**4.1. The volatility and fluidity of water**

Water is indispensable for human life. As water is generally present surrounding us, the characteristics of water is hard to be considered as a strange matter. But, the truth that a boiling point of water is 100°C is peculiar comparing with the other elements with the similar molecular weight. For examples, though a sulfur is next of oxygen in the periodic table of the elements, the boiling point of hydrogen sulfate (H2S) is -60.7°C. But, as the molecular weight of oxygen is smaller than that of sulfur, the boiling point must be smaller than -60.7°C. It is supposed that the boiling point of water should be theoretically at about -80°C. Then, the coagulating point of water is appropriate theoretically at about - 110°C. Well, why the boiling and coagulating points are respectively at 100°C and 0°C? The reason is by the formation of cluster through the pulling against each other among the water molecules by a hydrogen bond et al. The imaginary molecular weight inferred from the boiling and coagulating points is about 100 by which 5, 6 water molecules gathered. Then, a water molecule has as much as four hydrogen bonds. Moreover, it was thought that the shapes of the cluster shows a straight chain, from a square to eleven-cornered shape, and the average shape is the pentagon. Moreover, it was thought that the cluster of water is not stable for long time, but it was formed and broken at the cycles of 10-12 seconds. But, this condition of cluster is only applied to the very pure water without any ion and impurity. The real water actually contained various impurities. Even if the very pure water is kept in air, the condition of water changes moment by moment by melting of a gas and molecules from air. Then, in order to estimate the present conditions of energy in the water, the change of the weight of water is exactly measured for the indicator of the volatility. When the difference of volatility between the tap water and the deionized water are compared, the deionized water is clearly volatilized more quickly than the tap water (Fig.9).

**Figure 9.** The change of the weight of the deionized water and the tap water

278 Blood Cell – An Overview of Studies in Hematology

**Figure 8.** The FIR animal raising apparatus

**4.1. The volatility and fluidity of water** 

**4. The effects of FIR energy radiation on water** 

Water is indispensable for human life. As water is generally present surrounding us, the characteristics of water is hard to be considered as a strange matter. But, the truth that a boiling point of water is 100°C is peculiar comparing with the other elements with the similar molecular weight. For examples, though a sulfur is next of oxygen in the periodic table of the elements, the boiling point of hydrogen sulfate (H2S) is -60.7°C. But, as the molecular weight of oxygen is smaller than that of sulfur, the boiling point must be smaller than -60.7°C. It is supposed that the boiling point of water should be theoretically at about -80°C. Then, the coagulating point of water is appropriate theoretically at about - 110°C. Well, why the boiling and coagulating points are respectively at 100°C and 0°C? The reason is by the formation of cluster through the pulling against each other among the water molecules by a hydrogen bond et al. The imaginary molecular weight inferred from the boiling and coagulating points is about 100 by which 5, 6 water molecules gathered. Then, a water molecule has as much as four hydrogen bonds. Moreover, it was thought that the shapes of the cluster shows a straight chain, from a square to eleven-cornered shape, and the average shape is the pentagon. Moreover, it was thought that the cluster of water is not stable for long time, but it was formed and broken at the cycles of 10-12 seconds. But, this condition of cluster is only applied to the very pure water without any

The high volatility means that a lot of water molecules spring out of the surface of water. Therefore, it is supposed that the energy of the water molecules is high and its cluster is small. This graph shows the change of weight of the deionized water radiated FIR energy through the grass layer by keeping in the FIR incubator for 12 hours and the untreated deionized water (Fig.10). Though the deionized water more quickly losses the weight than the tap water, the deionized water radiated FIR energy losses much bigger weight than the deionized water. In other words, it was made clear that the deionized water radiated FIR energy has high energy in the molecules to evaporate much easy. The results mean that the cluster of the water can become small by the FIR energy radiation. If that is the case, why the cluster formed by the water molecules changes by accepted the FIR energy? The water molecule consisting of two [O-H] bonds opening with 104°has three types of the vibration energies. One is the deformation vibration of 1594cm-1 and the others are the symmetrical and asymmetrical expansion and contraction energy of each 3656 cm-1 and 3756 cm-1. When the energy were convert to the wave length, the deformation vibration is 6.27μm and the expansion and contraction energy are 2.74μm and 2.66μm [10]. If the FIR energy of 5~20μm radiated the water molecules, the deformation vibration of the water molecule was activated by the transmission of the energy through the resonance effect because the energy of deformation vibration was coincident with the radiating energy. Consequently, it was thought that the kinetic energy

of the water molecule increased to rush out of a cluster. The cluster in the activated water by FIR energy radiation ought to raise the volatility. On the contrary, the strength of FIR energy radiation by any materials can be estimated by measuring the change of the volatility. Then, when the boiling points of water was measured, that of FIR radiated water was 97.4°C and that of control water was 98.9°C. The boiling point of water decreased clearly by FIR energy radiation. These results also suggested that the water molecules are at the condition to rush out easy from the surface of water. Still more, when the viscosity of water was measured, it was clarified that the viscosity clearly decreased by the FIR energy radiation (Fig.11). These results also suggest that the water become to flow easy by the same change of the cluster in water. It is thought that the water radiated FIR energy increases the volatility and fluidity at the same time. It is a very important finding. For example, it is thought that the blood and lymph flow in the human body become smoothly and activates by radiation of the FIR energy. It is only natural that the collection of lymph fluid into a lymph vessel rides the swelling in the body. Then, a tear, sweat and digestive juices become similarly to flow easy also. It is suggested that the activation of the secretion of a tear stimulate the para-sympathetic nerve system. Still more, the activation of the secretion of a sweat stimulates the various metabolisms in whole body. The activation of the secretion of the saliva and digestive juices stimulates the digestive function to make the body vigorously.

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 281

**\*\***

**Figure 11.** The change of the viscosity of water by the rhyolite as the FIR radiator

It was found that the water radiated FIR energy by the rhyolite become to dissolve variable products. The plastic culture dish adhered proteins as bovine serum albumin (BSA) were prepared for the estimation of the solubility of water. The FIR energy radiated ultrapure water by the rhyolite and non-treated same water poured into the dish, and was collected for measurement of the UV absorbance for protein. The results showed that the FIR energy radiated ultrapure water could dissolve much more BSA than the non-treated same water (Fig.12). Not only protein but also sugar and sodium hydroxide were checked the solubility. The results showed that the FIR energy radiated ultrapure water could dissolve much more them. When the rhyolite powder was inserted into a fish tank at about 0.5%, the water in the tank became clear just after 4 days. These results were considered by which the deposited protein and other products in the tank dissolved in water to raise the degree of transparency by the FIR energy radiation from the rhyolite. It was confirmed that the fishes and shell fishes become live longer than usual and keep the freshness in the tank with the rhyolite. For the application of these effects of the FIR energy radiation on water, the development of the contact lens cleaner was tried. The force of cleaning in the FIR energy radiated ultrapure water were estimated to the contact lens attached the BSA on the surface. The results suggested that the force of cleaning of water was surely raised by FIR energy radiation from rhyolite. But, as the force of FIR was defeated to a detergent, the development of the contact lens cleaner was given up. However, it was expected that the effects of the FIR energy radiation from rhyolite, on which the force of solubilizing in water was activated, will be applied in various fields in

**4.2. The solubility of water** 

future.

**Figure 10.** The change of the deionized water weight affected by the rhyolite as the FIR radiator

**Figure 11.** The change of the viscosity of water by the rhyolite as the FIR radiator

#### **4.2. The solubility of water**

280 Blood Cell – An Overview of Studies in Hematology

the digestive function to make the body vigorously.

of the water molecule increased to rush out of a cluster. The cluster in the activated water by FIR energy radiation ought to raise the volatility. On the contrary, the strength of FIR energy radiation by any materials can be estimated by measuring the change of the volatility. Then, when the boiling points of water was measured, that of FIR radiated water was 97.4°C and that of control water was 98.9°C. The boiling point of water decreased clearly by FIR energy radiation. These results also suggested that the water molecules are at the condition to rush out easy from the surface of water. Still more, when the viscosity of water was measured, it was clarified that the viscosity clearly decreased by the FIR energy radiation (Fig.11). These results also suggest that the water become to flow easy by the same change of the cluster in water. It is thought that the water radiated FIR energy increases the volatility and fluidity at the same time. It is a very important finding. For example, it is thought that the blood and lymph flow in the human body become smoothly and activates by radiation of the FIR energy. It is only natural that the collection of lymph fluid into a lymph vessel rides the swelling in the body. Then, a tear, sweat and digestive juices become similarly to flow easy also. It is suggested that the activation of the secretion of a tear stimulate the para-sympathetic nerve system. Still more, the activation of the secretion of a sweat stimulates the various metabolisms in whole body. The activation of the secretion of the saliva and digestive juices stimulates

**Figure 10.** The change of the deionized water weight affected by the rhyolite as the FIR radiator

It was found that the water radiated FIR energy by the rhyolite become to dissolve variable products. The plastic culture dish adhered proteins as bovine serum albumin (BSA) were prepared for the estimation of the solubility of water. The FIR energy radiated ultrapure water by the rhyolite and non-treated same water poured into the dish, and was collected for measurement of the UV absorbance for protein. The results showed that the FIR energy radiated ultrapure water could dissolve much more BSA than the non-treated same water (Fig.12). Not only protein but also sugar and sodium hydroxide were checked the solubility. The results showed that the FIR energy radiated ultrapure water could dissolve much more them. When the rhyolite powder was inserted into a fish tank at about 0.5%, the water in the tank became clear just after 4 days. These results were considered by which the deposited protein and other products in the tank dissolved in water to raise the degree of transparency by the FIR energy radiation from the rhyolite. It was confirmed that the fishes and shell fishes become live longer than usual and keep the freshness in the tank with the rhyolite. For the application of these effects of the FIR energy radiation on water, the development of the contact lens cleaner was tried. The force of cleaning in the FIR energy radiated ultrapure water were estimated to the contact lens attached the BSA on the surface. The results suggested that the force of cleaning of water was surely raised by FIR energy radiation from rhyolite. But, as the force of FIR was defeated to a detergent, the development of the contact lens cleaner was given up. However, it was expected that the effects of the FIR energy radiation from rhyolite, on which the force of solubilizing in water was activated, will be applied in various fields in future.

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 283

*5.1.2. The effect of the FIR energy radiation on the change of blood flow of an human* 

by the application of the Rhyolite containing cloths.

the rhyolite containing blanket compared with the control blanket.

The Rhyolite powder with a peak diameter of about 10μm was printed to the blanket cloths made in Japan at 15%, like polka dots of about 2 cm (MATERA Inc. Toon, Ehime, Japan). The Rhyolite containing blanket and the control blanket were applied to the healthy volunteers for application of FIR energy of 20 min at room temperature 22°C and room humidity 50%, after acclimation of the condition in the room for 10 min. The velocity and the quantity of blood were continuously measured by the laser Doppler blood perfusion monitor and imagers in the same way for 20 min. The order of the application between the FIR and the control blankets was often changed at the condition of double blind. The velocity and the quantity of blood flow were accelerated 32.2% (Fig.14) and 19.1% (Fig.15)

**Figure 14.** The change of the velocity of blood by the FIR blanket. This graph showed the velocity of the blood after 60 min of using the rhyolite containing blanket. The velocity of the blood increased 32.2% in

**Figure 15.** The change of the quantity of blood by the FIR blanket. This graph showed the quantity of

the blood after 60 min of using the rhyolite containing blanket. The quantity of the blood increased19.1% in the rhyolite containing blanket compared with the control blanket.

**Figure 12.** The solubility of BSA in the FIR energy radiated water

#### **5. Experiment**

#### **5.1. The effects of the FIR energy radiation on an animal**

#### *5.1.1. The effect of the FIR energy radiation on the change of body weight of an animal*

When the rats were raised by using the FIR animal raising equipment, the increase of the body weight of rats radiated FIR energy became gentler than that of control (Fig.13). Though the same experiments on the body weight of rats repeated five times, the results showed the same tendency that the FIR energy radiation inhibited the increase of the body weights. In some laboratories, the rats and the mice eat foods and drink water as much as wish. As a result, the rats and the mice naturally grow fat within a year. Therefore, it should be thought that the FIR energy radiation activates the exercise activity and keeps away the switchover to the obesity conditions.

**Figure 13.** The change of the body weight affected by FIR. The growth of the rats was inhibited by the rhyolite as the FIR radiator.

#### *5.1.2. The effect of the FIR energy radiation on the change of blood flow of an human*

282 Blood Cell – An Overview of Studies in Hematology

**5. Experiment** 

to the obesity conditions.

rhyolite as the FIR radiator.

**Figure 12.** The solubility of BSA in the FIR energy radiated water

**5.1. The effects of the FIR energy radiation on an animal** 

*5.1.1. The effect of the FIR energy radiation on the change of body weight of an animal* 

When the rats were raised by using the FIR animal raising equipment, the increase of the body weight of rats radiated FIR energy became gentler than that of control (Fig.13). Though the same experiments on the body weight of rats repeated five times, the results showed the same tendency that the FIR energy radiation inhibited the increase of the body weights. In some laboratories, the rats and the mice eat foods and drink water as much as wish. As a result, the rats and the mice naturally grow fat within a year. Therefore, it should be thought that the FIR energy radiation activates the exercise activity and keeps away the switchover

**Figure 13.** The change of the body weight affected by FIR. The growth of the rats was inhibited by the

The Rhyolite powder with a peak diameter of about 10μm was printed to the blanket cloths made in Japan at 15%, like polka dots of about 2 cm (MATERA Inc. Toon, Ehime, Japan). The Rhyolite containing blanket and the control blanket were applied to the healthy volunteers for application of FIR energy of 20 min at room temperature 22°C and room humidity 50%, after acclimation of the condition in the room for 10 min. The velocity and the quantity of blood were continuously measured by the laser Doppler blood perfusion monitor and imagers in the same way for 20 min. The order of the application between the FIR and the control blankets was often changed at the condition of double blind. The velocity and the quantity of blood flow were accelerated 32.2% (Fig.14) and 19.1% (Fig.15) by the application of the Rhyolite containing cloths.

**Figure 14.** The change of the velocity of blood by the FIR blanket. This graph showed the velocity of the blood after 60 min of using the rhyolite containing blanket. The velocity of the blood increased 32.2% in the rhyolite containing blanket compared with the control blanket.

**Figure 15.** The change of the quantity of blood by the FIR blanket. This graph showed the quantity of the blood after 60 min of using the rhyolite containing blanket. The quantity of the blood increased19.1% in the rhyolite containing blanket compared with the control blanket.

#### *5.1.3. The effect of the FIR energy radiation on the change of blood catecholamine*

The amount of adrenaline, noradrenaline and dopamine in blood of the 40 mice keeping in the FIR and control animal raising apparatus for 40 days were measured at the blood center. It was shown that the amount of the dopamine in blood catecholamine significantly decreased, and the adrenaline and the noradrenaline in the blood catecholamine tend to decrease by radiated the FIR energy (Fig.16). These results suggested that the FIR energy radiation decreased the amount of the blood catecholamine in mice to inhibit the sympathetic nerve and activate the parasympathetic nerve. A mamma as a human body has the autonomous nervous system with the motor nervous and sensory nervous system. The autonomous nervous system distributes all internal organs to work in order to sustain a person's life independent of his mind.

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 285

*5.1.4. The effect of the FIR energy radiation on the biological macromolecules* 

radiation has a diet effect.

A

bso

r

a

n

c

e

b

The effect of FIR energy radiation on the biological macromolecules is being made clear. The effects of FIR energy radiation on the activity of the enzyme can be estimated by using the FIR incubator developed by us. An enzyme is the special protein which has the various amino acids forming the three-dimensional structure with the various bonds to get a special role to a chemical reaction. In case that the activities of the 19 kinds of the enzymes appear in radiating FIR energy, it was made clear that the activity of some enzymes were accelerated or restrained. These results suggested that the efficiency of the enzyme activity changed because the various bond conforming the three-dimensional structure of the enzyme was affected by the FIR energy. In the enzymes measured by us, the results suggested that the activities of esterase, esterase lipase, lipase, leucin allylamidase, betaglucuronidase were accelerated and that of alkaline-phosphatase, acid-phosphatase, naphtol-AS-BI-phospho hydrase were restrained. Especially, esterase, esterase lipase and lipase are the enzymes to decompose a fat. Though the experiments were preliminary study, it was made clear that the FIR energy radiation changed the three-dimensional structure of the various enzymes to accelerate or restrain the activities. Still more, it was possible from the results that the increase of body weight restrained because some decomposition enzyme of fat were restrained by FIR energy radiation. These results suggested that the FIR energy

. **Figure 17.** The change of activity of lipase by FIR energy radiation. The activity in the FIR radiation

group was higher 40.5% than the control group at 530nm.

**Figure 16.** The change of the concentration of catecholamine in the blood affected by the FIR energy radiation. The concentration of the adrenaline, noradrenaline and dopamine in the FIR group were significantly lower 14.4%

There are the sympathetic nerves and the parasympathetic nerves in the autonomous nervous system which distribute every internal organ at the same times to control the function of each internal organ in spite of his mind. When the sympathetic nerves excited, the heart beat violently, the blood pressure increase, the breath become hard and the stress build up in the body. On the contrary, when the parasympathetic nerves excited, the feeling become confortable as at home to become feeling an appetite, sleep and sexual desire. It is thought that the rats and the mice radiate FIR energy become loss of the stress in the cage and lives the active and healthy life. In short, FIR energy radiation may avoid the stress and obesity.

#### *5.1.4. The effect of the FIR energy radiation on the biological macromolecules*

284 Blood Cell – An Overview of Studies in Hematology

person's life independent of his mind.

significantly lower 14.4%

obesity.

*5.1.3. The effect of the FIR energy radiation on the change of blood catecholamine* 

The amount of adrenaline, noradrenaline and dopamine in blood of the 40 mice keeping in the FIR and control animal raising apparatus for 40 days were measured at the blood center. It was shown that the amount of the dopamine in blood catecholamine significantly decreased, and the adrenaline and the noradrenaline in the blood catecholamine tend to decrease by radiated the FIR energy (Fig.16). These results suggested that the FIR energy radiation decreased the amount of the blood catecholamine in mice to inhibit the sympathetic nerve and activate the parasympathetic nerve. A mamma as a human body has the autonomous nervous system with the motor nervous and sensory nervous system. The autonomous nervous system distributes all internal organs to work in order to sustain a

**Figure 16.** The change of the concentration of catecholamine in the blood affected by the FIR energy radiation. The concentration of the adrenaline, noradrenaline and dopamine in the FIR group were

Adrenalin Noradrenalin Dopamine

**\***

There are the sympathetic nerves and the parasympathetic nerves in the autonomous nervous system which distribute every internal organ at the same times to control the function of each internal organ in spite of his mind. When the sympathetic nerves excited, the heart beat violently, the blood pressure increase, the breath become hard and the stress build up in the body. On the contrary, when the parasympathetic nerves excited, the feeling become confortable as at home to become feeling an appetite, sleep and sexual desire. It is thought that the rats and the mice radiate FIR energy become loss of the stress in the cage and lives the active and healthy life. In short, FIR energy radiation may avoid the stress and The effect of FIR energy radiation on the biological macromolecules is being made clear. The effects of FIR energy radiation on the activity of the enzyme can be estimated by using the FIR incubator developed by us. An enzyme is the special protein which has the various amino acids forming the three-dimensional structure with the various bonds to get a special role to a chemical reaction. In case that the activities of the 19 kinds of the enzymes appear in radiating FIR energy, it was made clear that the activity of some enzymes were accelerated or restrained. These results suggested that the efficiency of the enzyme activity changed because the various bond conforming the three-dimensional structure of the enzyme was affected by the FIR energy. In the enzymes measured by us, the results suggested that the activities of esterase, esterase lipase, lipase, leucin allylamidase, betaglucuronidase were accelerated and that of alkaline-phosphatase, acid-phosphatase, naphtol-AS-BI-phospho hydrase were restrained. Especially, esterase, esterase lipase and lipase are the enzymes to decompose a fat. Though the experiments were preliminary study, it was made clear that the FIR energy radiation changed the three-dimensional structure of the various enzymes to accelerate or restrain the activities. Still more, it was possible from the results that the increase of body weight restrained because some decomposition enzyme of fat were restrained by FIR energy radiation. These results suggested that the FIR energy radiation has a diet effect.

**Figure 17.** The change of activity of lipase by FIR energy radiation. The activity in the FIR radiation group was higher 40.5% than the control group at 530nm.

.

## **5.2. The effects of FIR energy radiation skin**

#### *5.2.1. The effect of FIR energy radiation on skin repair*

The wound of circle with 8mm diameter was artificially made at the skin of rats keeping the FIR and the control animal raising apparatus. Then, the area of the wound was measured each several days. And the surface condition was observed. The results showed that though the surface of the wound in the FIR radiation group wet and soft, that of the control group was covered the hard scab (Fig.18).

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 287

a fee for medical treatment becomes a social problem, if the bedsore could be suppressed to shorten the term of the admission to a hospital, the fee for the medical treatment would be reduced in the future. Still more, if the space of the residence was constructed with the FIR energy radiating building material as the rhyolite, the metabolism of skin would be promoted to hinder the aging of skin. Then, the FIR energy radiating ceramics as rhyolite may be tied together with a development of the creams and cosmetics to hinder the aging of

**Figure 19.** The repair area of wound hearing. The speed of repair was accelerated affected by the FIR

The mouse naturally induced a crisis of the inflammation of the skin as the atopic dermatitis with the application of a picric acid as the material raising the inflammation was imported from the United States. Then, the effects of the FIR energy radiation on the induction of the inflammation of the skin were analyzed by using the mouse. No an inflammation of skin in the mouse radiated FIR energy was detected after the application of a picric acid, though the atopic dermatitis of control mouse in the same condition were all induced without the FIR energy radiation (Fig.20). It was made clear that the area of the inflammation of skin and the number of the mast cells at the inflammation of skin as an atopic dermatitis clearly

These results suggested that the FIR energy radiation was effective for the prevention and the medical treatment to the inflammation of the skin as an atopic dermatitis. In the future, it is thought that the FIR ceramics as the rhyolite are tied together with the development of

the medicine or the cure for the inflammation of the skin as an atopic dermatitis.

*5.2.2. Effect of the FIR energy radiation on a atopic dermatitis* 

decreased by the FIR energy radiation (Fig.21).

skin.

energy radiation.

**Figure 18.** The surface of wound of skin affected by the FIR energy radiation. Control : (A) FIR energy radiation : (B)

The repair area of FIR radiation group increased each 30.54%, 28.27% and 14.22% after 5, 6 and 7days compared with control group (Fig.19). These results showed that the FIR energy radiation from out of body accelerated the wound healing at the surface of rat skin. Still more, the repair area of wound increased each 44.39% and 24.29% after 3 and 5 days by application of the ointment containing the 10% rhyolite as the FIR energy radiating ceramics. In other words, it was clarified that the FIR energy radiation promoted the wound hearing of skin, even if the radiation of FIR energy were by far from body or by contact with skin. If the blanket and sheet with FIR energy radiation effects were developed, the bedsore of the bedridden old persons or the sick persons in bed for a long time would be suppressed and restored to regenerate the skin and ease the pain of the patients. Now, though the rise in a fee for medical treatment becomes a social problem, if the bedsore could be suppressed to shorten the term of the admission to a hospital, the fee for the medical treatment would be reduced in the future. Still more, if the space of the residence was constructed with the FIR energy radiating building material as the rhyolite, the metabolism of skin would be promoted to hinder the aging of skin. Then, the FIR energy radiating ceramics as rhyolite may be tied together with a development of the creams and cosmetics to hinder the aging of skin.

286 Blood Cell – An Overview of Studies in Hematology

was covered the hard scab (Fig.18).

A

radiation : (B)

**5.2. The effects of FIR energy radiation skin** 

*5.2.1. The effect of FIR energy radiation on skin repair* 

B

The wound of circle with 8mm diameter was artificially made at the skin of rats keeping the FIR and the control animal raising apparatus. Then, the area of the wound was measured each several days. And the surface condition was observed. The results showed that though the surface of the wound in the FIR radiation group wet and soft, that of the control group

**Figure 18.** The surface of wound of skin affected by the FIR energy radiation. Control : (A) FIR energy

The repair area of FIR radiation group increased each 30.54%, 28.27% and 14.22% after 5, 6 and 7days compared with control group (Fig.19). These results showed that the FIR energy radiation from out of body accelerated the wound healing at the surface of rat skin. Still more, the repair area of wound increased each 44.39% and 24.29% after 3 and 5 days by application of the ointment containing the 10% rhyolite as the FIR energy radiating ceramics. In other words, it was clarified that the FIR energy radiation promoted the wound hearing of skin, even if the radiation of FIR energy were by far from body or by contact with skin. If the blanket and sheet with FIR energy radiation effects were developed, the bedsore of the bedridden old persons or the sick persons in bed for a long time would be suppressed and restored to regenerate the skin and ease the pain of the patients. Now, though the rise in

**Figure 19.** The repair area of wound hearing. The speed of repair was accelerated affected by the FIR energy radiation.

#### *5.2.2. Effect of the FIR energy radiation on a atopic dermatitis*

The mouse naturally induced a crisis of the inflammation of the skin as the atopic dermatitis with the application of a picric acid as the material raising the inflammation was imported from the United States. Then, the effects of the FIR energy radiation on the induction of the inflammation of the skin were analyzed by using the mouse. No an inflammation of skin in the mouse radiated FIR energy was detected after the application of a picric acid, though the atopic dermatitis of control mouse in the same condition were all induced without the FIR energy radiation (Fig.20). It was made clear that the area of the inflammation of skin and the number of the mast cells at the inflammation of skin as an atopic dermatitis clearly decreased by the FIR energy radiation (Fig.21).

These results suggested that the FIR energy radiation was effective for the prevention and the medical treatment to the inflammation of the skin as an atopic dermatitis. In the future, it is thought that the FIR ceramics as the rhyolite are tied together with the development of the medicine or the cure for the inflammation of the skin as an atopic dermatitis.

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 289

**5.3. The effects of the FIR energy radiation on bone** 

phase-contrast microscope (Fig.22).

oppositely dropped (Table 2).

*5.3.1. The effect of the FIR energy radiation on the cultured osteoblast-like cells* 

analysis the microarray hybridization method (http://www.godatabase.org).

The culturing osteoblast-like MC3T3-E1 cell (Riken Cell Banc, Tsukuba, Japan) were separated in the two groups in the FIR and the control CO2 incubator. After 1, 3, 5, 7, 10 and 14days, the cell number of living cells in both groups were measured by a hematocytometer and observed by phase contrast microscope CK40 (Olympus, Tokyo, Japan). Still more, the gene expression of the 4 days culture cells in both groups was analyzed by the cluster

The proliferation of the osteoblast-like MC3T3-E1 cells in FIR group significantly inhibited each 19.08% and 12.24% after 3day and 10days culture compared with control group. But, it was considered that the formation of the calcified nodules in the FIR group was more than the control group by the observation of the culture cells stained with von kossa and with a

**Figure 22.** The effects of the FIR energy radiation on the bone nodules formed by MC3T3-E1 cells. The cultured MC3T3-E1 cells in FIR group (A) and control group (B) was observed by the phase contrast microscope CK40 (Olympus, Tokyo, Japan). Some samples after 4weeks culture were stained with Von Kossa method and observed by the same method. FIR group (C) Control group (D) Bar: 100μm

The results showed that the number and the area of the calcified nodules of the FIR group were 148.8 and 158.8 μm2, though no calcified nodules was formed yet in control group after 2 weeks culture. Still more, that of FIR group increased each 2.92times and 4.91times compared with the control group after 4weeks (Fig.23). Consequently, the formation of the calcified nodules was clearly promoted by the FIR energy radiation. On the other hand, it was made clear by the results of cluster analysis after 4 day culture that the genes expression of Interferon activated gene 205, 203 and Interferon α-inducible protein 27 in the cell to cell signal field were reinforced and the gene expression of Interleukin 1 family member 6, Interleukin 17D, 17 receptor C, 17B, and Interleukin 3 related with inflammation were

It was also made clear that the genes expression of Platelet-derived growth factor (PDGF) D polypeptide, Transforming growth factor β (TGFβ) induced and TGFβ inducible early growth response 1 in the cell proliferation field were reinforced, and the gene expression of PDGF B polypeptide and Fibroblast growth factor (FGF) 2, 21, 8 were oppositely dropped (Table 2). It was clarified by these data that the cell differentiation as the function of osteoblast estimated by the formation of calcified nodules were promoted by FIR energy

**Figure 20.** The change of the inflammation of the skin as the atopic dermatitis. The induction of the inflammation of the skin on all mice was suppressed by the FIR energy radiation

**Figure 21.** The area of the inflammation of skin and the number of the mast cells at the inflammation. Though the area of the inflammation in control group was 1.96cm2, no inflammation was detected in the FIR energy radiation group (A). The number of must cells was smaller 26.9% in the FIR energy radiation group than the control group (B).

#### **5.3. The effects of the FIR energy radiation on bone**

288 Blood Cell – An Overview of Studies in Hematology

**Figure 20.** The change of the inflammation of the skin as the atopic dermatitis. The induction of the

**Figure 21.** The area of the inflammation of skin and the number of the mast cells at the inflammation. Though the area of the inflammation in control group was 1.96cm2, no inflammation was detected in the FIR energy radiation group (A). The number of must cells was smaller 26.9% in the FIR energy radiation

group than the control group (B).

inflammation of the skin on all mice was suppressed by the FIR energy radiation

B A

#### *5.3.1. The effect of the FIR energy radiation on the cultured osteoblast-like cells*

The culturing osteoblast-like MC3T3-E1 cell (Riken Cell Banc, Tsukuba, Japan) were separated in the two groups in the FIR and the control CO2 incubator. After 1, 3, 5, 7, 10 and 14days, the cell number of living cells in both groups were measured by a hematocytometer and observed by phase contrast microscope CK40 (Olympus, Tokyo, Japan). Still more, the gene expression of the 4 days culture cells in both groups was analyzed by the cluster analysis the microarray hybridization method (http://www.godatabase.org).

The proliferation of the osteoblast-like MC3T3-E1 cells in FIR group significantly inhibited each 19.08% and 12.24% after 3day and 10days culture compared with control group. But, it was considered that the formation of the calcified nodules in the FIR group was more than the control group by the observation of the culture cells stained with von kossa and with a phase-contrast microscope (Fig.22).

**Figure 22.** The effects of the FIR energy radiation on the bone nodules formed by MC3T3-E1 cells. The cultured MC3T3-E1 cells in FIR group (A) and control group (B) was observed by the phase contrast microscope CK40 (Olympus, Tokyo, Japan). Some samples after 4weeks culture were stained with Von Kossa method and observed by the same method. FIR group (C) Control group (D) Bar: 100μm

The results showed that the number and the area of the calcified nodules of the FIR group were 148.8 and 158.8 μm2, though no calcified nodules was formed yet in control group after 2 weeks culture. Still more, that of FIR group increased each 2.92times and 4.91times compared with the control group after 4weeks (Fig.23). Consequently, the formation of the calcified nodules was clearly promoted by the FIR energy radiation. On the other hand, it was made clear by the results of cluster analysis after 4 day culture that the genes expression of Interferon activated gene 205, 203 and Interferon α-inducible protein 27 in the cell to cell signal field were reinforced and the gene expression of Interleukin 1 family member 6, Interleukin 17D, 17 receptor C, 17B, and Interleukin 3 related with inflammation were oppositely dropped (Table 2).

It was also made clear that the genes expression of Platelet-derived growth factor (PDGF) D polypeptide, Transforming growth factor β (TGFβ) induced and TGFβ inducible early growth response 1 in the cell proliferation field were reinforced, and the gene expression of PDGF B polypeptide and Fibroblast growth factor (FGF) 2, 21, 8 were oppositely dropped (Table 2). It was clarified by these data that the cell differentiation as the function of osteoblast estimated by the formation of calcified nodules were promoted by FIR energy

radiation, though the proliferation was inhibited by it. In short, the FIR energy radiation shorts the term of cell proliferation and advances the timing of cell differentiation on the osteoblast. Finally, it was considered that the FIR energy radiation control these genes expression to inhibit the cell proliferation and promote the cell differentiation of osteoblast [8, 9]. It was made clear by these facts that the differentiation of cultured osteoblast-like cell as an osteoblast is promoted by the radiation of FIR energy.

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 291

**\*\***

**Figure 23.** The effects of the FIR energy radiation on the number and the area of the bone nodules formed by MC3T3-E1 cells. Each five samples stained with Von Kossa method after 1, 2, 3 and 4 weeks

Pure titanium post (RTP post titanium #Ti, Dentech, Tokyo) were treated with the snowtech O solution (Nissan Chemical Industries, Tokyo) for 48 hours and immersed in MEM medium (Minimum Essential Medium Eagle, Sigma, St. Louis, MO, USA) for 5 days. Control group was only immersed in MEM medium at same condition. The 20 S.D. rats of 6 weeks old were implanted the titanium post into the central position of the femur. Left side was control group and Right side was the snowtech O solution treated group. These rats were separated two groups which the FIR energy radiating group kept in the FIR animal raising apparatus and the control group was kept in the normal animal raising apparatus. The femur with the titanium post was extracted and measured the intensity between the titanium and the bone by the tension test with the small omnipotent testing machine (Autograph, Shimadzu, Kyoto) after 1, 2 and 4 weeks raising. The same experiments were repeated twice. Though the data were easy affected by the damage of the surgical operation and the extent of the inflammation, the identical tendency was shown. The tension intensity of the implanted titanium post with the snowtech O solution in the FIR energy radiating group significantly reinforced 2.68 times as much as the same implant in the control group

in both groups were measured the nodules (A) and the number of the area (B) by Bio-system microscope BX-51. The measurement was analyzed by the average of five field of visions of each

samples. Statistical analysis was carried out by t-test. \*P<0.01

after 2 weeks (Fig.24).

*5.3.1.1. The effects of FIR energy radiation on the titanium implant* 


**Table 2.** The cluster analysis of the gene expression in MC3T3-E1 cells affected by FIR energy radiation by the microarray hybridigestion method These genes were reinforced and repressed at 1.3 over by the FIR energy radiation in the cluster of the Cell-to signaling and the growth.

**Figure 23.** The effects of the FIR energy radiation on the number and the area of the bone nodules formed by MC3T3-E1 cells. Each five samples stained with Von Kossa method after 1, 2, 3 and 4 weeks in both groups were measured the nodules (A) and the number of the area (B) by Bio-system microscope BX-51. The measurement was analyzed by the average of five field of visions of each samples. Statistical analysis was carried out by t-test. \*P<0.01

#### *5.3.1.1. The effects of FIR energy radiation on the titanium implant*

290 Blood Cell – An Overview of Studies in Hematology

**Cell-cell signaling** 

**Interleukin 17D**

**Interleukin 17 receptor** 

**Interleukin 3**

**Interleukin 17B**

**Growth Gene Name**

**Interferon activated gene 205 Interleukin 15 receptor, alpha chain Interferon alpha-inducible protein 27 Mitogen activated protein 3K 7 Interferon activated gene 203**

**Interleukin 1 family, member 6**

**Growth differentiation factor 15**

**Fibroblast growth factor 8 Fibroblast growth factor 21**

**Fibroblast growth factor 2**

**Early growth response 3**

**1(Gab1)**

**Heparin-binding EGF-like growth factor** 

**Platelet-derived growth factor, D polypeptide Transforming growth factor, beta induced TGFb Inducible early growth response 1**

**Mitogen activated protein kinase kinase kinase 7**

**Epidermal growth factor receptor related gene**

**Platelet-derived growth factor, B polypeptide**

**Growth factor receptor binding protein2-ap** 

FIR energy radiation in the cluster of the Cell-to signaling and the growth.

**Heparin-binding EGF-like growth factor**

as an osteoblast is promoted by the radiation of FIR energy.

radiation, though the proliferation was inhibited by it. In short, the FIR energy radiation shorts the term of cell proliferation and advances the timing of cell differentiation on the osteoblast. Finally, it was considered that the FIR energy radiation control these genes expression to inhibit the cell proliferation and promote the cell differentiation of osteoblast [8, 9]. It was made clear by these facts that the differentiation of cultured osteoblast-like cell

**Gene Name Acc. No. Fold change**

NM\_172648 NM\_008358 AK010014 BC006665 NM\_008328

NM\_019450 NM\_145837 NM\_010415 NM\_010556 NM\_134159 NM\_019508

Acc. No.

AK003359 NM\_009369 NM\_013692 BC006665 NM\_011819

D12483 NM\_020013 M99623 NM\_008006 NM\_010415 NM\_018781 NM\_021356 NM\_011057

**Table 2.** The cluster analysis of the gene expression in MC3T3-E1 cells affected by FIR energy radiation by the microarray hybridigestion method These genes were reinforced and repressed at 1.3 over by the

1.597 1.422 1.388 1.365 1.328


1.555 1.505 1.383 1.365 1.322


Fold change

Pure titanium post (RTP post titanium #Ti, Dentech, Tokyo) were treated with the snowtech O solution (Nissan Chemical Industries, Tokyo) for 48 hours and immersed in MEM medium (Minimum Essential Medium Eagle, Sigma, St. Louis, MO, USA) for 5 days. Control group was only immersed in MEM medium at same condition. The 20 S.D. rats of 6 weeks old were implanted the titanium post into the central position of the femur. Left side was control group and Right side was the snowtech O solution treated group. These rats were separated two groups which the FIR energy radiating group kept in the FIR animal raising apparatus and the control group was kept in the normal animal raising apparatus. The femur with the titanium post was extracted and measured the intensity between the titanium and the bone by the tension test with the small omnipotent testing machine (Autograph, Shimadzu, Kyoto) after 1, 2 and 4 weeks raising. The same experiments were repeated twice. Though the data were easy affected by the damage of the surgical operation and the extent of the inflammation, the identical tendency was shown. The tension intensity of the implanted titanium post with the snowtech O solution in the FIR energy radiating group significantly reinforced 2.68 times as much as the same implant in the control group after 2 weeks (Fig.24).

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 293

and the left femur at the distal position grew 1.3% also. The width of the right femur at central and distal position significantly grew each 3.7% and 6.1%, and that of the right tibia at central position and the left tibia at the distal position grew 11.8% also. Then, on the length of the long bone, the left tibia significantly grew 2.4% alone. The special affected parts were shown at Fig.25. It was clarified that the width of the femur and tibia quickly grew as

**Figure 25.** The effect of the eating the feed containing rhyolite on the growth of the bone. The

measurement parts of the femur and the tibia were shown (A). The growth of the femur and the tibia of these three groups of 0.01% and 0.1%FIR treated and control were compared after 115days. Right : (B), Left : (C) It was clarified that the width of the femur and tibia quickly grew in the 0.01% FIR group.

It was made clear by these results that the bone formation was promoted by the inside radiation from the digestive organs by eating the feed containing the FIR energy radiating ceramics as same as the outside radiation of the FIR energy. These results suggest that the FIR energy radiation from the digestive organs arrives easy the deep posited bone of the living body. Still more, it was thought that the growth of length was a little inhibited and that of width was promoted. In other words, the FIR energy radiation does not promote the intrachondral ossification but the intra-membranous ossification that the osteoblast directly differentiates from the undifferentiated cells. As the effects of the bone affected more sensitive than the cartilage with the blood capillary, it was thought that the bone formation by the osteoblast is promoted by the FIR energy radiation. The results also prove that the

shown in Fig.25.

**Figure 24.** The effect of the FIR radiation on the strength of the bone bonding to titanium. The bone bonding to titanium was clearly promoted by the FIR energy radiation.

It was been thinking that FIR energy was too weak to permeate deep into the tissue of the living body. But, if the FIR energy radiation affects the water molecules in the blood as mentioned previously, the blood of the whole body was affected by FIR energy even if the deep position. The possibility is supported by the experimental results which the new bone formation inside of the femur was activated by the FIR energy radiation at the surface of the skin from outside of body.

It is suggested that the deformational vibration of the water molecules in the blood affected by the FIR energy radiation was activated by the transmission of the energy through the resonance effect because the energy of deformation vibration was coincident with the radiating energy. It was proved that the blood containing the activated water molecules become quickly flow into the bone to activate the new bone formation.

#### *5.3.1.2. The effects of the feed containing the FIR ceramics on the growth of bone*

The powder of the rhyolite mined at the vein of ore located on the Median Tectonic Line around Ehime prefecture was utilized for this experiment. The composition of the ingredient is SiO2 66.8%, Al2O3 13.52%, Fe2O3 6.98%, K2O 4.19%, Na2O 3.98%, CaO 2.55%. The 60 rats of SD strain at 4 weeks old were separated the three groups of 0.01% and 0.1%FIR treated and control. The each group was raised every 2 or 3 days with the standard feed containing 0.01% and 0.1% rhyolite and nothing for the each group in the measurement the amount of the eating feed. After 115days, all rats were killed by the over anesthesia and the femur and the tibia were measured at many parts of width and the length.

By the results of experiments, the amount of the eating feed of 0.01% FIR treated group increased up to 6.3%, that of 0. 1% FIR treated group decreased on the contrary compared with the control group. The width of the right femur at distal, central and postal position significantly grew each 3.2%, 21.4% and 12.3% and that of the right tibia at central position and the left femur at the distal position grew 1.3% also. The width of the right femur at central and distal position significantly grew each 3.7% and 6.1%, and that of the right tibia at central position and the left tibia at the distal position grew 11.8% also. Then, on the length of the long bone, the left tibia significantly grew 2.4% alone. The special affected parts were shown at Fig.25. It was clarified that the width of the femur and tibia quickly grew as shown in Fig.25.

292 Blood Cell – An Overview of Studies in Hematology

skin from outside of body.

**Figure 24.** The effect of the FIR radiation on the strength of the bone bonding to titanium. The bone

It was been thinking that FIR energy was too weak to permeate deep into the tissue of the living body. But, if the FIR energy radiation affects the water molecules in the blood as mentioned previously, the blood of the whole body was affected by FIR energy even if the deep position. The possibility is supported by the experimental results which the new bone formation inside of the femur was activated by the FIR energy radiation at the surface of the

It is suggested that the deformational vibration of the water molecules in the blood affected by the FIR energy radiation was activated by the transmission of the energy through the resonance effect because the energy of deformation vibration was coincident with the radiating energy. It was proved that the blood containing the activated water molecules

The powder of the rhyolite mined at the vein of ore located on the Median Tectonic Line around Ehime prefecture was utilized for this experiment. The composition of the ingredient is SiO2 66.8%, Al2O3 13.52%, Fe2O3 6.98%, K2O 4.19%, Na2O 3.98%, CaO 2.55%. The 60 rats of SD strain at 4 weeks old were separated the three groups of 0.01% and 0.1%FIR treated and control. The each group was raised every 2 or 3 days with the standard feed containing 0.01% and 0.1% rhyolite and nothing for the each group in the measurement the amount of the eating feed. After 115days, all rats were killed by the over anesthesia and

By the results of experiments, the amount of the eating feed of 0.01% FIR treated group increased up to 6.3%, that of 0. 1% FIR treated group decreased on the contrary compared with the control group. The width of the right femur at distal, central and postal position significantly grew each 3.2%, 21.4% and 12.3% and that of the right tibia at central position

bonding to titanium was clearly promoted by the FIR energy radiation.

become quickly flow into the bone to activate the new bone formation.

*5.3.1.2. The effects of the feed containing the FIR ceramics on the growth of bone* 

the femur and the tibia were measured at many parts of width and the length.

**Figure 25.** The effect of the eating the feed containing rhyolite on the growth of the bone. The measurement parts of the femur and the tibia were shown (A). The growth of the femur and the tibia of these three groups of 0.01% and 0.1%FIR treated and control were compared after 115days. Right : (B), Left : (C) It was clarified that the width of the femur and tibia quickly grew in the 0.01% FIR group.

It was made clear by these results that the bone formation was promoted by the inside radiation from the digestive organs by eating the feed containing the FIR energy radiating ceramics as same as the outside radiation of the FIR energy. These results suggest that the FIR energy radiation from the digestive organs arrives easy the deep posited bone of the living body. Still more, it was thought that the growth of length was a little inhibited and that of width was promoted. In other words, the FIR energy radiation does not promote the intrachondral ossification but the intra-membranous ossification that the osteoblast directly differentiates from the undifferentiated cells. As the effects of the bone affected more sensitive than the cartilage with the blood capillary, it was thought that the bone formation by the osteoblast is promoted by the FIR energy radiation. The results also prove that the

FIR energy radiation affected the living body though the blood containing many water molecules. It was clarified that the FIR energy radiation promotes the bone formation though the energy transmission to water molecules.

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 295

**Table 3.** The analysis of the extracted genes induced by the FIR energy radiation. The 10 most

promoted genes and the 10 most inhibited genes in these changed genes showed

## **5.4. The Effects of the FIR energy radiation on the cancer**

#### *5.4.1. The Effects of the FIR energy radiation on the culture cancer cells*

In order to clarify the effects of the FIR energy radiation on the culture cancer cells, A431 human epithelial vulva carcinoma cells, Sa3 human gingival squamous carcinoma cells, HSC3 human tongue squamous carcinoma cells, A549 human lung carcinoma cells, and MCF7 human breast carcinoma cells were cultured for the analysis of the effects on the proliferation, the shape of cells and the induction of apoptosis.

A431, A549, and MCF7 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture (Sigma, St. Louis, MO, USA). HSC3 and Sa3 cells were cultured in Eagle's basal medium (Sigma). All culture medium was supplemented with 10% heatinactivated fetal bovine serum. The cells (5×104) were plated and measured on days 8 using 0.2% trypan blue and the hemocytometer. Incorporation of 5-bromo-2'-deoxyuridine (BrdU) was used to determine the amount of DNA synthesis. Cells were observed with a CK40 phase contrast microscope (Olympus, Tokyo, Japan), fixed, and stained with hematoxylin and eosin. Still more, apoptotic cells were identified using an Apo-BrdU in situ DNA Fragmentation Assay Kit (Bio Vision, Mountain View, CA, USA).

Although the proliferation of A549, HSC3, and Sa3 cells was significantly suppressed from day 6 of culture (59.0%, 75.4%, and 76.2%, respectively) up to at least day 10, FIR irradiation had little effect on the growth of A431 or MCF7 cells. Measurement of BrdU incorporation on day 4 of culture also showed a significant suppression of growth by FIR irradiation in A549, HSC3, and Sa3 cells but not in A431 or MCF7 cells. Observation of the morphology by the phase contrast microscopy revealed that the cytoplasm and nucleus was enlarged in A549 cells. Some of the HSC3 cells also showed hypertrophy of the cytoplasm and nucleus and others tended to show atrophy. Finally, some of the Sa3 cells showed hypertrophy of the cytoplasm (to refer JEMAA 2010, 2, 382-394, Fig.1) [10]

To determine whether the inhibition of proliferation by FIR energy radiation was associated with apoptosis or necrosis, cancer cells were subjected to FIR irradiation of 48 h and analyzed on day 4 by staining with annexin V-FITC and PI and by TUNEL (TdT-mediated dUTP-biotin nick end labeling) (to refer JEMAA, 2010, 2, 382-394, Fig.3)[10]. These results indicated that FIR did not induce apoptosis in A431, HSC3, or Sa3 cells. A few of the Sa3 cells showed significant necrosis. These results indicate that the inhibition of proliferation by FIR in the cancer cells, particularly in HSC3 and Sa3 cells, was not due to apoptosis. Still more, in order to analyze all expressing genes induced by FIR energy radiation, the control and the FIR-irradiated samples after four days culture was monitored using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) with elimination of low signal feature of background signal + 2.6 × SD, and P value < 0.01. Genes were further classified for process and function according to their

though the energy transmission to water molecules.

**5.4. The Effects of the FIR energy radiation on the cancer** 

proliferation, the shape of cells and the induction of apoptosis.

Fragmentation Assay Kit (Bio Vision, Mountain View, CA, USA).

the cytoplasm (to refer JEMAA 2010, 2, 382-394, Fig.1) [10]

and function according to their

*5.4.1. The Effects of the FIR energy radiation on the culture cancer cells* 

FIR energy radiation affected the living body though the blood containing many water molecules. It was clarified that the FIR energy radiation promotes the bone formation

In order to clarify the effects of the FIR energy radiation on the culture cancer cells, A431 human epithelial vulva carcinoma cells, Sa3 human gingival squamous carcinoma cells, HSC3 human tongue squamous carcinoma cells, A549 human lung carcinoma cells, and MCF7 human breast carcinoma cells were cultured for the analysis of the effects on the

A431, A549, and MCF7 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture (Sigma, St. Louis, MO, USA). HSC3 and Sa3 cells were cultured in Eagle's basal medium (Sigma). All culture medium was supplemented with 10% heatinactivated fetal bovine serum. The cells (5×104) were plated and measured on days 8 using 0.2% trypan blue and the hemocytometer. Incorporation of 5-bromo-2'-deoxyuridine (BrdU) was used to determine the amount of DNA synthesis. Cells were observed with a CK40 phase contrast microscope (Olympus, Tokyo, Japan), fixed, and stained with hematoxylin and eosin. Still more, apoptotic cells were identified using an Apo-BrdU in situ DNA

Although the proliferation of A549, HSC3, and Sa3 cells was significantly suppressed from day 6 of culture (59.0%, 75.4%, and 76.2%, respectively) up to at least day 10, FIR irradiation had little effect on the growth of A431 or MCF7 cells. Measurement of BrdU incorporation on day 4 of culture also showed a significant suppression of growth by FIR irradiation in A549, HSC3, and Sa3 cells but not in A431 or MCF7 cells. Observation of the morphology by the phase contrast microscopy revealed that the cytoplasm and nucleus was enlarged in A549 cells. Some of the HSC3 cells also showed hypertrophy of the cytoplasm and nucleus and others tended to show atrophy. Finally, some of the Sa3 cells showed hypertrophy of

To determine whether the inhibition of proliferation by FIR energy radiation was associated with apoptosis or necrosis, cancer cells were subjected to FIR irradiation of 48 h and analyzed on day 4 by staining with annexin V-FITC and PI and by TUNEL (TdT-mediated dUTP-biotin nick end labeling) (to refer JEMAA, 2010, 2, 382-394, Fig.3)[10]. These results indicated that FIR did not induce apoptosis in A431, HSC3, or Sa3 cells. A few of the Sa3 cells showed significant necrosis. These results indicate that the inhibition of proliferation by FIR in the cancer cells, particularly in HSC3 and Sa3 cells, was not due to apoptosis. Still more, in order to analyze all expressing genes induced by FIR energy radiation, the control and the FIR-irradiated samples after four days culture was monitored using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) with elimination of low signal feature of background signal + 2.6 × SD, and P value < 0.01. Genes were further classified for process







**Table 3.** The analysis of the extracted genes induced by the FIR energy radiation. The 10 most promoted genes and the 10 most inhibited genes in these changed genes showed

By the results that the significant changed genes of the 5 cancer cells by the FIR energy radiation were search, the cells inhibited the proliferation and showed that the many genes related the proliferation changed. The number of the inhibited genes by FIR energy radiation is more than that of the promoted gene except for the HSC3 cells. The 10 most promoted genes and the 10 most inhibited genes in these changed genes were shown at Table 3. There is no common gene in the expressed gene of 5 cells. These genes contained mainly the transcriptional factor and the control factor of the transcriptional factor.

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 297

stress induces HSPs. Moreover, chemotherapeutic agents such as cisplatin, adriamycin, and bleomycin, as well as γ-radiation induce HSPs. HSP70 participates in cytoprotection and is associated with cellular resistance to lethal external effects [18-21]. However, in the present study, HSP70 was never induced by FIR. These results suggested that FIR has anti-tumor activity without inducing HSP70 as an anti-stress factor. This characteristic indicates that

We next examined the effect of knocking down HSP70A and HSP70C mRNA and HSP70 protein expression using siRNA. Transfection with HSP70A/C siRNA effectively decreased HSP70A and HSP70C mRNA (Fig. 4A) and protein levels in both A431 and HSC3 cells without affecting the level of HSP70B mRNA or protein. HSP70A/C siRNA did not suppress BrdU incorporation in unirradiated A431 cells, but it suppressed BrdU incorporation in cells irradiated with FIR. Similarly, the HSP70A/C siRNA enhanced the suppression of BrdU incorporation by FIR irradiation. FIR irradiation also significantly suppressed BrdU incorporation in HSC3 cells transfected with the negative control siRNA. These results indicate that a decrease in HSP70 protein mediates the ability of limited FIR to inhibit the proliferation of A431 and HSC3 cells (to refer Med.Oncol 2008,

*5.4.3. The effects of the FIR energy radiation on the implanted cancer cell to mice* 

volume (mm3) =1/2× (long diameter) × (short diameter) 2.

organization change after 30 days.

For experiments on body weight change, 10-week-old male skid mice (CB17/Icr-Prkdc) and 6- to 8-week-old male nude mice (Crlj:CD1-Foxn1) purchased from Charles River Japan (Yokohama, Japan) were housed with temperature control and a 12-h light-dark cycle. Male skid mice were weighed every 5 days, beginning at 10 weeks of age. FIR treatment by our developed animal raiser (FIR group), n=7 and control (control group),

The log-phase cancer cells cultured in 10% FBS and DMEM/HamF-12 medium (Sigma, St. Louis, USA) resuspended at a cell density of 1×107 ml in PBS containing 500 μg/ml of MatrigelTM basement membrane matrix (Becton Dickinson Labware, Two Oak Park, Bedford, MA). The tumor cells (1×106) were suspended in 0.1 ml of PBS containing 50 μg of MatrigelTM and were slowly hypodermically injected in the back using a 25-gauge needle. After injecting tumor cells, mice were separated two groups: FIR group and control group. During the experiments, the long and short diameters of the resulting tumor were measured in five-day intervals, and tumor volume was calculated by the following formula: tumor

For purpose of the ascertaining tumor organization change by the FIR energy radiation, the excised tumor was observed after stained with hematoxylin and eosin to detect tumor

Then we carried out immunohistochemical analyses to detect matrix metalloproteinases (MMP) members MMP-1, MMP-9, MMP-10 and MMP-13 [9]. The increase in tumor volume

FIR may be suitable for medical treatment.

25, 229-237, Fig.4)[9].

n=8.

#### *5.4.2. The control system in the cancer cells for the FIR energy radiation*

At the process of the analysis of the gene expression induced by the FIR energy radiation, it was made clear that the gene expression of HSP (Heat Shock Protein) 70 was reinforced by the FIR energy radiation. Therefore, the hyper expression cells and the knockdown cells of HSP70 were formed to analyze the effect on the proliferation.

 In order to directly determine whether HSP70 can protect cells from FIR-induced cell death, we developed A431 and HSC3 cell lines stably expressing human HSP70A (A431-HSP70A and HSC3-HSP70A cells, respectively). Control cells were transfected with empty pcDNA3.1 (A431-Neo and HSC3-Neo). In our initial experiments, we found that the exposure of HSC3 and Sa3 cells but not A431 cells to the FIR energy radiation causes G2/M arrest and induces partial hypertrophy to the necrosis (data not shown) [12]. To determine whether the increased expression of HSP70A confers protection against the FIR energy radiation, the cell survival was examined in FIR-irradiated A431-HSP70, A431- Neo, A431-wt, and HSC3-HSP70, HSC3-Neo, and HSC3-wt cells. We found that over expression of HSP70A increased the cell proliferation in A431 and HSC3 cells. Furthermore, the proliferation of FIR-irradiated and control (unirradiated) A431-HSP70A cells was similar. The survival rate after 6 days of FIR irradiation was significantly higher in HSC3-HSP70A cells than in HSC3-Neo or HSC3-wt cells. In addition, the proliferation of FIR-treated HSC3-HSP70A cells was similar to that of control HSC3-HSP70A cells. The BrdU incorporation was significantly higher in FIR-irradiated or control A431-HSP70A cells than in A431-Neo or A431-wt cells. Although the BrdU incorporation of FIRirradiated HSC3-wt and HSC3-Neo cells was lower than in unirradiated HSC3-wt and HSC3-Neo cells, it was similar in FIR-irradiated and unirradiated HSC3-HSP70A cells (to refer Med.Oncol 2008, 25, 229-237, Fig.3)[9].

HSP70 appears to be present in a variety of normal cell types and its expression may be induced by several stressors, such as hyperthermia, cardiac ischemia, infection, UV radiation, endotoxin, and nitric oxide to suppress or denature any foreign protein and restore an injured protein from lethal effects [13]. HSP70 seems particularly important for cancer cells. In human breast cancer, the expression of HSP70 correlates with increased cell proliferation, poor differentiation, lymph node metastases, and poor therapeutic outcome [14]. In vivo animal studies and clinical trials have revealed that hyperthermia may serve as a powerful tool in the treatment of prostate cancer [15-20]; at the cellular level, hyperthermic stress induces HSPs. Moreover, chemotherapeutic agents such as cisplatin, adriamycin, and bleomycin, as well as γ-radiation induce HSPs. HSP70 participates in cytoprotection and is associated with cellular resistance to lethal external effects [18-21]. However, in the present study, HSP70 was never induced by FIR. These results suggested that FIR has anti-tumor activity without inducing HSP70 as an anti-stress factor. This characteristic indicates that FIR may be suitable for medical treatment.

296 Blood Cell – An Overview of Studies in Hematology

factor.

By the results that the significant changed genes of the 5 cancer cells by the FIR energy radiation were search, the cells inhibited the proliferation and showed that the many genes related the proliferation changed. The number of the inhibited genes by FIR energy radiation is more than that of the promoted gene except for the HSC3 cells. The 10 most promoted genes and the 10 most inhibited genes in these changed genes were shown at Table 3. There is no common gene in the expressed gene of 5 cells. These genes contained mainly the transcriptional factor and the control factor of the transcriptional

At the process of the analysis of the gene expression induced by the FIR energy radiation, it was made clear that the gene expression of HSP (Heat Shock Protein) 70 was reinforced by the FIR energy radiation. Therefore, the hyper expression cells and the knockdown cells of

 In order to directly determine whether HSP70 can protect cells from FIR-induced cell death, we developed A431 and HSC3 cell lines stably expressing human HSP70A (A431-HSP70A and HSC3-HSP70A cells, respectively). Control cells were transfected with empty pcDNA3.1 (A431-Neo and HSC3-Neo). In our initial experiments, we found that the exposure of HSC3 and Sa3 cells but not A431 cells to the FIR energy radiation causes G2/M arrest and induces partial hypertrophy to the necrosis (data not shown) [12]. To determine whether the increased expression of HSP70A confers protection against the FIR energy radiation, the cell survival was examined in FIR-irradiated A431-HSP70, A431- Neo, A431-wt, and HSC3-HSP70, HSC3-Neo, and HSC3-wt cells. We found that over expression of HSP70A increased the cell proliferation in A431 and HSC3 cells. Furthermore, the proliferation of FIR-irradiated and control (unirradiated) A431-HSP70A cells was similar. The survival rate after 6 days of FIR irradiation was significantly higher in HSC3-HSP70A cells than in HSC3-Neo or HSC3-wt cells. In addition, the proliferation of FIR-treated HSC3-HSP70A cells was similar to that of control HSC3-HSP70A cells. The BrdU incorporation was significantly higher in FIR-irradiated or control A431-HSP70A cells than in A431-Neo or A431-wt cells. Although the BrdU incorporation of FIRirradiated HSC3-wt and HSC3-Neo cells was lower than in unirradiated HSC3-wt and HSC3-Neo cells, it was similar in FIR-irradiated and unirradiated HSC3-HSP70A cells (to

HSP70 appears to be present in a variety of normal cell types and its expression may be induced by several stressors, such as hyperthermia, cardiac ischemia, infection, UV radiation, endotoxin, and nitric oxide to suppress or denature any foreign protein and restore an injured protein from lethal effects [13]. HSP70 seems particularly important for cancer cells. In human breast cancer, the expression of HSP70 correlates with increased cell proliferation, poor differentiation, lymph node metastases, and poor therapeutic outcome [14]. In vivo animal studies and clinical trials have revealed that hyperthermia may serve as a powerful tool in the treatment of prostate cancer [15-20]; at the cellular level, hyperthermic

*5.4.2. The control system in the cancer cells for the FIR energy radiation* 

HSP70 were formed to analyze the effect on the proliferation.

refer Med.Oncol 2008, 25, 229-237, Fig.3)[9].

We next examined the effect of knocking down HSP70A and HSP70C mRNA and HSP70 protein expression using siRNA. Transfection with HSP70A/C siRNA effectively decreased HSP70A and HSP70C mRNA (Fig. 4A) and protein levels in both A431 and HSC3 cells without affecting the level of HSP70B mRNA or protein. HSP70A/C siRNA did not suppress BrdU incorporation in unirradiated A431 cells, but it suppressed BrdU incorporation in cells irradiated with FIR. Similarly, the HSP70A/C siRNA enhanced the suppression of BrdU incorporation by FIR irradiation. FIR irradiation also significantly suppressed BrdU incorporation in HSC3 cells transfected with the negative control siRNA. These results indicate that a decrease in HSP70 protein mediates the ability of limited FIR to inhibit the proliferation of A431 and HSC3 cells (to refer Med.Oncol 2008, 25, 229-237, Fig.4)[9].

#### *5.4.3. The effects of the FIR energy radiation on the implanted cancer cell to mice*

For experiments on body weight change, 10-week-old male skid mice (CB17/Icr-Prkdc) and 6- to 8-week-old male nude mice (Crlj:CD1-Foxn1) purchased from Charles River Japan (Yokohama, Japan) were housed with temperature control and a 12-h light-dark cycle. Male skid mice were weighed every 5 days, beginning at 10 weeks of age. FIR treatment by our developed animal raiser (FIR group), n=7 and control (control group), n=8.

The log-phase cancer cells cultured in 10% FBS and DMEM/HamF-12 medium (Sigma, St. Louis, USA) resuspended at a cell density of 1×107 ml in PBS containing 500 μg/ml of MatrigelTM basement membrane matrix (Becton Dickinson Labware, Two Oak Park, Bedford, MA). The tumor cells (1×106) were suspended in 0.1 ml of PBS containing 50 μg of MatrigelTM and were slowly hypodermically injected in the back using a 25-gauge needle. After injecting tumor cells, mice were separated two groups: FIR group and control group. During the experiments, the long and short diameters of the resulting tumor were measured in five-day intervals, and tumor volume was calculated by the following formula: tumor volume (mm3) =1/2× (long diameter) × (short diameter) 2.

For purpose of the ascertaining tumor organization change by the FIR energy radiation, the excised tumor was observed after stained with hematoxylin and eosin to detect tumor organization change after 30 days.

Then we carried out immunohistochemical analyses to detect matrix metalloproteinases (MMP) members MMP-1, MMP-9, MMP-10 and MMP-13 [9]. The increase in tumor volume

after implantation was significantly suppressed by whole-body FIR starting from day 15, compared to the control group (to refer J Table1). The cDNA microarray analysis of tumor samples of control and FIR-treated showed that MMP-1, MMP-9, MMP-10, and MMP-13 in the MMP family were significantly down-regulated in the FIR group compared with control group (to refer ITEL 2005, 6(6), 597-601, Table1) [9]. The expression profiles obtained from cDNA microarray analyses as analyzed by quantitative real-time RT-PCR showed the control group to have highly significant overexpression of the genes for MMP-1, MMP-9, MMP-10, and MMP-13 compared to the FIR group (to refer ITEL 2005, 6(6), 597-601, Fig.4) [9].

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 299

degradative processes involved in breast cancer progression [29]. In the present study, it was suggested that MMP-1, MMP-9, MMP-10, and MMP-13 are important for the invasion and metastasis of epithelial cancer of the human vulva A431 cell line in vivo and that the activities of MMP-1, MMP-9, MMP-10, and MMP-13 are selectively inhibited by FIR

In conclusion, FIR irradiation can suppress tumor growth and invasion of epithelial cancer of the human vulva tumor by inhibiting the expression of MMP-1, MMP-9, MMP-10, and MMP-13 without critical side effects. Inhibition of MMP expression was considered to be one of the mechanisms by which multiplication of A431 tumor cells was suppressed in FIR

It was made clear that the motion of the water molecules was surely and physically activated by the radiation of FIR with the CO2 incubator and the animal raising apparatus. The analysis of the change of blood circulation conducted the conclusion that the FIR energy radiation activated not only the motion of water molecules but also the blood circulation in the living body. Still more, it was made clear that the activation effects on the water molecules developed the activation effects of the skin regeneration and the new bone formation. Moreover, the proliferation or metastasis of the various cancer cells were inhibited by the FIR energy radiation connected with the activation of blood circulation. It is expected that the present studies on the FIR energy radiation connect with the development of medical treatment for the regeneration of the tissue and organ as a skin and a bone, and

*Department of Oral and Maxillofacial Anatomy, Institute of Health Biosciences,* 

[1] Inoué S, Kabaya M. Biological activities caused by far-infrared radiation. Int J

[2] Honda K, Inoué S. Sleep-enhancing effects of far-infrared radiation in rats. Int J

[3] Udagawa Y, Nagasawa H. Effects of far-infrared ray on reproduction, growth, behaviour

and some physiological parameters in mice. In Vivo 2000;14(2) 321-326.

*The University of Tokushima Graduate School, Tokushima, Japan* 

irradiation.

irradiation.

**6. Conclusion** 

**Author details** 

Kikuji Yamashita

**7. References** 

the prevention medicine for cancer.

Biometeorol 1989;33(3) 145-150.

Biometeorol 1988;32(2) 92-94.

These results suggest that suppression of tumor volume increase by in vivo FIR radiation was due to inhibition of MMPs by FIR radiation. In other words, FIR suppresses invasion and metastasis of tumor cells by inhibiting the expression of MMP-1, 9, 10 and 13.

As shown in (to refer ITEL 2005, 6(6), 597-601, Fig 5) [9], extensive portions of tumor tissue in the FIR group was encapsulated and necrotized in intra tumor division. On the other hand, in the control group tumor cells showed active proliferation and invasion into the surrounding muscular tissue. In addition, evaluation of the immunohistochemical expression of MMP-1, MMP-9, MMP-10 and MMP-13 using tumor tissues of control and FIR groups on day 30 after implantation showed MMP-1 and MMP-9 to be positively expressed in the tumor stroma of the control group, while MMP-10 and MMP-13 were strongly positive in tumor parenchyma (the part positively dyed at Cytokeratin10) (to refer ITEL 2005, 6(6), 597-601, Fig. 6) [9]. However, these MMPs in the FIR group were not significantly expressed. The result of immunohistological detection for these MMPs was concordant with the results of cDNA microarray and QRT-PCR. That is, FIR radiation seemed to suppress invasion of tumor cells by inhibiting the expression of MMP-1, MMP-9, MMP-10, and MMP-13.

The increased expression and activity of MMPs is associated with tumor invasion, metastasis, and angiogenesis [22]. The role of MMP-1 in tumor invasion and metastasis has only recently been determined [23]. MMP-1 has been shown to cleave entactin, thus contributing to the degradation of basement membranes and hence potentially contributing to the transitioning across epithelial barriers by tumor cells [24]. Immunohistochemical detection of MMP-1 expression is also associated with increased invasive potential and poor prognosis in colon and esophageal cancers [25, 26]. The first barrier to tumor invasion is the basement membrane, and because one of its principle constituents is type I collagen, the gelatinases (MMP-2 and MMP-9) are thought to play important roles in its degradation [27]. MMP-10 (stromelysin-2) is known to degrade various components of the extracellular matrix, and though it has been reported that MMP-10 (stromelysin-2) expression by lymphoma cells accelerates the growth of thymic lymphoma [28], the role of MMP-10 in other types of tumor growth is relatively unknown. MMP-13 (collagenase-3) is expressed in breast carcinoma and in articular cartilage of arthritic patients [29]. In addition, MMP-13 has high collagenolytic and gelatinolytic activity, and MMP-13 may be of importance in degradative processes involved in breast cancer progression [29]. In the present study, it was suggested that MMP-1, MMP-9, MMP-10, and MMP-13 are important for the invasion and metastasis of epithelial cancer of the human vulva A431 cell line in vivo and that the activities of MMP-1, MMP-9, MMP-10, and MMP-13 are selectively inhibited by FIR irradiation.

In conclusion, FIR irradiation can suppress tumor growth and invasion of epithelial cancer of the human vulva tumor by inhibiting the expression of MMP-1, MMP-9, MMP-10, and MMP-13 without critical side effects. Inhibition of MMP expression was considered to be one of the mechanisms by which multiplication of A431 tumor cells was suppressed in FIR irradiation.
