Competing Mechanistic Hypotheses of Acetaminophen-Induced Hepatotoxicity Challenged by Virtual Experiments.

Acetaminophen-induced liver injury in mice is a model for drug-induced liver injury in humans. A precondition for improved strategies to disrupt and/or reverse the damage is a credible explanatory mechanism for how toxicity phenomena emerge and converge to cause hepatic necrosis. The Target Phenomenon in mice is that necrosis begins adjacent to the lobule's central vein (CV) and progresses outward. An explanatory mechanism remains elusive. Evidence supports that location dependent differences in NAPQI (the reactive metabolite) formation within hepatic lobules (NAPQI zonation) are necessary and sufficient prerequisites to account for that phenomenon. We call that the NZ-mechanism hypothesis. Challenging that hypothesis in mice is infeasible because 1) influential variables cannot be controlled, and 2) it would require sequential intracellular measurements at different lobular locations within the same mouse. Virtual hepatocytes use independently configured periportal-to-CV gradients to exhibit lobule-location dependent behaviors. Employing NZ-mechanism achieved quantitative validation targets for acetaminophen clearance and metabolism but failed to achieve the Target Phenomenon. We posited that, in order to do so, at least one additional feature must exhibit zonation by decreasing in the CV direction. We instantiated and explored two alternatives: 1) a glutathione depletion threshold diminishes in the CV direction; and 2) ability to repair mitochondrial damage diminishes in the CV direction. Inclusion of one or the other feature into NZ-mechanism failed to achieve the Target Phenomenon. However, inclusion of both features enabled successfully achieving the Target Phenomenon. The merged mechanism provides a multilevel, multiscale causal explanation of key temporal features of acetaminophen hepatotoxicity in mice. We discovered that variants of the merged mechanism provide plausible quantitative explanations for the considerable variation in 24-hour necrosis scores among 37 genetically diverse mouse strains following a single toxic acetaminophen dose.

Effect of transgenic extrahepatic expression of betaine-homocysteine methyltransferase on alcohol or homocysteine-induced fatty liver.

BACKGROUND: Chronic alcohol feeding induces hyperhomocysteinemia (HHcy). Previously, we reported a protective role of betaine-homocysteine methyltransferase (BHMT) in homocysteine-induced injury in cultured hepatocytes. In this study, we investigated the direct role of BHMT in alcohol or homocysteine-induced liver injury. METHODS: Betaine-homocysteine methyltransferase transgenic (Tg) mice were generated. Comparisons were made between the Tg and wild type (WT) mice in their response to intragastric alcohol infusion or to oral feeding of a high methionine low folate diet (HMLF). RESULTS: Expression of the Tg BHMT was increased in organs peripheral to the liver. The alcohol infusion for 4 weeks increased: plasma ALT by 5-fold in WT mice and 2.7-fold in Tg mice; plasma homocysteine by 7-fold in WT mice and 2-fold in Tg mice; liver triglycerides by 4-fold in WT mice and 2.5-fold in Tg mice. The alcohol-induced fatty liver was more severe in WT than in Tg mice based on H&E staining. The HMLF feeding for 4 weeks increased plasma ALT by 2-fold in WT mice and 1-fold in Tg mice; plasma homocysteine by 21-fold in WT mice and 3.3-fold in Tg mice; liver triglycerides by 2.5-fold in WT mice and 1.5-fold in Tg mice. HMLF induced accumulation of macro fat droplets in WT but not Tg mice. Betaine supplementation decreased partially the alcohol or HMLF-induced increase of ALT, homocysteine and liver lipids in WT mice. However, Tg mice were normal when fed both HMLF and betaine. In WT mice, both alcohol and HMLF induced moderate increase of sterol regulatory element binding protein 1 (SREBP1) protein which was partially reduced by betaine supplementation. In Tg mice, alcohol but not HMLF increased SREBP1. Carbohydrate responsive element-binding protein was increased by alcohol in either WT or Tg mice which was not affected by betaine supplementation. Ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) was reduced by 50% in WT and by 20% in Tg mice fed alcohol. Ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was reduced in WT but not Tg mice fed alcohol. Changes in PE methyltransferase activities were not detected in response to alcohol or HMLF feeding but were increased by betaine. CONCLUSIONS: The BHMT Tg mice are resistant to alcohol or HMLF-induced HHcy and liver steatosis indicating that peripheral metabolism of homocysteine protected the liver without a direct effect of BHMT in the liver. Multiple mechanisms are involved in protection by betaine including increased SAM/SAH and PC/PE ratios.

3alpha-Hydroxysteroid dehydrogenase type III deficiency: a novel mechanism for hirsutism.

CONTEXT: Dihydrotestosterone (DHT), the primary active androgen in peripheral target tissues, is metabolized by 3alpha-hydroxysteroid dehydrogenase type III (3alpha-HSD), encoded by the AKR1C2 gene, forming 5alpha-androstane-3alpha,17beta-diol (3alpha-diol). 3alpha-HSD may play a role in the pathogenesis of hirsutism. OBJECTIVES: Our objective was to evaluate the role of 3alpha-HSD in hirsutism by comparing 1) tissue levels of active androgens, 2) relative gene expression of AKR1C2, and 3) activity of 3alpha-HSD in genital skin from normal and hirsute women. DESIGN: Genital skin was obtained from normal and hirsute women. After homogenization, testosterone (T) and DHT levels were quantified by conventional RIA. From isolated RNA, relative expression of AKR1C2 was determined by real-time PCR. In addition, minced genital skin was incubated with [(3)H]DHT, and the product, [(3)H]3alpha-diol, was quantified by radio-HPLC. SETTING: The study took place at an inner-city hospital. PATIENTS: PATIENTS included women undergoing posterior colporrhaphy. MAIN OUTCOME MEASURES: We assessed 1) tissue levels of T, DHT, and 3alpha-diol; 2) relative expression of AKR1C2; and 3) conversion ratio of [(3)H]3alpha-diol to [(3)H]DHT. RESULTS: In genital skin, tissue DHT and T concentrations in hirsute women were 1.90-fold and 1.84-fold higher than in normal women (P =0 .002 and 0.03), and relative expression of AKR1C2 mRNA was reduced approximately 7-fold (P = 0.04). Genital skin from hirsute women showed less metabolism of [(3)H]DHT to [(3)H]3alpha-diol (conversion ratio, 0.24 +/- 0.19 vs. 0.85 +/- 0.55, P = 0.01). CONCLUSIONS: In genital skin of hirsute women, reduced AKR1C2 gene expression and 3alpha-HSD activity results in decreased DHT metabolism and elevated tissue levels of DHT. Diminished DHT metabolism may play an important role in the pathogenesis of hirsutism.

Impaired dihydrotestosterone catabolism in human prostate cancer: critical role of AKR1C2 as a pre-receptor regulator of androgen receptor signaling.

We previously reported the selective loss of AKR1C2 and AKR1C1 in prostate cancers compared with their expression in paired benign tissues. We now report that dihydrotestosterone (DHT) levels are significantly greater in prostate cancer tumors compared with their paired benign tissues. Decreased catabolism seems to account for the increased DHT levels as expression of AKR1C2 and SRD5A2 was reduced in these tumors compared with their paired benign tissues. After 4 h of incubation with benign tissue samples, (3)H-DHT was predominantly catabolized to the 5alpha-androstane-3alpha,17beta-diol metabolite. Reduced capacity to metabolize DHT was observed in tumor samples from four of five freshly isolated pairs of tissue samples, which paralleled loss of AKR1C2 and AKR1C1 expression. LAPC-4 cells transiently transfected with AKR1C1 and AKR1C2, but not AKR1C3, were able to significantly inhibit a dose-dependent, DHT-stimulated proliferation, which was associated with a significant reduction in the concentration of DHT remaining in the media. R1881-stimulated proliferation was equivalent in all transfected cells, showing that metabolism of DHT was responsible for the inhibition of proliferation. PC-3 cells overexpressing AKR1C2 and, to a lesser extent, AKR1C1 were able to significantly inhibit DHT-dependent androgen receptor reporter activity, which was abrogated by increasing DHT levels. We speculate that selective loss of AKR1C2 in prostate cancer promotes clonal expansion of tumor cells by enhancement of androgen-dependent cellular proliferation by reducing DHT metabolism.

Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells.

Rat multidrug resistant protein 2 (Mrp2; Abcc2), an ATP-driven pump located on the canalicular domain of hepatocytes, exports glutathione S-conjugates (GS-X) and GSH among its wide variety of substrates. Previous studies have shown that chelerythrine (CHEL), a quaternary benzophenanthridine cation, reacts with GSH to form a reversible adduct under physiological conditions. Here we report that CHEL can strongly stimulate GSH efflux by Mrp2, when it is constitutively expressed in polarized canine kidney cells, thereby leading to the depletion of cellular GSH. Transepithelial transport experiments indicate that Mrp2 transports GSH and CHEL with a 1:1 stoichiometry, which can be readily inhibited by GS-bimane, a GS-X substrate for Mrp2. Moreover, CHEL can block Mrp2-mediated leukotriene C4 uptake by membrane vesicles with an IC50 approximately 100 microM in the presence of GSH, but not S-methyl GSH or ophthalmic acid. Thus the thiol group of GSH is required for inhibition of Mrp2 in the presence of CHEL. Our results suggest that CHEL stimulates GSH efflux by forming a reversible GS-CHEL adduct, which is transported by Mrp2 and dissociates extracellularly.