Comparative genotoxic effects of the cooked-food-related mutagens Trp-P-2 and IQ in bacteria and cultured mammalian cells

As part of a major study to evaluate the mutagenicity of chemicals produced during the cooking of foods, we examined the responses of bacteria and cultured Chinese hamster cells to the compounds Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and IQ (2-amino-3-methylimidazo[4,5-f]quinoline), constituents identified in cooked beef and fish. In the Ames/Salmonella tester strain TA1538, both compounds were confirmed to be extremely potent mutagens that were active at levels below 1 ng/plate in the presence of hamster-liver S9 microsomal fraction. 50-fold higher doses of both compounds were required for mutagenicity in the uvr+ tester strain TA1978. Trp-P-2 also behaved as a strong mutagen in CHO cells using the standard exogenous activation with hamster-liver S9 fraction. At concentrations below 1 microgram/ml it produced dose-dependent increases in cell killing, mutations at the hprt and aprt loci, sister-chromatid exchanges, and chromosomal aberrations. An excision-repair-deficient strain was about 2-fold more sensitive than the normal CHO cells with respect to these genotoxic effects of Trp-P-2. IQ had unexpectedly weak activity for all genetic endpoints in the CHO cells, and it produced clear-cut responses only in the repair-deficient cells and only above a concentration of 10 micrograms/ml. The toxicity that was observed with IQ was not affected by the repair capacity of the cells and was not associated with chromosomal aberrations, indicating that damage to cellular structures other than nuclear DNA was likely the predominant pathway for cell killing. Because the culture conditions normally used for CHO cell exposure were shown to be competent in producing bacterial mutagenicity with IQ, it was concluded that the active metabolite of IQ was present in the medium but was somehow ineffective in reaching the DNA of CHO cells and/or reacting with it. These results suggest that the relative mutagenic potency of compounds in Salmonella may bear no direct relationship to relative mutagenicity in CHO cells, emphasizing precaution in attempting to extrapolate microbial data to mammalian somatic cells. This study illustrates the use and merits of a multi-endpoint assay for genetic damage in CHO cells, the utility of using CHO cells that are defective in excision repair of DNA, and the importance of comparative testing between bacterial and mammalian systems.     

Peptidoglycan structure of Salmonella typhimurium growing within cultured mammalian cells

The cell wall structure of Salmonella typhimurium has been studied for the first time during transit from free-living to parasitic lifestyles. Peptidoglycan of S. typhimurium proliferating within human epithelial cells contains a high proportion of previously unidentified muropeptides (5–10-fold higher than in extracellular bacteria). Amino acid and mass-spectrometry analyses showed that these new components consist of dimeric cross-linked muropeptides lacking one of the two disaccharide ( N -acetyl-glucosamine-β-(1→4)- N -acetyl-muramic acid) molecules. This unique structure suggests an active role for an N -acetyl-muramyl- l -alanine-amidase in remodelling the peptidoglycan of intracellular S. typhimurium . Additional alterations observed included: (i) the absence of glycine-containing muropeptides; (ii) the increase in the relative proportion of muropeptides cross-linked by l ( meso )-diaminopimelyl- d ( meso )-diaminopimelic acid ( l – d ) peptide bridges; and, (iii) the decrease in the global cross-linkage of the macromolecule. The structural alterations observed in the peptidoglycan of intracellular bacteria do not produce loss of the cell envelope. These results show that intracellular residence of S. typhimurium within epithelial cells is accompanied by significant changes in the bacterial cell wall. Remodelling of peptidoglycan structure may constitute another sophisticated strategy of this pathogen for adapting to and colonizing the intracellular niche of eukaryotic cells.     

Enhancement of the mutagenicity of IQ and MeIQ by nitrite in the Salmonella system.

The fried food mutagens IQ, MeIQ, Glu-P-1 and Trp-P-2 were treated with nitrite at pH 3.0 for 1 h at 37 degrees C. The resulting reaction mixtures were tested for mutagenicity towards Salmonella typhimurium TA97, TA98, TA100 and TA1535. Glu-P-1 and Trp-P-2 were readily converted to weak or non-mutagenic deaminated compounds, whereas IQ and MeIQ were converted to extremely strong mutagenic derivatives in both the presence and the absence of rat liver S9 mix. The mutagenicity of MeIQ in TA98 was enhanced by nitrite up to 3-fold, while that of nitrosated MeIQ was further enhanced by S9 mix up to 15-fold. The nitrosation products of MeIQ were resolved into 7 bands by TLC on silica gel plate. Bands I, III, V and VI were highly mutagenic to both TA98 and TA100. The experimental results suggest that the non-enzymatic formation of direct-acting mutagens from indirect-acting mutagens such as IQ or MeIQ might be physiologically important, especially with regard to the etiology of human gastrointestinal tract tumors.     

Genotoxicity and genotoxic enhancing effect of tetrandrine in Salmonella typhimurium

Tetrandrine has been used for the treatment of silicosis in China. The potential genotoxic and carcinogenic hazards of this drug were studied using the Salmonella/histidine reversion assay and the SOS/Umu test. The results show that tetrandrine was weakly mutagenic to Salmonella typhimurium TA98 with metabolic activation and did not induce SOS response. However, tetrandrine increased the mutagenic activity of benzo[alpha]pyrene, trinitrofluorenone (TNF), 2-aminoanthracene (2AA), diesel emission particles, airborne particles, and cigarette smoke condensate by more than 100%; the activity of aflatoxin B1 and fried beef was increased by over 75%. It also increased the 2AA and TNF-induced SOS response by more than 300%. These results indicated that tetrandrine was a weak promutagen inducing frameshift mutations and was a potent genotoxic enhancer. The mechanism for the genotoxic enhancement is not known. However, the fact that the increase in mutagenicity was noted only in TA98 and not in TA1538 suggested that the enhancement of genotoxicity by tetrandrine may result from an increase in error-prone DNA repair.     

Comparative mutagenicity of halogenated pyridines in the Salmonella typhimurium/mammalian microsome test

The Salmonella/microsome assay with strains TA97, TA98, TA100 and TA102 was used to examine the potential mutagenicity and structure-activity of 16 mono- and di-halogenated pyridines. The chemical reactivity of the halopyridines suggests that nucleophilic displacement of halogens can occur with halogens at positions 2, 4 and 6 being displaced in addition-elimination reactions. 2-Chloropyridine gave a positive result with rat-liver metabolic activation, and 2-fluoropyridine gave equivocal results under these conditions. Mutagenic responses were also obtained with 2-chloromethyl pyridine and 3-chloromethyl pyridine, in both the presence and absence of rat-liver S9. These results suggest that the halogenated pyridines, especially with halogens at the 2-position, and singly on a methyl substituent, have mutagenic activity in the Salmonella assay.     

Genotoxic activity of caramel on Salmonella and cultured mammalian cells

The genetic activity of 2 commercial caramel preparations, manufactured either by heating the malt sugar solution directly (non-ammoniated caramel) or by heating it with ammonia (ammoniated caramel) was studied in the Salmonella mutagenicity test and UDS assay in cultured mammalian cells. The non-ammoniated caramel was found to be mutagenic to S. typhimurium TA100, while the ammoniated one was genetically active in all the tester strains used, namely TA100, TA97 and TA98. It was also demonstrated that non-ammoniated caramel was capable of inducing UDS in cultured human amnion FL cells, but for the ammoniated one, no such activity was observed. Furthermore, based on the results obtained in the DNA synthesis inhibition assay, it was suggested that the DNA synthesis inhibition seen in our experiments with the ammoniated caramel was probably not of DNA damage in origin. These data indicate that the mutagenic fractions formed during ammoniated and non-ammoniated caramelization were quite different.     

An investigation of the genotoxic effects of N-nitrosomorpholine in mammalian cells

N-nitrosomorpholine (NMOR) is a well-known hepatocarcinogen. Since this compound is representative of the group of indirect-acting N-nitrosamines, its metabolic activation should be essential. However, the mechanism of NMOR-induced carcinogenesis is still not completely clear. In this paper we tried to further our understanding of the genotoxic effects of NMOR. The central aim of this study was to elucidate to what extent NMOR requires metabolic activation. For evaluation of the mutagenicity of NMOR, V79 cells were used either in the presence or absence of the microsomal S9 fraction in the mutation assay and formation of reactive oxygen/nitrogen species (ROS/RNS) in Caco-2 cells treated with NMOR was measured by a fluorescent assay. A very weak rise of 6-thioguanine resistant mutations was observed in both NMOR-treated model cells, V79/-S9 and V79/+S9. A significant difference between the level of mutations in V79/-S9 and V79/+S9 cells was recorded on the 7th day of expression only. Data obtained by the fluorescent assay confirmed that NMOR caused generation of ROS/RNS. In summary, the presented results showed that NMOR might induce DNA damage not only indirectly by its activation by drug-metabolizing enzymes but also via direct formation of ROS/RNS.     

Metabolic conversion of IQ and MeIQ to bacterial mutagens

The metabolic conversion of 2-amino-3-methyl- and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (IQ and MeIQ respectively) to bacterial mutagens was studied using a bacterial mutation assay. Studies were performed using S9 fractions derived from either corn oil (uninduced) or Aroclor-1254-treated Sprague-Dawley rats. Aroclor 1254 treatment lowered the S9 protein concentration required for optimum levels of mutagenesis, enhanced the numbers of mutants observed and altered the effects of metabolic inhibitors and cofactors added to the assay. Studies with uninduced preparations revealed that IQ and MeIQ exhibited similar responses to the effects of metabolic inhibitors and cofactors involved in detoxication reactions. Both IQ and MeIQ activation appeared to be inhibited by the biogenic amines tryptamine and tyramine and inactivated by conjugation with either acetyl coenzyme A or glutathione.     

Dose-rate effects of gamma-ray-induced mutations in cultured mammalian cells

The dose-rate dependency of three radiobiological parameters, cell killing and mutations resistant to 6-thioguanine (6-TG^r) and to methotrexate (MTX^r), were studied in populations of mouse L5178Y cells exposed to gamma-rays. when the dose rate was reduced from 50 rad/min to 0.8 rad/min, the shape of the dose—response curves changed from sigmoidal to exponential for cell killing, from upward concave to linear in 6-TG^r mutations and remained linear in MTX^r mutations. A linear quadratic model appears capable of explaining the cell killing and 6-TG^r mutations but not the MTX^r mutations.The declining patterns of induced mutation frequencies of 6-TG^r and MTX^r with decreasing dose rate seem to be similar. The addition of DMSO resulted in protection of cells from cell killing, 6-TG^r and MTX^r mutations with acute exposure, but had little effect with chronic exposure. The reduction of mutation frequency of the 6-TG^r marker with chronic exposure was eliminated by holding cells in ice-cold condition during irradiation. These results suggest that there may be two components of induced mutation. One results primarily from repairable damage induced by the indirect action of radiation and shows a clear dose-rate dependency. The other is mainly from non-repairable damage by the direct action of radiation and is only slightly dose rate-dependent. Under chronic exposure conditions, the latter may predominate.     

The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells

The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C-terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118-142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity.     

Uptake and genotoxic effects of ochratoxin A in cultured porcine urinary bladder epithelial cells

Uptake of radiolabelled ochratoxin A (OTA) into porcine urinary bladder epithelial cells (PUBEC) was measured at neutral (pH 7.5) or acidic (pH 5.0) conditions. Genotoxicity of OTA was evaluated with the Comet assay and cytotoxicity with the neutral red uptake assay. At acidic pH-conditions, the bladder cells were able to take up more OTA than at neutral conditions. Cytotoxic effects were not increased at pH 5.0 compared to pH 7.5, but higher OTA uptake correlated with stronger genotoxic effects in the Comet assay at pH 5.0 compared to pH 7.5. These results demonstrate that uptake of OTA has to be regarded as an important factor for the toxicity of OTA as adverse effects depend on the amount of OTA taken up by the cells. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Cytotoxic and genotoxic effects of energetic compounds on bacterial and mammalian cells in vitro.

The mutagenicity and toxicity of energetic compounds such as 2,4, 6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), hexahydro-1,3, 5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3, 5,7-tetrazocine (HMX), and of amino/nitro derivatives of toluene were investigated in vitro. Mutagenicity was evaluated with the Salmonella fluctuation test (FT) and the V79 Chinese hamster lung cell mutagenicity assay. Cytotoxicity was evaluated using V79 and TK6 human lymphoblastic cells. For the TK6 and V79 assays, TNB and 2, 4,6-triaminotoluene were more toxic than TNT, whereas RDX and HMX were without effect at their maximal aqueous solubility limits. The primary TNT metabolites (2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2, 6-diamino-4-nitrotoluene) were generally less cytotoxic than the parent compound. The FT results indicated that TNB, TNT and all the tested primary TNT metabolites were mutagenic. Except for the cases of 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene in the TA98 strain, addition of rat liver S9 resulted in either no effect, or decreased activity. None of the tested compounds were mutagenic for the V79 mammalian cells with or without S9 metabolic activation. Thus, the FT assay was more sensitive to the genotoxic effects of energetic compounds than was the V79 test, suggesting that the FT might be a better screening tool for the presence of these explosives. The lack of mutagenicity of pure substances for V79 cells under the conditions used in this study does not preclude that genotoxicity could actually exist in other mammalian cells. In view of earlier reports and this study, mutagenicity testing of environmental samples should be considered as part of the hazard assessment of sites contaminated by TNT and related products.     

Comparative genotoxicity of nitrosamine drinking water disinfection byproducts in Salmonella and mammalian cells

Nitrosamine water disinfection byproducts (DBPs) are an emerging class of non-halogenated, nitrogen-containing water contaminants. Five nitrosamine DBPs were analyzed for genotoxicity (N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) and N-nitrosodiphenylamine (NDPhA). Using Salmonella typhimurium strain YG7108 the descending rank order of mutagenicity was NDMA>NPIP>NMOR>NPYR; NDPhA was not mutagenic. We developed and calibrated an exogenous S9 mix that was highly effective in activating NDMA in Chinese hamster ovary (CHO) cells using the SCGE (Comet) assay. The descending rank order for genotoxicity was NDMA>NPIP>NMOR. NDPhA was genotoxic only at one concentration and NPYR was not genotoxic. The genotoxic potencies in S. typhimurium and CHO cells were highly correlated. Based on their comparative genotoxicity attention should be focused on the generation and occurrence of NDMA, NPIP and NMOR. Current drinking water disinfection processes may need to be modified such that the generation of nitrosamine DBPs is effectively limited in order to protect the environment and the public health.     

Mutagenic activity of 4 active-principle forms of pharmaceutical drugs. Comparative study in the Salmonella typhimurium microsome test, and the HGPRT and Na+/K+ ATPase systems in cultured mammalian cells

The nitroimidazole derivatives used in human therapy have shown a strong mutagenic activity in bacterial tests using Ames strains of Salmonella typhimurium. Our study compared the response of 4 products of this family on bacterial target cells as well as on mammalian target cells (Chinese hamster V79 cells). The strong positive response on TA100 was greatly reduced on the nitroreductase-deficient strain TA100 Frl. Furthermore, no mutagenic activity was found in V79 mammalian cells that we examined for ouabain and 6-thioguanine resistance.     

Toxic and mutagenic effects of formaldehyde in Salmonella typhimurium

Toxic and mutagenic activities of formaldehyde were studied in Salmonella typhimurium strain TM677, using forward mutation to 8-azaguanine (8-AG) resistance both in the absence and in the presence of Aroclor-induced rat-liver postmitochondrial supernatant (PMS). The results showed that formaldehyde was toxic and mutagenic to the bacteria in both systems, but toxicity and mutagenicity were reduced in the presence of PMS. The minimum concentration required to induce toxicity and mutagenicity was 0.17 mM in the absence of PMS and 0.33 mM in the presence of PMS.     

Radiation equivalent of genotoxic chemicals: Validation in cultured mammalian cell lines

Published data on mutations induced by ionizing radiation and 6 monofunctional alkylating agents, namely EMS, MMS, ENNG, MNNG, ENU and MNU, in different cell lines (Chinese hamster ovary, Chinese hamster lung V79, mouse lymphoma L5178 and human cells) were analysed so that radiation-equivalent chemical (REC) values could be calculated.REC values thus obtained for a given alkylating agent with different cell lines fall within a narrow range suggesting its validation in cultured mammalian cell systems including human.     

Comparative studies of the effects of carcinogenic and antitumor agents on the DNA replication of cultured mammalian cells

Cultured mouse L₅₁₇8Y cells were exposed to several carcinogenic and antitumor agents. After exposure to one of the agents, the cells were label with [³H]-thymidine for 20 min, and the DNA was subjected to alkaline sucrose gradient centrifugation immediately or after a chase period. This led us to classify the agents into 3 groups: (1) UV, 4-nitroquinoline-1-oxide (4NQO), N-methyl-N′-nitrosoguanidine (MNNG), nitrogen mustard and Mitomycin C. These were characterized by 20-min DNA labeling patterns showing the formation of small DNA and by the slowing down of their subsequent elongation. Replicated DNA strands would have gaps where “damage” was present on the parental strands. Subsequently, gap-filling replication would occur with or without repairing damage. (2) γ-rays. The 20-min DNA labeling profile displayed a larger size of DNA pieces and the subsequent elongation of this DNA was slightly affected. This probably due to a preferential depression of initiation DNA replication. (3) Methyl methanesulfonate (MMS) and low temperature (28°). The 20-min DNA labeling patterns were qualitatively similar to, but quantitatively different from those of non-irradiated control. The rate of DNA elongation was slightly retarded.     

Mutagenicity of pyrrolizidine alkaloids in the Salmonella typhimurium/mammalian microsome system

The mutagenicity of a series of pyrrolizidine alkaloids, and of extracts from several Italian Senecio species containing pyrrolizidine alkaloids, including S. inaequidens, S. fuchsii and S. cacaliaster, were tested using the Salmonella typhimurium/mammalian microsome system. Retrorsine, senecivernine, seneciphylline and the Senecio extracts showed a weakly mutagenic activity.