Molecular modelling of the human glucocorticoid receptor (hGR) ligand-binding domain (LBD) by homology with the human estrogen receptor alpha (hERalpha) LBD: quantitative structure-activity relationships within a series of CYP3A4 inducers where induction is mediated via hGR involvement.

The results of homology modelling of the human glucorticoid receptor (hGR) ligand-binding domain (LBD) based on the ligand-bound domain of the human estrogen receptor alpha (hERalpha) are reported. It is shown that known hGR ligands which induce the human cytochrome P450 enzyme CYP3A4 are able to fit the putative ligand-binding site of the nuclear hormone receptor and form hydrogen bonds with key amino acid residues within the binding pocket. Quantitative structure-activity relationships (QSARs) have been derived for hGR-mediated CYP3A4 induction which involve certain molecular structural and physicochemical properties of the ligand themselves, yielding good correlations (R=0.96-0.98) with fold induction of CYP3A4 known to be mediated via hGR involvement.

The interleukin-6 -174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men.

AIMS: Inflammation is a key component of coronary heart disease, and genes coding for cytokines are candidates for predisposing to coronary heart disease risk. We have examined the effect of two polymorphisms (-174G>C and -572G>C) in the promoter of the interleukin-6 (IL-6) gene on risk of coronary heart disease, and on intermediate risk traits including fibrinogen and systolic blood pressure, in 2751 middle-aged healthy U.K. men. RESULTS: The -174C allele (frequency 0.43, 95% CI 0.42-0.44) was not associated with significant effects on fibrinogen levels, but was associated with a significantly (P=0.007) higher systolic blood pressure (mean mmHg (95% CI): GG=135.5 (134.3-136.7); GC=137.9 (136.9-138.9); CC= 138.0 (136.3-139.8)). This effect was of similar magnitude in smokers and non-smokers, and was greater in men in the top two tertiles of body mass index (>24.86 kg x m(-2)) than in those in the bottom tertile. Compared to those with the genotype GG, men carrying the -174C allele had a relative risk of coronary heart disease of 1.54 (95% CI 1.0-2.23, P=0.048) and this effect was greatest in smokers (compared to GG non-smokers, RR 2.66, CI 1.64-4.32). These effects remained statistically significant after adjusting for classical risk factors including blood pressure (P=0.04). The -572C allele (frequency 0.05, 0.04-0.06) was not associated with a significant effect on blood pressure, fibrinogen or relative risk of coronary heart disease. In a subset of the genotyped men (n=494), carriers of the -174C allele had higher levels of C-reactive protein than non-carriers. CONCLUSIONS: These data confirm the importance of the inflammatory system in the development of coronary heart disease. They suggest that, at least in part, the effect of the IL-6 -174G>C polymorphism on blood pressure is likely to be operating through inflammatory mechanisms, but the genotype effect on coronary heart disease risk is largely unexplained by its effect on blood pressure. The molecular mechanisms whereby genetically determined differences in plasma levels of IL-6 are having these effects remain to be determined.

A reporter gene assay to assess the molecular mechanisms of xenobiotic-dependent induction of the human CYP3A4 gene in vitro.

1. A plasmid containing 1 kb of the CYP3A4 regulatory (promoter) region coupled to a reporter gene for secretary placental alkaline phosphatase (SPAP) was transfected into HepG2 cells. Transfected cells were dosed with several known inducers of CYP3A4 and the levels of SPAP were measured. The effect of co-transfecting a plasmid encoding the human glucocorticoid receptor on reporter gene activity was also examined. 2. Dexamethasone induced CYP3A4-dependent reporter gene expression in a concentration-dependent manner and induction was approximately doubled in the presence of the glucocorticoid receptor. Dexamethasone-dependent induction was blocked by RU-486 (a glucocorticoid receptor antagonist), in the presence of the co-transfected glucocorticoid receptor. 3. Induction of CYP3A4-dependent reporter gene expression and enhancement of the induction by the glucocorticoid receptor was also observed with pregnenolone-16alpha-carbonitrile (PCN), rifampicin, phenytoin, carbamazepine, phenylbutazone and phenobarbitone, all known in vivo inducers of CYP3A4 in man. 4. Metyrapone and sulfinpyrazone induced CYP3A4-dependent reporter gene expression, but induction was not enhanced by the glucocorticoid receptor. 5. Clotrimazole, erythromycin and triacetyloleandomycin (TAO) did not induce CYP3A4-dependent reporter gene expression, consistent with the observation that these inducers act through post-transcriptional mechanisms. 6. These results highlight differences in the molecular mechanisms of induction of CYP3A4 by the xenobiotics studied and indicate that the glucocorticoid receptor is involved in the induction of the CYP3A4 gene by some, but not all, CYP3A4 inducers. 7. We propose that the approach described here provides a useful in vitro approach for the identification of transcriptional regulators of the CYP3A4 gene.

Development of an in vitro reporter gene assay to assess xenobiotic induction of the human CYP3A4 gene.

The current work concerns the development and validation of an in vitro reporter gene assay system for the assessment of induction of human CYP3A4. A plasmid containing approximately 1 kb of the CYP3A4 regulatory region (which contains several recognised regulatory elements including glucocorticoid responsive elements) coupled to the reporter gene for human secreted placental alkaline phosphatase (SPAP) was transfected into the human hepatoblastoma cell line HepG2. Calcium phosphate precipitation was the method of choice for transfection. The transfected cells were dosed with known inducers of CYP3A4 and the levels of SPAP in the medium were subsequently measured using a chemiluminescent assay, as an indirect measure of CYP3A4 induction. The inducers used in this study included dexamethasone, phenytoin, triacetyloleandomycin (TAO), rifampicin, carbamazepine, phenylbutazone and sulfinpyrazone. These compounds activated CYP3A4 by between 1.5-4.5-fold thus representing a major advance in assessing the induction of human CYP genes in vitro.

Studies on the differential sensitivity between diabetic and non-diabetic human erythrocytes to monoacetyl dapsone hydroxylamine-mediated methaemoglobin formation in vitro.

Methaemoglobin generation by monoacetyl dapsone hydroxylamine in non-diabetic and diabetic erythrocytes was investigated in vitro. Methaemoglobin formation in purified haemoglobin isolated from both types of erythrocytes as well as haemolysates from both diabetic and non-diabetic erythrocytes did not differ. Prior to 18 h incubation with 10 and 20 mM glucose diabetic erythrocytes were significantly less sensitive to monoacetyl dapsone-induced methaemoglobinaemia. After pre-incubation the differential was lost although significant change in glutathione concentrations could not be shown between the two cell types. NADH-diaphorase levels measured in diabetics and non-diabetics did not significantly differ. It is possible that diabetic cells display reduced hydroxylamine-mediated methaemoglobin generation due to differences in glutathione metabolism.