Indian Journal of Experimental Biology Vol. 42. February 2004. pp. 220-223

Vitamin E prevents nonylphenol-induced oxidative stress in testis of rats

K C Chitra & P P Mathur*

School of Life Sciences. Pondicherry University. Pondicherry 605 014. India

Received 3 July 2003; revised 1 October 2003

In the present study we have investigated if administration of nonylphenol-induced oxidative stress in various subcellular fractions of adult rat testis and the effect of vitamin E on reactive oxygen species mediated nonylphenol toxicity . Male rats were ad~nistered orally with nonylphenol at 1. 10 and 100 ~glkg body weight per day for 45 days with and without supplementation of vitamin E (20 mglkg body weight). In nonylphenol-treated rats the activities of antioxidant enzymes superoxide dismutase and glutathione reductase decreased significal1tly while the levels of lipid peroxidation increased significantly in the crude homogenate and in the mitochondrial and microsome-rich fractions of testis. Co-administration of nonylphenol and vitamin E did not cause changes in the activities of antioxidant enzymes in various subcellular fractions of rat testis. The results suggest that graded doses of nonylphenol elicit depletion of antioxidant defence system in rat testis. indicating nonylphenol induced oxidative stress in the testis of rats which could be reversed by the administration of vitamin E.

Keywords: Antioxidant enzymes. Nonylphenol. Oxidative stress. Polyvinyl chloride. Rat. Testis. Vitamin E

Nonylphenol, a final degradation product of alkylphenol polyethoxylate surfactants is found in polyvinyl chloride (PVC) used in the food processing and packaging industry that contaminates the water flowing through PVC pipes. The persistent and lipophilic nature of nonylphenol leads to its accumulation in . animal tissues. Nonylphenol possesses estrogenic properties and has been shown to impair reproduction of male and female rats. Nonylphenol also showed a reduction in the activities of 17oc-hydroxylase/C17,20-lyase in testicular homogenates of neonatal, pubertal and adult rats 1

•

Nonylphenol has been shown to inhibit the activities of cytochrome P450-1A (CYPIA) in rat hepatic microsomal fractions2

• Inhibition of cytochrome P450 has been shown to provoke reactive oxygen species (ROS) generation that impairs both

*Correspondent author : Tel: +91-413-2655212 Fax : +91 -413-2655211 E-mail: ppmathur@hotmail.com

epididymal and testicular sperm fur..ctions. In subcellular fractions, mitochondria have been shown to be a major site of ROS generation3

• Subcellular membranes are equipped with antioxidant enzymes like superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase, and they dispose ROS generated by the cells thereby protecting cells or tissues from oxidative stress. Normally a balance is maintained between oxidative stress of free radicals and antioxidative defence system. Nonylphenol has been shown to generate ROS in epididymal sperm of rats by decreasing the activities of antioxidant enzymes and increasing the levels of lipid peroxidation4

• Previous studies from our laboratory have shown that environmental contaminants such as lindane, methoxychlor, bisphenol A and dioxin induce ROS generation in testis5

•6, epididymis and epididymal sperm of rats7

•1O

•

Several reports have suggested that vitamin E possesses antioxidant properties to protect against oxidative damage caused by free radicals. Vitamin E, a lipophilic antioxidant; eliminates free oxygen radicals in the reproductive tissues of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity in male rats ll

. However, the protective role of vitamin E on nonylphenol-induced toxicity in the testis of rats is not elucidated. The present study was undertaken to evaluate the effect of nonylphenol and vitamin E on the testicular antioxidant system of rats.

Male albino rats of Wistar strain (45 days) were purchased from the Central Animal House, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India. Animals were housed in plastic cages under a well-regulated light and dark (12 hr: 12 hr) schedule at 24°±3°C and fed with standardized pellet food and tap water ad libitum. Experiments were carried out according to the ethical guidelines. p-Nonylphenol (-98% pure) was a gift from Dr. Werner Kloas, University of Karlsruhe, Germany. Thiobarbituric acid and malondialdehyde were obtained from Merck­Schuchardt, Germany. All other chemicals were of analytical grade and were purchased from local commercial sources.

Animals were divided into three groups of 6 rats each. Group I was administered orally with nonylphenol dissolved in olive oil at 1, 10 and 100 /lglkg body weight/day, respectively for 45 days. Group II

NOTES 221

received 1, 10 and 100 Ilg nonylphenol along with 20 mg of vitamin E for 45 days. This dose of vitamin E was considered appropriate since it has been cited as therapeutic dose and was used by us previouslyll. Group m was administered orally with olive oil alone and served as control. The animals were fasted overnight, weighed and sacrificed 24 hr after the last treatment using overdose of anesthetic ether.

A 10% testicular homogenate was prepared in cold normal saline with the help of glass teflon homogenizer. The homogenate was centrifuged at 800 g for 20 min at 4°C and the crude homogenate obtained was used for biochemical estimation of antioxidant enzymes. Mitochondrial and microsome-rich fractions obtained by the method of differential centrifugation12

were used for biochemical estimation of antioxidant enzymes. Protein was estimated by the method of Lowry et al. 13

• Activity of superoxide dismutase was estimated by the method of Marklund and

. Marklundl4. Briefly, the assay mixture contained 2.4 rnl of tris hydrochloric acid buffer (50 rnM) containing 1 mM EDT A (PH 7.6), 300 III of pyrogallol (0.2 rnM) and 100 III enzyme source. Increase in the absorbance was measured immediately at 420 nm against enzyme blank at 10 sec intervals for 3 min on a Systronics Spectrophotometer. The activity of enzyme was expressed as nmole pyrogallol oxidised/minlmg protein at 35°C. Glutathione reductase was assayed by the method of Carlberg and Mannervikl5. Briefly, the assay mixture contained 1.75 rnl of phosphate buffer (100 mM, pH 7.6), 100 III of NADPH (200 rnM), 100 III of EDT A (10 rnM), 50 III of glutathione oxidized (20 rnM) and 50 III of enzyme source. Disappearance of NADPH was measured immediately at 340 nm against enzyme blank at 10 sec intervals for 3 min on a Systronics Spectrophotometer. Activity of enzyme was expressed as nmole of NADPH oxidised/minlmg protein at 35°C. Levels of lipid peroxidation were measured by the method of Ohkawa et al. 16

• Briefly, the stock solution contained trichloroacetic acid 15% (w/ v) in 0.25 N hydrochloric acid and 2-thiobarbituric acid (0.37%). One volume of the test sample and two volumes of stock reagent were mixed in a screw­capped centrifuge tube, vortexed and heated for 15 min in a boiling water bath. After cooling on ice the precipitate was removed by centrifugation at 1000 g for 15 min and absorbance was measured at 532 nm against enzyme blank. The values were expressed as Ilmole of malondialdehyde formed/minlmg protein. A standard curve was prepared with the known amount

of malondialdehyde and all the above reagents except enzyme source.

Data were expressed as mean ± SO for six animals per group. Statistical analyses were performed using an analysis of variance (one-way ANOVA) followed by unpaired Student's t-test. Differences were considered to be significant at P <0.05 against control group.

Nonylphenol-treated rats exhibited significantly (P < 0.05) decreased activities of superoxide dismutase and glutathione reductase in crude homogenate as well as in the mitochondrial and microsome-rich fractions of testis (Fig. 1A,B). In the testis, in addition to undergoing complex cellular changes, germ cells migrate progressively from the basal to the adluminal compartment of the seminiferous epithelium where elongate spermatids are eventually released into the tubular lumen at spermiation. During this restructuring process production of reactive oxygen species (ROS) such as superoxide (02'), hydroxyl (OH"), peroxyl (R02"),

hydroperoxyl (H02"), nitric oxide (NO"), and nitrogen dioxide (N02") has been reported 17. ROS have been shown to damage macromolecules of the cell including membrane polyunsaturated fatty acids, causing impairment of cellular functions. ROS formation in mitochondria increased by uncouplers of oxidative phosphorylation, hyperbaric oxygen treatment, pathological conditions such as ischemia! reperfusion syndrome, ageing and during alterations of mitochondrial polyunsaturated fatty acids and lipoperoxidation process lS

• Mitochondrial respiration has been shown to generate free oxygen radicals suggesting a possible link between oxidative stress, mitochondrial integrity and cell deathl9. In the subcellular fractions, including mitochondria 'and microsomes, several antioxidant enzymes are known to operate. Superoxide dismutase and catalase are two rate-limiting enzymes of free oxygen radical metabolism. Superoxide dismutase, an important antioxidant molecule in the testis, has been shown to be under the germ cell regulation2o

• Glutathione peroxidase/reductase directly acts as antioxidant enzymes to inhibit lipid peroxidation.

ROS have been shown to produce in Leydig cells through mitochondrial electron transport chain III

particular, during steroid hydroxylations to cytochrome P450 enzymes, which are localized in mitochondria and in smooth endoplasmic reticulum21

•

Therefore, any impairment in the activities of antioxidant enzymes in mitochondria and microsomes

222

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homogenate fractions fractions homogenate fractions fractions

Nonylphenol (pg/ Kg body weight) Nonylphenol (pg) + Vitamin E(20 mg/ Kg body weight)

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Nonylphenol (pg/ Kg body weight) Nonylphenol (pg) + Vitamin E (20 mg/ Kg body weight)

[JControl [] 1 JIg E!l10 JIg • 100 j.lQ

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Nonylphenol (pg/ Kg body weight) NonyJphenoJ (pg) + Vitamin E (20 mgI Kg body weight)

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Fig. 1-Effect of nonylphenol and co-administration of nonylphenol and vitamin E on (A) superoxide dismutase, (B) glutathione reductase and (C) Lipid peroxidation of six rats in various subcellular fractions of testis. The values are expressed as mean ± SD. *P<O.05 against controls (nmol pyrogallol oxidized! mini mg protein at 35°C).

NOTES 223

reflects an adverse effect of nonylphenol on the antioxidant defence system of rat testis . The levels of lipid peroxidation were increased significantly in crude homogenate as well as in the mitochondrial and microsome-rich fractions in testis of nonylphenol­treated rats (Fig. IC). In the present study increased levels of lipid peroxidation along with decreased actIvity of antioxidant enzymes indicate that nonylphenol induces oxidative stress in mitochondrial and microsome-rich fractions of rat testis.

Co-administration of nonylphenol and vitamin E did not show any significant changes in the activities of antioxidant enzymes and in the levels of lipid peroxidation in various subcellular fractions of rat testis (Fig. lA-C). The results suggested that nonylphenol elicit depletion of the antioxidant defence system and increased lipid peroxidation in various subcellular fractions in the testis of rat while vitamin E imparts protective effects against them.

The authors thank the staff of Bioinformatics Centre, Pondicherry University, Pondicherry for providing various facilities. KCC acknowledges the Lady Tata Memorial Trust, Mumbai, India for Junior Scholarship. PPM acknowledges the receipt of financial support from the Population Council, New York, USA (Grant Nos. B99.047P-9/ ICMC and B99.048 RlICMC). The authors declare that the experiments done during these studies comply with the current laws of their country.

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