P.A. Lefevre

An assessment of the in vivo rat hepatocyte DNA-repair assay

The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described by Mirsalis et al, and its in vitro counterpart described earlier by Williams have been employed by us for 4 years. Our experience is that the in vivo assay performs as described in the literature. We have therefore concentrated in this initial paper on the key practical factors we have found to govern the assay sensitivity and reproducibility. This has been achieved by a discussion of the assay performance with two potent rat hepatocarcinogens [the novel azo compound 6-dimethylaminophenylazobenzthiazole (6BT) and the reference agent 2-acetylaminofluorene (2AAF)] and a non-carcinogen of similar structure to 6BT [5-dimethylaminophenylazoindazole (51)]. Assay responses were compared with the effect of these chemicals in the Salmonella mutation assay. We conclude that the in vivo liver UDS assay has a critical role to play as a complement to rodent bone marrow cytogenic assays when conducting assessment studies on agents defined as genotoxic in vitro. However, the in vivo assay is resource-consuming and false results could consequently arise due to incomplete evaluations. Methods to counteract this danger are discussed and criteria for assessing weak UDS responses are suggested.

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.

Non-genotoxicity of 2,4,6-Trinitrotoluene (TNT) to the mouse bone marrow and the rat liver: Implications for its carcinogenicity

Abstract2,4,6-Trinitrotoluene (TNT) is structurally related to the rat liver carcinogen 2,4-dinitrotoluene (technical grade), and both compounds are known to be mutagenic to bacteria in vitro. TNT is therefore established as a potential rodent carcinogen; the present paper describes experiments designed to assess if this potential is likely to be expressed in appropriately exposed animals. TNT gave a negative response in the mouse bone marrow micronucleus assay and in an in vivo/in vitro rat liver assay for unscheduled DNA synthesis (UDS). In the latter assay animals are exposed to the test chemical in vivo and their hepatocytes subsequently evaluated for UDS in vitro. The negative response observed for TNT in the liver assay at dose-levels up to 1000 mg/kg was accompanied by a positive response for the hepatocarcinogen 2,4-dinitrotoluene at the lower dose-level of 200 mg/kg. In contrast, the dinitro compound gave a negative response in the micronucleus assay, as was also observed for TNT. It is concluded that the negative response observed for TNT in the liver assay indicates that it is unlikely to be a rat hepatocarcinogen. Nonetheless, high levels of methaemoglobin were observed in the TNT-treated rats and their urine was coloured red. These facts, together with the known toxicities of this agent suggest a possible carcinogenic hazard to the haemopoetic and urinary tissues of animals exposed chronically to it at toxic dose-levels.

Potent mitogenic activity of 4-acetylaminofluorene to the rat liver

Administration of 4-acetylaminofluorene (4AAF) to rats by oral gavage (1000 mg/kg) produces a wave of S-phase activity in the liver 36 h later, followed by a wave of mitoses at 48 h. These events were monitored by autoradiography of isolated hepatocytes and by histopathology, respectively. DNA-labelling was shown to occur following both in vivo and in vitro radiolabelling. The level of S-phases observed approached that reported following partial hepatectomy. These effects were not accompanied by unscheduled DNA synthesis (UDS) nor was any frank histopathological damage to the liver evident. 2-Acetylaminofluorene (2AAF) elicited a very weak S-phase response at a dose level of 50 mg/kg, but gave marked UDS between 12 and 48 h.

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.

Cyclic amines as less mutagenic replacements for dimethyl amino (-NMe2) substituents on aromatic organic compounds: Implications for carcinogenicity and toxicity

Replacement of the dimethylamino (--NMe2) group of the rodent liver carcinogens 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT) with a pyrrolidinyl group leads to a marked attenuation of their mutagenicity to S. typhimurium in vitro. Replacement with the 6-membered piperidinyl group leads to a virtual loss of mutagenic activity. These results are discussed within the context of a possible, albeit limited correlation between carcinogenic potency to rodents and mutagenic potency to S. typhimurium. Based on these observations, it is suggested that replacement of the --NMe2 group of a toxic/carcinogenic/mutagenic aromatic chemical by a cyclic amine substituent may produce a less toxic (etc.) analogue with similar gross molecular properties. The significance of weak (less than 2-fold increase) mutagenic responses is discussed in relation to potential carcinogenicity.

Mutagenicity to Salmonella of the mono methylamino and N-cyanoethyl analogues of 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT)

Replacement of one of the methyl groups of the carcinogens 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT) with a cyanoethyl (-CH2CH2CN) substituent dramatically increases their mutagenic potency to Salmonella (strain TA98). The corresponding monomethylamino derivatives (-NHCH3) are more mutagenic than the parent dimethylamino [-N(CH3)2] compounds, but substantially less mutagenic than the cyanoethyl derivatives. All of these mutagenic activities are liver-S-9-dependent. The very similar dose response curves observed for the two cyanoethyl compounds argues for the formation of a common electrophilic intermediate from each.