Elsevier

Life Sciences

Volume 74, Issue 7, 2 January 2004, Pages 897-907
Life Sciences

Antioxidant and antiapoptotic activities of idoxifene and estradiol in hepatic fibrosis in rats

https://doi.org/10.1016/j.lfs.2003.08.004Get rights and content

Abstract

Oxidative stress plays a causative role in the development of hepatic fibrosis and apoptosis. Estradiol (E2) is an antioxidant, and idoxifene is a tissue-specific selective estrogen receptor modulator. We have previously demonstrated that E2 inhibits hepatic fibrosis in a rat model of hepatic fibrosis induced with dimethylnitrosamine (DMN), and suppresses activation of the nuclear factor (NF)-κB proinflammatory transcription factor in cultured rat hepatocytes undergoing oxidative stress. This study reports on the antioxidant and antiapoptotic role of idoxifene and E2 in the DMN model of hepatic fibrosis. The DMN model rats were administered with idoxifene or E2, and were examined activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and expression of Bcl-2 family proteins in the liver. During the course of hepatofibrogenesis after DMN treatment, serum levels of lactate dehydrogenase (LDH), a biomarker for necrosis, and hepatic levels of malondialdehyde (MDA), an end product of lipid peroxidation, increased rapidly for 3 days. On day 14, serum LDH levels normalized, and hepatic fibrosis developed with increased levels of MDA and collagen and decreased production of SOD and GPx in the liver. Fibrotic liver also showed downregulation of Bcl-2 and Bcl-XL expression and upregulation of Bad expression. Idoxifene and E2 suppressed DMN-mediated necrosis, lipid peroxidation, the loss of antioxidant enzyme activity, and proapoptotic status in Bcl-2 family protein expression as well as hepatic fibrosis. These findings indicate that, in addition to their antiinflammatory and antifibrotic action, idoxifene and E2 could enhance antioxidant and antiapoptotic activity in hepatic fibrosis in rats.

Introduction

Parenchymal cell membrane damage could result in the release of oxygen-derived free radicals and other reactive oxygen species (ROS) derived from lipid peroxidative processes, which represent a general feature of sustained inflammatory response and liver injury, leading to apoptosis and necrosis (Houglum et al., 1990). Fortunately, cells possess antioxidant protective mechanisms, including enzymatic defense molecules such as superoxide dismutase (SOD) and glutathione peroxidase (GPx), which can react with ROS and neutralize them before inflict damage on vital components. There is large body of evidence that in humans and animals oxidative stress is implicated in chronic liver disease and serves as a link between hepatic injury and fibrosis Shimizu, 2001, Shimizu, 2003 It should be noted that estradiol (E2) is a potent endogenous antioxidant, which reduces lipid peroxide levels in liver and serum Yoshino et al., 1987, Lacort et al., 1995. We have already reported that E2 suppressed hepatic fibrosis in rat models of hepatic fibrosis induced with pig serum and dimethylnitrosamine (DMN) Shimizu et al., 1999a, Shimizu et al., 1999b, Yasuda et al., 1999, and attenuated iron-induced lipid peroxidation in rat liver mitochondria (Omoya et al., 2001). It is well known that overexpression of Bcl-2 suppresses lipid peroxidation (Hockenbery et al., 1993), prevents apoptosis and is up-regulated by estrogen in several tissues Wang and Phang, 1995, Garcia-Segura et al., 1998, Gohel et al., 1999 Bcl-2, Bcl-XL, Bad, and Bax are members of the Bcl-2 family proteins that play an important role in regulating cell survival and apoptosis. In addition to Bcl-2, Bcl-XL also prevents apoptosis in response to a wide variety of stimuli. Conversely, proapoptotic proteins, Bad and Bax can accelerate death and in some instances are sufficient to cause apoptosis. The effects of E2 on the SOD and GPx production and the Bcl-2 family protein expression in the liver, however, remain to be elucidated.

It should be pointed out that the administration of estrogen per se in women poses some potential risks including breast cancer and endometrial abnormalities (Collaborative Group on Hormonal Factors in Breast Cancer, 1997). Idoxifene is a tissue-specific selective estrogen receptor modulator (Omoya et al., 2001), which acts in breast tissue as an estrogen antagonist through the same estrogen receptor element as the natural steroid hormone. Idoxifene acts in bone as an estrogen agonist for osteoblasts, and shows negligible agonist activity in human endometrial cells (Nuttall et al., 1998). Idoxifene was originally developed for the treatment of advanced breast cancer and the prevention of postmenopausal osteoporosis. Our recent report suggests that idoxifene and E2 protect hepatocytes from inflammatory cell injury by inhibiting activation of the nuclear factor (NF)-κB proinflammatory transcription factor (Omoya et al., 2001). However, little information is available relative to the antioxidant and antiapoptotic role of idoxifene as well as E2 in the liver.

The present study was undertaken to explore the antioxidant and antiapoptotic role of idoxifene in the DMN-induced fibrotic liver in direct comparison to the natural hormone E2. In this study, in addition to their antifibrogenic role, idoxifene and E2 suppressed DMN-mediated necrosis, lipid peroxidation, the loss of SOD and GPx production, and downregulation of Bcl-2 and Bcl-XL expression and upregulation of Bad expression in the liver.

Section snippets

Preparation of animals

Adult male Wistar rats weighing about 200 g were used for the DMN model of hepatic fibrosis. The animals (n=6 in each group), which comprised groups 2 through 6 were administered a single intraperitoneal injection of 40 mg/kg DMN (Sigma, St. Louis, MO), diluted with saline Shimizu et al., 1999a, Shimizu et al., 1999b, Yasuda et al., 1999. The controls in group 1 (n=6) received a single dose of saline. The rats in groups 4, 5, and 6 received daily oral gavage of idoxifene, which was provided by

Effects of idoxifene and E2 on hepatic necrosis, serum E2 levels, and body weights in the animal model of hepatic fibrosis

When animals were given a single injection of 40 mg/kg DMN, serum LDH levels and hepatic MDA levels increased rapidly for 3 days. Serum LDH levels normalized after 14 days, and hepatic MDA levels remained significantly higher than control values for 14 days. The serum LDH level on day 3 after DMN treatment was 5.8 times the initial value of 367 ± 49 U/L (Table 1). Hepatic collagen contents gradually rose, remaining significantly higher than control values for 14 days.

Histological examination of

Discussion

It is conceivable that cellular damage, leading to apoptosis and necrosis, might be attributed to the presence of oxygen-derived free radicals and other ROS, which are by-products of the inflammatory response. Whereas cells are well equipped to neutralized the effects of ROS, by means of a series of antioxidant protective systems. Our preliminary studies have showed that idoxifene and E2 are able to inhibit ROS generation and lipid peroxidation (Inoue et al., 2003) and inactivate NF-κB (Omoya

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    Present address: Department of Anatomy, Nantong Medical College, Nantong 226001, P.R. China.

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