In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.     

Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora

To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100. Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with beta-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic beta-D-glucuronides of phenolphthalein and/or p-nitrophenol. These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial beta-glucuronidases to potent mutagenic aglycone(s).     

Evidence for caffeine-sensitive damage in methylazoxymethanol acetate-treated L5178Y cells.

The effects of methylazoxymethanol (MAM) acetate on colony survival, cell proliferation and DNA synthesis of murine lymphoma L5178Y cells are studied. Decreased sensitivity and immediate depression of cell proliferation and DNA synthesis were found in L5178Y cells in contrast to the reports on HeLa cells. Pre-labelling with 5-bromodeoxyuridine (BUdR) did not enhance significantly the carcinogen-induced cell lethality. Post-treatment with caffeine greatly enhanced cell lethality and depression of cell proliferation. These effects of caffeine were diminished when the cells had passed through two generations following the MAM acetate treatment. Experiments with synchronized cells showed that the action of caffeine was located primarily in S phase following the MAM acetate-treatment. These results strongly suggest that in L5178Y cells, MAM acetate induces damage, which is repaired by a mechanism analogous to post-replication repair of UV light-induced damage.     

Effect of cell growth rate and dose fractionation on chemically-induced ouabain-resistant mutations in Chinese hamster V79 cells.

Chinese hamster V79 cells were grown in medium containing either 10% or 2% FCS during the expression time following exposure to MNNG. The lower serum concentration was used to reduce the rate of cell replication, thereby allowing more time for DNA repair prior to "fixation" of the mutagenic lesion. In addition, fractionated and continuous exposures to MNNG and MAM, respectively, were carried out to determine their effect on the number of induced ouabain-resistant mutants. The results indicated that lowering the rate of cell growth effectively reduces the mutation frequency at low, but not at high doses of MNNG. Fractionated doses of MNNG result in a potentiation of their mutagenic effects compared to single doses. Also, continuous exposures to MAM result in an exponential increase in the mutation frequency. Collectively, these results suggest the importance of a repair process in Chinese hamster V79 cells which is dependent upon cell growth rate and the dose of the mutagen for its effectiveness.     

Inhibition of cell proliferation in black‐capped chickadees suggests a role for neurogenesis in spatial learning

Following development, the avian brain continues to produce neurons throughout adulthood, which functionally integrate throughout the telencephalon, including the hippocampus. In food‐storing birds like the black‐capped chickadee (Poecile atricapillus), new neurons incorporated into the hippocampus are hypothesized to play a role in spatial learning. Previous results on the relation between hippocampal neurogenesis and spatial learning, however, are correlational. In this study, we experimentally suppressed hippocampal neuronal recruitment and tested for subsequent effects on spatial learning in adult chickadees. After chickadees exhibited significant learning, we treated birds with daily injections of either saline or methylazoxymethanol (MAM), a toxin that suppresses cell proliferation in the brain and monitored subsequent spatial learning. MAM treatment significantly reduced cell proliferation around the lateral ventricles and neuronal recruitment in the hippocampus, measured using the cell birth marker bromodeoxyuridine. MAM‐treated birds performed significantly worse than controls on the spatial learning task 12 days following the initiation of MAM treatment, a time when new neurons would begin functionally integrating into the hippocampus. This difference in learning, however, was limited to a single trial. MAM treatment did not affect any measure of body condition, suggesting learning impairments were not a product of non‐specific adverse effects of MAM. This is the first evidence of a potential causal link between hippocampal neurogenesis and spatial learning in birds. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1002–1010, 2014     

Animal models of brain maldevelopment induced by cycad plant genotoxins

Cycads are long‐lived tropical and subtropical plants that contain azoxyglycosides (e.g., cycasin, macrozamin) and neurotoxic amino acids (notably β‐N‐methylamino‐l ‐alanine l ‐BMAA), toxins that have been implicated in the etiology of a disappearing neurodegenerative disease, amyotrophic lateral sclerosis and parkinsonism‐dementia complex that has been present in high incidence among three genetically distinct populations in the western Pacific. The neuropathology of amyotrophic lateral sclerosis/parkinsonism‐dementia complex includes features suggestive of brain maldevelopment, an experimentally proven property of cycasin attributable to the genotoxic action of its aglycone methylazoxymethanol (MAM). This property of MAM has been exploited by neurobiologists as a tool to study perturbations of brain development. Depending on the neurodevelopmental stage, MAM can induce features in laboratory animals that model certain characteristics of epilepsy, schizophrenia, or ataxia. Studies in DNA repair‐deficient mice show that MAM perturbs brain development through a DNA damage‐mediated mechanism. The brain DNA lesions produced by systemic MAM appear to modulate the expression of genes that regulate neurodevelopment and contribute to neurodegeneration. Epigenetic changes (histone lysine methylation) have also been detected in the underdeveloped brain after MAM administration. The DNA damage and epigenetic changes produced by MAM and, perhaps by chemically related substances (e.g., nitrosamines, nitrosoureas, hydrazines), might be an important mechanism by which early‐life exposure to genotoxicants can induce long‐term brain dysfunction. Birth Defects Research (Part C) 99:247–255, 2013. © 2013 Wiley Periodicals, Inc.