Enhancement of dimethylnitrosamine metabolism and activation to a mutagen following chronic ethanol consumption.

Chronic ethanol ingestion in rats results in an increase in hepatic microsomal dimethylnitrosamine (DMN) demethylase activity and in an increase in hepatic microsomal activation of DMN to a mutagen. These effects of ethanol on DMN metabolism were detectable in vitro at DMN concentrations as low as 0.3 to 1 mM and as high as 100 mM. This ability of ethanol to increase the rate of DMN metabolism over such a broad range of DMN concentrations is in marked contrast to the effects of other microsomal enzyme inducers, such as phenobarbital and 3-methylcholanthrene, which increase the rate of DMN metabolism only at relatively high DMN concentrations and repress its metabolism at low DMN concentrations.

Alcohol-related diseases and carcinogenesis.

Possible mechanisms whereby alcohol abuse and alcohol-related diseases may promote the development of cancer are analyzed. The mechanisms discussed include: (a) contact-related local effects on the upper gastrointestinal tract; (b) the presence of low levels of carcinogens in alcoholic beverages; (c) induction of microsomal enzymes involved in carcinogen metabolism; (d) various types of cellular injury produced by ethanol and its metabolites and their relationship to cancer, particularly in the liver; (e) the nutritional disturbances frequently associated with alcohol abuse. The relationship between alcohol-induced cirrhosis and hepatocellular carcinoma is also discussed, and case histories of patients seen at the Bronx Veterans Administration Medical Center with hepatocellular carcinoma in the absence of cirrhosis are reviewed. Data are presented demonstrating the induction, by chronic ethanol consumption, of microsomal enzymes which convert procarcinogens to carcinogens. These data were derived from experiments in which the ability of microsomes isolated from liver, intestine, and lung tissues of ethanol-fed and control rats to activate several test carcinogens was examined in the Ames Salmonella-mutagenicity test. The hypothesis is presented that ethanol-mediated induction of enzyme systems which activate procarcinogens to carcinogens in various tissues contributes to the enhanced incidence of cancer in the alcoholic.

Nucleotide sequence of the immunity region of Bacillus subtilis bacteriophage phi 105: identification of the repressor gene and its mRNA and protein products.

A gene involved in the regulation of lysogeny in the temperate Bacillus subtilis phage phi 105 has been identified and isolated. A plasmid, pDC4, was constructed that contains a 740-bp HindIII-PvuII fragment that is derived from the phi 105 immunity region and is capable of rendering B. subtilis immune to infection by phi 105. Three different hybrid plasmids that contain the 740-bp fragment, pAG101 [Cully and Garro, J. Virol. 34 (1980) 789-791], pDC1 and pDC2, were found to synthesize a common 18-kDal polypeptide in B. subtilis minicells and Escherichia coli maxicells. The nucleotide (nt) sequence of this region revealed three open reading frames (ORFs) that predict proteins with Mrs of 16521, 7332, and 5516. In vivo synthesized phi 105 prophage RNA was mapped by primer extension and shown to be transcribed from the DNA strand coding for the Mr 16521 protein. The 5' end of the phi 105 lysogen RNA was mapped to a region that contains conserved sequences for RNA polymerase recognition.

Urinary mutagen levels in smokers.

Urinary mutagen levels were measured over an 8-week period in a group of 13 smokers. Individual urinary mutagen levels were observed to be relatively constant and while there was a general correlation between mutagen excretion levels and cigarette consumption, individuals' excretion levels could not be predicted on the basis of either the numbers of cigarettes smoked or the tar content of the cigarettes.

Mutagenic activity in gastric juice and urine of subjects with chronic atrophic gastritis, gastric epithelial dysplasia and gastric cancer.

Gastric juice and urine samples from consecutive patients who underwent endoscopy for upper GI tract complaints were examined for the presence of mutagens. Patients endoscopically and histologically diagnosed as having either chronic atrophic gastritis (CAG) or gastric cancer (GC) had higher than normal levels of mutagens in their gastric juice and urine. The gastric juice pH of these patients was also elevated and, in the case of the CAG patients, contained detectable levels of nitrites. No correlation was however found between gastric mutagen levels and urinary mutagen excretion in the individuals examined.

Sex dependent effect of chronic ethanol consumption in rats on hepatic microsome mediated mutagenicity of benzo[a]pyrene.

The effect of chronic ethanol consumption by rats on hepatic microsomal metabolism of the procarcinogen benzo[a]pyrene (B[a]P) was investigated both with respect to induction of microsomal arylhydrocarbon hydroxylase (AHH) activity and activation of B[a]P to a mutagen. In female rats, chronic ethanol ingestion produced a 42% increase in AHH activity (P less than 0.01), as measured in isolated microsomes, and also resulted in a significantly enhanced capacity (P less than 0.01) of these microsomes to activate B[a]P to a mutagen detectable in the Ames bacterial mutagenesis assay. Hepatic microsomes from male rats on the other hand did not exhibit any significant differences, either in AHH activity or in their capacity to activate B[a]P to a mutagen after chronic ethanol feeding.

Mutagenicity of the halogenated olefin, 2-bromo-2-chloro-1,1-difluoroethylene, a presumed metabolite of the inhalation anesthetic halothane.

The presumed halothane metabolite, 2-bromo-1,1-difluoroethylene, produces both base substitution and frameshift mutations in Salmonella typhimurium. Direct mutagenesis of isolated DNA also was observed by using a Bacillus subtils transformation assay to score the production of mutagenic lesions in transforming DNA.

Effects of cytochrome p-448 and p-450 inducers on microsomal dimethylnitrosamine demethylase activity and the capacity of isolated microsomes to activate dimethylnitrosamine to a mutagen.

The relationship between microsomal dimethylnitrosamine (DMN) demethylase activity and the capacity of isolated hepatic microsomes to activate DMN to a mutagen was examined using microsomes from C57 and DBA/2 mice which had been exposed to three different types of microsomal enzyme inducers: phenobarbital, which induces cytochrome P-450, 3-methylcholanthrene, which induces cytochrome P-448, and the polychlorinated biphenyl, Aroclor 1254 which appears to induce both types of cytochromes. DNM induced mutagenesis was assayed by a Salmonella auxotroph reversion test. With the C57 mice all three inducers increased both the activity of microsomal DMN demethylase and the capacity of the microsomes to activate DMN mutagenicity. In each case, however, the increase in mutagenicity was disproportionately greater than the increase in DMN demethylase activity. This was particularly evident with microsomes prepared from Aroclor induced mice. Microsomes from 3-methylcholanthrene treated DBA/2 mice were not induced for DMN demethylase or the activation of DMN mutagenicity. In addition the capacity of Aroclor to function as an inducer was relatively poor in this strain. Both DMN demethylation and mutagenesis were inhibited by the addition of either SKF 525-A or benzo (a)pyrene to the reaction mixtures. Thus microsomal activation of DMN to a mutagen and DMN demethylase appear to involve both cytochromes P-450 and P-448.

Vinyl chloride dependent mutagensis: effects of liver extracts and free radicals.

The mutagenic effects of vinyl chloride (VC) on Salmonella typhimurium strain TA1530 are enhanced by mouse or rat liver extracts. The extracts prepared from mice pretreated either with vinyl chloride or the microsomal enzyme inducer, Aroclor 1254, did not produce any greater stimulation of VC-dependent mutagenesis than extracts from untreated animals. These same extracts, however, differed markedly in their capacity to stimulate the mutagenicity of dimethylnitrosamine (DMN), a compound which is converted to a mutagen by an NADPH dependent microsomal mixed function oxidase. The order of activity of the extracts with DMN was Aroclor pretreated is greater than untreated is greater than VC pretreated. Furthermore, the stimulatory effect of the liver extracts on VC mediated mutagenesis did not require NADPH and was still evident in liver extracts in which the microsomal mixed function oxidase system had been heat inactivated. The mutagenic activity of VC also was found to be stimulated by riboflavin in the presence of light suggesting that free radicals may be involved in VC dependent mutagenesis.

Chronic ethanol feeding alters the structure and function of the epidermal growth factor receptor in rat stomach.

We investigated the effect of chronic ethanol feeding on the EGF receptor in rat stomach. Adult male rats were fed either an isocaloric control or ethanol (EtOH)-containing liquid diet (36% total calories as EtOH) for 4 weeks EtOH significantly reduced the specific binding of 125I-EGF to the gastric mucosal membrane (control vs. EtOH, 2.07 +/- 0.2 vs. 0.94 +/- 0.16 fmol/mg protein; p < 0.01). Scatchard analysis suggested that the lower binding might be due to the reduction of EGF receptor number, and/or the affinity of the high-affinity binding site. Western blot analysis, using anti-EGF receptor antibody, revealed four immunoreactive protein bands (180, 150, 60, and 50 kDa) in the lectin-purified gastric membrane prepared from both groups. However, the intensities of these protein bands in the EtOH-fed animals were 90% lower compared to the controls. In the EGF-responsive protein kinase assay, 32P-ATP was incubated with lectin-purified samples in the absence or presence of 1 microM EGF. EGF stimulated autophosphorylation of the EGF receptor (180 kDa) in stomach from the control groups, but not the EtOH-fed animals. This EtOH-related alteration of the gastric EGF receptor may be one of the mechanisms underlying the gastric pathology associated with alcohol abuse.

Cytochrome P-450 in the activation and inactivation of carcinogens.

The capacity of isolate mouse liver microsomes to alter the mutagenicity for bacteria of the primary carcinogen N-methyl-N'-nitro-N-nitrosoguanisine (MNNG) and the secondary one dimethylnitrosamine (DMN) was studied. Microsomal activation of DMN and inactivation of MNNG were decreased by protein- and protein-cholinedeficient diets and were increased by pretreatment with microsomal enzyme inducers. The decrease and increase paralleled the content of cytochrome P-450 present in the different microsomal preparations. With human liver microsomes of differing cytochrome P-450 contents similar correlation was obtained, whereas normal rat liver microsomes did not activate or inactivate DMN or MNNG. Oxidative demethylation of DMN by mouse liver microsomes and the activation of DMN to a mutagen followed similar kinetics. Both reactions were inhibited by carbon monoxide and the inhibition was maximally reversed by monochromatic light at 450 nm. These observations indicate that at least some carcinogens are activated or inactivated by the unspecific cytochrome P-450 dependent enzyme system, suggesting that the extent of this biotransformation may be one factor influencing human carcinogenesis.