John D. Hayes

Polymorphism in cytochrome P450 CYP2D6, CYP1A1, CYP2E1 and glutathione S-transferase, GSTM1, GSTM3, GSTT1 and susceptibility to tobacco-related cancers: studies in upper aerodigestive tract cancers.

Glutathione S-transferase GSTM1, GSTM3 and GSTT1 and cytochrome P450 CYP2D6, CYP1A1 and CYP2E1 loci are susceptibility candidates for cancers of the upper aerodigestive tract because putatively protective and risk genotypes have been identified from studies in other diseases associated with alcohol and tobacco consumption. We describe genotype frequencies in 398 oral, pharyngeal and laryngeal squamous cell carcinoma patients and 219 control individuals. Of the genotypes presumed to be protective, only GSTM1 A/B influenced susceptibility; the GSTM1 A/B frequency was lower in the patients than the control individuals both before [odds ratio = 0.3, 95% confidence interval (CI) 0.1-0.7] and after correction for imbalances in age, sex, smoking and alcohol consumption (odds ratio = 0.2, 95% CI 0.1-0.5). Of the putatively risk genotypes, GSTM3 AA, previously associated with susceptibility to skin cancer, was higher in the cases (odds ratio = 1.6, 95% CI 1.1-2.4). Dividing cases into oral/pharyngeal and laryngeal squamous cell carcinoma showed the GSTM3 AA frequency was higher in laryngeal squamous cell carcinoma than control individuals (odds ratio = 1.6, 95% CI 1.1-2.5) and the difference between control individuals and oral/pharyngeal squamous cell carcinoma approached significance (odds ratio = 1.7, 95% CI 1.0-2.8). The putatively protective GSTM3 BB genotype was lower in patients with glottic (1.0%) than supraglottic (3.0%) squamous cell carcinoma. We identified no differences between patients and control individuals in the frequencies of presumed risk genotypes (e.g. CYP2D6 EM, CYP1A1 m1/m1, CYP1A1 Ile/Ile, CYP2E1 DD, CYP2E1 c1c1, GSTT1 null) or, interactions between genotypes and smoking or alcohol consumption. We conclude, first, that mu class glutathione S-transferase influence risk of upper aerodigestive tract cancers thereby complementing studies in skin cancer patients showing GSTM1 A/B is protective, while GSTM3 AA moderately increases risk. The influence of GSTM1 A/B, but not GSTM1 A or GSTM1 B (mostly heterozygotes with GSTM1*0) suggests that two expressed alleles may attenuate risk. While we found immunohistochemical evidence of GSTM3 expression in the cilia lining the larynx, the biochemical consequences of the polymorphism are unclear. Indeed, the influence of the gene may reflect linkage disequilibrium with another gene. However, we did not find an association with GSTM1 genotypes. Second, we conclude that the CYP2D6, CYP2E1, CYP1A1 and GSTT1 alleles studied, although putatively good candidates, either do not determine the effectiveness of detoxification of tobacco-derived carcinogens in the upper aerodigestive tract or, that chronic consumption of tobacco and alcohol overwhelms enzyme defences, irrespective of genotype.

Purification of acidic glutathione S-transferases from human lung, placenta and erythrocyte and the development of a specific radioimmunoassay for their measurement

Human acidic glutathione S-transferases (GST) have been purified from placenta, lung and erythrocytes. The purification protocol resulted in a high yield of pure protein for each tissue, when compared to previous procedures. An apparent subunit Mr of 24,800 was calculated for each of the acidic GST and each enzymes had a pI of 4.75. No immunochemical differences were detected between the acidic GST isolated from the three tissues. A specific and sensitive radioimmunoassay suitable for the measurement of human acidic GST in plasma or tissues is described.

Glutathione S-transferase in human bile

Glutathione S-transferase (GST) isoenzymes have been measured by specific radioimmunoassay in human bile samples. GST Mu was found in 50% of samples while GST Pi, GST B1 and GST B2 were present in all samples; GST Pi constituted the major isoenzyme identified. The findings of the radioimmunoassay were confirmed by a one-step purification of GST from bile, using affinity chromatography, followed by their identification using sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). Inhibition studies showed that, at the concentrations of bile salts found in bile, GST Pi would have little or no enzymic activity. It is proposed that GST Pi acts as a carrier protein of toxic, non-substrate, ligands to remove as yet unidentified substances from biliary epithelial cells and prevent their reabsorption.

Nrf2-mediated neuroprotection against recurrent hypoglycemia is insufficient to prevent cognitive impairment in a rodent model of type 1 diabetes

It remains uncertain whether recurrent nonsevere hypoglycemia (Hypo) results in long-term cognitive impairment in type 1 diabetes (T1D). This study tested the hypothesis that specifically in the T1D state, Hypo leads to cognitive impairment via a pathological response to oxidative stress. Wild-type (Control) and nuclear factor–erythroid 2 p45–related factor 2 (Nrf2) null mice were studied. Eight groups of mice (Control and Nrf2−/− ± T1D and ± Hypo) were subject to recurrent, twice-weekly, insulin or saline injections over 4 weeks, after which cognitive function was assessed and brain tissue analyzed. Recurrent moderate hypoglycemia in T1D, but not Control, mice significantly impaired cognitive performance, and this was associated with hippocampal oxidative damage and inflammation despite an enhanced expression of Nrf2 and its target genes Hmox1 and Nqo1. In Nrf2−/− mice, both T1D and Hypo independently resulted in impaired cognitive performance, and this was associated with oxidative cell damage and marked inflammation. Together, these data suggest that Hypo induces an Nrf2-dependent antioxidant response in the hippocampus, which counteracts oxidative damage. However, in T1D, this neuroprotective mechanism is insufficient to prevent neuronal oxidative damage, resulting in chronic deficits in working and long-term memory.