The activity of 4CMB, 4HMB and BC in Saccharomyces cerevisiae JD1

3 structurally related compounds, 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB), and benzyl chloride (BC) were assayed for their ability to induce mitotic gene conversion in stationary phase cultures of the yeast, Saccharomyces cerevisiae JD1. This strain allows gene conversion to be scored at 2 independent loci, trp 5 and his 4.The results reported in this paper indicate that both 4CMB and BC are genetically active in yeast, producing dose-related increases in mitotic gene conversion at both the loci tested; 4HMB showed no such activity. At high survival levels 4CMB and BC showed comparable activity. However, as toxicity increased BC showed much more potent convertogenic activity, whereas with 4CMB a reduction in induced gene conversion was observed. The presence of a microsomal activation system derived from the livers of Aroclor-induced male rats did not significantly affect the activity of any of the compounds.     

A comparison of the response to 4CMB, 4HMB and BC of 5 yeast strains differing in their radiosensitivities

The 3 test compounds 4CMB, 4HMB and BC were assayed for their genotoxicity using stationary phase cultures of 5 yeast strains which differ in their mutagen sensitivity. It was found that 4HMB produced no differences in survival between the 5 strains whereas 4CMB and BC caused more lethality in the triple rad strain than the other 4 strains. The results indicate that both BC and 4CMB are capable of inducing DNA damage which results in cell lethality in the repair-deficient triple mutant.     

Bacterial mutagenicity tests on 4-chloromethylbiphenyl and 2 structural analogues

4CMB, 4HMB and BC were tested in 5 strains of S. typhimurium and 2 strains of E. coli without S9. 4HMB was negative in all strains. 4CMB was a strong positive mutagen in TA1535, TA1537, TA1538, TA98, TA100 and WP2uvrA⁻(pKM101), and BC was a weak mutagen in TA100 and WP2uvrA⁻(pKM101). Positivity was determined as a dose response over 3 or more points, in repeat experiments, giving a significant correlation coefficient.     

Fluctuation test data on 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB) and Benzyl Chloride (BC) using Salmonella typhimurium TA98 and TA100

Bacterial fluctuation tests (Green et al., 1976) were performed both with and without metabolic activation using the ‘Ames’ Salmonella typhimurium strains TA98 and TA100 (Ames et al., 1975) to assay the mutagenic potential of 4CMB, 4HMB and BC.4CMB and 4HMB were tested on the same occasion. However, 4CMB was only compared to BC in one assay. The results also show an independent test of BC.     

Assay of the induction of mitotic crossing-over and aneuploidy in yeast by BC, 4CMB and 4HMB

Both BC and 4CMB but not 4HMB were shown to be capable of inducing mitotic crossing-over in exponential phase cells of the D6 strain of the yeast Saccharomyces cerevisiae. In contrast, none of the 3 test compounds were capable of inducing mitotic chromosome aneuploidy.     

UKEMS trial: Bacterial mutation tests of 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyl,and benzyl chloride,using E. coli WP2uvrA(pKM101) and S. typhimurium TA98 and TA100

4CMB, 4HMB and BC were assayed in plate tests, using E. coli WP2uvrA(pKM101), and S. typhimurium TA98 and TA100, in the presence or absence of microsomal activation. 4CMB was also assayed in fluctuation tests using E. coli WP2uvrA(pKM101). 4HMB was uniformly negative, and 4CMB was mutagenic to all 3 strains. BC was negative in TA98 and positive in TA100 and WP2uvrA(pKM101). The presence or absence of S9 made no substantial difference to the mutagenicity of 4CMB or BC.     

An assay for mutagenic activity of 4CMB, 4HMB and BC,using the “microtitre” fluctuation test

4CMB, 4HMB and BC were assayed for mutagenic activity using the ‘microtitre’ bacterial fluctuation test without metabolic activation. 4CMB was positive in strains of Salmonella typhimurium detecting both base-substitution and frameshift mutation. BC was weakly positive only in the strain which detects base-substitution mutation. 4HMB was negative in both strains. 4CMB and 4HMB were equally toxic to the strains, whilst BC was comparatively less toxic.     

Chromosome analysis of cultured rat-liver epithelial cells (RL4) treated with 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyl and benzyl chloride

Cultured rat-liver epithelial cells, RL₄, were exposed to maximum concentrations of 24 μg/ml 4CMB, 100 μg/ml 4HMB and 30 μg/ml BC. 4CMB and BC induced highly significant numbers of aberrations; 4HMB did not cause an increase in aberration levels.     

Testing for mitotic crossingover and induced aneuploidy using Aspergillus nidulans as part of the UKEMS test programme

4CMB and BC were shown to induce mitotic crossingover in Aspergillus nidulans, whereas no such activity was shown in the presence of 4HMB. None of the 3 test compounds were able to induce chromosome aneuploidy.     

The induction of mitotic gene conversion in the yeast,Saccharomyces cerevisiae JD1 by 4-chloromethylbiphenyl (4CMB), Benzyl Chloride (BC) and 4-hydroxymethylbiphenyl (4HMB)

The induction of mitotic gene conversion by 4CMB, BC and 4HMB was studied in both log-phase and stationary-phase cultures of the yeast, Saccharomyces cerevisiae JD1. Assays were performed both in the presence and in the absence of S9 microsomal fraction obtained from a liver homogenate from rats pretreated with Aroclor 1254.Exposure of both stationary-phase and log-phase cultures to 4CMB and BC resulted in an increase in mitotic gene conversion, both in the presence and in the absence of a microsomal activation system; the magnitude of response was greater in stationary-phase cultures. 4HMB did not increase the gene conversion frequency in log-phase or stationary-phase cultures.     

The effects of 4CMB, 4HMB and BC on SCE,chromosome aberration and point mutation in cultured Chinese hamster ovary cells

Cultured Chinese hamster ovary cells (CHO-KI-BH₄) were treated for 2 h with 4CMB, 4HMB and BC, in the absence of any exogenous metabolic activation system. The cells were subjected to tests for survival, sister-chromatid exchange, chromosome aberration and mutation to thioguanine resistance.4HMB had no effect in any test at concentrations up to 100 μg/ml. 4CMB was slightly more toxic than BC. Both 4CMB and BC induced SCE and chromosome aberrations, but the effects were more marked with BC. With 4CMB, SCE increased with dose only up to about 7 μg/ml and then levelled off. A weak mutagenic effect was observed with both BC and 4CMB, but in each case, the response reached a peak and was not evident at higher doses.     

UKEMS trial compounds: In vitro bacterial mutagenicity

4CMB, 4HMB and BC were examined in the Ames test using Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100. 4CMB was mutagenic for all of the indicator strains, 4HMB was inactive and BC was weakly mutagenic for TA100 only.     

Additional data in support of the quadruplicated white-ivory reversion system to test for somatic genotoxicity in Drosophila melanogaster

Cyclophosphamide, ethyl methanesulfonate, propyleneimine and tritiated water were tested in a new short-term somatic mutation bioassay, previously described by Green and coworkers (1986), to evaluate the suitability of the quadruplicated white-ivory system of Drosophila melanogaster for genotoxicity testing of chemicals. A 2.9-kb tandemly duplicated sequence of w⁺ within a w⁺ gene is responsible for the white-ivory phenotype. Reversion of wⁱ to w⁺ is in general, associated with the loss of the appended 2.9 kb or other alterations affecting this duplicated region. The appearance of light (white or nearly white) phenotypes could be due to the loss of some wⁱ copies. Thus, in the eyes of adult males resulting from treated larvae, we can detect 2 types of sectors (red and light) on the orange-yellow background. Our results indicate that the genetic system used in this somatic assay is sensitive to the genotoxic effects of the 4 compounds tested. All 4 compounds tested were positive. Tritiated water had weak effects, cyclophosphamide was characterized by small revertant clones and ethyl methanesulfonate and propyleneimine by large clone size. From the available data, we feel that the wⁱ system can be considered of potential value for genotoxicity testing.     

Mutation at the TK locus of mouse lymphoma L5178Y cells by 4CMB, 4HMB and BC

4CMB, 4HMB and BC were tested for their ability to increase the mutation frequency at the thymidine kinase locus of mouse lymphoma L5178Y cells.     

4-chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethylbiphenyl (4HMB): Reverse mutation tests with Salmonella typhimurium

In this study 4CMB was shown to be a strong, direct-acting, mutagen for S. typhimurium strains TA1538, TA1537, TA98 and TA100. However, for strain TA1535 the compound was only weakly mutagenic. No conclusive evidence of mutagenic activity was seen in tests with BC or 4HMB.     

The testing of 4CMB, 4HMB and BC for mutagenicity in a new Neurospora crassa heterokaryon

This genetic system permits both point mutations and deletions to be detected in the same experimental material. 4CMB and BC are regarded as being mutagenic in this system. 4HMB induced mutations in only 1 of the 3 treatment protocols used, and its mutagenicity is questionable.     

The biological activity of 4-chloromethylbiphenyl, benzyl chloride and 4-hydroxymethylbiphenyl in 4 short-term tests for carcinogenicity. A report of an individual study in the UKEMS genotoxicity trial 1981

4-Chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethyl-biphenyl (4HMB) were tested for biological activity in the following assays: (i) the Salmonella/microsome assay; (ii) a bacterial 'fluctuation' assays; (iii) a DNA repair assay in Hela cells, and (iv) a mouse lymphoma mutation assay. 4CMB was active in assays (i), (ii) and (iii) but not in (iv); BC was active in assays (i), (ii), (iii) but not in (iv) while 4HMB was inactive in all assays. Where biological activity was seen this did not require addition of a liver S9 preparation. 4CMB was more active than BC in all the test systems in which a positive response was obtained. The implication of these results for a test battery approach to in vitro testing is discussed.     

The biological activity of 4-chloromethylbiphenyl,benzyl chloride and 4-hydroxymethylbiphenyl in 4 short-term tests for carcinogenicity: A report of an individual study in the UKEMS genotoxity trial 1981

4-Chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethyl-biphenyl (4HMB) were tested for biological activity in the following assays: (i) the Salmonella/microsome assay; (ii) a bacterial ‘fluctuation’ assays; (iii) a DNA repair assay in Hela cells, and (iv) a mouse lymphoma mutation assay. 4CMB was active in assays (i), (ii) and (iii) but not in (iv); BC was active in assays (i), (ii), (iii) but not in (iv) while 4HMB was inactive in all assays. Where biological activity was seen this did not require addition of a liver S9 preparation. 4CMB was more active than BC in all the test systems in which a positive response was obtained. The implication of these results for a test battery approach to in vitro testing is discussed.     

Molecular analysis of large transposable elements carrying the white locus of Drosophila melanogaster

A large transposable element (TE) comprising the white-apricot and roughest genes has been found to transpose to well over a hundred sites scattered over the Drosophila genome. We report the cloning of the essential parts of several TEs. TE98 and TE28 sequences were cloned by `walking' along the chromosome from the previously cloned heatshock genes. The ends of the TEs are characterized by dispersed repetitive elements belonging to the foldback (FB) family. FB elements are also associated with two independently isolated transposable elements originating from the white locus, Tp w^c-1 and Tp w^+IV. The strong correlation between FB elements and large composite transposons suggests that a pair of these elements can mobilize large intermediary DNA segments. One particular FB family member, FB-NOF, is associated with TE28, the white-crimson (w^c) mutant, the w^c-derived Tp w^c-1 and probably also with Tp w^+IV. A unique sequence located close to the white end of TE28 was used to clone the borders of TE77 and the surrounding sequences in the bithorax region, indicating that the TE can be used as a probe for gene isolation. Some evolutionary implications of the large composite transposons are discussed.     

Attempts to initiate skin tumours in mice in the 2-stage system using 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB), and benzyl chloride (BC). Report of the experiment at 10 months

Groups of T.O. mice (Theiler's Original, derived from Swiss albino mice) were treated topically, with a single dose of 1.0 mg of 4CMB, 4HMB, or BC, or with 0.4 mg benzo[a]pyrene (B[a]P). Twice-weekly promotion of the treated skin area with croton oil was begun 1 week later. At 10 months the skin-tumour incidence in the positive control (B[a]P) was 8/19, with a mean latent period of 20 weeks. Both 4CMB and 4HMB have so far produced 1 papilloma each (1/36 at 40 weeks, and 1/39 at 34 weeks, respectively), while BC has produced none. Further time is required in order to ascertain whether these single papillomas will develop into carcinomas and thereby herald weak initiating activity for 4CMB and 4HMB.