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MicroRNAs (miRNA) are short single-stranded RNA sequences that have a role in the post-transcriptional regulation of genes. The identification of tissue specific or enriched miRNAs has great potential as novel safety biomarkers. One longstanding goal is to associate the increase of miRNA in biofluids (e.g., plasma and urine) with tissue-specific damage. Next-generation sequencing (miR-seq) was used to analyze changes in miRNA profiles of tissue, plasma and urine samples of rats treated with either a nephrotoxicant (cisplatin) or one of two hepatotoxicants (acetaminophen [APAP] or carbon tetrachloride [CCL 4 ]). Analyses with traditional serum chemistry and histopathology confirmed that toxicant-induced organ damage was specific. In animals treated with cisplatin, levels of five miRNAs were significantly altered in the kidney, 14 in plasma and six in urine. In APAP-treated animals, five miRNAs were altered in the liver, 74 in plasma and six in urine; for CCL 4 the changes were five, 20 and 6, respectively. Cisplatin treatment caused an elevation of miR-378a in the urine, confirming the findings of other similar studies. There were 17 in common miRNAs elevated in the plasma after treatment with either APAP or CCL 4 . Four of these (miR-122, -802, -31a and -365) are known to be enriched in the livers of rats. Interestingly, the increase of serum miR-802 in both hepatotoxicant treatments was comparable to that of the well-known liver damage marker miR-122. Taken together, comparative analysis of urine and plasma miRNAs demonstrated their utility as biomarkers of organ injury. Copyright © 2016 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd., (Copyright © 2016 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.)

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of diagnoses ranging from simple fatty liver (SFL), to non-alcoholic steatohepatitis (NASH). This study aimed to determine the effect of moderate and severe NAFLD on hepatic transporter expression and function in vivo. Rats were fed a high-fat diet (SFL model) or a methionine-choline-deficient diet (NASH model) for eight weeks. Hepatic uptake transporter function was determined by bromosulfophthalein (BSP) disposition. Transporter expression was determined by branched DNA signal amplification assay and western blotting; inflammation was identified by immunostaining of liver slices for interleukin 1 beta (IL-1beta). MC- rats showed significant retention of BSP in the plasma when compared to control rats. Hepatic NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 mRNA levels were significantly decreased in high-fat and MC- diet rats when compared to control. Protein expression of OATP1a1 was significantly decreased in high-fat animals, while OATP1a1 and OATP1b2 expressions were significantly lower in MC- rats when compared to control. Liver tissue from high-fat and MC- rats stained positive for IL-1beta, a pro-inflammatory cytokine known to decrease expression of NTCP, OATP and OAT transporters, suggesting a plausible mechanism for the observed transporter alterations. These data suggest that different stages of NAFLD result in altered hepatic uptake transporter expression that can lead to a functional impairment of xenobiotic uptake from the blood. Furthermore, NAFLD may alter the plasma retention time of clinically relevant drugs that are reliant on these transporters and may increase the potential drug toxicity.

Garlic oil (GO) contains several linear sulfur compounds, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), that induce drug-metabolizing enzymes such as CYP2B and NAD(P)H quinone oxidoreductase 1 (NQO1). CYP2B and NQO1 are primarily regulated by constitutive androstane receptor (CAR) and nuclear factor E2-related factor 2 (Nrf2) transcription factors, respectively. The purpose of this study was to determine whether GO and its specific constituents induce these two enzymes via CAR and Nrf2 activation. Female Wistar-Kyoto (WKY) rats express little CAR protein and exhibit less induction of CYP2B1/2 than males. GO, DAS, and DADS, but not DATS, induced CYP2B1/2 mRNA levels to a greater extent in WKY males than in females, suggesting CAR activation. Conversely, DAS induced NQO1 levels equally in WKY males and females, indicating CAR-independent induction in rats. DAS, but not GO, DADS, or DATS, induced CYP2B10 mRNA levels 530-fold in wild-type (WT) mice, whereas this induction was attenuated in CAR(-/-) mice. DAS induced NQO1 in WT and CAR(-/-) mice equally, suggesting CAR-independent induction in mice. DAS induced NQO1 5-fold in WT mice, whereas induction was completely absent in Nrf2(-/-) mice, indicating DAS also activates Nrf2. DAS induction of CYP2B10 mRNA was independent of Nrf2 presence or absence. In in vivo transcription assays, DAS activated the human CYP2B6 promoter, and the antioxidant response element of the human NQO1 promoter, respectively. These studies indicate that GO constituents, particularly DAS, activate CAR and Nrf2 to induce drug-metabolizing enzymes.

trans-Stilbene oxide (TSO) is a synthetic proestrogen that induces phase I and II drug-metabolizing enzymes in rat liver. The purpose of this study was to determine whether TSO also induces transporter expression in rat liver and whether gene induction in rat liver after TSO occurs in a constitutive androstane receptor (CAR)-dependent manner. Total RNA was isolated from male rat livers after treatment with TSO for up to 4 days (200 mg/kg, i.p., twice daily), and the mRNA levels for each gene were quantified. CYP2B1/2, CYP3A1, epoxide hydrolase, heme oxygenase-1, UGT1A6, UGT2B1, multiple drug resistance protein (Mdr) 1a and 1b, as well as multidrug resistance-associated protein (Mrp) 2, 3, and 4 mRNA were increased in livers after TSO treatment. To determine whether TSO activates gene expression in a CAR-dependent manner, male and female Wistar-Kyoto (WKY) rats were treated with TSO for 3 days. TSO induced CYP2B1/2, UGT2B1, and Mdr1b in males more than in females, suggesting that TSO could increase their expression via CAR. Conversely, TSO induced CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 similarly in both genders, indicating that induction of these genes occurs independently of CAR. TSO treatment also increased the activity of a CAR binding element luciferase reporter construct in HepG2 cells transfected with rat CAR and in mouse liver. Additionally, TSO increased antioxidant response element/electrophile response element luciferase reporter construct activity in HepG2 cells. In conclusion, in WKY rat liver, TSO increases CYP2B1/2, UGT2B1, and Mdr1b mRNA expression in a gender-dependent manner and CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 in a gender-independent manner.