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.     

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.     

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)     

Genotoxic effects of heterocyclic aromatic amines in human derived hepatoma (HepG2) cells

In order to study the mutagenic effects of heterocyclic aromatic amines (HAAs) in cells of human origin, five compounds, namely 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-3, 4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), the pyridoimidazo derivative 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), were tested in micronucleus (MN) assays with a human derived hepatoma (HepG2) cell line. All HAAs caused significant, dose-dependent effects. The activities of IQ, MeIQ, MeIQx and PhIP were similar (lowest effective concentrations 25-50 microM), whereas Trp-P-1 was effective at a dose of >/=2.1 microM. In addition, the HAAs were tested in MN assays with Chinese hamster ovary (CHO) cells and in Salmonella strain YG1024 using HepG2 cell homogenates as an activation mix. In the CHO experiments, positive results were obtained with Trp-P-1 and PhIP, whereas the other compounds were devoid of activity under all experimental conditions. The discrepancy in the responsivity of the two cell lines is probably due to differences in their acetylation capacity: enzyme measurements with 2-aminofluorene as a substrate revealed that the cytosolic acetyltransferase activity in the HepG2 cells is approximately 40-fold higher than that of the CHO cells. In the bacterial assays all five HAAs gave positive results but the ranking order was completely different from that seen in the HepG2/MN experiments (IQ > MeIQ > Trp-P-1 >/= MeIQx > PhIP) and the mutagenic potencies of the various compounds varied over several orders of magnitude. The order obtained in bacterial tests with rat liver S9 mix was more or less identical to that seen in the tests with HepG2 cell homogenates but the concentrations of the amines required to give positive results were in general substantially lower (10(-5)-10(-1) microM). Overall, the results of the present study indicate that MN/HepG2 tests might reflect the mutagenic effects of HAAs more adequately than other in vitro mammalian cell systems due to the presence of enzymes involved in the metabolic conversion of the amines.     

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.     

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.     

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.     

Metabolic activation of 2-amino-3-methylimidazo(4, 5-f)quinoline by hepatic preparations -- contribution of the cytosolic fraction and its significance to strain differences

In accordance with previous studies the bioactivation of 2-amino-3-methylimidazo(4,5-f)quinoline(IQ) to mutagens in the Ames test was preferentially catalysedby the 3-methyl-cholanthrene-induced cytochromes P-448, in contrastto the phenobarbital-induced forms of the cytochrome. The mutagenicityof IQ catalysed by microsomes, in the absence of cytosol, wasmuch lower when compared with that observed with S9 fractions.Cytosol itself could not activate IQ but markedly potentiatedthe microsome-mediated mutagenicity of the carcinogen. The effectof the cytosol was still evident when micro-somal metabolismwas terminated, indicating that the cytosol contains enzyme(s)that can further convert the microsome-generated metabolitesof IQ to more potent mutagens. The cytosolic enzyme(s) wereinducible by pre-treatment of the rats with Aroclor 1254. Thehigher efficiency of activation of IQ to mutagens by Sprague—DawleyS9 mixes when compared with similar preparations from the Wistarrat could be attributed not only to differences in the rateof microsomal metabolism but also to the higher ability of theSprague-Dawley cytosolic fraction in further metabolizing themicro-some-generated metabolite(s). The present study demonstratesclearly that the mutagenic response of this compound in theAmes test may be profoundly modulated by the cytosolic fractionand its role in the metabolic activation of pre-mutagens meritsfurther investigation. ¹To whom correspondence should be addressed     

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.     

The food pyrolysis product IQ enhances its own activation

The metabolic activation of the food pyrolysis product 2-amino-3-methylimidazo (4,5-f) quinoline (IQ) to mutagenic intermediates in the Ames test was studied using hepatic activation systems from control and IQ-treated rats. Hepatic S9 preparations from IQ-treated rats were more efficient than control in converting IQ to mutagens. An increase was also seen when isolated microsomes were employed as activation systems but this was less pronounced. The microsome-mediated mutagenicity of IQ was potentiated by addition of the cytosolic fraction from control and IQ-treated rats, the latter being more effective. It is concluded that IQ, at the doses employed in the present study, enhances its own bioactivation to genotoxic metabolites by stimulating both its microsomal and cytosolic metabolism.     

Effects of Maillard reaction products on mutagen formation in boiled pork juice

The three IQ (2-amino-3-methylimidazo[4,5-f]quinoline) compoundsIQ, MeIQx (2-amino-3, 4-dimethyl[4, 5-f]quinoxaline) and MeIQ(2-amino-3, 4-dimethylimidazo[4, 5-f]quinoline) have been foundin boiled pork juice. To determine which Maillard reaction productsare important in the formation of IQ-type mutagens in boiledpork juice, six Maillard reaction products were separately addedto the pork juice before reflux boiling and then the mutagenicityof each sample was examined with Salmonella typhe murium TA98in the presence of S9 mix. The addition of four Maillard reactionproducts enhanced the mutagenicity of pork juice 1.2–2.9-foldafter reflux boiling. The highest level of enhancement was observedwith tetrahydrothiophene, followed by 2, 3-dimethylpyrazine,3-methylpyridine and 2-methylpyridine. However, the additionof 2-acetylpyrrole and imidazole greatly inhibited the mutagenicityof pork juice. To confirm which IQ-type mutagenes were changed,four major mutagenic fractions were monitored after HPLC separationby their mutagenicity with Salmonella typhimurium TA98. By comparingthe retention time of authentic IQ compounds from boiled porkjuice with added tetrahydrothiophene or 2, 3-dimethylpyrazine,we show that four major HPLC fractions have significantly increasedmutagenicity compared with the same fractions in boiled porkjuice alone. In contrast, the mutagenicity of these fractionswas considerably reduced with the addition of imidazole or 2-acetylpyrroleto the pork juice before refluxing. The residual amounts oftetrahydrothiophene and 2, 3-dimethylpyrazine added to the boiledpork juice after heating were measured by gas chromatographyand found to be inversely correlated with the mutagenicity ofthe pork juice. These data suggest that these two Maillard reactionproducts are probably involved in the formation of IQ-type mutagensin boiling pork juice. ¹To whom correspondence should be addressed     

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.     

Activation of MeIQ (2-amino-3,4-dimethylimidazo- [4,5-f]quinoline) by sequence variants of recombinant human cytochrome P450 1A2

Understanding the relationships between the sequences and catalytic activities of P450 enzymes that catalyze the bioactivation of mutagens and carcinogens is an important goal in mutation research. Escherichia coli strain DJ4309 expresses recombinant human P450 1A2 and activates promutagens such as MeIQ (2-amino-3, 4-dimethylimidazo[4,5-f]quinoline), as measured by induction of reverse mutations detected as lacZ(+) colonies on minimal lactose (ML) plates. Pools of P450 1A2 mutants were constructed by polymerase chain reaction (PCR) mutagenesis of putative substrate recognition sites (SRSs). Cultures of individual clones were patched onto MeIQ/ML plates and the growth of revertant microcolonies within each patch was inspected after 2 days of incubation. Beginning with a pool of several thousand clones, we identified 25 distinct P450 1A2 SRS variants with altered activities. In this study, the MeIQ dose-responses of all the variants are reported. The implications of the results are considered with reference to published models of the protein's structure.     

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.     

The hepatic conversion of some heterocyclic amines to bacterial mutagens is modified by dietary fat and cholesterol

Groups of Sprague—Dawley rats were fed on diets containingincreasing amounts of beef dripping, but having a constant cholesterolcontent. One group of rats was fed on a diet containing no drippingand no added cholesterol (control). We have studied the abilityof individual hepatic S9 preparations to activate the cookedfood mutagens 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ),2-amino-3, 4-dimethyl-imidazo[4, 5-f]quinoline (MeIQ) and 2-amino-3,8-dimethyl-imidazo[4, 5-f]quinoxaline (MeIQ_x) to bacterial mutagensusing Salmonella typhimurium TA98 as indicator. Hepatic preparationsfrom animals fed on a high cholesterol, low-fat diet were lesseffective in activating the mutagens than preparations fromrats fed on the control diet. It was also observed that thecapacity of hepatic preparations to activate these mutagensincreased as the amount of dripping in the diet increased. Theseresults suggest that it is the triglyceride rather than thecholesterol content of beef dripping which promotes increasedmutagen activation capacity in the liver. ¹To whom correspondence should be addressed     

Activation of 2-amino-3-methylimidazo (4,5-f) quinoline in rat and human hepatocyte/Salmonella mutagenicity assays: the contribution of hepatic conjugation

The capacity of human liver S9 and hepatocytes to metabol-icallyactivate 2-amino-3-methylimidazo (4,5-f) quinoline (IQ) in Salmonellamutagenicity assays more closely resembles that of preparationsfrom Aroclor-induced rat than control rat. The extent to whichhepatocyte conjugating enzymes contribute to activation in theseassays has been studied. Omission of sulphate or addition ofa ‘specific’ sulphotransferase inhibitor (2,6-DCNP)did not significantly reduce mutagenicity, nor did PAPS enhanceS9-mediated bacterial mutagenicity. Conversely, mutagenicitywas significantly inhibited by PCP (an inhibitor of both sulphotransferaseand acetyltranferase) and an acetylation-deficient Salmonella(TA98/1,8DNP₆) was unresponsive to the mutagenicity of IQ. Thesedata suggest that acetylation but not sulphation is importantin IQ bacterial mutagenesis. The addition of acetyl CoA, PAPS-generatingsystem or ATP paradoxically reduces the mutagenicity of IQ inS9/Salmonella TA98 assays. Therefore, activation by esterificationin hepatocytes does not contribute to the mutagenicity of IQin Salmonella typhimurium possibly due to restricted accessof conjugates into the bacterial cell.     

Antimutagenic activity of spearmint

The antimutagenic activity of spearmint (Mentha spicata), a popular food flavoring agent, was studied in the Salmonella assay. Spearmint leaves were brewed in hot water for 5 min at concentrations up to 5% (w/v), and the water extracts were tested against the direct-acting mutagens 4-nitro-1,2-phenylenediamine (NPD) and 2-hydroxyamino-3-methyl-3H-imidazo[4,5-f]quinoline (N-OH-IQ) using Salmonella typhimurium strain TA98. Nontoxic concentrations of spearmint extract inhibited the mutagenic activity of N-OH-IQ in a concentration-dependent fashion, but had no effect against NPD. These experiments by design focused on the water extract consumed commonly as an herbal tea, but chloroform and methanol extracts of spearmint also possessed antimutagenic activity against N-OH-IQ. Water extract of spearmint inhibited the mutagenic activity of the parent compound, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), in the presence of rat liver S9; however, the concentration for 50% inhibition (IC50) against IQ was approximately 10-fold higher than in assays with N-OH-IQ minus S9. At concentrations similar to those used in the Salmonella assays, spearmint extract inhibited two of the major enzymes that play a role in the metabolic activation of IQ, namely, cytochromes P4501A1 and 1A2, based on ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase assays in vitro. In vivo, rats were given spearmint water extract (2%; w/v) as the sole source of drinking fluid before, during, and after 2-week treatment with IQ; colonic aberrant crypt foci were inhibited significantly at 8 weeks (P < 0.05, compared with rats given IQ alone). Collectively, these findings suggest that spearmint tea protects against IQ and possibly other heterocyclic amines through inhibition of carcinogen activation and via direct effects on the activated metabolite(s).     

Effects of cigarette smoke and a heterocyclic amine,MeIQx on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver

In order to elucidate the mechanism underlying enhancement by cigarette smoke (CS) of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced rat hepatocarcinogenesis, hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of various carcinogens and UDP-glucuronyltransferase (UDPGT) activities were assayed in male F344 rats. Immunoblot analyses for microsomal CYP proteins revealed induction of CYP1A1 and constitutive CYP1A2 (2.3- to 2.7-fold), but not CYP2B1/2, 2E1 or 3A2, by CS exposure for 1, 12 or 16 weeks using a Hamburg type II smoking machine; the enhancement of CYP1A2 was 4.7-5.7 times that of CYP1A1. CS exposure also elevated the mutagenic activities of MeIQx and five other heterocyclic amines (HCAs) 1.4- to 3.7-fold, but not those of benzo[a]pyrene (BP) and aflatoxin B(1) in strain TA98 and N-nitrosodimethylamine, N-nitrosopyrrolidine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in strain TA100. In contrast, feeding 300 p.p.m. MeIQx in the diet for 1 or 16 weeks produced no significant alterations in the levels of these CYP species and mutagenic activities. However, i.g. administration of 50 or 100 mg/kg MeIQx in a single dose selectively increased CYP1A1 and 1A2 (2.6-fold) levels and mutagenic activities of five HCAs (1.7- to 3.3-fold), but not BP. On the other hand, feeding of MeIQx for 16 weeks enhanced UDPGT activities towards 4-nitrophenol and testosterone (2.9- and 1.5-fold, respectively), but not bilirubin, while CS exposure induced that towards 4-nitrophenol (1.6-fold); combined treatment with CS and MeIQx showed a summation effect on induction of UDPGT1A6 activity (3.5-fold). Consequently, these results demonstrate that CS and MeIQx have a bifunctional action, with similar induction patterns of specific CYP proteins, mutagenic activity and UDPGT activity. In conjunction with the finding of N-hydroxy-MeIQx being a poor substrate for rat liver UDPGT, our results clearly indicate that enhancement by CS of MeIQx-induced hepatocarcinogenesis in F344 rats can be attributed to an increase in metabolic activation of MeIQx by hepatic CYP1A2 during the initiation phase.     

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.