Glutathione (GSH)-driven lipid peroxidation (LPO) in vitro was catalyzed by gamma-glutamyltranspeptidase (GGT; EC 2.3.2.2.). The reaction required iron, iron chelators and oxygen, was accelerated by glycylglycine (gly)2, a GGT enhancer, and was inhibited by the GGT inhibitors serine--borate and acivicin. LPO occurred at rat plasma concentrations of GSH and transferrin, and in the presence of putative physiological chelators such as citrate and ADP. GSH-driven LPO was inhibited by butylated hydroxytoluene, but not by catalase, peroxidase or superoxide dismutase. These results suggest that metabolism of GSH initiated by GGT may lead to oxidative damage. Such oxidative damage may be induced in vivo by GSH in proximity to GGT-rich preneoplastic foci in rat liver.

Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N-acetylcysteine or by co-culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte-neuron c-glutamyl cycle is up-regulated by Ethanol, enhancing control of neuron GSH in response to the pro-oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of c-glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrosoyte up to 517% (p < 0.05). Ethanol increased both c-glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p ¼ 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the c-glutamyl cycle that are up-regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanolmediated oxidative stress and apoptotic death.

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.

Previous studies from our laboratories have shown that catabolism of glutathione (GSH) by gamma-glutamyl transpeptidase (GGT) in the presence of transition metals leads to oxidative damage (OD). This damage is exemplified in vitro by GGT-dependent GSH mutagenesis which involves reactive oxygen species and by GGT-dependent accumulation of lipid peroxidation (LPO) products in systems containing polyunsaturated fatty acid and GSH. In order to test whether catabolism of GSH by membranal GGT in enzyme-altered preneoplastic hepatic lesions can induce oxidative damage in situ, and to test whether the OD is localized in these lesions, 21 day old Fischer rats were treated with 12 mg/kg diethylnitrosamine (DEN) followed by 0.1% or 0.25% phenobarbital (PB) in the diet. Cryostat sections were examined histochemically for GGT-rich hepatic lesions. Adjacent sections were incubated with GSH and iron and examined for areas staining for lipid peroxidation. Distinct LPO-positive areas were shown to correspond well with the GGT-positive hepatic lesions. Promotion with 0.25% PB led to increasing proportions of LPO-positive lesions with time among GGT-positive lesions. The visualization of LPO in GGT-rich hepatic lesions depended on the presence of GSH and iron, and was not observed following chelation of iron by diethyl triaminopentaacetic acid (DTPA), in the presence of acivicin, an inhibitor of GGT, or in the presence of the radical scavenger butylated hydroxytoluene (BHT). The factors affecting GSH-GGT-dependent LPO in the GGT-rich foci were identical to those affecting GSH-GGT-driven LPO in vitro, and were similar to those affecting oxidative GSH-mutagenesis catalyzed by GGT. The results indicate that metabolism of GSH by GGT in preneoplastic liver foci can initiate an oxidative process leading to a radical-rich environment and to oxidative damage. Such damage may contribute to the processes by which cells within such foci progress to malignancy.

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