An emerging powerful technique for distinguishing isomers: Trapped ion mobility spectrometry time-of-flight mass spectrometry for rapid characterization of estrogen isomers.

RATIONALE: Isomer metabolites are involved in metabolic pathways, and their characterization is essential but remains challenging even using high-performance analytical platforms. The addition of ion mobility prior to mass analysis can help to separate isomers. Here, the ability of a recently developed trapped ion mobility spectrometry system to separate metabolite isomers was examined. METHODS: Three pairs of estrogen isomers were studied as a model of isomeric metabolites under both negative and positive electrospray ionization (ESI) modes using a commercial trapped ion mobility spectrometry-TOF mass spectrometer. The standard metabolites were also spiked into human urine to evaluate the efficiency of trapped ion mobility spectrometry to separate isomers in complex mixtures. RESULTS: The estradiol glucuronide isomers (E2 ß-3G and E2 ß-17G) could be distinguished as deprotonated species, while the estradiol epimers (E2 ß and E2 α) and the methoxyestradiol isomers (2-MeO-E2 ß and 4-MeO-E2 ß) were separated as lithiated adducts in positive ionization mode. When performing analyses in the urine matrix, no alteration in the ion mobility resolving power was observed and the measured collision cross section (CCS) values varied by less than 1.0%. CONCLUSIONS: The trapped ion mobility spectrometry-TOF mass spectrometer enabled the separation of the metabolite isomers with very small differences in CCS values (ΔCCS% = 2%). It is shown to be an effective tool for the rapid characterization of isomers in complex matrices.

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.

Loss of hepatocyte nuclear factor 1alpha function in human hepatocellular adenomas leads to aberrant activation of signaling pathways involved in tumorigenesis.

UNLABELLED: Hepatocellular adenomas (HCAs) are benign liver tumors that usually develop in women who are taking oral contraceptives. Among these tumors, biallelic inactivating mutations of the hepatocyte nuclear factor 1alpha (HNF1A) transcription factor have been frequently identified and in rare cases of hepatocellular carcinomas developed in noncirrhotic liver. Because HNF1A meets the genetic criteria of a tumor suppressor gene, we aimed to elucidate the tumorigenic mechanisms related to HNF1alpha inactivation in hepatocytes. We searched for signaling pathways aberrantly activated in human HNF1A-mutated HCA (H-HCA) using a genome-wide transcriptome analysis comparing five H-HCA with four normal livers. We validated the main pathways by quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting in a large series of samples. Then, we assessed the role of HNF1alpha in the observed deregulations in hepatocellular cell models (HepG2 and Hep3B) by silencing its endogenous expression using small interfering RNA. Along with the previously described induction of glycolysis and lipogenesis, H-HCA also displayed overexpression of several genes encoding growth factor receptors, components of the translation machinery, cell cycle, and angiogenesis regulators, with, in particular, activation of the mammalian target of rapamycin (mTOR) pathway. Moreover, estradiol detoxification activities were shut down, suggesting a hypersensitivity of H-HCA to estrogenic stimulation. In the cell model, inhibition of HNF1alpha recapitulated most of these identified transcriptional deregulations, demonstrating that they were related to HNF1alpha inhibition. CONCLUSION: H-HCA showed a combination of alterations related to HNF1alpha inactivation that may cooperate to promote tumor development. Interestingly, mTOR appears as a potential new attractive therapeutic target for treatment of this group of HCAs.