Stereospecific Deuterium Substitution Attenuates the Tumorigenicity and Metabolism of the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Abstract

Stereochemical determinants of the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were investigated using the stereospecifically deuterated isotopomers (4R)-[4-(2)H(1)]NNK and (4S)-[4-(2)H(1)]NNK. Upon ip administration to groups of 20 female A/J mice, NNK and (4S)-[4-(2)H(1)]NNK exhibited similar lung tumorigenicity at three different doses, whereas (4R)-[4-(2)H(1)]NNK was 2-fold less tumorigenic at all three doses. In a parallel experiment, levels of O(6)-methylguanine and 7-methylguanine were 2-fold lower in lung DNA of mice treated with (4R)-[4-(2)H(1)]NNK than in mice treated with NNK or (4S)-[4-(2)H(1)]NNK. To corroborate these in vivo data, the in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda (Sf9)-expressed mouse cytochrome p450s 2A4 and 2A5. Kinetic isotope effects on the apparent V(max) ((D)V) for the product of NNK 4-hydroxylation, OPB, were 2.7 +/- 0.2 and 2.8 +/- 0.4 when (4R)- and (4S)-[4-(2)H(1)]NNK were incubated with mouse lung microsomes, respectively. The (D)V values for OPB formation were 3.2 +/- 0.2 and 2.2 +/- 0.2 when (4R)-[4-(2)H(1)]NNK was the substrate for p2A4 and 2A5, respectively, whereas they were 1.3 +/- 0.1 and 1.1 +/- 0.1 when (4S)-[4-(2)H(1)]NNK was the substrate for these respective enzymes. Analysis of an OPB derivative (10) for deuterium content by LC/MS confirmed the results from the kinetic assays and indicated that p450s 2A4 and 2A5 preferentially abstract the pro-R 4-hydrogen of NNK. The results obtained using Sf9-expressed p450s provide a rationale for the differences observed in the lung tumor and DNA adduct experiments, namely, that the attenuated tumorigenicity of (4R)-[4-(2)H(1)]NNK relative to (4S)-[4-(2)H(1)]NNK is due to prochiral selectivity during p450-catalyzed metabolic activation.

Progress and Challenges in Selected Areas of Tobacco Carcinogenesis

“…Lung tumor multiplicity is highly correlated with levels of persistent O 6 -methyl-dGuo in DNA (22). This DNA adduct causes G f A mutations in codon 12 of the k-ras oncogene leading to lung tumor formation (23,24 (21). Earlier, we demonstrated a high correlation between persistent O 6 -methyl-dGuo in this model and the number of lung tumors per mouse, as well as the presence of G f A transition mutations in the k-ras gene isolated from the lung tumors (22,23).…”

“…Human P450s 2A13 and 2B6, rat P450 2A3, and mouse P450 2A5 may be the most important catalysts of NNK metabolic activation in these species, based on their kinetic parameters. The critical role of mouse P450 2A5 has been clearly shown by deuterium-labeling studies, which demonstrated the stereoselective abstraction of the 4(R)-hydrogen of NNK, leading to a cascade of events resulting in lung tumor induction in A/J mice (21) (Figure 1 (21). The initially formed intermediate shown here is converted to R-methylene-hydroxyNNK (7, Scheme 1), which spontaneously yields methanediazohydroxide and the methyl diazonium ion (shown).…”

Molecular and Cytogenetical Alterations Induced by Environmental Cigarette Smoke in Mice Heterozygous for Fhit

“…Although in that study DNA adducts were not investigated, NNK, a tobacco-specific lung carcinogen, is known to function by initiating a cascade of reactions that results in production of the adduct O 6 -methylguanine, among other DNA adducts (49). A strong correlation between persistent O 6 -methylguanine levels in pulmonary DNA and lung tumors has been observed in mice (50). At this point, it is probably worth studying more in detail the relationship among Fhit protein, DNA adducts of different types, and lung tumor incidence in future investigations.…”

Determination of the Role of Target Tissue Metabolism in Lung Carcinogenesis Using Conditional Cytochrome P450 Reductase-Null Mice

“…Lung tumor formation was induced with NNK according to the protocol of Jalas et al (12). All animals, at the age of 2 months, with or without the doxycycline treatment from E0, were given a single i.p.…”

“…NNK-induced lung carcinogenesis is believed to occur via P450-mediated a-hydroxylation, which leads to production of reactive metabolites that induce the formation of DNA adducts, including O 6 -methylguanine (O 6 -mG; refs. 10,12). The in vivo role of P450 in NNK-induced carcinogenesis has been indicated in studies using structurally diverse P450 inhibitors (10,(13)(14)(15).…”