J.A. Styles

Saccharin: An epigenetic carcinogen/mutagen?

Saccharin, together with four of the impurities found in the commercial material and a further nine functional analogues gave negative results in the Salmonella reverse mutation assay of Ames et al. (Mutation Res. 1975, 31, 347) and the cell-transformation assay of Styles (Br. J. Cancer 1977, 36, 558). The significance of these and related in vitro results is evaluated within the context of the ability of commercial saccharin to cause bladder cancer in rats and dominant lethal effects in mice. In particular, evidence that the carcinogenicity and the dominant lethal effect associated with saccharin may be mediated by an epigenetic mechanism is evaluated, as is the possible role played by impurities in the generation of these and other biological effects. The case for separating carcinogens into two classes, namely those that operate by a genotypic mechanism and those that elicit their effects via an epigenetic mechanism, is discussed. The compounds evaluated in the current study were saccharin base, sodium saccharin, o-toluenesulphonamide, o-sulphonamidobenzoic acid, 5-chlorosaccharin, 3-iminosaccharin, 6-chlorosaccharin, 5-chlorobenzisothiazoline-1,1-dioxide, 6-aminosaccharin. 6-nitrosaccharin, 3-chloro-ψ-saccharin, 3,6-dichloro-ψ-saccharin, 3-dimethylamino-ψ-saccharin, 3-piperidinyl-ψ-saccharin and 3-pyrrolidino-ψ-saccharin.

Cytogenetic effects of benzene: dosimetric studies on rats exposed to benzene vapour

Rats were exposed to benzene vapour at nominal concentrations in air of 1, 10, 100 and 1000 ppm acutely for 6 h. Bone marrow cells from each animal were examined for chromosomal abnormalities 24 h after the end of the exposure period. This analysis was carried out on 250 metaphases per animal where possible and showed a significant increase in the percentage of cells with chromosomal abnormalities, excluding gaps, in the groups of animals exposed to 100 and 1000 ppm benzene. In the 10-ppm and 1-ppm exposure groups there were elevated levels of cells with abnormalities which showed evidence of being dose-related, although they were not statistically significant.

Chloracetamide-N-metholol: an example of an in vitro and in vivo clastogen which is non-mutagenic to Salmonella

The industrial biocide chloracetamide-N-metholol (CAM) has been shown to be non-mutagenic to 6 strains of Salmonella using both the plate-incorporation and a pre-incubation test protocol. Its biocidal activity is unlikely to have influenced these results since Kathon 886, a more potent biocide, was concomitantly detected as mutagenic to strain TA100. In contrast, CAM was weakly clastogenic to human lymphocytes cultured in vitro and elicited a positive response in the mouse bone marrow micronucleus test when assayed using the intraperitoneal, but not the oral route of administration. A positive response was concomitantly observed for the rodent carcinogen and formaldehyde-releasing agent hexamethylphosphoramide (HMPA) in these 2 clastogenicity assays. Data are presented showing the slow hydrolysis of CAM to formaldehyde in vitro, and both [carbonyl-14C]CAM and [metholol-14C]CAM have been shown to interact covalently with calf-thymus DNA in vitro. It is concluded that CAM may be a direct-acting carcinogen to rodents, but that both the qualitative and quantitative outcome of its bioassay for carcinogenicity will be influenced critically by the bioassay protocol adopted; in particular, by the route of administration selected. These findings emphasize the need to complement the Salmonella gene-mutation assay with an in vitro assay for the induction of chromosomal aberrations if in vivo genotoxins are to be detected efficiently in vitro.

A comparison of the incidence of micronuclei in blood and bone marrow in 3 strains of mouse dosed with cyclophosphamide or hexamethylphosphoramide (HMPA)

The response of 3 strains of mouse (C57Bl/6J, C3H/C57 hybrid and BALBC/CBA) to cyclophosphamide (75 mg/kg) and hexamethylphosphoramide (HMPA) (1.28 ml/kg) were compared in the micronucleus test. Each compound was administered by intraperitoneal injection on two consecutive days and samples of bone marrow and blood taken for examination at 48 and 72 h after the first injection. Both test chemicals produced a statistically significant increase (P 0.001) in the incidence of micronuclei in bone marrow cells in all strains at both sampling times but the response with HMPA in C57Bl/6J mice appears to occur earlier than in the other two strains. Significant increases in micronuclei were seen in circulating erythrocytes only at 48 h in C57Bl/6J mice with both test chemicals and in C3H/C57 mice only with cyclophosphamide.

Genotoxicity and carcinogenicity of fluorocarbons: Assessment by short-term in vitro tests and chronic exposure in rats

Two short-term in vitro tests for mutagenicity (Salmonella reverse mutation and BHK21 cell transformation) were conducted on a series of fluorocarbons. Some of these materials (FC22, FC31, FC142b, FC143, and FC143a) were found to be positive in one or both of the tests and could therefore be considered as being potentially carcinogenic to animals. Such activity was not anticipated for what were previously considered inert materials and in consequence several examples of these fluorocarbons, which represented different combinations of short-term test results, were tested for carcinogenicity in limited in vivo bioassays. In these studies, rats were dosed for 1 year by gavage 5 days a week with either FC22, FC31, FC133a, FC134a, or FC143a dissolved in a corn-oil at a single dosage of 300 mg/kg body weight. The animals were then observed until week 125 with detailed necropsy at termination. The study revealed that FC31 was a potent carcinogen (to the rat stomach), a result which reflected the short-term test predictions, but FC133a, which gave a negative response in both the in vitro assays, induced a high incidence of reproductive tract tumors. The weak bacterial mutagens FC22 and FC143a did not induce tumors in this study, and the nonmutagenic FC134a was without overt carcinogenic activity. It is concluded that, while recognizing the limitations of the in vivo component of this study, the short-term tests were only partially successful in identifying potential carcinogens for this series of chemicals. Fluorocarbon 31 was a potent carcinogen which was first identified by bacterial mutation and cell transformation, whereas the equally potent carcinogen FC133a was not so identified. The lack of genotoxic activity with this particular compound leads us to believe that the carcinogenic activity may be due to mechanisms other than those which involve direct DNA interactions.

Norharman and ellipticine: A comparison of their abilities to interact with DNA in vitro

The anti-tumor agent ellipticine has been compared in vitro with the bacterial co-mutagen norharman, a compound which it resembles superfically in chemical structure. Ellipticine was shown to stabilize the structure of double stranded calf-thymus DNA, to induce mutations in strain TA153 of Salmonella tryhimurium and to cause BHK cells to transform. Further, the major absorbance in its visible spectrum underwent a red shift of approximately 40 nm in the presence of native DNA. It is concluded that ellipticine intercalates with dna, and from this, that its action as an anti-tumor agent may, as has been previously suggested, be dependent upon this property. In contrast, norharman, a chemical suspected initially of being an intercalating agent, failed to stabilize the structure of DNA, was non-mutagenic to the same strain of S. typhimurium and was inactive as cell-transforming agent. In addition, its visible spectrum was not affected by the presence of DNA. The last observation is contrary to the conclusion of other workers, and an explanation of this difference is given. It is concluded that norharman is not capable of intercalating with DNA, and consequently, its mode of action as a co-mutagen is probably dependent upon its ability to inhibit certain mixed-function oxidase enzymes present in the liver activation system employed with in vitro mutagenicity assays.

Activity of 2,4,6-trinitrotoluene in an in vitro mammalian gene mutation assay

In the present study both 2,4,6-trinitrotoluene (TNT) and pure 2,4-dinitrotoluene (2,4-DNT) gave positive responses in the P388 mouse lymphoma gene mutation assay in the absence of auxiliary metabolic activation. Both chemicals gave negative results when an activation system was included. Technical grade DNT (consisting of an 80:20 mixture of 2,4- and 2,6-DNT) and pure 2,6-DNT gave negative responses in the assay both in the presence and absence of auxiliary metabolism. These observations in mammalian cells support the bacterial mutagenesis data indicating the TNT is a potential rodent liver carcinogen and suggest that the activity of TNT should be investigated in vivo to assess whether its potential hepatocarcinogenicity is realised in rats.

An overview of the chemical and biological reactivity of 4CMB and structurally related compounds: Possible relevance to the overall findings of the UKEMS 1981 study

Abstract Studies are described that may help to elucidate why the ability of 4CMB to mutate bacteria in vitro appears not predictive of genotoxicity in rodents.

Evaluation of some formaldehyde-release compounds and other biocides in the mouse micronucleus test

A number of biocidal chemicals were tested for clastogenic activity in the micronucleus test using C57Bl/6J mice. The materials tested were: 5-chloro-2-methyl-4-isothiazolin-3-one (I), N-methyl-isothiazolone hydrochloride (II), Glokill 77 and Parmetol A23. Two of the biocides (Glokill and Parmetol) depend on the release of formaldehyde for their activity while the other two compounds are the active chemicals in the biocide Kathon. Hexamethylphosphoramide (HMPA) was tested as the positive control for the series and N,N-dinitrosopentamethylenetetramine (DNPT) as the negative control. HMPA produced significant dose-related increases in the incidence of micronuclei whereas DNPT, I, II, Glokill and Parmetol A23 were without effect.

Activity of vinyl chloride monomer in the mouse micronucleus assay

C57Bl/6J mice of both sexes were exposed to 50 000 ppm vinyl chloride monomer (VCM) for 6 h. Animals were killed 24 and 48 h after cessation of exposure and examined for the presence of micronuclei in bone marrow cells. At 24 h the control incidences of micronuclei per 1000 polychromatic erythrocytes (PCEs) were 2.6 (male) and 1.2 (female), while in animals exposed to VCM the incidences were 24.6 (male) and 25.0 (female). At 48 h the control incidences were 2.2 (male) and 1.6 (female) and in the VCM exposed animals 7.2 (male) and 4.4 (female).