Faculty Bibliography

Mutagenesis induced by N-nitroso-N-ethylurea (NEU) was assayed in four strains of Salmonella typhimurium which are known to be reverted to histidine prototrophy by mutations at A-T base pairs and by extragenic suppression. NEU-induced revertants were characterized for the presence of extragenic suppressors by their sensitivities to the histidine analogue, thiazolealanine. In strains carrying the plasmid, pKM101, only a small percentage of the revertants was due to suppressors, indicating that NEU gives rise to a major premutagenic adenine or thymidine-DNA adduct. In strains without plasmid, mutagenesis was much less efficient and resulted mainly from suppressors. Apparently error-prone DNA-repair plays an important role in mutagenesis via the A or T-DNA adduct in the plasmid-containing strains. Ethylmethanesulfonate (EMS), a mutagen known to form ethyladenines but not ethylthymidines, induced mutagenesis that resulted mainly from suppressors in all strains, and there was little inter-strain difference in the sensitivity to EMS. Since NEU, but not EMS, forms ethylthymidines in appreciable yield, and only NEU induced high percentages of revertants with mutations at A-T base pairs, it appears that at least one ethylthymidine is a major premutagenic adduct in NEU-induced mutagenesis.

Mutagenesis induced by the N-nitroso compounds: N-nitrosomethylurea, N-nitrosoethylurea, N-nitrosodi-n-propylamine and N-nitrosopyrrolidine was measured in Salmonella typhimurium TA100, TA102 and TA104. TA100 detects damage mainly at G-C base pairs while TA102 and TA104 can detect damage at A-T base pairs. In general all strains were similarly sensitive, except that TA104 was much less sensitive to high doses of N-nitroso-N-methylurea. In TA104 a significant percentage of the revertants induced by all agents except NMU resulted from point mutations at A-T base pairs, indicating that adenine or thymine DNA adducts are important premutagenic adducts formed by certain N-nitroso compounds.

Cisplatin, an effective antineoplastic agent, is toxic to the kidney. Since the kidney's vulnerability to cisplatin may originate in its ability to accumulate and retain platinum to a greater degree than other organs, we studied the characteristics of the renal accumulation of platinum and investigated the nature of intracellular platinum. Cisplatin and ethylenediamminedichloroplatinum, nephrotoxic and antineoplastic liganded platinum compounds, were concentrated in rat renal cortical slices fivefold above medium concentration. Platinum uptake was energy- and temperature-dependent and could be inhibited by drugs which inhibit base transport. The organic anions para-aminohippurate and pyrazinoate did not reduce renal slice platinum uptake. Unbound platinum in the blood and urine was predominantly cisplatin but unbound platinum in kidney cytosol was not. This latter compound, in contrast to cisplatin, was not active as a mutagen. These studies suggest that the kidney accumulates platinum in part by transport or specific binding to the base transport system in the kidney and biotransforms it intracellularly. Unbound platinum in the cell is not cisplatin and may no longer be toxic.

The dose-responses for N-nitroso-N-ethylurea (NEU)-induced mutagenesis in the hisG46 mutant, Salmonella typhimurium TA1535, and for the formation of O6-ethylguanine (O6-EtGua) and 7-ethylguanine in the DNA isolated from these cells were measured. Mutagenesis and O6-EtGua formation exhibited threshold-like behavior, whereas the formation of 7-ethylguanine was linear with dose. These results are consistent with a dependence of mutagenesis on O6-EtGua. There was no threshold in the production of O6-EtGua in isolated DNA treated with NEU. The failure of O6-EtGua to appreciably accumulate in the cellular DNA at low doses of NEU was attributed to a saturable, constitutive repair activity in the bacteria. Based on (i) the ratio of O6-EtGua in DNA to revertant fraction, (ii) published values for the size of the Salmonella genome and (iii) the target size and target bases (guanine-cytosine base pairs) for reversion of the hisG46 (missense) mutation, it was calculated that about 1/3 of the O6-EtGua's in the DNA led to mutations. Using the same calculations and data from previous experiments, a mutational efficiency for O6-methylguanine of 2/3 was obtained. Threshold-type responses in NEU-induced mutagenesis were observed in the other hisG46 mutants, TA100 and TA1975, but not in the frameshift mutant, TA98 where the dose response was linear. As TA98 contains the same DNA repair systems as TA100, frameshift mutations induced by NEU may result from DNA adducts produced linearly with dose.