J Occup Health 2009; 51: 169172
Short Communication
Preventive Effects of Taurine and Vitamin C
on Renal DNA Damage of Mice Exposed to
Arsenic
Zongyuan LI1, Fengyuan PIAO1, Shuang LIU1, Lin SHEN1,
Ning SUN1, Bin LI1 and Shuxian QU2
1
Department of Occupational and Environmental Health and
2
Central 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 well
water and contaminated soil. There is also exposure to
arsenic in some industries, such as non-ferrous smelting,
wood preservation, and electronics. The risk of As-
induced human diseases is high in some developing
countries such as Bangladesh, India, and China 1, 2).
Epidemiologic investigations and animal experiments
have demonstrated that acute and chronic exposure to As
can cause injury to the kidney and increase the risk of
renal cancer3), so the kidney may be a major target of
toxicities induced by As. It was reported that As depresses
the functions of the antioxidant defense system and led
to oxidative damage to cellular macromolecules4, 5). The
above results show that increase of oxidative stress and
subsequent oxidative DNA damage of cells may be
involved in the renal disorders induced by As. Taurine,
an end product of l-cysteine metabolism, is the most
abundant free amino acid in many tissues and protects
against the adverse effects of reactive oxygen species
(ROS) caused by various toxic substances6). Vitamin C
(Vit C), as a kind of water-soluble antioxidant, also
prevents oxidative damage to many important
macromolecules7).
8-OHdG is formed from deoxyguanosine (dG) in DNA
by hydroxy free radicals. Because of its stability, 8-OHdG
is known as one of the most reliable markers of oxidative
DNA damage 8) . In the present study, in order to
investigate the protective effects of taurine and Vit C
against As-induced nephrotoxicity, we examined the
expression of 8-OHdG and the histopathological changes
in the renal tissues of mice administered As or both As
Received Mar 15, 2008; Accepted Nov 21, 2008
Published online in J-STAGE Feb 3, 2009
Correspondence to: F. Piao, Department of Occupational and
Environmental Health, Dalian Medical University, No 9 Western
Section of Lushun South Road, Dalian, LiaoNing 116044, P.R.
China (e-mail: piaofy_dy@yahoo.com.cn)
and taurine or Vit C.
Materials and Methods
Chemicals
Arsenic trioxide, taurine and Vit C were purchased from
the Sigma Chemical Company (St. Louis, USA). Mouse
monoclonal anti-8-OHdG antibody was obtained from
the Japan Institute for the Control of Aging (Fukuroi,
Japan). Diaminobenzidine (DAB) color reagent kit and
UltrasensitiveTM S-P kit were purchased from MAIXIN-
Bio (Fuzhou, China).
Animal and treatment
Forty Kunming mice (20 male and 20 female) weighing
20 2 g were purchased from the Experimental Animal
Center, Dalian Medical University. These mice were
randomly divided into 4 groups of 5 mice in each gender.
Group 1 received drinking water alone as controls. Group
2 received 4 mg/l arsenic trioxide. Group 3 and group 4
received 4 mg/l arsenic trioxide with 150 mg/kg taurine
or 45 mg/kg Vit C (as combined treatment groups),
respectively. Arsenic trioxide was given through drinking
water for 60 days. Taurine and Vit C were administered
by gavage twice a week. The animals were maintained
on a standard diet and water ad libitum. They were caged
under a 12-h dark-light cycle under standard conditions
of temperature (1822C) and humidity (50%). After
the last administration of arsenic trioxide, the thoraxes
of the mice were opened under urethane (1 g/kg, ip)
anesthesia, and tissue fixative (10% formalin) was
injected via a needle inserted into the left ventricle of the
heart; and then, the kidneys were removed and placed in
fixative. The animal experiment was performed in
accordance with the Animal Guideline of Dalian Medical
University and in agreement with the Ethical Committee
of Dalian Medical University.
Histopathological examination
The formalin-fixed kidney tissues were embedded in
paraffin, sliced at 5 m, mounted on glass slides coated
with poly L-lysine, and subjected to hematoxylin and
eosin staining according to routine histopathological
methods. Histopathological changes were observed under
a light microscope.
Immunohistochemistry for 8-OHdG formation
For immunohistochemical staining with the
streptavidin-biotin-peroxidase method, 3- m tissue
sections were deparaffinized, dehydrated in graded
alcohols and then subjected to antigen retrieval, followed
by 1% hydrogen peroxide (15 min) and 5% rabbit serum
in buffered solution (15 min). The specimens were then
incubated with mouse monoclonal anti-8-OHdG antibody
(1:300, 4C, overnight) followed by incubation with
biotin-labeled rabbit anti-mouse IgG for 60 min, and then
 170 J Occup Health, Vol. 51, 2009

Fig. 1. 8-OHdG immunoreactivity in the renal tissues of mice given various arsenic treatments. Mice of four
groups 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 1.67

Group2 4 mg/l As2O3 10 5,522.71 686.34a,b,c
Group3 4 mg/l As2O3 + 150 mg/kg taurine 10 25.83 5.90
Group4 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 each
group, 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.

with diaminobenzidine (DAB) chromogen solution.

Finally, sections were counterstained with hematoxylin
and mounted in xylene-based mountant. Five fields
selected randomly were observed for each section and
the total optical density value of 8-OHdG was analyzed
quantitatively using an image analyzer (Image-Pro Plus
4.5, Media Cybernetics).
Statistical analysis
Values were expressed as mean SD for the 10 mice
of each group, and the significances of the differences
between mean values were determined by one-way
analysis of variance (ANOVA) followed by the Duncan
test for multiple comparison using the Statistical Package
for Social Sciences l1.5 (SPSS l1.5) computer package.
Results
Expression of 8-OHdG in kidney tissue of mice
As shown in Fig. 1, intensive 8-OHdG expression was
found in the kidney tissues of mice exposed to As. The
8-OHdG expression was mainly distributed in the regions
of the glomerulus and renal tubules. Especially, high
expression of 8-OHdG was show in the proximal
convoluted tubule and Bowmans capsule. In contrast,
in the two combined treatment groups receiving taurine
or Vit C, weak expression of 8-OHdG was observed in
the kidney tissues. In controls, no 8-OHdG expression
was seen. Optical density values of 8-OHdG were
quantitatively evaluated using an image analyzer and the
results are shown in Table 1. Because there was no
significant difference between the genders (data not
shown) in each group, the optical density values of 8-
 Zongyuan LI, et al.: Protection of Taurine and Vitamin C against Renal DNA Damage 171

Fig. 2. Histopathological changes in the renal tissues given various arsenic

treatments. The treatment and abbreviations are the same as in the legend to
Fig.1. Histopathological changes were examined by H&E staining. Original
magnification 200.

OHdG of both genders were pooled into one group for
calculation of the mean SD. The optical density value
of the As-exposed group was significantly higher than
those of the other three groups (p<0.01).
Histopathological changes in kidney tissues of mice
Histopathological changes in kidney tissues of mice
are shown in Fig. 2. Cellular swelling and tubular
dilatation were observed in kidney tissues of mice
exposed to As. Especially, more evident structural
damage, such as loss of cell-cell contacts and loss of
microvilli were seen in the epithelium of proximal
convoluted tubule. In the glomerulus, pathological
changes were mainly found in the area of Bowmans
capsule. Bowmans capsules were diminished or
disappeared in the group exposed to As. However,
dilation and hyperemia of glomerular capillaries and mild
cellular proliferation were also observed in the
glomerulus. In contrast, the pathological changes in the
two combined treatment groups were mild and showed
only slight swelling of the cell body in the proximal
convoluted tubules. Moreover, the cell boundary was
clear and without any changes in the cytoplasm and
nucleus. In controls, there were no histopathological
changes in the proximal convoluted tubules and
glomerulus. The regions where these histopathological
changes presented in the kidneys of mice exposed to As
were consistent with the distribution of 8-OHdG
expression.
Discussion
Epidemiologic investigations and animal experiments
have shown that acute and chronic exposure to As induces
dysfunction of the renal system and renal diseases,
indicating that As possesses nephrotoxicity. It has been
reported that As generates ROS, inducing lipid
peroxidation and the oxidative damage of proteins as well
as DNA. As DNA is more sensitive to oxidative stress,
8-OHdG an oxidative product of DNA, is used for
evaluating oxidative damage in vivo8).
In the present study, obvious expression of 8-OHdG
in kidney tissues was observed in the group which
received As alone. The 8-OHdG expression was mainly
concentrated in the regions of the glomerulus and renal
tubules. Especially, high expression of 8-OHdG was seen
in the proximal convoluted tubules and Bowmans
capsule. Histopathological changes such as cellular
swelling, dilation and hyperemia of glomerular capillaries
in kidney tissue were also found in the As-exposed group.
In particular, the more evident pathological changes were
seen in the proximal convoluted tubules and Bowmans
capsule. Moreover, the regions where these
histopathological changes presented in the kidneys of the
mice exposed to As were consistent with the distribution
 172
of 8-OHdG expression. These results suggest that the
kidney may be a major target of As toxicity, and that the
epithelial cells of proximal convoluted tubules and
Bowmans capsule seem to be more sensitive to As-
induced nephrotoxicity. Many recent studies have
provided experimental evidence that As exposure can
result in the generation of ROS and cause cell damage or
death through the ROS signaling pathway9). These studies
and the result of our immunohistochemistry analysis
indicate that the pathological changes in the kidney tissues
of mice exposed to As may be related to the As-induced
oxidative stress. The proximal convoluted tubules are
particularly sensitive due to their high reabsorptive
activity and anatomical position as the first renal epithelial
cell to be exposed to filtered toxicants10). It was reported
that chronic toxicity of As to kidney resulted in
proteinuria11). However, the mechanism is still unclear.
Recent studies by many investigators have shown that
the podocyte is important for maintaining
permselectivity12, 13). Podocytes line the outer aspect of
the glomerular basement membrane and serve as the final
defense against urinary protein loss. Once the podocytes
are damaged, loss of permselectivity follows. Therefore,
we suspect that proteinuria resulting from chronic As
poisoning may be associated with As-induced damage to
podocytes.
In the two combined treatment groups receiving taurine
or Vit C, weak expression of 8-OHdG in the kidney tissues
was observed. Moreover, the optical density value of
the combined treatment 8-OHdG expression of these two
groups was significantly lower than that of the As-exposed
group (p<0.01). In these two groups, the pathological
changes showed slight swelling of the cell body in the
proximal convoluted tubule. However, the cell boundary
was clear. These results indicate that taurine and Vit C
protect the kidney against arsenic-induced oxidative DNA
damage and pathologic changes. It is well known that
taurine is the major intracellular free -amino acid and
plays an important physiological role in the prevention
of oxidant-induced injury to many tissues 6) . The
preventive effect of taurine as an antioxidant for As-
induced damage may be attributed to its ability to stabilize
biomembranes, scavenge ROS, and reduce the production
of lipid peroxidation14, 15). In addition, many studies have
demonstrated that Vit C, as a water-soluble antioxidant,
can readily scavenge ROS, reactive nitrogen species
(RNS), increase As excretion7, 16) and prevent oxidative
damage to many important biological macromolecules
such as DNA, lipids, and proteins 16, 17). Hence, the
preventive effect of Vit C on renal damage induced by
As may be associated with its antioxidation capacity. In
summary, the above results show that taurine and Vit C
protect against As-induced nephrotoxicity. Further studies
are required to explaining the molecular mechanisms of
their preventive action.
J Occup Health, Vol. 51, 2009
Acknowledgments: This study was the supported by
the National Natural Science Foundation of China (No.
30571584).
References
1) Singh N, Kumar D, Sahu AP. Arsenic in the
environment: effects on human health and possible
prevention. J Environ Biol 2007; 28: 35965.
2) International Program on Chemical Safety (IPCS).
Arsenic and arsenic compounds, 2nd. Environmental
Health Criteria. Geneva: World Health Organization;
2001. p.2673l8.
3) Ratnaike RN. Acute and chronic arsenic toxicity.
Postgrad Med J 2003; 79: 3916.
4) Wang TC, Jan KY, Wang AS, Gurr JR. Trivalent
arsenicals induce lipid peroxidation, protein
carbonylation, and oxidative DNA damage in human
urothelial cells. Mutat Res 2007; 615: 7586.
5) Ding W, Hudson LG, Liu KJ. Inorganic arsenic
compounds cause oxidative damage to DNA and
protein by inducing ROS and RNS generation in human
keratinocytes. Mol Cell Biochem 2005; 279: 10512.
6) Chesney RW. Taurine: its biological role and clinical
implications. Adv Pediatr 1985; 32: 142.
7) Sohini, Rana SV. Protective effect of ascorbic acid
against oxidative stress induced by inorganic arsenic
in liver and kidney of rat. Indian J Exp Biol 2007; 45:
3715.
8) Leinonen J, Lehtimaki T, Toyokuni S. New biomarker
evidence of oxidative DNA damage in patients with
moninsulin dependent diabetes mellitus. Febs Lett
1997; 409: 1502.
9) Honglian S, Xianglin S, Ke JL. Oxidative mechanism
of arsenic toxicity and Carcinogenesis. Molecular and
Cellular Biochemistry 2004; 255: 6778.
10) Brown MM, Rhyne BC, Goyer RA. Intracellular ejects
of chronic arsenic administration on renal proximal
tubule cells. J Toxicol Environ Health 1976; 1: 50514.
11) Buchet JP, Heilier JF, Bernard A, et al. Urinary protein
excretion in humans exposed to arsenic and cadmium.
Int Arch Occup Environ Health 2003; 76: 11120.
12) Endlich K, Kriz W, Witzgall R. Update in podocyte
biology. Curr Opin Nephrol Hypertens 2001; 10: 331
40.
13) Kerjaschki D. Caught flat-footed: podocyte damage and
the molecular bases of focal glomerulosclerosis. J Clin
Invest 2001; 108: 15837.
14) Yildirim Z, Kili N, Ozer C, Babul A, Take G, Erdogan
D. Effects of taurine in cellular responses to oxidative
stress in young and middle-aged rat liver. Ann N Y
Acad Sci 2007; 1100: 55361.
15) Balkan J, Kanbagli O, Aykac-Toker G, Uysal M.
Taurine treatment reduces hepatic lipids and oxidative
stress in chronically ethanol treated rats. Biol Pharm
Bull 2002; 25: 12313.
16) Sohini, Rana SV. Amelioration of arsenic toxicity by
L-Ascorbic acid in laboratory rat. J Environ Biol 2007;
28: 37784.
17) Konopacka M. Role of vitamin C in oxidative DNA
damage. Postepy Hig Med Dosw 2004; 58: 3438.