J Occup Health 2009; 51: 169–172

Short Communication

Received Mar 15, 2008; Accepted Nov 21, 2008Published online in J-STAGE Feb 3, 2009Correspondence to: F. Piao, Department of Occupational andEnvironmental Health, Dalian Medical University, No 9 WesternSection of Lushun South Road, Dalian, LiaoNing 116044, P.R.China (e-mail: piaofy_dy@yahoo.com.cn)

Preventive Effects of Taurine and Vitamin Con Renal DNA Damage of Mice Exposed toArsenic

Zongyuan LI1, Fengyuan PIAO1, Shuang LIU1, Lin SHEN1,Ning SUN1, Bin LI1 and Shuxian QU2

1Department of Occupational and Environmental Health and2Central Laboratory, Dalian Medical University, P.R. China

Key words: Arsenic trioxide, Ascorbic Acid, 8-hydroxy-2-deoxyguanosine (8-OHdG), Kidney, Oxidative stress,Taurine

Arsenic (As), as a pollutant, ubiquitously exists in wellwater and contaminated soil. There is also exposure toarsenic in some industries, such as non-ferrous smelting,wood preservation, and electronics. The risk of As-induced human diseases is high in some developingcountries such as Bangladesh, India, and China1, 2).Epidemiologic investigations and animal experimentshave demonstrated that acute and chronic exposure to Ascan cause injury to the kidney and increase the risk ofrenal cancer3), so the kidney may be a major target oftoxicities induced by As. It was reported that As depressesthe functions of the antioxidant defense system and ledto oxidative damage to cellular macromolecules4, 5). Theabove results show that increase of oxidative stress andsubsequent oxidative DNA damage of cells may beinvolved in the renal disorders induced by As. Taurine,an end product of l-cysteine metabolism, is the mostabundant free amino acid in many tissues and protectsagainst the adverse effects of reactive oxygen species(ROS) caused by various toxic substances6). Vitamin C(Vit C), as a kind of water-soluble antioxidant, alsoprevents oxidative damage to many importantmacromolecules7).

8-OHdG is formed from deoxyguanosine (dG) in DNAby hydroxy free radicals. Because of its stability, 8-OHdGis known as one of the most reliable markers of oxidativeDNA damage8). In the present study, in order toinvestigate the protective effects of taurine and Vit Cagainst As-induced nephrotoxicity, we examined theexpression of 8-OHdG and the histopathological changesin the renal tissues of mice administered As or both As

and taurine or Vit C.

Materials and Methods

ChemicalsArsenic trioxide, taurine and Vit C were purchased from

the Sigma Chemical Company (St. Louis, USA). Mousemonoclonal anti-8-OHdG antibody was obtained fromthe Japan Institute for the Control of Aging (Fukuroi,Japan). Diaminobenzidine (DAB) color reagent kit andUltrasensitiveTM S-P kit were purchased from MAIXIN-Bio (Fuzhou, China).

Animal and treatmentForty Kunming mice (20 male and 20 female) weighing

20 ± 2 g were purchased from the Experimental AnimalCenter, Dalian Medical University. These mice wererandomly divided into 4 groups of 5 mice in each gender.Group 1 received drinking water alone as controls. Group2 received 4 mg/l arsenic trioxide. Group 3 and group 4received 4 mg/l arsenic trioxide with 150 mg/kg taurineor 45 mg/kg Vit C (as combined treatment groups),respectively. Arsenic trioxide was given through drinkingwater for 60 days. Taurine and Vit C were administeredby gavage twice a week. The animals were maintainedon a standard diet and water ad libitum. They were cagedunder a 12-h dark-light cycle under standard conditionsof temperature (18–22°C) and humidity (50%). Afterthe last administration of arsenic trioxide, the thoraxesof the mice were opened under urethane (1 g/kg, ip)anesthesia, and tissue fixative (10% formalin) wasinjected via a needle inserted into the left ventricle of theheart; and then, the kidneys were removed and placed infixative. The animal experiment was performed inaccordance with the Animal Guideline of Dalian MedicalUniversity and in agreement with the Ethical Committeeof Dalian Medical University.

Histopathological examinationThe formalin-fixed kidney tissues were embedded in

paraffin, sliced at 5 µm, mounted on glass slides coatedwith poly L-lysine, and subjected to hematoxylin andeosin staining according to routine histopathologicalmethods. Histopathological changes were observed undera light microscope.

Immunohistochemistry for 8-OHdG formationFor immunohistochemical staining with the

streptavidin-biotin-peroxidase method, 3-µm tissuesections were deparaffinized, dehydrated in gradedalcohols and then subjected to antigen retrieval, followedby 1% hydrogen peroxide (15 min) and 5% rabbit serumin buffered solution (15 min). The specimens were thenincubated with mouse monoclonal anti-8-OHdG antibody(1:300, 4°C, overnight) followed by incubation withbiotin-labeled rabbit anti-mouse IgG for 60 min, and then

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with diaminobenzidine (DAB) chromogen solution.Finally, sections were counterstained with hematoxylinand mounted in xylene-based mountant. Five fieldsselected randomly were observed for each section andthe total optical density value of 8-OHdG was analyzedquantitatively using an image analyzer (Image-Pro Plus4.5, Media Cybernetics).

Statistical analysisValues were expressed as mean ± SD for the 10 mice

of each group, and the significances of the differencesbetween mean values were determined by one-wayanalysis of variance (ANOVA) followed by the Duncantest for multiple comparison using the Statistical Packagefor Social Sciences l1.5 (SPSS l1.5) computer package.

Results

Expression of 8-OHdG in kidney tissue of miceAs shown in Fig. 1, intensive 8-OHdG expression was

found in the kidney tissues of mice exposed to As. The8-OHdG expression was mainly distributed in the regionsof the glomerulus and renal tubules. Especially, highexpression of 8-OHdG was show in the proximalconvoluted tubule and Bowman’s capsule. In contrast,in the two combined treatment groups receiving taurineor Vit C, weak expression of 8-OHdG was observed inthe kidney tissues. In controls, no 8-OHdG expressionwas seen. Optical density values of 8-OHdG werequantitatively evaluated using an image analyzer and theresults are shown in Table 1. Because there was nosignificant difference between the genders (data notshown) in each group, the optical density values of 8-

Fig. 1. 8-OHdG immunoreactivity in the renal tissues of mice given various arsenic treatments. Mice of fourgroups were treated respectively with drinking water (a, as control), 4 mg/l arsenic trioxide (b), 4 mg/l arsenic trioxide and 150 mg/kg taurine (c), and 4 mg/l arsenic trioxide and 45 mg/kg vitamin C (d).PCT: proximal convoluted tubule, DCT: distal convoluted tubule. G: glomerulus. Each 8-OHdG-positive cell appears brown, and had stained nuclei. Original magnification ×200.

Table 1. Optical density value of 8-OHdG in kidney tissues of mice

Groups Chemicals No. of Animals* Optical density value (mean ± SD)

Group1(controls) drinking water 10 4.94 ± 1.67Group2 4 mg/l As2O3 10 5,522.71 ± 686.34a,b,c

Group3 4 mg/l As2O3 + 150 mg/kg taurine 10 25.83 ± 5.90Group4 4 mg/l As2O3 + 45 mg/kg Vit C 10 23.32 ± 12.12

* Five mice of each gender. Because there was no significant difference between genders (data not shown) in eachgroup, the optical density values of 8-OHdG of both genders were pooled into one group for calculation of the mean± SD. ap<0.01, compared with controls; bp<0.01, compared with group 3; cp<0.01, compared with group 4.

171Zongyuan LI, et al.: Protection of Taurine and Vitamin C against Renal DNA Damage

OHdG of both genders were pooled into one group forcalculation of the mean ± SD. The optical density valueof the As-exposed group was significantly higher thanthose of the other three groups (p<0.01).

Histopathological changes in kidney tissues of miceHistopathological changes in kidney tissues of mice

are shown in Fig. 2. Cellular swelling and tubulardilatation were observed in kidney tissues of miceexposed to As. Especially, more evident structuraldamage, such as loss of cell-cell contacts and loss ofmicrovilli were seen in the epithelium of proximalconvoluted tubule. In the glomerulus, pathologicalchanges were mainly found in the area of Bowman’scapsule. Bowman’s capsules were diminished ordisappeared in the group exposed to As. However,dilation and hyperemia of glomerular capillaries and mildcellular proliferation were also observed in theglomerulus. In contrast, the pathological changes in thetwo combined treatment groups were mild and showedonly slight swelling of the cell body in the proximalconvoluted tubules. Moreover, the cell boundary wasclear and without any changes in the cytoplasm andnucleus. In controls, there were no histopathologicalchanges in the proximal convoluted tubules andglomerulus. The regions where these histopathologicalchanges presented in the kidneys of mice exposed to As

were consistent with the distribution of 8-OHdGexpression.

Discussion

Epidemiologic investigations and animal experimentshave shown that acute and chronic exposure to As inducesdysfunction of the renal system and renal diseases,indicating that As possesses nephrotoxicity. It has beenreported that As generates ROS, inducing lipidperoxidation and the oxidative damage of proteins as wellas DNA. As DNA is more sensitive to oxidative stress,8-OHdG an oxidative product of DNA, is used forevaluating oxidative damage in vivo8).

In the present study, obvious expression of 8-OHdGin kidney tissues was observed in the group whichreceived As alone. The 8-OHdG expression was mainlyconcentrated in the regions of the glomerulus and renaltubules. Especially, high expression of 8-OHdG was seenin the proximal convoluted tubules and Bowman’scapsule. Histopathological changes such as cellularswelling, dilation and hyperemia of glomerular capillariesin kidney tissue were also found in the As-exposed group.In particular, the more evident pathological changes wereseen in the proximal convoluted tubules and Bowman’scapsule. Moreover, the regions where thesehistopathological changes presented in the kidneys of themice exposed to As were consistent with the distribution

Fig. 2. Histopathological changes in the renal tissues given various arsenictreatments. The treatment and abbreviations are the same as in the legend toFig.1. Histopathological changes were examined by H&E staining. Originalmagnification ×200.

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of 8-OHdG expression. These results suggest that thekidney may be a major target of As toxicity, and that theepithelial cells of proximal convoluted tubules andBowman’s capsule seem to be more sensitive to As-induced nephrotoxicity. Many recent studies haveprovided experimental evidence that As exposure canresult in the generation of ROS and cause cell damage ordeath through the ROS signaling pathway9). These studiesand the result of our immunohistochemistry analysisindicate that the pathological changes in the kidney tissuesof mice exposed to As may be related to the As-inducedoxidative stress. The proximal convoluted tubules areparticularly sensitive due to their high reabsorptiveactivity and anatomical position as the first renal epithelialcell to be exposed to filtered toxicants10). It was reportedthat chronic toxicity of As to kidney resulted inproteinuria11). However, the mechanism is still unclear.Recent studies by many investigators have shown thatthe podocy te i s impor t an t fo r ma in t a in ingpermselectivity12, 13). Podocytes line the outer aspect ofthe glomerular basement membrane and serve as the finaldefense against urinary protein loss. Once the podocytesare damaged, loss of permselectivity follows. Therefore,we suspect that proteinuria resulting from chronic Aspoisoning may be associated with As-induced damage topodocytes.

In the two combined treatment groups receiving taurineor Vit C, weak expression of 8-OHdG in the kidney tissueswas observed. Moreover, the optical density value ofthe combined treatment 8-OHdG expression of these twogroups was significantly lower than that of the As-exposedgroup (p<0.01). In these two groups, the pathologicalchanges showed slight swelling of the cell body in theproximal convoluted tubule. However, the cell boundarywas clear. These results indicate that taurine and Vit Cprotect the kidney against arsenic-induced oxidative DNAdamage and pathologic changes. It is well known thattaurine is the major intracellular free β-amino acid andplays an important physiological role in the preventionof oxidant-induced injury to many tissues6). Thepreventive effect of taurine as an antioxidant for As-induced damage may be attributed to its ability to stabilizebiomembranes, scavenge ROS, and reduce the productionof lipid peroxidation14, 15). In addition, many studies havedemonstrated that Vit C, as a water-soluble antioxidant,can readily scavenge ROS, reactive nitrogen species(RNS), increase As excretion7, 16) and prevent oxidativedamage to many important biological macromoleculessuch as DNA, lipids, and proteins16, 17). Hence, thepreventive effect of Vit C on renal damage induced byAs may be associated with its antioxidation capacity. Insummary, the above results show that taurine and Vit Cprotect against As-induced nephrotoxicity. Further studiesare required to explaining the molecular mechanisms oftheir preventive action.

Acknowledgments: This study was the supported bythe National Natural Science Foundation of China (No.30571584).

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