony Potassium Tartrate 28300-74-5 0.132183908 46 naringin 10236-47-2 0.131016043 49 SB 203580 0.13022508 81 Mercury 7439-97-6 0.129606099 102 Rutin 153-18-4 0.129518072 43 alpha-Tocopherol 59-02-9 0.126262626 50 cobaltiprotoporphyrin 14325-03-2 0.124338624 47

Luteolin 491-70-3 0.122395833 47

**Index**

0.122615804 45

**Common Interacting Genes for Respiratory disease**

**Table 4.**

*Transcription factors for the chromium interacting genes involved in the Respiratory diseases.*

ZMIZ1 and ESR1 are significant for respiratory disease influencing DEGs. The regulated genes for each of these TFs for chromium toxicity are shown in **Table 4** followed by the miRNAs identified for chromium interacting genes involved in the

*Trace Elements and Their Effects on Human Health and Diseases*

Information about biological effects of a chemical at genetic level can be exten-

This highly contributes in identifying similar contaminants responsible for specific diseases. Comparable chemicals extracted from CTD for the possible sharing with many of the networks common to chromium in respiratory disease are given in **Table 5**. Mercury, SB 203580, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4 one, 2,3-dimethoxy-1,4-naphthoquinone, were found interacting with 102,

> **Combined Score**

TRIM28 9/210 1.02E-05 9.92E-04 82.94725092 EFNB2;EGR1;JUN;PARP1;ERBB3;

NFE2L2 19/1022 3.76E-05 0.001413873 32.51065582 CDKN1A;GSTM1;WNT5A;FN1;

EGR1 10/315 4.37E-05 0.001413873 53.21069483 EGR1;JUN;SERPINA1;STAT3;AKT1;

GATA2 15/772 1.64E-04 0.003970477 28.45933865 JUN;CDKN1A;EDN1;GPX1;MMP2;

PPARG 12/535 2.10E-04 0.004075587 31.58677746 EFNB2;PDGFRA;JUN;CDKN1A;

ZMIZ1 16/914 3.19E-04 0.005162124 23.65905038 EGR1;TGFB1;CDKN1B;GSTP1;

ESR1 6/154 5.36E-04 0.006683025 48.17527072 EDN1;SERPINA1;STAT3;CYP1B1;

CTCF 24/1790 5.51E-04 0.006683025 17.25236108 PDGFRA; EGR1; JUN; EDN1; TGFB1;

MYC 11/573 0.001387281 0.014951805 20.72253353 GJA1; GCLC; PCNA; TFRC; CCND1;

RAD21 17/1265 0.003726145 0.036105828 12.44311845 PDGFRA; JUN; EDN1; PCNA;

*Transcription factors for the chromium interacting genes involved in the Respiratory diseases.*

**Gene**

WNT5A;SOX9;HIF1A;SOD1

PTGS2;ESR1;PRDX6;VEGFA;EFNB2; GJA1;GCLC;PRDX1;DPYD;CAT; CYP1B1;HMOX1;FYN;PTX3;AVPI1

MAPK1;ESR1;MMP9;EGFR;SOD1

LAMB1;FOS;MAPK14;IGF1R;IL4; CHEK2;PDCD4;IDS;IER2;BCL2L1

CASP8;INSR;HILPDA;CYP1B1;FOS; BCL2L1;SOD1;VEGFA

MIR21;SOD1;VEGFA;GCLC;MYC; PRDX1;CAT;IDS;MAPK1;CTNNB1; IER2;AVPI1

FOS;ESR1

PCNA; CAV1; APOA1; EEF2; MAPK14; IGF1R; VEGFA; EFNB2; GCLC; IL6; CEACAM1;CASP8; ERBB3;MYC;CYP1B1;MAP2K7;TP53; BCL2L1;NFE2L2

TERT;PARP1;EEF2;TP53;IER2;SOD1

APOA1; EEF2; MAPK14; SOD2; VEGFA; EFNB2; IL6; CEACAM1; CASP8; MYC; TP53; BCL2L1;NFE2L2

sively extracted from CTD to create new hypotheses with a lot of interaction pathways and networks among genes-contaminants and diseases [22].

81,77and 61 chromium-iInteracting genes in Respiratory disease.

**P-value**

Respiratory diseases in **Figure 1**.

**Term Overlap P-value Adjusted**

**3.3 Comparable chemicals**

**Table 4.**

**70**

**Figure 1.** *Gene-TFs-miRNA Interaction Network.*



**Table 5.**

*Chemicals having comparable sets of interacting genes to chromium.*

#### **4. Conclusions**

Chromium (VI) is a vital toxic environmental pollutant having various sources including mine tailings. This chapter enlighten respiratory disease accelerated as well as caused due to chromium exposure at genetic level following bioinformatics method that leverages curated data from the public database CTD to generate novel sets of information. This strategy does not require a priori knowledge of the toxicant, biological system, or adverse outcome, and it can be used to identify potential molecular and biological intermediary steps that help fill in knowledge gaps connecting chemical exposures with outcomes for environmentally influenced diseases. With the existed data libraries (mainly CTD, GO, pathway, TFs and miRNA relate databases), bioinformatics web-based tools (David and EnrichR), BPs, CCs, MFs, KEEG signal pathways and gene regulation in the chromium-gene-disease networks were presented. In this study, 127 genes are identified as affected by exposure CR(VI), which are majorly regulated by 10 TFs and 10 very high target miRNAs. The Gene-TFs-miRNAs network recognises maximum interacted genes (EFNB2, IGF1R, CYP1B1, INSR, and VEGFA) and TFs (ZMIZ1, NFE2L2, CTCF and RAD21) and miRNAs (hsa-miR-4506, hsa-miR-379, hsa-miR-3529, hsa-miR-4535, hsa-miR-3684, and hsa-miR-409-5p). The significant biological process (positive regulation of gene expression and positive regulation of nitric oxide biosynthetic process), Cellular Component (extracellular space and protein complex) and Molecular Function (identical protein binding and enzyme binding) are influenced by chromium exposures. From pathway analysis of Cr (VI) influence on respiratory disease, maximum of DEGs are identified to be involved in various pathways in cancer (41 nos.) followed by proteoglycans in cancer (30 nos.), and HTLV-I infection (23 nos.) and so on. Comparable contaminants analysis has recognised Mercury, SB 203580, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one to have maximum common DEGs with Cr (VI) exposure.

**Author details**

**73**

Jyoti Kant Choudhari1,2, Jyotsna Choubey2

provided the original work is properly cited.

Anand Kumar Jayapal<sup>3</sup> and Biju Prava Sahariah<sup>1</sup>

*Chromium Genotoxicity Associated with Respiratory Disease*

*DOI: http://dx.doi.org/10.5772/intechopen.97336*

2 Raipur Institute of Technology Raipur, C.G, India

3 National Institute of Technology Raipur, C.G, India

\*Address all correspondence to: biju.sahariah@gmail.com

, Mukesh Kumar Verma1,3,

\*

1 Chhattisgarh Swami Vivekanand Technical University, Bhilai, C.G, India

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Chromium Genotoxicity Associated with Respiratory Disease DOI: http://dx.doi.org/10.5772/intechopen.97336*

#### **Author details**

**4. Conclusions**

**Table 5.**

common DEGs with Cr (VI) exposure.

**72**

Chromium (VI) is a vital toxic environmental pollutant having various sources including mine tailings. This chapter enlighten respiratory disease accelerated as well as caused due to chromium exposure at genetic level following bioinformatics method that leverages curated data from the public database CTD to generate novel sets of information. This strategy does not require a priori knowledge of the toxicant, biological system, or adverse outcome, and it can be used to identify potential

**Index**

154447-36-6 0.122222222 77

0.120385233 75

**Common Interacting Genes for Respiratory disease**

molecular and biological intermediary steps that help fill in knowledge gaps connecting chemical exposures with outcomes for environmentally influenced diseases. With the existed data libraries (mainly CTD, GO, pathway, TFs and miRNA relate databases), bioinformatics web-based tools (David and EnrichR), BPs, CCs, MFs, KEEG signal pathways and gene regulation in the chromium-gene-disease networks were presented. In this study, 127 genes are identified as affected by exposure CR(VI), which are majorly regulated by 10 TFs and 10 very high target miRNAs. The Gene-TFs-miRNAs network recognises maximum interacted genes (EFNB2, IGF1R, CYP1B1, INSR, and VEGFA) and TFs (ZMIZ1, NFE2L2, CTCF and RAD21) and miRNAs (hsa-miR-4506, hsa-miR-379, hsa-miR-3529, hsa-miR-4535, hsa-miR-3684, and hsa-miR-409-5p). The significant biological process (positive regulation of gene expression and positive regulation of nitric oxide biosynthetic process), Cellular Component (extracellular space and protein complex) and Molecular Function (identical protein binding and enzyme binding) are influenced by chromium exposures. From pathway analysis of Cr (VI) influence on respiratory disease, maximum of DEGs are identified to be involved in various pathways in cancer (41 nos.) followed by proteoglycans in cancer (30 nos.), and HTLV-I infection (23 nos.) and so on. Comparable contaminants analysis has recognised Mercury, SB 203580, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one to have maximum

**Chemical CAS RN Similarity**

*Trace Elements and Their Effects on Human Health and Diseases*

*Chemicals having comparable sets of interacting genes to chromium.*

Thioctic Acid 62-46-4 0.121890547 49 Cholesterol, Dietary 0.120943953 41

pyrazolanthrone 0.117117117 65 Docosahexaenoic Acids 25167-62-8 0.117073171 48

2-(4-morpholinyl)-8-phenyl-4H-1-

2-(2-amino-3-methoxyphenyl)-4H-1-

benzopyran-4-one

benzopyran-4-one

Jyoti Kant Choudhari1,2, Jyotsna Choubey2 , Mukesh Kumar Verma1,3, Anand Kumar Jayapal<sup>3</sup> and Biju Prava Sahariah<sup>1</sup> \*

1 Chhattisgarh Swami Vivekanand Technical University, Bhilai, C.G, India


\*Address all correspondence to: biju.sahariah@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 2

Trace Elements in

Environmental Problems

Section 2
