Time-matched analysis of DNA adduct formation and early gene expression as predictive tool for renal carcinogenesis in methylazoxymethanol acetate treated Eker rats

Abstract

Genotoxic carcinogens pose great hazard to human health. Uncertainty of current risk assessment strategies and long latency periods between first carcinogen exposure and diagnosis of tumors have raised interest in predictive biomarkers. Initial DNA adduct formation is a necessary step for genotoxin induced carcinogenesis. However, as DNA adducts not always translate into tumorigenesis, their predictive value is limited. Here we hypothesize that the combined analysis of pro-mutagenic DNA adducts along with time-matched gene expression changes could serve as a superior prediction tool for genotoxic carcinogenesis. Eker rats, heterozygous for the tuberous sclerosis (Tsc2) tumor suppressor gene and thus highly susceptible towards genotoxic renal carcinogens, were continuously treated with the DNA alkylating carcinogen methylazoxymethanol acetate (MAMAc). Two weeks of MAMAc treatment resulted in a time-dependent increase of O⁶-methylguanine and N7-methylguanine adducts in the kidney cortex, which was however not reflected by significant expression changes of cyto-protective genes involved in DNA repair, cell cycle arrest or apoptosis. Instead, we found a transcriptional regulation of genes involved in the tumor-related MAPK, FoxO and TGF-beta pathways. Continuous MAMAc treatment for up to 6 months resulted in a mild but significant increase of cancerous lesions. In summary, the combined analysis of DNA adducts and early gene expression changes could serve as a suitable predictive tool for genotoxicant-induced carcinogenesis.

Keywords: Biomarkers of effect; Biomarkers of exposure; DNA adducts; Eker rat; Kidney cancer; Methylazoxymethanol acetate.

Figure 1

Acute (a) or chronic (b) MAMAc dosing regimen in Eker rats and follow-up analyses on gene expression, DNA adduct formation, renal pathology and cell proliferation. Body weights of male (c) and female (d) Eker rats before and after 6 months of MAMAc or vehicle treatment. Data represent means ± SEM. Student's t-tests comparing body weights at either day 0 or day 180 revealed no significant differences between MAMAc and vehicle treated groups.

Figure 2

Numbers of O⁶-methyl-2′-deoxyguanosine (O⁶-me-dG) (a) and N7-methylguanine (N7MG) (b) adducts per 10⁶ deoxyguanosine (dG), detected via LC-MS/MS in DNA of male and female Eker rats after 1, 3 and 14 days of MAMAc treatment (combined DNA pooled from renal cortices of three replicate animals).

Figure 3

(a) Heatmap showing significant gene expression changes in the kidney cortex after 1, 3, 7 or 14 days of MAMAc exposure (n = 3 per time point). Blue: down-regulated; red: up-regulated, white: no regulation compared to the vehicle treated controls. (b) STRING interaction network of proteins encoded by genes associated with cancer-related pathways. (c) Selected KEGG pathways sorted by functional classification. Total numbers of genes per pathway are shown as horizontal bars, the significance level is indicated by the color scheme ranging from red (p < 0.01) to blue (p < 0.16). (d) Boxplots indicating microarray log expression levels of DNA repair gene Mgmt in rats treated for 1, 3, 7 and 14 days with vehicle (controls), MAMAc or the positive control aristolochic acid (AA). (e) Quantitative PCR-based Mgmt expression normalized to the housekeeping gene Hprt in male and female Eker rats treated for 14 days with MAMAc or vehicle (n = 3). Data represent means ± SEM. One-way ANOVA with Tukey's post-hoc test for multiple comparisons revealed no significant effects of MAMAc or sex on Mgmt expression levels.

Figure 4

(a) Basophilic atypical tubule (bAT), (b) basophilic atypical hyperplasia (bAH) and (c) invasive carcinoma in H&E stained renal sections of Eker rats. Total numbers of all pre-cancerous and cancerous lesions in male and female Eker rats after 3 months (d) or 6 months (e) of MAMAc exposure. Zonal occurrence of pre-neoplastic lesions (bATs and bAHs) in the renal cortex (f, g) or medulla / papilla (h, i) of male and female Eker rats following 3 months (f, h) or 6 months (g, i) of MAMAc exposure. Data represent means ± SEM. N = 10 per group in 3 and 6 months MAMAc and 3 months vehicle treated control rats. N = 9 for the female 6 months treated control group.

Figure 5

(a-c) Assessment of MAMAc effects on proliferation via (a) proliferating-cell-nuclear-antigen (PCNA) S-phase labeling indices (LI %) in the renal cortex of 14-days MAMAc treated male and female Eker rats (n=3), or via (b, c) BrdU S-phase labeling indices of male and female Eker rats treated with MAMAc for 3 (b) and 6 months (c), respectively (n=5). (d) Pearson correlation analysis of BrdU labeling indices versus the total number of renal lesions determined by renal histopathology in rats (N = 5 per group), either 3 months (white) or 6 months (black) treated with vehicle (circles), MAMAc (squares), ochratoxin A (OTA, diamonds), and aristolochic acid (AA, triangles). Data represent means ± SEM. One-way ANOVA with Tukey's post-hoc test (a-c) was used to test for statistical significance.