Heterocyclic amine induced apoptotic response in the human lymphoblastoid cell line TK6 is linked to mismatch repair status.

The human lymphoblastoid cell, TK6, exhibited a dose-dependent cytotoxic and apoptotic response following treatment with the food borne heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Augmentation of the p53 protein and increases in p21-WAF1 levels were also observed. Comparison of the survival by clonogenic assays and the percentage of apoptotic cells (cells containing subG1 DNA or condensed nuclei) revealed that only 10-20% of the PhIP-induced cell death could be attributed to apoptosis that occurred in the first 24h after treatment. MT1, a derivative of TK6 that contains mutations in both alleles of its hMSH6 gene and is mismatch repair deficient, showed a decreased apoptotic response. A significant increase (P<0.05) in apoptosis was observed in TK6 and not in MT1 following treatment with 2.5microg/ml PhIP. A five- to six-fold increase and less than a two-fold increase in the fraction of apoptotic cells were observed in TK6 and MT1, respectively. Treatment with 5microg/ml PhIP resulted in significant increases in apoptosis (P<0.05) in TK6 and MT1. The percentages of apoptotic cells were, however, two- to three-fold higher in TK6 than in MT1. HCT116, a hMLH1 defective mismatch repair deficient colorectal carcinoma cell line, also exhibited lower PhIP-induced apoptosis than its mismatch repair proficient chromosome transfer cell line (HCT116+chr3) following PhIP treatment. These results show that PhIP-induced apoptosis is mediated through a mismatch repair dependent pathway. Accumulation of p53 in TK6 and MT1 were evident in samples taken 24h after PhIP treatment. Increases in p21-WAF1 were also observed in both cell lines confirming that the p53 was functional. The lower apoptotic response of MT1 but similar p53 accumulation in TK6 and MT1 suggest that the mismatch repair protein(s) are involved downstream of p53 or that PhIP-induced apoptosis is p53-independent.

Comparison of the mutagenic responses of mismatch repair-proficient (TK6) and mismatch repair-deficient (MT1) human lymphoblast cells to the food-borne carcinogen PhIP.

Heterocyclic amines are ubiquitously present in cooked meats and fish. They represent an important class of food-borne carcinogens. We describe the cytotoxic, apoptotic, and mutagenic responses of mismatch repair-proficient (TK6) and mismatch repair-deficient (MT1) human lymphoblastoid cells to PhIP, the most abundant heterocyclic amine. Dose-dependent increases in cytotoxicity, in apoptosis, and in mutant fractions at the hprt locus were observed following PhIP treatment. We present a statistical method that is useful for comparing two populations. With this method, we show that the data fitted a model that assumes that the PhIP-induced mutation rate is dependent on the cell line. Estimated rates of increase of 22.8 x 10(-6) and 2.2 x 10(-6) mutation per cell per microg PhIP were found in MT1 and TK6, respectively, showing that MT1 is hypermutable to PhIP. MT1 also exhibited lower PhIP-induced apoptosis. We conclude from these results that mismatch repair-deficient cells are hypermutable to the food-borne carcinogen PhIP and that the PhIP-DNA adducts, when not eliminated by apoptosis, can be transformed into mutations.

Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in human lymphoblastoid cells.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a heterocyclic aromatic amine that has been identified in cooked meats and cigarette condensates, is mutagenic in human lymphoblastoid TK6 cells at the thymidine kinase and hypoxanthine-guanine phosphoribosyl transferase (hprt) loci. Treatment of the cells with IQ following activation with either an exogenous metabolizing mixture (S9) or following photoactivation of the azido-derivative of IQ (N3-IQ) showed that the photolytic-derivative of N3-IQ was more active. This observation is consistent with other reports that indicate that the weak mutagenicity of IQ in mammalian cells is caused by the lack of enzymes required for the ultimate activation of the compound within the cells. Two DNA adducts were found by 32P-post-labelling in the cells treated with the photoactivated N3-IQ. The major adduct was identified as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) and the minor adduct as 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ). The ratio of the dG-C8IQ to the dG-N2-IQ adducts was approximately 3:1 and did not significantly change in cultures treated with different concentrations of the mutagen. Approximately 50% of the adducts were removed 9 h after treatment with IQ and <10% of these adducts remained after 24 h. There was no significant preferential repair of either adduct under the experimental conditions used. The identification of 15 mutations induced at the hprt locus (of the 44 mutants analysed) showed IQ to be efficient at inducing single base deletions in a run of guanines. Six single guanine deletions were observed in the run of six guanines in exon III and one deletion of a single guanine was observed in a non-repetitive sequence in exon VI. Other mutations observed were two GC-->TA transversions, two GC-->CG transversions, one AT-->TA transversion and one GC-->AT transition. In addition, two multiple mutations were found. The majority of the identified mutations (12/15) occurred at GC base pairs and suggests either the dG-C8-IQ or the dG-N2-IQ adduct to be the pre-mutagenic lesion.

Genotoxicity of ethyl carbamate (urethane) in Salmonella, yeast and human lymphoblastoid cells.

Ethyl carbamate is a known carcinogen occurring in fermented food and beverages and is therefore of interest for food safety assurance. We studied the genotoxicity of ethyl carbamate in Salmonella typhimurium, in Saccharomyces cerevisiae and in human lymphoblastoid TK6 cells. In absence of cytochrome P450 enzymes, no ethyl carbamate-mediated genotoxicity was observed in any of the three test systems in the non-cytotoxic range. In the presence of an activating system, ethyl carbamate was found to be mutagenic in Salmonella typhimurium strain TA100 but not in strains TA98 and TA102, indicating base-pair substitutions at G-C base pairs. In contrast, no significant mutagenicity of ethyl carbamate could be detected in human lymphoblastoid TK6 cells. However, applied in cytotoxic concentrations, ethyl carbamate was genotoxic for Saccharomyces cerevisiae in the absence of P450-mediated metabolic activation. Inhibitors of P450IIE1 (DMSO, ethanol and dithiodiethylcarbamate) diminished ethyl carbamate-mediated mutagenicity in Salmonella typhimurium strain TA100 in a dose dependent manner, suggesting that P450IIE1 is the activating enzyme.

The inhibitory effects of coffee on radical-mediated oxidation and mutagenicity.

Hydrogen peroxide (H2O2) has been implicated as a major contributor to coffee mutagenicity and genotoxicity in vitro. We have used three assays to show the gradual formation of H2O2 in freshly prepared roasted ground coffee and in instant coffees over time reaching levels of 400-450 microM after a 1-h incubation period. Formation of H2O2 occurs through an auto-oxidation process where polyphenolics, in the presence of transition metals, reduce atmospheric oxygen. However, because of these polyphenolics, coffee also possesses in vitro antioxidant activity as shown by its capacity to inhibit lipid peroxidation in Fenton-catalysed hydroxylation reactions. The pro- and antioxidative effects of coffee are also reflected in its mutagenic and antimutagenic activity in the Ames test. Coffee is directly mutagenic in strains TA100 and TA102 due to H2O2 formation. However, coffee is also an antioxidant and antimutagen. This beverage exerts a strong protective effect against the mutagenicity and cytotoxicity induced by the oxidant t-butylhydroperoxide (t-BOOH). Thus, coffee, like many antioxidants, exhibits dual effects in vitro which are highly dependent upon parameters such as dose, atmospheric oxygen, transition metals as well as the biological and chemical endpoints used for measurement. Consequently, the data obtained on the pro- and antioxidant properties of foods and beverages from in vitro bioassays must be interpreted with caution and the results are not easily extrapolated in vivo to assess the impact on human health.