Douglas B. McGregor

Endogenous xenobiotic enzyme levels in mammalian cells

The response of mammalian cell lines to chemicals depends, in part, on the exogenous activation system used for the induction of a biological response. This could be attributed to differences in the expression of enzymes involved in xenobiotic metabolism. We have measured the activities of benzo[a]pyrene hydroxylase, dimethylaminoazobenzene N-demethylase, catalase, superoxide dismutase, peroxidase and glutathione-S-transferase in human lymphoblast TK6, mouse lymphoma L5178Y, Chinese hamster ovary (CHO) and lung (V79) and mouse C3H10T1/2 cell lines as well as in primary hepatocytes and S9 preparations of liver from male F344 rats. Nitroreductase was also measured in some of these preparations. Human lymphoblast TK6 and mouse C3H10T1/2 cells had the capacity to metabolize dimethylaminoazobenzene and the latter cell line also metabolized benzo[a]pyrene, indicating the presence of constitutive mono-oxygenase activity. Cytochrome P450 could not be detected spectrophotometrically in the cell lines. Western blot analysis indicated that P450 from the P450IIA family is expressed in C3H10T1/2 cells. Reactivity was also observed with an antibody to P450IA2; however, the identity of this protein remains uncertain. Superoxide dismutase, catalase and peroxidase, which protect cells against oxygen radical damage, were found in all the cell lines and in rat hepatocytes and S9. The human lymphoblast TK6 cell line, however, had the least of each of these three enzymes. Glutathione-S-transferase activity was detected at varying levels in all cell types. Nitroreductase activity was high in S9 and Chinese hamster ovary cells and lower in mouse lymphoma and Chinese hamster V79 cells.

Mutagenic activity of fluorides in mouse lymphoma cells.

The L5178Y mouse lymphoma cell forward-mutation assay was used to test for the mutagenic activity of sodium and potassium fluoride at the thymidine kinase locus. Mutants were detected by colony formation in soft agar in the presence of trifluorothymidine. Mutagenic and toxic responses were observed in the concentration range of 300-600 micrograms/ml with both sodium and potassium fluoride. Approximately 3-fold increases in mutant frequency were observed for concentrations in the 500-700 micrograms/ml range that reduced the relative total growth to approximately 10% in the absence or presence of a rat-liver S9 activation system. A sample of 30% sodium fluoride-70% sodium bifluoride (NaHF2) induced a similar mutagenic response but was more toxic with respect to the fluoride concentration. A specificity for fluoride ions in causing mutagenesis was indicated by the fast that much higher concentrations of sodium or potassium chloride were necessary to cause toxicity and increases in the mutant frequency. The possible involvement of chromosomal changes was signaled by the predominant increase in the small colony class of mutants.

Dominant lethal studies with paraquat and diquat in male CD-1 mice

The herbicides paraquat and diquat were tested for dominant lethal activity in male CD-1 mice. No mutagenic effects were detected on any stage of spermatogenesis in treated males if these compounds were administered orally up to 4 mg paraquat/kg/body weight/day of 10 mg diquat/kg/body weight/day for 5 days. There was no increase in the number of post-implantation losses as shown by the number of females with one or more early deaths or number of early deaths/pregnancy or the number of early deaths/total implants/pragnancy. There was no evidence of pre-implantation losses as indicated by the total implants/pregnant female. There was no anti-fertility effect on the treated males as measured by the pregnancy frequency or successful mating frequency. The lack of dominant lethal effect was not due to particular insensitivity of the animals used since such effects were amply demonstrated in mice doses orally on 5 days with ethyl methanesulphonate at 100 mg/kg/body weight/day, or intraperitoneally on one day with cyclophosphamide at 200 mg/kg/body weight.

Dominant lethal study of ribavirin in male rats

Ribavirin, a new synthetic antiviral agent, was studied for dominant lethal effects in male CD rats. The drug was administered intraperitoneally at doses of 50, 100 and 200 mg/kg/day for 5 days. Males were mated weekly with 8 consecutive batches of female rats. Marginal increase in early foetal death detected in Assessment Weeks 3 and 8 in females mated with the low-dose and high-dose males were not dose-related and were most probably chance events caused by the particularly low vehicle control frequencies for these 2 weeks. Also, the slightly reduced pregnancy proportion among females mated with the high-dose treated males was to a substantial extent the effect of a single male rate which failed to fertilize any females. Ribavirin was, therefore, regarded as being devoid of any mutagenic potential demonstrable by a rat dominant lethal assay.

Genotoxic activity in microorganisms of tetryl, 1,3‐dinitrobenzene and 1,3,5‐trinitrobenzene

N-Methyl-N,2,4,6-tetranitroaniline (tetryl), 1,3-dinitrobenzene, and 1,3,5-trinitrobenzene were subjected to DNA repair assays using the Escherichia coli W3110/polA+, p3478/polA- system, reverse mutation assays with His-Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, and mitotic recombinogenic tests with the yeast Saccharomyces cerevisiae D5. Tests were carried out in the absence of an exogenous activation system and in tissue homogenate-mediated assays using Aroclor 1254-induced, male rat-liver-derived S9 mix. Mutagenic activity of tetryl was demonstrated with S typhimurium strains TA1537, TA1538, TA98, and TA100. The responses were particularly strong in the absence of S9 mix. Tetryl also induced increases in recombinant numbers and frequencies in the S cerevisiae test without the S9 mix, but not in its presence. 1,3-Dinitrobenzene was demonstrated to be mutagen with S typhimurium strains TA1538, TA98, and TA100. Slight activity was also seen with TA1537. The S9 mix reduced the magnitude of the responses. 1,3,5-Trinitrobenzene was also demonstrated to be mutagenic with S typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100. Again, the S9 mix reduced the magnitude of the responses. In this segment of a programme initiated by military authorities, the genotoxic potential of three nitroaromatic compounds, which have found significant use in explosive preparations, has been demonstrated. Twelve other compounds used in ordnance were not active in any of the test systems. These were octahydro-1-acetyl-3,5,7-trinitro-S-tetramine (SEX), hexahydro-1,3-dinitro-5-acetyl-S-triamine (TAX), ethyl centralite, 2-nitrodiphenylamine, N-nitrosodiphenylamine, diphenylamine, diethyleneglycoldinitrate, nitroguanidine, lead salicylate, lead resorcylate, red phosphorus, and zinc chloride.

The relationship between early deaths and implants in control and mutagen-treated CD-1 mice in dominant lethal assays

In dominant lethal assays it has been assumed that the number of early deaths per pregnancy is a function of the germ cells and is independent of maternal influence. It has also been assumed that there is a direct relationship between the number of early deaths and the total number of implants, which in turn has influenced dominant lethal studies. The present communication supports the view that at least this second assumption is erroneous. Data from dominant lethal assays using CD-1 mice in 2 laboratories have been compared and early deaths per implantation as a function of implantations were examined and the implications of the results generated have been considered. It was found in negative control data that a direct relationship between the numbers of early deaths and total implantations was true only at very low, possibly, very high total implantation numbers. Between 4 and 15 implant (based on over 7500 females) the average number of early deaths remained constant. After treatments the direct relationship seen at low implant numbers extended over a larger implant range, but above this range the average number of early deaths again tended to remain constant. It followed, therefore, that, when early deaths per implantation as a function of implantations was considered the average values were dependent on total implantation number over the whole range decreasing as implantations per pregnancy increased. The effect of the mutagens on the frequency of early deaths per implant was greater at low implant numbers. Approaches to statistical analysis of the data from these studies are discussed in the light of the findings and a model is proposed whereby these unexpected relationships between the number of early deaths and the numbers of implantations are explicable in terms of the properties of the germ cells in CD-1 mice, but they may also be affected by some aspect of the females physiology.