Roles of cytochrome P450IIE1 in the dealkylation and denitrosation of N-nitrosodimethylamine and N-nitrosodiethylamine in rat liver microsomes

Abstract

N-Nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are widely occurring nitrosamines and require enzyme-catalyzed activation for their carcinogenic actions. The low Km forms of the enzyme are generally considered to be important in the activation of environmental carcinogens. In this work we examined the role of cytochrome P450IIE1--a constitutive enzyme that is also inducible by acetone, ethanol, fasting and other factors--in catalyzing the dealkylation and denitrosation of these two carcinogens. The experimentally determined Km value of NDMA demethylase depended upon the experimental conditions and was lower when lower protein concentrations were used. Low Km values of 15-20 microM were observed for NDMA demethylase with different preparations of microsomes. In the deethylation of NDEA, a low Km of approximately 40 microM was observed for both control and acetone-induced microsomes. Immunoinhibition studies indicated that P450IIE1 was responsible for almost all the low Km NDMA demethylase activity in acetone-induced microsomes and greater than 80% in control microsomes. This enzyme was also responsible for about three-quarters of the low Km NDEA deethylase activity in acetone-induced microsomes and about half in control microsomes. The denitrosation of NDMA and NDEA was inhibited to approximately the same extents as the dealkylation reactions under different experimental conditions, suggesting the involvement of the same enzyme and perhaps a common initial intermediate in these two types of reactions. The relevance of this work and its relationship to related information in the literature are discussed.

Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole

“…Our results indicate a metabolism-mediated interaction with cinnamon or t-CA would be markedly more probable for CYP2A6 substrates than for drugs metabolized by the other P450 isoforms typically evaluated during drug discovery (Food and Drug Administration, 2012). Even though CYP2A6 and CYP2E1 exhibit overlap in substrate selectivity (Yoo et al, 1990;Yamazaki et al, 1992;Chen et al, 1998;Harrelson et al, 2007), t-CA was 10.5-fold more selective for CYP2A6. The IC 50 values for other major drug-metabolizing P450s were at least 16-fold higher or more, and in some cases, substantially higher (i.e., 2D6 and 2C19).…”

Effects of N‐acetylcysteine in an esophageal carcinogenesis model in rats treated with diethylnitrosamine and diethyldithiocarbamate

“…18,19 Other studies have shown that DEDTC and its parental compound disulfiram, whose molecule is formed of 2 DEDTC moieties, affect the metabolic activation of DEN and cause a shift of the carcinogenic response from the liver to the esophagus. 21,22 On the other hand, it was also demonstrated that injection of DEDTC, after a single necrogenic dose of DEN, significantly inhibited both liver toxicity and induction of ␥-glutamyltranspeptidase (␥-GT)-positive foci, without affecting ethylation of liver DNA.…”

The binding affinity of carcinogenic N-nitrosodimethylamine and N-nitrosomethylaniline to cytochromes P450 2B4, 2E1 and 3A6 does not dictate the rate of their enzymatic N-demethylation

“…Even though the CYP2E1 and CYP2B enzymes were shown to oxidize two members of this group of carcinogens, NDMA Amelizad et al 1988;Yang et al 1990Yang et al , 1994Yoo et al 1990;Encell et al 1996;Stiborova et al 1996;Smith et al 1998;Sulc et al, 2004) and NMA Stiborova et al 1996aStiborova et al ,b, 1999Kushida et al 2000;Sulc et al 2004), respectively, there are still some discrepancies found in earlier studies about their efficiencies in the reactions. Likewise, the knowledge on the detailed kinetics of reactions catalyzed by these enzymes is still limited.…”

“…To avoid any potential interference found in former studies utilizing perfused livers, hepatocytes, microsomes and/or partially purified enzymes (Skipper et al 1983;Tu and Yang 1985;Yang et al 1985Yang et al , 1990Yoo et al 1990;Encell et al 1996;Stiborova et al 1996a,b;Smith et al 1998;Sulc et al 2004), the CYP2E1 and CYP2B4 enzymes, isolated from rabbit livers to homogeneity, were used. The enzymatic experiments were performed under the reaction conditions minimizing any inhibition effects that might be caused by some compounds present in incubation mixtures of former studies.…”

“…This discrepancy is likely caused by various artificial effects such as high concentrations of glycerol, thiol-compounds or residual detergents from purification procedures, which are present in in-vitro experiments, whereas other compounds such as proteins and nucleic acids, influencing the kinetics of N-nitrosamine oxidation, are present in tissue cells in vivo (Yoo et al 1987. Furthermore, when microsomes were used to study a demethylation of NDMA and NMA, the multiplicity of CYP enzymes in these subcellular systems resulted in determination of more than one K m Yoo et al 1990;Stiborova et al 1996a;Sulc et al 2004).…”