Enhancing the in vitro and in vivo detection of aneuploidy by fluorescence in situ hybridization with the use of bromodeoxyuridine as a proliferation marker

alpha,beta-Unsaturated aldehydes are ubiquitous environmental pollutants, important industrial chemicals, have mani-fold biological functions in plants and insects and are natural products in food. They are endogenously formed in animals and humans during lipid peroxidation and arachidonic acid oxidation and are genotoxic, mutagenic and carcinogenic. Crotonaldehyde and 2-hexenal in food may contribute to general carcinogenicity in humans. The high bacterial toxicity of these compounds leads to problems in genotoxicity testing in bacterial systems. Recently, we have shown that using ethanol as solvent instead of dimethylsulfoxide (DMSO) results in an increase in the induction factors and the SOS-inducing potency of alpha,beta-unsaturated ketones in the SOS chromotest. Here, we demonstrate that utilization of ethanol as solvent also improves the testing of alpha,beta-unsaturated aldehydes. Five aldehydes out of nine tested were clearly positive in the SOS chromotest according to the criteria of Quillardet, i.e. acrolein, crotonaldehyde, 2,4-hexadienal, 2-methylacrolein and 2-ethylacrolein, three further, 2-hexenal, 2-heptenal and 2-propylacrolein showed a dose dependent increase of the induction factors which was however lower than 1.5 times that of the background. Only 2-butylacrolein did not lead to an increase in the induction factors. With DMSO as solvent only the three aldehydes acrolein, crotonaldehyde and 2,4-hexadienal showed an increase in the induction factor, which was however lower than 1.5 that of the background. Utilization of ethanol allows to establish structure genotoxicity relationships for alpha,beta-unsaturated aldehydes in the SOS chromotest. Genotoxicity decreases with increasing degree of substitution. The decreasing genotoxicities can be explained (a) by increasing bacterial toxicity due to increasing lipophilicities of the higher substituted aldehydes and (b) by decreasing reactivity due to steric hindrance by the alkyl substituents.     

Structural requirements for the induction of the SOS repair in bacteria by nitrated polycyclic aromatic hydrocarbons and related chemicals.

The CASE (computer-automated structure evaluation) methodology was used to investigate the structural basis of the SOS-inducing activity of 56 nitrated polycyclic aromatic hydrocarbons (nitroarenes, nPAH) and the unsubstituted parent PAH molecules. Based upon the presence and/or absence of structural features, CASE identified 5 activating (biophores) and 4 inactivating (biophobes) fragments responsible for the SOS-inducing activity. Based upon these fragments, CASE correctly calculated the genotoxicity of 94.6% of the molecules in the training set (sensitivity = 0.85, specificity = 1.0). Disregarding the questionable experimental results of the unexpected very weak direct-acting activity of the unsubstituted benzo[a]pyrene, dibenzo[a,h]anthracene and 7,12-dimethylbenz[a]anthracene, the concordance of the prediction was 100%, i.e., sensitivity = 1.0, specificity = 1.0. Additionally, the quantitative analysis of the SOS-inducing potency showed a good correlation between the experimental and predicted results. The present analyses indicate an identity in the structural determinants responsible for SOS induction in E. coli PQ37 (SOS chromotest) and mutagenicity in Salmonella typhimurium.     

Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons

To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E. coli PQ37 density and centrifugation steps on the expression of β-galactosidase (βg) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs). Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37°C before use.Initially, we used 0–5000 ng (=0–20 nmole) benzo[a]pyrene (B[a]P) as a reference compound for the test procedure in the presence of standard S9 mix. Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol.We observed the highest βg and ap activities and/or IFs using only 11–27 μl 9000 × g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20–50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8–8.0. 60–300 μg NADP/assay (10–50% of standard) was sufficient for optimum activation of PAHs. In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12–18 × 10⁶ cfu/assay). Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts. The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.     

Chemical carcinogenicity: can it be predicted from knowledge of mutagenicity and allergic contact dermatitis?

Naturally occurring substances were tested for genotoxicity using a modified laboratory protocol of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) in the presence and in the absence of an exogenous metabolizing system from rat liver S9-mix. Aristolochic acid I, II, the plant extract aristolochic acid and psoralene were genotoxic; cycasine, emodine, monocrotaline and retrorsine were classified as marginal genotoxic in the SOS chromotest in the absence of S9-mix. In the presence of an exogenous metabolizing system from rat liver S9-mix aristolochic acid I, the plant extract, beta-asarone, cycasin, monocrotaline, psoralen and retrorsine showed genotoxic effects; aristolochic acid II marginal genotoxic effects. Arecoline, benzyl acetate, coumarin, isatidine dihydrate, reserpine, safrole, sanguinarine chloride, senecionine, senkirkine, tannin and thiourea revealed no genotoxicity in the SOS chromotest either in the presence or in the absence of an exogenous metabolizing system from rat liver S9-mix. For 17 of 20 compounds, the results obtained in the SOS chromotest could be compared to those obtained in the Ames test. It was found that 12 (70.6%) of these compounds give similar responses in both tests (6 positive and 6 negative responses). The present investigation and those reported earlier, the SOS chromotest, using E. coli PQ37, was able to detect correctly most of the Salmonella mutagens and non-mutagens.     

Induction of the SOS system in a dam-3 mutant: a diagnostic strain for chemicals causing DNA mismatches

We have developed a strain of E. coli in which expression of the SOS function sfiA, monitored by means of a sfiA::lacZ operon fusion, is efficiently triggered by the two base analogues 2-aminopurine and 5-bromo-2'-deoxyuridine. This strain resulted from introduction of a dam-3 mutation into a Uvr+, Rfa+ derivative of strain PQ37 used in the SOS chromotest, a bacterial colorimetric assay for genotoxins (Quillardet et al., 1982). The dam-3 mutation affects the mismatch correction system in E. coli. We show that the SOS-inducing capacity of a weak SOS inducer such as the alkylating agent ethyl methanesulfonate was also increased in the dam-3 strain. We provide evidence that the increase in SOS inducibility due to the dam-3 mutation is specific for compounds causing DNA mismatches and propose the use of the dam-3 derivative of PQ37 as a diagnostic strain for such agents. This diagnostic strain can be a useful addition to the SOS chromotest.     

Impact of dimethyl sulfoxide and examples of combined genotoxicity in the SOS chromotest.

The bacterial SOS chromotest with Escherichia coli PQ37 was used for the assessment of genotoxicity of combined xenobiotic treatments. The modulation of test compound genotoxicity by dimethyl sulfoxide (DMSO), a common solvent for test compounds, was assessed as well. It was shown that DMSO modulated SOS chromotest genotoxicity of several xenobiotics: in comparison to test compound dissolution in water, the commonly used addition of 3.2% (v/v) DMSO as solvent lead to a significant increase in the genotoxicity of K(2)RhCl(5) and beta-propiolactone (BPL). However, the effects of cisplatin decreased significantly when DMSO was added. Thus, albeit DMSO is not genotoxic in this test itself, it can interfere with SOS chromotest responses. Further experiments were performed in the absence of DMSO. BPL and cisplatin in combination showed an over-additive synergism in SOS genotoxicity as well as K(2)RhCl(5) and cisplatin did. Addition of Pd(NH(3))(4)Cl(2) and NaAsO(2), which are non-genotoxic in the SOS chromotest, did not enhance the K(2)RhCl(5)- or BPL-mediated SOS sfiA induction. Nevertheless, at the highest subcytotoxic dose of NaAsO(2) tested (200 microM), a slight yet significant suppression of BPL-mediated SOS genotoxicity was observed. These results confirm that the SOS chromotest is a useful tool for the rapid evaluation of the combined genotoxicity of compound mixtures. However, the use of DMSO as test solvent has to be taken with caution.     

Genotoxicity assessment of low-level doses of gamma radiation with the SOS chromotest and the Ames test

This is the first study to present data on the genotoxicity of low γ-irradiation doses for E. coli and S. typhimurium cells obtained using the SOS chromotest and the Ames test. The most pronounced effect was recorded in the first 24 h of γ-irradiation. After 72 h in the Ames test and after 96 h in the SOS chromotest, a significant effect of γ-irradiation on bacterial cells was detected. The absence of genotoxicity at the later stages can be explained by the adaptation of bacterial cells to the conditions of exposure. The findings allow the bacterial test system to be used for studying the effects of low doses at the early stages of exposure to radiation.     

Quinolone effects in the SOS chromotest and in the synthesis of biomacromolecules

The genotoxicity of quinolone antibiotics (ciprofloxacin, enoxacin, nalidixic acid, norfloxacin, ofloxacin, pefloxacin) was studied on the selected mutantE. coli strain PQ37 (SOS chromotest). The genotoxicity was expressed by SOS-inducing potential (SOSIP) values. The highest SOSIP values were found with ciprofloxacin (SOSIP=1967 δIF/nmol), the lowest value was observed with nalidixic acid (SOSIP=0.3 ΔIF/nmol). Similar results were also found with the biosynthesis of nucleic acids, as indicated by incorporation of¹⁴C-adenine into TCA-insoluble fractions ofS. typhimurium cells (ciprofloxacin IC₅₀=0.39, nalidixic acid IC₅₀=400). DNA-damaging effects were tested in the absence of an exogenous metabolizing system.     

The influence of organic solvents on estimates of genotoxicity and antigenotoxicity in the SOS chromotest

In this work, the toxicity and genotoxicity of organic solvents (acetone, carbon tetrachloride, dichloromethane, dimethylsulfoxide, ethanol, ether and methanol) were studied using the SOS chromotest. The influence of these solvents on the direct genotoxicity induced by the mutagens mitomycin C (MMC) and 4-nitroquinoline-1-oxide (4-NQO) were also investigated. None of the solvents were genotoxic in Escherichia coli PQ37. However, based on the inhibition of protein synthesis assessed by constitutive alkaline phosphatase activity, some solvents (carbon tetrachloride, dimethylsulfoxide, ethanol and ether) were toxic and incompatible with the SOS chromotest. Solvents that were neither toxic nor genotoxic to E. coli (acetone, dichloromethane and methanol) significantly reduced the genotoxicity of MMC and 4-NQO. When these solvents were used to dissolve vitamin E they increased the antigenotoxic activity of this compound, possibly through additive or synergistic effects. The relevance of these results is discussed in relation to antigenotoxic studies. These data indicate the need for careful selection of an appropriate diluent for the SOS chromotest since some solvents can modulate genotoxicity and antigenotoxicity.     

A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells.

A genetically controlled luminescent bacterial reporter assay, the SOS lux test, was developed for rapid detection of environmental genotoxins. The bioassay is based on the recombinant plasmid pPLS-1, which was constructed as a derivative of pBR322, carrying the promoterless luxCDABFE genes of Photobacterium leiognathi downstream of a truncated cda gene from ColD with a strong SOS promoter. E. coli recA+ strains containing this construction are inducible to high levels of light production in the presence of substances or agents that cause damage to the DNA of the cells. The light signal, reflecting the SOS-inducing potency, is recorded from the growing culture within 1 s, and the test results are available within 1 to 2 h. Induction of bioluminescence was demonstrated by treatment of E. coli C600(pPLS-1) with 6 genotoxic chemicals (mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, nalidixic acid, dimethylsulfate, hydrogen peroxide, and formaldehyde) and with UV and gamma radiation. A clear dose-response relationship was established for all eight genotoxins. The sensitivity of the SOS lux test is similar to that of other bioassays for genotoxicity or mutagenicity, such as the SOS chromotest, umu test, and Ames mutatest. These results indicate that the SOS lux test is potentially useful for the in situ and continuous detection of genotoxins.     

A comparative study of mutagenic and SOS-inducing activity of biphenyls, phenanthrenequinones and fluorenones

A total of 23 chemicals--biphenyls, phenanthrenequinones and fluorenones--were tested for mutagenicity towards Salmonella typhimurium strains TA1538, TA1535 and TA98. SOS-inducing activity of the same chemicals was studied in terms of the SOS-inducing potency in Escherichia coli PQ37, using an automated instrument controlled by a dedicated computer program for the SOS Chromotest. Of the 23 chemicals studied 14 induced His+ revertants in S. typhimurium TA1538 hisD305 (-1 frameshift); none induced His+ reversions in TA1535 (base-pair substitution). The mutagenicity of the chemicals in S. typhimurium TA98 (pKM 101) was lower than in TA1538. There was a close correlation between mutagenicity and SOS-inducing activity of fluorenones and phenanthrenequinones. None of the biphenyls tested induced SOS response and this property does not depend upon the mutagenic activity of the chemicals. SOS Chromotest is particularly valid in detecting chemicals which give rise to base-pair substitutions through SOS induction. If positive results are obtained, the Salmonella assay may be omitted. However, this test cannot replace the Ames test especially for the primary screening of mutagenicity of chemicals with unknown structure.     

Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. A comparison with the mutagenicity in S. typhimurium TA100

17 monofunctional methanesulphonates of widely varying structures were investigated in the SOS chromotest using the E. coli strain PQ37. All compounds tested were positive in this assay. The monofunctional methanesulphonates in general possess low SOSiP values. Five of the compounds tested i.e. iBMS, NpMS, 2 PhPMS, PkMS and 1,3-DC12PMS (for abbreviations see Table 1) did not show increasing beta-galactosidase activity and both the positive induction factors and the positive SOSiP values resulted from the toxicity correction as performed according to Quillardet and Hofnung (1985). In general methanesulphonates with a higher SN1 reactivity, in particular the secondary compounds, showed clear genotoxic activities whereas those possessing low SN1 reactivities (primary compounds) induced a low SOS repair indicating that the alkylation of O-atoms in the DNA bases contributes more to the induction of SOS repair in strain PQ37 than N-alkylations. The only exception was methyl methanesulphonate (MMS) which possessed a very high SN2 reactivity but a rather low SN1 reactivity. It had the highest SOSiP value of all tested methanesulphonates. No dependence of the genotoxicity on the SN2 reactivity could be found in this series. In general the phenyl-substituted methanesulphonates showed higher SOSiP values, which is presumably due to their relatively high SN1 reactivities and their relatively long life times in aqueous systems. There is a clear relationship between SN1 reactivities and the SOSiP values: the SOSiP values increase with rising SN1 reactivities reaching a maximum at iPMS after which the genotoxicities decrease due to the decreasing life times. The compounds with very high SN1 reactivities also possess very high hydrolysis rates. A good correlation could be established between the mutagenicities in S. typhimurium TA100 and the SOS chromotest (strain PQ37). Only 4 small deviations from this correlation could be found. The reasons for these deviations are discussed.     

Levels of direct-acting mutagens, total N-nitroso compounds in nitrosated fermented fish products, consumed in a high-risk area for gastric cancer in southern China.

A high gastric cancer mortality in Fujian province (Peoples Republic of China) has been associated with the consumption of certain salted fermented fish products such as fish sauce (FS). We have investigated the levels and nature of N-nitroso compounds (NOC) and genotoxins present, before and after nitrosation, in 49 FS samples collected from villages in this high-risk area, pooled into six samples. The concentrations of total NOC before nitrosation ranged from 0.2 to 16 mumoles/l, and after nitrosation at pH 2 and pH 7, they rose by up to 4800- and 100-fold, respectively. In nitrosated samples, 40-50% of total NOC was not extractable into organic solvents; volatile N nitrosamines accounted for 1-2% and N-nitrosamino acids for 8-16% of total NOC. None of the FS samples exhibited genotoxic activity, but after nitrosation all were weakly active in the SOS chromotest. The highest SOS-inducing potency was observed with nitrosated ethyl acetate extracts of most samples. The formation of methylating agents was measured by incubation of nitrosated FS with DNA and subsequent analysis of 7-methylguanine adduct. 2 of the 6 nitrosated FS samples caused a slight increase in DNA methylation. 1 pooled home-made FS sample (the only one tested) contained tumour promoter-like substances, as measured by expression of certain EBV genes in Raji cells. HPLC fractionation of ethyl acetate extracts of FS samples allowed identification of three UV-absorbing peaks that, upon nitrosation, produced direct-acting genotoxins. This genotoxicity was partly ascribed to the formation of nitrite-derived arene diazonium cations that were characterized by a coupling reaction with N-ethyl-1-naphthylamine and thin-layer chromatography.     

Survival and induction of SOS in Escherichia coli treated with cisplatin, UV-irradiation, or mitomycin C are dependent on the function of the RecBC and RecFOR pathways of homologous recombination

Resistance of tumors to drugs such as cisplatin and mitomycin C (MMC) is an important factor limiting their usefulness in cancer chemotherapy. The antitumor effects of these drugs are due to the formation of bifunctional adducts in DNA, with cisplatin causing predominantly intrastrand-crosslinks and MMC causing interstrand-crosslinks. The SOS chromotest was used to study the cellular mechanisms that process DNA damage in Escherichia coli exposed to cisplatin, ultraviolet irradiation (UV) and MMC and subsequently facilitate the production of a molecular signal for induction of the SOS response. Strains used in the SOS chromotest have a fusion of lacZ with the sfiA (sulA) gene so that the amount of SOS inducing signal, which is modulated by the ability of the cell to repair DNA, is measured by assaying beta-galactosidase activity. SOS induction in a strain proficient in homologous recombination (HR) was compared with that in isogenic strains deficient in HR due to a blocked RecBC pathway caused by a recB mutation or a blocked RecFOR pathway caused by a recO mutation. The effect of cisplatin treatment in a uvrA mutant strain blocked at the first step of NER was compared with that in an isogenic strain proficient in NER. Cellular resistance was measured as percent colony forming units (cfu) for cells treated with increasing doses of cisplatin, MMC and UV relative to that in untreated control cultures. The importance of both HR pathways for resistance to these treatments was demonstrated by decreased survival in mutants with the recB mutant being more sensitive than the recO mutant. SOS induction levels were elevated in the sensitive recB strain relative to the HR proficient strain possibly due to stalled and/or distorted replication forks at crosslinks in DNA. In contrast, induction of SOS was dependent on RecFOR activity that is thought to act at daughter strand gaps in newly synthesized DNA to mediate production of the signal for SOS induction. Proficiency in NER was necessary for both survival and high levels of SOS induction in cisplatin treated cells.     

Antioxidative activity of thiophane [bis(3-(3,5-di-tret-butyl-4-hydroxyphenyl)propyl)sulfide]

The antioxidant activity, mutagenicity, and genotoxicity of bis(3-(3,5-di-tret-butyl-4-hydroxyphenyl)propyl)sulfide (thiophane) were studied using bacterial tests. The results of both an Ames test and SOS chromotest, as well as those studying the survival of E. coli cells deficient in enzymes responsible for the repair of DNA oxidative damage, testify to the fact that thiophane is not mutagenic and genotoxic, and it protects Salmonella typhimurium cells better than the well-known antioxidant trolox.     

Genotoxicity of six pesticides by Salmonella mutagenicity test and SOS chromotest

Two in vitro tests (Ames test and SOS chromotest), one for bacterial mutagenicity and one for primary DNA damage, were assayed to determine the genotoxic activity of 6 pesticides (atrazine, captafol, captan, chlorpyrifosmethyl, molinate and tetrachlorvinphos). Assays were carried out both in the absence and presence of S9 fractions of liver homogenate from rat (Sprague–Dawley) pretreated with Aroclor 1254. Captan and captafol were genotoxic on both the Ames test and the SOS chromotest. Comparisons with mutagenesis data in Salmonella indicated that the SOS assay detected as genotoxic the pesticides that were mutagenic on the Salmonella test. Non-genotoxic effects were not detected in vitro either in the Salmonella/microsome assay nor in the SOS chromotest when bacterial tester strains were exposed to atrazine, molinate, chlorpyrifosmethyl and tetrachlorvinphos in the absence or presence of S9 mix.     

Induction of SOS-response of cells exposed to autoregulatory factors of microorganisms

Among examined microbial growth regulators of alkyl hydroxybenzene group (hexylresorcinol, methylresorcinol, and hydroxyethylphenol), only hexylresorcinol induces cellular SOS response, demonstrating a dose-dependent increase of the induction factor in the SOS chromotest with the Escherichia coli PQ37 strain. At the highest of used concentrations (100 micrograms/ml), hydroxyethylphenol and nonalkylated resorcinol were shown to exert a weak toxic effect, reducing the activity of constitutive alkaline phosphatase, but did not induce SOS response. Nontoxic methylresorcinol did not induce genome damage, which can trigger SOS functions. It is concluded that substitutions in phenolic ring affect genotoxic activity of alkylresorcinols.     

Mutagenic effect on Escherichia coli bacteria of 8-hydroxy-2'-deoxyguanosine--a DNA base damage product induced by oxygen radicals and ionizing radiation]

The effect of 8-oxo-2'-deoxyguanosine (8-oxo-dG) (8-hydroxydeoxyguanosine)--a DNA base damage product induced by oxygen radicals and irradiation on survival and mutagenesis in Escherichia coli strains C-600 and P-687 was investigated. Survival and mutagenesis curves, in dependence of 8-oxo-dG concentrations in the medium, ranging from 0.2 through 10 mM, were obtained. Bacterial survival at all 8-oxo-dG concentrations tested was shown to be no lesser than in the control. The mutagenic effect of 8-oxo-dG was tested by frequency of reversions in the absence of leucine and threonine. A non-linear dependence of mutagenesis on the concentration was observed. Linear increase in the amount of revertants took place at concentrations of 8-oxo-dG lower than 1 mM, and being kept constant at higher concentrations. Induction of SOS repair under the action of 8-oxo-dG in E. coli PQ37 strain was estimated according to alteration of activity of beta-galactosidase in the SOS chromotest. Weak induction of the SOS response was observed within the wide range of 8-oxo-dG concentration values, which points to a lack of genotoxicity and independence of mutagenesis on SOS repair.     

Antimutagenic activity of flavonoids from Chrysanthemum morifolium

A methanol extract from the flower heads of Chrysanthemum morifolium showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract was re-extracted with hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction showed a suppressive effect. Suppressive compounds in the ethyl acetate fraction were isolated by silica gel column chromatography and identified as the flavonoids acacetin (1), apigenin (2), luteolin (3), and quercetin (4) by EI-MS, IR, and (1)H and 13C NMR spectroscopy. Compounds 1-4 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1-4 suppressed 60.2, 75.7, 90.0, and 66.6% of the SOS-inducing activity at a concentration of 0.70 micromol/ml. The ID50 (50% inhibitory dose) values of 1-4 were 0.62, 0.55, 0.44, and 0.59 micromol/ml. These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver-metabolizing enzymes. These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver-metabolizing enzymes, and UV irradiation. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S. typhimurium TA100.