Investigation of a Hepatotoxicity Screening System in Primary Cell Cultures-"What Biomarkers Would Need to Be Addressed to Estimate Toxicity in Conventional and New Approaches?"-

Abstract

-High throughput toxicological estimation is required for safety evaluation in the early stage of drug discovery. In this context, establishment of an in vitro screening system reflecting in vivo toxicity is demanded for earlier safety assessment. We investigated LDH release and mitochondrial respiration (WST-1 reduction assay; WST-1) to detect cytotoxicity, morphological evaluation, and proteomics for estimating the reliable and sensitive biomarkers by using rat primary hepatocytes exposed to the compounds (acetaminophen, amiodarone, tetracycline and carbon tetrachloride) that are known to induce hepatotoxicity. In LDH release, no significant difference was detected between the control and compound exposed cells after exposure for 3 or 6 hr, but a dose-dependent increase was observed after exposure for 24 hr. Regarding the WST-1 assay, a dose-dependent reduction was detected after exposure for 6 and 24 hr to all of the compounds evaluated. In the proteomics analysis, 31 candidate proteins were identified from among the 103 demonstrating altered expression spots after exposure to acetaminophen. It was concluded that the cytotoxicity was detected earlier by measuring WST-1 than by measuring LDH release because the reduction of mitochondrial respiration is an expressions of earlier toxicity for cellular function, while the measured increase in the LDH release occurs after the failure of the cell membrane. Mitochondrial respiration ability was a useful parameter for cytotoxicity in in vitro hepato-toxicity screening, as cytotoxicity can be detected during the early stage of exposure. In addition to the conventional biomarkers, several protein biomarkers which relate to oxidative stress and metabolism-regulation were detected. Further comprehensive analysis of defined proteins would be necessary to estimate the more sensitive toxicology biomarker.

Identification of Oxidative Stress-Related Proteins for Predictive Screening of Hepatotoxicity Using a Proteomic Approach

“…The expression of glutathione peroxidase (P04041, GPX) was significantly decreased by more than 2-fold after treatment with APAP and AD for 24 hr (Fig. 1), whose expression change by APAP exposure was also detected in our previous study reported (Kikkawa et al, 2005). GPX is an antioxidative enzyme that scavenges various peroxides and prevents apoptotic cell death (Hockenbery et al, 1993;Fujii and Taniguchi, 1999).…”

“…Based on the results of our previous study, the dose levels and exposure term were determined as a high dose (over the EC 50 value) with the exposures of 6 and 24 hr, which would be expected to cause the toxicities concerned (Kikkawa et al, 2005). In terms of the usability of primary cultures of rat hepatocytes, the advantages have been reported in comparison with a cell line screening system such as HepG2 (Fry et al, 1990;Paillard et al, 1999;Wang et al, 2002).…”

“…Concentrations of the test compounds (APAP: 10 mM, AD: 50 µM, TC: 500 µM) were determined based on our previous cytotoxicity study in primary rat hepatocytes (Kikkawa et al, 2005). Protein expression was examined by two-dimensional gel electrophoresis (2DE), and proteins that expressed differentially in control and treated cells were identified by mass spectrometric analysis following In-gel tryptic digestion.…”

“…Primary hepatocytes were isolated from SpragueDawley male rats (Charles River Japan, Yokohama, Japan) using the two-step collagenase perfusion method (Seglen, 1976;Kikkawa et al, 2005). Rat hepatocytes were cultured in Williams' E culture medium (WEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, Invitrogen) buffer, pH7.4, 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen), and insulin-transferring-selenium-A supplement (Invitrogen).…”

“…After pre-incubation, the medium was replaced with a medium containing test compounds, and hepatocytes cultured in 6-well plates were exposed to test compounds for 6 and 24 hr. Based on the results of our previous cytotoxicity study (Kikkawa et al, 2005), the concentrations of the test compounds were set at 10 mM, 50 µM and 500 µM for APAP, AD and TC, respectively. At the end of the exposure periods, hepatocytes were harvested using a clean cell scraper and recovered into 100 mM HEPES buffer.…”