Graduate research project: N-Nitroso compound mutagenicity mechanisms and comparison with carcinogenicity
Since the project with mutagen-treated transforming DNA wasn’t going to work, I ended up testing some nitrosamines. [Willie was always recruiting people to work on nitrosamines. He gave Bill Russell ENU and that became the animal super mutagen]. Willie had said Don’t go to medical school, science is much more interesting”; and later: “Just go into the lab and do experiments; things will happen!”.
It was fortuitous that Evelyn Witkin’s graduate student, Donna George, provided some of Evelyn’s DNA repair deficient E. coli strains when she joined Jane’s lab as a new post-doc. Thus, Willie’s nitrosamines and Evelyn’s E. coli strains, along with Jane’sHaemophilus influenzae, morphed naturally into my thesis project[a sequence of random events] .
I had a set of methylating agents, MNU, MNNG and Nitrosocarbaryl (nitrosated insecticide Sevin) and some ethyl homologs. Nitrosocarbaryl was mutagenic and carcinogenic. It was likely the first nitrosamine considered a possible human environmental risk (Elespuru et al. 1974). Why were the mutagenicities of diverse methylating agents so different? They broke down to the same intermediate, in principle, but mutagenicity and carcinogenicity varied a lot. Also, how did mutagenicity and carcinogenicity compare?
Differences greater than 3 orders of magnitude in the mutagenic potency of methylating agents (MNU, MNNG and N-nitrosocarbaryl) could be accounted for by differences in their uptake into Haemophilus influenzae (using 3H-labeled nitroso compounds) (Elespuru, 1979). The results correlated roughly with the octanol/water coefficient, indicating the critical effect of lipophilicity on uptake. This result was surprising and likely important, but uninteresting from a mechanistic standpoint, as it was not based on a function of active intermediate generation. There goes our hypothesis . Moreover, the differences we found did not correlate with carcinogenicity (Lijinsky and Elespuru 1976; Lijinsky and Schmael 1978). Nitrosocarbaryl was the most mutagenic but not the most carcinogenic methylating agent. Was lipophilicity and distribution an issue with different effects in mammalian systems? We have no idea.
As a contribution to mechanistic studies, I performed mutagenicity experiments with deuterium replacing the alpha carbon hydrogens of NDMA, for comparison with the carcinogenicity studies. Diminished mutagenicity matched diminished carcinogenicity for NDMA, and thus alpha carbon metabolism was key for both mutagenic and carcinogenic effects (Keefer et al. 1973; Elespuru RK 1978; Lijinsky 1986).
Post-doc Donna George brought E. coli WU3610 from Evelyn Witkin’s lab. This was a UV repair-deficient strain with two mutational markers at 2 amino acid auxotrophic loci, amber and ochre, both with AT base-pair targets. Using this strain with methylating and ethylating nitrosamines, I noticed something really interesting: mutations induced by ethylating agents appeared in one day, while those induced by methylating agents took two days. Thus, the E. coli strain was able to differentiate what was happening at the molecular level. DNA targets of ethylating and methylating agents were different (most likely direct alteration of AT base pairs vs. alteration of slower growing GC-based suppressors). Thus, I could differentiate between methylating and ethylating intermediate-caused mutations. With this tool we could examine the effects of nitrosomethylethylamine (NMEA) as compared with nitroso dimethyl- and diethyl-amine. Would the mutagenicity results resemble that of NDMA or NDEA, neither or both? The mutagenicity results in E. coli at AT base pairs indicated a preponderance of ethylation, a result which it seems I didn’t publish. However, alkylation of DNA in vitro or in vivo indicated an overwhelming bias toward methylation vs. ethylation of N7-alkylguanine by NMEA alkylation (von Hofe et al., 1986, 1991). [Although O6 alkylation was not always studied, differences between methylation and ethylation would not alter the conclusions]. Deuterium isotope effects for carcinogenicity of NMEA indicated complex results, including targeting of esophagus, an organ targeted by neither NDMA or NDEA (lijinsky and Reuber, 1980, Streeter et al. 1990). A later paper expanding on the idea of studying differential chemical interactions using mutational endpoints was probably the first use of the term “mutational fingerprints”, a paper I thought novel and worthy of PNAS, but not accepted by Dick Albertini (Elespuru et al. 1991).