Genes and enzymes of metabolic activation of xenobiotics in chemical carcinogenesis

In the initial stage of chemical carcinogenesis the primary key event is metabolic activation of exogenic carcinogenic substances. The main enzymes of carcinogen's biotransformation (microsomal hydroxylation, reactions of conjugation) and genes which controlling the activity of these enzymes, has been characterized. The tissue(organ)specificity of expression of gene products (isoforms of su-perfamilies of CYPs and GSTs, family of NATs) as well as genetic polymorphism of enzymes involving into the biotransformation of carcinogenic xenobiotics were demonstrated

Structural requirements for the utilization of ascorbate analogues in the prolyl 4-hydroxylase reaction

The ability of structural analogues of ascorbate to serve as substitutes for this reducing agent in the prolyl 4-hydroxylase reaction was studied. In experiments using the purified enzyme, variations of the compounds′ side chain were compatible with co-substrate activity. The presence of very large hydrophobic substituents or a positively charged group caused an increase in the observed Km values. A negative charge and smaller modifications did not change the affinity to the enzyme when compared with L-ascorbate. 6-Bromo-6-deoxy-L-ascorbate had a lower Km than the physiological reductant. Substitution at the -OH group in ring position 3 prevented binding to the enzyme. The same pattern of activity was observed when the full and uncoupled prolyl 4-hydroxylase reactions were studied. The Vmax. values with all compounds were similar. The reaction of microsomal prolyl 4-hydroxylase was supported by D-isoascorbate, O6-tosyl-L-ascorbate and 5-deoxy-L-ascorbate, giving the same dose-response behaviour as L-ascorbate itself. Again, 6-bromo-6-deoxy-L-ascorbate gave a lower Km and a similar Vmax. value. L-Ascorbic acid 6-carboxylate produced substrate inhibition at concentrations above 0.3 mM. The Km and Vmax. values calculated from concentrations up to 0.2 mM were similar to those of L-ascorbate. The enzyme activity observed with 6-amino-6-deoxy-L-ascorbate was very low in the microsomal hydroxylation system. The calculated Vmax. value was lower than that of L-ascorbate, suggesting a restriction of the access of this compound to the enzyme.

The characteristics of the microsomal hydroxylation of tolbutamide

The in vitro metabolism of tolbutamide to the hydroxymethyl derivative was studied using hepatic microsomal homogenates. The hydroxymethyl metabolite was quantitated by HPLC. The hepatic microsomal hydroxylase was completely inhibited by carbon monoxide and was NADPH dependent. Metyrapone, α-naphthoflavone, phenelzine, mercuric chloride, and nitrogen significantly inhibited the reaction indicating the involvement of the cytochrome P-450 monooxygenase. Species variation showed that the order of hepatic microsomal activity was rat > rabbit >> guinea pig >> mouse and hamster. The reaction increased with time up to 40 min and followed Michaelis–Menten kinetics in rat liver microsomes with apparent Km and Vmax values of 224.4 μM and 359.9 pmol∙mg−1∙min−1, respectively. The reaction was induced by phenobarbital but was depressed after pretreatment with 3-methylcholanthrene and isosafrole. However, expression of the hydroxylase activity per nanomoles of cytochrome P-450 showed that the activity was much higher in liver microsomes of isosafrole pretreated rats. These results indicate the involvement of different isozymes of cytochrome P-450 in the microsomal hydroxylation of tolbutamide.Key words: tolbutamide metabolism, tolbutamide hydroxylation, microsomal hydroxylation, microsomal metabolism of tolbutamide, hepatic metabolism of tolbutamide.