Cyproterone acetate induces a cellular tolerance to cadmium in rat liver epithelial cells involving reduced cadmium accumulation

Abstract

Several reports indicate that some steroids, in particular sex steroid hormones, can modify cadmium toxicity. We recently reported that cyproterone acetate (CA), a synthetic steroidal antiandrogen that is closely related in structure to progesterone, affects cadmium toxicity in mice. In the present study, we investigated the effect of CA on cadmium toxicity in a rat liver epithelial cell line (TRL 1215) in vitro. Cells were exposed to various concentrations of CA (0, 1, 10, or 50 μM) for 24 h and subsequently exposed to cadmium (0, 50, or 100 μM; as CdCl₂) for an additional 24 h. CA pretreatment resulted in a clear decrease in the sensitivity to cadmium. Additional time course study showed CA pretreatment provided protection against cadmium toxicity but only when given for 6 or more hours prior to cadmium exposure. Cellular cadmium accumulation was markedly reduced (60% decrease) in cells pretreated for 6 or more hours with CA. In the presence of protein synthesis inhibitors the protective effect of CA toward cadmium toxicity was abolished. However, in the presence of the GSH synthesis inhibitor, l-buthionine (S,R)-sulfoximide (BSO), the protective effect of CA toward cadmium toxicity remained. CA alone increased metallothionein (MT) levels 2.4-fold, while cadmium (50 μM) alone resulted in a 8.9-fold increase over control. However, cadmium-induced MT synthesis was markedly decreased by CA pretreatment probably because of reduced cadmium accumulation. Analysis of various metal transporters by bDNA signal amplification assay revealed that the ZnT-1 transporter gene, which encodes for a membrane protein associated with zinc efflux, was expressed three-fold more in CA treated cells than control. These data show that CA pretreatment provides protection against cadmium toxicity in vitro and indicate that this protection is due to a decreased accumulation of cadmium rather than through activation of MT synthesis. This decrease of cellular cadmium accumulation appears to be related to events that require protein synthesis and may be due to activation of the genes associated with zinc efflux.

Introduction

Cadmium is a highly toxic, potentially carcinogenic, transition metal which is a continuing cause for concern due to increasing exposure in humans (Goering et al., 1994). In animal models, acute exposure to cadmium can result in toxic lesions in liver, kidney, lung, central nervous system, ovaries, placenta, and testes (Waalkes et al., 1991, Goering et al., 1994). Hepatic effects, characterized by hepatocellular necrosis, are thought to be the primary cause of death from acute cadmium exposure (Dudley et al., 1982).

Metallothionein (MT) is a metal-binding protein (Kägi and Schäfferr, 1988) that is often associated with cellular tolerance to cadmium (Rugstad and Norseth, 1975, Rugstad and Norseth, 1978, Waalkes and Goering, 1990). After exposure to cadmium, the de novo synthesis of MT is induced (Winge and Rajagopalan, 1972, Probst et al., 1977), and the presence of intracellular MT, because of its high affinity for cadmium (Kägi and Vallee, 1960, Waalkes et al., 1984), appears to provide a mechanism by which the metal can be sequestered in a relatively inert, and thus non-toxic state (Webb, 1975, Leber and Miya, 1976). Thus, MT levels may play a critical role in defining the sensitivity of a given tissue or cell to various aspects of cadmium toxicity.

Although MT is clearly associated with tolerance to cadmium toxicity, the exact nature of this association is a matter of some controversy. Acquisition of tolerance to cadmium lethality by low-dose cadmium pretreatment has been shown to be directly related to the concentration of hepatic MT (Probst et al., 1977, Goering and Klaassen, 1984). Similarly, in cell culture, the development of cadmium-resistant cell lines shows a good correlation between such resistance and cellular MT content (Rugstad and Norseth, 1975, Rugstad and Norseth, 1978). On the other hand, 5-azacytidine (AZA) pretreatment also prevents cadmium cytotoxicity in a rat liver cell line, TRL 1215 (Waalkes et al., 1985) even though AZA pretreatment causes only a modest induction of MT in these cells. It is thought that the AZA treatment ‘primes’ the machinery for MT synthesis by hypomethylating DNA and allowing more copies of the MT gene to be expressed (Waalkes et al., 1985). Thus, subsequent exposure to cadmium results in a more rapid accumulation of MT. Therefore, tolerance to cadmium can be due to increased capacity to synthesize MT as well as high quantities of preexisting MT at the time of cadmium exposure. In any event, enhanced levels of MT or increase MT gene expression are, with few exceptions, clearly associated with tolerance to cadmium.

Several reports indicate that some steroids, including sex steroid hormones such as estradiol, progesterone and testosterone, can modify cadmium toxicity (Gunn et al., 1965, Gunn et al., 1966, Wolkowoski-Tyl and Preston, 1979, Shiraishi et al., 1993, Shimada et al., 1997a, Shimada et al., 1997b). For instance, testosterone pretreatment can protect against cadmium-induced toxicity in mice (Shimada et al., 1997b). In this case, testosterone increases cadmium-induced hepatic MT level above that seen with cadmium alone. It has also recently been found that pretreatment with progesterone markedly increases cadmium toxicity in vivo (Shiraishi et al., 1993) and in vitro (Shimada et al., 1997a). Of interest in this case is the fact that progesterone exacerbates cadmium-induced cytotoxicity in vitro despite also markedly enhancing expression of the MT gene, an event typically associated with acquisition of tolerance to cadmium. Recently, we reported that cyproterone acetate (CA), a synthetic steroidal antiandrogen that is closely related in structure to progesterone, reduces cadmium-induction of hepatic MT synthesis and accumulation of liver cadmium in mice (Shimada and Waalkes, 1998). Thus, the precise nature of the altered sensitivity to cadmium and its relationship to MT induction by steroid or antisteroid compounds must be considered as ambiguous and still poorly defined.

In the present study we sought to further define the mechanisms of the CA-induced reduction in cadmium toxicity using cultured liver cells. Studies examined the possibility that CA pretreatment modifies cadmium toxicity by modulating cadmium accumulation, MT levels, or cellular redox status.