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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., (Copyright 2003 Wiley-Liss, Inc.)

Low-level cytotoxicity may affect low-dose dose-response relations for cancer and other endpoints. Conventional colony-forming assays are rarely sensitive enough to examine small changes in cell survival and growth. Automated image-analysis techniques are limited to ca. 10(4) cells/plate. An alternative method involves encapsulation of single proliferating cells into ca. 35-75-microm-diameter agarose gel microdrops (GMDs) that are randomly grouped, differential exposure of these groups, culture at 37 degrees C for 3-5 days, and finally GMD analysis by flow cytometry (FC) to determine the ratio of GMDs containing multiple versus single cells as a measure of clonogenic survival. This GMD/FC assay was used to examine low-dose cell killing induced by a cooked-meat mutagen/rodent-carcinogen (MeIQx) in DNA-repair-deficient/metabolically-sensitive CHO cells. Results of conventional colony-forming assays using up to 30 replicate plates indicate a shouldered, threshold-like dose-response; in contrast, those obtained using the GMD/FC assay suggest "hypersensitivity"-like nonlinearity in dose-response. The GMD/FC assay was also applied to human A549 lung cells after GMD-encapsulation and gamma radiation followed by culture for a total of 4 days, to examine survival after exposure to > or =100 cGy delivered at a relatively low dose rate (0.18 cGy/min). Dose-response for clonogenic growth was again observed to be reduced with apparent nonlinear suggesting hypersensitivity between 0 and 50 cGy, insofar as doses of 5 and 10 cGy appear to be ca. fivefold more effective per unit dose than the 50- or 100-cGy doses used. The GMD/FC assay may thus reveal low-dose dose-response relations for chemical and radiation effects on cell proliferation/killing with implications for low-dose risk assessment.

Background: Heterocyclic amine carcinogens are formed during the cooking of a number of foods, especially well-done meats. Lower temperatures and shorter cooking times can minimize the formation of these carcinogens, yet a major food safety concern is that pathogens in the meat must be thermally inactivated. This study investigated cooking techniques that minimize heterocyclic amine formation while simultaneously destroying contaminating bacteria., Methods: Ground beef patties were inoculated with Escherichia coli K12 bacteria and fried to internal temperatures ranging from 35 degrees C to 70 degrees C in a skillet preheated to 160 degrees C, 180 degrees C, or 200 degrees C. Each patty was then analyzed for four common heterocyclic amines and for surviving bacteria. Additionally, the frequency of turning of the beef patty during cooking was varied (a single turn or multiple turns), length of time required for each patty to reach 70 degrees C was recorded, and heterocyclic amine levels were determined. An additional pan temperature of 250 degrees C was tested for its effect on heterocyclic amine formation but not on bacterial killing. Statistical tests were two-sided., Results: Colony-forming bacteria were reduced by five orders of magnitude at internal temperatures greater than 60 degrees C, regardless of cooking method, and were completely inactivated at 70 degrees C. For patties turned just once, heterocyclic amine levels increased as the cooking temperatures increased. However, levels of heterocyclic amines were statistically significantly lower with turning every minute. For each pan temperature, patties reached 70 degrees C internal temperature sooner when they were turned every minute than when they were turned just once during cooking., Conclusion: Lowering the pan temperature and turning the patties frequently can greatly reduce the formation of heterocyclic amines and can simultaneously achieve bacterial inactivation with little or no increase in cooking time, ensuring a product that is safe for human consumption.

To test the hypothesis that the sulfotransferase gene plays a role in the phase II bioactivation of PhIP, a heterocyclic amine found in cooked meats, we transfected the UV5P3 cell line with cDNA plasmids of human aryl sulfotransferases (HAST1 and HAST3). UV5P3 is a nucleotide excision repair-deficient and P4501A2-expressing CHO cell line that we have previously developed. Functionally transformed clones were identified by the differential cytotoxicity (DC) assay that used PhIP as the cytotoxic agent. Two clones designated 5P3H1 and 5P3H3, expressing HAST1 and HAST3, respectively, were chosen for further characterization. Correct fragment sizes of the sulfotransferase cDNAs were identified in both cell lines by polymerase chain reaction. Immunoblot analysis confirmed the expression of the sulfotransferase proteins. The addition of the sulfotransferase inhibitor DCNP decreased the cytotoxic effects of PhIP in a dose-dependent manner. The increase in cell growth was 6. 5-fold for 5P3H1 and 2.4-fold for 5P3H3, relative to values obtained without DCNP. Based on D(50) values, the dose that reduced the survival to 50% relative to untreated controls, the cytotoxic effect of PhIP was increased threefold for 5P3H1 and 1.87-fold for 5P3H3 cell lines over the parental UV5P3 line. There was also a small increase in the mutation response at the aprt locus. These newly established 5P3H1 and 5P3H3 sulfotransferase-expressing cells provide valuable mechanistic information of the bioactivation of PhIP and related compounds. Environ. Mol. Mutagen. 35:57-65, 2000. Published 2000 Wiley-Liss, Inc.