Phenotype of the Cyp1a1/1a2/1b1-/- triple-knockout mouse. Crossing the Cyp1a1/1a2(-/-) double-knockout mouse with the Cyp1b1(-/-) single-knockout mouse, we generated the Cyp1a1/1a2/1b1(-/-) triple-knockout mouse. In this triple-knockout mouse, statistically significant phenotypes (with incomplete penetrance) included slower weight gain and greater risk of embryolethality before gestational day 11, hydrocephalus, hermaphroditism, and cystic ovaries. Oral benzo[a]pyrene (BaP) daily for 18 days in the Cyp1a1/1a2(-/-) produced the same degree of marked immunosuppression as seen in the Cyp1a1(-/-) mouse; we believe this reflects the absence of intestinal CYP1A1. Oral BaP-treated Cyp1a1/1a2/1b1(-/-) mice showed the same """"""""rescued"""""""" response as that seen in the Cyp1a1/1b1(-/-) mouse; we believe this reflects the absence of CYP1B1 in immune tissues. Urinary metabolite profiles were dramatically different between untreated triple-knockout and wild-type; principal components analysis showed that the shifts in urinary metabolite patterns in oral BaP-treated triple-knockout and wild-type mice were also strikingly different. Liver microarray cDNA differential expression (comparing triple-knockout with wild-type) revealed at least 89 genes up- and 62 genes down-regulated (P-value less than or equal to 0.00086). Gene Ontology """"""""classes of genes"""""""" most perturbed in the untreated triple-knockout (compared with wild-type) include lipid, steroid, and cholesterol biosynthesis and metabolism; nucleosome and chromatin assembly; carboxylic and organic acid metabolism; metal-ion binding; and ion homeostasis. In the triple-knockout compared with the wild-type mice, response to zymosan-induced peritonitis was strikingly exaggerated, which may well reflect down-regulation of Socs2 expression. If a single common molecular pathway is responsible for all of these phenotypes, we suggest that functional effects of the loss of all three Cyp1 genes could be explained by perturbations in CYP1-mediated eicosanoid production, catabolism and activities. Evaluation of melatonin metabolism using metabolomics. Metabolism of melatonin in mouse was evaluated through a metabolomic analysis of urine samples from control and melatonin -treated mice. Besides identifying seven known melatonin metabolites (6-hydroxymelatonin glucuronide, 6-hydroxymelatonin sulfate, N-acetylserotonin glucuronide, N-acetylserotonin sulfate, 6-hydroxymelatonin, 2-oxomelatonin, 3-hydroxymelatonin), principal components analysis of urinary metabolomes also uncovered seven new melatonin metabolites, including melatonin glucuronide, cyclic melatonin, cyclic N-acetylserotonin glucuronide, cyclic 6-hydroxymelatonin; 5-hydroxyindole-3-acetaldehyde, di-hydroxymelatonin and its glucuronide conjugate. However, N(1)-acetyl-N(2)-formyl-5-methoxy-kynuramine and N(1)-acetyl-5-methoxy-kynuramine, known as melatonin antioxidant products, were not detected in mouse urine. Metabolite profiling of melatonin further indicated that 6-hydroxymelatonin glucuronide was the most abundant melatonin metabolite in mouse urine, which comprised 75, 65, and 88% of the total melatonin metabolites in CBA, C57/BL6, and 129Sv mice, respectively. Chemical identity of 6-hydroxymelatonin glucuronide was confirmed by deconjugation reactions using beta-glucuronidase and sulfatase. Compared with wild-type and CYP1A2-humanized mice, Cyp1a2-null mice yielded much less 6-hydroxymelatonin glucuronide (approximately 10%) but more N-acetylserotonin glucuronide (approximately 195%) and melatonin glucuronide (approximately 220%) in urine. In summary, melatonin metabolism in mouse was recharacterized by using a metabolomic approach, and the melatonin metabolic map was extended to include seven known and seven novel pathways. This study also confirmed that 6-hydroxymelatonin glucuronide was the major melatonin metabolite in the mouse, and suggested that there was no interspecies difference between humans and mice with regard to CYP1A2-mediated metabolism of melatonin, but a significant difference in phase II conjugation, yielding 6-hydroxymelatonin glucuronide in the mouse and 6-hydroxymelatonin sulfate in humans. Mechanism of toxicity of the acetaminophen-induced liver toxicity. CYP2E1 is recognized as the most important enzyme for initiation of acetaminophen-induced toxicity. In this study, the resistance of Cyp2e1-null mice to acetaminophen treatment was confirmed by comparing serum aminotransferase activities and blood urea nitrogen levels in wild-type and Cyp2e1-null mice. However, unexpectedly, profiling of major known acetaminophen metabolites in urine and serum revealed that the contribution of CYP2E1 to acetaminophen metabolism decreased with increasing acetaminophen doses administered. Measurement of hepatic glutathione and hydrogen peroxide levels exposed the importance of oxidative stress in determining the consequence of acetaminophen overdose. Subsequent metabolomic analysis was capable of constructing a principal components analysis model that delineated a relationship between urinary metabolomes and the responses to acetaminophen treatment. Urinary ions high in wild-type mice treated with 400 mg/kg acetaminophen were elucidated as 3-methoxy- acetaminophen glucuronide (VII) and three novel acetaminophen metabolites, including S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid (VI, formed by a Cys- acetaminophen transamination reaction in kidney), 3,3'-biacetaminophen (VIII, an acetaminophen dimer), and a benzothiazine compound (IX, originated from deacetylated acetaminophen), through mass isotopomer analysis, accurate mass measurement, tandem mass spectrometry fragmentation, in vitro reactions, and chemical treatments. Dose-, time-, and genotype-dependent appearance of these minor acetaminophen metabolites implied their association with the acetaminophen -induced toxicity and potential biomarker application. Overall, the oxidative stress elicited by CYP2E1-mediated acetaminophen metabolism might significantly contribute to acetaminophen -induced toxicity. The combination of genetically modified animal models, mass isotopomer analysis, and metabolomics provides a powerful and efficient technical platform to characterize acetaminophen -induced toxicity through identifying novel biomarkers and unraveling novel mechanisms.

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