Species and sex differences in genotoxicity of heterocyclic amine pyrolysis and cooking products in the hepatocyte primary culture/DNA repair test using rat, mouse, and hamster hepatocytes

Eleven mutagenic heterocyclic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3]indole (Trp-P-2), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (A alpha C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoline (MeIQX), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-diMeIQX), and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-diMeIQX), were studied for genotoxicity in the hepatocyte/DNA repair test employing hepatocytes of male rats, male and female mice, and male hamsters. In these four assay systems, all compounds elicited DNA repair in at least three systems, except Trp-P-2, which was uniformly inactive. However, there were several significant differences in the responses of different systems. Rat and hamster hepatocytes responded to nine of the ten genotoxic compounds with the exception of Glu-P-2. Male and female mouse hepatocytes responded to Glu-P-2, whereas female, but not male, mouse hepatocytes responded to MeIQX and 4,8-diMeIQX. These results illustrate species and sex differences in response to these heterocyclic amines and suggest that a number of these compounds are carcinogenic in hamsters, as they have been in rats and mice.     

Organ-specific distribution of genotoxic effects in mice exposed to cooked food mutagens.

The induction of organ-specific genotoxic effects of five cooked food mutagens in Swiss albino mice was investigated in microbial animal-mediated assays. The indicator of the induction of DNA damage was a pair of Escherichia coli K12 strains, differing vastly in repair capacity (uvrB/recA versus uvr+/rec+). All compounds gave positive results in the tested dose range between 2.5 and 40 mg/kg body weight (i.p. administration, exposure time 120 min). 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were slightly more genotoxic than 2-amino-3,8-dimethylimidazo[4,5-f]quinoline (MeIQx), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) which caused similar effects. When the compounds were administered orally, higher doses were required to induce repairable DNA damage. The pattern of organ-specific effects was essentially similar for all compounds; genotoxicity was most pronounced in livers and lungs, whereas in kidneys, spleen and testes comparatively lower effects were measured. The activity of PhIP, MeIQ and IQ in the blood was similar to that observed in the liver. The results obtained in vivo were compared with data gained in vitro with subcellular organ fractions. Our findings indicate the following. (i) The concentrations required to induce repairable DNA damage in microbial animal-mediated assays are substantially higher than might be expected on the basis of the liquid suspension tests. (ii) The ranking order of the genotoxicity of the various compounds in vitro is similar to that measured in vivo, but the differences in genotoxic potencies are less pronounced in the living animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genotoxicity of the cooked-food mutagens IQ and MeIQ in primary cultures of rat, hamster, and guinea pig hepatocytes

To investigate possible interspecies differences in the hepatocellular genotoxicity of the food-borne mutagens 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), primary cultures of rat, hamster, and guinea pig hepatocytes were established. The induction of DNA repair activity in cultures exposed to various concentrations of IQ and MeIQ was determined by liquid scintillation spectrometry. DNA repair responses to MeIQ were, in general, greater than those elicited by IQ. In all three preparations of rat and hamster hepatocytes and in two of three preparations of guinea pig cells, MeIQ produced statistically significant (p less than 0.05) repair responses. IQ stimulated significant levels of repair in all three rat hepatocyte preparations and in two of three hamster cell preparations. In guinea pig cells exposed to IQ, no significant repair activity was observed. These results indicate that the genotoxicity of IQ and MeIQ in hepatic cells in species-dependent.     

Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains

Three recombinant human P450 enzymes, forms 1A1, 1A2, and 1B1, were coexpressed with NADPH-cytochrome P450 reductase in an E. coli lacZ strain suitable for detection of the mutagenicity of heterocyclic and aromatic amines. The resulting strains expressed the recombinant P450 holoenzymes at high levels. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) was activated effectively by P450 1A2, weakly by P450 1A1, and not detectably by P450 1B1. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) were activated by all three enzymes, with form 1A2 the most effective. These strains facilitate analysis of the substrate specificity of human P450 forms that participate in the metabolic activation of carcinogens.     

Genotoxicity of compounds from cooked beef in repair-deficient CHO cells versus Salmonella mutagenicity

A series of compounds isolated on the basis of their muta-genicityin the Ames/Salmonella reversion assay were previously identifiedin fried beef and chemically synthesized for further evaluation.In this study three of these compounds were tested for genotoxiceffects in the UV5 line of Chinese hamster ovary (CHO) cells,which is deficient in nucleotide excision repair. Both 2-amino-3,4-dimethyl-imidazo]4, 5-f]quinoline (MeIQ) and 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline (MelQx) gave very weak responses for cell killing,hprt mutation induction and sister chromatid exchange. Theseeffects occurred at doses in the range of 100–800 µg/ml( solubility limit), and dose-dependent increases were notobserved. Induction of chromosomal aberrations did not occurwith either compound. Nor did either of these compounds producedifferential cytotoxicity in normal CHO cells versus UV5 cells,indicating that potentially repairable DNA damage was not responsiblefor the observed cell killing. In contrast to these results,2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP), whichconstitutes > 90% of the mass of bacterial mutagens in beef,was strongly positive for all endpoints at doses in the range1–3 µg/ml. PhIP also gave marked differential cytotoxicity(ratio of 6) and cell survival curves that were strongly dependenton repair capacity. Because PhIP is 50- to 300-fold less mutagenicthan MelQ and MelQx in Salmonella TA1538, these results pointto major differences between the bacterial and mammalian assaysin terms of the relative potency of these food-related compounds.     

Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113-120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S. typhimurium TA1535. In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S. typhimurium TA1535/1,8-DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo[4,5-f]quinol ine(MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a::3,2'-d]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-pyrido[4, 3-a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase.     

Effect of tryptamine on the mutagenic activity of 2-amino-3-methylimidazo(4,5-f) quinoline (IQ) and related azaarenes in the Ames test

The bioactivation of the azaarenes 2-amino-3-methylimid-azo(4,5-f) quinoline (IQ), 2-amino-3, 4-dimethylimidazo(4, 5-f) quinoline(MeIQ) and 2-amino-3, 8-dimethylimidazo(4, 5-f) quinoxaline(MeIQx) to mutagens by hepatic S9 preparations derived fromAroclor-pretreated Wistar rats was inhibited by tryptamine (2–50µM).However, with similar prepations derived from Sprague-Dawleyrats, bioactivation of IQ and MeIQx was less markedly inhibitedby tryptamine while metabolic activation of MeIQ was enhanced.In the absence of cytosol, activation of IQ by microsomal preparationsof both rat strains was inhibited by tryptamine. Cytosolic fractionsfrom both rat strains were incapable of activation of IQ perse but increased the mutagenicity of the microsomal metabolite(s).This potentiation of the mutagenic activity by cytosol derivedfrom Wistar rats was also inhibited by tryptamine whereas nosignificant inhibition was observed with cytosolic preparationsfrom Sprague-Dawley rats. There appear to be two alternativepathways of microsomal metabolism of IQ: a tryptamine-sensitivepathway, probably involving the formation of the N-hydroxymetabolite;and a tryptamine-insensitive pathway producing weakly mutagenicor non-mutagen metabolites which are activated to a potent mutagenby the cytosol. The tryptamine-insensitive pathway appears tobe the major route of activation of the azaarenes in microsomalpreparations from Sprague-Dawley rats and the principal activationroute for MeIQ in both rat strains.     

Studies on mammary carcinogenesis induced by a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, in mice and rats

Ten heterocyclic amines (HCAs) that are produced by heating amino acids, proteins, or proteinaceous food such as fish and meat were examined for carcinogenicity in rats and mice. Three of them, 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), have been shown to induce mammary cancer in female F344 and/or SD rats, but none of the HCAs induced mammary cancer in CDF(1) mice. This report reviews our recent studies on mammary carcinogenesis of PhIP in various strains of mice and on the roles of genomic instability in the rat mammary carcinogenesis of PhIP. We demonstrated that the survival time from mammary adenocarcinomas was shorter in PhIP-treated BALB/c mice than that in the untreated control, and with a significantly higher incidence in the C.B-17 strain of mice compared with that of the control. To clarify mechanisms of mammary carcinogenesis, we examined genomic instability in rat mammary cancer induced by PhIP. Mammary cancers were induced in F344 x SD F(1) rats harboring the lacI transgene, and two cell lines were established from two adenocarcinomas. They showed a greater than 10-fold higher frequency of spontaneous mutations than that of the primary culture of normal mammary epithelial cells, in the lacI transgene and the hprt endogenous gene during cell replication. Nucleotide sequencing revealed that almost all types of mutations were increased, with a remarkable increase of A:T --> C:G mutation. This genomic instability was not attributed either to alterations of mismatch-repair enzymes or to p53. These mutational characteristics were also observed in the original tumors. Single-nucleotide instability (SNI) might be implicated in the mammary cancer induced by PhIP.     

Mechanism of the in vitro antimutagenic action of retinol

The antimutagenic action of retinoids against three amino-imidazoazaarene pre-carcinogens, i.e. 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), was investigated using the Ames test and hepatic activation systems derived from rats pretreated with Aroclor 1254. Both retinol and retinal, when incorporated into the S9 activation system, gave rise to a concentration-dependent decrease in the mutagenicity of all three mutagens, retinol being generally the more effective. Retinol suppressed the mutagenic activity of IQ even when isolated microsomes were used as activation systems. Moreover, retinol gave rise to a concentration-dependent inhibition of the microsomal dealkylations of pentoxy- and benzyloxy- and, especially, ethoxy-resorufin, but had no effect on the NADPH-dependent reduction of cytochrome c. Exposure of the bacteria to retinol with subsequent removal of the vitamin did not influence the mutagenicity of IQ. It is concluded that retinoids suppress the mutagenicity of aminoimidazoazaarenes and this is achieved through inhibition of their cytochrome P450-dependent metabolic activation. Retinol is a non-selective in vitro inhibitor of the hepatic cytochrome P450-dependent mixed function oxidase system as predicted by a computer graphic analysis of its molecular shape.     

Genotoxicity of 2-amino-3-methylimidazo 4,5-f]quinoline (IQ) and related compounds in Drosophila.

The potent food mutagen and carcinogen 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) and the structurally related heterocyclic aromatic amines 2-aminoimidazo[4,5-f]quinoline (demethyl-IQ) and 2-amino-1-methylimidazo[4,5-f]quinoline (iso-IQ) were assayed for genotoxicity in the wing somatic mutation and recombination test (SMART) as well as in the sex-linked recessive lethal (SLRL) test in Drosophila melanogaster. In addition, 3-methyl-2-nitroimidazo[4,5-f]quinoline (nitro-IQ), 2-nitrofluorene and 1,8-dinitropyrene were also assayed in the wing spot test. IQ was clearly mutagenic in the SLRL test with highest activity in spermatids. Iso-IQ was more active than IQ whereas demethyl-IQ was inactive in this test. The same pattern of results was obtained in the wing SMART: iso-IQ produced greater than 2-fold higher frequencies of spots than IQ and demethyl-IQ was clearly negative. In addition, nitro-IQ exhibited an approximately equal genotoxic activity as IQ. 2-Nitrofluorene and 1,8-dinitropyrene were both inactive in the wing spot test. These data provide good evidence for a correlation of genotoxic effects in germinal and somatic cells, and for the practical advantage of the wing spot test in Drosophila. Moreover, the results show structure-activity relationships among the heterocyclic aromatic amines and nitro compounds similar to those found in Salmonella.     

Inhibition of human brain aromatic L-amino acid decarboxylase by cooked food-derived 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and other heterocyclic amines

A carcinogenic, food-derived heterocyclic amine, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), was found to inhibit aromatic L-amino acid decarboxylase isolated from human brainstem. Trp-P-2 inhibited the enzyme activity toward L-DOPA more markedly than that toward 5-hydroxytryptophan. The inhibition was competitive to a cofactor of the enzyme, pyridoxal-5-phosphate, and the Ki value of Trp-P-2 was 163 microM. The enzyme activity could be fully recovered after removal of Trp-P-2 by gel filtration, which indicates that the inhibition was reversible. Among a series of heterocyclic amines examined for their effects on the activity toward L-DOPA, Trp-P-2 was the most potent inhibitor, followed by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, then Trp-P-1. Another heterocyclic amine, 2-amino-3-methyl-9H-pyrido[2,3-b]indole also inhibited the enzyme. The inhibition of the decarboxylase activity by these heterocyclic amines may affect the catecholamine metabolism in human brain.     

Possible application of human c-Ha-ras proto-oncogene transgenic rats in a medium-term bioassay model for carcinogens

With the aim of developing a medium-term assay for screening of environmental carcinogens, we exposed mammary carcinogen sensitive human c-Ha-ras proto-oncogene transgenic (Hras128) rats to various carcinogens, including compounds that do not normally induce mammary tumors. Seven-week-old Hras128 rats and wild-type littermates received administrations of 3-methylcholanthrene (3-MC), benzo[a]pyrene (B[a]P), anthracene, pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), dimethylarsinic acid (DMA), diethylnitrosamine (DEN) or azoxymethane (AOM) and were sacrificed at week 12 (females) (at week 10 for the 3-MC group) or week 20 (males). Female Hras128 rats receiving NNK, DEN, or DMA showed a significant increase in mammary tumor incidence and/or multiplicity compared to the respective values with olive oil or deionized distilled water (DDW) vehicles. In male Hras128 rats, a significant increase in mammary tumors was also observed in groups administered 3-MC, B[a]P, anthracene, IQ, and NNK. Mutations of transgenes were observed in codons 12 and/or 61 in the induced tumors by PCR-RFLP except in the DEN group in female and in the MeIQx group in male Hras128 rats. Thus various carcinogens, not necessarily limited to those normally targeting the breast, were found to induce mammary carcinomas in Hras128 rats, especially in females, pointing to potential use for medium-term screening.     

Metabolic activation and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in Chinese hamster ovary cells expressing murine cytochrome P450 IA2

We have utilized Chinese hamster ovary cell lines which stably express a murine cytochrome P450IA2 (P(3)450) cDNA to characterize more fully the mechanisms of genotoxicity of heterocyclic amines derived from cooked meats. To verify that these cell lines were capable of converting promutagens into active metabolites, we studied the microsomal metabolism and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pridine (PhIP). Microsomal preparations derived from excision repair-deficient Chinese hamster ovary cells expressing the mouse cytochrome P(3)450 cDNA (UV5P3) converted PhIP to the genotoxic N-hydroxy-PhIP metabolite. Cytotoxic activity in UV5P3 was observed at concentrations of PhIP as low as 1 microM. Cytotoxicity of PhIP was an order of magnitude lower in a matched repair-proficient cell line (5P3R2) expressing the P(3)450 cDNA. PhIP produced a concentration-dependent increase in sister chromatid exchange (SCE) in UV5P3. N-Hydroxy-PhIP, at concentrations as low as 0.1 microM, produced an increase in SCE in both UV5P3 and in UV5 cells which lack the P(3)450 cDNA. Incubation of PhIP with UV5P3 cells increased the frequency of micronuclei (MN) in cytokinesis-blocked cells. Chromatid gaps, but not aberrations also were induced by treatment with PhIP. N-Hydroxy-PhIP produced increases in MN and chromatid gaps in both UV5 and UV5P3 cell lines; chromosomal aberrations were induced in UV5P3 cells. These results suggested that UV5P3 cells metabolize sufficient quantities of PhIP to produce cytogenetic damage and further indicated that N-hydroxylation of PhIP was requisite for mammalian genotoxicity.     

In vitro reaction of hydroxyamino derivatives of MelQx, Glu-P-1 and Trp-P-1 with DNA: 32P-postlabelling analysis of DNA adducts formed in vivo by the parent amines and in vitro by their hydroxyamino derivatives

The synthetic hydroxyamino derivatives of three mutagenic andcarcinogenic heterocyclic amines present in cooked foods andamino acid pyrolysates, 2-amino-3,8-dimethylimidazo-[4,5-f)quinoxaIine(MeIQx), 2-amino-6-methyldipyrido[l,2-a:3',2'-d]imidazole (Glu-P-1)and 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), werereacted with DNA in vitro. Their reactivities were increasedby addition of 10-fold excess of acetic anhydride. ³²P-Postlabellinganalysis of the adducts formed in these in vitro reactions revealedthat almost all the adducts of the hydroxyamino derivativesof MeIQx and Glu-P-1 were the same as those formed in liverDNA of rats intragastrically treated with the parent amines.In contrast, analysis of Trp-P-1 -DNA adducts showed that theadducts formed in vitro were minor components of those formedin vivo; the two main adducts formed in vivo were not formedin vitro. Thus, MeIQx and Glu-P-1 may be metabolized in vivoto hydroxyamino derivatives and/or their esterified forms, suchas N-acetoxy derivatives that form DNA adducts. Formation ofadducts by Trp-P-1, however, may occur through more complicatedmetabolic pathways. Elucidation of the structures of DNA adductsin vivo is necessary to clarify this problem. ¹Present addresses: Department of Chemistry, Swedish Universityof Agricultural Sciences, PO Box 7015, S-750 07 Uppsala, Sweden      

Mammary gland carcinogenesis by food-derived heterocyclic amines: metabolism and additional factors influencing carcinogenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

The heterocyclic amines (HCAs) are a family of mutagenic/carcinogenic compounds found in cooked meats. Several HCAs are mammary gland carcinogens in rats. Of these compounds, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the major one present in the human diet. This report reviews the studies on rat mammary gland carcinogenesis by HCAs; discusses what is currently known regarding mechanisms of mammary gland carcinogenesis of PhIP, especially the significance of metabolic processing; and further highlights the evidence for the possible role of PhIP in human breast cancer.     

The modulatory effects of tryptamine and tyramine on the S9-mediated mutagenesis of IQ and MeIQ in Salmonella strain TA98.

The S9-mediated mutagenesis of IQ and MeIQ in Salmonella strain TA98 was modulated by introduction to the assay of tryptamine or tyramine. Both biogenic amines inhibited or enhanced the mutagenic response as a function of amine concentration, strain of rat used as the S9 source, and the IQ-type mutagen tested. Enhancement of IQ mutagenesis by tryptamine (10-80 microM) was observed in the presence of S9 preparations derived from Aroclor 1254-pretreated Fischer rats; the enhancing effect ceased at tryptamine concentrations > 160 microM. When Sprague-Dawley-S9 or Wistar-S9 were used for activation, the enhancement of IQ mutagenesis by tryptamine shifted to inhibition at tryptamine concentrations > 40 microM, with Sprague-Dawley-S9, and > 20 microM, with Wistar-S9. By contrast, MeIQ-mutagenesis was enhanced by tryptamine (10-160 microM), regardless of the rat strain used as S9 source. Tyramine was a weaker enhancer of MeIQ mutagenesis than was tryptamine and, unlike tryptamine, its inhibitory effects on IQ mutagenesis were observed only with Wistar-S9. Tryptamine (10-80 microM) inhibited cytochromes P450IA1 and P450IA2 activities, monitored by the O-deethylation of ethoxyresorufin and Glu-P-1 mutagenesis in TA98, respectively. These data suggest that the effects of biogenic amines on IQ and MeIQ bioactivation are complex. Furthermore, this study demonstrates that tryptamine and tyramine act both as enhancers (comutagens) and as inhibitors (antimutagens) of IQ and MeIQ mutagenesis, depending on the testing conditions.     

Genotoxic activity of the N-acetylated metabolites of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3, 4-dimethylimidazo[4,5-f]quinoline (MeIQ)

The genotoxic potential of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline(IQ) and 2-amino-3,4-di-methylimidazo[4,5-f]quinoline (MelQ)and their N-acetylated metabolites (AcIQ and AcMelQ, respectively)has been studied, in order to evaluate whether an initial N-acetylationof IQ or MelQ is important for the overall in vivo genotoxicityof the compounds. When incubated with uninduced (control) rathepatocytes, both the acetylated and the unacetylated compoundsappeared to be relatively stable, whereas water-soluble metabolites(i.e. not extractable by ethyl acetate at alkaline pH) wererapidly formed with hepatocytes from PCB-induced animals. NoDNA damage was induced by IQ or MelQ in hepatocytes isolatedfrom control rats, as measured by alkaline elution. In hepatocytesfrom PCB-pretreated rats, IQ, MelQ, AcIQ and AcMelQ inducedDNA damage at low (10^–6 M) concentrations, with AcIQ beingmore potent than IQ whereas AcMelQ was less potent than MelQ.Similar patterns were observed when unscheduled DNA synthesiswas measured in hepatocytes. The compounds induced sister chromatidexchanges in Chinese hamster V79 cells with PCB-induced hepatocytesas activation system; IQ and AcIQ were equal while AcMelQ hadless activity than MelQ. The compounds were also compared inbacterial mutagenesis test systems (Salmonella typhimurium TA98).With hepatocyte activation, AcIQ was slightly more potent thanIQ, whereas AcMelQ was markedly less mutagenic than MelQ. Withsub-cellular fractions as activation system (rat liver S9 ormicro-somes), the N-acetylated compounds were similar to orless mutagenic than their parent compounds. The mutagenic effectsof AcIQ and AcMelQ in bacteria with microsomal activation weremarkedly reduced by the deacetylase inhibitor paraoxon. Paraoxonalso reduced the DNA strand breaks induced by AcIQ or AcMelQin PCB-induced hepatocytes, but did not affect IQ- or MelQ-inducedDNA damage. The results show that an initial N-acetylation ofIQ or MelQ does not dramatically change the overall genotoxicityof these hetrocyclic aromatic amines.     

A new modification of the 32P-post-labeling method to recover IQ-DNA adducts as mononucleotides

To obtain accurate estimates of DNA adduct levels yielded by genotoxic compounds, it is essential to completely digest adducted nucleotides to mononucleotides. We previously developed a suitable method, called modified method I, to obtain DNA adducts of heterocyclic amines as 32P-labeled-mononucleoside adduct 5'-phosphate forms, by use of nuclease P1 (NP1) and phosphodiesterase I (PDEI) to digest adducted oligonucleotides. In this study, we applied method I to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA adduct analysis and found that one of the IQ-DNA adducts, 5-(deoxyguanosin-N2-yI)-2-amino-3-methylimidazo[4,5-f]quinoline 3',5'-diphosphate (pdGp-N2-IQ), was resistant to the 3'-phosphatase activity of NP1, but sensitive to that of T4 polynucleotide kinase (PNK). DNA obtained from the liver of rats fed IQ was 32P-labeled by the standard method and the 32P-labeled nucleotides obtained were incubated with PNK and NP1 to remove 3'-phosphate groups and then digested with PDEI. Three spots were obtained. One major spot was identified as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 5'-phosphate (pdG-C8-IQ) and a second abundant adduct as pdG-N2-IQ. The third spot, of which the structure is unknown, was minor. The new method is called modified method II. Modified method II could be applicable to a wide variety of chemicals.     

Modification of in vivo heterocyclic amine genotoxicity by dietary flavonoids

Female BALB/c mice were fed diets containing equimolar amountsof quercetin or its glycoside, rutin, for 5 weeks. These micewere used either in host-mediated bacterial mutation assaysor as sources of hepatic microsomes. In host-mediated bacterialmutation assays using radiolabelled mutagens, the heterocyclicamines 2-amino-3,5-dimethyl [4,5-f]imidazoquin-oline (MeIQ)and 3-amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-P-2) inducedgreater numbers of revertants in mice fed either of the flavonoiddiets compared with control. Experiments using hepatic microsomesrevealed that although feeding mice either flavonoid producedslight changes in some parameters of hepatic xenobiotic metabolism(mixed function oxidase and glutathione transferase activities),microsomes from quercetin-fed mice were more potent activatorsof both MeIQ and Trp-P-2 compared with microsomes from controlor rutin-fed mice. This difference in microsomal ability maybe due to the different biological availability of the two flavonoidswithin the gastrointestinal tract. ¹To whom correspondence to be addressed     

Development and characterization of CHO repair-proficient cell lines for comparative mutagenicity and metabolism of heterocyclic amines from cooked food

In order to understand the role of repair and metabolism in the mutagenicity of heterocyclic amines from cooked foods, we previously developed the nucleotide excision repair-deficient CHO 5P3NAT2 cell line engineered to coexpress the mouse CYP1A2 and human N-acetyltransferase genes. In the present study, we have made a matched repair-competent cell line by mutagenizing 5P3NAT2 cells with ethyl methanesulfonate and selecting for resistance to cytotoxicity by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In the differential cytotoxicity (DC) assay, 4 out of 15 clones showed no cytotoxic effect with IQ at the highest dose (30 microg/ml) tested, in contrast to repair-deficient 5P3NAT2 cells, which showed approximately 100% cytotoxicity at 0.3 microg/ml. Subsequently, these IQ-resistant clones were examined for resistance to killing by UV irradiation. All four IQ-resistant clones, which show resistance to UV similar to that of repair-proficient AA8 cells, still express both the CYP1A2 and N-acetyltransferase genes. Sequence analysis of CXPD cDNA from the 5P3NAT2R9 clone revealed an A:T-->G:C reversion event at the site of the UV5 mutation. This base change results in reversion of the codon 116 tyrosine in UV5 cells back to the original cysteine in AA8 cells, thereby restoring wild-type CXPD activity and repair function. In contrast to 5P3NAT2 cells, the repair-proficient 5P3NAT2R9 revertant cell line shows little IQ-induced cell killing, and dramatically lower levels of induced mutation at the adenine phosphoribosyltransferase (Aprt) gene locus over the range of 2-40 microg/ml IQ. This matched pair of repair-proficient/deficient cell lines can provide insight not only into the genotoxicity of heterocyclic amine dietary carcinogens such as IQ and PhIP, but also into the effects of nucleotide excision repair on the ultimate mutagenicity of these compounds.