Genotoxicity and mutagenicity of melanoidins isolated from a roasted glucose-glycine model in human lymphocyte cultures, intestinal Caco-2 cells and in the Salmonella typhimurium strains TA98 and TA102 applying the AMES test.

Melanoidins are formed during household cooking procedures and are part of our daily diet, but data on their toxicological potential are still scarce. Therefore, the mutagenic, cytotoxic and genotoxic activity of the water soluble total fraction (sol A), the water soluble high molecular weight fraction (HMW; Molecular weight>12,400 Da) and the remaining water soluble low molecular weight fraction (LMW) isolated from a glucose-glycine model system roasted at 125 degrees C was comprehensively studied in human lymphocytes (genetic end point: sister chromatid exchange (SCE)), Caco-2 cells (SCE, cell viability, cell proliferation) and in the Salmonella typhimurium strains TA98 and TA102 (Ames test). Tests were performed in a dose- and time-dependent manner. The results indicate a significant increase in SCE formation in human lymphocytes after the exposure to 0.05% and 0.1% of the melanoidin fractions. In Caco-2 cells, only the exposure to LMW increased the SCE formation as a matter of concentration. Cell's proliferation and viability decreased significantly after exposure to melanoidins. In the AMES test, melanoidins did not show a mutagenic potential, neither using the TA98 nor the TA102 strain. These results show that melanoidins isolated from the glucose-glycine mixture exhibited modest but significant genotoxic effects in human lymphocytes and, in particular the LMW, in Caco-2 cells, but they induce neither in low nor in very high concentrations mutagenicity in bacteria strains.     

Mutagenicity and anti-mutagenicity of Acanthopanax divaricatus var. albeofructus

The beneficial effects of Acanthopanax divaricatus var. albeofructus (ADA) extracts have been assessed by mutagenic and anti-mutagenic activities by Ames test. Mutation of Salmonella typhimurium strains TA 98, TA 100, TA1535, TA1537, and Escherichia coli WP2 uvr A was assayed in duplicates by the procedure of Maron and Ames in the presence or absence of S9 mix. As a result, ADA extracts were not mutagenic for S. typhimurium strains TA 98, TA 100, TA1535, TA1537, and E. coli by the Ames assay. Anti-mutagenic activity was assayed by the Ames mutagenicity assay using histidine mutant of S. typhimurium strains TA 98 and TA 100, using the plate-incorporation method. 2-Aminoanthrancene (2-AA), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2), and sodium azide (NaN(3)) were used as the mutagens. ADA extracts showed a strong anti-mutagenic activity against 2-AA-induced mutagenesis which requires liver-metabolizing enzymes, and the same extract exhibited inhibitory effects on AF-2 and NaN(3)-induced mutagenesis in the absence of liver-metabolizing enzymes. The data indicate that ADA extracts contain anti-mutagenic activities against typical mutagens. The anti-mutagenic property of ADA provides additional health supplemental value to the other claimed therapeutic properties of the plant.     

Partial chemical/structural elucidation of anti-mutagenic compounds from corn.

In this study, corn fractions obtained from an isolation process of anti-mutagenic factors in our previous research work (Burgos-Hernández et al., 2001), were subjected to several analyses for chemical/structural elucidation. The anti-mutagenic activity of these fractions was tested against aflatoxin B(1) (AFB(1)) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), a mutagen that does not require bioactivation. Two concentrations of this agent in the corn fractions were tested for anti-mutagenicity in the Salmonella/microsomal mutagenicity assay, using tester strain TA100 with no metabolic activation. Corn fractions tested showed evidence of anti-mutagenic activity by producing a dose-response type of relationship between a constant amount of MNNG and several concentrations of tested corn fraction. Five different varieties of yellow corn were tested in order to determine if the anti-mutagenic factors were intrinsic to corn. Variety of the corn did not show an effect on the reduction of the mutagenic potential of AFB(1) suggesting that anti-mutagenic compounds are intrinsic to corn. Four corn fractions, previously obtained after the isolation process were analyzed by MALDI-MS and GC-MS. MALDI-MS showed the presence of two groups of molecules or molecular fragments. The molecular mass of one group ranged from 250 to 370 m/z, the other ranged from 540 to 640 m/z. GC-MS identified linoleic acid as one of the compounds responsible for the anti-mutagenic activity present in corn.     

Comparison of the cytotoxic and mutagenic potential of liquid smoke food flavourings, cigarette smoke condensate and wood smoke condensate.

Although products of pyrolysis are often cytotoxic and mutagenic, the relationship between the type of material pyrolysed and the toxicity of the resulting pyrolysis products is poorly understood. The objective of this study was to evaluate and compare the cytotoxicity and mutagenicity of several types of common pyrolysis products. The cytotoxicity and mutagenicity of these products were assessed by using neutral red uptake and Ames mutagenicity assays, respectively. The biological activities of four liquid smoke food flavourings (LSF) were compared with two other pyrolysis-derived materials; cigarette smoke condensate (CSC) and a wood smoke condensate (WSC). Results indicated all of the mixtures exhibited a concentration-dependent cytotoxic response. The CSC and WSC were less cytotoxic than three of the LSFs, but more cytotoxic than one of the brands. The CSC was mutagenic in two Salmonella strains; however, none of the LSFs or WSC was mutagenic using TA98, and only three of the LSFs were positive with TA100. The six pyrolysis-derived materials evaluated in this study showed differing patterns and magnitudes of cytotoxicity and mutagenicity. These results indicate that the cytotoxicity and mutagenicity of complex mixtures derived from pyrolysis products are affected by the type of material pyrolysed and/or the method used to prepare the mixture. The cytotoxic potential of some commercial smoke flavourings is greater than cigarette smoke condensate and several of the food flavourings are mutagenic in one Salmonella strain.     

Mutagenic activity promoted by amentoflavone and methanolic extract of Byrsonima crassa Niedenzu

Byrsonima crassa is a plant pertaining to the Brazilian central savannah-like belt of vegetation and popularly used for the treatment of gastric dysfunctions and diarrhoea. The methanol extract contains catechin, tannins, terpenes and flavonoids; both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study the mutagenic activity of the methanol, chloroform and 80% aqueous methanol extracts, as well as acetate and aqueous sub-fractions, of this medicinal plant were evaluated by Salmonella typhimurium assay, using strains TA100, TA98, TA102 and TA97a, and in mouse reticulocytes. The results showed mutagenic activity of the methanolic extract in the TA98 strain without S9, but no mutagenicity to mouse cells in any of the extracts. The acetate fraction showed strong signs of mutagenicity without S9, suggesting that in this enriched fraction were concentrated the compounds that induced mutagenic activity. The aqueous fraction showed no mutagenic activity. The TLC and HSCCC analyses of the acetate fraction with some standard compounds permitted the isolation of the quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, methyl gallate and (+)-catechin, of which only the amentoflavone exhibited positive mutagenicity to TA98 (+S9, -S9).     

Mutagenicity of Mouriri pusa Gardner and Mouriri elliptica Martius

Mouriri pusa Gardner and Mouriri elliptica Martius are fruit-bearing plants of the Melastomataceae family, popularly known in Brazil as puçá-preto or jaboticaba-do-cerrado, and they are used in folk medicine for the treatment of gastric ulcers. In this study, we employ the Ames test to assess the mutagenicity of compounds obtained from the leaves of these species. The methanol extract of the M. pusa was mutagenic to the Salmonella typhimurium strains TA98, TA97a and TA100, with or without metabolic activation. The methanol extract of M. elliptica induced mutagenic activity in TA98 when metabolized with S9 fraction and TA97a with and without S9, but with lower mutagenicity index (MI) and potencies values than those for M. pusa. Enriched fractions of flavonoids and tannins of M. pusa were also evaluated and they demonstrated positive mutagenicity. The highest values of MI and potency were obtained with the flavonoid fraction, which contains large amounts of quercetin, quercetin glycosides and myricetin. These compounds are probably related to the mutagenicity observed in the Ames test. The dichloromethane extract was not mutagenic in any of the test conditions employed.     

Ames mutagenicity tests of products from a heated potato-starch system

A charred sample was prepared from potato starch heated with ammonium carbonate at 600°C in a flask under a nitrogen stream. The water produced was collected and extracted with methylene chloride. The basic fraction obtained from the extract exhibited strong mutagenicity in Ames assays using Salmonella typhimurium strains TA98 or TA100 with metabolic activation (rat-liver S-9 mix). The basic fraction was further fractionated by silica gel column chromatography and subsequently by Scphadex column chromatography. Some of the resulting fractions exhibited strong mutagenic activities in S. typhimurium strain TA98 with S-9 mix.     

Mutagen formation in the reaction of maillard browning products, 2-acetylpyrrole and its analogues, with nitrite

Three 2-substituted pyrroles (2-acetylpyrrole, pyrrole-2-carboxaldehyde and pyrrole-2-carboxylic acid), which are products of the Maillard browning reaction, were reacted with nitrite in buffer solution (pH 3) at 50°C for 24 hr. The reaction mixtures were extracted with methylene chloride and the extracts were tested for mutagenicity using Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104, with and without S-9 metabolic activation. The methylene chloride extract of the 2-acetylpyrrole-nitrite reaction mixture showed strong mutagenicity to all the tester strains, both in the presence and absence of S-9 mix. The reaction product of pyrrole-2-carboxaldehyde with nitrite only gave a weak mutagenic response with strain TA100, while the pyrrole-2-carboxylic acid-nitrite reaction product did not produce a mutagenic response in any of the tester strains. Two mutagenically active fractions, separated by thin-layer chromatography, were found in the reaction of 2-acetylpyrrole with nitrite. The formation of mutagenic products in the latter reaction was found to vary with reaction pH, time and temperature, with nitrite level and with 2-acetylpyrrole concentration.     

Mutagencity and antimutagencity studies of lipidic extracts from yellowtail fish (Seriola lalandi), lisa fish (Mugil cephalus) and cazón fish (Mustelus lunulatus).

Organic extracts from fresh and smoked yellowtail fish (Seriola lalandi), lisa fish (Mugil cephalus) and cazon fish (Mustelus lunulatus) were tested for mutagenicity using Ames Salmonella tester strains TA98 and TA100 with metabolic activation (S9). Also, the antimutagenicity of the organic extract from yellowtail fish was tested against aflatoxin B1 (AFB1). Yellowtail fish extract was sequentially fractionated by thin-layer chromatography (TLC) and each fraction was also tested for antimutagenicity. None of the fresh species showed mutagenicity. Extract from smoked yellowtail showed the highest mutagenic potential among the smoked species tested. Organic extract from fresh yellowtail reduced the number of revertants caused by AFB1 showing a dose response type of relationship. Sequential TLC fractionation of the antimutagenic extract produced four antimutagenic fractions from fresh yellowtail fish. These results that the lipidic fraction of the species tested contains at least four compounds with chemoprotective properties that reduce the mutagenicity of AFB1.     

Synthesis,characterization and mutagenicity of new cis-[Pt(2-substituted-benzimidazole)2Cl2] complexes

In this study, four new platinum(II) complexes with the structures cis-[Pt(Ligand)2Cl2] (ligand = 2-(p-methoxy-/or-p-chlorobenzyl or p-methoxyphenyl)benzimidazol (1, 2, 4 respectively) and 5(6)-methyl-2-phenoxymethylbenzimidazole (3) were synthesized and characterized by their elemental analysis, and IR and 1H NMR spectra. The potentials of the Pt(II) complexes for short-term bacterial mutagenicity were tested in reverse-mutation assays using Salmonella typhimurium frame-shift strain T 98 and S. typhimurium TA 100 and TA 102 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution. The tests were performed in the absence of S9 rat liver fraction. Among the complexes tested 1 had no mutagenic activity. Complex 4 was found to be weakly mutagenic in TA 98 only. The Pt(II) complexes 2 and 3 were found to be mutagenic in TA 98, TA 100 and TA 102.     

Sources of mutagenicity in cooked Finnish foods

The mutagenic activities of the alkaline fractions derived from various heat-processed, ready-made meat, fish and poultry foods were studied using the Salmonella mutagenicity assay with strain TA98 and S-9 mix to provide information about the mutagenicity of everyday Finnish foods. The majority of the food samples tested were mutagenic. The mutagenic activities of various commercial ready-made products. Mutagenicity varied remarkably between different samples. The cooking temperature clearly affected the mutagenicities of fried or reheated food samples, mutagenicity increasing with increasing temperature. The results indicate that the main sources of cooked-food mutagens in everyday Finnish foods are grilled and broiled products and foods fried at restaurants and at home. By comparison, commercial ready-made fried foods are only a minor source of mutagenicity. Variations between equivalent food samples indicated that heat processing has a marked effect on the mutagenic activity of the product, which might therefore be reduced by modifying the cooking process.     

Amplification of doxorubicin mutagenicity by cupric ion.

There is a presumption that copper and anthracycline drugs will interact with DNA to produce genotoxicity. This is of concern because serum copper levels are increased in certain neoplastic diseases. To test the interaction, it was determined if the metal ion could alter the mutagenesis of doxorubicin and related drugs in the Salmonella microsome test. In the standard form of the test, doxorubicin was strongly mutagenic against frameshift-sensitive strain TA98. When cupric acetate was added with doxorubicin it amplified the mutagenesis of the antineoplastic with an increase of approximately 19% in peak mutagenic values. This apparent "chemoactivation" was evaluated by additional applications of the Salmonella test. Preincubation of cupric acetate, drug, and bacteria (+/- rat liver S9 fraction) also resulted in a copper-amplifying effect. In the preincubation method copper produced a drug concentration-related increase of more than 700% in the mutagenicity of doxorubicin. This large an increase occurred without S9 in the test. The effects observed in TA98 were not seen with TA102, a strain sensitive to oxidation mechanisms. Copper amplification in the mutagenicity of a positive control, aflatoxin B1, was also observed with TA98 but these effects were not seen with the chelator, EDTA, the antifolate antineoplastic drug, methotrexate, or a test negative amino acid, methionine. Results point to a direct frameshift mechanism to explain the increase in mutagenicity with copper. Amplification of mutagenicity found in this study provides initial experimental support that anthracycline-metal ion-DNA associations might contribute to genotoxicity as has been inferred in the literature.     

霍山石斛类原球茎免疫调节活性的有效部位及其毒理安全性评价

目的研究霍山石斛类原球茎对小鼠免疫调节活性的有效部位及其毒理安全性。方法制备霍山石斛类原球茎冷冻干燥物、醇提物、水提物、醇溶物、醇沉物、多糖等不同提取部位;检测不同提取部位对ConA诱导的小鼠脾淋巴细胞增殖反应及免疫器官指数的影响;采用急性毒性试验、Ames试验、大鼠30d喂养实验考察多糖的毒性及致突变作用。结果水提物、醇沉物和多糖各剂量组均可以促进ConA诱导的小鼠脾淋巴细胞增殖反应和免疫器官指数的增加,且具有浓度依赖性,其中,多糖的促进作用最强;多糖对小鼠ig给药的最大耐受量大于15.6g/kg,属基本无毒物质;无论有无S9活化系统,霍山石斛类原球茎多糖各剂量组对鼠伤寒沙门菌株TA97、TA 98、TA 100、TA102无致突变作用;30d喂养期间,霍山石斛类原球茎多糖各剂量组对大鼠生长发育无不良影响。结论霍山石斛类原球茎对小鼠免疫调节活性的有效部位是多糖,其无毒,无致突变作用。     

Mutagenicity of cysteine and penicillamine and its enantiomeric selectivity

We previously observed that postmitochondrial supernatant (S9) from rat liver and kidney homogenates transforms L-cysteine into a mutagen that reverts bacteria of the strain Salmonella typhimurium TA100 to histidine independence. In the present study the enantiomers of cysteine and penicillamine (beta, beta-dimethylcysteine) have been investigated for mutagenicity. The Salmonella typhimurium strain TA92 was found to be more sensitive than TA100 to the mutagenic action of L-cysteine and was therefore also included. This strain allowed the unambiguous realization of a (weak) mutagenic effect of L-cysteine even in the absence of mammalian enzyme preparations. D-cysteine did not show mutagenicity under any experimental conditions. However, it was strongly bacteriotoxic. On the other hand, both enantiomers of penicillamine exerted clear mutagenic effects. Qualitatively, their mutagenicity was similar to that of L-cysteine in the following respects: the penicillamines were directly mutagenic, their mutagenicity was enhanced by S9, kidney S9 enhanced the mutagenicity more than did liver S9, TA92 was more sensitive than TA100. Thereby it is noteworthy that the ratios of the specific mutagenicities in the two strains were virtually identical in the direct, kidney-S9-mediated and liver-S9-mediated tests suggesting that the ultimate mutagens under these different metabolic conditions were identical. On the other hand, substantial quantitative differences in the mutagenicity between the beta-thiol amino acids were observed. L-penicillamine was about eight times more mutagenic than the clinically used enantiomer, D-penicillamine. In the direct tests, the mutagenic potency of L-cysteine was equal to that of D-penicillamine. In the S9-mediated experiments, the mutagenic potency of L-cysteine was intermediate between those of L- and D-penicillamine.     

Mutagenicity evaluation of phthalic acid esters and metabolites in Salmonella typhimurium cultures

The mutagenic potential of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEPH), as well as metabolites of DEHP--i.e., mono-2-ethylhexyl phthalate (MEHP), 2-ethylhexanol (2-EH), and phthalic acid (PA)--were tested in Salmonella typhimurium cultures using the Ames test procedure. The compounds were tested on strains TA98, TA100, TA1535, TA1537, TA1538, and TA2637 for base-pair substitution or frameshift-type mutations. Spot tests yielded negative responses for all compounds with the strains tested. Each compound was tested for a dose-effect relationship in the TA98, TA100, TA1535, and TA1538 systems. DEP and DBP exhibited a mildly positive response in both TA100 and TA1535 cultures, and DMP showed a similar response in TA1535. Normalization of the data for cytotoxicity of DMP suggests TA100 has a mildly positive effect. The higher doses of these compounds exhibited some cytotoxic effects. The mutagenic effects were apparently abolished by the addition of S9 fraction in TA100 and TA1535 cultures, while no effect, other than cytotoxicity, was observed in the TA98 and TA1538 systems. DEHP, MEHP, 2-EH, and PA exhibited no mutagenicity in any of the strains of Salmonella typhimurium tested, with or without S9 metabolic activation. MEHP and 2-EH, however, exhibited a moderate cytotoxic effect in most cultures.     

Fruit extract of the medicinal plant Crataegus oxyacantha exerts genotoxic and mutagenic effects in cultured cells

Crataegus oxyacantha, a plant of the Rosaceae family also known “English hawthorn, haw, maybush, or whitethorn,” has long been used for medicinal purposes such as digestive disorders, hyperlipidemia, dyspnea, inducing diuresis, and preventing kidney stones. However, the predominant use of this plant has been to treat cardiovascular disorders. Due to a lack of studies on the genotoxicity of C. oxyacantha, this investigation was undertaken to determine whether its fruit extract exerts cytotoxic, genotoxic, or clastogenic/aneugenic effects in leukocytes and HepG2 (liver hepatocellular carcinoma) cultured human cells, or mutagenic effects in TA100 and TA98 strains of Salmonella typhimurium bacterium. Genotoxicity analysis showed that the extract produced no marked genotoxic effects at concentrations of 2.5 or 5 µg/ml in either cell type; however, at concentrations of 10 µg/ml or higher significant DNA damage was detected. The micronucleus test also demonstrated that concentrations of 10 µg/ml or higher produced clastogenic/aneugenic responses. In the Ames test, the extract induced mutagenic effects in TA98 strain of S. typhimurium with metabolic activation at all tested concentrations (2.5 to 500 µg/ml). Data indicate that, under certain experimental conditions, the fruit extract of C. oxyacantha exerts genotoxic and clastogenic/aneugenic effects in cultured human cells, and with metabolism mutagenicity occurs in bacteria cells.     

Mutagenicity of paepalantine dimer and glycoside derivatives from Paepalanthus bromelioides.

The first isocoumarin isolated from the methylene chloride extract of Paepalanthus bromelioides, named paepalantine (isocoumarin 1), was found to have antimicrobial activity; but, it is mutagenic clastogenic and cytotoxic. Two other isocoumarins, paepalantine-9-O-beta-D-glucopyranoside (isocoumarin 2) and paepalantine-9-O-beta-D-allopyranosyl(1 --> 6) glucopyranoside (isocoumarin 3) were isolated from the ethanolic extract. A fourth new isocoumarin, also isolated from the methylene chloride extract of the capitula of P. bromelioides, was characterized as an 8-8' dimer of paepalantine and denominated isocoumarin 4. The abilities of isocoumarins 2, 3 and 4 to induce mutations in Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 were investigated. Mutagenic activity was observed in strain TA97a treated with isocoumarin 2 in the presence of S9 mixture. The substitution of H at position 9 by glucose or glucose-allose caused reductions in the mutagenic activities of paepalantine, indicating this to be an important site for these properties.     

Release of mutagens after chemical or microbial degradation of beech wood lignin

The microbial or chemical degradation of lignin from untreated samples of beech wood dusts (Fagus silvatica) resulted in the release of different mutagenic responses in the Salmonella/mammalian plate incorporation assay. In the first experiment using chemical degradation of lignin, dust samples were pre-extracted using acetone-water; the lignin portions were degraded into simpler compounds which were further fractionated on a Sephadex-LH20 column. The compounds isolated from the second phase of Sephadex, representing substances with a 3-4 ring structure and/or those of the same molecular weight, were highly mutagenic towards Salmonella typhimurium TA100 in the presence of metabolic activation. These substances were also active to some extent in strain TA1537 both in the presence and absence of Aroclor-induced rat liver homogenates. In contrast, no direct- or indirect-acting mutagenicity was found when testing with strains TA97 and TA98. Strain TA1535 responded positively only to direct-acting mutagens in the fraction tested. The mutagenic fraction was found to be toxic to the cells when tested in a histidine-rich medium. Repurification of this mutagenic fraction, using silica-gel column chromatography, revealed much higher mutagenic activity than the test material towards strain TA100. In the second pilot experiment, Phanerochaete chrysosporium and Chaetomium globosum, which are known for their ability to degrade lignin, were each incubated with wood dusts in a mixture of physiological saline and nutrient broth for either 3 or 30 days. Significant mutagenic activity was observed with the dust extract after incubation with Ph. chrysosporium but not with Ch. globosum which is a known degrader of beech lignin. These results are discussed regarding hypotheses on the carcinogenicity of beech wood dusts.     

Enzyme-mediated mutagenicity in Salmonella typhimurium of contaminants of synthetic indigo products

The mutagenic potential of two natural and seven synthetic, commercial indigo dye products was investigated. The natural products showed no mutagenicity in Salmonella typhimurium stains TA98 and TA100. In the presence of rat-liver homogenate from Aroclor 1254 pretreated rats all of the synthetic products were mutagenic towards strain TA98 but not towards strain TA100. The mutagenic effect produced was highly dependent on the amount of rat-liver homogenate added. Because of its high mutagenic potential, one product was further investigated. In the presence of rat-liver homogenate this product was weakly mutagenic towards strain TA1537 and strongly mutagenic towards strain TA1538. No mutagenicity was observed in strain TA1535. Experiments with purified synthetic indigo and natural indigo revealed that the mutagenic activity of the synthetic commercial products can be ascribed to one or more contaminants.     

Mutagenicity of nitroxyl compounds: structure-activity relationships.

Three piperidinoxyl radicals were found to be directly mutagenic in Salmonella typhimurium TA 100, one pyrrolidinoxyl compound had weaker activity, and two other pyrrolidinoxyl derivatives did not produce an increase of the spontaneous revertants. The tester strain TA 100 was selected in preliminary tests for its higher sensitivity compared to TA 98 and TA 102. The mutagenic activity of the three active compounds was abolished by partial reduction with ascorbic acid, suggesting that the mutagenicity was linked to the free radical nature of these compounds, and reduced in the presence of a cofactor supplemented rat liver subcellular fraction. The mutagenicity of the tested compounds was correlated to the resistance of the nitroxyl spin labels to reduction: the more reactive radicals were found to possess higher mutagenic activity.