Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in patas monkey lung and liver microsomes.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.
Smith
T J
TJ
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ 08855, USA.
Liao
A M
AM
Liu
Y
Y
Jones
A B
AB
Anderson
L M
LM
Yang
C S
CS
D000818:Animals; D001241:Aspirin / Q000494:pharmacology; D001711:Biotransformation; D002248:Carbon Monoxide / Q000494:pharmacology; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology; D004901:Erythrocebus patas; D005260:Female; D017879:Isothiocyanates / Q000494:pharmacology; D008084:Lipoxygenase / Q000502:physiology; D008168:Lung / Q000378:metabolism*; D009637:Masoprocol / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D009602:Nitrosamines / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010105:Oxygenases / Q000502:physiology; D011451:Prostaglandin-Endoperoxide Synthases / Q000502:physiology
D003374:Coumarins; D017879:Isothiocyanates; D009602:Nitrosamines; C058305:phenethyl isothiocyanate; D009637:Masoprocol; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D010105:Oxygenases; D008084:Lipoxygenase; C020066:dimethylaniline monooxygenase (N-oxide forming); D011451:Prostaglandin-Endoperoxide Synthases; D001241:Aspirin