Epistane, an anabolic steroid used for recreational purposes, causes cholestasis with elevated levels of cholic acid conjugates, by upregulating bile acid synthesis (CYP8B1) and cross-talking with nuclear receptors in human hepatocytes.

Anabolic-androgenic steroids are testosterone derivatives, used by body-builders to increase muscle mass. Epistane (EPI) is an orally administered 17α-alkylated testosterone derivative with 2a-3a epithio ring. We identified four individuals who, after EPI consumption, developed long-lasting cholestasis. The bile acid (BA) profile of three patients was characterized, as well the molecular mechanisms involved in this pathology. The serum BA pool was increased from 14 to 61-fold, basically on account of primary conjugated BA (cholic acid (CA) conjugates), whereas secondary BA were very low. In in vitro experiments with cultured human hepatocytes, EPI caused the accumulation of glycoCA in the medium. Moreover, as low as 0.01 µM EPI upregulated the expression of key BA synthesis genes (CYP7A1, by 65% and CYP8B1, by 67%) and BA transporters (NTCP, OSTA and BSEP), and downregulated FGF19. EPI increased the uptake/accumulation of a fluorescent BA analogue in hepatocytes by 50-70%. Results also evidenced, that 40 µM EPI trans-activated the nuclear receptors LXR and PXR. More importantly, 0.01 µM EPI activated AR in hepatocytes, leading to an increase in the expression of CYP8B1. In samples from a human liver bank, we proved that the expression of AR was positively correlated with that of CYP8B1 in men. Taken together, we conclude that EPI could cause cholestasis by inducing BA synthesis and favouring BA accumulation in hepatocytes, at least in part by AR activation. We anticipate that the large phenotypic variability of BA synthesis enzymes and transport genes in man provide a putative explanation for the idiosyncratic nature of EPI-induced cholestasis.

Effect of organochlorine pesticides on human androgen receptor activation in vitro.

Many persistent organochlorine pesticides (OCs) have been implicated in adverse effects, that is, reproductive and developmental effects, in man and in wildlife alike. It has been hypothesized that these so-called xeno-hormones could be responsible for the increased incidence in various male sexual differentiation disorders such as hypospadias, cryptorchidism, low sperm counts and quality. In this report, OCs, called endocrine disrupters, were tested for their interaction with the androgen receptor. The stable prostatic cell line PALM, which contains a human androgen receptor (hAR) expression vector and the reporter MMTV-luciferase, was used to characterize the response of hAR to OC and was compared with synthetic androgen compound R1881. We found that all the OC pesticides tested were able to shift the agonist [(3)H]-R1881 from its binding site to the AR in competitive binding assays. In addition, these compounds antagonize-in a dose-dependent manner-the AR-mediated transcription by synthetic AR ligand R1881. None of the pesticides reacted as agonists. These results demonstrate that OC endocrine activities in vivo probably result from direct and specific binding to the AR ligand-binding domain. Although the antagonistic potential of OC pesticides is lower than that of hydroxyflutamide, they are capable of disrupting the male hormone signaling pathway. Because these chemicals are extremely persistent and tend to bioaccumulate, these results support the hypothesis that the recent increase in the incidence of male sexual disorders could be due to long exposure to ubiquitous OC pesticides found in the environment.

Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro.

Twenty-four pesticides were tested for interactions with the estrogen receptor (ER) and the androgen receptor (AR) in transactivation assays. Estrogen-like effects on MCF-7 cell proliferation and effects on CYP19 aromatase activity in human placental microsomes were also investigated. Pesticides (endosulfan, methiocarb, methomyl, pirimicarb, propamocarb, deltamethrin, fenpropathrin, dimethoate, chlorpyriphos, dichlorvos, tolchlofos-methyl, vinclozolin, iprodion, fenarimol, prochloraz, fosetyl-aluminum, chlorothalonil, daminozid, paclobutrazol, chlormequat chlorid, and ethephon) were selected according to their frequent use in Danish greenhouses. In addition, the metabolite mercaptodimethur sulfoxide, the herbicide tribenuron-methyl, and the organochlorine dieldrin, were included. Several of the pesticides, dieldrin, endosulfan, methiocarb, and fenarimol, acted both as estrogen agonists and androgen antagonists. Prochloraz reacted as both an estrogen and an androgen antagonist. Furthermore, fenarimol and prochloraz were potent aromatase inhibitors while endosulfan was a weak inhibitor. Hence, these three pesticides possess at least three different ways to potentially disturb sex hormone actions. In addition, chlorpyrifos, deltamethrin, tolclofos-methyl, and tribenuron-methyl induced weak responses in one or both estrogenicity assays. Upon cotreatment with 17beta-estradiol, the response was potentiated by endosulfan in the proliferation assay and by pirimicarb, propamocarb, and daminozid in the ER transactivation assay. Vinclozolin reacted as a potent AR antagonist and dichlorvos as a very weak one. Methomyl, pirimicarb, propamocarb, and iprodion weakly stimulated aromatase activity. Although the potencies of the pesticides to react as hormone agonists or antagonists are low compared to the natural ligands, the integrated response in the organism might be amplified by the ability of the pesticides to act via several mechanism and the frequent simultaneous exposure to several pesticides.

Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas).

Due to the time and expense associated with full life-cycle testing, most current toxicity tests with fish do not explicitly consider reproductive output as an endpoint but, rather, focus on early life-stage survival and development. However, some classes of chemicals could adversely impact reproduction at concentrations below those that affect development. Further, estimates of the effects of toxic compounds on reproductive output can be critical to the ecological risk assessment process. In this manuscript, we describe a short-term reproduction test with the fathead minnow (Pimephales promelas) and evaluate the test using two model reproductive toxicants, methoxychlor (an estrogenic compound) and methyltestosterone (an androgenic chemical). The test is initiated with reproductively mature animals and is comprised of a pre-exposure phase of 14 to 21 d, followed by a chemical exposure of up to 21 d. During and at completion of the test, several endpoints related to reproductive fitness and endocrine function are assessed. Both chemicals evaluated in our study caused a significant decrease in fecundity of the fish at nominal concentrations of 5.0 micrograms/L (methoxychlor) and 0.2 mg/L (methyltestosterone). Methoxychlor decreased plasma concentrations of one or more steroids (testosterone, 11-ketotestosterone, beta-estradiol) in both sexes and caused a significant induction of plasma vitellogenin in males, a response consistent with activation of the estrogen receptor by the pesticide (or its metabolites). Methyltestosterone decreased plasma concentrations of sex steroids and adversely affected gonadal status (as evaluated by relative weight and histopathology) in both sexes. The androgenic nature of methyltestosterone was clearly expressed as masculinization of exposed females via formation of nuptial tubercles, structures normally present only in reproductively active males. The chemical also caused a significant induction of plasma vitellogenin in both males and females; this unexpected estrogenic response was most likely due to aromatization of the androgen to a form capable of binding to the estrogen receptor. These studies demonstrate the utility of this short-term assay for identifying chemicals that exert reproductive toxicity through alterations in endocrine systems controlled by estrogens and androgens.