James P. Hardwick

Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic fatty liver of rats†

Increased oxidative/nitrosative stress is a major contributing factor to alcohol‐mediated mitochondrial dysfunction. However, which mitochondrial proteins are oxidatively modified under alcohol‐induced oxidative/nitrosative stress is poorly understood. The aim of this study was to systematically investigate oxidized and/or S‐nitrosylated mitochondrial proteins and to use a biotin‐N‐maleimide probe to evaluate their inactivation in alcoholic fatty livers of rats. Binge or chronic alcohol exposure significantly elevated nitric oxide, inducible nitric oxide synthase, and ethanol‐inducible CYP2E1. The biotin‐N‐maleimide‐labeled oxidized and/or S‐nitrosylated mitochondrial proteins from pair‐fed controls or alcohol‐fed rat livers were subsequently purified with streptavidin‐agarose. The overall patterns of oxidized and/or S‐nitrosylated proteins resolved by 2‐dimensional polyacrylamide gel electrophoresis were very similar in the chronic and binge alcohol treatment groups. Seventy‐nine proteins that displayed differential spot intensities from those of control rats were identified by mass spectrometry. These include mitochondrial aldehyde dehydrogenase 2 (ALDH2), ATP synthase, acyl‐CoA dehydrogenase, 3‐ketoacyl‐CoA thiolase, and many proteins involved in chaperone activity, mitochondrial electron transfer, and ion transport. The activity of 3‐ketoacyl‐CoA thiolase involved in mitochondrial β‐oxidation of fatty acids was significantly inhibited in alcohol‐exposed rat livers, consistent with hepatic fat accumulation, as determined by biochemical and histological analyses. Measurement of activity and immunoblot results showed that ALDH2 and ATP synthase were also inhibited through oxidative modification of their cysteine or tyrosine residues in alcoholic fatty livers of rats. In conclusion, our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol‐mediated liver damage. (HEPATOLOGY 2006;44:1218–1230.)

Specificity of cDNA‐expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12‐dimethylbenz[a]anthracene

7,12‐Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6‐ to 33‐fold more activity than other P450s. 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0–2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0‐ to 37‐fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9‐diol, but human 2B6 and rat 281 preferentially formed the 5,6‐diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7‐hydroxymethyl‐12‐methylbenz[a]anthracene (7HOM12MBA) than 12‐hydroxymethyl‐7‐methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans‐8,9‐dihydrodiol > 7HOM12MBA ≥ DMBA trans‐5,6‐dihydrodiol ≥ 7,12‐dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans‐3,4‐dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver.

Role of peroxisome proliferator-activated receptor-α in fasting-mediated oxidative stress

The peroxisome proliferator-activated receptor-alpha (PPARalpha) regulates lipid homeostasis, particularly in the liver. This study was aimed at elucidating the relationship between hepatosteatosis and oxidative stress during fasting. Fasted Ppara-null mice exhibited marked hepatosteatosis, which was associated with elevated levels of lipid peroxidation, nitric oxide synthase activity, and hydrogen peroxide accumulation. Total glutathione (GSH), mitochondrial GSH, and the activities of major antioxidant enzymes were also lower in the fasted Ppara-null mice. Consequently, the number and extent of nitrated proteins were markedly increased in the fasted Ppara-null mice, although high levels of protein nitration were still detected in the fed Ppara-null mice while many oxidatively modified proteins were only found in the fasted Ppara-null mice. However, the role of inflammation in increased oxidative stress in the fasted Ppara-null mice was minimal based on the similar levels of tumor necrosis factor-alpha change in all groups. These results with increased oxidative stress observed in the fasted Ppara-null mice compared with other groups demonstrate a role for PPAR alpha in fasting-mediated oxidative stress and that inhibition of PPAR alpha functions may increase the susceptibility to oxidative damage in the presence of another toxic agent.