Dose-dependent effects on rat liver miRNAs 200a/b and 429: potential early biomarkers of liver carcinogenesis

Abstract

An increased incidence of liver tumours in the long term rodent bioassay is not an uncommon finding, invariably as a result of a non-genotoxic mode of action. Non-genotoxic liver carcinogenesis has been found to involve activation of certain nuclear hormone receptors (NHR) including the constitutive androstane receptor (CAR), peroxisome proliferator activated receptor alpha (PPARalpha) and arylhydrocarbon receptor (AHR) and more recently the induction of specific microRNAs (miRs), has also been demonstrated following CAR activation in studies up to 90 days (Koufaris et al., 2012). The stable induction of these tissue specific miRs, namely miR200a, 200b and 429, by liver non-genotoxic carcinogens may serve as early predictors (biomarkers) of heptocarcinogenic potential. To test this hypothesis we used RT-PCR to measure the levels of these miRs in the livers from Wistar rats treated with two rat hepatocarcinogenic and one non hepatocarcinogenic pyrazole carboxamide succinate dehydrogenase inhibitors, Isopyrazam, Sedaxane and Benzovindiflupyr, respectively. The miRs were quantified by RT-PCR in liver RNA samples from three 90 day repeat dose toxicity studies performed at the low, mid and high doses relative to control. In Isopyrazam treated rats a statistically significant (p < 0.01) dose-dependent increase in miR 200a, 220b and 429 in both males and females was observed, whilst for Sedaxane a significant (p < 0.05) increase in miR200b in males and females at the high dose was seen. Benzovindiflupyr treatment did not cause any dose related changes in miR 200a, 200b and 429 relative to control. Our results suggest that assessment of miR 200a/200b/429 levels has potential as a biomarker of the perturbation of pathways involved in hepatocarcinogenesis in Wistar rats. Further work is required to establish the possible relationship between miR200 cluster induction and CAR-mediated hepatocarcinogenesis in a more diverse range of compounds.

Keywords: Biomarker; Hepatocarcinogesis; Liver; microRNA.

Fig. 1

Relative Expression of MiR 200a, 200b, 429 in Liver of Rats Treated for 90 days with Isopyrazam. Relative expression of miR 200a, 200b and 429 in liver (left lobe) of rats treated (dietary) for 90 days with Isopyrazam (low dose: 300 ppm; mid dose: 1500 ppm; high dose: 6000 ppm). Results are mean (n = 5) ± SD fold changes relative to sex matched controls. *, ** significantly different from control by Student’s T test, p < 0.05 or p < 0.01, respectively. NB: Data are normalised to the respective sex matched controls in order to aid visual comparison of the data. Dashed red line shows control value = 1.

Fig. 2

Relative Expression of MiR 200a, 200b, 429 in Liver of Rats Treated for 90 days with Sedaxane. Relative expression of miR 200a, 200b and 429 in liver (left lobe) of rats treated (dietary) for 90 days with Sedaxane (low dose: 300 ppm; mid dose: 2000 ppm; high dose: 4000 ppm). Results are mean (n = 5) ± SD fold changes relative to sex matched controls. *, ** significantly different from control by Student’s T test, p < 0.05 or p < 0.01, respectively. NB: Data are normalised to the respective sex matched controls in order to aid visual comparison of the data. Dashed red line shows control value = 1.

Fig. 3

Relative Expression of MiR 200a, 200b, 429 in Liver of Rats Treated for 90 days with Benzovindiflupyr. Relative expression of miR 200a, 200b and 429 in liver (left lobe) of rats treated (dietary) for 90 days with Benzovindiflupyr (low dose: 100 ppm; med dose: 750 ppm; high dose; 1500 ppm). Results are mean (n = 5) ± SD fold changes relative to sex matched controls. *significantly different from control by Student’s T test, p < 0.05. NB: Data are normalised to the respective sex matched controls in order to aid visual comparison of the data. Dashed red line shows control value = 1.