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.     

In vitro induction of micronuclei by monofunctional methanesulphonic acid esters: possible role of alkylation mechanisms

Six monofunctional alkylating methanesulphonates of widely varying structures were investigated in the in vitro micronucleus assay with Syrian hamster embryo fibroblast cells. The results were compared with the alkylating activities measured in the 4-(nitrobenzyl)pyridine test (NBP-test) and the N-methyl mercaptoimidazole (MMI-test) as measures for S(N)2 reactivity as well as in the triflouoroacetic acid (TFA) solvolysis and the hydrolysis reaction as measures for S(N)1 reactivity in order to provide insights into the role of alkylation mechanisms on induction of micronuclei. Moreover we compared the results of micronucleus assay with those of the Ames tests in strain TA 100 and TA1535 and with those of the SOS chromotest with the strains PQ37, PQ243, PM21 and GC 4798. The potency of methanesulphonates to induce micronuclei depended only to a certain degree, on the total alkylating activity (S(N)1 and S(N)2 reactivity). An inverse, significant correlation between the Ames test and the micronucleus assay was observed and an inverse correlation between the micronucleus assay and the SOS chromotest with the different strains. The results indicate that the primary mechanism leading to induction of micronuclei is not O-alkylation in DNA as it is the case in the Ames test with the hisG46 strains TA1535 and TA100 and not N-alkylation as with the SOS chromotest. There is evidence that protein alkylation, e.g. in the spindle apparatus in mitosis is decisive for induction of micronuclei by alkylating compounds. The structurally voluminous methanesulphonates 2-phenyl ethyl methanesulphonate and 1-phenyl-2-propyl methanesulphonate show a clear higher micronuclei inducing potency than the other tested though the bulky methanesulphonates possess a lower total alkylating activity than the others. This effect can be explained by a higher disturbance during mitosis after alkylation of the spindle apparatus with the structurally more bulky methanesulphonates.     

Chemical reactivity and mutagenicity of some dihalomethanes

Four dihalomethanes; dichloromethane, bromochloromethane, dibromomethane and diiodomethane, have been studied with respect to their reactivities towards nucleophilic compounds of different strengths in water solution and with respect to their toxicities and mutagenic effectiveness in bacterial test systems. The correlation between biological activity (toxicity and mutagenic effectiveness in Salmonella TA100) and reactivity towards strong nucleophiles indicates that reactions with nucleophilic groups of high reactivity in the biological material, possibly SH or amino groups in proteins, are involved in their mechanism of action.     

Mutagenic and alkylating activities of organophosphate impurities of commercial malathion

The purpose of this study was to determine if 4 major organophosphate impurities of malathion were active as alkylators of nitrobenzylpyridine (NBP) or as mutagens in the Salmonella typhimurium bioassay. Malathion, isomalathion, O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate, and O,S,S-trimethyl phosphorodithioate produced alkylated NBP at varying rates. In order of increasing NBP reactivity, the compounds ranked: O,O,O-trimethyl phosphorothioate = O,O,S-trimethyl phosphorothioate less than O,S,S-trimethyl phosphorodithioate less than isomalathion = malathion. At 37 degrees C, the most reactive compounds produced an NBP alkylation rate equal to approximately 25% of the rate produced by methyl methanesulfonate, a potent Salmonella mutagen. However, none of the organophosphates were mutagenic in S. typhimurium TA97, TA98 and TA100 when tested by the standard plate-incorporation method or by the preincubation modification of the plate-incorporation method. The possible relationships between NBP reactivity and the biological activities of these organophosphates are discussed.     

Xanthine oxidase-mediated mutagenicity of the bladder carcinogen 4-nitrobiphenyl

Xanthine oxidase catalyzed mutagenicity of 4-nitrobiphenyl (NBP), a dog-bladder carcinogen, was tested in Ames assay using Salmonella typhimurium TA98 strains. NBP was active as a mutagen in the parent strain TA98 which is proficient in nitroreductase, while it was inactive in the strain TA98NR which is deficient in nitroreductase. However, preincubation of NBP at 37 degrees C with NADH and commercial preparations of xanthine oxidase for 30 min resulted in a dose-dependent increase in the mutagenic activity in TA98NR. Allopurinol blocked the xanthine oxidase catalyzed mutagenicity of NBP in TA98NR and the extent of inhibition was dependent upon the concentration of the inhibitor. Rat-liver and dog-bladder cytosol preparations also enhanced the mutagenic activity of NBP in TA98NR in a dose-dependent manner. In addition, the cytosol-mediated activity was also inhibited by allopurinol, implying that the cytosolic enzyme activity might be due to xanthine oxidase. In vitro enzymatic reduction of NBP using bacterial cell lysates of TA98 and TA98NR revealed the major product of reduction to be 4-aminobiphenyl. The transient intermediates of reduction were not detected during the in vitro incubation. The reduction intermediate N-hydroxylaminobiphenyl showed direct and equal mutagenic activity in both TA98 and TA98NR, in contrast to NBP. These results suggest that N-hydroxylaminobiphenyl is generated during the preincubation of NBP with xanthine oxidase or cytosolic preparations and the former might account for the mutagenicity of NBP. Furthermore, the occurrence of such enzyme(s) in the target tissue for NBP carcinogenesis, support the hypothesis that metabolic activation of the bladder carcinogen NBP could occur within the target organ by virtue of its intrinsic metabolic potential.     

A straight correlation between mutagenic activity and beta-galactosidase activity induced by monofunctional alkylating agents.

Eight monofunctional alkylating agents were examined for their ability to induce mutation in Salmonella typhimurium. The assay was carried out in S. typhimurium TA100 with the preincubation method. The SN1-type agents were more mutagenic than the SN2-type ones; besides, methylating agents exerted more mutagenic activity than ethylating ones. Those responses in the reversion assay were quite similar to the results obtained previously with the beta-galactosidase assay in Escherichia coli CSH26/pMCP1000 (alkA'-lacZ') as to the induction of the adaptive response. A good correlation was found between mutagenic potency in the reverse mutation assay and inducing potency in the beta-galactosidase assay.     

A sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM1011 having a high nitroreductase activity.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC'-'lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the beta-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.     

Methyl methanesulphonate (MMS) is clearly mutagenic in S. typhimurium strain TA1535; a comparison with strain TA100

No mutagenicity or an uncertain mutagenic response has been reported in the literature for methyl methanesulphonate (MMS) in S. typhimurium strain TA1535 when using the plate assay. In our studies we found a reproducible mutagenic activity of 62 revertants/mumole and plate for MMS in strain TA1535 when using the preincubation assay. A dose-dependent increase in revertants was, however, observed only at fairly high doses (exceeding 4 mumole). Two different slopes were observed in the dose-response curve when testing MMS with strain TA100. Slope A is dependent on the error-prone response, possible only in strain TA100 due to the pKm101 plasmid (R factor) but not possible in strain TA1535 due to its umuDC deficiency. Slope B observed at higher doses (as in strain TA1535) could be explained through a GC----AT transition initiated by the O6-methylation of guanine. Our findings demonstrate that MMS induces back mutation in S. typhimurium strains carrying the hisG46 missense mutation due to the formation of O6-methylguanine. In the case of strain TA100 the pKm101 plasmid-mediated error-prone mechanism is, however, the predominant process in MMS mutagenesis which leads to a higher mutagenic response at much lower doses than the GT----AT transition in strain TA1535.     

Mutagenicity of five food additives in Ames/Salmonella/microsome test

The mutagenic activity of five food additives (K₂S₂O₅: potassium metabisulphite, KMB; K₂SO₄: potassium sulphate, KS; Na₂SO₃: sodium sulphite, SS; KNO₃: potassium nitrate, KN; NaNO₃: sodium nitrate, SN) were investigated using histidin auxotrophs TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix. The test substance were investigated for their mutagenic effects at non toxic concentrations of 0.83, 1.66, 3.33 and 5.00 mg/plate with and without S9 mix. All the test substances were not mutagenic on TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix except KS and SN. KS and SN showed a weak mutagenic effect on TA100 strain in the absence of S9 mix.     

On the role of alkylating mechanisms, O-alkylation and DNA-repair in genotoxicity and mutagenicity of alkylating methanesulfonates of widely varying structures in bacterial systems

The Ames test and the SOS-chromotest are widely used bacterial mutagenicity/genotoxicity assays to test potential carcinogens. Though the molecular mechanisms leading to backmutations and to the induction of SOS-repair are in principle known the role of alkylation mechanisms, of different DNA-lesions and of DNA-repair is in parts still unknown. In this study we investigated 14 monofunctional methanesulfonates of widely varying structures for mutagenicity in Salmonella typhimurium strain TA 1535 sensitive for O(6)-guanine alkylation for comparison with strain TA 100 in order to obtain additional information on the role of alkylation mechanisms, formation of the procarcinogenic DNA-lesion O(6)-alkylguanine and the role of DNA-repair in induction of backmutation. The substances were also tested in the SOS-chromotest with Escherichia coli strain PQ 37 and strain PQ 243 lacking alkyl base glycosylases important for base excision repair in order to examine the role of alkylation mechanisms, of base excision repair and the role of O-alkyl and N-alkyl DNA-lesions on the induction of SOS-repair. The secondary methanesulfonates with very high S(N)1-reactivity isopropyl methanesulfonate and 2-butyl methanesulfonate showed highest mutagenicities in both strains. The higher substituted methanesulfonates with very high S(N)1-reactivity had lower mutagenic activities because of reduced half lives due to their high hydrolysis rates. A clear increase in mutagenicities in strain TA 100 was observed for the primary compounds methyl methanesulfonate and allyl methanesulfonate with very high S(N)2-reactivity. The primary compound phenylethyl methanesulfonate has a relatively high mutagenicity in both Salmonella strains which can be explained by an increased S(N)1-reactivity and by low repair of the O(6)-phenylethylguanine. Highest SOSIPs (SOS inducing potency) in strains PQ 37 and PQ 243 were found for methyl methanesulfonate and for the secondary compounds with high S(N)1-reactivity. The ratios in the SOSIPs between strain PQ 243 and PQ 37, indirectly indicative for the role of O- and N-alkylation in the induction of SOS-repair, was high for the primary methanesulfonates and lower for the secondary, indicating that the SOS-repair is, to a certain extent, also induced by other lesions than O(6)-alkylation. The results indicate that O(6)-alkylation is also a predominant lesion for backmutation in strain TA 100 and that in the case of monofunctional alkylating agents high S(N)2-reactivities are required to induce error prone repair mediated backmutations. The O(6)-alkylguanine lesion is also important for induction of SOS-repair in the SOS-chromotest, however, other sites of alkylation which are repaired by the base pair excision repair system can also efficiently contribute to the induction of SOS-repair.     

Mutagenicity of nitrofurans in Salmonella typhimurium TA98, TA98NR and TA98/1,8-DNP6

8 representative 2-substituted 5-nitrofurans were assayed for mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6. The tested compounds were: 5-nitro-2-furanacrylic N-(5-nitro-2-furfurylidene)hydrazide (1); furazolidone (2); 5-nitro-2-furanacrolein (3); 5-nitro-2-furaldehyde semicarbazone (4); 5-nitro-2-furaldehyde (5); nitrofurantoin (6); 5-nitro-2-furaldehyde diacetate (7); and 5-nitro-2-furoic acid (8). These compounds exhibited markedly different mutagenic activities in TA98, and these mutagenicities were similar both in the presence and the absence of rat-liver hepatic S9 activation enzymes. The mutagenic responses ranged from potent (90-300 revertants/nmole, compounds 1-3), to medium (about 10 revertants/nmole, compounds 4 and 6), to weak (0-4 revertants/nmole, compounds 5, 7 and 8). The mutagenicity of 3 was similar in all 3 tester strains, while compound 8 was essentially inactive. The mutagenicities of 1, 4, 5 and 7 were decreased 30-75% in TA98NR, while 2 and 6 showed an even greater depression of activity in this strain. Compound 6 with S9 was about equally mutagenic in TA98 and TA98/1,8-DNP6, while the activities of 6 without S9 and 2 and 7 both with and without S9 were 50-75% lower in TA98/1,8-DNP6. Compounds 1, 4 and 5 were only about 5-10% as mutagenic in TA98/1,8-DNP6 as in TA98. These results suggest that: (i) nitrofurans and their S9-mediated metabolites have similar mutagenic potencies; (ii) with the possible exception of No. 3, nitroreduction is the major route of mutagenic activation for these nitrofurans; and (iii) for compounds 2, 6 and 7, both the presumed N-hydroxy and N,O-ester derivatives of the corresponding aminofuran metabolites appear to lead to mutations.     

Salmonella typhimurium mutagenicity tester strains that overexpress oxygen-insensitive nitroreductases nfsA and nfsB

We have designed and constructed a series of plasmids that contain the major and/or minor Escherichia coli nitroreductase genes, nfsA and nfsB, in different combinations with R plasmid mucA/B genes and the Salmonella typhimurium OAT gene. The plasmid encoded gene products are necessary for both the metabolic activation of a range of structurally diverse nitrosubstituted compounds, and for mutagenic translation bypass. Introduction of these plasmids into S. typhimurium TA1538 and TA1535 has created several new tester strains which exhibit an extremely high mutagenic sensitivity and a broad substrate specificity towards a battery of nitrosubstituted test compounds that included 4-nitroquinoline-1-oxide (4-NQO), nitrofurazone (NF), 1-nitropyrene (1-NP), 2-nitronaphthalene (2-NN), 2-nitrofluorene (2-NF), and 1,6-dinitropyrene (1,6-DNP). Our studies show that the nfsA gene encodes a product that is extremely effective in the metabolic activation of a range of structurally diverse nitrosubstituted compounds. Several of the new tester strains are more than two orders of magnitude more sensitive to nitrosubstituted compounds than the Ames tester strains TA100 or TA98. In addition to enhancing mutagenic sensitivity, plasmids encoding both metabolic and mutagenesis functions on a single plasmid provide considerable flexibility for future mechanistic studies or tester strain development, in which it may be necessary to introduce additional plasmids containing different antibiotic resistance markers.     

Mutagenicity of benzylic acetates, sulfates and bromides of polycyclic aromatic hydrocarbons

Studies were performed to determine the direct mutagenicity of the acetates and some bromides and sulfates of hydroxymethyl polycyclic aromatic hydrocarbons in S. typhimurium strains TA98 and TA100. Benzylic acetates, bromides and sulfates were synthesized and characterized. The compounds tested were benzyl alcohol, 5-hydroxymethylchrysene, 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, 6-(2-hydroxyethyl)benzo[a]pyrene, 6-hydroxymethylanthanthrene, 9-hydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, 7-hydroxymethylbenz[a]anthracene, 7-(2-hydroxyethyl)benz[a]anthracene, 12-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, 12-hydroxymethyl-7-methylbenz[a]anthracene, 1-hydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene, 3-hydroxy-3, 4-dihydrocyclopental[cd]pyrene and 4-hydroxy-3, 4-dihydrocyclopental[cd]pyrene. The benzylic sulfate esters of 6-hydroxymethylbenzo[a]pyrene and 7-hydroxymethylbenz[a]anthracene were the most mutagenic compounds, whereas the aliphatic sulfate ester of 7-hydroxyethylbenz[a]anthracene did not cause an increase in mutations above background. All meso-anthracenic benzylic acetate esters were mutagenic in both strains with various degrees of activity, whereas the corresponding non-benzylic esters were inactive, as expected. Of the non-meso-benzylic acetate esters, only the 3-acetoxy-3, 4-dihydrocyclopenta[cd]pyrene was mutagenic. In the benzylic bromide series, only the eight mesoanthracenic were mutagenic, whereas benzyl bromide and 5-bromomethylchrysene were inactive. The aliphatic bromides, 6-(2-bromoethyl)benzo[a]pyrene and 7-(2-bromoethyl)benz[a]anthracene did not display significant activity. The potencies of the acetate esters more accurately reflect the mutagenicity because the rate of solvolysis did not compete with the reactivity of the esters with bacterial DNA. In the case of benzylic sulfates and bromides, the rate of solvolysis was very rapid and could have diminished the level of mutagenicity, depending on the assay conditions. These results demonstrate that meso-anthracenic benzylic acetates, sulfates and bromides are mutagenic, whereas benzylic acetate esters attached to other carbon atoms are inactive.     

The mutagenicities of safrole, estragole, eugenol, trans-anethole, and some of their known or possible metabolites for Salmonella typhimurium mutants.

Safrole, estragole, anethole, and eugenol and some of their known or possible metabolites were tested for mutagenic activity for S. typhimurium TA1535, TA100, and TA98. Highly purified 1'-hydroxyestragole and 1'-hydroxysafrole were mutagenic (approximately 15 and 10 revertants/micromole, respectively) for strain TA100 in the absence of fortified liver microsomes; trans-anethole and estragole appeared to have very weak activity. 3'-Hydroxyanethole was too toxic for an adequate test. Supplementation with NADPH-fortified rat-liver microsomes and cytosol converted 3'-hydroxyanethole to a mutagen(s) and increased the mutagenic activities for strain TA100 of 1'-hydroxyestragole, 1'-hydroxysafrole, estragole, and anethole. No mutagenicity was detected for safrole or eugenol with or without added NADPH-fortified liver preparations. The electrophilic 2',3'-oxides of safrole, 1'-hydroxysafrole, 1'-acetoxysafrole, 1'-oxosafrole, estragole, 1'-hydroxyestragole, and eugenol showed dose-dependent mutagenic activities for strain TA1535 in the absence of fortified liver microsomes. These mutagenic activities ranged from about 330 revertants/micromole for 1'-oxosafrole-2',3'-oxide to about 7000 revertants/micromole for safrole-2',3'-oxide. The arylalkenes, their hydroxylated derivatives, or their epoxides did not show mutagenic activity for strain TA98, except for 1'-oxosafrole-2',3'-oxide, which had weak activity. Since the arylalkenes are hydroxylated and/or epoxidized by hepatic microsomes, hydroxy and epoxide derivatives appear to be proximate and ultimate mutagenic metabolites, respectively, of the arylalkenes.     

Assays for phosphotriester formation in the reaction of bacteriophage R17 with a group of alkylating agents.

The interaction of bacteriophage R17 with 8 compounds has been studied, comparing the contribution of degradation of ribonucleic acid to the total toxicity. Breaks in the RNA chain result from the hydrolysis of phosphotriesters and thus are a measure of the extent of O-alkylation and of the SN1-type mechanism of the reaction. With many alkylating agents mutagenicity and carcinogenicity increase with increasing SN1 character of the reaction. In experiments with methyl methanesulphonate no evidence of degradation was observed at up to 19 times the mean lethal dose (620 methylations/RNA molecule). Breaks in the RNA chain accounted for 1 in 10 of the lethal lesions with beta-hydroxyethyl methanesulphonate, 1 in 60 with bis-(2-chloromethyl)methylamine (nitrogen mustard, HN2), less than 1 in 125 with 2,2-dichlorvinyl dimethyl phosphate (dichlorovos, DDVP), and 1 in 200 with propylene oxide. The hydrolysis rate of bis-(2 chloroethyl)ether was too slow for any reaction to be detected. In reactions with the carcinogen bis-(2-chloromethyl)ether the toxicity observed could be accounted for by the formaldehyde produced on hydrolysis. Cross-linking of the bacteriophage components by formaldehyde reduced the survival range over which the physical state of the RNA could be studied. No evidence of RNA degradation was observed. Reaction of the formaldehyde led to a progressive loss of biological activity over 24 h, a loss which was partially reversed by dialysis.     

The mutagenicity of halogenated alkanols and their phosphoric acid esters for Salmonella typhimurium.

9 halogenated alkanols, 9 corresponding tris (haloalkyl)phosphates, and 2 bis-(2,3-dibromopropyl)phosphate salts were evaluated for mutagenicity against Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538, with and without rat liver in vitro metabolic activation system (S9 mix). Most of the test samples showed mutagenic activity in the strains TA100 and TA1535, but not in the strains TA98, TA1537 and TA1538. In general, the mutagenic activities of the phosphates obtained with S9 mix were greater than the activities obtained without S9 mix. Among the phosphates, several structure--activity relationships were found; i.e., (i) the bromoalkyl derivatives were more mutagenic than the corresponding chloroalkyl derivatives, (ii) the beta-haloethyl derivatives were more mutagenic than the gamma-halopropyl derivatives, (iii) the phosphates having adjacent beta and gamma halogen atoms in the alkyl moiety, e.g., tris-(2,3-dibromopropyl)phosphate, were particularly potent mutagens, (iv) the branched carbon chain reduced the mutagenic activities in spite of the presence of beta-halogen atoms, e.g., tris(1-bromomethyl-2-bromoethyl)phosphate. However, such relations did not necessarily apply to the halogenated alkanols. It is concluded that the metabolic activation pathway via haloalkanols to mutagens must not be in common with all tris-BP-like phosphates.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

Mutagenic action of methyl 2-cyanoacrylate vapor

Alkyl 2-cyanoacrylate adhesives were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1538. Both a normal spot test and a spot test specially designed to test volatile compounds were used. The adhesives were also tested in the plate incorporation assay. These investigations showed that methyl 2-cyanoacrylate adhesives are mutagenic in strain TA100. The spot test for volatile compounds showed that it is the vapors from the methyl 2-cyanoacrylate monomer that are responsible for the mutagenic effect. One can conclude that working with methyl 2-cyanoacrylate adhesives entails exposure to vapors with a mutagenic effect and may therefore pose a carcinogenic hazard. Because the adhesives are used in industry, their mutagenic effect has a special importance in work environment.     

Diethylstilbestrol and 11 derivatives: a mutagenicity study with Salmonella typhimurium.

Diethylstilbestrol was tested for mutagenicity with his- S. typhimurium strains under 10 different matabolic situations (no exogenous metabolizing system; S9 mix from liver homogenate of rats induced with Aroclor 1254, with or without inhibition of epoxide hydratase; liver and/or kidney S9 mix from control or hamsters treated with Aroclor 1254; horse-radish peroxidase + H2O2). Under none of these conditions did diethylstilbestrol give any indication of a mutagenic effect. Furthermore, 11 metabolites and other derivatives of diethylstilbestrol, 2 of them potent inducers of sister-chromatid exchange in cultured fibroblasts, were not mutagenic with any of the 4 tester strains (S. typhimurium TA100, TA98, TA1537, TA1535) in the presence or absence of S9 mix from liver homogenate of rats induced with Aroclor 1254. Thus, one of the few known human carcinogens is very resistant to detection by the mammalian enzyme-mediated Salmonella typhimurium mutagenicity test (Ames test). This is especially remarkable since the metabolizing systems used included: (1) some of very high metabolic activity (S9 mix from liver homogenate of rats and hamsters induced with Aroclor 1254); (2) metabolizing systems from organs susceptible to the carcinogenic activity of diethylstilbestrol (hamster kidney); as well as (3) a mixture of (1) and (2) in case both activities are required for the carcinogenic effect in the whole animal.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.