Genotoxicity Of Heterocyclic Amines Research Articles

LP-12: The role of N-acetyltransferase 2 genetic polymorphism on the O-acetylation and genotoxicity of heterocyclic amines

LP-12: The role of N-acetyltransferase 2 genetic polymorphism on the O-acetylation and genotoxicity of heterocyclic amines

The effect of the rs1799931 G857A (G286E) polymorphism on N-acetyltransferase 2-mediated carcinogen metabolism and genotoxicity differs with heterocyclic amine exposure.

Human N-acetyltransferase 2 (NAT2) is subject to genetic polymorphism in human populations. In addition to the reference NAT2*4 allele, two genetic variant alleles (NAT2*5B and NAT2*7B) are common in Europe and Asia, respectively. NAT2*5B possesses a signature rs1801280 T341C (I114T) single-nucleotide polymorphism (SNP), whereas NAT2*7B possesses a signature rs1799931 G857A (G286E) SNP. NAT2 alleles possessing the T341C (I114T) or G857A (G286E) SNP were recombinant expressed in yeast and tested for capacity to catalyze the O-acetylation of the N-hydroxy metabolites of heterocyclic amines (HCAs). The T341C (I114T) SNP reduced the O-acetylation of N-hydroxy-2-amino-3-methylimidazo [4,5-f] quinoline (N-OH-IQ), N-hydroxy-2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (N-OH-MeIQx) and N-hydroxy- 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (N-OH-PhIP), whereas the G857A (G286E) SNP reduced the O-acetylation of N-OH-IQ and N-OH-MeIQx but not N-OH-PhIP. The G857A (G286E) SNP significantly (p < 0.05) reduced apparent Km toward N-OH-PhIP but did not significantly (p > 0.05) affect apparent Vmax. Cultures of DNA repair-deficient Chinese hamster ovary (CHO) cells transfected with human CYP1A2 and NAT2*4, NAT2*5B or NAT2*7B alleles were incubated with various concentrations of IQ, MeIQx or PhIP and double-stranded DNA damage and reactive oxygen species (ROS) were measured. Transfection with human CYP1A2 did not significantly (p > 0.05) increase HCA-induced DNA damage and ROS over un-transfected cells. Additional transfection with NAT2*4, NAT2*5B or NAT2*7B allele increased both DNA damage and ROS. The magnitude of the increases was both NAT2 allele- and substrate-dependent showing the same pattern as observed for the O-acetylation of the N-hydroxylated HCAs suggesting that both are mediated via NAT2-catalyzed O-acetylation. The results document the role of NAT2 and its genetic polymorphism on the O-acetylation and genotoxicity of HCAs.

Genotoxicity of heterocyclic amines (HCAs) on freshly isolated human peripheral blood mononuclear cells (PBMC) and prevention by phenolic extracts derived from olive, olive oil and olive leaves

In this study we investigated the genotoxic potential of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, (PhIP); 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline, (IQ); 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline, (MeIQx) and 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (DiMeIQx) on human freshly isolated peripheral blood mononuclear cells (PBMC) by the comet assay. The preventive ability of three different phenolic extracts derived from olive (O-PE), virgin olive oil (OO-PE) and olive leaf (OL-PE) on PhIP induced DNA damage was also investigated.PhIP and IQ induced a significant DNA damage at the lowest concentration tested (100 μM), while the genotoxic effect of MeIQx and DiMeIQx become apparent only in the presence of DNA repair inhibitors Cytosine b-D-arabinofuranoside and Hydroxyurea (AraC/HU). The inclusion of metabolic activation (S9-mix) in the culture medium increased the genotoxicity of all HCAs tested. All three phenolic extracts showed an evident DNA damage preventive activity in a very low concentration range (0.1–1.0 μM of phenols) which could be easily reached in human tissues “in vivo” under a regular intake of virgin olive oil. These data further support the observation that consumption of olive and virgin olive oil may prevent the initiation step of carcinogenesis. The leaf waste could be an economic and simple source of phenolic compounds to be used as food additives or supplements.

Differential effect of acetyltransferase expression on the genotoxicity of heterocyclic amines in CHO cells

We earlier developed the Chinese hamster ovary UV5P3 cell line that expresses cytochrome P4501A2 and lacks nucleotide excision repair for studying metabolism and mutagenicity of heterocyclic amines. The Chinese hamster ovary UV5P3 cells are approximately 50-fold more sensitive to the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) than 2-amino-3-methylimidazo[4,5- f]quinoline (IQ), another genotoxic compound found in cooked food, with respect to cytotoxicity and mutation induction at the adenine phosphoribosyltransferase ( aprt) locus. To test the hypothesis that the important missing activity in our CHO system for IQ genotoxicity was acetyltransferase, we transfected the UV5P3 cells with cDNA plasmids of either the human NAT2 N-acetyltransferase gene or a bacterial O-acetyltransferase gene. Functionally transformed clones were determined by the differential cytotoxicity assay using IQ, and confirmed by measuring the enzyme activity with isoniazid as substrate. Two clones designated 5P3NAT2 and 5P3YG (expressing human and bacterial transferases, respectively) were characterized. Both cell lines were sensitive to killing by IQ at concentrations as low as 4 ng/ml. Based on the D 37 value, the dose that reduced the survival to 37% relative to untreated controls, the acetyltransferase expressing lines showed ∼1000-fold increase in sensitivity to the killing effects of IQ over the parental UV5P3 cell line. The same dramatic change in sensitivity was also seen in mutation response at the aprt locus and with chromosomal aberrations and sister chromatid exchanges. In contrast, these cell lines showed cytotoxicity to PhIP similar to that of the parental line UV5P3. These results suggest that PhIP does not require acetyltransferase for metabolic activation leading to genotoxicity in these cells. These new cell lines constitute a sensitive cell system for assessing genotoxicity of compounds requiring metabolic activation by both P450IA2 and acetyltransferase, as well as for studying the molecular processes by which DNA damage can lead to mutation and cancer.

Genotoxicity of heterocyclic amines in the hepatocyte/DNA repair assay using hepatocytes of rats or mice pretreated with 3-methylcholanthrene

The genotoxicity of 4 heterocyclic amines, 3-amino-1,4-dimethyl-5 H-pyrido[4,3- b]indole (Trp-P-1), 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2- a:3′,2′- d]imidazole (Glu-P-1) and 2-aminodipyrido[1,2- a:3′,2′- d]imidazole (Glu-P-2) was examined in the hepatocyte primary culture (HPC)/DNA-repair assay using hepatocytes of rats and mice pretreated with 3-methylcholanthrene (MC), and the results were compared with those obtained in a previous assay with hepatocytes of normal rodents. All of these heterocyclic amines clearly elicited positive responses of DNA repair in the assay with hepatocytes of the rodents given MC, although in the regular HPC/DNA-repair test without MC pretreatment Tyr-P-2 and Glu-P-2 were negative in rat hepatocytes, and Try-P-2 was also negative in mouse hepatocytes. The level of unscheduled DNA synthesis induced by these positive compounds in the assay with hepatocytes of MC-treated rodents was much higher than that in the assay with hepatocytes of normal rodents. These results are basically in agreement with the reports that this class of compounds is highly activated metabolically by the cytochrome P-450 system from liver microsomes.