Anthony J. Garro

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.

Mutagenicity of primary and secondary carcinogens altered by normal and induced hepatic microsomes.

To establish the role of the activity of the microsomal biotransformation system in chemical carcinogenesis, the mutagenic effect of primary (ultimate) and secondary (potential) carcinogens after exposure to isolated hepatic microsomes was studied. The principal enzyme system, suggested to be active in the metabolism of carcinogenic compounds, is the nonspecific cytochrome P-450 dependent mixed-function oxidase (1). This microsomal enzyme system is inducible by many substrates, including some environmental contaminants (2). The involvement of the microsomal enzymes in carcinogenesis is suggested by the observed increased carcinogen biotransformation in animals (3) and isolated cells (4) following exposure to inducing substrates. Most carcinogenic compounds are mutagenic for microorganisms (5). The primary are themselves mutagenic, the secondary are inactive until metabolized to a mutagenic form (6). Metabolism of primary carcinogens may reduce mutagenic and carcinogenic properties. Both types of metabolic conversion, activation and deactivation, have been studied by host-mediated assay, which tests the ability of laboratory animals to alter the mutagenicity of carcinogens (6). Similar activity also has been demonstrated in the 30,000g supernatant of mice liver homogenates (7). To demonstrate the precise relationship between the activity of the microsomal enzyme system and the biological activity of carcinogens, an assay is required in which both activities can be measured. For this purpose the ability of isolated normal or induced microsomes to alter the mutagenic activity of two carcinogens, dimethylnitrosamine (DMN) (8) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (9) was studied. These compounds were chosen as examples of primary (MNNG) and secondary (DMN) carcinogens. Methods. Hepatic microsomes were isolated from male Swiss-Webster mice untreated or pretreated four days before sacrifice from a single ip injection of 500 mg/kg of the polychlorinated biphenyls (PCB), Arochlor 1254, an inducer of the microsomal enzyme system (10) and a wide-spread polluting agent (1l).

Chronic ethanol consumption inhibits repair of dimethylnitrosamine-induced DNA alkylation

Chronic ethanol consumption causes a DNA repair deficiency. This was demonstrated in Sprague-Dawley rats injected with 14C-labeled dimethylnitrosamine after being pair-fed isocaloric, ethanol, or carbohydrate control diets for 4 weeks. Hepatic DNA was isolated from rats killed at intervals over a 36 hour period after administration of the nitrosamine and concentrations of alkylated guanine derivatives were measured. While N7-methylguanine was lost at equivalent rates from the DNA of both diet groups, 06methylguanine, a promutagenic lesion, persisted at higher levels for longer periods of time in the DNA from the alcohol-fed animals.

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.

Alcohol Abuse: Carcinogenic Effects and Fetal Alcohol Syndrome

Alcohol abuse is associated with an increased risk of cancer and birth defects, two disease processes that at some stage involve alterations in gene expression and cell development. The biochemical, molecular, and physiological mechanisms through which alcohol consumption produces these pathological effects are not thoroughly understood and seem to be multifactorial in nature. This chapter focuses largely on the experimental systems that have provided insights into potential mechanisms underlying the carcinogenic and teratogenic effects of alcohol abuse. The epidemiologic studies that established the association between alcohol abuse and cancer and birth defects have recently been reviewed (Garro and Lieber, 1990; Abel and Sokol, 1989) and are not discussed in detail here.

Detection of mutagen-induced lesions in isolated DNA using a new Bacillus subtilis transformation-based assay

A new mutagenesis assay based on the induction of mutations in isolated transforming DNA of Bacillus subtilis is described. The assay tests the ability of chemical to induce lesions in isolated DNA which lead to reversion of the ilvB2 base-substitution or leuA169 frameshift mutations which are carried by the recipient cells that are transformed with the treated DNA. This report demonstrates the capacity of the assay to detect as mutagens the following DNA-reactive chemicals: hydroxylamine (HA); N-methyl-N′-nitrosoguanidine (MNNG); chloroacetaldehyde (CAA); propylene oxide (PO) and N-acetyl-N-acetoxy-2-aminofluorene (AAAF). The later 4 chemicals were reacted with the test DNAs in a dimethyl formamide-containing buffer which was used both to maintain the denatured DNA in an extended single-stranded state and to increase the solubilities of the chemicals. The relative mutagenic activities of the compounds in this buffer system were PO < MNNG < AAAF < CAA. In addition to mutagenesis, the capacity of the test compounds to inactivate transforming activity and disrupt gene linkages in the treated DNAs also was assayed. PO, although a much weaker mutagen than CAA, produced more extensive disruption of gene linkage than CAA when compared at concentrations which produced comparable levels of inactivation of transforming activity. The new mutagenesis assay offers 2 advantages over previously described transformation-based assays. (1) In contrast to assays based on the induction of ribosome-associated drug resistances, the new assay can detect frameshift as well as base-substitution type mutagens. (2) The mutants generated can be detected at high cell-plating densities. The assay thus may be useful for general mutagen screening especially with highly bactericidal compounds which are not readily tested in other microbial assays.

Detection of mutagen-induced lesions in isolated DNA by marker rescue of Bacillus subtilis phage φ105

Abstract A new mutagenesis assay is described which detects the induction of forward mutations in isolated DNA. The assay utilizes replicative from DNA of the temperate Bacillus subtilis phage φ105 and tests the ability of chemicals to induce lesions which inactivate phage genes involved in lysogen formation. There is a cluster of such genes tightly linked to the φ105 genetic marker Jsus11 which restricts the host range of the phage to cells capable of suppressing sus mutations. In the actual assay chemically treated DNA, from wild-type J + phage, is added to competent cells which are infected with φ105 Jsus11 . Wild-type phage, capable of producing plaques on cells which are nonpermissive for φ105 Jsus11 , are produced by recombination between the added chemically-treated DNA and infecting φ105 Jsus11 DNA. If the added DNA also carried mutagenic lesions in any of the genes controlling lysogeny, clear plaque mutants are produced which are readily distinguishable from the turbid plaquing wild-type phage. This report demonstrates the capacity of this marker rescue-based assay to detect as mutagens the following DNA-reactive chemicals: hydroxylamine (HA); N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG); chloroacetaldehyde (CAA); propylene oxide (PO) and N -acetyl- N -acetoxy-2-amino-fluorene (AAAF). The effect of using a host cell, defective for excision repair, on the sensitivity with which the assay detected the mutagenic activities of CAA, PO and AAAF also was examined. The new mutagenesis assay offers 2 advantages over several other previously described transformation-based assays: (1) in contrast to assays based on the induction of ribosome-associated drug resistances, the new assay can detect frameshift as well as base-substitution-type mutagens and (2) the mutants generated can be detected at high plating densities. The assay thus may be useful for general mutagen screening especially with highly bactericidal compounds which are not readily tested in other microbial assays.