Detection of exocyclic guanine adducts in hydrolysates of hepatic DNA of rats treated with N-nitrosopyrrolidine and in calf thymus DNA reacted with alpha-acetoxy-N-nitrosopyrrolidine. (9/22)

This report describes the isolation and characterization of DNA adducts formed in vitro from alpha-acetoxy-N-nitrosopyrrolidine and in rats treated with the hepatocarcinogen N-nitrosopyrrolidine. Esterase-catalyzed hydrolysis of alpha-acetoxy-N-nitrosopyrrolidine in the presence of calf thymus DNA, followed by neutral thermal hydrolysis of the DNA, resulted in formation of three previously unknown Adducts 1-3. They were isolated and characterized by their UV, mass, and proton magnetic resonance spectra as the exocyclic 7,8-guanine adducts 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purine-4(3H)-one (Adduct 1), and cis- and trans-2-amino-7,8-dihydro-8-hydroxy-6-methyl-3H-pyrrolo[2,1-f] purine-4(6H)-one (Adducts 2 and 3). Adduct 1 was formed by addition of 4-oxobutyl diazohydroxide, or a related carbonium ion, to the 7 and 8 positions of guanine. Adducts 2 and 3 resulted from Michael addition of 2-butenal to the 7 and 8 positions of guanine. Esterase-catalyzed hydrolysis of alpha-acetoxy-N-nitrosopyrrolidine in the presence of DNA also produced the exocyclic 1,N2-propanodeoxyguanosine Adducts 4a and 4b which we have previously described. Neutral thermal hydrolysates of hepatic DNA isolated from rats treated with N-nitrosopyrrolidine contained a fluorescent adduct, as previously reported (E.J. Hunt and R.C. Shank, Biochem. Biophys. Res. Commun., 104: 1343, 1982). This fluorescent adduct was shown to be identical to Adduct 1. Adducts 2, 3, 4a, and 4b were not detected in hepatic DNA hydrolysates from these animals. The results of this study provide the first example of a structurally characterized DNA adduct formed in vivo from a cyclic nitrosamine and support the alpha-hydroxylation hypothesis of cyclic nitrosamine metabolic activation.  (+info)

Induction of liver tumors in F344 rats by crotonaldehyde. (10/22)

The tumorigenic activities in F344 rats of crotonaldehyde, a representative alpha, beta-unsaturated carbonyl compound, and N-nitrosopyrrolidine, which could produce crotonaldehyde upon metabolism, were compared. Groups of rats were treated with either crotonaldehyde (0.6 mM or 6.0 mM) or N-nitrosopyrrolidine (0.6 mM) in their drinking water for 113 or 84 weeks, respectively. At the 0.6 mM dose, crotonaldehyde induced neoplastic lesions of the liver in 9 of 27 rats; 2 rats had hepatocellular carcinomas, and 9 rats had neoplastic nodules. It also caused altered liver cell foci in 23 of 27 rats. The incidences of tumors and foci were significantly higher than those of the control group. N-Nitrosopyrrolidine induced hepatocellular carcinomas in 20 of 23 rats, liver neoplastic nodules in 16 of 23 rats, and altered liver cell foci in 23 of 23 rats. Thus, crotonaldehyde appears to be a weaker tumorigen than N-nitrosopyrrolidine. At the 6.0 mM dose, crotonaldehyde treatment caused moderate to severe liver damage in 10 of 23 rats. No preneoplastic or neoplastic lesions were observed in these rats. The remaining 13 rats of this group developed altered liver cell foci. The tumorigenicity of crotonaldehyde suggests that alpha, beta-unsaturated carbonyl compounds, which are ubiquitous in the human environment and can be formed endogenously, may be an important class of potential carcinogens.  (+info)

Reconstitution of rabbit liver microsomal N-nitrosopyrrolidine alpha-hydroxylase activity. (11/22)

The in vitro alpha-hydroxylation of N-nitrosopyrrolidine (NPYR) by both isolated rabbit liver microsomes and purified cytochrome P-450 isozymes was investigated. Microsomes from untreated rabbits catalyzed the alpha-hydroxylation of NPYR at rates similar to those reported previously for rats, mice, and hamsters. The effect of established inducers of microsomal P-450 caused complex changes in apparent rates of alpha-hydroxylation of NPYR which made interpretation of responses to inducer pretreatment difficult and suggested the participation of multiple cytochrome P-450 isozymes in the metabolism of NPYR. Partial inhibition of alpha-hydroxylase activity by antibodies against rabbit isozymes 2, 3a, and 5 indicated the participation of at least these three isozymes in microsomal catalysis. Reconstitution studies using purified rabbit isozymes 2, 3a, 3b, 3c, 4, and 6 indicated that isozymes 2, 3a, 4, and 6 possessed significant alpha-hydroxylase activity with isozymes 3a and 6 exhibiting the highest activity when assayed at 20 mM NPYR. As NPYR concentrations were decreased, the rates of catalysis for the reconstituted systems were differentially decreased such that isozyme 3a exhibited the highest activity at low NPYR concentrations. These data indicate that isozyme 3a is the preferred catalyst for the alpha-hydroxylation of NPYR at low substrate concentrations and suggest that conditions such as chronic ethanol consumption which lead to the induction of isozyme 3a in rabbits or its orthologue in other species can account for enhanced rates of alpha-hydroxylation and metabolic activation of NPYR.  (+info)

Comparative study of DNA damage and repair induced by ten N-nitroso compounds in primary cultures of human and rat hepatocytes. (12/22)

Ten carcinogenic N-nitroso compounds were assayed for DNA-damaging activity in primary cultures of human and rat hepatocytes. DNA fragmentation was measured by the alkaline elution technique, and unscheduled DNA synthesis by quantitative autoradiography. Positive dose-related responses in the range of subtoxic concentrations indicated were obtained in cells of both species with N-nitrosodiethylamine (10-32 mM), N-nitrosodi-n-propylamine (1.8-10 mM), N-nitrosomorpholine (1-3.2 mM), N-nitrosopiperidine (1-3.2 mM), N-nitrosopyrrolidine (3.2-18 mM), N-nitroso-N-methylurea (0.32-1.8 mM), N-nitroso-N-ethylurea (0.32-1.8 mM), and N-nitroso-N-butylurea (0.1-0.32 mM). N-nitrosodi-n-butylamine was practically inactive at the maximal soluble concentration (1 mM). The responses of human hepatocytes were qualitatively similar to those of rat hepatocytes, but statistically significant differences between the two species in the amounts of DNA damage and/or unscheduled DNA synthesis were observed with N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosopiperidine, N-nitrosopyrrolidine, and N-nitroso-N-butylurea. On the other hand, quantitative differences in the genotoxic effects induced by 5 mM N-nitrosodimethylamine in cultures derived from 20 human donors and from 20 rats were greater than average interspecies differences displayed by this nitrosamine and by other N-nitroso compounds. These results indicate that the rat hepatocyte DNA repair assay is a valid model for predicting the genotoxic potential of N-nitroso compounds in human hepatocytes.  (+info)

Carcinogenic effects of sequential administration of two nitrosamines in Fischer 344 rats. (13/22)

The carcinogenic effects of sequential treatment of female F344 rats with two nitrosamines were studied. The animals received either methylethylnitrosamine (NMEA), a strong liver carcinogen, N-nitrosomethylaniline (NMA), a moderately strong esophageal carcinogen, or N-nitrosopyrrolidine, (NPyr), a weaker liver carcinogen. The sequentially treated groups were given NMEA followed by NMA and vice versa, NPyr followed by NMEA and vice versa. The dose and duration for each chemical in the sequentially treated groups were identical for the individual treatments. The animals were allowed to die or were killed when moribund. The animals surviving longer than 110 weeks were sacrificed. The NMEA-NPyr and NPyr-NMEA groups had a tumor spectrum characteristic for NMEA alone (a mixture of hepatic carcinomas and sarcomas with extensive metastases to the lungs). The survival was reduced in the NMEA-NPyr group compared to the NMEA alone group. The time to death of the NMA-NMEA group was not affected by the NMA treatment, but many of the animals had esophageal neoplasms. The NMEA-NMA group survival was reduced when compared to the NMEA alone group but the tumor spectrum was dominated by NMEA. The data indicate that when the target organ is the same, the effect of two nitrosamines is additive with the stronger carcinogen dominating the tumor spectrum. When the target organs are different, the initial exposure influences the tumor spectrum, although the treatment with the second nitrosamine enhances the tumorigenicity of the initial nitrosamine.  (+info)

Plate diffusion assay as a rapid method for dosimetry of mutagens. (14/22)

This paper presents a method for determining mutagenic concentrations of chemicals by using an agar diffusion assay. The method is based on the linear relationship between the amount of chemical placed at the center of the dish and the radius of the mutagenic zone. A brief theoretical discussion and experimental data confirming this relationship are given. Alkylating agents and mycotoxins were used to test the system. This method can be used to follow up decreased mutagenic potencies of solutions of unstable mutagens and to follow the production of mutagenic substances throughout fermentation.  (+info)

Effects of butylated hydroxyanisole on the tumorigenicity and metabolism of N-nitrosodimethylamine and N-nitrosopyrrolidine in A/J mice. (15/22)

Female A/J mice were maintained on NIH-07 diet or on NIH-07 diet containing butylated hydroxyanisole (BHA, a mixture of 2- and 3-tert-butyl-4-hydroxyanisole), 5 mg/g, for 1 week prior to and during 10 weeks of treatment with N-nitrosodimethylamine (NDMA) or N-nitrosopyrrolidine (NPYR), administered in the drinking water. Twenty weeks after nitrosamine treatment ended, mice were sacrificed and lung adenomas were counted. BHA inhibited NDMA tumorigenesis but enhanced NPYR tumorigenesis. Treatment of A/J mice for three weeks with BHA (5 mg/g) added to semisynthetic diet increased whole lung microsomal alpha-hydroxylation of NDMA and NPYR, as measured by aldehyde production, and increased lung and hepatic glutathione-S-transferase activities. No evidence was found for formation of S-methylglutathione in incubations with NDMA and hepatic supernatants obtained from BHA treated or control mice. Four h after gavage of NDMA, levels of 7-methylguanine in the lung DNA of mice treated with BHA were higher than in the lung DNA of control mice, but levels of O6-methylguanine in the two groups were the same. The results of this study indicate that BHA treatment increases the microsomal metabolic alpha hydroxylation of both NDMA and NPYR, but has differential effects on their tumorigenic activities.  (+info)

Metabolism of the liver carcinogen N-nitrosopyrrolidine by rat liver microsomes. (16/22)

This report represents a study of the total metabolism of the hepatocellular carcinogen, N-nitrosopyrrolidine (NO-PYR), by rat liver microsomes and postmicrosomal supernatant. [2,5-14C]NO-PYR, which is totally extractable from aqueous solution with methylene chloride, is converted to radioactive nonmethylene chloride-extractable products by these fractions. The initial rate of conversion to nonmethylene chloride-extractable products follows simple Michaelis-Menten kinetics with an apparent Km of 3.6 x 10(-4) M NO-PYR. The major products of NO-PYR metabolism by rat liver microsomes and postmicrosomal supernatant have been isolated and identified. One product of metabolism of NO-PYR is 2-hydroxytetrahydrofuran formed by alpha-hydroxylation by the microsomes. In the presence of postmicrosomal supernatant enzymes, this compound exists only as a transient intermediate which is rapidly converted to 1,4-butanediol or gamma-hydroxybutyrate. These compounds may be cycled into general cellular metabolism resulting in the production of CO2. Two minor pathways of metabolism have also been found.  (+info)