The metallohormone cadmium modulates AhR-associated gene expression in the small intestine of rats similar to ethinyl-estradiol.

Cadmium (Cd) affects the expression of estrogen receptor (ER) and aryl hydrocarbon receptor (AhR)-associated genes in rat uterus and elicits estrogen-like activity in vitro. The small intestine is highly exposed to dietary Cd which may mimic or antagonize estrogen action in this tissue. We investigated the effects of Cd and 17-alpha-ethinylestradiol (EE2) on AhR-associated gene expression after oral exposure of ovariectomized female Wistar rats, and metallothionein (Mt1a) expression as a typical metal-response marker. Mt1a in the small intestine was strongly induced by co-treatment with CdCl2 at 2 mg/kg b.wt (Cd 2) and 0.1 mg/kg b.wt EE2 than by the single compound (3-day gavage). The Cd 2-induced down-regulation of Cyp1a1, Gsta2, and Nqo1 mRNA was not antagonized by pure anti-estrogen (2.5 mg/kg b.wt ZK191703 s.c., ZK). Interestingly, the EE2-induced down-regulation of Cyp1a1, Gsta2, and Nqo1 mRNA was antagonized by Cd 2 in vivo and in colon cancer cell lines (HT-29 and CaCo-2, treated 5 days with Cd 1 µM and/or E2 0.01 µM) with low or no ER-beta expression. Dose dependency was studied after Cd exposure with drinking water (5 and 50 ppm CdCl2 equivalent to 0.4 and 4 mg/kg b.wt; Cd 0.4, Cd 4) for 28 days and EE2 as reference. Intestinal Mt1a expression was dose dependently induced, while AhR target genes were down-regulated by Cd 0.4 similar to EE2 and more pronounced than by Cd 4. We propose that Cd modulates intestinal AhR-associated gene expression similar to estrogens, but (contrary to its effects in uterus) via ER-independent and/or ER-beta-mediated mechanisms. Our new data suggest interference of Cd with estrogen and AhR signaling in the small intestine.

Cadmium modulates expression of aryl hydrocarbon receptor-associated genes in rat uterus by interaction with the estrogen receptor.

Estrogen-like effects of the heavy metal cadmium have been reported in both in vitro and in vivo studies. Yet, the molecular mechanisms involved in the hormonal activity of cadmium ions have not been fully elucidated. There are extensive data on cross-talk between aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). Recently, 17ß-estradiol (E(2)) was found to modulate the expression of AhR and AhR-regulated genes in rat uterus (Kretzschmar et al. in Mol Cell Endocrinol 321:253-257, 2010). Thus, we hypothesized that cadmium may also affect AhR signaling and examined whether cadmium or E(2) modulate AhR-associated genes via the ER in rat uterus. Ovariectomized Wistar rats received E(2) (0.5 mg/kg bw) or cadmium chloride (0.05 and 2 mg/kg bw i.p.) alone and in combination with the pure anti-estrogen ZK191703. We also co-treated a group with E(2) and cadmium 2 mg/kg bw to assess how they act in concert. Uterus wet weight, uterus epithelial height, complement C3 mRNA, and progesterone receptor (PR) protein expression served as estrogen response parameters, and expression of Mt1a mRNA was analyzed as a cadmium responsive gene. The expression of AhR protein and AhR-associated gene expression, i.e., Ahr, Arnt1, Arnt2, Cyp1a1, and Gsta2, were analyzed to examine effects on AhR-mediated signaling pathways in the uterus of all groups. Both, E(2) and cadmium induced C3 and PR expression, and this was antagonized by ZK191703. Mt1a expression was clearly induced by cadmium but slightly reduced by E(2) compared to controls. Uterine Ahr, Arnt1, Arnt2, and Cyp1a1 expression was modulated by E(2) via the ER since down-regulation by E(2) was reversed by anti-estrogen. Cadmium apparently also modulated Cyp1a1 expression via the ER. Furthermore, cadmium-induced AhR was antagonized by E(2,) and anti-estrogen-induced Gsta2 expression was antagonized by cadmium. Together our findings provide evidence for cross-talk of ER and AhR in the rat uterus.

Investigations on the estrogenic activity of the metallohormone cadmium in the rat intestine.

Cadmium (Cd), a toxic heavy metal and an important environmental pollutant, is now also regarded as potential endocrine disruptor. Its estrogenic effects have been examined so far just in classical target tissues, e.g. uterus, and mostly upon intraperitoneal (i.p.) injection of CdCl(2). Yet, estrogen receptors are also expressed in the gut, and food is the main source of cadmium intake in the general population. Therefore, possible estrogenic effects were now investigated in the intestine of ovariectomized Wistar rats after oral short- and long-term administration of CdCl(2) (0.05-4 mg/kg bw on 3 days by gavage and 0.4-9 mg/kg bw for 4 weeks in drinking water) or upon i.p. injection (0.00005-2 mg CdCl(2)/kg bw), and compared to steroid estrogen (estradiol or ethinylestradiol) treated groups. Analysis of Cd in kidneys and small intestine by atomic absorption spectrometry showed dose-dependent increases in tissue levels with rather high Cd concentrations in the gut, both after oral and i.p. administration. Expression of metallothionein (MT1a), a typical metal response parameter, was clearly induced in kidney and small intestine of several CdCl(2) treated groups, but also notably increased by steroid estrogens. Levels of estrogen-regulated genes, i.e. pS2/TFF1, vitamin D receptor (VDR), and estrogen receptor alpha and beta (ER alpha/beta) were studied as parameters of hormonal activity: The intestinal mRNA expression of pS2/TFF1 was significantly decreased in the estrogen reference groups, but also after single i.p. injection and oral long-term administration of CdCl(2). In contrast, the mRNA and protein expression of the VDR were unaffected by long-term administration of Cd via drinking water. We detected expression of ERbeta, but not ERalpha in the small intestine of OVX rats. ERbeta mRNA and protein expression were significantly down-regulated by Cd, similar to the ethinylestradiol reference group. The mRNA expression and immunostaining of proliferating cell nuclear antigen (PCNA), as an index for cell proliferation, revealed decreases after long-term administration of Cd and ethinylestradiol. In summary, cadmium exposure was shown to modulate molecular and functional parameters of estrogenicity in the intestinal tract of OVX rats. As the intestine is known to express predominantly ERbeta, and is an important site of interaction with dietary contaminants, it is indicated to further investigate specific molecular mechanisms of cadmium and estrogen receptor interactions in more detail.

Dose- and route-dependent hormonal activity of the metalloestrogen cadmium in the rat uterus.

The toxic heavy metal cadmium (Cd) is regarded as a potential endocrine disruptor, since Cd exerts estrogen-like activity in vitro and can elicit some typical estrogenic responses in rodents upon intraperitoneal (i.p.) injection. But estrogenic effects have not been documented in vivo with other more relevant routes of exposure, although it is known that Cd absorption and distribution in the body is strongly affected by the application route. Therefore, we investigated its hormonal activity in ovariectomized Wistar rats after oral administration of CdCl(2) (0.05-4 mg/kg b.w. on 3 days by gavage and 0.4-9 mg/kg b.w. for 4 weeks in drinking water) in comparison with i.p. injection of CdCl(2) (0.00005-2 mg/kg b.w.). Uterus wet weight, height of uterine epithelium, and modulation of estrogen-regulated gene expression, i.e. uterine complement component 3 (C3), were determined, and also Cd-levels in uterus and liver were measured by atomic absorption spectrometry. The analysis revealed pronounced differences in Cd tissue levels and hormonal potency for the two routes of administration: a single i.p. injection of Cd increased dose-dependently uterine wet weight and thickness of the uterine epithelium. Interestingly, C3 mRNA expression in the uterus was down regulated at low doses of CdCl(2) (0.00005-0.05 mg/kg b.w.), but strongly stimulated at the highest dose of 2 mg/kg b.w. Other than i.p. injection, oral treatment with Cd, by gavage or in drinking water, did neither increase uterine wet weights nor epithelial thickness. But, both 3-day- and 4-week oral Cd administration resulted in a dose-dependent stimulation of C3 expression in the uterus, significant at and above 0.5 mg/kg b.w. In summary, our data demonstrate an estrogenic effect in the uterus upon i.p. injection of Cd, but considerably lower hormonal potency with oral administration: short and long-term oral treatment with Cd did not affect uterus weight or histology, whilst on the molecular level, an induction of estrogen sensitive uterine gene expression was observed, albeit at dose levels far exceeding those of dietary exposure in humans.

Anabolic and androgenic activity of 19-norandrostenedione after oral and subcutaneous administration--analysis of side effects and metabolism.

One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (estr-4-ene-3,17-dione, NOR). Recently we have show that NOR stimulates skeletal muscle growth after s.c. administration in a highly selective manner but exhibits only weak androgenic activity in rats. Because most abusers take NOR orally, the aim of this study was to compare the anabolic and androgenic potency of NOR between s.c. and oral application. Orchiectomised rats were treated with NOR either s.c. (1 mg/kg BW/day) or orally (0.1, 1 and 10 mg/kg BW/day). The tissue weights of the levator ani, the seminal vesicle and the prostate were analysed to determine the anabolic and androgenic activity. Heart and liver wet weights were examined to identify side effects. Serum concentrations of NOR and its metabolite nandrolone (NT) were determined. GCMC analysis revealed that free and glucuronidated NOR and NT were detectable in the serum after oral and s.c. administration and that NOR was converted to NT in comparable amounts independent of the route of administration. In agreement to our previous study s.c. application of NOR stimulates skeletal muscle growth but has only weak androgenic effects. In contrast, after oral administration of NOR neither stimulation of the prostate nor the levator ani could be observed in the doses administered in this study. Interestingly, and in contrast to s.c. treatment, oral administration of NOR resulted in a dose-dependent decrease of body weight. In summary, oral administration of NOR, at least in the rat, seems to be a very ineffective strategy for stimulating skeletal muscle mass increases but may be associated with side effects.