Toxicology Mechanisms and Methods, 16:347–352, 2006Copyright c© Informa HealthcareISSN: 1537-6516 print / 1537-6524 onlineDOI: 10.1080/15376520600616800

Methyl Parathion-Induced Changes in Freeand Protein-Bound SH Levels in Rat Tissues

Deniz Yildiz, Semih Dalkilic, Hasan Yildiz, and Haydar OztasMustafa Kemal University, Biology Department, Antakya/Turkey

The main objective of this study was to investigate the changes infree and protein-bound SH contents in methyl parathion-exposedrat tissues. The free and protein-bound SH levels are usuallyaffected and depleted by oxidative stress-inducing agents. Resultswould indicate if methyl parathion toxicity partly results fromdepletion of sulfhydryl content of tissues. Six-week-old maleWistar albino rats were used in this study. Following exposureto methyl parathion for 3 months, the liver, the brain, andthe kidney tissues were removed from the rats. The free andprotein-bound SH contents were determined in these tissues. Inaddition, plasma lactate dehydrogenase levels were determined.Our results showed that methyl parathion exposure significantlylowers the free and protein-bound SH levels in rat tissues. However,lactate dehydrogenase activity in the blood plasma did not displayany differences compared to the control group. The free SHconcentrations in the control rat liver, brain, and kidney tissueswere 3.78 ± 0.1 µmol/100 mg tissue, 1.56 ± 0.08 µmol/100 mg tissue,and 2.16 ± 0.08 µmol/100 mg tissue, respectively, whereas thefree SH concentrations in rats exposed to methyl parathion weredetermined as 0.536 ± 0.1 µmol/100 mg tissue in the liver, 1.06 ±0.1 µmol/100 mg tissue in the brain, and 0.108 ± 0.03 µmol/100 mgtissue in the kidney. The protein-bound SH concentrations inthe liver and in the kidney in rats exposed to methyl parathiondisplayed a significant decrease also. However, the protein-boundSH level in the brain did not change significantly. These resultsindicate that methyl parathion exposure partially depletes the freeand protein-bound SH levels. Thus, it was concluded that methylparathion toxicity may partly result from oxidative stress.

Keywords Free and Protein-Bound SH, Methyl Parathion, OxidativeStress, Rat Tissues

INTRODUCTIONMethyl parathion, (0,0-dimethyl 0-(p-nitrophenyl) phospho-

rothioate) is an organophosphorus compound that is widely usedas an insecticide in agriculture and hygenic control of insects. Ithas been known that methyl parathion displays a high toxicity

Received 14 August 2005; accepted 14 September 2005.Address correspondence to Deniz Yildiz, PhD, Mustafa Kemal

University, Biology Department, Antakya/Turkey. E-mail: dyildiz@mku.edu.tr

toward humans and several other mammals. It has also beenknown that several people are poisoned by methyl parathioneach year. It is reported that 1.274 persons are poisoned frompesticides including methyl parathion in 1996 (Leveridge 1998).Methyl parathion exposure may occur through several routes in-cluding inhalation, dermal contact, and the mouth (Atsdr 1990).Methyl parathion and other organophosphorous insecticidesand their metabolites partly display their toxic affects throughinhibition of acetylcholinesterase enzyme activity at the motorendings of the nerve cells. Methyl paraoxon, an active metaboliteof methyl parathion, phosphorylates the active site of the enzymeand inhibits its activity (Wills 1972). Once the enzyme is inhib-ited, acetylcholine accumulates and overstimulates the receptors(Sultatos 1994; Taylor 1996). Most of the clinical symptomsdisplayed by methyl parathion exposure have been described.These symptoms may include vomiting, increased salivation,and diarrhea. In addition, some behavioral and immune systemdepression symptoms are also reported (WHO 1986).

The methyl parathion metabolism has been extensivelyinvestigated. It has been shown that cytochrome P450, esterases,and glutathione play important roles in methyl parathionmetabolism (Chambers et al. 1991; Sultatos 1987; Nielsen1991). The involvement of cytochrome P450 enzymes andglutathione in methyl parathion metabolism may suggest a rolefor oxidative stress in methyl parathion toxicity, because it isknown that agents that can overactivate microsomal cytochromeP450 enzymes and agents that deplete glutathione reservoir mayinduce oxidative stress. Glutathione is a soluble antioxidantcomposed of glutamic acid, cysteine, and glycine. It is atripeptide with a relatively low molecular weight. It plays animportant role in the maintenance of a proper redox statusin cells and in tissues (Freya 2001). Glutathione also formsconjugates with many different xenobiotics and thus contributesto the removal of foreign substances from the cell or organism.It has been shown that glutathione also forms conjugates withmethyl parathion. This conjugation is carried out by cytoplasmicglutathione S-transferases (Bammler et al. 2001). GlutathioneS-transferase involves in dealkylation of methyl parathionforming S-methylglutathione (Fukami 1980). Depletion ofglutathione from the cells by these ways may increase the

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348 D. YILDIZ ET AL.

amount of free radicals produced inside the cells. Althoughit has long been known that glutathione is extensively involvedin methyl parathion metabolism, glutathione depletion and theconsequences of glutathione depletion by methyl parathion havenot been investigated in detail. Overutilization of glutathionein methyl parathion metabolism may significantly decreasethe intracellular glutathione content and induce free radicaldamage. Thus, the possibility that some of the toxic effectsof methyl parathion may occur through induction of oxidativestress should not be ruled out. Studies demonstrating that methylparathion exposure results in increased lipid peroxidation andoxidative DNA damage also support this view (Hasan and Khan1985; Lodovici et al. 1994). A mixture of pesticides includingmethyl parathion has been shown to induce 8-OH-dG formation(Lodovici et al. 1994). However, this type of direct studyis very limited. Previous studies also accumulated evidencesupporting the view that methyl parathion toxicity may involvefree radical generation and oxidative stress. These studiesinclude retinal degeneration and cataract formation (Suba1984), induction of inflammation in stomach and in intestine(Fazekas 1971), necrotic liver cell death and elevated serumalanine amino transferase activity (Sonnenschein et al. 1989),and chromosome aberrations in lymphocytes from humansexposed to methyl parathion (Van Bao et al. 1974). Studiesare also present indicating that methyl parathion exposureproduces cardiovascular effects characterized by heart andblood vessel lesions, and also significant lesions in the liver,kidney, and muscle were observed (Fazekas 1971). All of thesedescribed parameters could be accepted as an indicator for thepossibility that methyl parathion toxicity may involve oxidativestress.

To support the above-mentioned hypothesis, we investigatedwhether methyl parathion treatment results in depletion offree and protein-bound SH content of tissues, thus renderingthe tissues more vulnerable to free radical attacks. Free SHdepletion and consequent decrease in the amount of protein-bound SH levels could be considered as an indicator for thepresence of oxidative stress. We also compared the responsesof different tissues to methyl parathion treatment in this respectand determined lactate deghydrogenase activities in the bloodplasma as a measure of cell membrane damage.

MATERIALS METHODS

MaterialsPyruvic acid (sodium salt), β-nicotinamide adenine din-

ucleotide, reduced form (β-NADH), and Trisma base wereobtained from Sigma Chemical Co., USA. 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB) was obtained from Fluka Chemie,Switzerland. Trichloroacetic acid (TCA) was obtained fromMerck Company, Germany. Parathion methyl (0,0-dimethyl0-(p-nitrophenyl) phosphorothioate) was obtained from ClausHuth GmbH, Germany.

MethodsIn the present study we used 6-week-old male Wistar albino

rats. The control and the methyl parathion group includedfive rats each. The control rats were fed with standard diet.The diet and the water were supplied ad libitum. The methylparathion group was fed with standard diet containing methylparathion (rats received 4 mg methyl parathion/kg/day) andthe diet and the water were supplied ad libitum. Rats werethen kept at 12 hours light and 12 hours dark cycle. Ratswere continuously fed as described for a period of 3 months.Most of the in vivo studies with rats are carried out by usingmethyl parathion at concentrations between 2 to 4 mg/kg/dayand intermediate exposure time (15 to 365 days). Thus, wechose the concentration of methyl parathion we used and theexposure time to provide a correlation with previous studies.At the end of the 3-month period, rats were anesthetized byusing carbon dioxide. The blood was taken from the hearth byusing heparinized injectors with wide needles. Blood sampleswere immediately transferred to ice water. Samples werethen centrifuged at 2000 g for 5 min and the resulting clearsupernatant was transferred to clean tubes and further storedat −70◦C until the enzyme activity measurements. In the samemanner, the liver, the kidney, and the brain tissues were removedand stored at −70◦C until the measurements.

Measurement of Free SH Concentrations in TissuesMeasurements of tissue free and protein-bound SH levels

were carried out as described by Sedlak (1968). The tissues wereremoved and thawed at 37◦C. One hundred milligrams of tissueswere added to 2 mL of ice-cold Tris-EDTA (262 mM Tris base,13 mM EDTA, pH 7.4) and homogenized on ice at 10-secondintervals. Then, 200 µL of homogenate was transferred to atube containing 20% TCA prepared in sodium phosphate-EDTAbuffer (0.01 M sodium phosphate/0.005 M EDTA). The mixturewas then vortexed for 1 min and the resulting mixture wascentrifuged at 12,000 g for 5 min. The resulting supernatant wastransferred to a clean tube and the pH was adjusted to 8.0 byaddition of Tris-EDTA. Then DTNB dissolved in methanolwas added to obtain a concentration of 0.6 µM/mL DTNBand the mixture was allowed for 5 min to develop color. Theabsorbance of the samples were then measured at 412 nm andthe concentrations of free-SH were calculated by using the mMextinction coefficient of 13.6.

Measurement of Total SH Concentrations in TissuesThe total SH concentrations were measured from the same

homogenates. Briefly, homogenates were taken to clean tubes,and to these tubes we added tris-EDTA, DTNB at the sameconcentration as above described, and methanol. The sampleswere then centrifuged at 4000 g for 5 min. The absorbance of thesamples was then measured at 412 nm and the concentrationsof total SH were calculated by using the mM extinctioncoefficient of 13.6. The protein-bound SH concentrations were

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METHYL PARATHION-INDUCED CHANGES IN –SH GROUPS 349

later calculated by subtracting the free SH concentration fromthe total-SH concentration.

Protein bound − SH = Total SH − Free SH

Lactate Dehydrogenase Activity MeasurementsBlood plasma samples were removed from −70◦C and

thawed at 37◦C. Lactate dehydrogenase activity measurementswere carried out as described by Welder (1994). Briefly, 100µL of the plasma samples were transferred to clean tubes and200 µL of NADH solution (2.5 mg/mL of NADH was dissolvedin 10 mL of 0.1 M phosphate buffer), 2500 µL of warm 0.1 Mphosphate buffer, and 200 µL of sodium salt of pyruvic acid(1 mg/mL of sodium pyruvate was prepared in 25 mL of 0.1M phosphate buffer) were added. The content was mixed andtransferred to cuvettes and absorbance changes were taken at1-min intervals for 4 min at a wavelength of 340 nm.

Statistical AnalysisOne-way analysis of variance (ANOVA) and Student-

Newman-Keuls multiple comparison tests were applied toprocess the data statistically. Results were expressed as mean ±S.D. p < 0.05 values were considered to be significant.

RESULTSFigure 1 displays the effect of methyl parathion on free

and protein-bound SH levels. As seen, methyl parathionexposure resulted in a complete depletion of the free SH

FIG. 1. Free and protein bound SH levels were determined in control andparathion methyl-exposed rat kidneys. Results are the mean and S.D. of fivedifferent measurement (n = 5). p < 0.05. ∗Significantly different from thecontrol group.

FIG. 2. Free and protein-bound SH levels were determined in control andparathion methyl-exposed rat livers. Results are the mean and S.D. of fivedifferent measurement (n = 5). p < 0.05. ∗Significantly different from thecontrol group.

content of kidneys. In a similar manner, protein-bound SHlevel was also significantly decreased. Protein-bound SH levelwas decreased 52% in methyl parathion-exposed Rat kidneyscompared to control kidneys. Figure 2 displays the changes infree and protein-bound SH levels in the liver of rats followingtreatment with methyl parathion. Similarly, methyl parathiondecreased both free and protein-bound SH levels in the liveralso. Protein-bound SH level was decreased 25% in methylparathion-exposed rat livers compared to control livers. Figure 3shows the effect of methyl parathion treatment on SH levelsin the brain. As seen in the figure, free SH levels weresignificantly decreased in methyl parathion-exposed rat braintissue. However, protein-bound SH levels remained statisticallythe same in the control and in the methyl parathion-exposedrat brain tissue. Figure 4 shows the results of the lactatedehydrogenase activity measurements. There were no statisticaldifferences between lactate dehydrogenase activities in thecontrol and methyl parathion-treated rat blood plasma.

DISCUSSIONThe main objective of the present study was to investigate

the changes in free and protein-bound SH levels in rats exposedto methyl parathion. Previous studies have already accumulatedsignificant evidence to suggest that methyl parathion toxicitymay partly result from increased free radical generation andconsequent induction of oxidative stress. Depletion of freeSH content and the consequent decrease in protein-bound SH

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FIG. 3. Free and protein-bound SH levels were determined in control andparathion methyl-exposed rat brains. Results are the mean and S.D. of fivedifferent measurement (n = 5). p < 0.05. ∗Significantly different from thecontrol group.

FIG. 4. LDH activities were determined in control and parathion methyl-exposed rat blood plasma. Results are the mean and S.D of four differentmeasurement (n = 5).

levels are good indicators for the presence of oxidative stress.Thus, we measured the changes in these parameters in vivo inmethyl parathion-exposed rats to provide evidence for methylparathion-induced oxidative stress.

Our results demonstrated that exposure of rats to methylparathion results in a significant decrease in free and protein-bound SH levels. Although there was a decrease in either free SHlevel or protein SH level in each tissue investigated, the extents ofSH depletion in different tissues varied. Free and protein-boundSH depletion in response to methyl parathion treatment wasmore dramatic in the kidney tissue compared to the liver and thebrain tissues. The liver followed the kidney tissue results in thisrespect. Methyl parathion was least effective in the brain tissuein decreasing the SH levels. These results suggest that differenttissues respond to methyl parathion exposure in a differentmanner or cope differentially with methyl parathion. Based onthese different responses of different tissues to methyl parathionexposure, it could be suggested that the rate of activity ofcytochrome P450 systems in these tissues primarily determinesthe extent of SH depletion. Methyl parathion metabolismand detoxification requires the activity of cytochrome P450enzymes. A sustained increased activity of cytochrome P450enzymes has long been known to generate free radicals (Meister1981). Although the liver, kidney, and brain tissues all possessthe cytochrome P450 enzyme system, the rate of activity ofthis enzyme system may differ in these tissues. In this respect,it will be plausible to suggest that brain tissue SH levels aremore preserved compared to the kidney and the liver becausethe cytocrome P450 activity is less in this tissue, and thus lessfree radicals are produced. The other route of SH depletion in thebrain might be through conjugation by glutathione S-transferase.The intracellular free SH is mainly glutathione, and free SHdepletion represents the glutathione depletion also. When weconsider both route and see the nonefficient depletion of SHgroups, it could be suggested that brain is not significantlyinvolved in methyl parathion metabolism. However, still there isa significant decrease in free SH levels in the brain, which maybe sufficient to propose that methyl parathion disturbs the properredox status in this tissue and contributes to toxicity. The viewthat the rate of activity of cytochrome P450 determines the extentof SH seems to oppose the results reported for the kidney andthe liver. Because the decrease in SH levels in the kidneys wasmore pronounced compared to the liver, it is expected that thecytocrome P450 activity rate is higher in the liver and thus SHlevels should decrease more efficiently in the liver compared tothe kidney. This contradiction might be partly explained by thefact that the liver tissue generally has two to three times more ofa glutathione level compared to the kidney, which may alleviatethe SH depletion (Glosli et al. 2002). It could be suggestedthat liver cells are more efficiently equipped against oxidativestress-inducing conditions and can cope more easily with thiskind of xenobiotics compared to the kidney. In addition to ahigher glutathione level in the liver, SH-regenerating enzymes

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METHYL PARATHION-INDUCED CHANGES IN –SH GROUPS 351

and other enzymes with antioxidant function may be upregulatedin the liver to provide an advantage over the kidney. Supportingthis view is the fact that sustained generation of free radicals inmitochondria establishes a more powerful defense compared toother cellular compartments such as the endoplasmic reticulum,which does not have as much oxidant flow. Liver is the maindetoxification site in the organism and must be equipped withstrong defense systems also. It has been known that glutathionemay reach up to 10 mM, the highest concentrations attained ina tissue, in the parenchymal cells of healthy liver, and liver cellsare highly specialized in glutathione synthesis and regeneration(Deleve et al. 1990).

It is clear from our results that parathion methyl significantlydepletes both free and protein-bound SH levels in differenttissues. Depletion of free SH levels is a strong indication fortissue glutathione depletion also. Glutathione plays a role incell division, differentiation, and carcinogenesis (Alexander andBoyer 1971; Brachet 1962; Egyud and Szentgyorgyi 1966).Glutathione also plays a role in conjugation with severalxenobiotics that result in depletion of SH groups (Shivapurkarand Bhide 1978). Glutathione not only plays as a solubleantioxidant, but also contributes in recycling of importantcellular molecules with antioxidant function. Glutathione issuggested to regenerate oxidized forms of antioxidants such asvitamin C and E and carotenoids (Meister 1994). In addition, itis known that glutathione plays a significant role in preservationand maintenance of protein SH groups. Several enzymes andproteins with crucial intracellular functions have been shownto possess a critical SH group required for activity (Ondarza1989; Crane et al. 1988; Hidalgo et al. 1990). Some transportfunctions and proper receptor activities are also regulated by theredox status. Thus, depletion of free and protein-bound SH mayconsequently disturb the SH-dependent activities of the cellsand contribute to adverse toxicity induced by parathion methyl.The consequences of a sustained free and protein-bound SHdepletion may be deleterious for the cells and tissues. Cellsmay start to die because of insufficient defense against the freeradicals generated by free SH depletion, and then the death maypropagate in tissue by a chain reaction of radicals destroyingthe most essential functions of the tissue. In our study we didnot use the lethal doses of methyl parathion; however, resultssuggest that oxidative stress-induced tissue degeneration maycontribute significantly to methyl parathion-induced toxicityand death. Increase in lipid peroxidation by methyl parathionand induction of oxidative DNA damage by a mixture ofpesticides including parathion methyl support our results andsuggestions also (Hasan and Khan 1985; Lodovici et al. 1994).

In our study we also investigated the possibility thatmethyl parathion may induce deteriorating membrane damages.Previously, it has been shown that decreased membrane SHcontributes to several changes in the membrane, includingosmotic fragility and fluidity. Methyl parathion itself has alsobeen shown to cause a change in lipid composition and lipidperoxidation in rat brain. We measured the plasma lactate

dehydrogenase levels to evaluate whether methyl parathiondamaged the cellular membranes and caused an enzymeleakage. However, there were no significant changes in lactatedehydrogenase activities, indicating that the dose of methylparathion and maybe the exposure time was not sufficient for asignificant membrane damage in tissues.

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