Induction of cytochrome P450 1A1 in MCF-7 human breast cancer cells by 4-chlorobiphenyl (PCB3) and the effects of its hydroxylated metabolites on cellular apoptosis.

Several studies suggest an involvement of PCBs in breast cancer formation, but the results are ambiguous and the mechanisms not clear. We propose that local activation of cytochrome P450 enzymes, CYP1A1 and CYP1B1 by PCB3, may generate active metabolites which affect apoptosis and thereby promote mammary carcinogenesis. To test this hypothesis MCF-7 human breast cancer cells were exposed to 300 nM PCB3 and its hydroxylated metabolites, 4OH-PCB and 3,4diOH-PCB3. The enzyme activity for CYP1A1 was assayed using the EROD assay, and CYP1A1 and CYP1B1 protein expression by western blotting. PCB3 increased CYP1A1 activity (~1.5fold) and protein levels within 6h after exposure. No effect on CYP1B1 protein expression was observed. The effects of PCB3 and both its metabolites on staurosporine-induced apoptosis were determined by measuring DNA fragmentation using ELISA and TUNEL assays, and by measuring caspase-8 and caspase-9 activity. We found that PCB3 and both of its hydroxylated metabolites had no effect on caspase-8 and caspase-9 activity when cells were grown in medium deprived of estrogen, but reduced caspase-9 activity when cells were grown in medium supplemented with serum containing estradiol. Interestingly, a decrease of DNA fragmentation was observed upon treatment with 3,4diOH-PCB3 in both culture conditions, suggesting that 3,4diOH-PCB3 affects a caspase-independent pathway of cell death. In summary, interactions of PCB3 and its metabolites with estradiol by yet unknown mechanisms inhibit caspase 9-related apoptosis and additional, other death pathways are affected by the catechol metabolite 3,4diOH-PCB3. These anti-apoptotic effects and the change in metabolic activity may contribute to the carcinogenic effect of PCBs.

Steroid secretion following exposure of ovarian follicular cells to three different natural mixtures of persistent organic pollutants (POPs).

This study investigated in vitro endocrine disrupting effects of three mixtures of POPs: 'Marine mix' extracted from Atlantic cod liver, and two mixtures extracted from burbot liver, 'Mjøsa mix' and 'Losna mix'. The POP mixtures were chemically characterized. Co-culture of theca and granulosa cells, were exposed for 48h with different doses of 'Marine mix', 'Mjøsa mix' or 'Losna mix'. As an end point cell viability was determinated by LDH test, steroid analysis by EIA and caspase-3 by colorimetric substrate. Chemical characterization of the mixtures demonstrated that the 'Marine mix' contained high levels of DDTs and PCBs. In the 'Mjøsa mix', the dominant pollutants were BDEs and HBCD. The concentrations of POPs measured in the 'Losna mix' were considerably lower. All mixtures used in the present study had a stimulatory effect on testosterone and estradiol secretion with 'Marine mix'>'Mjøsa mix'>'Losna mix'. These results show that even a mixture containing background concentrations of POPs significantly affected steroid secretion. A higher steroidogenic response in low dose ranges, compared with high dose ranges indicated xenobiotic-conditioning hormesis. This could complicate predictions of effects in risk assessments.

Effects of dioxin (2,3,7,8-TCDD) and PCDDs/PCDFs congeners mixture on steroidogenesis in human placenta tissue culture.

OBJECTIVE: The aim was to compare the direct effect of most toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as well as of naturally occurring congener mixture of polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) extracted from fly ash on the placental steroidogenesis. The concentration of all 17 toxic congeners was reported and the toxic equivalent (TEQ) was calculated as a 27.7 micrograms-TEQ/kg of fly ash. METHODS: Placental cotyledons were harvested immediately after expulsion of placenta. The cells were prepared according to KLIMAN et al. (1986). To examine TCDD and PCDDs/PCDFs mixture action on cytochrome P450 side change cleavage enzyme (P450 scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity the placental cells were cultured either in basal conditions or with the addition of 25-hydroxycholesterol (25-OH) or pregnenolone (P5). RESULTS: TCDD in all doses used decreased basal P4 secretion, while did not show any effect on 25-hydroxycholesterol (25-OH) and pregnenolone (P5) supplemented cultures. In all variants of culture PCDDs/PCDFs mixture was without effect on basal and substrate supplemented progesterone (P4) secretion suggesting a reduction in the activity of cytochrome P450scc or 3 beta-HSD. To examine TCDD and PCDDs/PCDFs mixture action on aromatase cytochrom P450 (P450 arom) activity the placental cells were cultured in basal condition or with the addition of dehydroepi-androsterone (DHEA) or testosterone (T). Significant increase of estradiol secretion under the influence of TCDD in DHEA and T supplemented cultures suggests its action on the activity of P450 arom. CONCLUSION: The discrepancy found between the action of pure TCDD and dioxin mixture on placental steroids secretion is possibly due to an additional effect of pentachlorodibenzo-p-dioxin (PeCDD) and pentachlorodibenzo-furan (PeCDF) which covered > 50% of the total toxic equivalents (TEQ) present in this mixture.