Heterocyclic aromatic amines induce DNA strand breaks and cell transformation
Wolfgang Pfau2,
Francis L. Martin1,
Kathleen J. Cole1,
Stanley Venitt1,
David H. Phillips1,
Philip L. Grover1 and
Hans Marquardt
Fraunhofer Society, Department of Toxicology and Environmental Medicine and Department of Toxicology, Hamburg University Medical School, D-20146 Hamburg, Germany and
1 Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, UK
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Abstract
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Heterocyclic aromatic amines (HAAs), formed during the cooking of foods, are known to induce tumours in rodent bioassays and may thus contribute to human cancer risk. We tested six HAAs in a morphological transformation assay and in three in vitro genotoxicity assays. The morphological transforming abilities of HAAs were tested, in the presence of rat-liver S9, in the C3H/M2 fibroblast cell line. Concentration levels of 50 µM 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 100 µM 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (4,8-DiMeIQx), 50 µM 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 100 µM 2-amino-9H-pyrido[2,3-b]indole (A
C), 100 µM 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA
C) and 15 µM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced maximum transformation potencies of 5.5, 6.6, 6.3, 5.2, 7.3 and 9.2 transformed foci per 104 surviving cells, respectively. Bacterial mutagenic activity was determined in the presence of rat-liver S9 using the Salmonella typhimurium reverse-mutation assay employing strain YG1019. Mutagenic potencies of 3800 revertants (revs)/ng with 8-MeIQx, 2900 revs/ng with 4,8-DiMeIQx, 3480 revs/ng with IQ, 1.6 revs/ng with A
C, 2.9 revs/ng with MeA
C and 5 revs/ng with PhIP were observed. Clastogenic activity in vitro was analysed by the micronucleus assay in metabolically competent MCL-5 cells. Dose-dependent induction of micronuclei was observed for all HAAs tested with 15.4% of cells containing micronuclei at 10 ng/ml. Micronucleus induction was in the order 4,8-DiMeIQx > 8-MeIQx > IQ > MeA
C > PhIP > A
C. DNA strand-breaking activity in MCL-5 cells was measured by the alkaline single cell-gel (comet) assay. The lowest effect doses for significant increases (P
0.0007, MannWhitney test) in comet tail length (µm) were 45.5 µg/ml (200 µM) for PhIP, 90.9 µg/ml (410510 µM) for 4,8-DiMeIQx, IQ, MeA
C and A
C, and 454.5 µg/ml (2130 µM) for 8-MeIQx. It is not yet clear which of these assays most accurately reflects the genotoxic potential to humans of compounds of this class of environmental carcinogens.
Abbreviations: AAF, 2-acetylaminofluorene; A
C, 2-amino-9H-pyrido-[2,3-b]indole; CA, chromosomal aberration; CHL, Chinese hamster lung; CHO, Chinese hamster ovary; 4,8-DiMeIQx, 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline; DMSO, dimethyl sulphoxide; HAA, heterocyclic aromatic amine; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MCA, 3-methylcholanthrene; MeA
C, 2-amino-3-methyl-9H-pyrido[2,3-b]indole; 8-MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; rev, revertant; SCE, sister chromatid exchange.
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Introduction
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Among the environmental factors that may contribute to the genesis of human cancer, diet is regarded as a major determinant (13). Heterocyclic aromatic amines (HAAs) (see Figure 1
for structures) are formed at parts-per-billion levels in fried or grilled meats as products of protein pyrolysis or Maillard reactions (4,5) and have been shown to be carcinogenic in rodent bioassays (3,6). Much effort has gone into investigating the factors affecting the formation, yield and structures of HAAs (4,6). However, much remains to be done to determine the mechanisms by which HAAs exert their effects and to discover which HAAs have the greatest relevance to human cancer incidence.
The aminoimidazo-quinoxaline HAA derivatives, e.g. 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), and the aminoimidazo-quinoline HAA derivatives, e.g. 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), are strongly mutagenic to bacteria (4), whereas the pyrido-indole HAAs, 2-amino-9H-pyrido[2,3-b]indole (A
C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA
C) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are much less potent (6). However, A
C, MeA
C and PhIP may possess greater genotoxicity in test systems involving mammalian cells (711) than 8-MeIQx, 4,8-DiMeIQx and IQ (12). Specifically, PhIP induced more gene mutations in Chinese hamster ovary (CHO) cells than IQ or 8-MeIQx (7), and it was mutagenic in vivo in the mouse small intestine whereas 8-MeIQx was not (8,9). PhIP also induced mutations, as did A
C, in the lacZ transgene of the Mutamouse® (10) and in the lacI transgene isolated from the colon of Big Blue® mice (11). In the last two test systems, transversion mutations (GC
TA) were the most common, which is in agreement with in vitro studies of mutational spectra produced by HAAs or of mutations observed in oncogenes or tumour suppressor genes isolated from HAA-induced rodent tumours (11).
In common with other genotoxic aromatic amines, HAAs are thought to be metabolically activated via oxidation of the exocyclic amino group, a reaction mediated mainly by the cytochrome P-450 isoenzyme CYP1A2 (5), but subsequent conjugating reactions such as acetylation or sulphation (4,5) may also be required. Recent studies suggest that human hepatic microsomes possess greater bioactivation potential than microsomes isolated from the livers of rats or mice (1315). There may have been an underestimation of the potential risks to humans of HAAs. Following metabolic activation, most HAAs form covalent DNA adducts both in vivo and in vitro, the major adducts formed linking the C8 position of guanine to the exocyclic amino group of the HAA (16,17).
Despite the substantial number of investigations into the genotoxicity of HAAs, little is known about their cell-transforming activities. In this study, the abilities of six different HAAs (Figure 1
) to transform mammalian C3H/M2 mouse fibroblasts in vitro have been examined. The same HAAs were also tested for microbial mutagenicity and for their abilities to induce micronuclei and DNA strand breaks in MCL-5 cells. The transforming and strand-breaking activity of the six HAAs examined did not appear to be directly related to their mutagenic potencies.
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Materials and methods
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Chemicals
HAAs (>97% pure) were obtained from Toronto Research Chemicals (Toronto, Canada). 3-Methylcholanthrene (MCA) and 2-acetylaminofluorene (AAF) were obtained from Sigma (Deisenhofen, Germany).
Morphological transformation of C3H/M2 mouse fibroblasts in vitro
This assay was carried out as described previously (18). Briefly, cells of the C3H/M2 clone (fibroblasts established from C3H mouse prostate cells) harvested from logarithmically growing stock cultures (between passages 5 and 20) were plated on day 0 into 60 mm dishes in basal Eagle's medium supplemented with 10% fetal calf serum to determine the plating efficiency (100 cells/dish) or the transformation rate (1000 cells/dish). After 24 h, the cultures were treated for 2 h with MCA (10 µg/ml), AAF (10 µg/ml), the test compound or the solvent [dimethyl sulphoxide (DMSO), 0.5%] in the presence of rat liver S9 (0.4 mg protein/ml medium) from Aroclor 1254-induced rat liver and cofactors (final concentrations 0.23 mM NADP+, 0.28 mM glucose-6-phosphate, 0.45 mM MgCl2, 0.45 mM KCl). Thereafter the medium was changed twice weekly. The cells were fixed and stained after 2 weeks to determine plating efficiency or after 8 weeks to determine the transformation rate.
Bacterial mutagenicity
HAAs were assessed for bacterial mutagenic potency towards the frameshift-detecting strains, Salmonella typhimurium TA1538 and YG1019. Bacterial strains such as YG1019, that carry genes for N-acetyltransferases on multicopy plasmids, catalyse the N-acetylation of aromatic amines and the O-acetylation of hydroxylamines, and are consequently more sensitive to such compounds than are conventional strains such as TA1538 (19). Pre-incubation assays, with Aroclor 1254-induced rat-liver S9 prepared from male Wistar rats, were performed with a 30 min pre-incubation at 37°C (20). S9 mix contained 30% v/v S9, 33 mM KCl, 8 mM MgCl2, 5 mM glucose-6-phosphate, 3.3 mM NADP+, 0.1 M phosphate buffer, pH 7.4. Revertant colonies (in triplicate plates) were scored after incubation for 72 h at 37°C. Assays were performed in the 10150 pg/plate range for 8-MeIQx, 4,8-DiMeIQx and IQ, and 10300 ng/plate range for A
C, MeA
C and PhIP. Mutagenic potencies for individual HAAs were obtained from the linear parts of their doseresponse curves and expressed as revertants (revs)/ng. The bacterial strains used in this study, S.typhimurium TA1538 and YG1019, were generous gifts from B.N.Ames (Berkeley, CA, USA) and P.D.Josephy (University of Guelph, Ontario, Canada), respectively.
Micronucleus assay
MCL-5 cells, a line of human lymphoblastoid cells, were obtained under licence from Gentest (Woburn, MA). They constitutively express a high level of native CYP1A1 and also four other human cytochromes (CYP1A2, CYP2A6, CYP3A4 and CYP2E1) and microsomal epoxide hydrolase that are carried as cDNAs in plasmids (21). Micronucleus induction in MCL-5 cells, blocked at cytokinesis by cytochalasin-B, was used as an indicator of chromosomal damage (22). Cell cultures were treated with a minimum of two concentrations of an HAA in DMSO (maximum 100 µl DMSO in 10 ml medium) or with DMSO alone (solvent control) for 24 h at 37°C. They were then treated with 6 µg/ml cytochalasin-B for a further 24 h, fixed and stained (22). The incidence of micronucleated cells was recorded by scoring 500 binucleate cells with intact cytoplasm. The percentage of binucleate cells was used as an index of cytotoxicity. Increases in the numbers of micronucleated cells were tested for statistical significance using a
2 test for trend (22).
Single cell-gel electrophoresis (comet) assay
The assay was carried out exactly as described previously (23). Briefly, MCL-5 cell suspensions in phosphate-buffered saline (~1x105 cells/75 µl) were incubated with or without DNA-repair inhibitors, hydroxyurea (HU; 10 mM) and cytosine arabinoside (ara-C; 1.8 mM) at 37°C for 30 min in the presence of varying concentrations of individual HAAs. Cells embedded in an agarose sandwich on frosted microscope slides (Curtin Matheson Scientific, Houston, TX) were lysed. Exposed nuclei were allowed to unwind in freshly prepared alkaline electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH 12.3) prior to electrophoresis at 0.8 V/cm and 300 mA for 36 min. Nuclear material was stained with ethidium bromide (20 ng/ml) and visualized by epifluorescence using a Leitz Laborlux S microscope. DNA migration quantified from digitized images was expressed as comet tail length (µm). A total of 50 nuclei/datum point from two slides were scored. Differences in comet tail lengths were assessed for significance using the MannWhitney test.
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Results
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Cell-transforming activity
In order to determine the cytotoxic activity of HAAs, C3H/M2 fibroblasts were incubated for 2 h with concentrations of 1250 µM (0.252 µg/ml) of an HAA, with or without the addition of rat-liver S9. No cytotoxic effect was observed in the absence of rat-liver S9. Cytotoxicity data for the HAAs and the positive controls MCA and AAF are presented in Table I
as cloning efficiencies relative to the solvent control. While 8-MeIQx, 4,8-DiMeIQx, A
C and MeA
C were only weakly cytotoxic over this concentration range, IQ (250 mM, 50 µg/ml) resulted in a 45% reduction of the cloning efficiency and PhIP significantly reduced cell survival at 15 µM (3.3 µg/ml) with no surviving cells detectable at 25 mM (5.6 µg/ml).
In the transformation assay, 1000 cells/dish were incubated for 2 h with various concentrations of an HAA and in the presence of S9-mix as described in Materials and methods. The medium containing the HAA and S9-mix was then removed, fresh medium added and the cells maintained in culture for 8 weeks before staining and scoring for the presence of transformed foci. All six HAAs were positive in this assay (Table I
). The most active compound was PhIP, which induced 15 transformed foci in 43 treated dishes at a concentration of 1.1 µg/ml. When the cytotoxicity is taken into account, a concentration-dependent increase in the number of foci per 104 surviving cells in the range of 0.043.3 µg PhIP/ml was observed. A broader range was tested for the other HAAs since these were less cytotoxic. For IQ and 4,8-DiMeIQx, a dose-dependent increase in transformed foci per 104 surviving cells was observed. For 8-MeIQx, A
C and MeA
C, transforming activity was detectable at 2.0 µg/ml with a maximum at ~10 µg/ml. No cell transformation by the HAAs was detectable in the absence of rat-liver S9.
Bacterial mutagenicity
The mutagenic potencies of the six HAAs in TA1538 and YG1019, listed in Table II
, were derived from the linear part of doseresponse curves (data not shown). There were clear differences in mutagenic potency between the results obtained with TA1538 and the genetically engineered strain, YG1019, that overexpresses bacterial N,O-acetyltransferase. The elevations in mutagenic potencies towards S.typhimurium YG1019, compared with S.typhimurium TA1538, were by factors of 13.3 (8-MeIQx), 5.7 (4,8-DiMeIQx), 21.8 (IQ), 6.3 (A
C), 16.8 (MeA
C) and 1.4 (PhIP). Bacterial mutagenic potencies were in the following order: 4,8-DiMeIQx > 8-MeIQx > IQ > PhIP > A
C > MeA
C with TA1538; and 8-MeIQx > IQ > 4,8-DiMeIQx > PhIP > MeA
C > A
C with YG1019.
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Table II. Bacterial mutagenicity of HAAs in the Salmonella/mammalian microsome test using tester strains TA1538 and YG1019
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Micronucleus formation by HAAs in MCL-5 cells
Treatment of MCL-5 cells in the concentration range 2.022.5 ng HAA/ml medium (10100 nM) resulted in cytotoxicity and micronucleus formation in a dose-dependent fashion (Figure 2
). For all six HAAs, micronucleus formation was significant as judged by a
2 test for trend in a doseresponse. Cytotoxicity, expressed as a reduction in the percentage of binucleate cells, was observed in the following order: 4,8-DiMeIQx > 8-MeIQx > IQ > PhIP > MeA
C > A
C. Similarly, the aminoimidazo-quinoline and aminoimidazo-quinoxaline HAAs induced the highest net frequencies of micronuclei at a concentration of 10 ng HAA/ml medium, with 5.4% binucleate cells with micronuclei following treatment with 4,8-DiMeIQx, 4.0% with 8-MeIQx and 3.0% with IQ. At 10 ng HAA/ml medium 2.4% binucleate cells possessed micronuclei following treatment with MeA
C, 1.4% with PhIP and 1.0% with A
C.

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Fig. 2. Doseresponse curves for induction of micronuclei in MCL-5 cells by individual HAAs. Micronucleus formation (micronuclei/500 binucleate cells, ) and cytotoxicity (% binucleate cells, ) were determined in MCL-5 cells following treatment with one of the six HAAs studied as follows: (a) 8-MeIQx; (b) 4,8-DiMeIQx; (c) IQ; (d) A C; (e) MeA C; and (f) PhIP. Assays were carried out as described in Materials and methods. Significance in the 2 test for trend was 8-MeIQx, P = 0.0003; 4,8-DiMeIQx, P < 0.000001; IQ, P = 0.0007; A C, P = 0.01; MeA C, P = 0.004; PhIP, P = 0.002.
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Comet formation in MCL-5 cells
Distributions of comet tail lengths observed when 50 nuclei/datum point were scored following incubation of MCL-5 cells with 90.9 µg/ml (410510 µM) of each of the six individual HAAs in the presence of HU/ara-C are shown in Figure 3
. Incorporation of HU/ara-C into the assay has been shown to increase its sensitivity in other applications without increasing tail length in control cells (23). Median comet tail lengths in control cell populations ranged from 5.0 to 7.5 µm and 6.0 to 10.0 µm with or without HU/ara-C, respectively. Individual median comet tail lengths in MCL-5 cells induced with 90.9 µg/ml of HAA in the presence of HU/ara-C were: 12.0 µm with 8-MeIQx, 16.0 µm with IQ, 10.5 µm with 4,8-DiMeIQx, 83.5 µm with PhIP, 47.5 µm with MeA
C and 26.0 µm with A
C. Induction of DNA strand breaks by HAAs was weak when these inhibitors were omitted. Median comet tail lengths observed in the absence of HU/ara-C were 11.0 µm with 8-MeIQx, 11.0 µm with IQ, 10.0 µm with 4,8-DiMeIQx, 11.5 µm with PhIP, 12.0 µm with MeA
C and 7.5 µm with A
C.

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Fig. 3. Frequency distributions of comet-forming activities of the six HAAs tested in MCL-5 cells. MCL-5 cells were treated in the presence of HU/ara-C with designated HAAs at a concentration of 90.9 µg/ml (410510 µM). Assays were carried out as described in Materials and methods.
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In order to obtain doseresponse data MCL-5 cells were incubated at final concentrations of 45.5 µg/ml (200250 µM), 90.9 µg/ml (410510 µM) and 454.5 µg/ml (20002480 µM) of an HAA with HU/ara-C and the median comet tail lengths observed are given in Figure 4
. The data show that PhIP was the most active HAA with an increase in median comet tail length even at the lowest concentration tested (P < 0.0001, MannWhitney test); 4,8-DiMeIQx (P = 0.0007), IQ (P < 0.0001), A
C (P < 0.0001) and MeA
C (P < 0.0001) significantly increased comet tail lengths at 90.9 µg/ml, whereas for 8-MeIQx the increase was significant only at the highest concentration tested (P = 0.0002).
Comparison between morphological transformation and genotoxicity
No correlation between morphological transformation (in C3H/M2 mouse fibroblasts) and bacterial mutagenic potency (in S.typhimurium TA1538 and YG1019), or between morphological transformation and micronucleus formation in MCL-5 cells, was observed (Figure 5
). However, an association between morphological transformation and comet formation (in MCL-5 cells) was evident.

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Fig. 5. Comparative biological activities of HAAs in each of three genotoxicity assays and the morphological transformation assay. Values are normalized to the most active HAA in each assay, assigned an arbitrary value of 100 and shown on the vertical axis.
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Discussion
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HAAs are mutagenic substances formed during the high-temperature cooking of proteinaceous foodstuffs such as meat, fish and poultry. In this study we have compared the activities of six HAAs in a morphological transformation assay with their activities in the Ames, micronucleus and comet genotoxicity assays.
In long-term feeding studies with rodents, tumour induction was reported at similar dose levels for all HAAs tested (100800 p.p.m.) with a variable organ specificity that included liver, small and large intestine, zymbal's or mammary gland, lung and prostate (5,24,25). IQ has also been shown to induce liver tumours in primates (26). IQ and PhIP were strong inducers of liver tumours in the neonatal mouse assay whereas 8-MeIQx was less active (27). Subchronic treatment with HAAs also induces preneoplastic foci in the livers of F344 rats (28).
In vitro cell transformation assays are well established short-term predictive tests of tumorigenicity (18). IQ induces cell transformation in BALB 3T3 mouse embryo fibroblasts (29), but the transforming activities of HAAs as a class of carcinogens have not been thoroughly investigated. The transformation assay using the C3H/M2 mouse fibroblast cell line has been employed with a wide range of test compounds and morphologically transformed foci have been shown repeatedly to induce tumours in vivo. Our initial experiments indicated that HAAs required an external metabolic activation system in order to exert their cytotoxic and transforming potential in the C3H/M2 cell line, as does the reference carcinogen AAF. All six HAAs tested were potent inducers of transformed foci in this assay with PhIP being the most potent and the order of activity being PhIP > A
C > MeA
C > 8-MeIQx > 4,8-DiMeIQx > IQ. The C3H/M2 cell line was originally derived from a mouse prostate and, perhaps coincidentally, PhIP has been shown to be an effective inducer of tumours in the rodent prostate (24).
The extraordinarily high mutagenicity of aminoimidazo-quinoxaline and aminoimidazo-quinoline HAAs in the Salmonella/mammalian microsome test has been reported previously (4,6). The two amino-
-carbolines and PhIP are several orders of magnitude less active in this assay than are 8-MeIQx, 4,8-DiMeIQx and IQ. The bacterial mutagenicity of HAAs is elevated when Salmonella strains engineered to overexpress the N-acetyltransferase are employed as test organisms (19). Compared with S.typhimurium TA1538, mutagenicities were elevated by a factor of 13.3 for 8-MeIQx, 5.7 for 4,8-DiMeIQx, 21.8 for IQ, 6.3 for A
C, 16.8 for MeA
C and 1.4 for PhIP (Table II
). N,O-acetylation thus appears to be a more important step in the metabolic activation of aminoimidazo-quinoline and aminoimidazo-quinoxaline-type HAAs than it is for PhIP (7,19). Involvement of N,O-acetylation in the metabolic activation of MeA
C and, to a lesser extent, of A
C has not been described before. Differential expression of conjugating enzymes has been related to the organotropic carcinogenicity of HAAs in experimental animals and polymorphisms of these enzymes have been associated with alterations in human cancer risk (30,31).
The MCL-5 cell line, which expresses a battery of human enzymes known to metabolize many xenobiotics, is well suited for the detection of micronuclei induced by a broad range of clastogenic compounds, including 8-MeIQx (22). Cytotoxicity in the micronucleus assay was highest for 8-MeIQx, 4,8-DiMeIQx and IQ, which were also the most active in inducing micronulei. Whereas PhIP was more cytotoxic than MeA
C, the clastogenic activity was higher for MeA
C. A
C was the weakest compound tested here, with regard to both cytotoxicity and induction of micronuclei. These results are broadly similar to those obtained in the bacterial mutagenicity assays. Micronucleus induction has also been observed in a human hepatoma cell line treated with IQ, 8-MeIQx or PhIP (32), albeit at high concentrations (120 µg/ml). PhIP induced micronuclei in Chinese hamster lung (CHL) cells and this activity was enhanced when these cells were transfected with an N-acetyltransferase gene (33). However, micronuclei were not found in peripheral lymphocytes or in the bone marrow of mice treated with 8-MeIQx or PhIP (34,35). Chromosomal aberrations (CAs) were not observed when CHO cells or human lymphocytes were treated with IQ (36), but sister chromatid exchanges (SCEs) and CAs were detected in an engineered cell line transfected with cytochrome P4501A2 and N-acetyltransferase (37). CAs were observed in CHL cells at 20 µg IQ/ml (38). IQ induced CAs and SCEs in a human fibroblast cell line and in human lymphocytes in the presence of rat-liver S9 (33).
In contrast to the induction of micronuclei in MCL-5 cells, PhIP was the most potent of the HAAs tested when the comet assay was carried out using these cells. The amino-
-carbolines (A
C and MeA
C) were moderately active while 8-MeIQx, 4,8-DiMeIQx and IQ were only active at the highest concentrations tested. Strand-breaking activity of HAAs was less pronounced, or required higher concentrations of the test compound, when inhibitors of DNA repair (hydroxyurea and cytosine arabinoside) were omitted. This refinement has allowed the detection of genotoxic activity that could otherwise have been overlooked. The profile of HAA-induced genotoxicity provided by the comet assay differed from that given by the micronucleus assay in the same cell system. Moreover, the micronucleus assay (which gave positive results at doses not exceeding 25 ng/ml) appeared to be much more sensitive than the comet assay (in which positive results required doses of HAAs of 50100 µg/ml). This 2000-fold difference in sensitivity might be due in part to the fact that cells were incubated for 24 h in the micronucleus assay, in contrast to the 30 min incubation employed in the comet assay. It might also reflect differences in the rates at which various kinds of DNA damage are induced and repaired, and the fact that cell division, which is an intrinsic feature of the micronucleus assay (but not of the comet assay), might amplify the effects of some of the damage produced by the test compounds. In other studies, PhIP has been shown to induce DNA strand breaks in V79 cells in the presence of an external metabolic activation system (39). However, neither PhIP nor IQ induced DNA strand breaks in human colonic epithelial cell populations whereas PhIP alone was active in rat colonic epithelial cells (40).
In summary, the results of the present study demonstrate that for the six HAAs tested, there is an association between morphological transformation and DNA strand-breaking activity, but not between transformation and either bacterial mutagenicity or micronucleus formation. PhIP is the most active in the comet and transformation assays, but among the least active in the micronucleus and bacterial mutagenicity assays. It is not yet clear which of these assays most accurately reflects the genotoxic potential to humans of compounds of this class of environmental carcinogens.
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Acknowledgments
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We thank Chris Crofton-Sleigh for helpful discussions regarding the micronucleus and comet assays and Anne Ruge and Angelika Piasecki for technical assistance with the transformation assays. Supported by research grants from the DFG (grant no. Pf283/1-3) and the Association for International Cancer Research.
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Notes
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2 To whom correspondence should be addressed Email: pfau{at}uke.uni-hamburg.de 
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Received July 22, 1998;
revised November 18, 1998;
accepted November 18, 1998.