Meso 2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA effect on gallium arsenide induced pathological liver injury in rats

Toxicology Letters 132 (2002) 9 – 17 www.elsevier.com/locate/toxlet

Meso 2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA effect on gallium arsenide induced pathological liver injury in rats S.J.S. Flora a,*, Rupa Dubey b, G.M. Kannan a, R.S. Chauhan b, B.P. Pant c, D.K. Jaiswal c a

Di6ision of Pharmacology and Toxicology, Defence Research and De6elopment Establishment, Jhansi Raod, Gwalior 474 002, India b Di6ision of Electron Microscopy, Defence Research and De6elopment Establishment, Jhansi Road, Gwalior 474 002, India c Di6ision of Synthetic Chemistry, Defence Research and De6elopment Establishment, Jhansi Road, Gwalior 474 002, India Received 1 October 2001; received in revised form 21 January 2002; accepted 24 January 2002

Abstract The effect of meso 2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA (MiADMSA) on gallium arsenide (GaAs) induced liver damage was studied. The oral feeding rat model was used in this study. The animals were exposed to 10 mg/kg GaAs, orally, once daily, 5 days a week for 24 weeks and treated thereafter with single oral daily dose of either 0.3 mmol/kg DMSA or MiADMSA for two course of 5 days treatment. The animals were sacrificed thereafter. Lipid peroxidation was assessed by measuring liver thiobarbituric acid reactive substance (TBARS). Liver damage was assessed by number of biochemical variables and by light microscopy. The activity of superoxide dismutase (SOD) and d-aminolevulinic acid dehydratase (ALAD) beside reduced glutathione (GSH) concentration was measured in blood. Exposure to GaAs produced a significant reduction in GSH while, increased the oxidized glutathione (GSSG) concentration. Hepatic glutathione peroxidase (GPx) and catalase activity increased significantly while level of serum transaminase increased moderately. Gallium arsenide exposure also produced marked hepatic histopathological lesions. Overall, treatment with MiADMSA proved to be better than DMSA in the mobilization of arsenic and in the turnover of some of the above mentioned GaAs sensitive biochemical alterations. Histopathological lesions also, responded more favorably to chelation treatment with MiADMSA than DMSA. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Gallium arsenide exposure; Liver damage; Biochemical and histopathological changes; Chelation therapy; Succimers

1. Introduction * Corresponding author. Tel.: + 91-751-341980 Ext. 365; fax: +91-751-341148. E-mail address: [email protected], [email protected] (S.J.S. Flora).

In the semiconductor industry a number of new materials are being used for which even the most basic toxicological information is lacking. Conse-

0378-4274/02/$ - see front matter © 2002 Published by Elsevier Science Ireland Ltd. PII: S0378-4274(02)00034-6

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quently, the health hazard to individual exposed to these materials is not known. Among these novel materials is a group IIIA and VA compound, gallium arsenide (GaAs) which has found an increased use in the electronic industry (Carter and Sullivan, 1992). Although, this material poses a very small risk to the public; however, accidental release from the production units may occur besides, the workers are at the increased risk to the toxic effects of GaAs (Flora, 2000). It is now well known that GaAs dissociates into its two constitutive moieties gallium (Ga) and arsenic (As) on exposure suggesting that one should not take toxicity of GaAs lightly. Number of studies have reported solubility of GaAs particles in vivo and the tissue distribution and excretion patterns of Ga and As over time after the administration of GaAs particles via a variety of routes (Webb et al., 1984, 1986). These results lead to the conclusion that particles of GaAs are degraded in vivo to release their constitutive elements, which are then distributed to major target organs. Intratracheal or oral exposure to GaAs resulted in a decreased body weight as well as quantitative and qualitative changes in porphyrin excretion (Webb et al., 1984, 1986; Flora et al., 1997, 1998). Toxic effects from GaAs appear to occur primarily after inhalation exposure. However, oral exposure to high dose of GaAs may also result in toxicity (Carter and Sullivan, 1992). Other recent studies indicate that in experimental animals at least one step in the haem along with several immune system variables are significantly altered by GaAs (Flora et al., 1998; Sikorski et al., 1989, 1991; Burns et al., 1993). Both humoral and cell mediated sites of immunity are specifically affected (Flora and Kumar, 1996; Burns et al., 1993). We also reported the effects of multiple oral GaAs exposure on some brain and hepatic biochemical variables (Flora et al., 1994; Flora, 1996). Beside these studies little is known about the toxicity of GaAs on extrapulmonary tissues i.e. liver and kidneys. Webb et al. (1984) reported impaired liver function due to arsenic dissociated from GaAs, as indicated by increased urinary excretion of uroporphyrin. We reported changes in some key biochemical variables in the liver of rats exposed to various doses of GaAs but the

changes were only moderate (Flora, 1996; Flora et al., 1998). As the toxicology of GaAs is still not very well understood and clearly defined, the treatment also remains to be doubtful. Administration of meso 2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane 1sulfonate (DMPS) was reported to be moderately effective in rats exposed sub-acutely to GaAs (Flora and Kumar, 1996; Burns et al., 1993). Attempts were thus made in this study to assess the (i) possible hepatic pathological lesions and their relationship with few selected variables of hepatic oxidative stress; and (ii) efficacy of DMSA and one of its analogue (monoester), monoisoamyl DMSA, in the mobilization of gallium and arsenic from hepatic tissues and to assess the recovery in the altered biochemical variables.

2. Materials and methods

2.1. Chemicals All the chelating agents except MiADMSA were procured commercially from Sigma Chemicals (St. Louis, MO, USA) or Merck (Germany) and were approximately 98% pure. MiADMSA was synthesized in our Synthetic Chemistry division, by the controlled esterification of DMSA with the corresponding alcohol (isoamyl alcohol) in acidic medium (Jones et al., 1992). The product was purified (purity 99.9%) and characterized using spectral and analytical methods before experimentation. The samples were stored, refrigerated in a dessicator to avoid oxidation and thermal decomposition. Both the chelators were dissolved in saline; DMSA was dissolved in 10% sodium bicarbonate while MiADMSA was dissolved in 5% sodium bicarbonate solution. All the antidote solutions were prepared immediately before use. The injection volume amounted to 4-ml/kg-body weight.

2.2. Animals and treatments Male wistar albino rats weighing approximately 120 g were obtained from the Defence Research

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and Development Establishment (DRDE) animal facility and prior to use they were acclimatized for 7 days. The Animal Use Committee of the DRDE, Gwalior, India, approved the protocols for the experiments. Rats were divided into two groups of five and 15 rats each and were exposed to distilled water and 10 mg/kg GaAs, respectively, orally once daily 5 days a week for 24 consecutive weeks. After 24 weeks of exposure, GaAs exposed animals were divided into three groups and treated as below for 5 consecutive days: Group IIA: saline Group IIB: 0.3 mmol/kg, DMSA, orally once daily Group IIC: 0.3 mmol/kg, MiADMSA, orally once daily. After 5 days of treatment they were given a rest of 7 days and then subjected to a second course of 5 days treatment. GaAs exposure was stopped during the course of chelation treatment. The doses and time intervals selected for the chelators were appropriate not to cause any lethal/toxic effects keeping in view their metabolism/half-life (Flora et al., 2001, unpublished data). We have already reported that male rats exposed orally for 21 consecutive days to 25, 50 or 100 mg/kg MiADMSA do not produce any marked changes in body weight, tissue weight or alterations in parameters indicative of liver, kidneys or haematological damage (Flora et al., 2002, under communication). Thus, a single dose of 0.3 mmol/kg for DMSA and MiADMSA was selected based on the clinical application and on results from previous experiments in human and experimental animals against heavy metal poisoning (Flora and Kumar, 1996; Flora et al., 1995; Gong and Evans, 1997; Lifshitz et al., 1997). Throughout the experiment the animals were kept on standard pellet diet (Amrut Feeds, Pranav Agro, New Delhi, metal contents of diet, in ppm dry weight Zn 45, Cu 10, Mn 55, Fe 70, Co 5). After the last administration animals were given 1-day rest period and were sacrificed under light ether anesthesia. Blood was collected from heart in heparinized tubes. Liver was removed rinsed in cold saline,

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blotted, weighed and used for various biochemical variables and metal analysis.

2.3. Biochemical assay 2.3.1. Blood l-aminole6ulinic acid dehydratase (ALAD) The activity of blood d-aminolevulinic acid dehydratase (ALAD) was assayed according to the procedure of Berlin and Schaller (1974). The assay system consisted of 0.2 ml of heparinized blood and 1.3 ml of distilled water. After 10 min of incubation at 37 °C for complete hemolysis, 1 ml of standard d-aminolevulinic acid was added to the tubes and incubated for 60 min at 37 °C. The reaction was stopped after 1 h by adding 1 ml of trichloroacetic acid (TCA). To the supernatant, an equal volume of Ehrlich reagent was added and the absorbance was recorded at 555 nm after 5 min. 2.3.2. Alanine aminotransferase and aspartate aminotransferase Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) activities were measured in serum following the method of Reitman and Frankel (1957). The assay system contained 1 ml of buffer/substrate solution, 0.1 ml of serum and incubated for exactly 60 min (for ALT) and 30 min for AST at 37 °C in water bath. One milliliter of chromogen solution was added, mixed and allowed to stand for 20 min at room temperature and 10 ml of 0.4 N NaOH was added subsequently. The extinction was read at 505 nm against blank. The controls were run in parallel, the substrate being added after deproteinization. 2.3.3. Blood glutathione (GSH) and hepatic GSH and oxidized glutathione (GSSG) For the determination of blood glutathione (GSH) concentration (Ellman 1959), 200 ml of whole blood was added to 200 ml of 10 mM solution of DTNB in phosphate buffer (pH 7.5) containing 17.5 mM Na2EDTA. Samples were centrifuged at 2000× g for 6 min and the supernatant used for assay. For the GSH assay in liver (Hissin and Hilf, 1976), 0.5 ml supernatant and 4.5 ml phosphate

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buffer (pH 8.0) were mixed. The final assay mixture (2.0 ml) contained 100 ml supernatant, 1.8 ml phosphate—EDTA buffer and 100 ml O-phthaldehyde (OPT; 1000 ml/ml in absolute methanol, prepared fresh). After mixing, fluorescence was determined at 420 nm with an excitation wavelength of 350 nm using a spectroflurometer (Model RF 5000 Shimadzu, Japan).

2.3.4. Glutathione peroxidase Liver Glutathione peroxidase (GPx) was determined by the method of Flohe and Gunzler (1984) at 37 °C. The reaction mixture consisted of 500-ml phosphate buffer, 100-ml glutathione reductase (0.24 units). The 100-ml tissue extract was added to the reaction mixture and incubated at 37 °C for 10 min. The 50 ml of 12 mM t-butyl hydroperoxide was added to 450 ml tissue reaction mixture and measured at 340 nm for 180 s. The molar extinction coefficient of 6.22× 103 cm − 1 was used to determine the activity. 2.3.5. Superoxide dismutase Superoxide dismutase (SOD) activity was assayed spectrophotometrically as described by Durak et al., (1996). Briefly, 2.8 ml of reactive mixture (xanthine 9.13 mg/200 ml distilled water, EDTA 25 mg/100 ml, sodium carbonate, 2.54 g/60 ml, bovine albumin 30 mg/30 ml) is added to 0.1 ml sample and 50 ml xanthine oxidase (10 ml in 2 M ammonium sulphate), incubated at 25 °C for 20 min and mixed with 0.1 ml copper chloride (108 mg/100 ml). The colour reaction is detected at 560 nm. 2.3.6. Catalase Catalase activity was assayed following the procedure of Aebi (1984) at room temperature. One hundred microliter of tissue extract was placed on ice bath for 30 min and then for another 30 min at room temperature. Ten microliter Triton-X 100 was added to the each tube. In a cuvette containing 200 ml phosphate buffer and 50 ml of tissue extract, was added 250 ml of 0.066 M H2O2 (in phosphate buffer) and decrease in optical density was measured at 240 nm for 60 s. The molar extinction coefficient of 43.6 M cm − 1 was used to

determine CAT activity. One unit of activity is equal to the moles of H2O2 degraded/min/mg protein.

2.3.7. Lipid peroxidation (MDA) Hepatic lipid peroxidation was measured by shaking the liver homogenate in 150 mM KCl for 30 min at 37 °C and measuring the malondialdehyde (MDA) formed with the thiobarbituric acid reaction (Wilber et al., 1949). The amount of MDA was calculated using a molar extinction coefficient of 1.56×105/M/cm. 2.3.8. Metal estimation For blood and liver metal determination, wet tissue weight and volume of blood was recorded. After digestion with concentrated nitric acid using a microwave digestion system (model MDS-2100, CEM, USA), samples were brought to a constant volume and determination of tissue metal contents was performed. Arsenic concentration in blood and liver were measured after wet acid digestion using a Microwave Digestion System (CEM, USA, model MDS-2100). Arsenic was estimated using a Hydride Vapour Generation System (Perkin–Elmer model MHS-10) fitted with an atomic absorption spectrophotometer (AAS, Perkin–Elmer model AAnalyst 100). Gallium contents were also measured in the digested tissue samples using AAS. 2.3.9. Histopathological obser6ations Liver was removed, rinsed with normal saline and cut into small bits. Tissue bits were fixed in aqueous Bouins fluid and 3 –4 mm thin sections were stained with haematoxylin and eosin. Histopathological changes were observed by light microscopy. 2.3.10. Statistical analysis Data are expressed, as means9 SEM. Data comparisons were carried out using one-way analysis of variance followed by Student’ t-tests to compare means between the different treatment groups. Difference between unexposed (with or without chelation) with PB0.05 were considered significant.

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of the thiol chelators were able to alter the changes in the above-described variables except for a marginal beneficial effect on GPx activity. Interestingly, serum GOT and GPT activities were higher in animals treated with chelating agents indicating mild hepatotoxic effect of these succimers. Blood ALAD activity was significantly reduced in GaAs treated rats compared to normal control (Table 3). SOD activity also inhibited significantly on GaAs exposure while blood GSH level remained unchanged. DMSA was unable to restore the inhibited ALAD activity while; MiADMSA was comparatively effective in increasing ALAD activity towards normal. Both DMSA and MiADMSA were able to significantly increase GaAs induced inhibition of SOD activity while, GSH level remained unchanged on treatment with DMSA and MiADMSA.

3. Results There was no difference in the weight gain of animals in the different groups during the study. Liver weight of the animals exposed to GaAs although, increased marginally (data not shown). Exposure to GaAs lead to a significant fall in hepatic GSH level while, GSSG and MDA levels increased significantly (Table 1). DMSA proved ineffective while MiADMSA produced a moderate increase (non-significant) in the inhibited GSH level. No effect of any of the chelator on GSSG was observed. On the other hand, both DMSA and MiADMSA effectively reduced increase in the MDA level. Table 2 shows the effect of DMSA and MiADMSA on hepatic GPx, catalase and serum transaminase activities in GaAs pre-exposed rats. GaAs exposure produced significant elevation of GPx, catalase and transaminase activities. None

Table 1 Chelating agents effect on gallium arsenide-induced hepatic oxidative stress in rats

Normal animals GaAs (control) GaAs+DMSA GaAs+MiADMSA

GSH (nmol/mg protein)

GSSG (nmol/mg protein)

MDA (nmol/mg protein)

40.6 93.5 26.1 93.0* 26.0 95.2 31.5 94.6

2.94 9 0.08 3.71 90.15* 3.52 90.25 3.94 9 0.77

1.39 90.03 2.33 90.24* 1.20 90.04** 1.65 90.14**

Values are mean 9SE; n= 5; GSH, glutathione; GSSG, oxidized glutathione; MDA, malonialdehyde. * PB0.05 compared to negative control (normal animals). ** PB0.05 compared to positive control (GaAs exposed).

Table 2 Chelating agents effect on gallium arsenide-induced changes in hepatic glutathione peroxidase (GPx), catalase and serum transaminase activities in rats

Normal animals GaAs (control) GaAs+DMSA GaAs +MiADMSA

GPx (m mol/min/mg protein)

Catalase (m mol/min/mg protein)

S-GOT (m mol/min/mg protein)

S-GPT (m mol/min/mg protein)

0.379 90.003 0.473 90.032* 0.426 90.012 0.397 90.015**

70.69 5.3 123.8 9 21.5* 110.7 9 8.9 123.69 9.5

3.87 9 0.32 6.12 9 0.34* 8.12 9 0.18 6.87 9 0.51

5.12 9 0.24 9.12 9 0.45* 11.8 9 0.87 10.3 9 0.43

Values are mean 9 SE; n= 5; GPx, glutathione peroxidase; S-GOT, serum glutamic oxaloacetic transaminase; S-GPT, serum glutamic pyruvic transaminase. * PB0.05 compared to negative control (normal animals). ** PB0.05 compared to positive control (GaAs exposed).

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Table 3 Chelating agents effect on gallium arsenide-induced changes in some hematological variables

Normal animals GaAs (control) GaAs+DMSA GaAs+MiADMSA

ALAD (nmol/min/ml rbc)

GSH (mg/ml)

SOD (nmol/ mg protein)

6.169 0.15 2.6190.30* 3.109 0.12 4.159 0.46**

3.94 9 0.08 3.61 9 0.15 3.42 9 0.35 3.979 0.24

78.6 9 0.32 30.8 9 6.78* 54.4 9 1.12** 50.7 9 1.19**

Values are mean 9 SE; n=5; ALAD, delta aminolevulinic acid dehydratase; GSH, glutathione; S-SOD, serum superoxide dismutase. * PB0.05 compared to negative control (normal animals). ** PB0.05 compared to positive control (GaAs exposed). Table 4 Chelating agents effect on gallium and arsenic concentration in rat blood and liver Gallium

Normal animals GaAs (control) GaAs+DMSA GaAs+MiADMSA

Arsenic

Blood (ng/dl)

Liver (mg/g)

Blood (mg/ml)

Liver (mg/g)

N.D. 32.619 5.30* 26.229 3.12 33.429 7.46

N.D 2.99 9 0.35* 3.35 90.65 2.37 9 0.24

0.14 9 0.01 15.81 9 1.69* 8.44 9 2.12** 4.879 0.91**

0.26 90.03 9.12 90.74* 4.05 90.23** 2.34 90.16**

Values are mean 9SE; n= 5. * PB0.05 compared to negative control (normal animals). ** PB0.05 compared to positive control (GaAs exposed).

Table 4 shows gallium and arsenic concentration in blood and liver. Animals exposed to GaAs had significantly higher gallium and arsenic level in blood and liver. Gallium level in blood and liver remained uninfluenced by chelators but arsenic concentration reduced significantly on treatment with DMSA and MiADMSA. MiADMSA was being more effective than DMSA in mobilizing arsenic. Fig. 1a represents the liver of control animals. The hepatic cord is well organized and the sinusoids are well separated. The cells show round nuclei with abundant cytoplasm. At higher magnification cells show well dispersed chromatin’s materials with prominent nucleolus. Fig. 1b shows GaAs exposed liver cells. It indicates disturbed hepatic architecture at places. Though the outer epithelial boundary appears to be intact the cytoplasm has become granulated. At places nucleus have picknotic appearance. The nuclear diameter shows reduction as compared to the control. Fig. 1b also shows cyto-

plasmic vacuoles and margination of nuclear chromatids as a result of GaAs exposure. Fig. 1c shows liver cell of DMSA treated animals. Though hepatic architecture is preserved a large number of inflammatory and the endothelial cells are present. The sinusoid space is dilated to an extent. Since these endothelial cells perform a broad range of metabolic and secretory task, their presence indicated the hepatic response to counter the injurious insult to GaAs. At higher magnification a range of nuclear deformation noticed. Fig. 1d represents a general view of hepatic parenchyma treated with MiADMSA. Though, the inflammatory cells are present their number has reduced compared to DMSA exposed animals. The most striking changes that occurs after MiADMSA treatment is the rupture of nuclear membrane resulting in the fragmented chromatin’s. This inturn lead to rupture of nucleus. MiADMSA because of its lipophilic character might thus be crossing the membrane wall.

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4. Discussion The present study was carried out to compare the effectiveness of DMSA and MiADMSA on the chronic hepatotoxicity of GaAs in rats. Exposure to GaAs increased the lipid peroxidation, level of GSSG, glutathione peroxidase (GPx), catalase with a concomitant increase in arsenic concentration in liver and blood indicating GaAs induced toxic manifestation on liver and a possible role of oxidative stress in mediating these changes. Webb et al. (1984) and Flora (1996) have reported impaired liver function due to arsenic dissociated from GaAs as an increased urinary excretion of uroporphyrin and changes in serum and liver transaminase. We recently suggested

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possible production of reactive oxygen species (ROS) and oxidative stress as the possible mechanism of GaAs induced alterations in blood, liver and renal tissue (Flora et al., 2002). Inorganic arsenic compounds are known to have hepatotoxic effects (Fowler, 1977). The hepatotoxic effects of GaAs would also be explained by the fact that there was a simultaneous glutathione reduction. GaAs induced MDA production could be due to the impairment of cells’ natural protective system by As and could be directly related to GSH depletion. Glutathione is well known for its pivotal role on the intrinsic anti-oxidant defence system of mammalian cells. The oxidized form of GSH was also marginally increased following GaAs exposure while; GSH/GSSG ratio de-

Fig. 1. (a) Liver of control rat showing well organized hepatic architecture with normal cellular structure. (b) Liver of GaAs exposed animals. Disrupted hepatic cord with granulated cytoplasm and picknotic nuclei seen. Cytoplasmic vacuoles are also present. (c) Liver of DMSA treated rats. Overall hepatic architecture is maintained. The presence of large number of inflammatory cells is indicative of recovery process. (d) MiADMSA treated rat shows a prominent histopathological changes resulting in membrane rupture and fragmented chromatin.

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creased significantly from 14 (in normal animals) to seven in GaAs exposed rats. It is now well known that ratio of GSH – GSSG is considered to be a sensitive indicator of oxidative stress and from the present study it is evident that arsenic moiety in GaAs may be causing oxidative stress possibly due to the disruption of intracellular prooxidant/antioxidant ratio. Glutathione peroxidase activity also increased profoundly in the liver. Perhaps to cope up with the GaAs induced oxidative stress the activity of this enzyme is activated. Catalase (CAT) activity increased due to GaAs indicating enhanced CAT-mediated metabolism. Chelating agents have been the mainstays of arsenic poisoning. Ercal et al. (1996) pointed out that there is validity in the notion that these chelating agents may participate in enhancing oxidative damage to organs. Chelating properties of DMSA is well known while MiADMSA is one of the most effective of the vicinal class of metal mobilizing agent (Jones et al., 1992; Xu et al., 1995). Although, this compound is more toxic than the parent diacid DMSA (Jones et al., 1992), its structural feature suggests that it might well be effective in binding arsenic. The present results indicate that MiADMSA shows more pronounced beneficial effects compared to those exhibited by succimer (DMSA) under similar circumstances. Both DMSA and MiADMSA are capable of mobilizing arsenic (but not gallium) and preventing the characteristic damage to liver and haematopoietic system caused by GaAs (Flora, 1996). Few earlier reports clearly indicate access of MiADMSA to ion, which have attained intracellular sites. Thus it can be concluded that MiADMSA because of its high lipophilic character might be facilitating extraction of arsenic from the cell component by crossing the membrane wall. Apoptosis is known to induce changes in the cellular structure. Enhanced apoptosis during regression is suggestive of readjustment of the tissue to normal size. The histopathological data recorded in the present study on nuclear diameter suggests that the observed hepatic histopathological lesions are due to GaAs, which showed marked recovery following chelation with MiADMSA. The nuclear diameter that was reduced

from 7.109 0.41 mm in normal control to 6.319 0.38 mm in GaAs exposed rats. It is interesting to note that the nuclear diameter reduced (5.609 0.50 mm) significantly after treatment with DMSA compared to both normal controls and GaAs exposed control, while, in MiADMSA treated rats it was almost the same (6.689 0.29 mm) as in normal animals. In conclusion, the results reported in the present study indicated that DMSA and MiADMSA significantly protects increase in oxidative stress and the increase in the concentration of arsenic in liver caused by GaAs exposure. MiADMSA is comparatively more effective than DMSA in the GaAs induced pathological liver injury.

Acknowledgements Authors thank K. Sekhar, Director of the establishment for his support and encouragement.

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