Mycelial biomass estimation and metabolic quotient of Lentinula edodes using species-specific qPCR.

Lentinula edodes, commonly known as shiitake, is an edible mushroom that is cultivated and consumed around the globe, especially in Asia. Monitoring mycelial growth inside a woody substrate is difficult, but it is essential for effective management of mushroom cultivation. Mycelial biomass also affects the rate of wood decomposition under natural conditions and must be known to determine the metabolic quotient, an important ecophysiological parameter of fungal growth. Therefore, developing a method to measure it inside a substrate would be very useful. In this study, as the first step in understanding species-specific rates of fungal decomposition of wood, we developed species-specific primers and qPCR procedures for L. edodes. We tested primer specificity using strains of L. edodes from Japan and Southeast Asia, as well as related species of fungi and plant species for cultivation of L. edodes, and generated a calibration curve for quantification of mycelial biomass in wood dust inoculated with L. edodes. The qPCR procedure we developed can specifically detect L. edodes and allowed us to quantify the increase in L. edodes biomass in wood dust substrate and calculate the metabolic quotient based on the mycelial biomass and respiration rate. Development of a species-specific method for biomass quantification will be useful for both estimation of mycelial biomass and determining the kinetics of fungal growth in decomposition processes

Preliminary Evaluation of Three-Dimensional Primary Human Hepatocyte Culture System for Assay of Drug-Metabolizing Enzyme-Inducing Potential

Drug-induced liver injury (DILI) is a common reason for withdrawal of candidate drugs from clinical trials, or of approved drugs from the market. DILI may be induced not only by intact parental drugs, but also by metabolites or intermediates, and therefore should be evaluated in the enzyme-induced state. Here, we present a protocol for assay of drug-metabolizing enzyme-inducing potential using three-dimensional (3D) primary cultures of human hepatocytes (hepatocyte spheroids). Hepatocyte spheroids could be used up to 21 d after seeding (pre-culture for 7 d and exposure to inducer for up to 14 d), based on preliminary evaluation of basal activities of CYP subtypes and mRNA expression of the corresponding transcription factor and xenobiotic receptors (aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR)). After 2 d exposure of hepatocyte spheroids to omeprazole, phenobarbital and rifampicin (typical inducers of CYP1A2, 2B6 and 3A4, respectively), CYP1A2, 2B6 and 3A4 mRNA expression levels were significantly increased. The mRNA induction of CYP2B6 remained reasonably stable between days 2 and 14 of exposure to inducers, while induction of both CYP1A2 and 3A4 continued to increase up to day 14. These enzyme activities were all significantly increased compared with the control until day 14. Our findings indicate that our 3D hepatocyte spheroids system would be especially suitable for long-term testing of enzyme activity induction by drugs, either to predict or to verify clinical events

Utility of human hepatocyte spheroids without feeder cells for evaluation of hepatotoxicity

We investigated the utility of three-dimensionally cultured hepatocytes (spheroids) without feeder cells (Sph(f-)) for the prediction of drug-induced liver injury (DILI) in humans. Sph(f-) and spheroids cultured on feeder cells (Sph(f+)) were exposed to the hepatotoxic drugs flutamide, diclofenac, isoniazid and chlorpromazine at various concentrations for 14 days, and albumin secretion and cumulative leakages of toxicity marker enzymes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (γ-GTP), were measured. The cumulative AST, LDH or γ-GTP leakages from Sph(f-) were similar to or greater than those from Sph(f+) for all drugs tested, although ALT leakages showed no consistent difference between Sph(f+) and Sph(f-). In the case of Sph(f-), significant correlations among all the toxicity markers except for γ-GTP were observed. As regards the drug concentrations causing 1.2-fold elevation of enzyme leakage (F1.2), no consistent difference between Sph(f+) and Sph(f-) was found, although several F1.2 values were undetermined, especially in Sph(f+). The IC50 of albumin secretion and F1.2 of AST leakage from Sph(f-) were equal to or lower than those of Sph(f+) for all the tested drugs. These results indicate that feeder cells might contribute to resistance to hepatotoxicity, suggesting DILI could be evaluated more accurately by using Sph(f-). We suggest that long-term exposure of Sph(f-) to drugs might be a versatile method to predict and reproduce clinical chronic toxicity, especially in response to repeated drug administration