Antioxidant enzyme level in the testes of cirrhotic rats

Abstract

Objectives: An understanding of the tissue and organ level of antioxidant enzymes that scavenge reactive oxygen species may provide an indication of their susceptibility to free radical-related cytotoxic damage. A direct association between testicular production of excessive reactive oxygen species and male infertility has been noted. We measured the activities of superoxide dismutase and glutathione peroxidase in the testes of thioacetamide-induced cirrhotic rats.

Methods: Antioxidant enzyme activities and trace element levels (copper, zinc, manganese, and selenium) in the testes of thioacetamide-induced cirrhotic and control rats were measured. The statistical difference between the experimental and control groups with regard to the activities of superoxide dismutase and glutathione peroxidase and levels of trace elements was analyzed with Student’s t test.

Results: Our results showed a significant decrease in the activity of these enzymes in the testes of cirrhotic rats. The testicular levels of copper, zinc, and manganese, which are associated with these antioxidant enzymes, increased, whereas selenium decreased slightly in cirrhotic rats; that decrease was not statistically significant.

Conclusions: Our studies showed a drastic decrease in the level of antioxidant enzymes in the testes of cirrhotic rats that could have deleterious effects on sperm function in these animals. Further studies are necessary to understand the exact pathways of trace element metabolism in the testes of cirrhotic rats.

Introduction

Oxidative stress is believed to be a major cause of sperm dysfunction.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 Highly reactive oxygen species (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH⁻), and singlet oxygen are formed as byproducts of normal aerobic metabolism.11 These factors react with a variety of biomolecules including nucleic acids, proteins, carbohydrates, and lipids. The products of these reactions are usually cytotoxic, mutagenic, and carcinogenic.11, 12 Spermatocytes, because of the a high percentage of polyunsaturated fatty acids within their membranes, are quite vulnerable to the attacks of the reactive oxygen species.13

Spermatocytes require a minimal level of reactive oxygen species for normal capacitation and acrosome reaction.14 However, because the concentration of reactive oxygen species is critical to spermatogenesis and sperm maturation, there should be some tight mechanisms for the regulation of the level of reactive oxygen species within the male reproductive tract and seminal fluid.13, 14

Various investigators have found a direct correlation between infertility and elevated level of reactive oxygen species in semen.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 This increase in the level of reactive oxygen species is due to an imbalance between its production and degradation in the spermatozoa and seminal plasma. Degradation of reactive oxygen species is mediated by various antioxidant scavengers that are present in the cell.11, 12 The major reactive oxygen species generated by human spermatozoa appears to be the superoxide anion, which produces hydrogen peroxide through the action of superoxide dismutase (SOD). Further, they generate hydroxyl radical through the Haber-Weiss reaction with the iron and copper available in the seminal plasma.5 Hydrogen peroxide rather than superoxide anion is cytotoxic to human spermatozoa.5

The etiology of various diseases such as cancer, heart diseases, and diabetes may be related to the generation of free radicals.11, 12 For many years our laboratory has been interested in the study of the factors that lead to the development of liver cirrhosis.15, 16, 17, 18, 19, 20, 21 It is believed that increased oxidative stress also may be a major contributing factor that leads to the development of liver cirrhosis. Treatment with antioxidant scavengers alleviated the accumulation of fat in the liver of ethanol-treated rats.24

To protect against the adverse effects of reactive oxygen species, mammalian cells are equipped with various enzymatic and non-enzymatic antioxidant scavengers.9, 11, 12 The major enzymatic antioxidants are SOD, glutathione peroxidase (GPX), and catalase and enzymes such as glutathione reductase and glucose-6-phosphate dehydrogenase, which are involved in the recycling of oxidized glutathione.11 Various studies have shown that the level of antioxidant enzymes in tissues might provide a clear indication on the extent of cytotoxic damage that occurs in various tissues.9, 11, 12

In thioacetamide-induced cirrhotic rat models, we found, in addition to liver changes, marked pathologic changes in other organs such as kidney and spleen.19, 20 These pathologies also were associated with considerable alterations in the level of trace elements in these organs. The testis of the cirrhotic rat is also subjected to abnormal trace element metabolism,22 which could affect the normal spermatogenesis, maturation, and fertilization in the cirrhotic rat.

Considering our previous observations on the association between abnormal trace element level and structural damage in different organs, we assumed that trace element level would cause structural and functional alteration in testes. Further, various studies have indicated that masculine infertility diseases are related to altered trace element metabolism in the testis. However, the mechanism by which changes in trace element level leads to structural and functional abnormalities in different organs is not understood. In this regard, various investigators have considered changes in the level of natural antioxidant enzymes that protect cells from free radical damage. Therefore, we investigated the antioxidant status of thioacetamide (CH₃CSNH₂)-induced cirrhotic rats.

We compared the levels of the major antioxidant enzymes SOD and GPX in the testes of cirrhotic rats with those of their normal age-matched counterparts. In addition, we measured the testiscular levels of copper, zinc, manganese, and selenium because these trace elements act as functional components of SOD and GPX.