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?"-

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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.