Genotoxicity tests with 6-acetyl-1,1,2,4,4,7-hexamethyltetraline and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyr an

6-Acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB), synthetic fragrance ingredients, were evaluated for potential genotoxicity in a battery of short-term tests. Salmonella typhimurium/Escherichia coli plate incorporation and liquid preincubation assays were conducted on AHTN using tester strains TA97, TA98, TA100, TA102, TA1535, TA1537 and WP2 uvrA +/- S9 activation at doses from 8 to 5000 micrograms/plate. The plate incorporation mutagenicity assay was conducted on HHCB using tester strains TA98, TA100, TA1535, TA1537, TA1538 and WP2 uvrA +/- S9 activation at doses from 10 to 5000 micrograms/plate. An in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells was conducted with AHTN and HHCB at three concentrations each with +/- S9 activation. In the non-activated study, the exposure/harvest periods were 4/20-, 20/20- and 44/44-h. In the S9 activated study, the exposure/harvest periods were 4/20- and 4/44-h. In vitro unscheduled DNA synthesis (UDS) assays were conducted in primary rat hepatocytes at concentrations between 0.15 and 50 micrograms/ml for AHTN and HHCB. In vivo mouse micronucleus assays were conducted with high doses of 1600 mg AHTN/kg and of 1500 mg HHCB/kg in corn oil. No positive responses were observed in any of the tests with HHCB. With AHTN, no positive responses were observed except for cells with structural aberrations in the in vitro cytogenetics assay in CHO cells with S9 activation at the treatment/harvest time of 4/20 h. In initial studies with AHTN, the high dose of 7.8 micrograms/ml showed 0.5% aberrant cells, with the mitotic index at 41% relative to vehicle control and cell growth inhibition in the range of 25-50%. Thus the genotoxicity findings with AHTN were limited to this one positive response; all other genotoxicity tests with AHTN were considered as negative. In particular, the negative finding in the in vivo assay supports AHTN as not likely to be mutagenic in mammalian systems. These considerations, along with other negative published data, lead to the conclusion that both AHTN and HHCB do not have significant potential to act as genotoxic carcinogens.     

Mutagenicity studies of human fibroblast interferon (HuIFN-beta)

Human fibroblast interferon (HuIFN-beta) was studied for mutagenicity using the Ames method and in vitro cytogenetics. HuIFN-beta had no mutagenic effect on S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) and E. coli (WP2 uvrA) at concentrations of 3, 30, 300, 3000, 30 000 or 300 000 IU/plate. In the cytogenetic study, HuIFN-beta had no clastogenic effect on human peripheral blood lymphocytes at concentrations of 3, 30, 300, 3000, or 30 000 IU/ml. These results suggest that HuIFN-beta has no mutagenic potential.     

Mutagenicity studies with N6, O2'-dibutyryl cyclic adenosine 3',5'-monophosphate (DBcAMP)

N6,O2'-Dibutyryl cyclic adenosine 3,5-monophosphate (DBcAMP) was studied for mutagenicity using the rec assay, the Ames method, and in vitro cytogenetics. DBcAMP had no mutagenic effect on B. subtilis in the rec assay, or on S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) or E. coli (WP2 uvrA). In the cytogenetic study, a significant increase in chromosomal aberrations was observed at a concentration of 50,000 micrograms/ml, but it was considered that this effect could be attributed to the secondary effect of the high osmotic pressure in the culture medium. These results suggest that DBcAMP has no mutagenic potential.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.     

Aspects of chloroquine mutagenicity

Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP2, WP2hcr, WP6 and WP67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP2, i.e. EE97 and EE102, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EE102 from tryptophan dependence to independence. The E. coli strains WP2, WP2hcr; WP6 and WP67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50 micrograms/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.     

Lack of activity of the bacterial mutagen emodin in HGPRT and SCE assay with V79 Chinese hamster cells

Genotoxicity of emodin was studied in the Salmonella/microsome assay, the sister-chromatid exchange (SCE) assay and the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) forward mutation assay with V79 Chinese hamster cells. In the Salmonella/microsome assay, emodin was found to be positive in TA97, TA100 and TA1537 in the presence of liver homogenate. In TA1537 a weak direct mutagenicity was also observed. In both mammalian test systems, no genotoxicity was found either with or without metabolic activation.     

Mutagenicity of antiamebic and anthelmintic drugs in the Salmonella typhimurium microsomal test system

4 amebicides (chloroquine diphosphate, diiodohydroxyquin, iodochlorohydroxyquin and dehydroemetine) and 6 anthelmintics (bephenium hydroxynaphthoate, 4-hexylresorcinol, mebendazole, niclosamide, pyrantel pamoate and pyrvinium pamoate) were tested for mutagenicity in the Salmonella typhimurium microsomal test system. Frameshift mutations were induced by dehydroemetine and niclosamide following activation by microsomal enzymes, while pyrvinium pamoate induced both frameshift and base-pair substitution mutations with or without metabolic activation. The urine of mice treated with dehydroemetine or pyrvinium pamoate showed no mutagenic activity. However, urine obtained from mice treated with niclosamide was mutagenic in strains TA98 and TA1538. The fluctuation assay showed chloroquine diphosphate to be mutagenic in TA1537, a strain which detects frameshift mutations.     

A new Salmonella tester strain,TA97, for the detection of frameshift mutagens: A run of cytosines as a mutational hot-spot

We have developed a new Salmonella tester strain, TA97, for use in the Salmonella/microsome mutagenicity test. DNA sequencing has shown that this strain contains and added cytosine, resulting in a run of six cytosines at the mutated site in the histidine D gene. Its mutagenic specificity is similar to that of the frameshift mutagen tester strain, TA1537, which also contains an added cytosine in a run of cytosines and is currently among the five standard tester strains used for general mutagen screening. We assessed the mutagenic potency of 21 frameshift mutagens for TA1537 and TA97. TA97 was considerably more sensitive than TA1537 to reversion by these frameshift mutagens. In addition, one agent, PR toxin (from Penicillium roqueforti), which was not detected by any of the previously existing standard tester strains, did revert TA97; and two substituted aryl-alkyl triazenes, which had not been reported previously to be frameshift mutagens, were mutagenic in this new tester strain. We suggest that TA1537 be replaced by TA97 for general screening of mutagenicity.     

Mutagenicity of anthraquinone and hydroxylated anthraquinones in the Ames/Salmonella microsome system.

The mutagenicity of anthracene, anthraquinone, and four structurally similar compounds of each was evaluated in the Ames/Salmonella microsome assay. Anthraquinone was shown to be mutagenic for strains TA1537, TA1538, and TA98 in the absence of rat liver homogenate. The four anthraquinone derivatives tested were mutagenic for TA1537 exclusively. None of the anthracenes exhibited mutagenic activity.     

Absence of mutagenic activity of benorylate, paracetamol and aspirin in the Salmonella/mammalian microsome test

Benorylate and its two major hydrolysis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Lack of mutagenicity and putative carcinogenicity of several novel benzopyrene derivatives.

Several novel benzopyrene derivatives with the same gross structure and the same electronic periphery as benzo(a)pyrene, but with some alteration in the complete electronic structure, when tested in the Ame's Salmonella/microsome test (TA 1537, TA 100 and TA 98], were found to lack mutagenicity and, therefore, putative carcinogenicity.     

Absence of genotoxic activity of penbutolol in bacterial and mammalian cell screening systems

The genotoxic potential of the beta-adrenergic blocker penbutolol was assessed using the Ames and HGPRT tests, unscheduled DNA synthesis (UDS) and alkaline elution assays. In the Ames test, penbutolol was tested for cytotoxicity and genotoxic activity in concentration ranges of 0.8-500 micrograms/plate and 0.1-125 micrograms/ml in the HGPRT, UDS and alkaline elution assays. In the Ames test penbutolol showed significant toxicity above 500 micrograms/plate. In the mammalian cells (V79) used for the HGPRT test and A459 cells used for alkaline elution and UDS assays, penbutolol was cytotoxic at concentrations above 30 micrograms/ml. In another series of experiments, male Wistar rats were treated i.p. with penbutolol (1, 10 and 100 mg/kg) and after 2 h liver nuclei were isolated and formation of single DNA-strand breaks was measured. The results of the present study demonstrate the absence of genotoxic activity of penbutolol in the 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and in the strain of Escherichia coli WP2 uvrA in the presence or absence of metabolic activation. In V79 cells, penbutolol showed no mutagenic effects at the HGPRT locus in the presence or absence of metabolic activation. Additionally, no significant incorporation of [3H]thymidine into the DNA in the UDS test or formation of DNA-strand breaks in the alkaline elution assay was detected in the non-toxic concentration range of penbutolol with or without metabolic activation. Furthermore, penbutolol did not cause DNA damage in liver nuclei isolated from penbutolol-treated rats.     

Frameshift mutagenesis by chloroquine in Escherichia coli and Salmonella typhimurium

Chloroquine can be detected as a direct-acting mutagen in plate-incorporation assays using the excision-deficient Salmonella typhimurium strain TA97, but very much more effectively using the repair-proficient Escherichia coli strain DG1669 which carries the lacZ19124 marker. When tested at concentrations of 200-1000 micrograms/plate with strain DG1669, the mutagenicity of chloroquine is enhanced by the addition of Aroclor-induced rat-liver S9. Further experiments indicated that chloroquine-induced reversion frequencies were essentially identical in wild-type, recA, umuC and uvrC derivatives of DG1669, as well as in strains carrying the mutation enhancing plasmid pKM101, over a wide range of doses (0-1200 micrograms/plate). These results suggest that neither excision repair nor SOS-type repair are important in chloroquine-induced frameshift mutagenesis.     

Absence of mutagenic activity of benorylate,paracetamol and aspirin in the Salmonella//mammalian microsome test

Benorylate and its two major hydroyssis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Ames Salmonella/mammalian-microsome testing of peptides and peptide synthesis reagents

The Ames Salmonella/mammalian-microsome assay was used to evaluate the bacterial mutagenicity of 6 bioactive peptides and of 11 chemical reagents used in peptide synthesis. Samples of 2 reagents, bis(2-oxo-3-oxazolidinyl)phosphinic chloride and fluoren-9-ylmethyl chloroformate, showed mutagenic activity with strains TA100 and TA1535, and with TA1537, respectively. No mutagenic activity was found with the bioactive peptides or with the other 9 peptide synthesis reagents.     

Mutagenicity studies on fenitrothion in bacteria and mammalian cells

The mutagenicity of fenitrothion was determined in strains of Salmonella typhimurium and Escherichia coli. Fenitrothion was found to be non-mutagenic in Salmonella typhimurium strains of TA98, TA1535 and TA1537 and in Escherichia coli WP2uvrA both with and without S9 mix, while weak mutagenicity was observed only in Salmonella typhimurium TA100 and enhanced by the addition of S9 mix. The mutagenicity observed in the TA100 strain was not expressed in a nitroreductase-deficient strain, TA100 NR, and decreased in a transacetylase-deficient strain, TA100 1,8-DNP6. The mutagenicity of fenitrothion was also examined by a gene mutation assay using the gene for hypoxanthine-guanine phosphoribosyltransferase (hgprt) in V79 Chinese hamster lung cells. Fenitrothion did not induce any increment of 6-thioguanine-resistant mutant cells at doses ranging from 0.01 to 0.3 mM regardless of the presence or absence of S9 mix. These results suggest that reduction of fenitrothion by a bacterial nitroreductase of TA100 to an active form is essential for the expression of the mutagenicity of fenitrothion in TA100 and that a bacterial transacetylase of TA100 also has an important role in the process of mutagenic activation.     

Relationships between structures and mutagenic potencies of 16 heterocyclic nitrogen mustards (ICR compounds) in Salmonella typhimurium

16 heterocyclic nitrogen mustards (ICR compounds), which were synthesized for use as possible antitumor agents by Creech and coworkers, were tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1536, TA1537, TA1538, TA98 and TA100. The compounds were incorporated into the top agar at 5 doses: 0.5, 1, 2.5, 5 and 10 micrograms/plate. All of the compounds were negative in TA1535 except ICR 449, which was positive in all 6 strains. The other 15 compounds were positive in the remaining strains with the following exceptions: ICR 371 and 355 were negative in TA100; ICR 445 was negative in TA98 and TA100; and ICR 360 was negative in TA1537, TA1538, TA98 and TA100. Good qualitative agreement was observed between the mutagenic and antitumor activities of the 16 compounds, and between the mutagenic and carcinogenic activities of the 5 compounds that have been tested for carcinogenicity by Peck and coworkers. However, no significant correlation was found between mutagenic potency in Salmonella and antitumor potency in mice for the 16 compounds. Also, for the 5 compounds that have been tested for carcinogenicity, no significant correlation was found between their mutagenic potency in Salmonella and their carcinogenic potency in mice. In Salmonella, the secondary (2 degrees) amines generally were more mutagenic than their tertiary (3 degrees) amine homologs, although the opposite result has been reported in certain eukaryotes. Relationships between structures and potencies for the different nuclei of the 16 ICR compounds are discussed, as are similarities and differences in strain sensitivities. We conclude that the Salmonella his reversion test is not a good predictor of the antitumor and carcinogenic potencies of these ICR compounds.     

The bacterial mutagenicity of three naturally occurring indoles after reaction with nitrous acid

Three naturally occurring indoles were evaluated for potential nitrosatability using the Nitrosation Assay Procedure (NAP test) as recommended by the World Health Organisation. All three indoles i.e. tryptophan, tryptamine and 5-hydroxy-tryptamine were nitrosated to products which were directly mutagenic for S. typhimurium TA1537. In addition, the products of nitrosation of tryptamine and 5-hydroxytryptamine were also mutagenic for strains TA1538, TA98 and TA1535 without the need for metabolic activation. The sensitivities of the frameshift-detecting strains TA1537, TA1538 and TA98 were of particular interest, since nitroso compounds are characteristically base-substitution mutagens. The mutagenic effects of the products formed after nitrosation of each indole at pH 3.6, were eliminated in the presence of S9 mix. This was not the case when the nitrosation assay was carried out at pH 2.6. At this pH the mutagenicity of the nitrosated products varied in the presence of S9 mix and depended upon the nature of the indole undergoing nitrosation, and the bacterial test strain utilised for the mutagenicity assay. This indicated that more than one mutagenic product was responsible for the observed effects. As well as pH, a number of other factors influenced the formation of mutagenic nitroso products. Most notably, the concentrations of precursor compounds (sodium nitrite, and indole) present in the NAP test were of critical importance. As the sodium nitrite concentration was reduced from that recommended by the W.H.O. (40 mM), so the mutagenicity decreased. For all three compounds significant mutagenic effects were lost at sodium nitrite concentrations below 15 mM. In conclusion the data presented in this paper clearly demonstrates that individuals are chronically exposed to naturally occurring substances which readily nitrosate in excess nitrous acid and yield bacterial mutagens.     

Comparative analysis of mutagenic potency of 1-nitro-acridine derivatives

The anticancer effect of 1-nitro-9-hydroxyethylamino acridine (C-857), a compound belonging to the 1-nitroacridine class, has been well documented. Despite its therapeutic efficacy, the clinical development of C-857 has been impeded partly due to its high systemic toxicity. In an effort to enhance antitumor efficacy and lower toxicity, derivatives of C-857 have been synthesized with substitutions made at position C-4 and/or an esterified hydroxyl group in side chain at the C-9 position. The introduction of a methyl group at C-4 resulted in C-1748, which has a significantly higher therapeutic efficacy and is being clinically developed as an anticancer agent for solid tumors. The present study was undertaken to correlate the mutagenicity of C-857, C-1748, C-1790, C-1872 and C-1873 with their cytotoxicity and their anti-tumor efficacy. The mutagenicity of these drugs was determined using three Ames Salmonella typhimurium strains TA1537, TA98 and TA102. The bacteria were treated with different molar concentrations, ranging from 10(-3) to 10(-12) M, of the drugs and drug-induced histidine revertants were then counted after a 48 h incubation. C-1748 did not induce any revertants in both TA1537 and TA98 at a dose of 10(-6) M, whereas, C-857 at the same dose induced approximately 842 and approximately 1034 revertants respectively. In TA102, mutagenicity was lower than observed with TA98 and TA1537 with highest revertants observed at 10(-5) M with C-857 (approximately 606) and C-1748 (approximately 108). Higher mutagenicity was observed in the derivatives C-1790, C-1872 and C-1873 compared to C-1748, but lower than C-857. These studies demonstrate that C-1748 has the least mutagenic potential, with a much higher antitumor effect in prostate cancer and is a promising chemotherapeutic agent for clinical development.     

Mutagenicity of pyrene in Salmonella

Pyrene was tested for mutagenicity in Salmonella typhimurium strains TA97, TA98, TA100 and TA1537. Mutagenicity was seen in all strains when S9 was present.