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Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(−/−) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by 32P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography–mass spectrometry (GC–MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(−/−) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(−/−) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechani, SCOPUS: ar.j, info:eu-repo/semantics/published

Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure.

A positive family history, present in about 30% of breast cancer cases, has been shown to double a woman's risk of breast cancer. The genetic factors responsible are largely unknown, although the autosomal dominant, relatively high penetrant genes BRCA1/2 may account for 3%. It has been hypothesized that susceptibility genes of lower penetrance may also affect breast cancer risk, and a likely group of such genes are those that regulate the production, intracellular transport, and metabolism of estrogen. Previous studies of these susceptibility genes have not been conducted with women with high familial risk. This study is being conducted with identical twins with differing genetic risks (i.e. concordant for breast cancer pairs vs. discordant pairs) as well as unaffected controls. We have chosen to focus on those genes related to estrogen metabolism and carcinogen metabolism. In the estrogen metabolism pathway, polymorphisms have been described related to the CYP17 gene, the CYP 19 gene, the COMT gene, and the HSD17B1 gene. Genes related to carcinogen metabolism which have been linked to breast cancer risk include GSTM1 and Pt and CYP1A1. We will compare the frequency of selected polymorphisms in these genes in 200 breast cancer concordant, 200 discordant, and 200 control women. We currently have tissue or buccal smears and informed consents from 130 concordant, 152 discordant, and 133 control women. Laboratory analyses of the CYP17 gene have shown some inconsistencies with repeat testing and additional testing is being done to assure that the results are accurate. Once the assay method has been validated, additional genes will be tested..