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ABSTRACT:Immunohistochemistry and autoradiography were used to identify sites of the cytochrome P450 enzymes (P450) 1A1 and 1B1 expression and activation of 7,12-dimethylbenz (
7,12-Dimethylbenz[a]anthracene (DMBA) is an adrenocorticolytic agent that causes apoplexy (haemorrhage) and massive necrosis in the adrenal cortex in rat. Several explanations regarding the origin of toxicity have been proposed. Huggins and Morii (J Exp Med 114:741-60, 1961) suggested that the cells of the inner adrenal cortex are the primary target, whereas Horváth and Kovács (Pathol Eur 8:43-59, 1973) suggested the vascular endothelium as being the origin of toxicity. In the present study, cultured precision-cut tissue slices were used to localize target cells for irreversible [(3)H]DMBA binding in rat and mouse adrenal cortex. The sites of binding were confirmed by autoradiography in vivo. Irreversible [(3)H]DMBA binding was confined to zona fasciculata/reticularis cells in rat (but not in mouse) adrenal cortex. Pronounced binding was observed in clusters of cells (focal binding), localized predominantly in zona reticularis of rat. [(3)H]DMBA binding in zona fasciculata/reticularis cells was inhibited by the cytochrome p450 1A/B (CYP1A/B) inhibitors ellipticine, alpha-naphthoflavone, and 1-ethynylpyrene. The CYP11B1-inhibitor metyrapone did not reduce [(3)H]DMBA binding. In CYP1-induced (PCB 126-treated) rats and mice, intense irreversible [(3)H]DMBA binding was found also in endothelial cells of the adrenal cortex. The endothelial binding was abolished by the CYP1 inhibitors but remained unaffected by metyrapone. We conclude that the metabolic activation in adrenal parenchymal cells is presumably catalysed by CYP1B1, whereas CYP1A1 presumably catalyses the activation in endothelial cells. We suggest that the adrenocorticolytic effect of DMBA is the result of a dual mode of action, targeting both endothelial and parenchymal cells in the rat adrenal cortex.
Polycyclic aromatic hydrocarbons (PAHs) are widespread in the environment and birds may be exposed to PAHs via diet, from preening feathers contaminated with oil, or through contamination of the eggshell during embryo development. In the present study, tissue distribution and the cell-specific binding of two labeled PAHs, benzo[a]pyrene ([3H]BaP) and 7,12-dimethylbenz[a]anthracene ([3H]DMBA), were examined in chicken embryos exposed in ovo to CYP1A inducers. Tape-section autoradiograms revealed high concentrations of radioactivity in the bile, liver, kidneys, heart, and leptomeninges. Light microscopy autoradiography of solvent-extracted tissue slices showed a high and selective binding in endothelial cells in certain blood vessels in brain, heart, lung, and chest muscle. Binding was also observed in blood vessel endothelial cells in the chorioallantoic membrane (CAM), an extraembryonal tissue lining the eggshell. Endothelial binding was confirmed in CAM exposed in vitro, implying that tissue-binding metabolites were formed in situ. The CYP1A inhibitor ellipticine abolished bleeding in the target endothelial cells in CAM. It is thus concluded that blood vessel endothelia in various tissues in birds can bioactivate environmental contaminants and be targets for their toxicity. In view of its critical position beneath the shell, the CAM could be an important target for toxicants following external exposure in oviparous species.
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