Faculty Bibliography

Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to alpha-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., alpha-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 X g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

Many N-nitrosamines have been assayed for mutagenic activity in bacteria but few have been systematically compared in a series of strains. In this study through the use of several Salmonella tester strains, we have examined the effects of Uvr repair, error-prone repair, and the critical site for mutation (GC or AT base pair) on the mutagenic activities of a diverse group of N-nitrosamines. We have employed the histidine autotrophs, TA1975 (uvrB+), TA1535 (uvrB-) and TA100 (uvrB-/pKM101) which are hisG46 strains, sensitive mainly to G-C base damage, and TA104 (uvrB-/pKM101), a hisG428 strain, which can be reverted at the hisG428 locus by damage to A-T base-pairs, or by suppression at G-C base pairs. The N-nitrosamines studied were, N-nitroso: dimethylamine, diethylamine, dipropylamine, dibutylamine, pyrrolidine, piperidine, morpholine, methylbenzylamine, bis-(2-hydroxypropyl)amine, bis-(2-oxopropyl)amine and 3,4-dichloropyrrolidine. For all of the nitrosamines larger than diethylnitrosamine (except for methylbenzylnitrosamine) mutagenesis was greatly enhanced (3-20 X) by the lack of uvrB activity, indicating that the DNA adducts produced by these nitrosamines can be classified as "bulky adducts". For most nitrosamines the plasmid, pKM101, enhanced mutagenesis in hisG46 strains, several fold, suggesting that error-prone DNA repair plays a role in mutagenesis by these compounds. All of the compounds tested were more mutagenic in TA100 than TA104 except diethylnitrosamine and methylbenzylnitrosamine which were more potent in TA104. Revertants induced by all of the nitrosamines in TA100 were due predominantly to damage at G-C base pairs. Revertants induced by all the nitrosamines except diethylnitrosamine and dibutylnitrosamine resulted mainly from damage to G-C base pairs in TA104.

Aqueous extracts of smokeless tobacco products (snuff, chewing tobacco, smokeless tobacco) sold in the USA are mutagenic in the base-pair substitution mutant, Salmonella typhimurium TA100, with potencies of 8000-16,000 revertants/g tobacco. Most of the activity failed to extract into organic solvents at neutral pH and a portion of the activity extracted into organic solvents at acidic pH. The tobacco-specific nitrosamines, N-nitrosonornicotine and (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone did not exhibit this behavior. Mutagenesis required metabolic activation (liver S-9 fraction plus NADPH) and a weakly acidic liquid preincubation. The mutagenesis behavior is typical of nitrosamines, and suggests that N-nitrosamines are responsible for at least some of the mutagenic activity in the extracts.

Cis-dichlorodiammine platinum (II), or cisplatin, is currently among the most widely used agents in the chemotherapy of cancer. The chief limit to its greater efficacy is its nephrotoxicity. Acute and chronic nephrotoxicity of cisplatin occurs in man and animals especially after repeated administration. Morphological damage is restricted to the P3 segment of the proximal tubule. Abnormalities of water and solute reclamation and transglomerular passage of fluid are commonly associated with cisplatin nephrotoxicity. The vulnerability of the kidney to cisplatin may be related to its function as the primary excretory organ for platinum. Platinum binds to multiple cellular organelles and macromolecules, yet the precise mechanism of its cytotoxicity has not been delineated. Because abnormalities in renal function are preceded by a period where gross renal function appears normal, it is an ideal model to study the early physiological and biochemical determinants of metal induced acute renal failure.

The metabolism of a series of nitrosamines in vitro was monitored by measuring nitrogen production and was compared with mutagenesis by the same compounds, allowing separation of mutagenic potencies into metabolic and postmetabolic terms. The rate of nitrogen production from symmetrical di-n-alkyl and methylalkyl nitrosamines increased with increasing molecular weight. The cyclic nitrosamines N-nitrosopiperidine and N-nitrosopyrrolidine were metabolized slightly less rapidly than the most hydrophobic compounds, and N-nitrosomorpholine was metabolized at about half this rate. N-Nitrosomethylaniline was metabolized to nitrogen relatively slowly. Branching at the alpha-carbons reduced alpha-oxidative metabolism several fold. Substitution at the beta-carbon of N-nitrosodiethylamine or N-nitrosodi-n-propylamine with hydroxyl, cyano, oxo and methoxyl groups reduced metabolism to an even greater extent. Carboxyl substitution at the 4-position of N-nitrosopiperidine greatly reduced nitrogen formation, but 4-tert-butyl substitution had little effect. Effects of structure on mutagenic activities in Salmonella followed a different pattern. Higher homologue di-n-alkyl nitrosamines were more potent than lower homologues at lower doses, when potencies were taken from slopes of dose-response curves. However, when mutagenic potencies were expressed as 'mutagenic efficiencies' (revertants/mumol nitrogen), regardless of dose, the order of potency was N-nitrosodimethylamine greater than N-nitrosodiethylamine greater than N-nitrosodi-n-propylamine greater than N-nitrosodibutylamine. For the series of methylalkyl nitrosamines, mutagenic potencies were greatest for the higher molecular weight compounds, but they were all similar to that of N-nitrosodimethylamine when expressed as mutagenic efficiencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Cis-dichlorodiammine platinum (II), or cisplatin, has emerged as a principal chemotherapeutic agent in the treatment of otherwise resistant solid tumors and is currently among the most widely used agents in the chemotherapy of cancer. The chief limit to its greater efficacy is its nephrotoxicity, which has made it necessary both to lower its dosage and actively hydrate patients to reduce it. The vulnerability of the kidney to cisplatin is almost certainly related to its primary role in the excretion of cisplatin. Cisplatin enters renal cells by a process that depends on normal oxygen utilization and is specifically inhibited by organic bases. Greater localization of platinum to the S3 segment of the proximal tubules suggests that the vulnerability of this segment may depend on its specific uptake of the drug. The majority of intracellular platinum is bound to macromolecules, including protein and DNA, yet a significant portion of cell platinum is biotransformed to a nonmutagenic and possibly nontoxic compound. Polyuria and hypomagnesemia, which are commonly associated with cisplatin nephrotoxicity, may be due to defects in deep nephron or collecting duct fluid and solute transport. Low single nephron glomerular filtration rates (SNGFR) during early cisplatinum-induced acute renal failure is accompanied by reduced renal blood flow and transglomerular hydrostatic pressure without elevated intratubular hydrostatic pressure, suggesting preglomerular vasoconstriction as an important determinant of renal failure.(ABSTRACT TRUNCATED AT 250 WORDS)