**4.1.1 Prevention of phthalate genotoxicity by selenocompounds**

Phthalate esters are a widespread class of peroxisome proliferators (PPs) and endocrine disruptors. They have attracted substantial attention due to their high production volume and use in a variety of polyvinyl chloride (PVC)-based consumer products **(Akingbemi et al., 2001; Grande et al., 2006)**.

Uses of the various phthalates mainly depend on their molecular weight (MW). Higher MW phthalates, such as di(2-ethylhexyl) phthalate, (DEHP), are used in construction materials and in numerous PVC products including clothing (footwear, raincoats), food packaging, children products (toys, grip bumpers), and medical devices **(Heudorf et al., 2007)**, while relatively lower MW phthalates like di-methyl phthalate (DMP), di-ethyl phthalate (DEP), and di-n-butyl phthalate (DBP) are mainly used as odor/color fixatives or as solvents and in cosmetics, insecticides and pharmaceuticals, but are also used in PVC **(Heudorf et al., 2007)**.

Phthalate migrate out from PVC-containing items into food, air, dust, water, and soils and create human exposure in various ways **(Clark et al., 2003)**. Increasing number of studies on human blood and urine have revealed the ubiquitous phthalate exposure of consumers in industrialized countries **(Wormuth et al., 2006, Frederiksen et al., 2008; Frederiksen et al., 2010; Janjua et al., 2011 , Durmaz et al., 2010)**.

DEHP is the most important phthalate derivative with its high production, use and occurrence in the environment. It is mainly used in PVC plastics in the form of numerous

A larger difference in the reduced death rates was reported for men than for women in regions with high levels of Se, and mortality was significantly lower for some types of cancer **(Shamberger et al., 1976; Clark et al., 1991)**. Higher blood levels of Se have been associated with a lower risk of many types of neoplasia, including prostate, lung, colorectal, and possibly bladder, although the data are inconsistent. A significant 39% decreased risk of bladder cancer associated with high levels of Se by combining results from seven epidemiologic studies, conducted in different populations, which applied individual levels

Supra-physiological levels of sodium selenite (SS) in the presence of polythiols have oxidative properties that might have an anticancer effect by increasing the vulnerability of cancer cells to destruction. It was stated that Se, independent of type (organic/inorganic), can alter several genes to prevent cancer. High doses of Se might upregulate phase II detoxification enzymes, some Se-binding proteins, and some apoptotic genes, and downregulate phase I activating enzymes and cell proliferation genes **(El Bayoumy & Sinha, 2005)**. Inhibition of carcinogen–DNA adducts formation and induction of apoptosis by high doses of Se suggests that protection occurs at both the initiation and post-initiation phases of carcinogenesis **(El Bayoumy & Sinha, 2005)**. However, at lower physiological

The literature agrees on the protective effect of Se evaluated with the Comet assay towards a variety of chemical or physical toxic agents. However, it remains inconclusive which is/are the most suitable Se compound/s to prevent DNA damage and which doses should be used to observe protection. In this chapter, the protective effects of both inorganic and organic

Phthalate esters are a widespread class of peroxisome proliferators (PPs) and endocrine disruptors. They have attracted substantial attention due to their high production volume and use in a variety of polyvinyl chloride (PVC)-based consumer products **(Akingbemi et** 

Uses of the various phthalates mainly depend on their molecular weight (MW). Higher MW phthalates, such as di(2-ethylhexyl) phthalate, (DEHP), are used in construction materials and in numerous PVC products including clothing (footwear, raincoats), food packaging, children products (toys, grip bumpers), and medical devices **(Heudorf et al., 2007)**, while relatively lower MW phthalates like di-methyl phthalate (DMP), di-ethyl phthalate (DEP), and di-n-butyl phthalate (DBP) are mainly used as odor/color fixatives or as solvents and in cosmetics, insecticides and pharmaceuticals, but are also used in PVC **(Heudorf et al., 2007)**. Phthalate migrate out from PVC-containing items into food, air, dust, water, and soils and create human exposure in various ways **(Clark et al., 2003)**. Increasing number of studies on human blood and urine have revealed the ubiquitous phthalate exposure of consumers in industrialized countries **(Wormuth et al., 2006, Frederiksen et al., 2008; Frederiksen et al.,** 

DEHP is the most important phthalate derivative with its high production, use and occurrence in the environment. It is mainly used in PVC plastics in the form of numerous

of Se measured in serum or toenails **(Brinkman et al., 2006)**.

doses, Se prevents apoptosis, and induces DNA repair **(Longtin, 2003)**.

selenocompounds, against phthalate and radiation toxicity will be discussed.

**4.1.1 Prevention of phthalate genotoxicity by selenocompounds** 

**al., 2001; Grande et al., 2006)**.

**2010; Janjua et al., 2011 , Durmaz et al., 2010)**.

consumer and personal care products and medical devices **(Doull et al., 1999)**. The biological effects of DEHP are hence of major concern but so far elusive. Although, the main mechanism underlying hepatocarcinogenicity of phthalates is not fully elucidated, ROS are thought to be associated with the mechanism of tumorigenesis by PPs, including DEHP. This assumption is based to a fact that various proteins that are induced by DEHP in liver parenchymal cells (peroxisomes, mitochondria and microsomes) are prone to formation of H2O2 and other oxidants. Besides, activation of metabolizing enzymes and peroxisome proliferator-activated receptor α (PPARα) might be other substantial factors leading to high intracellular ROS production **(O'Brien et al., 2005; Gazouli et al., 2002)**. However, the mechanisms by which phthalates and particularly DEHP exert toxic effects in reproductive system are not yet fully elucidated. Irreversible and reversible changes in the development of the male reproductive tract like vimentin collapse of Sertoli cells as well as apoptosis of germ cells, effects on sex hormones (mainly on testosterone) as well as follicle stimulating hormone (FSH) and luteinizing hormone (LH), histopathological changes in testis and sperm anomalies were observed with phthalate exposure **(Corton & Lapinskas, 2005; Foster et al., 2001; Erkekoglu et al., 2011a; Erkekoglu et al., 2011b; Kasahara et al, 2002; Noriega et al., 2009)**. Most of the toxic effects were related to its antiandrogenic potential **(Ge et al., 2007)**. A PPARα-mediated pathway based on its peroxisome proliferating (PP) activity **(Gazouli et al., 2002)**, and activation of metabolizing enzymes have also been suggested **(O'Brien et al., 2005)**. While the induction of an oxidative stress may represent a common mechanism in endocrine disruptor–mediated dysfunction, especially on testicular cells **(Latchoumycandane et al., 2002)**, recent studies are also providing supporting evidences for such an effect with DEHP and its major metabolite, mono(2-ethylhexyl)phthalate (MEHP) **(Erkekoglu et al. 2010a; Erkekoglu et al. 2010b; Erkekoglu et al. 2011c; Fan et al. 2010)**. Thus, the primary targets for the DEHP and MEHP are the Sertoli and Leydig cells of testis. In several studies, it was shown that DEHP caused disruption in the function of both cell types. In fact, **Richburg and Boekelheide (1996)** demonstrated histopathological disturbances and alterations of cytoplasmatic distribution of vimentin in Sertoli cells in testis of 28-day-old Fisher rats after a single oral dose of MEHP (2000 mg/kg). Administration of MEHP to Wistar rats at a single oral dose (400 mg/kg bw) was toxic to Sertoli cells and caused detachment of germ cells **(Dalgaard et al., 2000). Tay et al. (2007)** reported vimentin disruption in MEHP-treated C57Bl/6N mice, and gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. We have also reported that in DEHP-treated rats, significant disruption and collapse of vimentin filaments and disruption of seminiferous epithelium in Sertoli cells was observed **(Erkekoglu et al., 2011b)**. Among several others, an earlier data has demonstrated the increase of ROS generation and depletion in antioxidant defenses by DEHP treatment in rat testis **(Kasahara et al., 2002)**. Our recent studies on MA-10 Leydig **(Erkekoglu et al., 2010b)** and LNCaP human prostate cells **(Erkekoglu et al., 2010a)** have also produced comprehensive data suggesting that at least one of the mechanisms underlying the reproductive toxicity of DEHP is the induction of intracellular ROS. The data of **Fan et al. (2010)** have also suggested oxidative stress as a new mechanism of MEHP action on Leydig cells steroidogenesis *via* CYP1A1-mediated ROS stress. On the other hand, in rats exposed to 1000 mg/kg DEHP for 10 days, we observed that this particular phthalate induced oxidative stress in rat testis, as evidenced by significant decrease in GSH/GSSG redox ratio (10-fold) and marked increase in TBARS levels **(Erkekoglu et al., 2011d)**.

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 423

and 18% with SM supplementations, and the tail moment induced by MEHP was reduced 24% with SS supplementation; however, none of these were statistically significant. Only SM supplementation provided a significant (34%) reduction in the tail moment induced by MEHP. But again, tail moments remained 64 and 95% higher than that of NT-C in SS/DEHP-T and SM/DEHP-T cells, respectively; similarly in SS/MEHP-T and SM/MEHP-T cells, tail moments were still 94 and 69% high compared to NT-C cells. In all cases, protective effects of SS and SM were not significantly different than each other **(Erkekoglu et** 

For Leydig MA-10 cells, The IC50 values for DEHP and MEHP were again found to be 3 mM and 3 μM, respectively. Se supplementation of the cells with either SS (30 nM) or SM (10 μM) was protective against the cytotoxic effects of DEHP, and MEHP. Intracellular ROS production showed substantial increases with both of the phthalates where the effect of MEHP was much more pronounced. SS and SM showed partial protection against the ROS increment for both the phthalates. In cells exposed to DEHP or MEHP, GPx1 and TrxR activities decreased significantly. Se supplementation either with SS or SM in DEHPexposed cells was able to enhance the both of the selenoenzyme activities. Moreover, GST activity also decreased significantly with both of the phthalates. However, Se supplementation in both of the forms was not effective in restoring GST activity. GSH levels also decreased significantly in DEHP and MEHP treated Leydig cells while Se supplementation in both forms provided significant restoration in both groups. On the other hand, both DEHP and MEHP produced high level of DNA damage as evidenced by significantly increased tail % intensity (~3.4-fold and ~3.8-fold, respectively), and tail moment (~4.2-fold and ~3.8-fold, respectively) compared to non-treated MA-10 cells. The difference between the DNA damaging effects of the parent compound and the metabolite was insignificant. Se supplementation itself did not cause any alteration on the steady state levels of the DNA damage biomarkers of MA-10 cells. But Se was highly effective to decrease the genotoxic effects of phthalate esters. Increased tail % intensities by DEHP and MEHP exposure were lowered ~50–55% with SS supplementation, whereas SM treatment provided ~30–40% protection. SS decreased the tail moments of the DEHP- or MEHPexposed cells by ~55–65%, whereas the protective effect of SM on tail moments was significantly lower than SS as being ~45% and ~34% for the effects of DEHP and MEHP, respectively. However, both SS and SM reduced the tail moments of the DEHP- and MEHPexposed cells down to the levels that were not significantly different than that of control

**al., 2010a)**.

cells **(Erkekoglu et al., 2010b)**.

**al., 1998; Grossman et al., 1988)**.

**4.1.2 Prevention of radiation genotoxicity by selenocompounds** 

Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10-400 nm, and energies from 3 -124 eV. UV light is found in sunlight, can be emitted by electric arcs and specialized lights such as black lights. It can cause chemical reactions, and it causes many substances to glow or fluoresce. Most UV is classified as non-ionizing radiation **(Müller et al., 1998, Griffiths et** 

The toxic effects of UV from natural sunlight and therapeutic artificial lamps are a major concern for human health. The major acute effects of UV irradiation on normal human skin

Several strategies have been attempted to prevent the oxidative stress caused by toxic chemicals and the use of antioxidant vitamins has been the most common approach. **Ishihara et al. (2000)** showed that supplementation of rats with vitamin C and E protected the testes from DEHP-gonadotoxicity. **Fan et al. (2010)** reported that the increase in ROS generation with MEHP exposure in MA-10 cells was inhibited by N-acetylcysteine (NAC). In the above mentioned *in vitro* studies **(Erkekoglu et al., 2010a; Erkekoglu et al., 2010b)**, we demonstrated that Se supplementation in either organic form (SM, 10 M) or in inorganic form (SS, 30 nM) was highly protective against the cytotoxicity, ROS producing and antioxidant status-modifying effects of DEHP and MEHP in both MA-10 Leydig and LNCaP cells.

Concerning LNCaP cells, we observed that DEHP had a flat dose–cell viability response curve while MEHP showed a very steep dose–response curve and the cytotoxicity of the MEHP was much higher than that of the parent compound. On the other hand, we determined that both organic and inorganic Se supplementation increased resistance to DEHP and MEHP cytotoxicity. From these data, the doses of DEHP and MEHP to be used for the antioxidant status measurements and Comet assay were chosen as close to IC50 values and were 3 mM for DEHP and 3 μM for MEHP. We demonstrated that MEHP was the main active form in LnCAP cells with an almost ~1000- fold higher cytotoxicity than the parent compound. Intracellular ROS production showed marked increases with both DEHP and MEHP treatment; however the effect of MEHP was much higher. Both selenocompounds were partially effective in reducing intracellular ROS production. For the antioxidant enzymes, both DEHP and MEHP caused substantial decreases in GPx1 activity (3-fold, and 4-fold, respectively) compared to control cells. However, there was no significant difference between the effects of the two phthalate derivatives. Se supplementation with either SS or SM effectively countered the effect of DEHP by completely restoring the activity up to the control level (NT-C) or even higher. In the case of MEHP treatments, both SS and SM supplementations significantly restored the effect of 3 μM MEHP on GPx1 activity, providing 2-fold increase. For thioredoxin reductase (TrxR) activity, DEHP did not cause a change compared to control; however, MEHP caused a marked increase. Se supplementation in both organic and inorganic forms increased the TrxR activity almost up to the levels of SS and SM supplemented cells alone. However, no changes were observed with both of the phthalates in glutathione S-transferase (GST) activity and total glutathione (GSH) levels. On the other hand, using alkaline Comet assay, we have demonstrated that in LnCAP cells both DEHP and MEHP produced significant DNA damage as evidenced by increased tail % intensity (2.9-fold and 3.2-fold, respectively), and tail moment (2.4-fold and 2.6-fold, respectively) compared to NT LNCaP cells. The overall difference between the DNA damaging effects of the parent compound and the metabolite was insignificant. Se supplementation itself did not cause any alteration in the steady-state levels of the biomarkers of DNA damage in LNCaP cells, whereas the presence of Se either in SS or SM form reduced the genotoxic effects of DEHP and MEHP as evidenced by significant (30%) decreases in tail % intensity. These results thus indicated that the Se with the doses and forms used in this study was not genotoxic, but showed antigenotoxic activity against the genotoxicity of DEHP and MEHP. However, the protective effect of Se with the doses used in this study was not complete. Tail intensity remained 90% and 80% higher than that of NT-C in SS/DEHP-T and SM/DEHP-T cells, respectively. Similarly, in SS/MEHP-T and SM/ MEHP-T cells, tail intensities were still 95% and 120% high compared to NT-C cells. On the other hand, the extent of tail moment increase induced by DEHP was reduced 30% with SS

Several strategies have been attempted to prevent the oxidative stress caused by toxic chemicals and the use of antioxidant vitamins has been the most common approach. **Ishihara et al. (2000)** showed that supplementation of rats with vitamin C and E protected the testes from DEHP-gonadotoxicity. **Fan et al. (2010)** reported that the increase in ROS generation with MEHP exposure in MA-10 cells was inhibited by N-acetylcysteine (NAC). In the above mentioned *in vitro* studies **(Erkekoglu et al., 2010a; Erkekoglu et al., 2010b)**, we demonstrated that Se supplementation in either organic form (SM, 10 M) or in inorganic form (SS, 30 nM) was highly protective against the cytotoxicity, ROS producing and antioxidant status-modifying effects of DEHP and MEHP in both MA-10 Leydig and

Concerning LNCaP cells, we observed that DEHP had a flat dose–cell viability response curve while MEHP showed a very steep dose–response curve and the cytotoxicity of the MEHP was much higher than that of the parent compound. On the other hand, we determined that both organic and inorganic Se supplementation increased resistance to DEHP and MEHP cytotoxicity. From these data, the doses of DEHP and MEHP to be used for the antioxidant status measurements and Comet assay were chosen as close to IC50 values and were 3 mM for DEHP and 3 μM for MEHP. We demonstrated that MEHP was the main active form in LnCAP cells with an almost ~1000- fold higher cytotoxicity than the parent compound. Intracellular ROS production showed marked increases with both DEHP and MEHP treatment; however the effect of MEHP was much higher. Both selenocompounds were partially effective in reducing intracellular ROS production. For the antioxidant enzymes, both DEHP and MEHP caused substantial decreases in GPx1 activity (3-fold, and 4-fold, respectively) compared to control cells. However, there was no significant difference between the effects of the two phthalate derivatives. Se supplementation with either SS or SM effectively countered the effect of DEHP by completely restoring the activity up to the control level (NT-C) or even higher. In the case of MEHP treatments, both SS and SM supplementations significantly restored the effect of 3 μM MEHP on GPx1 activity, providing 2-fold increase. For thioredoxin reductase (TrxR) activity, DEHP did not cause a change compared to control; however, MEHP caused a marked increase. Se supplementation in both organic and inorganic forms increased the TrxR activity almost up to the levels of SS and SM supplemented cells alone. However, no changes were observed with both of the phthalates in glutathione S-transferase (GST) activity and total glutathione (GSH) levels. On the other hand, using alkaline Comet assay, we have demonstrated that in LnCAP cells both DEHP and MEHP produced significant DNA damage as evidenced by increased tail % intensity (2.9-fold and 3.2-fold, respectively), and tail moment (2.4-fold and 2.6-fold, respectively) compared to NT LNCaP cells. The overall difference between the DNA damaging effects of the parent compound and the metabolite was insignificant. Se supplementation itself did not cause any alteration in the steady-state levels of the biomarkers of DNA damage in LNCaP cells, whereas the presence of Se either in SS or SM form reduced the genotoxic effects of DEHP and MEHP as evidenced by significant (30%) decreases in tail % intensity. These results thus indicated that the Se with the doses and forms used in this study was not genotoxic, but showed antigenotoxic activity against the genotoxicity of DEHP and MEHP. However, the protective effect of Se with the doses used in this study was not complete. Tail intensity remained 90% and 80% higher than that of NT-C in SS/DEHP-T and SM/DEHP-T cells, respectively. Similarly, in SS/MEHP-T and SM/ MEHP-T cells, tail intensities were still 95% and 120% high compared to NT-C cells. On the other hand, the extent of tail moment increase induced by DEHP was reduced 30% with SS

LNCaP cells.

and 18% with SM supplementations, and the tail moment induced by MEHP was reduced 24% with SS supplementation; however, none of these were statistically significant. Only SM supplementation provided a significant (34%) reduction in the tail moment induced by MEHP. But again, tail moments remained 64 and 95% higher than that of NT-C in SS/DEHP-T and SM/DEHP-T cells, respectively; similarly in SS/MEHP-T and SM/MEHP-T cells, tail moments were still 94 and 69% high compared to NT-C cells. In all cases, protective effects of SS and SM were not significantly different than each other **(Erkekoglu et al., 2010a)**.

For Leydig MA-10 cells, The IC50 values for DEHP and MEHP were again found to be 3 mM and 3 μM, respectively. Se supplementation of the cells with either SS (30 nM) or SM (10 μM) was protective against the cytotoxic effects of DEHP, and MEHP. Intracellular ROS production showed substantial increases with both of the phthalates where the effect of MEHP was much more pronounced. SS and SM showed partial protection against the ROS increment for both the phthalates. In cells exposed to DEHP or MEHP, GPx1 and TrxR activities decreased significantly. Se supplementation either with SS or SM in DEHPexposed cells was able to enhance the both of the selenoenzyme activities. Moreover, GST activity also decreased significantly with both of the phthalates. However, Se supplementation in both of the forms was not effective in restoring GST activity. GSH levels also decreased significantly in DEHP and MEHP treated Leydig cells while Se supplementation in both forms provided significant restoration in both groups. On the other hand, both DEHP and MEHP produced high level of DNA damage as evidenced by significantly increased tail % intensity (~3.4-fold and ~3.8-fold, respectively), and tail moment (~4.2-fold and ~3.8-fold, respectively) compared to non-treated MA-10 cells. The difference between the DNA damaging effects of the parent compound and the metabolite was insignificant. Se supplementation itself did not cause any alteration on the steady state levels of the DNA damage biomarkers of MA-10 cells. But Se was highly effective to decrease the genotoxic effects of phthalate esters. Increased tail % intensities by DEHP and MEHP exposure were lowered ~50–55% with SS supplementation, whereas SM treatment provided ~30–40% protection. SS decreased the tail moments of the DEHP- or MEHPexposed cells by ~55–65%, whereas the protective effect of SM on tail moments was significantly lower than SS as being ~45% and ~34% for the effects of DEHP and MEHP, respectively. However, both SS and SM reduced the tail moments of the DEHP- and MEHPexposed cells down to the levels that were not significantly different than that of control cells **(Erkekoglu et al., 2010b)**.

#### **4.1.2 Prevention of radiation genotoxicity by selenocompounds**

Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10-400 nm, and energies from 3 -124 eV. UV light is found in sunlight, can be emitted by electric arcs and specialized lights such as black lights. It can cause chemical reactions, and it causes many substances to glow or fluoresce. Most UV is classified as non-ionizing radiation **(Müller et al., 1998, Griffiths et al., 1998; Grossman et al., 1988)**.

The toxic effects of UV from natural sunlight and therapeutic artificial lamps are a major concern for human health. The major acute effects of UV irradiation on normal human skin

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 425

protected against oxidative damage to DNA as measured by formation of formamidopyrimidine (FaPy) glycosylase-sensitive sites, which are indicative of 8-oxoGua photoproduct formation. Preincubation for 18 h with 50 nM SS or with 200 nM SM abolished the UVB-induced increase in comet length. The researchers concluded that both of selenocompounds were protective against UVB-induced oxidative damage in human keratinocytes; however they did not protect from formation of UVB-induced excision repair

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic Se compounds. Studies have shown its antioxidant, hepatoprotective, neuroprotective, anti-inflammatory, and antinociceptive effects. In a study by **Rosa et al. (2007)**, the researchers used a permanent lung fibroblast cell line derived from Chinese hamsters and investigated the antigenotoxic and antimutagenic properties of DPDS. In the clonal survival assay, at concentrations ranging from 1.62 to 12.5 μM, DPDS was not cytotoxic, while at concentrations up to 25 μM, it significantly decreased survival. The treatment with this DPDS at non-cytotoxic dose range increased cell survival after challenge with H2O2, methyl-methanesulphonate, and UVC radiation, but did not protect against 8-methoxypsoralen plus UVA-induced cytotoxicity. In addition, the treatment prevented induced DNA damage, as verified in the Comet assay. The mutagenic effect of these genotoxic agents, as measured by the micronucleus test, similarly attenuated or prevented cytotoxicity and DNA damage. Treatment with DPDS also decreased lipid peroxidation levels after exposure to H2O2, MMS, and UVC radiation, and increased GPx1 activity in the cells. The results of this study demonstrated that DPDS at low concentrations presents antimutagenic properties, which are most probably due to its antioxidant

Diet should include components such as vitamins and flavonoids and the antioxidant capacity of body is directly linked to the diet. Vitamins like ascorbic acid (vitamin C, AA) are important antioxidants. About 90% of AA in the average diet comes from fruits and

AA is a water soluble dietary antioxidant that plays an important role in controlling oxidative stress **(Vallejo et al., 2002)**. Most importantly, AA is a mild reducing agent. For this reason, it degrades upon exposure to oxygen, especially in the presence of metal ions and light. It can be oxidized by one electron to a radical state or doubly oxidized to the stable form called "dehydroascorbic acid". Typically it reacts with oxidants such as ROS, such as the •OH formed from H2O2. Hydroxyl radical is the most detrimental species, due to its high interaction with nucleic acids, proteins, and lipids. AA can terminate these chain radical reactions by electron transfer. AA is special because it can transfer a single electron, owing to the stability of its own radical ion called "semidehydroascorbate". The oxidized forms of AA are relatively unreactive, and do not cause cellular damage. However, being a good electron donor, high concentrations of AA in the presence of free metal ions can not only promote, but also initiate free radical reactions, thus making it a potentially dangerous pro-oxidative compound in certain metabolic contexts **(Choe and** 

sites **(Rafferty et al., 2003)**.

properties **(Rosa et al., 2007)**.

vegetables **(Vallejo et al., 2002)**.

**Min, 2006; Blokhina et al., 2003)**.

**4.2.1 Ascorbic acid** 

**4.2 Prevention of genotoxicity by vitamins** 

comprise sunburn inflammation erythema, tanning, and local or systemic immunosuppression. On the other hand, UV irradiation present in sunlight is an environmental human carcinogen. There is considerable evidence that UV is implicated in skin carcinogenesis and the risk of cutaneous cancers has increased during the last decade due to increase of sun exposure. For a long time, ultraviolet B radiation (UVB: 290-320 nm) have been considered to be the more efficient wavelength in eliciting carcinogenesis in human skin. It is today clear that ultraviolet A (UVA, 320-400 nm), especially UVA1 (340-400 nm) also participate to photo-carcinogenesis. It penetrates deeply, but it does not cause sunburn. One of molecular mechanisms in the biological effects of UV is the induction of ROS directly or through endogenous photosensitization reactions. UVA radiation mainly acts *via* this production of ROS and the subsequent oxidative stress seems to play a crucial role in the deleterious effects of UVA. UVA does not damage DNA directly like UVB and UVC, but it can generate highly reactive chemical intermediates, such as hydroxyl and oxygen radicals, which in turn can damage DNA and lead to the formation of 8-oxoGua **(Ridley et al., 2009)**. UVB light can cause direct DNA damage. The radiation excites DNA molecules in skin cells, causing aberrant covalent bonds to form between adjacent cytosine bases, producing a dimer. When DNA polymerase comes along to replicate this strand of DNA, it reads the dimer as "AA" and not the original "CC". This causes the DNA replication mechanism to add a "TT" on the growing strand. This mutation can result in cancerous growths, and is known as a "classical C-T mutation". The mutations caused by the direct DNA damage carry a UV signature mutation that is commonly seen in skin cancers. The mutagenicity of UV radiation can be easily observed in bacterial cultures. This cancer connection is one reason for concern about ozone depletion and the ozone hole. UVB causes some damage to collagen, but at a very much slower rate than UVA. Fortunately, the skin possesses a wide range of inter-linked antioxidant defense mechanisms to protect itself from damage by UV-induced ROS. However, the capacity of these systems is not unlimited; they can be overwhelmed by excessive exposure to UV and then ROS can reach damaging levels. An interesting strategy to provide photoprotection would be to support or enhance one or more of these endogenous systems **(Béani, 2001)**.

There is limited number of studies in literature concerning the protective effect of selenocompounds on UV-caused genotoxicity. In a study by **Emonet-Piccardi et al. (1998)**, the researchers determined the protective effects of NAC (5 mM), SS (0.6 M) or zinc chloride (ZnCl2, 100 M) against UVA radiation in human skin fibroblasts using Comet assay. The cells were incubated with NAC, SS or ZnCl2 and then UVA was applied as 1 to 6 J/cm2 to the cells. The tail moment increased by 45% (1 J/cm2) to 89% (6 J/cm2) in nonsupplemented cells (p<0.01). DNA damage was significantly prevented by NAC, SS and ZnCl2, with similar efficiency from 1 to 4 J/cm2. For the highest UVA dose (6 J/cm2), SS and ZnCl2 were more effective than NAC.

In a study assessing the effects of pretreatment of primary human keratinocytes with Se on UV-induced DNA damage, cells were irradiated with UVB from FS-20 lamps and were subjected to Comet assay. Comet tail length due to UVB-induced T4 endonuclease Vsensitive sites (caused by cyclopyrimidine dimers, CPDs) increased to 100% immediately after irradiation (time 0). After 4 h, 68% of the damage remained and after 24 h, 23% of the damage was still present. Treatment with up to 200 nM SM or 50 nM SS had no effect on CPD formation or rates of repair, or on the number of excision repair sites as measured by cytosine arabino furanoside and hydroxyurea treatment. However, both SS and SM protected against oxidative damage to DNA as measured by formation of formamidopyrimidine (FaPy) glycosylase-sensitive sites, which are indicative of 8-oxoGua photoproduct formation. Preincubation for 18 h with 50 nM SS or with 200 nM SM abolished the UVB-induced increase in comet length. The researchers concluded that both of selenocompounds were protective against UVB-induced oxidative damage in human keratinocytes; however they did not protect from formation of UVB-induced excision repair sites **(Rafferty et al., 2003)**.

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic Se compounds. Studies have shown its antioxidant, hepatoprotective, neuroprotective, anti-inflammatory, and antinociceptive effects. In a study by **Rosa et al. (2007)**, the researchers used a permanent lung fibroblast cell line derived from Chinese hamsters and investigated the antigenotoxic and antimutagenic properties of DPDS. In the clonal survival assay, at concentrations ranging from 1.62 to 12.5 μM, DPDS was not cytotoxic, while at concentrations up to 25 μM, it significantly decreased survival. The treatment with this DPDS at non-cytotoxic dose range increased cell survival after challenge with H2O2, methyl-methanesulphonate, and UVC radiation, but did not protect against 8-methoxypsoralen plus UVA-induced cytotoxicity. In addition, the treatment prevented induced DNA damage, as verified in the Comet assay. The mutagenic effect of these genotoxic agents, as measured by the micronucleus test, similarly attenuated or prevented cytotoxicity and DNA damage. Treatment with DPDS also decreased lipid peroxidation levels after exposure to H2O2, MMS, and UVC radiation, and increased GPx1 activity in the cells. The results of this study demonstrated that DPDS at low concentrations presents antimutagenic properties, which are most probably due to its antioxidant properties **(Rosa et al., 2007)**.
