The Danish contribution to the European DEMOCOPHES project: A description of cadmium, cotinine and mercury levels in Danish mother-child pairs and the perspectives of supplementary sampling and measurements.

Human biomonitoring (HBM) is an important tool, increasingly used for measuring true levels of the body burdens of environmental chemicals in the general population. In Europe, a harmonized HBM program was needed to open the possibility to compare levels across borders. To explore the prospect of a harmonized European HBM project, DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) was completed in 17 European countries. The basic measurements performed in all implemented countries of DEMOCOPHES included cadmium, cotinine and phthalate metabolites in urine and mercury in hair. In the Danish participants, significant correlations between mothers and children for mercury in hair and cotinine in urine were found. Mercury in hair was further significantly associated with intake of fish and area of residence. Cadmium was positively associated with BMI in mothers and an association between cadmium and cotinine was also found. As expected high cotinine levels were found in smoking mothers. For both mercury and cadmium significantly higher concentrations were found in the mothers compared to their children. In Denmark, the DEMOCOPHES project was co-financed by the Danish ministries of health, environment and food safety. The co-financing ministries agreed to finance a number of supplementary measurements of substances of current toxicological, public and regulatory interest. This also included blood sampling from the participants. The collected urine and blood samples were analyzed for a range of other persistent and non-persistent environmental chemicals as well as two biomarkers of effect. The variety of supplementary measurements gives the researchers further information on the exposure status of the participants and creates a basis for valuable knowledge on the pattern of exposure to various chemicals.

Is bupropion a more specific substrate for porcine CYP2E than chlorzoxazone and p-nitrophenol?

Porcine microsomes are able to hydroxylate chlorzoxazone and p-nitrophenol, the most commonly used human test substrates for CYP2E1. However, in pigs, CYP2E appears not to be the only enzyme involved in the hydroxylation of chlorzoxazone and p-nitrophenol, as the enzyme capacity and immunochemical level of the apoprotein do not correlate. The present study shows that the hydroxylation of chlorzoxazone and p-nitrophenol is inhibited 50-65% by anti-human CYP2A6, suggesting that these substrates are metabolized almost equally well by CYP2A and CYP2E in pigs. To find an alternative probe to porcine CYP2E, bupropion, another human substrate, was examined. Incubation with bupropion concentrations ranging from 0.05 to 20 mM and with various inhibitors revealed that this substrate is metabolized by both CYP2A and CYP2E. At the high substrate concentration (5 mM), however, the CYP2A6 inhibition decreased compared to inhibition percentages found using the low substrate concentration (0.5 mM). The opposite was found for CYP2E, as inhibition studies with antibodies and diethyldithiocarbamate indicate that it catalysed a negligible part of the reaction at the low substrate concentration and up to 84% at the high concentration. Thus, hydroxylation of bupropion follows the same pattern in pigs as in human beings and the activity measured in pigs is comparable with the human counterpart. Furthermore, bupropion is a more specific substrate for CYP2E than chlorzoxazone and p-nitrophenol although not perfect.

Regulation of gender-dependent CYP2A expression in pigs: involvement of androgens and CAR.

The expression of drug metabolizing cytochrome P4502A (CYP2A) is highly gender-dependent in minipigs with the highest activity in females. In other species, orthologs of CYP2A have been shown to be under the regulation of nuclear receptor constitutive androstane receptor, whereas little is known about regulation in pigs. To investigate the effect of sex hormones on porcine cytochrome P450 CYP2A and CYP3A expression was assessed in liver samples taken before and after castration of sexually mature minipig boars. Removal of the primary androgen source resulted in significant increases of CYP2A mRNA, protein and enzyme activity levels. Likewise, expression of CYP3A was increased, although to a lesser extent. To examine the involvement of constitutive androstane receptor in the regulation of CYP2A, primary porcine hepatocytes were exposed to modulators of murine constitutive androstane receptor and human constitutive androstane receptor activity. The CYP2A activity was significantly increased by exposure to phenobarbital, an indirect activator of constitutive androstane receptor, and the human constitutive androstane receptor-ligand CITCO. In contrast, no effect was seen following exposure to the potent murine constitutive androstane receptor-ligand TCPOBOP and the hormonal murine constitutive androstane receptor-ligands androstenol and oestrone. Thus, the results support that 1) porcine CYP2A is reversibly inhibited by androgens on a transcriptional basis in vivo; 2) the induction profile of CYP2A in vitro shares similarity with that of human constitutive androstane receptor-regulated CYPs, indicating an involvement of a porcine constitutive androstane receptor in the regulation of CYP2A.

Porcine CYP2A polymorphisms and activity.

CYP2A6 in man catalyzes the oxidation of nicotine-forming cotinine and 7-hydroxylation of coumarin, which is used as test substrate for CYP2A6 in man. Large interindividual differences are found in man and some are due to genetic polymorphism. The 7-hydroxylation of coumarin is present in pigs, and an inter-individual variation has been found that might be due to polymorphisms. To enable the finding of polymorphism in pigs, the minipig cDNA was sequenced. Two cDNAs were found and translated to a 494 and a 487 amino acid long protein, both cDNAs were found in all but one pig. The 494 a.a. protein showed high homology to the human and 100% homology to the conventional pig CYP2A19 protein. In the wild type protein, all 6 substrate recognition sites were found, whereas the short protein only contained the first 5 substrate recognition sites. SSCP analysis revealed 3 polymorphisms. In order to study the effect of these polymorphisms on enzyme activity, microsomes were incubated with nicotine and coumarin. The polymorphisms appeared to have no effect on either enzyme activity as the specific enzyme activity towards nicotine and coumarin were approximately the same for all pigs. The specificity of pig CYP2A was investigated and it was found that the formation of cotinine correlated with the immunochemical level of CYP2A as did the coumarin hydroxylation. Anti-human CYP2A inhibitory antibody inhibited coumarin 7-hydroxylation by about 90% and formation of cotinine by 44--60% and 85--100% at substrate concentrations of 500 microM and 50 microM respectively, showing that coumarin and nicotine (50 microM) are very specific substrates for CYP2A in pigs, whereas the CYP2A only is responsible for about 50% of the cotinine formation at a 500 microM nicotine incubation concentration. These results show that the large interindividual differences in porcine CYP2A activity are not caused by polymorphisms but transcriptional regulation and the coumarin 7-hydroxylation is as specific a reaction for porcine CYP2A as for human CYP2A6.

2-heptyl-formononetin increases cholesterol and induces hepatic steatosis in mice.

Consumption of isoflavones may prevent adiposity, hepatic steatosis, and dyslipidaemia. However, studies in the area are few and primarily with genistein. This study investigated the effects of formononetin and its synthetic analogue, 2-heptyl-formononetin (C7F), on lipid and cholesterol metabolism in C57BL/6J mice. The mice were fed a cholesterol-enriched diet for five weeks to induce hypercholesterolemia and were then fed either the cholesterol-enriched diet or the cholesterol-enriched diet-supplemented formononetin or C7F for three weeks. Body weight and composition, glucose homeostasis, and plasma lipids were compared. In another experiment, mice were fed the above diets for five weeks, and hepatic triglyceride accumulation and gene expression and histology of adipose tissue and liver were examined. Supplementation with C7F increased plasma HDL-cholesterol thereby increasing the plasma level of total cholesterol. Supplementation with formononetin did not affect plasma cholesterol but increased plasma triglycerides levels. Supplementation with formononetin and C7F induced hepatic steatosis. However, formononetin decreased markers of inflammation and liver injury. The development of hepatic steatosis was associated with deregulated expression of hepatic genes involved in lipid and lipoprotein metabolism. In conclusion, supplementation with formononetin and C7F to a cholesterol-enriched diet adversely affected lipid and lipoprotein metabolism in C57BL/6J mice.

The influence of bovine milk high or low in isoflavones on hepatic gene expression in mice.

Isoflavones have generated much attention due to their potential positive effects in various diseases. Phytoestrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phytoestrogens. The aim of this study was to analyze the changes in hepatic gene expression after dietary intake of milk high and low in isoflavones. In addition to pelleted feed female NMRI mice were offered water, water added either 17beta-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array qPCR kit. It was revealed that Tween 80 and 17beta-estradiol upregulated both phase I and phase II genes to the same extent whereas equol alone, high and low isoflavone milk did not alter the expression of phase I genes but decreased the expression of phase II genes. This study shows that dietary isoflavones can regulate the transcription of especially phase II liver enzymes which potentially could give rise to an increase in reactive oxygen metabolites that may contribute to the development of cancer.

Analyses of CYP2C in porcine microsomes.

The cytochrome P450 2C (CYP2C) subfamily in human beings includes four different isoenzymes that metabolize different substrates although with some cross reactivity. Some of these substrates (e.g. diclofenac and tolbutamide), have been investigated in porcine microsomes, but without identifying the specific CYP catalysing the reactions. The objective of this study was therefore to test some CYP2C substrates and identify the porcine CYP2C responsible for the reaction. Three substrates, paclitaxel, tolbutamide and omeprazole, were chosen, as they are metabolized by three different CYP2C isoenzymes in human beings. Microsomes, isolated from 20 different pigs, 12 conventional, and 8 minipigs, were incubated with these compounds, and correlations between the metabolism rates of these three substrates were found indicating that the reactions are catalysed by the same enzyme. Male minipigs tend to have higher average activity than female minipigs, which is in contrast to the gender-dependent expression seen for other CYP isoenzymes. The metabolic activities correlated with the protein level determined in Western blotting, using anti-human CYP2C9, indicating that this enzyme is responsible for the reaction. The expression of the CYP2C enzymes was analysed by real-time polymerase chain reaction, using a primer set that could amplify CYP2C8, CYP2C9 and CYP2C19. The melting curves (peaks) revealed that all three genes were present, showing very different expression levels in the various types of pigs. The area of one of the peaks, however, correlated with the CYP2C9-like enzyme concentration, suggesting that this peak represents CYP2C9. Among paclitaxel, tolbutamide and omeprazole, omeprazole is the best probe of CYP2C9-like enzyme in the pig.

Is cytochrome P450 CYP2D activity present in pig liver?

The presence of CYP2D in pig livers has been studied using different strains of pig, different CYP2D test substrates and monoclonal and polyclonal antibodies. The results of the studies lacked consistency, therefore the aim of this study was to identify the reasons for these inconsistencies. Liver microsomes isolated from conventional pigs and minipigs were tested in Western blotting using both monoclonal and polyclonal antibodies against human CYP2D6. The microsomes were also incubated with three different CYP2D tes t substrates.'The immunoblotting only gave a positive response when hybridised with polyclonal antibody. The pig microsomes did not metabolise debrisoquine, but metabolised two other test substrates, dextromethorphan and bufuralol. No correlation was found between the two enzyme assays and CYP2D apoprotein level. On the other hand positive correlations were found between dextromethorphan and bufuralol metabolism and the CYP2B immunochemical protein level, indicating that the CYP2B isoenzyme may be involved in the metabolism of these substrates. Further, assays using immunoinhibition and chemical inhibition of these reactions were performed. No response was obtained in the immunoinhibition assay. When using chemical inhibition, however, an average inhibition percentage of 83 were obtained with orphenadrine, a human CYP2B inhibitor. Average Ki values of 26.9 microM and 43.6 microM for orphenadrine indicate that it was a potent inhibitor. A rat and a mouse CYP2B inhibitor, resveratrol and pilocarpine, inhibited the reaction with an average of 40 and 70 percentage respectively. Orphenadrine did not inhibit CYPIA, CYP2A, CYP2E and CYP3A activities up to more than maximum 12 percentage, showing that it was almost selective for dextromethorphan metabolism. These results indicate that dextromethorphan and bufuralol metabolism may be catalysed by CYP2B and not CYP2D.