Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells

The heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are pyrolysis products formed whenmeat is cooked and are rodent mammary carcinogens. They are thought to bemetabolically activated by N-hydroxylation, catalysed by cytochrome P450(CYP), followed by O-acetylation catalysed by N- acetyltransferases.Primary cultures of human mammary epithelial cells (HMECs) prepared from upto 26 individuals for each compound, were treated with IQ, MeIQ, or PhIP(500 microM) or with N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) or N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ) (20 microM) and the levels ofadduct formation in their DNA analysed by 32P-post-labelling. In order toinvestigate whether pharmacogenetic polymorphisms influence DNA adductformation, the NAT2 genotype of each individual was determined by apolymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method that distinguishes between the wild-type and four variantalleles. Presence of two variant alleles designates a slow NAT2 acetylator,whereas individuals with one or two wild-type alleles are designated fastNAT2 acetylators. Interindividual variations in total DNA adduct levelsranged for IQ from 0.64-63.1 DNA adducts per 10(8) nucleotides (mean 7.80),for MeIQ from 1.99-17.8 (mean 6.63), for PhIP from 0.13-4.0 (mean 0.96),for N-OH-PhIP from 6.32-497 (mean 176) and for N-OH-IQ from 0.92-30.6 (mean9.24). The higher adduct levels observed in cells treated with the N-OHmetabolites suggests that N- hydroxylation is the rate-limiting step inHMECs and this may be due to low CYP levels. In contrast, the Phase IIreaction catalysed by N- acetyltransferases is probably the major step inthe metabolic activation of heterocyclic amines that occurs in the breast.Higher mean levels of heterocyclic amine-DNA adduct formation were detectedin the cells of NAT2 fast acetylators compared with slow acetylators, withmean adduct levels per 10(8) nucleotides following IQ treatment, of 12.74and 3.57 respectively, following PhIP treatment, of 1.20 and 0.74,respectively, following MeIQ treatment, of 7.90 and 5.08, respectively andfollowing N-OH-PhIP-treatment, of 243.1 and 130.0, respectively. However,due to the large variations in adduct levels, these differences in meanvalues were not statistically significant with the limited number ofindividuals studied. This appears to be the first pilot study todemonstrate interindividual variations in the metabolic activation ofheterocyclic amines and their metabolic intermediates in primary culturesof HMECs in vitro.