Effects of folate on arsenic toxicity in Chang human hepatocytes: Involvement of folate antioxidant properties

Toxicology Letters 195 (2010) 44–50

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Effects of folate on arsenic toxicity in Chang human hepatocytes: Involvement of folate antioxidant properties Yuanyuan Xu, Huihui Wang, Yi Wang, Yi Zheng, Guifan Sun ∗ Department of Occupational and Environmental Health, College of Public Health, China Medical University, Shenyang, Liaoning, PR China

a r t i c l e

i n f o

Article history: Received 5 November 2009 Received in revised form 16 February 2010 Accepted 18 February 2010 Available online 25 February 2010 Keywords: Arsenic Apoptosis Folate Hepatocytes Methylation Oxidative stress

a b s t r a c t We investigated the effects and modes of action of the nutritional factor folate on arsenic-induced toxicity in Chang human hepatocytes. Cells were cultured in folate-deficient medium, normal folate medium or folate-supplemented medium for 1 h and then co-treated with or without 20-␮M sodium arsenite (NaAsO2 ) for 24 h. The results showed that folate deficiency significantly aggravated the NaAsO2 -induced apoptotic progression [evidenced by phosphatidylserine externalization, cleavage of capspase-3 and poly (ADP-ribose) polymerase (PARP), collapse of mitochondrial potential, and release of cytochrome c from the mitochondria] and decrease of cell viability. Folate supplementation significantly attenuated all the above mentioned NaAsO2 -induced effects except phosphatidylserine externalization. The NaAsO2 -induced generation of intracellular reactive oxygen species and malondialdehyde was aggravated, to some extent, by folate deficiency, but these phenomena were significantly suppressed by folate supplementation. In contrast, NaAsO2 -induced elevation of reduced glutathione levels was significantly suppressed by folate deficiency, but significantly enhanced by folate supplementation. In addition, folate deficiency significantly decreased the arsenic methylation capacity of the hepatocytes, but had no effects on cellular retention of arsenic. Folate supplementation had no significant effect on cellular retention or methylation of arsenic. These results indicate that folate deficiency aggravates arsenic-induced toxicity and apoptosis, while folate supplementation attenuates these effects. Folate, which plays a role in arsenic metabolism, also exerts its effect on arsenic toxicity at least partly because of its antioxidant property. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Arsenic is a metalloid that exists naturally in soil, water, and food (NRC, 2001). Natural occurrence of arsenic in drinking water is a serious worldwide public-health problem in the 21st century. Chronic exposure to high level of arsenic in drinking water has been associated with the development of skin, lung, and bladder cancers as well as dermal lesions, cardiovascular diseases, hepatotoxicity, diabetes, intellectual function impairment, and respiratory disorders (Mazumder et al., 2005; NRC, 2001; Wasserman et al., 2007; Abernathy et al., 2003; Yoshida et al., 2004; Lam et al., 2007). Although environmental exposure to the toxic substance is an important cause of such diseases, certain other mitigating factors, such as genetic or environmental factors, also contribute to the development of these diseases. For example, nutritional status has

∗ Corresponding author at: Department of Occupational and Environmental Health, College of Public Health, China Medical University, No. 92 Bei Er Road, Heping District, Shenyang 110001, PR China. Tel.: +86 24 2326 1744; fax: +86 24 2326 1744. E-mail address: [email protected] (G. Sun). 0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2010.02.015

been suggested to contribute to the variations in the susceptibility to arsenic toxicity (Smith et al., 1992). Folate, a water-soluble vitamin B found in green leafy vegetables, citrus fruits, and legumes, has recently attracted much attention as a nutritional factor that influences arsenic-induced toxicity. Folate is one of the most important dietary sources of methyl groups and has been suggested to influence arsenic metabolism, which in turn is associated with the excretion rate of arsenic in the body (Vahter, 2000) and the risk of arsenic-related diseases (Tseng et al., 2005; Chen et al., 2003a,b, 2005). Mice that were fed diets containing inadequate folate levels showed decreased biotransformation and urinary excretion of arsenic (Spiegelstein et al., 2003). A study conducted on 300 adults in Bangladesh reported a positive association between the percentage of dimethylated arsenic (DMA) in the urine and folate concentration in the plasma (Gamble et al., 2005a). The relationship between folate and arsenic metabolism was further supported by a double-blind, placebo-controlled, folate-supplemented trial conducted in Bangladesh (Gamble et al., 2006). In this study, 200 adults with low plasma folate concentrations were randomly assigned to receive folate or a placebo. After 12 weeks, the urinary percentage of DMA in the folate-supplemented group was significantly higher than that in the control group, and the blood percentages of monomethylated arsenic (MMA) and inorganic arsenic (iAs) in the

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folate-supplemented group were lower than those in the control group. Oxidative stress plays an important role in arsenic pathogenesis (Kitchin and Ahmad, 2003). Folate has been shown to exhibit certain antioxidant properties. It is known to be an efficient in vitro scavenger of free radicals (Joshi et al., 2001). At the physiological pH level, folate can not only scavenge thiol radicals but also repair the thiol, which play an important role in cellular redox processes and scavenging of free radicals (Joshi et al., 2001). Although folate is water soluble, it plays an important inhibitory role in lipid peroxidation (Joshi et al., 2001). The antioxidant effects of folate have also been observed in patients with coronary artery disease (Doshi et al., 2001; Mayer et al., 2002). Therefore, folate may play an important role in arsenic-induced toxicity by influencing arsenic metabolism and cellular oxidative stress. However, the evidence supporting this hypothesis is relatively sparse. The effects of folate on arsenic toxicity and the mechanism of these effects must be further elucidated. Recent studies have indicated that the liver is not only the primary site for arsenic methylation but also a potential target of arsenic toxicity (Liu and Waalkes, 2008). In this study, we investigated the effects of folate on arsenic-induced cytotoxicity, apoptotic progression, and oxidative stress in Chang human hepatocytes. We also performed a preliminary analysis of the effect of folate on arsenic metabolism by determining the distribution of arsenicals (iAs, MMA, and DMA) in the cells and the medium at different concentrations of folate. 2. Materials and methods 2.1. Reagents Sodium arsenite (NaAsO2 , ≥99.0%), folate (folic acid), N-acetyl-l-cysteine (NAC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2 ,7 dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, USA). 5,5 ,6,6 -Tetrachloro-1,1 ,3,3 -tetraethylbenzimidazol carbocyanine iodide (JC-1) was from Molecular Probes (Eugene, OR, USA). Folate-free RPMI 1640 medium, standard RPMI 1640 medium, trypsin, penicillin and streptomycin were from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) assay kits were all from Keygen Biotech, Co. Ltd. (Nanjing, Jiangsu, China). Mitochondria isolation kit was purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). Polyclonal antibodies of cytochrome c and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) were from Cell Signaling Technology (Beverly, MA, USA). Enhanced chemiluminescence (ECL) plus kit was from Amersham Life Science (Buckinghamsire, UK). All reagents used were the highest grade obtainable. Water used in all preparations was distilled and deionized.

2.2. Cell culture and treatment Chang human hepatocytes were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were grown in standard RPMI 1640 medium, pH 7.4, supplemented with 2.0 g/l sodium bicarbonate, 100 units/ml penicillin, 100 ␮g/ml streptomycin and 10% FBS. The cells were grown in T-75 culture flasks at 37 ◦ C in a humidified 5% CO2 atmosphere until 85% confluence and were subcultured approximately twice a week. To analyze effects of folate, cells undergoing exponential growth were washed with phosphate buffered saline (PBS), and grown in folate-free 1640 medium (folatedeficient “FD” medium, containing no folate), standard RPMI 1640 medium (normal folate “NF” medium, containing 2.3 ␮M folate) and standard RPMI 1640 medium supplemented with folate (folate-supplemented “FS” medium, containing 10 ␮M folate) for 1 h, respectively. After these treatments, the cells cultured in different folate-containing medium were co-treated with or without 20 ␮M-NaAsO2 for another 24 h. In a previous study, we observed that 20-␮M-NaAsO2 treatment for 24 h induced significant oxidative stress and apoptosis in Chang hepatocytes (Wang et al., 2009). Meanwhile, the cell viability was above 80%. NAC-treated cells were used as the positive control, in which the protective role of antioxidant has been observed. In this group, cells were pretreated with 5 mM NAC 1 h before NaAsO2 treatment and kept with NAC in the medium during 20-␮M-NaAsO2 treatment for 24 h. All experiments were carried out at least in triplicate.

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2.3. MTT cell viability assay Cell viability based on mitochondrial enzyme functions was assayed by MTT conversion to formazan. For the assay, cells (5 × 104 cells per well) were seeded in 96-well plates. At the end of treatment, 100 ␮l of MTT solution (0.5 mg/ml in the medium) was added to each well, and the plates were incubated at 37 ◦ C for additional 4 h. Afterwards, the medium containing MTT was removed and the crystals were dissolved in 150 ␮l of 100% dimethyl sulfoxide (DMSO). The cell viability was quantified using a microplate reader (Multiscan Ascent, Labsystem, Finland) at 570 nm after subtracting the appropriate blank values. 2.4. Double-staining with annexin V/FITC and propidium iodide (PI) Detection of apoptotic cells was performed using annexin V/PI staining assay. Cells were seeded in 6-well plates. At the end of the treatment, cells were harvested by trypsin and labeled with annexin V-FITC and PI using an apoptosis detection kit according to the manufacturer’s protocol. Then cells were pelleted and analyzed with a flow cytometer (FACSCalibur, BECTON DICKINSON, USA). Excitation wave was set at 488 nm. The emitted green fluorescence of annexin V (FL1) and red fluorescence of PI (FL2) were detected using 525 nm and 595 nm emission filters, respectively. For each sample, 10,000 cells were analyzed. The amount of early apoptosis and late apoptosis was determined as the percentage of annexin V+ /PI− and annexin V+ /PI+ , respectively. 2.5. Measurement of mitochondrial membrane potential (

m)

For measurement of  m , the cationic dye JC-1 was used. JC-1 is capable of selectively entering mitochondria, where it aggregates and gives red fluorescence when  m is high, and forms monomers and emits green fluorescence when  m is relatively low. Briefly, after treatment mentioned above, cells grown in 12-well plates were trypsinized, washed in ice-cold PBS and incubated in 1 ml of serumfree medium containing 2.5 ␮M JC-1 dye at 37 ◦ C for 20 min in darkness. After washing twice with PBS, fluorescence in cells was immediately measured using a flow cytometer (FACSCalibur, BECTON DICKINSON, USA). Excitation wave was set at 488 nm for JC-1 analysis. Emission filters of 525 nm and 595 nm were used to quantify the population of cells with green (JC-1 monomers) and red (JC-1 aggregates) fluorescence, respectively. 2.6. Preparation of protein extracts for Western blot analysis Cells were seeded in 10-cm-diameter dishes and allowed to grow to 80% confluence. At the end of treatment, cells were washed 3 times with ice-cold PBS, scraped by “policeman” and disrupted in cell lysis buffer [50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), pH 7.4] supplemented with protease inhibitors [0.1 mM phenylmethyl sulfonyl fluoride (PMSF), 1% aprotinin, 1 mM leupeptin, 1 ␮g/ml pepstatin A] and 1% phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany) at 4 ◦ C for 30 min. Then lysates were cleared by centrifugation at 4 ◦ C. For cytochrome c analysis, mitochondria and cytosol fractions were separated using mitochondria isolation kit according to the manufacturer’s instructions. Protein concentrations were determined by the method of Bradford (1976). All the protein fractions were stored at −70 ◦ C until use. 2.7. Western blot analysis Protein extracts (30 ␮g per lane) were separated by 8% or 15% SDSpolyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking in 5% nonfat dry milk in Tween 20 Tris-buffered saline (TTBS), the membranes were incubated with primary antibodies (rabbit anti-human cleaved caspase-3, rabbit anti-human cleaved PARP, mouse anti-human cytochrome c and rabbit anti-human actin) at 1:800–5000 dilutions overnight at 4 ◦ C, and then secondary antibodies conjugated with horseradish peroxidase at 1:10,000 dilution for 1 h at room temperature. Protein bands were detected by ECL plus kit. 2.8. Determination of ROS generation ROS generation was evaluated by measuring dichlorofluorescein (DCF) fluorescence as described by Rothe and Valet (1990). Cells were seeded in 24-well plates. At the end of the treatment, cells were washed and resuspended in serum-free medium containing 10 ␮M DCFH-DA. After a further 30 min of incubation at 37 ◦ C, cells were washed twice with ice-cold PBS and harvested by trypsin. Then the cells were immediately analyzed with flow cytometer (exc = 488 nm, em = 525 nm) (FACSCalibur, BECTON DICKINSON, USA) to determine the ROS generation. 2.9. Measurement of intracellular GSH, CAT, SOD and lipid oxidation For determination of intracellular GSH, CAT, SOD and lipid oxidation, cells were cultured in 10-cm-diameter dishes and treated as indicated. At the end of treatment, cells were washed 3 times with ice-cold PBS, scraped off the dishes with a silicone ‘policeman’ and harvested into Eppendorf tubes. Then cells were lyzed in PBS by

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Fig. 1. Viability of Chang human hepatocytes. Results are expressed as mean ± SD, n = 6. *P < 0.05, compared with NaAsO2 -un-treated cells grown in normal folate (NF) medium (the control cells); # P < 0.05, compared with 20-␮M-NaAsO2 -treated cells grown in NF medium. sonication followed by centrifugation at 15,000 × g for 5 min at 4 ◦ C. The resulting supernatants were used immediately for the measurement. The intracellular GSH levels were measured with improved 5,5 -dithiobis-(2nitrobenzoic acid) (DTNB) method according to the manufacturer’s protocol. DTNB reacts with GSH to form a yellow product. Absorbance was measured at 412 nm and GSH levels in cellular extracts were quantified on the basis of a calibration curve generated using GSH as a standard. The CAT activity was determined on the basis of the rate constant of hydrogen peroxide (H2 O2 ) decomposition according to the manufacturer’s protocol. Briefly, cellular extracts were added to 3 ml of H2 O2 solution, and the absorbance was measured at 240 nm by spectrophotometry at the 0th second and the 60th second. The SOD activity was determined by hydroxylamine assay, which is a modification of the xanthine oxidase assay according to the manufacturer’s protocol. The unit of SOD activity was defined as the amount of enzyme required to give 50% inhibition of the nitrite reduction reaction compared with enzyme control. The lipid peroxidation was assessed by measuring MDA levels. The quantification was based on measuring formation of thiobarbituric acid (TBA) reactive substances (TBARS) according to the manufacturer’s protocol. TBA was added to each sample tube and vortexed. The reaction mixture was incubated at 95 ◦ C for 40 min. After cooling, the chromogen was read spectrophotometrically at 532 nm. Protein concentrations were determined by the method of Bradford (1976) to normalize the levels of GSH, CAT, SOD and MDA. 2.10. Speciation analysis of arsenic Cells were seeded in 10-cm-diameter dishes and allowed to grow to 80% confluence. After incubation, medium was collected, and cells were harvested with a silicone “policeman” and lyzed in PBS by sonication at 4 ◦ C. Determination of arsenic species, including iAs, MMA, DMA and trimethylated arsenic (TMA) was conducted using cold-trap hydride-generation atomic absorption spectrometry (HG-AAS) as we reported previously (Xu et al., 2008). Cells and culture medium were analyzed separately for each treatment. Arsines were generated, cold-trapped, separated by their boiling points, and analyzed using an atomic absorption spectrometer (AA6800, SHIMAZU, JAPAN). Under our analytical conditions, differentiation of the trivalent forms from the pentavalent forms of arsenic cannot be performed.

Fig. 2. Apoptosis of Chang human hepatocytes. (A) Apoptosis was assessed by measuring the exposure of phosphatidylserine residues on the cell surface, as described in materials and methods (double-staining with annexin V/FITC and PI). The percentages of early apoptotic (annexin V+ and PI− ) and late apoptotic (annexin V+ and PI+ ) cells are shown in the histogram. Data are presented as the mean ± SD, n = 3, *P < 0.05, compared with NaAsO2 -un-treated cells grown in normal folate (NF) medium (the control cells); # P < 0.05, compared with 20-␮M-NaAsO2 -treated cells grown in NF medium. (B) Caspase-3 cleavage and (C) poly (ADP-ribose) polymerase (PARP) cleavage in Chang human hepatocytes; the cleavage was detected by western blot analysis using total cell lysates. Proteins were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with anticleaved-caspase-3 or anti-cleaved-PARP antibodies. All the gel lanes were loaded with equal amounts of protein. The representative images of 3 experiments are shown.

that of NAC supplementation. We observed that folate deficiency or supplementation alone had no toxic effects on hepatocyte viability. 3.2. Apoptosis

2.11. Statistical analysis Statistical analyses were performed using the SPSS software (version 11.5; SPSS Inc., Chicago, IL, USA). All experiments were performed at least in triplicate and the results were expressed as means ± standard deviations (SD). Differences between treatment groups were analyzed by one-way analysis of variance (ANOVA) with post hoc analysis using Dunnett’s test. Differences were considered significant when P < 0.05.

3. Results 3.1. Cell viability After the indicated treatment, the MTT assay was performed to determine the cellular survival rates. As shown in Fig. 1, the viability of the NaAsO2 -treated cells cultured in NF medium was significantly lower than that of the control cells (P < 0.05). The arsenic-induced decrease in cell viability was significantly aggravated by folate deficiency (P < 0.05), but this decrease was attenuated by folate or NAC supplementation (P < 0.05). However, the protective effect of folate supplementation was not as strong as

Annexin V/PI-double-staining was used to investigate the apoptosis of Chang human hepatocytes (Fig. 2A). Compared to the percentage of cells in the control, the percentage of cells in either early apoptosis (annexin V+ /PI− ) or late apoptosis (annexin V+ /PI+ ) was significantly increased after a 24-h 20-␮M-NaAsO2 treatment in NF medium (P < 0.05). Among the cells treated with NaAsO2 , the percentages of early and late apoptosis in the cells grown in FD medium were significantly higher than those in the cells grown in NF medium (P < 0.05). Folate supplementation attenuated the induction of early and late apoptosis by NaAsO2 ; however, the differences were not significant. In addition, NAC supplementation exerted a significant beneficial effect by inhibiting NaAsO2 -induced apoptosis (P < 0.05). Under the culture conditions used in our study, folate deficiency or supplementation did not show any significant effects on early or late apoptosis in the NaAsO2 -un-treated cells. We performed an additional set of experiments to evaluate the cleavage of PARP and caspase-3. Western blot analysis showed that NaAsO2 induced the cleavage of caspase-3 (17-kDa fragments) (Fig. 2B) and PARP (89-kDa fragments) (Fig. 2C); this cleavage was

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Fig. 3. Depolarization of the mitochondrial membrane potential and cytochrome c release from the mitochondria. (A) The percentages of cells with collapsed mitochondrial membrane potential ( m ) were determined by flow cytometric analysis with JC-1 staining. *P < 0.05, compared with NaAsO2 -un-treated cells grown in normal folate (NF) medium (the control cells); # P < 0.05, compared with 20-␮MNaAsO2 -treated cells grown in NF medium. (B) The cytochrome c levels in the mitochondria and cytosol were analyzed by performing western blot analysis. The proteins were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed using anti-cytochrome c antibodies. All the gel lanes were loaded with equal amounts of protein. The representative images of 3 experiments are shown.

exacerbated by folate deficiency but improved by folate or NAC supplementation. 3.3. Depolarization of 

m

and cytochrome c release

The changes in  m were examined by JC-1 staining, and these changes are shown in Fig. 3A. After a 24-h treatment with 20␮M-NaAsO2 in NF medium, the number of cells that showed a  m collapse was 5.0 times higher than the corresponding number among control cells (P < 0.05). The percentage of cells showing NaAsO2 -induced  m collapse was significantly increased by folate deficiency (P < 0.05) and decreased by folate or NAC supplementation (P < 0.05). Western blotting was performed to analyze cytochrome c release from the mitochondria into the cytosol. In the cells treated with 20-␮M-NaAsO2 and incubated in NF medium, the cytochrome c levels increased in the cytosol but decreased in the mitochondria (Fig. 3B); this finding coincided with the changes in  m . The release of cytochrome c from the mitochondria into the cytosol of the NaAsO2 -treated cells grown in FD medium was markedly higher than the release in the NaAsO2 -treated cells grown in NF medium. The NaAsO2 -induced release of cytochrome c was reduced to some extent by folate supplementation, but the reduction was lower than that induced by NAC supplementation. 3.4. ROS and MDA As shown in Fig. 4A, the intracellular ROS level in the NaAsO2 treated cells cultured in NF medium was 1.8 times higher than that in the control cells (P < 0.05). The production of ROS in the NaAsO2 treated cells cultured in FD medium was 1.2 times higher than that in the NaAsO2 -treated cells cultured in NF medium (P < 0.05). Mean-

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Fig. 4. The generation of reactive oxygen species (ROS) and malondialdehyde (MDA) in Chang human hepatocytes. (A) The relative levels of ROS were assessed by measuring the average fluorescence intensity in the cells by using a flow cytometer. (B) The relative MDA amount was assessed by measuring the formation of thiobarbituric acid reactive substances (TBARS) in the cells. The results are expressed as mean ± SD, n = 3. *P < 0.05, compared with NaAsO2 -un-treated cells grown in normal folate (NF) medium (the control cells); # P < 0.05, compared with 20-␮M-NaAsO2 -treated cells grown in NF medium.

while, both folate and NAC supplementation significantly reduced the production of ROS in the NaAsO2 -treated cells (P < 0.05). Among the cells grown in NF medium, the production of MDA in the NaAsO2 -treated cells was significantly higher than that in the control cells (P < 0.05) (Fig. 4B). Folate deficiency aggravated the increase of MDA in the NaAsO2 -treated cells; however, the MDA elevation in the NaAsO2 -treated cells incubated in FD medium was not significantly higher than that in the NaAsO2 -treated cells grown in NF medium. Both folate and NAC supplementation significantly reduced the production of MDA in the NaAsO2 -treated cells (P < 0.05) to 84.5% and 71.8%, respectively, of the MDA production in the NaAsO2 -treated cells grown in NF medium (Fig. 4B). The levels of ROS or MDA in the NaAsO2 -un-treated cells did not significantly vary according to the concentrations of folate. 3.5. Antioxidants As shown in Fig. 5A, the intracellular GSH level in the NaAsO2 treated cells cultured in NF medium was 1.8 times higher than that in the control cells (P < 0.05). In the NaAsO2 -treated cells, folate deficiency reduced the GSH level to 82.0% of that in the cells grown in NF medium (P < 0.05). The GSH levels in the NaAsO2 -treated cells supplemented with folate and NAC were 1.2 and 1.5 times higher, respectively, than those in the NaAsO2 -treated cells grown in NF medium (P < 0.05). As shown in Fig. 5B, the CAT activity in the NaAsO2 -treated cells cultured in NF medium was 1.7 times higher than that in the control cells (P < 0.05). The CAT activity in the NaAsO2 -treated cells grown in FD medium was significantly lower than that in the NaAsO2 treated cells grown in NF medium (P < 0.05). The CAT activity in the NaAsO2 -treated cells supplemented with folate or NAC was significantly higher than that in the control cells (P < 0.05), but not significantly higher than that in the NaAsO2 -treated cells grown in NF medium.

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Fig. 6. Retention and methylation of arsenic in Chang human hepatocytes. Arsenic speciation was determined using cold-trap hydride-generation atomic absorption spectrometry (HG-AAS). Cellular retention of arsenic was calculated as the content of intracellular total arsenic (TAs), including inorganic arsenic (iAs), monomethylated arsenic (MMA), and dimethylated arsenic (DMA). Methylated arsenicals were calculated as the sum of MMA and DMA in both the cells and the medium. *P < 0.05, compared with 20-␮M-NaAsO2 -treated cells grown in normal folate (NF) medium.

NaAsO2 -treated cells grown in FD medium increased to 15.7 ng/mg of protein, the difference in cellular arsenic retention between the NaAsO2 -treated cells grown in NF medium and FD medium was not significant. Folate deficiency significantly decreased the amount of methylated arsenicals to 3.3 ng/mg protein (P < 0.05). Folate supplementation had no significant effect on cellular arsenic retention or arsenic methylation. 4. Discussion

Fig. 5. Antioxidants in Chang human hepatocytes. (A) Intracellular level of reduced glutathione (GSH). (B) Intracellular catalase (CAT) activity. (C) Intracellular superoxide dismutase (SOD) activity. Results are expressed as mean ± SD, n = 3. *P < 0.05, compared with NaAsO2 -un-treated cells grown in normal folate (NF) medium (the control cells); # P < 0.05, compared with 20-␮M-NaAsO2 -treated cells grown in NF medium.

The SOD activity in the NaAsO2 -treated cells cultured in NF medium significantly decreased to 29.0% of that in the control (P < 0.05). Folate deficiency or supplementation did not have a significant effect on SOD activity in the NaAsO2 -treated cells. However, NAC supplementation in the NaAsO2 -treated cells restored the NaAsO2 -inhibited SOD activity to 40.9% of that in the control (P < 0.05) (Fig. 5C). In addition, folate deficiency or supplementation did not have a significant effect on the intracellular GSH level, CAT activity, or SOD activity in the NaAsO2 -un-treated cells (Fig. 5). 3.6. Arsenic speciation To investigate the effects of folate on arsenic metabolism in the hepatocytes, we examined the distribution of arsenic species in the cells and the medium using cold-trap HG-AAS. We detected iAs, MMA, and DMA in Chang human hepatocytes, but only detected iAs and DMA in the medium. As shown in Fig. 6, the total arsenic (TAs, including iAs, MMA, and DMA) retained in the 20-␮M-NaAsO2 treated Chang human hepatocytes incubated in NF medium for 24 h was 13.6 ng/mg of protein. The level of methylated arsenic (DMA and MMA in the cells and DMA in the medium) in the culture was 3.8 ng/mg of protein. Although the level of arsenic retained in the

The natural occurrence of high arsenic levels in drinking water is a major health problem throughout the world. Inorganic arsenicals, including arsenite [iAs(III)] and arsenate [iAs(V)], are the most common arsenic species found in drinking water. The primary metabolic pathway of iAs in humans involves multiple methylation steps catalyzed by methyltransferase. This enzymatic process occurs mainly in the liver and results in the urinary excretion of iAs, MMA and DMA (Ford, 2002). Complete biomethylation of iAs to DMA has been suggested to be associated with a higher excretion rate of arsenic (Vahter, 2000) and a lower risk of arsenic-related diseases (Tseng et al., 2005; Chen et al., 2003a,b, 2005). Nutritional factors, particularly those involved in methylation, may be important determinants of arsenic methylation (Heck et al., 2007) and arsenic toxicity (Mitra et al., 2004). Folate is considered one such nutritional factor (Kile and Ronnenberg, 2008). Although folate is relatively ubiquitous in the foods, the limited availability of fresh vegetables in some areas and methods of food preparation involving prolonged cooking may result in inadequate folate intake. High prevalence of folate deficiency and lower arsenic methylation capacity has been reported in the rural areas of Bangladesh (Gamble et al., 2005a,b). In China, a cross-sectional study conducted with 2422 adults suggested that a large proportion of Chinese adults, particularly those living in the rural areas of North China, have a low folate status (Hao et al., 2003). Most of the identified areas with populations exposed to high level of arsenic in drinking water have also been reported from the rural areas in North China (Sun, 2004). These results indicate the presence of a large arsenic-exposed population with low folate status, and this scenario may increase the number of cases of arsenic-induced health defects. In a food-questionnaire study conducted in West Bengal, India, the patients in the lowest quantile of folate intake showed a modest increase in the risk of arsenic-induced skin lesions (Mitra et al., 2004). However, another study conducted in West Bengal, India, by Chung et al. (2006) found no association between serum folate levels and skin lesions. These contradictory results may be attributed to the differences in the methods used to assess nutritional status. Recently, Majumdar et al. (2009) found that folate supplementation in rats prevented arsenic trioxide-induced DNA smearing and oxidative stress in lymphocytes and in the mitochondria of pan-

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creatic islet cells. The study also showed that the protective role of folate might be attributed to its effects on the progress of arsenic methylation. However, they did not provide any direct evidence to substantiate this finding. In this study, we observed that folate deficiency aggravated the arsenic-induced cell-viability reduction and apoptotic progression and decreased arsenic methylation capacity. However, compared to the alteration of arsenic-induced cytotoxicity and apoptosis, the decrease in arsenic methylation capacity was not significant. In addition, folate supplementation did not have significant effects on arsenic methylation or cellular retention of arsenic, but it afforded significant protection from arsenic-induced apoptotic progression (cleavage of caspase-3 and PARP, collapse of  m , and release of cytochrome c from the mitochondria) and cellviability reduction. These results indicate that other mechanisms are also related to the effect of folate on arsenic-induced toxicity. The generation of ROS, which leads to oxidative stress, is considered to play an important role in arsenic toxicity (Flora et al., 2007). In a previous study, we observed that an increase in apoptosis and a decrease in cell viability in Chang human hepatocytes were related to the arsenic-induced generation of ROS (Wang et al., 2009). The direct antioxidant properties of folate have been observed in vitro (Joshi et al., 2001; Rezk et al., 2003) and in vivo (Doshi et al., 2001). In addition, folate deficiency has been shown to lead to an elevation in the levels of homocysteine (Mason, 2003), which is considered to have a pro-oxidant effect and play a role in the production of ROS (Eberhardt et al., 2000). In the present study, the levels of ROS, MDA, and GSH, and the activity of CAT in the arsenic-treated hepatocytes varied, to some extent, according to the folate levels in the medium; this finding suggests the antioxidant effects of folate though not as strong as NAC. Taken together, these results suggest that the effects of folate on arsenic-induced toxicity may be explained partially by its antioxidant property. In conclusion, the present study showed that folate deficiency aggravates arsenic-induced cytotoxicity and apoptosis, while folate supplementation attenuates these effects. The direct and/or indirect antioxidant properties of folate may also contribute to its effects on arsenic toxicity, though the importance of folate in arsenic toxicity is generally attributed to its essential roles in methylation reactions. Our study was limited by the analytical techniques, which could not differentiate between the trivalent and pentavalent forms of arsenic. Other studies on the metabolic pathway of inorganic arsenic have shown that both trivalent and pentavalent arsenic metabolites are generated during metabolism, and the trivalent forms are more toxic (Aposhian et al., 2004). Further investigations should aim to identify the effects of folate on the generation of arsenic metabolites with different valences, the cellular methyl metabolism such as the level of S-adenosyl methionine (SAM) and homocysteine, and the activity of Sadenosylhomocysteine hydrolase. Conflict of interest None declared. Acknowledgments The study was supported by research grants (Project Nos. 30530640 and 30600510) from National Natural Science Foundation of China (NSFC) and the National Science and Technology Pillar Program of China during the 11th Five-Year Plan Period (No. 2006BAI06B04). References Abernathy, C.O., Thomas, D.J., Calderon, R.L., 2003. Health effects and risk assessment of arsenic. J. Nutr. 133, 1536S–1538S.

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