Assessing Chemical-Induced Liver Injury In Vivo From In Vitro Gene Expression Data in the Rat: The Case of Thioacetamide Toxicity

Abstract

Consumers are exposed to thousands of chemicals with potentially adverse health effects. However, these chemicals will never be tested for toxicity because of the immense resources needed for animal-based (in vivo) toxicological studies. Today, there are no viable in vitro alternatives to these types of animal studies. To develop an in vitro approach, we investigated whether we could predict in vivo organ injuries in rats with the use of RNA-seq data acquired from tissues early in the development of toxicant-induced injury, by comparing gene expression data from RNA isolated from these rat tissues with those obtained from in vitro exposure of primary liver and kidney cells. We collected RNA-seq data from the liver and kidney tissues of Sprague-Dawley rats 8 or 24 h after exposing them to vehicle (control), low (25 mg/kg), or high (100 mg/kg) doses of thioacetamide, a known liver toxicant that promotes fibrosis; we used these doses and exposure times to cause only mild toxicant-induced injury. For the in vitro study, we treated two cell types from Sprague-Dawley rats, primary hepatocytes (vehicle; low, 0.025 mM; or high, 0.125 mM dose), and renal tube epithelial cells (vehicle; low, 0.125 mM; or high, 0.500 mM) dose) with the thioacetamide metabolite, thioacetamide-S-oxide, selecting in vitro doses and exposure times to recreate the early-stage toxicant-induced injury model that we achieved in vivo. RNA-seq data were collected 9 or 24 h after application of vehicle or thioacetamide-S-oxide. We found that our modular approach for the analysis of gene expression data derived from in vivo RNA-seq strongly correlated (R2 > 0.6) with the in vitro results at two different dose levels of thioacetamide/thioacetamide-S-oxide after 24 h of exposure. The top-ranked liver injury modules in vitro correctly identified the ensuing development of liver fibrosis.

Concordance between Thioacetamide-Induced Liver Injury in Rat and Human In Vitro Gene Expression Data

“…In our previous studies [19,25], we found that activation of the fibrogenesis module increased over time in the liver, an effect which we also observed here when comparing fibrogenesis module activation at 9 and 24 h in Table 1. The fibrogenesis module activation score would likely have continued to increase after 24 h, consistent with the observation that this phenotype takes longer than 24 h to become histologically visible.…”

“…Extensive efforts have been made to understand the frequently poor correspondence of experimental results between different species or between in vitro and in vivo studies [10,[29][30][31][32]. We have previously used our toxicogenomic approach to predict rat in vivo toxicity endpoints from rat in vitro RNA-seq data with good concordance [25]. A persisting challenge, however, has been to understand the often weak interspecies correlations.…”

“…To determine interspecies correlations, we compared the human in vitro results with results from our previous work on the effects of thioacetamide in cells from rats (in vivo) [19] and of thioacetamide-S-oxide in cultured rat cells (in vitro) [25]. Figure 2 presents an overview of the data collection we used for our analysis and all data are available in the supplementary files.…”

“…In both humans and rats, the numbers of DEGs in renal cells were less than those in hepatocytes, which suggests that the kidney was less affected by thioacetamide/thioacetamide-S-oxide toxicity ( Table 3). Some of the high numbers of DEGs in rat and human primary cells may be due to the higher dose used in the in vitro experiments as compared to the effective dose used in the rat in vivo studies [19,25]. Table 4 summarizes the correlations between the injury modules activated by exposing the liver or kidney to thioacetamide in rats or primary rat and human cells to thioacetamide-S-oxide, when comparing the human in vitro (human IVT) results with the rat in vitro (rat IVT) and in vivo (rat IVV) results (See Supplementary Material for all 24 graphs and tables).…”

“…Recently, we found that our modular approach could predict in vivo injury endpoints from in vitro RNA sequence (RNA-seq) data with a strong correlation (R 2 > 0.6) [25]. This study compared in vivo rat data with in vitro cellular data 24 h after treatment with thioacetamide-S-oxide, an intermediate metabolite of thioacetamide required to induce toxicity in vitro [24].…”