High doses of acetaminophen (APAP) result in hepatotoxicity that involves metabolic activation of the parent compound, covalent binding of the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) to liver proteins, and depletion of hepatic glutathione. Impaired fatty acid β-oxidation has been implicated in previous studies of APAP-induced hepatotoxicity. To better understand relationships between toxicity and fatty acid β-oxidation in the liver in APAP toxicity, metabolomic assays for long chain acylcarnitines were examined in relationship to established markers of liver toxicity, oxidative metabolism, and liver regeneration in a time course study in mice. Male B6C3F1 mice were treated with APAP (200 mg/kg IP) or saline and sacrificed at 1, 2, 4, 8, 24 or 48 h after APAP. At 1 h, hepatic glutathione was depleted and APAP protein adducts were markedly increased. Alanine aminotransferase (ALT) levels were elevated at 4 and 8 h, while proliferating cell nuclear antigen (PCNA) expression, indicative of hepatocyte regeneration, was apparent at 24 h and 48 h. Elevations of palmitoyl, oleoyl and myristoyl carnitine were apparent by 2–4 h, concurrent with the onset of Oil Red O staining in liver sections. By 8 h, acylcarnitine levels were below baseline levels and remained low at 24 and 48 h. A partial least squares (PLS) model suggested a direct association of acylcarnitine accumulation in serum to APAP protein adduct and hepatic glutathione levels in mice. Overall, the kinetics of serum acylcarnitines in APAP toxicity in mice followed a biphasic pattern involving early elevation after the metabolism phases of toxicity and later depletion of acylcarnitines.
HIF-1α is a nuclear factor important in the transcription of genes controlling angiogenesis including vascular endothelial growth factor (VEGF). Both hypoxia and oxidative stress are known mechanisms for the induction of HIF-1α. Oxidative stress and mitochondrial permeability transition (MPT) are mechanistically important in acetaminophen (APAP) toxicity in the mouse. MPT may occur as a result of oxidative stress and leads to a large increase in oxidative stress. We previously reported the induction of HIF-1α in mice with APAP toxicity and have shown that VEGF is important in hepatocyte regeneration following APAP toxicity. The following study was performed to examine the relative contribution of hypoxia versus oxidative stress to the induction of HIF-1α in APAP toxicity in the mouse. Time course studies using the hypoxia marker pimonidazole showed no staining for pimonidazole at 1 or 2 h in B6C3F1 mice treated with APAP. Staining for pimonidazole was present in the midzonal to periportal regions at 4, 8, 24 and 48 h and no staining was observed in centrilobular hepatocytes, the site of the toxicity. Subsequent studies with the MPT inhibitor cyclosporine A showed that cyclosporine A (CYC; 10 mg/kg) reduced HIF-1α induction in APAP treated mice at 1 and 4 h and did not inhibit the metabolism of APAP (depletion of hepatic non-protein sulfhydryls and hepatic protein adduct levels). The data suggest that HIF-1α induction in the early stages of APAP toxicity is secondary to oxidative stress via a mechanism involving MPT. In addition, APAP toxicity is not mediated by a hypoxia mechanism.
There is great interest in assessing the in vivo toxicity of chemicals using nonanimal alternatives. However, acute mammalian toxicity is not adequately predicted by current in silico or in vitro approaches. Mechanisms of acute toxicity are likely conserved across invertebrate, aquatic, and mammalian species, suggesting that dose-response concordance would be high and in vitro mechanistic data could predict responses in multiple species under conditions of similar bioavailability. We tested this hypothesis by comparing acute toxicity between rat, daphnia, and fish and by comparing their respective acute data to inhibition of mitochondria membrane potential (MMP) using U.S. Environmental Protection Agency ToxCast in vitro high-throughput screening data. Logarithmic scatter plots of acute toxicity data showed a clear relationship between fish, daphnia, and intravenous rat but not oral rat data. Similar plots versus MMP showed a well-delineated upper boundary for fish, daphnia, and intravenous data but were scattered without an upper boundary for rat oral data. Adjustments of acute oral rat toxicity values by simulating fractional absorption and CYP-based metabolism as well as removing compounds with hydrolyzable linkages or flagged as substrates for glucuronidation delineated an upper boundary for rat oral toxicity versus MMP. Mitochondrial inhibition at low concentrations predicted highly acutely toxic chemicals for fish and daphnia but not the rat where toxicity was often attenuated. This use of a single high-throughput screening assay to predict acute toxicity in multiple species represents a milestone and highlights the promise of such approaches but also the need for refined tools to address systemic bioavailability and the impact of limited absorption and first pass metabolism.
Acetaminophen (APAP) toxicity is responsible for approximately half of all cases of acute liver failure in the United States. The mouse model of APAP toxicity is widely used to examine mechanisms of APAP toxicity. Non-invasive approaches would allow for serial measurements in a single animal to study the effects of experimental interventions on the development and resolution of hepatocellular necrosis. The following study examined the time course of hepatic necrosis using small animal magnetic reasonance imaging (MRI) following the administration of 200 mg/kg ip APAP given to B6C3F1 male mice. Mice treated with saline served as controls (CON). Other mice received treatment with the clinical antidote N-acetylcysteine (APAP+NAC). Mouse liver pathology was characterized using T1 and T2 weighted sequences at 2, 4, 8 and 24 h following APAP administration. Standard assays for APAP toxicity (serum alanine aminotransaminase (ALT) levels and hemotoxylin and eosin (H&E) staining of liver sections) were examined relative to MRI findings. Overall, T2 sequences had a greater sensitivity for necrosis and hemorrhage than T1 (FLASH) images. Liver injury severity scoring of MRI images demonstrated increased scores in the APAP mice at 4, 8 and 24 h compared to the CON mice. APAP+NAC mice had MRI scores similar to the CON mice. Semi-quantitative analysis of hepatic hemorrhage strongly correlated with serum ALT. Small animal MRI can be used to monitor the evolution of APAP toxicity over time and to evaluate the response to therapy.
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