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Objective Characteristic pathological changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH) and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in this condition in humans, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD). Methods A principal component analysis for all 91 metabolites measured indicated that metabolome perturbation is greater and less varied for humans than for rodents. Results Metabolome changes in human and rat NAFLD were greatest for the amino acid and bile acid metabolite families (e.g., asparagine, citrulline, gamma-aminobutyric acid, lysine); although, in many cases, the trends were reversed when compared between species (cholic acid, betaine). Conclusions Overall, these results indicate that metabolites of specific pathways may be useful biomarkers for NASH progression, although these markers may not correspond to rodent NASH models. The MCD model may be useful when studying certain end points of NASH; however, the metabolomics results indicate important differences between humans and rodents in the biochemical pathogenesis of the disease.

In the present study, NMR-based urinary metabonomic profiles resulting from dosing with widely recognized microsomal enzyme inducers were evaluated in male rats. Wistar or Sprague–Dawley rats were dosed daily by oral gavage with phenobarbital (PB; 100 mg/kg), diallyl sulfide (DAS; 500 mg/kg), the investigational compound DMP-904 (150 mg/kg), or β-naphthoflavone (BNF; 100 mg/kg) for 4 days, and urine was collected daily for analysis. Compounds known to increase cytochrome P450 2B enzymes, including PB, DAS and DMP-904, increased the urinary excretion of gulonic and ascorbic acid in a time-dependent manner, reaching a maximum following 3–4 days of dosing. In contrast, BNF, an agent that induces primarily Cyp1A enzymes, did not increase gulonic or ascorbic acid excretion, despite inducing Cyp1A1 more than 200-fold. Given the metabonomic results, hepatic transcriptional changes in the regulation of ascorbic acid biosynthesis were determined by RT-PCR. All Cyp2B inducers increased hepatic mRNA levels of aldo-keto reductase 1A1, an enzyme that catalyzes the formation of gulonic acid from glucuronate with concurrent decreased expression of both regucalcin (Rgn), the enzyme responsible for conversion of gulonic acid to gulono-1, 4-lactone and gulonolactone oxidase (Gulo), the rate-limiting enzyme in ascorbate biosynthesis. These effects would be expected to increase levels of gulonic acid. In addition, Cyp2B inducers also increased hepatic expression of enzymes regulating ascorbic acid reutilization including glutaredoxin reductase (Glrx2) and thioredoxin reductase (Txnrd1). In contrast, BNF did not effect hepatic expression of any enzyme regulating gulonic or ascorbic acid biosynthesis. Thus, some microsomal enzyme inducers alter transcriptional regulation of ascorbic acid biosynthesis, and these changes are detected by noninvasive metabonomic profiling. However, not all microsomal enzyme inducers appear to alter ascorbic acid metabolism. Finally, the work illustrates how metabonomic results can direct additional studies to determine the biochemical mechanisms underlying changes in urinary metabolite excretion. Copyright © 2009 John Wiley & Sons, Ltd.

Musk xylene (MX) is a synthetic nitromusk perfume ingredient that, although uniformly negative in genotoxicity testing, causes liver tumors in B6C3F1 mice. MX is also capable of inducing cytochrome P450 enzymes in a manner similar to that of phenobarbital (PB), which suggests that epigenetic mechanisms may be involved in the carcinogenic response. At the same time, MX is metabolized in vivo by nitroreduction, a reaction catalyzed by intestinal flora that yields aromatic amine metabolites. These amine metabolites are also capable of inactivating CYP2B10, the major cytochrome P450 enzyme induced by MX treatment. In the study reported here, the monoamine metabolites of MX, o- and p-NH2-MX, were evaluated for their potential to induce CYP2B10 and CYP1A2 mRNAs. Northern blot analyses indicated that both amines markedly induced CYP2B10 mRNA, whereas CYP1A2 mRNA, the enzyme implicated in the bioactivation of aromatic amines and frequently induced by aromatic amines, was induced only slightly, a response that was not different from that seen with PB. Induction of CYP2B10 mRNA suggested that the amine metabolites may contribute to the enzyme induction profile seen with MX treatment. To test this hypothesis, mice were treated with broad-spectrum antibiotics (neomycin, tetracycline, and bacitracin) to eliminate the intestinal flora and prevent formation of o- and p-NH2-MX. In antibiotic-treated mice treated with MX (200 mg/kg) for 4 d, no evidence of microsomal enzyme induction was observed, including no increases in liver weight, total cytochrome P450 content, or CYP2B protein levels. These results indicate that the amine metabolites of MX are responsible for the enzyme induction seen after MX administration. Thus, the biochemical and molecular effects of amine metabolites of MX are markedly different from those of other aromatic amines but very similar to those of PB. Therefore, it appears that MX is a non-genotoxic chemical that may cause mouse liver tumors in a manner analogous to that of PB.