Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation.

Hepatocytes are dynamic cells that, upon injury, can alternate between nondividing differentiated and dedifferentiated proliferating states in vivo. However, in two-dimensional cultures, primary human hepatocytes (PHHs) rapidly dedifferentiate, resulting in loss of hepatic functions that significantly limits their usefulness as an in vitro model of liver biology, liver diseases, as well as drug metabolism and toxicity. Thus, understanding the underlying mechanisms and stalling of the dedifferentiation process would be highly beneficial to establish more-accurate and relevant long-term in vitro hepatocyte models. Here, we present comprehensive analyses of whole proteome and transcriptome dynamics during the initiation of dedifferentiation during the first 24 hours of culture. We report that early major rearrangements of the noncoding transcriptome, hallmarked by increased expression of small nucleolar RNAs, long noncoding RNAs, microRNAs (miRNAs), and ribosomal genes, precede most changes in coding genes during dedifferentiation of PHHs, and we speculated that these modulations could drive the hepatic dedifferentiation process. To functionally test this hypothesis, we globally inhibited the miRNA machinery using two established chemically distinct compounds, acriflavine and poly-l-lysine. These inhibition experiments resulted in a significantly impaired miRNA response and, most important, in a pronounced reduction in the down-regulation of hepatic genes with importance for liver function. Thus, we provide strong evidence for the importance of noncoding RNAs, in particular, miRNAs, in hepatic dedifferentiation, which can aid the development of more-efficient differentiation protocols for stem-cell-derived hepatocytes and broaden our understanding of the dynamic properties of hepatocytes with respect to liver regeneration. CONCLUSION: miRNAs are important drivers of hepatic dedifferentiation, and our results provide valuable information regarding the mechanisms behind liver regeneration and possibilities to inhibit dedifferentiation in vitro. (Hepatology 2016;64:1743-1756).

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease.

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

Furan in heat-treated foods: formation, exposure, toxicity, and aspects of risk assessment.

Furan is formed in a variety of heat-treated foods through thermal degradation of natural food constituents. Relatively high levels of furan contamination are found in ground roasted coffee, instant coffee, and processed baby foods. European exposure estimates suggest that mean dietary exposure to furan may be as high as 1.23 and 1.01 µg/kg bw/day for adults and 3- to 12-month-old infants, respectively. Furan is a potent hepatotoxin and hepatocarcinogen in rodents, causing hepatocellular adenomas and carcinomas in rats and mice, and high incidences of cholangiocarcinomas in rats at doses ≥ 2 mg/kg bw. There is therefore a relatively low margin of exposure between estimated human exposure and doses that cause a high tumor incidence in rodents. Since a genotoxic mode of action cannot be excluded for furan-induced tumor formation, the present exposures may indicate a risk to human health and need for mitigation. This review summarizes the current knowledge on mechanisms of furan formation in food, human dietary exposure to furan, and furan toxicity, and highlights the need to establish the risk resulting from the genotoxic and carcinogenic properties of furan at doses lower than 2 mg/kg bw.

Identification and pathway mapping of furan target proteins reveal mitochondrial energy production and redox regulation as critical targets of furan toxicity.

Furan, a heat-generated food contaminant, is hepatotoxic and carcinogenic in rodents. Furan is oxidized by cytochrome P450 2E1 to cis-2-butene-1,4-dial, a chemically reactive α,ß-unsaturated dialdehyde, which has been identified as the key toxic metabolite of furan based on its ability to interact with tissue nucleophiles. In addition to genotoxicity, sustained cytotoxicity mediated through covalent binding of cis-2-butene-1,4-dial to critical target proteins is thought to play a key role in furan carcinogenicity. To identify putative protein targets of reactive furan metabolites, male F344/N rats (n = 5 per dose) were administered a single dose of [3,4-(14)C]-furan (20 mCi/mmol) at doses associated with hepatotoxicity following long-term exposure (0.1 and 2 mg/kg body weight [bw]). Liver proteins were separated by two-dimensional gel electrophoresis and protein spots carrying radiolabel were located by fluorography. In total, 83 discrete protein spots containing (14)C were consistently detected in livers of animals given [3,4-(14)C]-furan at 2.0 mg/kg bw, accounting for 4-5% of the proteome covered by our analyses. Protein spots were excised and digested in gel with trypsin for identification by protein mass spectrometry. Protein database search and subsequent pathway mapping identified 61 proteins localized predominantly in the cytosol and mitochondria, including structural proteins, mitochondrial enzymes involved in glucose metabolism, mitochondrial ß-oxidation, and adenosine triphosphate synthesis, and proteins that participate in the maintenance of redox homeostasis and protein folding. Collectively, our data suggest that functional loss of several individual proteins and interference with pathways, most notably mitochondrial energy production, redox regulation, and protein folding, may combine to disrupt cell homeostasis and cause hepatocyte cell death.

Functional and proliferative effects of repeated low-dose oral administration of furan in rat liver.

SCOPE: Furan, a food contaminant formed during heat processing, induces hepatocellular tumors in rodents and high incidences of cholangiocarcinomas in rats even at the lowest dose (2 mg/kg b.w.) administered. Initial estimates suggested that human intake of furan may be as high as 3.5 µg/kg b.w./day, indicating a relatively narrow margin of exposure. The aim of this study was to establish dose-response data for cytotoxicity, regenerative cell proliferation and secondary oxidative DNA damage in livers of male F344 rats treated with furan at doses ≤2 mg/kg b.w. for 28 days. METHODS AND RESULTS: No significant signs of hepatotoxicity other than a mild, dose-dependent increase in serum cholesterol and unconjugated bile acids, and no evidence of oxidative DNA damage were seen. Histopathological alterations and proliferative changes were restricted to subcapsular areas of the left and caudate liver lobes. CONCLUSION: Although statistically significant effects were only seen at the 2 mg/kg b.w. dose during the course of our study, a ∼two and ∼threefold increase in 5-bromo-2'-deoxyuridine labeling index was observed at 0.1 and 0.5 mg/kg b.w., respectively, suggesting that chronic exposure to doses even below 2 mg/kg b.w. may cause proliferative changes in rat liver and highlighting the need to assess furan carcinogenicity at lower doses.