Validation of an in vitro model for assessment of androstenedione hepatotoxicity using the rat liver cell line clone‐9

Abstract

Androstenedione, a naturally occurring steroid hormone, has been used to enhance athletic performance. Little is known, however, about its hepatotoxicity. Clone-9 cells, a non-transformed epithelial cell line that was originally isolated from normal liver of a 4-week old Sprague-Dawley rat, were used as an in vitro model to assess the hepatotoxic potential of androstenedione. The cultures were treated with androstenedione for 24 h at 37 degrees C in 5% CO(2) at concentrations of 0-100 microg ml(-1). After the treatment period, the cells and the culture supernatants were assayed for markers of cytotoxicity which included: release of liver enzymes, cell viability, cellular double-stranded DNA content, oxidative stress, steatosis, cellular ATP content, caspase-3 activity, the mitochondrial permeability transition and induction of cytochrome P450 activity. Significant concentration-dependent differences from control were observed in some endpoints at medium concentrations of 10 microg ml(-1) and above. These in vitro findings were compared with comparable endpoints obtained from an in vivo study of androstenedione toxicity in female Sprague-Dawley rats. Of the eight endpoints that could be compared between the two studies, only three (lipid accumulation, ATP depletion and P450 activity) appeared to be concordant. This suggests that, under the experimental conditions used, the clone-9 cells were not a good model for androstenedione hepatotoxicity.

Effects of Synthetic Androgens on Liver Function Using the Rabbit as a Model

“…In some instances, investigators have been able to distinguish between hepatotoxic and nonhepatotoxic compounds with selected endpoints, as was determined for 17α‐alkylated steroids compared with nonalkylated steroids (Welder et al, 1995). In other cases, results from the rat liver–derived clone‐9 cells did not completely agree with data obtained from an in vivo rat model (Sahu et al, 2008). In this direct comparison, only 3 of the 8 endpoints evaluated were concordant, suggesting that the in vitro effects were not necessarily predictive of in vivo effects.…”

“…Development of model systems to detect hepatotoxicity of test compounds, particularly early in drug development, is desirable. Toward this end, investigators have been developing in vitro models using either primary liver cells or liver‐derived cell lines (Welder et al, 1995; Li et al, 1999; Li, 2001; Josse et al, 2008; Sahu et al, 2008). In some instances, investigators have been able to distinguish between hepatotoxic and nonhepatotoxic compounds with selected endpoints, as was determined for 17α‐alkylated steroids compared with nonalkylated steroids (Welder et al, 1995).…”