MiR-766-3p and miR-671-5p attenuate aristolochic acid-induced hepatotoxicity by directly targeting the key bioactivating enzyme NQO1.

Aristolochic acid (AA) as an emerging contaminant in herbal medicines or crops has been well-recognized for causing nephropathy since 1990s. Over the last decade, mounting evidence has linked AA to liver injury; however, the underlying mechanism is poorly elucidated. MicroRNAs respond to environmental stress and mediate multiple biological processes, thus showing biomarker potentials prognostically or diagnostically. In the present study, we investigated the role of miRNAs in AA-induced hepatotoxicity, specifically in regulating NQO1, the key enzyme responsible for AA bioactivation. In silico analysis showed that hsa-miR-766-3p and hsa-miR-671-5p were significantly associated with AAI exposure as well as NQO1 induction. A 28-day rat experiment of 20 mg/kg AA exposure demonstrated a 3-fold increase of NQO1 and an almost 50 % decrease of the homologous miR-671 that were accompanied with liver injury, which was consistent with in silico prediction. Further mechanistic investigation using Huh7 cells with IC50 of AAI at 146.5 µM showed both hsa-miR-766-3p and hsa-miR-671-5p were able to directly bind to and down-regulate NQO1 basal expression. In addition, both miRNAs were shown to suppress AAI-induced NQO1 upregulation in Huh7 cells at a cytotoxic concentration of 70 µM, and consequently alleviate AAI-induced cellular effects, including cytotoxicity and oxidative stress. Together, these data illustrate that miR-766-3p and miR-671-5p attenuate AAI-induced hepatotoxicity, and thus have monitoring and diagnostic potentials.

The path via pathway-based approaches towards safety assessment: A concise review.

For decades, chemical safety assessment has been proposed to shift from animal testing to in vitro testing systems in response to the call for the 3R. In Europe, the answer was to combine various information sources in integrated testing strategies (ITS); In the US, it was in 2007 when the landmark report by the National Research Council put forward a vision of in vitro toxicity testing paradigm. Since then, efforts to develop pathway-based assessment framework have been on the track. In 2010, systems biology brought out a conceptual framework called adverse outcome pathway (AOP), which took one step further from toxicity pathway to regulatory toxicology. Computational modeling, high-throughput screening, high-content omics have all been approached to facilitate this progress. This paper briefly reviewed the achievement of pathway-based chemical assessment since 2007, discussed potential pitfalls and challenges that mechanism-driven chemical assessment may undergo, and presented future perspectives of safety assessment that is to be based on computational system biology.

PIWI-interacting RNA-23210 protects against acetaminophen-induced liver injury by targeting HNF1A and HNF4A.

Acetaminophen (APAP) overdose is one of the leading causes of acute liver failure in the US and other developed countries, the molecular mechanisms of APAP-induced hepatotoxicity remain speculative. PIWI-interacting RNAs (piRNAs), a novel class of small non-coding RNAs, have been identified as epigenetic regulators of transposon silencing, mRNA deadenylation, and elimination. However, the functional role of piRNAs in APAP-induced liver injury remains unclear. In the current study, the piRNA profiles were constructed in HepaRG cells after APAP exposure, and the roles of piR-23210 in regulating nuclear receptors (NRs) expression, metabolizing enzymes expression, and consequently APAP-induced liver injury were systematically investigated. As a result, 57 upregulated piRNAs were identified after APAP exposure, indicating the stress-response characteristic of piRNA molecules. Subsequent in vitro and in vivo experiments proved that piR-23210 is a novel self-protective molecule that targets HNF1A and HNF4A transcripts by interacting with RNA binding protein Nucleolin (NCL), suppresses downstream CYPs (CYP2E1, CYP3A4, and CYP1A2) expression, and protects against APAP-induced liver injury. In conclusion, our findings provided new mechanistic clues revealing potential protective role of a piRNA against the hepatoxicity of APAP.

High throughput data-based, toxicity pathway-oriented development of a quantitative adverse outcome pathway network linking AHR activation to lung damages.

The quantitative adverse outcome pathway (qAOP) is proposed to inform dose-responses at multiple biological levels for the purpose of toxicity prediction. So far, qAOP models concerning human health are scarce. Previously, we proposed 5 key molecular pathways that led aryl hydrogen receptor (AHR) activation to lung damages. The present study assembled an AOP network based on the gene expression signatures of these toxicity pathways, and validated the network using publicly available high throughput data combined with machine learning models. In addition, the AOP network was quantitatively evaluated with omics approaches and bioassays, using 16HBE-CYP1A1 cells exposed to benzo(a)pyrene (BaP), a prototypical AHR activator. Benchmark dose (BMD) analysis of transcriptomics revealed that AHR gene held the lowest BMD value, whereas AHR pathway held the lowest point of departure (PoD) compared to the other 4 pathways. Targeted bioassays were further performed to quantitatively understand the cellular responses, including ROS generation, DNA damage, interleukin-6 production, and extracellular matrix increase marked by collagen expression. Eventually, response-response relationships were plotted using nonlinear model fitting. The present study developed a highly reliable AOP model concerning human health, and validated as well as quantitatively evaluated it, and such a method is likely to be adoptable for risk assessment.

A toxicity pathway-oriented approach to develop adverse outcome pathway: AHR activation as a case study.

With numerous new chemicals introduced into the environment everyday, identification of their potential hazards to the environment and human health is a considerable challenge. Developing adverse outcome pathway (AOP) framework is promising in helping to achieve this goal as it can bring In Vitro testing into toxicity measurement and understanding. To explore the toxic mechanism underlying environmental chemicals via the AOP approach, an integration of adequate experimental data with systems biology understanding is preferred. Here, we describe a novel method to develop reliable and sensible AOPs that relies on chemical-gene interactions, toxicity pathways, molecular regulations, phenotypes, and outcomes information obtained from comparative toxicogenomics database (CTD) and Ingenuity Pathway Analysis (IPA). Using Benzo(a)pyrene (BaP), a highly studied chemical as a stressor, we identified the pivotal IPA toxicity pathways, the molecular initiating event (MIE), and candidate key events (KEs) to structure AOPs in the liver and lung, respectively. Further, we used the corresponding CTD information of multiple typical AHR-ligands, including 2,3,7,8-tetrachlorodibenzoparadioxin (TCDD), valproic acid, quercetin, and particulate matter, to validate our AOP networks. Our approach is likely to speed up AOP development as providing a time- and cost-efficient way to collect all fragmented bioinformation in published studies. It also facilitates a better understanding of the toxic mechanism of environmental chemicals, and potentially brings new insights into the screening of critical paths in the AOP network.