Some Enzymatic/Nonenzymatic Antioxidants asPotential Stress Biomarkers of Trichloroethylene,Heavy Metal Mixture, and Ethyl Alcohol inRat Tissues

Shams Tabrez, Masood Ahmad

Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh 202002,Uttar Pradesh, India

Received 11 January 2009; revised 17 September 2009; accepted 30 September 2009

ABSTRACT: Enzymatic and nonenzymatic antioxidants serve as an important biological defense againstenvironmental pollutants. Various enzymatic and nonenzymatic antioxidants as a stress biomarker in liverand kidney of rat were investigated. The antioxidant enzymes that were analyzed included superoxidedismutase (SOD), catalase, glutathione reductase (GR), glutathione-S-transferase (GST), and glutathioneperoxidase. Levels of lipid peroxidation (LPO), reduced glutathione (GSH), as well as hydrogen peroxide(H2O2) were also measured in homogenates of the liver and kidney of the treated animals to determine oxi-dative stress induced by trichloroethylene (TCE), ethyl alcohol, and heavy metal mixture (H.M.M) individu-ally and in different combinations. An increase up to the extent of 382% in malonaldehyde, a marker ofLPO, was recorded in almost all the treatment groups in both the tissues. Similarly, a rise of 218% in GSTactivity was also recorded in kidney of TCE-treated animals. Although H.M.M ingestion resulted in signifi-cant change of 125% in SOD activity of hepatic tissue, the level of GR was increased by 93% in the renaltissue of the exposed rats. Solitary dose of alcohol in general did not show a significant change. Moreover,the changes in the levels of antioxidants were much more prominent when these toxicants were given incombination rather than alone. Overall, these results demonstrate the changes in the levels of antioxidantenzymes and GSH system, as well as alterations in the LPO and H2O2 levels as a result of test toxicants.# 2009 Wiley Periodicals, Inc. Environ Toxicol 26: 207–216, 2011.

Keywords: trichloroethylene; heavy metal; mixture; antioxidant enzymes; biomarkers; rats

INTRODUCTION

Increasing rate of urbanization coupled with industrializa-

tion has resulted in greater amount of wastewater discharge

as well as higher level of exposure to xenobiotics. Human

and animal exposure to environmental chemicals is rarely

limited to a single compound. Rather, they are exposed

concurrently or sequentially to multiple chemicals from a

variety of sources (Jadhav et al., 2007a). It is a well known

fact that toxic effects of a xenobiotic can be modified by

the presence of other substances (Brus et al., 1999; Gupta

and Gill, 2000). As simultaneous exposure to two or more

xenobiotics can take place in the environment and/or under

occupational conditions, the investigation of interactions

between toxic substances is an important problem in mod-

ern toxicology (Jurczuk et al., 2004).

Trichloroethylene (TCE) is a common metal degreasing

agent and has been used indiscriminately in the house-

based lock manufacturing and electroplating units. Like

many other chlorinated hydrocarbons, TCE has become an

Correspondence to: M. Ahmad; e-mail: smasood_ahmad@lycos.com

or masood_amua@yahoo.co.in

Contract grant sponsor: UGC, New Delhi (DRS-II programme).

Published online 12 November 2009 in Wiley Online Library

(wileyonlinelibrary.com). DOI 10.1002/tox.20548

�C 2009 Wiley Periodicals, Inc.

207

important environmental pollutant because of its toxic

properties and widespread occurrence as a soil, air, and

water contaminant (Candura and Faustman, 1991). The

United States Environmental Protection Agency (USEPA)

has set a maximum contaminated level of 5 lg/L of TCE in

drinking water (ACGIH, 1999).

Heavy metals are also important environmental pollu-

tants and their toxicity is a problem of increasing signifi-

cance for ecological, evolutionary, and environmental

reasons (Nagajyoti et al., 2008). Heavy metals cannot be

destroyed through biological degradation, and the ability

to accumulate in the environment make these toxicants

deleterious to the environment and consequently to

humans. Because of excessive ethanol (EtOH) consump-

tion by some people who may be exposed to different

xenobiotics environmentally and occupationally (Meyer

et al., 2000), interactions between different xenobiotics

and EtOH are important for epidemiological and medical

perspectives.

According to estimates of the Aligarh office of Uttar

Pradesh Pollution Control Board, there were at least 125

units using TCE in Aligarh (Dua, 2003). Surveys con-

ducted by Dua (2003) and Chiya Nivaran Samithi

(2009) have reported various ailments probably associ-

ated with TCE exposure in Aligarh city. In view of the

strategic position of Aligarh city that houses several

small electroplating industries, one should not expect

that its population is exposed only to TCE; exposure of

some people to heavy metals along with TCE also can

not be ruled out and some of them would certainly be

alcoholics. Thus, there was a need to study the effect of

these toxicants to find out some sensitive biomarkers in

the animal system. In our previous study (Fatima and

Ahmad, 2005), we reported a very high concentration of

heavy metals in wastewater of Aligarh city. Four heavy

metals namely cadmium, copper, lead, and iron were

selected for this study because of their relatively high

concentrations in Aligarh wastewater.

Present study was undertaken to assess the oxidative

stress and antioxidants status in liver and kidney after the

oral intake of rats with TCE, H.M.M, EtOH independently

or in combination in search of useful biomarkers. As our

knowledge goes, there is no available report on the effect

of TCE in the presence of heavy metals especially in

alcoholics.

MATERIALS AND METHODS

TCE, ethyl alcohol (EtOH), and heavy metal salts

(cadmium chloride, copper sulfate, lead nitrate, and

ferric chloride) were procured from Qualigens Fine

Chemicals, Mumbai, India. Substrates and reagents for

the enzyme analysis were obtained from Sisco

Research Laboratories, Mumbai, India.

Experimental Animals

Swiss albino rats weighing 100–150 g were housed under

standard laboratory conditions of temperature (25 6 58C)and natural 12-h light/dark cycle with free access of stand-

ard pellet diet (Aashirwad Industries, Chandigarh, India)

and water ad libitum. All animals were kept under condi-

tions that prevented them from experiencing unnecessary

pain and discomfort according to guidelines approved by

the institutional ethical committee.

Experimental Protocol

After 1 week of acclimatization, animals were randomly

selected into eight groups of six animals each. Dosing solu-

tions were prepared daily to minimize possible instability

of the chemicals in the mixture. The toxicants taken in

single or as mixture as the case may be were administered

via oral route for 15 consecutive days. Corn oil served as

control for TCE, whereas distilled water was the control for

EtOH and heavy metal mixture (H.M.M).

The treatment schedule was as follows:

Group I: Distilled Water only (10 mL/ kg/day)

Group II: Corn Oil only (10 mL/ kg/day)

Group III: TCE only (1000 mg/kg/day)

Group IV: Ethyl Alcohol only (1000 mg/kg/day)

Group V: Heavy Metal Mixture only (Cadmium, Copper,

Lead, and Iron 2.5 mg/kg/day)

Group VI: TCE and Ethyl Alcohol simultaneously

(1000 mg/kg/day each)

Group VII: TCE and Heavy Metal Mixture concurrently

(1000 mg/kg/day and Cadmium, Copper, Lead, and

Iron 2.5 mg/kg/day)

Group VIII: TCE, Heavy Metal Mixture, and Ethyl

Alcohol simultaneously (1000 mg/kg/day each for

TCE and EtOH and Cadmium, Copper, Lead, and Iron

2.5 mg/kg/day)

This treatment schedule was followed daily during the

experimental period of 15 days, at the end of which animals

were sacrificed under light ether anesthesia for biochemical

studies.

Preparation of Tissue Homogenates

The 10% (w/v) liver and kidney homogenates were pre-

pared in ice cold 10 mM Tris-HCl buffer, pH 7.5. The

homogenate was then centrifuged under cold at 9000 3 gfor 20 min and supernatant was stored at 2208C until

further analysis. On the day of experiment, the homoge-

nates (liver and kidney) were again centrifuged at

3000 3 g for 15 min at 48C and the supernatant was

used for assaying the test enzymes.

208 TABREZ AND AHMAD

Environmental Toxicology DOI 10.1002/tox

Enzymatic and Nonenzymatic AntioxidantInvestigations

Separate liver and kidney homogenates of experimental rats

were used for the estimation of following antioxidant

enzymes:

a. Superoxide dismutase (SOD): SOD activity in the tis-

sue samples was assayed by measuring its ability to in-

hibit the autooxidation of pyrogallol (Marklund and

Marklund, 1974).

b. Catalase (CAT): CAT activity was measured as the

decrease in H2O2 concentration by recording the absorb-

ance at 240 nm (Aebi, 1984).

c. Glutathione-S-transferase (GST): CDNB (1-chloro-2, 4-

dinitrobenzene) was used as the substrate, and activity was

measured as the amount of CDNB conjugate formed by

recording the absorbance at 340 nm (Habig et al., 1974).

d. Glutathione reductase (GR): Oxidized glutathione (GSSG)

was used as the substrate. Activity was measured as the

decrease in NADPH concentration by recording the ab-

sorbance at 340 nm (Carlberg and Mannervik, 1975).

e. Glutathione peroxidase (GPx): GPx activity was assayed

according to the method described by Mohandas et al.

(1984). The assay mixture consisted of phosphate buffer,

EDTA, sodium azide, GR (3 units), GSH, NADPH, and

H2O2. Oxidation of NADPH was recorded at 340 nm.

f. Glutathione (GSH): GSH levels were estimated by the

method as mentioned by Jollow et al. (1974).

g. Lipid peroxidation (LPO): Tissue samples of experimental

rats were analyzed for the level of malonaldehyde (MDA)

according to the method of Beuge and Aust (1978).

h. Hydrogen peroxide: The amount of H2O2 in the samples

was estimated by the horseradish peroxidase method

(Pick and Keisari, 1980).

Statistical Evaluation

Data were expressed as Mean 6 S.D of six values and ana-

lyzed by one-way ANOVA. Differences among controls and

treatment groups were determined using Student’s t test.

P values of less than 0.05 were considered statistically signif-

icant. All comparisons were made with control animals.

RESULTS

Effect of TCE Administration in Rats onEnzymatic/Nonenzymatic Antioxidants Levelsin Liver and Kidney

Figures 1–8(a,b) depict the levels of antioxidant enzymes

and nonenzymatic parameters as a result of TCE intake.

SOD response to TCE in liver showed an increase of 86%,

whereas no significant change was observed in kidney.

Activity of GR showed around 75% increase in liver as well

as in kidney in TCE-treated animals. Increase in GST activ-

ity was a mere 50% in liver, but a significant rise of 218%

was observed in kidney. The increase in enzymatic activity

of GPx was quite high in liver (127%) compared with kidney

(50%). A noteworthy decrease of around 38% in CAT activ-

ity was also observed in both the tissues. Furthermore, GSH

level too showed a considerable decrease of around 40%

both in liver and kidney due to TCE treatment. Level of

MDA, an indicator of LPO, showed an enhancement, that is,

187% in liver than in kidney (50%) in response to TCE treat-

ment. H2O2 content showed a decrease of 35% in both the

tissues as a result of TCE treatment.

Effect of EtOH Administration in Ratson Enzymatic/Nonenzymatic AntioxidantsLevels in Liver and Kidney

In this treatment system, SOD activity displayed a mere

increase of 45% in liver and it remained unchanged in

kidney. Although CAT activity remained unaltered in liver,

a slight decrease was seen in kidney. Similar increase of

Fig. 1. Effect of test toxicants taken alone or in combina-tion on SOD activity in liver (a) and kidney (b). Animals in thetreatment group received above mentioned toxicants for 15consecutive days. ���: P\ 0.001 compared with control byANOVA. n 5 6 rats/ group.

209POTENTIAL STRESS BIOMARKERS OF TCE, H.M.M, AND EtOH

Environmental Toxicology DOI 10.1002/tox

around 24% was observed in GR activity in liver as well as

in kidney. GST activity showed a meager increase of 13%

in liver, but no significant change was observed in kidney

after the treatment of alcohol. GPx activity was higher both

in liver and kidney by 42 and 27%, respectively. A small

decrease of around 25% in the level of GSH was also

observed in both the tissues compared to control. The level

of MDA was higher in liver (158%), but kidney did not

show any significant change. A drop of roughly 30% in

H2O2 level upon alcohol treatment was also observed in

liver as well as in kidney.

Effect of H.M.M Administration in Ratson Enzymatic/Nonenzymatic AntioxidantsLevels in Liver and Kidney

SOD profile in this treatment system showed much

enhanced activities in response to H.M.M ingestion of rats

in liver (125%), but the increase was only 76% in kidney.

Contrary to 79% enhancement in CAT activity in liver, a

significant decrease of around 50% was observed in kidney

of H.M.M-treated animals. GR activity in liver and kidney

were increased by 69 and 93%, respectively. A substantial

rise (�83%) in GST activity was also observed in these tis-

sues. Activity of GPx showed a considerable fall in

H.M.M-treated group in liver and an increase by 90% in

kidney. GSH level showed a considerable decline of around

55% in both the tissues as a result of H.M.M treatment.

MDA level rose up in liver and kidney by 229 and 100%,

respectively compared with control animals. A significant

drop of �55% in the H2O2 level was noticed in both the

tissues after H.M.M treatment.

Effect of TCE1 EtOH Administration in Ratson Enzymatic/Nonenzymatic AntioxidantsLevels in Liver and Kidney

Enzymatic activity of SOD showed a rise of 90% in liver,

but the change was insignificant in kidney of combined

Fig. 3. Effect of test toxicants taken alone or in combina-tion on GR activity in liver (a) and kidney (b). Animals in thetreatment group received above mentioned toxicants for15 consecutive days. �, ��, ���: P \ 0.05, P \ 0.01, P \0.001, respectively compared with control by ANOVA. n 5 6rats/ group.

Fig. 2. Effect of test toxicants taken alone or in combina-tion on CAT activity in liver (a) and kidney (b). Animals in thetreatment group received above mentioned toxicants for 15consecutive days. ��, ���: P\ 0.01, P\ 0.001, respectivelycompared with control by ANOVA. n 5 6 rats/ group.

210 TABREZ AND AHMAD

Environmental Toxicology DOI 10.1002/tox

treatment group of TCE and alcohol. CAT activity showed

an increase of 108% in liver, but in kidney it showed a

decline of 38%. Increase in the GR activity of liver was

106% and in kidney it was found to be 71%. GST showed

much higher increase of 171% in kidney contrary to liver

(131%). Elevation in GPx activity was found to be 100% in

liver and 72% in kidney. A decrease to the extent of around

50% in the level of GSH was observed in both the tissues.

Rise of 200% was observed in MDA level in the liver of

combined treated animals, but only 63% increase was found

in kidney compared to control animals. A considerable

decrease of 50% in H2O2 level was observed in liver [Fig.

8(a)] as well as in kidney [Fig. 8(b)] upon treatment.

Effect of TCE 1 H.M.M Administration in Ratson Enzymatic/Nonenzymatic AntioxidantsLevels in Liver and Kidney

Present system showed an increase in the SOD activity by

165% over and above the control value upon combined treat-

ment of TCE and H.M.M in liver, whereas an increase of

100% was observed in kidney. CAT activity was greatly

reduced in liver as well as in kidney. GR activity showed an

increase of 157% in liver and 117% in kidney compared to

control animals. An astonishing rise of 258% in GST activity

was observed in kidney as compared to 111% in liver upon

this combined treatment. GPx activity showed an increase of

92% in liver and 136% in kidney. Considerable decrease of

about 60% in the level of GSH was seen in this combination

groups compared to control. Level of MDA was increased

with a great deal, that is, 300% in liver but only 130% rise

was observed in kidney. Decrease of around 70% in H2O2

level was observed in liver as well as in kidney. The changes

in all the measured parameters were highly significant (P\0.001) in the entire treatment groups.

Effect of TCE1 EtOH1 H.M.M Administrationin Rats on Enzymatic/NonenzymaticAntioxidants Levels in Liver and Kidney

Activity of SOD was increased by 202% in liver compared

to 164% in kidney. A similar pattern of decrease in CAT

Fig. 5. Effect of test toxicants taken alone or in combinationon GPx activity in liver (a) and kidney (b). Animals in the treat-ment group received above mentioned toxicants for 15consecutive days. �, ��, ���: P \ 0.05, P \ 0.01, P \ 0.001,respectively compared with control by ANOVA. n5 6 rats/ group.

Fig. 4. Effect of test toxicants taken alone or in combina-tion on GST activity in liver (a) and kidney (b). Animals in thetreatment group received above mentioned toxicants for 15consecutive days. ���: P\ 0.001 compared with control byANOVA. n 5 6 rats/ group.

211POTENTIAL STRESS BIOMARKERS OF TCE, H.M.M, AND EtOH

Environmental Toxicology DOI 10.1002/tox

activity was recorded in liver and kidney tissues. GR activ-

ity showed a significant increase of around 220% in both

tissues. GST activity showed quite a high elevation in kid-

ney (1341%) rather than in liver (1163%). GPx showed

an increase of 135% in liver and 163% in kidney. However,

a significant drop of roughly 75% in the level of GSH was

observed in both the tissues. MDA levels were very high in

liver (1382%) as compared to kidney (1160%). A drop of

around 85% in H2O2 level was observed in both the tissues

in this combined treatment group. The change was highly

significant in the whole treatment groups in each and every

parameter.

DISCUSSION

To the best of our knowledge, the data presented in this

study demonstrate oxidative damage and alteration in anti-

oxidant enzyme activities for the first time in rat after

the treatment of TCE in the presence of heavy metals and

ethanol. Moreover, liver rather than kidney was found to be

more affected tissue of the rats orally administered by TCE,

EtOH, and/or H.M.M.

Increase in MDA level is frequently observed in various

mammalian tissues during oxidative stress and has gener-

ally been used as the marker of oxidative damage (Husain

et al., 2001; Jadhav et al., 2007a,b). LPO measured in terms

of MDA levels showed around threefold induction in liver,

whereas only a nominal increase in MDA was recorded in

the kidney of TCE-exposed animals [Fig. 7(b)]. These find-

ings are consistent with the previous reports (Toraason

et al., 1999; Watanabe and Fukui, 2000; Zhu et al., 2005).

Ethanol-exposed animals did not show any significant

change in MDA level in kidney. However, a small rise in

MDA level was recorded in liver [Fig. 7(a)]. Extensive

researches have shown an increased MDA equivalents

Fig. 6. Effect of test toxicants taken alone or in combina-tion on GSH level in liver (a) and kidney (b). Animals in thetreatment group received above mentioned toxicants for 15consecutive days. ��, ���: P\ 0.01, P\ 0.001, respectivelycompared with control by ANOVA. n 5 6 rats/ group.

Fig. 7. Effect of test toxicants taken alone or in combina-tion on MDA level in liver (a) and kidney (b). Animals in thetreatment group received above mentioned toxicants for 15consecutive days.�, ��, ���: P\ 0.05, P\ 0.01, P\ 0.001,respectively compared with control by ANOVA. n 5 6 rats/group.

212 TABREZ AND AHMAD

Environmental Toxicology DOI 10.1002/tox

resulting from ethanol ingestion (Jurczuk et al., 2004; Yao

et al., 2006; Lu and Cederbaum, 2008). Exposure of rats to

metals is characterized by accumulation, particularly in the

liver and kidney cortex (Novelli et al., 1997). We obtained

a rise in MDA level of more than threefold in liver and two-

fold in kidney as a result of H.M.M intake by rats [Fig.

7(a,b)]. It is quite evident from the available literatures, that

exposure to a single metal as well as to metal mixture

shows the similar pattern of MDA (Ozcelik et al., 2003;

Jurczuk et al., 2004; Jadhav et al., 2007a,b).

Primary biochemical components of the oxidative stress

response include elevation of LPO and suppression of GSH

and alteration of the activities of antioxidant enzymes. The

perturbation of antioxidant defense system is manifested by

marked increase in LPO and activities of SOD, GPx, GR,

and GST with concomitant decrease in CAT activity. The

increased tissue MDA levels indicate an increase pro-

oxidant status in the tissues that sets in when the generation

of free radicals increases or the capacity to scavenge the

free radicals and repair of oxidatively modified macromole-

cules decreases, or both (Holland et al., 1982; Sies, 1997).

The accumulation of ROS produces significant functional

alterations in lipids, protein, and DNA molecules. The oxi-

dative lipid damage (LPO) produces a gradual loss of

cell membrane fluidity and reduces the membrane potential

and increases the permeability of ions like Ca11 (Jacob

and Burri, 1996). The increased SOD activity is an adaptive

tissue response to detoxify the superoxide radicals giving

rise to H2O2; which is in turn handled by increased GPx ac-

tivity, a selenium containing enzyme that scavenge H2O2

and other peroxides in the present study. It appears that

treatment induced increase in LPO level is caused by the

increased SOD activity concomitant with reduced CAT,

which could be due to enzyme inactivation by the free radi-

cals generated due to oxidative stress. The increased GPx

activity to some extent can be attributed to the fall of H2O2.

As GPx functions in conjugation with reduced GSH and is

known to scavenge H2O2 and lipid peroxide (Bhattacharya

et al., 2003). Under severe oxidative stress, GSH levels are

suppressed due to the loss of compensatory response and

oxidative conversion of GSH to its oxidized form (Chen

and Lin, 1977). The depleted levels of GSH are compen-

sated by enhanced activities of GR that regenerates GSH

(Tabrez and Ahmad, 2009).

GSH is considered one of the most important antioxi-

dants involved in protection against ROS/free radicals

(Meister, 1989). GSH needs to be recycled back as it is the

key nonenzymatic antioxidant of the cell and also serves as

the substrate for the chief H2O2 scavenging enzyme,

namely GPx. Decrease in GSH level as a result of TCE,

EtOH, or heavy metal exposure has been reported by many

investigators (Jadhav et al., 2007a,b; Lu and Cederbaum,

2008). Our findings are consistent with these workers, since

we also recorded a significant decline in GSH level in both

the tissues compared to untreated animals [Fig. 6(a,b)]. Our

group has also reported a dose-dependent depletion in GSH

levels in Allium cepa exposed to heavy metal mixture

(Fatima and Ahmad, 2005). However, Goel et al. (1992)

had reported an increase in GSH level as a result of TCE

intake in mice.

A major cellular defense against ROS is provided by

SOD and CAT, which together convert superoxide radicals

first to H2O2 and then to water and molecular oxygen. Other

enzymes, such as GPx, use the thiol reducing power of

GSH for the reduction of oxidized lipids and protein targets

of ROS. Superoxide is considered as the central component

of the signal transduction that triggers the genes responsible

for antioxidant enzymes (Alavarez and Lamb, 1997). Since

SOD is induced by its own substrate, its activation in the

test tissues may be an indication that the animals are

Fig. 8. Effect of test toxicants taken alone or in combina-tion on H2O2 level in liver (a) and kidney (b). Animals in thetreatment group received above mentioned toxicants for 15consecutive days. �, ��, ���: P\ 0.05, P\ 0.01, P\ 0.001,respectively compared with control by ANOVA. n 5 6 rats/group.

213POTENTIAL STRESS BIOMARKERS OF TCE, H.M.M, AND EtOH

Environmental Toxicology DOI 10.1002/tox

experiencing the pollutant induced superoxide radical stress

(Allen and Tresini, 2000). In the present investigation, the

level of SOD increased around twofold in liver, but there

was no change in the kidney upon TCE treatment [Fig.

1(a,b)]. Such an increase in the level of SOD by TCE expo-

sure may be due to the formation of ROS like the superoxide

radical. Watanabe and Fukui (2000) also reported an

increase in SOD activity in mouse liver after TCE treatment.

Husain et al. (2001) also found an enhanced level of hepatic

SOD as a result of ethanol treatment that is in concurrence

with our result. However, Devipriya et al. (2007) reported a

decline in SOD after ethanol treatment. Moreover, a signifi-

cant increase in the activity of SOD in liver and kidney as a

result of heavy metal intake is in good agreement with the

results reported by other investigators (Ozcelik et al., 2003;

Jadhav et al., 2007a). Jadhav et al. (2007a) reported an

increase in SOD activity after 30 days subchronic exposure

to a mixture of eight metals in rats erythrocytes.

CAT and the peroxidases are the major enzymes

involved in H2O2 detoxification. Similar reduction in CAT

activity was found in both the tissues of TCE-treated ani-

mals [Fig. 2(a,b)]. This result confirms the earlier findings

of Elcombe et al. (1985), who reported such a decline as a

result of TCE treatment in rats. However, an increase in

CAT activity as a result of TCE intake in both liver and

kidney of mice was reported by several workers (Elcombe

et al., 1985; Goel et al., 1992; Watanabe and Fukui, 2000).

The difference in species may be the reason behind the

opposing results (Elcombe et al., 1985). Ethanol intake, on

the other hand, brought about a small decrease in CAT

activity in renal tissue concomitant with an insignificant

change in liver [Fig. 2(a,b)]. However, Ashakumary

and Vijayammal (1996) found a decrease in CAT activity

in liver as well as in kidney of rats after ethanol

administration.

The role of GST in detoxification is well documented

(Rees, 1993; Fernandes et al., 2002; Ferrat et al., 2003).

The fact that GST is ubiquitous also makes it a more suita-

ble stress biomarker (Stenersen et al., 1987). Our results are

in support of the previous report of Stajn et al. (1997).

Dose-dependent increase in GST levels as a result of heavy

metal mixture exposure has also been reported by our group

in Allium cepa system (Fatima and Ahmad, 2005). How-

ever, El-Demerdash et al. (2004) found a decrease in GST

activity after cadmium chloride intake in liver and plasma

of rat.

TCE as well as heavy metal-exposed animals showed

almost similar rise in GR level in both the tissues. Not so

significant change in GR activity was observed in both the

tissues of ethanol-exposed animals. All these findings are

consistent with earlier reports (Ashakumary and Vijayam-

mal, 1996; Watanabe and Fukui, 2000, Jadhav et al.,

2007a). Different variety of toxicants in combination

brought about a prominent change in GR activity

[Fig. 3(a,b)].

Increase in GPx activity was recorded in almost all treat-

ment groups of both the tissues. However, heavy metal

mixture alone and TCE and H.M.M-treated group showed a

decrease in GPx activity in liver. These results are in good

agreement with the previous report of Boccio et al. (1990).

It is known that GPx has higher affinity for H2O2 than CAT

and thus it is effective in decomposing H2O2 (Halliwell,

1974; Wassmann et al., 2004). However, Watanabe and

Fukui (2000) and Yao et al. (2006) found a decrease in GPx

activity as a result of TCE and alcohol treatment, respec-

tively. A highly significant fall in H2O2 level was recorded

by us in every treatment group.

CONCLUSION

From our result it is quite clear that the level of MDA, the

end product of LPO, can be used as the best biomarker for

these test toxicants in both the tissues of rats. Moreover, GST

can act as a biomarker for TCE in kidney of treated animals.

However, SOD and GR can acts as a potential biomarker for

H.M.M intoxication in hepatic and renal tissue, respectively.

Overall, the present results demonstrate appreciable changes

in the levels of antioxidant enzymes and GSH system as well

as alterations in the LPO and H2O2 levels as a result of TCE

or heavy metal mixture intake in rat tissues. Solitary dose of

alcohol in general did not show a significant change. This

may be due to the comparatively low dose of alcohol selected

in this study. The changes were much more prominent when

these toxicants were given in combination rather than alone.

The rise in the activities of antioxidant enzymes may be a

compensatory response to these test toxicants.

Shams Tabrez is a senior research fellow of ICMR, New Delhi.

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