Metabolic bioactivation of oestradiol-17beta (E2beta) in mouse colon epithelial cells bearing ApcMin mutation.

Epidemiological studies have revealed a protective role of oestrogens against the promotion of colorectal cancer (CRC). Therefore, the oestrogen metabolism status of colonic cells is studied to explain it. Loss of function of adenomatous polyposis coli (Apc) gene product is an early and frequent event in human colorectal carcinogenesis. Normal (Apc(+/+)) and premalignant (Apc(multiple intestinal neoplasia (Min)/+)) mouse colonic epithelial cells were used to compare their respective metabolic capabilities towards oestradiol-17beta (E(2)beta), with or without an inducer of the CYP1 family, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In both cell types, the major metabolite was oestradiol-17beta-3-glucuronide. The formation of catechol (CE) metabolites by cytochromes P450 of the CYP1 family and their derivatives was shown. Among these metabolites, several O-methyl-ether derivatives were detected, as unconjugated metabolites in Apc(+/+) cells and as glucuroconjugates in Apc(Min/+) cells, after TCDD treatment. Apc(Min/+) cells are metabolically more competent than Apc(+/+) cells to produce different hydroxylated metabolites as well as glucuroconjugates. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) experiments corroborate these results. Indeed, induction by TCDD has prevailing effects in gene expression of CYP1A1, CYP1A2 and CYP1B1 in Apc(Min/+) cells, compared with Apc(+/+) ones. Apc(Min/+) cells displayed higher rates of oestrogen metabolic biotransformation than Apc(+/+) ones, but exhibited two opposite tendencies. Apc(Min/+) cells were able to detoxify E(2)beta mainly by the formation of glucuronides and displayed at the same time a striking potential to bioactivate E(2)beta by producing only the electrophilic 2-CE derivatives, not the 4-CE ones, even though a significant CYP1B1 mRNA induction was noticed. These specific electrophilic metabolites may form DNA adducts but are not prone to generate new mutations. Interestingly, the ultimate 2-O-methyl-ether metabolite of E(2)beta may be an endogenous protective factor against CRC promotion given its recognised anti-angiogenic and pro-apoptotic properties.

High potency of bioactivation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mouse colon epithelial cells with Apc(Min) mutation.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a prominent heterocyclic aromatic amine (HAA) found in meat and fish cooked at moderate to high temperature. It is considered as a potent dietary factor promoting colon carcinogenesis. However, the role of intestinal cells in PhIP bioactivation has not been fully explained, particularly when cells are pre-malignant. Loss of function of the adenomatous polyposis coli (APC) gene product is an early and frequent event in human colorectal carcinogenesis. Normal (Apc(+/+)) and pre-malignant (Apc(Min/+), where Min=multiple intestinal neoplasia) colonic epithelial cells of mice can be used to study promotion of carcinogenesis, but these cells have not been characterized for bio-activation of HAA. We investigated the metabolism of (14)C-PhIP in these two murine cell lines. Cells induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) metabolized PhIP into 4'-OH-PhIP as the main metabolite in PhiP detoxification. Besides, 5-OH-PhIP was identified, revealing the formation of intermediary reactive metabolites, since it results from a degradation of conjugates of N-acetoxy-PhIP. Apc(Min/+) cells produce significantly higher amounts of these metabolites. Demethylated metabolites are also observed, indicating that the colon contains a significant CYP1 family dependent metabolic activity. A minor hydroxy-glucuronide-PhIP metabolite is observed in Apc(Min/+) cells, the glucuronidation being known as an important step in the detoxification pathway. Quantitative real-time reverse transcription polymerase chain reaction experiments demonstrate that induction by TCDD has prevailing effects in gene expression of CYP1A1, CYP1A2 and CYP1B1 in Apc(Min/+) cells. In these cells, N-acetyltransferase-2 is also expressed at higher levels. So, the more important potency to metabolically bio-activate PhIP, as measured in Apc(Min/+) cells, can be linked to a higher probability to generate new in situ mutations.

In vitro oxidative metabolism study of (-)-rhazinilam.

Metabolism studies were conducted in order to investigate the reasons for the in vivo lack of activity of (-)-rhazinilam 1, an original poison of the mitotic spindle. Bioconversion by Beauveria bassiana strains, rat and human liver microsomes allowed the identification of metabolites 2, 3, and 4 oxidized in positions 3 and 5 of rhazinilam. Further experiments indicated that CYP2B6 was the main CYP responsible for the oxidation of 1 by human liver microsomes. All isolated metabolites were markedly less active than rhazinilam in vitro, which might explain its in vivo inactivity.