Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity

Biliary Secretion of Glutathione in Estradiol 17␤-D-Glucuronide- Induced Cholestasis

ABSTRACT Estradiol-17␤-D-glucuronide (E2-17G) induces an acute but reversible inhibition of bile flow after its intravenous administration to rats, due in part to the endocytic retrieval of the canalicular multidrug resistance-associated transporter protein 2 and the bile salt export pump, transporters that contribute to bile flow. Decreased bile salt-independent bile flow (BSIF) is also involved and persists during the phase of recovery from cholestasis. Because glutathione and HCO 3 Ϫ are major contributors to BSIF, we evaluated changes in their biliary excretion and the hepatic content of total glutathione during E2-17G-induced cholestasis. E2-17G acutely decreased bile flow and biliary excretion of total glutathione by about 80%; glutathione excretion was still inhibited at 80 min and 120 min, even though bile flow was partially and totally restored, respectively. Neither liver glutathione content nor the proportions of oxidized glutathione in bile and liver were affected by E2-17G at any time. HCO 3 Ϫ concentrations in bile were unchanged, so that secretion paralleled variations in bile flow. In the isolated perfused liver, addition of E2-17G decreased both bile flow and the biliary concentration of glutathione, whereas addition of its noncholestatic isomer estradiol-3-D-glucuronide (E2-3G) did not inhibit bile flow, but significantly reduced the concentration of glutathione in bile. The bile:liver concentration ratios of glutathione were significantly decreased in vivo by E2-17G and in the perfused liver by E2-17G and E2-3G. These data indicate that E2-17G cis-inhibits the canalicular transport of glutathione and thus contributes to the cholestatic effect by inhibiting BSIF

Descriptions of selected candidate reference genes and genes of interest.

<p>Descriptions of selected candidate reference genes and genes of interest.</p

Workflow for reference gene selection.

<p>All the steps (S+number) followed for reference gene selection in this report are shown. The strategy (S) applied and the tools (T) used in each step are mentioned in each corresponding panel.</p

Effect of rat exposure to hepatotoxins on liver expressions of candidate reference genes.

<p>The expression of candidate reference genes for microRNA and mRNA normalization in the liver of rats treated with the hepatotoxins and their respective vehicles are shown. The livers were evaluated 24 h after rats were intraperitoneally administered with: acetaminophen (AA, 1.2 g/kg body weight) or its vehicle (1% carboxymethyl cellulose, 10 ml/kg body weight); carbon tetrachloride (CT, 1 ml/kg body weight) or its vehicle (corn oil, 4.4 ml/kg body weight), D-galactosamine (GA, 0.9 g/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight); thioacetamide (TA, 150 mg/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight). The bars represent the means of the relative quantity of transcript ± SD of 5 animals treated with hepatotoxins or their respective vehicle. An asterisk (*) indicates a significant expression difference with p<0.05 using Student's t-test of the log-transformed data between the control and treated groups.</p

Descriptions of gene-specific real-time PCR assays.

<p>Am: amplicon size. bp: numer of base pairs. E: Assay efficiency. Tm: melting temperature. Ref: references. RT: retro-transcription primer. R: Reverse primer. F: Forward primer. a: Primers were designed using Primer3 software.</p

Boxplot of RT-qPCR quantification cycles values of candidate reference genes.

<p>Boxplot of quantification cycles (Cq) values for each reference gene for microRNA and mRNA normalization in all liver samples assessed (n = 35) which belong to rats treated with each one of the four hepatotoxins studied and their respective control animals administered with vehicle. A line across the box depicts the median. The box indicates the 25% and 75% percentiles. Whiskers represent the maximum and minimum values, circles represent outliers. The livers were evaluated 24 h after rats were intraperitoneally administered with: acetaminophen (1.2 g/kg body weight) or its vehicle (1% carboxymethyl cellulose, 10 ml/kg body weight); carbon tetrachloride (1 ml/kg body weight) or its vehicle (corn oil, 4.4 ml/kg body weight), D-galactosamine (0.9 g/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight); thioacetamide (150 mg/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight).</p

Ranking of candidate reference genes according to their stability value.

<p>Each column refers to a gene stability ranking computed by one statistical tool, using all gene expression values measured for each candidate reference gene. The stability measurements produced by geNorm, NormFinder and BestKeeper were combined to establish a consensus rank of the genes applying the RankAggreg package.</p

Liver damage assessment in rat models of acute hepatotoxicity.

<p>Plasma alanine aminotransferase (ALT) levels and histological examination of the livers from rats after 24 h of intraperitoneal administration of acetaminophen (panel A), carbon tetrachloride (panel B), D-galactosamine (panel C) and thioacetamide (panel D) are shown. The change in the plasma ALT levels in response to increasing doses of each hepatotoxin was tested by a one-way ANOVA followed by the Newman–Keuls test for multiple comparisons. An asterisk (*) indicates a significant difference (p<0.05). Representative histological microphotographs of hematoxylin and eosin stained-liver sections after the administration of highest doses of the hepatotoxins and their respective vehicles (big and small image of each panel, respectively) are shown. The rats were intraperitoneally administered with: acetaminophen (1.2 g/kg body weight) or its vehicle (1% carboxymethyl cellulose, 10 ml/kg body weight); carbon tetrachloride (1 ml/kg body weight) or its vehicle (corn oil, 4.4 ml/kg body weight), D-galactosamine (0.9 g/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight); thioacetamide (150 mg/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight).</p