Role of CYP3A4 in acetaminophen hepatotoxicity: Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in the regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands. Metabolism of ifosfamide and cyclophosphamide. The differential metabolism of the cancer chemotherapeutic agents ifosfamide (IF) and cyclophosphamide (CP) has a central role in the differing toxicity profiles of these isomeric drugs. While the general pattern of intact oxazaphosphorine metabolites is similar for CP and IF, a key difference is the number and abundance of two-carbon metabolites that derive from side-chain dealkylation. It was proposed that CAL underlies both the neurotoxicity and nephrotoxicity of IF. Additionally, CEA, proposed as a toxic metabolite was reported to be derived directly from IF by chemical hydrolysis and also to reacting with bicarbonate to form 1,3-oxazolidin-2-one. CAL was reported to be converted to 2-chloroacetic acid (CAA) by human kidney and rabbit heart. In turn, CAA may react with cellular thiols to yield S-carboxymethylcysteine (SCMC) and its metabolite thiodiglycolic acid (TDGA). Other cysteine-derived metabolites have also been observed. What role this panoply of small IF metabolite plays in the selective toxicity and efficacy of IF is poorly understood. What is clear is that CP and IF are two potent drugs which are inactive per se and whose effects on both the tumor and the host are determined by metabolism. Despite the large number of published studies on the metabolism of CP and IF both in vitro and in vivo and in a variety of species including man, a comprehensive comparative analysis of their metabolism is indicated. The mouse was used for in vivo investigation because of the availability of mice humanized for various metabolic enzymes and the nuclear receptors that regulate their tissue expression. Ultra-performance chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLCESI-QTOFMS) has proven successful in studies involving metabolomic protocols to uncover unforeseen drug metabolites of a number of drugs and carcinogens. Interestingly, while CP and IF are isomers, only IF treatment is known to cause nephrotoxicity and neurotoxicity. Therefore, it was anticipated that a comparison of IF and CP drug metabolites in the mouse would reveal reasons for this selective toxicity. Drug metabolites were profiled UPLC-ESI-QTOFMS, and the results analyzed by multivariate data analysis. Of the total 23 drug metabolites identified for both IF and CP, five were found to be novel. IF preferentially underwent N-dechloroethylation, the pathway yielding 2-chloroacetaldehyde, while CF preferentially underwent ring-opening, the pathway yielding acrolein (AC). Additionally, S-carboxymethylcysteine and thiodiglycolic acid, two downstream IF and CP metabolites, were produced similarly in both IF- and CP-treated mice. This may suggest that other metabolites, perhaps precursors of thiodiglycolic acid, may be responsible for IF encephalopathy and nephropathy. This study illustrates the value of metabolomics to study metabolism and toxicity of chemotherapeutic drugs. Mechanism of acetaminophen hepatotoxicity: Metabolic bioactivation, glutathione depletion, and covalent binding are the early hallmark events after acetaminophen (APAP) overdose. However, the subsequent metabolic consequences contributing to APAP-induced hepatic necrosis and apoptosis have not been fully elucidated. In this study, serum metabolomes of control and APAP-treated wild-type and Cyp2e1-null mice were examined by liquid chromatography-mass spectrometry (LC-MS) and multivariate data analysis. A dose-response study showed that the accumulation of long-chain acylcarnitines in serum contributes to the separation of wild-type mice undergoing APAP-induced hepatotoxicity from other mouse groups in a multivariate model. This observation, in conjunction with the increase of triglycerides and free fatty acids in the serum of APAP-treated wild-type mice, suggested that APAP treatment can disrupt fatty acid beta-oxidation. A time-course study further indicated that both wild-type and Cyp2e1-null mice had their serum acylcarnitine levels markedly elevated within the early hours of APAP treatment. While remaining high in wild-type mice, serum acylcarnitine levels gradually returned to normal in Cyp2e1-null mice at the end of the 24 h treatment. Distinct from serum aminotransferase activity and hepatic glutathione levels, the pattern of serum acylcarnitine accumulation suggested that acylcarnitines can function as complementary biomarkers for monitoring the APAP-induced hepatotoxicity. An essential role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the regulation of serum acylcarnitine levels was established by comparing the metabolomic responses of wild-type and Ppara-null mice to a fasting challenge. The upregulation of PPARalpha activity following APAP treatment was transient in wild-type mice but was much more prolonged in Cyp2e1-null mice. Overall, serum metabolomics of APAP-induced hepatotoxicity revealed that the CYP2E1-mediated metabolic activation and oxidative stress following APAP treatment can cause irreversible inhibition of fatty acid oxidation, potentially through suppression of PPARalpha-regulated pathways.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005562-23
Application #
8157184
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2010
Total Cost
$1,080,050
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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