The cycad genotoxin MAM modulates brain cellular pathways involved in neurodegenerative disease and cancer in a DNA damage-linked manner

Abstract

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O⁶-methyldeoxyguanosine lesions, O⁶-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O⁶-mG DNA methyltransferase (MGMT) showed elevated O⁶-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimer's disease.

Figure 1. Heat map comparing the post-treatment (6 hr) transcriptional response of brain and liver (positive control) of wt mice to a single intraperitoneal dose of MAM.

The experiment was conducted at two independent laboratories using identical protocols. Green denotes down-regulation and red up-regulation of gene expression. OHSU: Oregon Health & Science University. FHCRC: Fred Hutchinson Cancer Research Center.

Figure 2. Time-course of O⁶-methylguanosine (O⁶-mG) DNA damage in the brain (A) and liver (B) of wt (blue) and Mgmt^−/− (red) mice following a single intraperitoneal dose of MAM.

Results for the two study sites are shown as separate red and blue lines. The plotting symbols (OHSU: circle; FHCRC: square) denote the estimated medians; lines extend ±2 standard errors from the medians. DNA damage (O⁶-mG) is three orders of magnitude higher in the liver than in brain, and the significantly elevated O⁶-mG levels in Mgmt^−/− vs. wt tissues at 48 hr are maintained at 168 hr post MAM treatment. Discontinuities in the red and blue lines are attributed to technical errors or where samples were not collected. The dashed gray line in B denotes the maximum observed O⁶-mG level in the brain (∼330 lesions per 10⁸ normal nucleotides).

Figure 3. MAM-modulated brain gene products for Mgmt^−/−.

Most significant brain expression sub-network modulated by MAM vs. vehicle in Mgmt^−/− mice (all time-points combined) composed of 362 differentially expressed genes.

Figure 4. MAM-modulated brain gene products (anchored) for Mgmt^−/−.

Most significant brain expression sub-network modulated by MAM vs. vehicle in Mgmt^−/− mice (all time-points combined) derived from 57 differentially expressed genes (a sub-set of the 362 genes) that were anchored to O ⁶-mG levels.

Figure 5. MAM-modulated brain gene products for Mgmt^−/− vs. wild type.

Most significant MAM-modulated expression sub-network in the brain of Mgmt^−/− vs. wild type mice (all time-points combined) composed of 153 differentially expressed genes. Note genes involved in epigenetic functions are also modulated: DNMT3A and SMARCC1 regulate chromatin function.

Figure 6. MAM-modulated brain gene products (anchored) for Mgmt^−/− vs. wild type.

Most significant MAM-modulated expression sub-network in the brain of Mgmt^−/− vs. wild type mice (all time-points combined) composed of 60 differentially expressed genes that were anchored to O ⁶-mG levels. Note the presence of NF-κB, ERK1, and p38-MAPK hubs, and the involvement of TP53 and glutamate receptors.

Figure 7. Transcription factor binding-site enrichment hierarchy.

Analysis of the promoter regions of the 60-anchored gene sub-set derived from the strain-specific differentially expressed genes between MAM and vehicle-treated animals for transcription regulatory elements. A heat map (interaction matrix) shows the genes (rows) and motifs (columns) that were individually clustered and found in >5% of all promoters. Note the HNF4 binding site is common to 60% of the 60 anchored genes.

Figure 8. Proposed relationship between MAM-induced colon cancer and brain disease/disorders.

Genotoxicants that induce O⁶-methylguanine lesions (DNA damage) (e.g. via methylazoxymethanol, MAM) disturb cell signaling pathways, including transforming growth factor-β (TGF-β), wingless and proto-oncogene Int-1 (Wnt), and mitogen-activated protein kinase (MAPK). In general, the literature supports up-regulation (green) and down-regulation (pink-red) in association with the two distinct phenoptypes. (Modified from Chen and Huang [40]).