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Assessing Concordance of Drug-Induced Transcriptional Response in Rodent Liver and Cultured Hepatocytes.

Sutherland JJ, Jolly RA, Goldstein KM, Stevens JL - PLoS Comput. Biol. (2016)

Bottom Line: We examined the concordance of compound-induced transcriptional changes using data from several sources: rat liver and rat primary hepatocytes (RPH) from Drug Matrix (DM) and open TG-GATEs (TG), human primary hepatocytes (HPH) from TG, and mouse liver/HepG2 results from the Gene Expression Omnibus (GEO) repository.Co-expression networks performed better than genes or GSA when comparing treatment effects within rat liver and rat vs. mouse liver.We observe that the baseline state of untreated cultured cells relative to untreated rat liver shows striking similarity with toxicant-exposed cells in vivo, indicating that gross systems level perturbation in the underlying networks in culture may contribute to the low concordance.

View Article: PubMed Central - PubMed

Affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, United States of America.

ABSTRACT
The effect of drugs, disease and other perturbations on mRNA levels are studied using gene expression microarrays or RNA-seq, with the goal of understanding molecular effects arising from the perturbation. Previous comparisons of reproducibility across laboratories have been limited in scale and focused on a single model. The use of model systems, such as cultured primary cells or cancer cell lines, assumes that mechanistic insights derived from the models would have been observed via in vivo studies. We examined the concordance of compound-induced transcriptional changes using data from several sources: rat liver and rat primary hepatocytes (RPH) from Drug Matrix (DM) and open TG-GATEs (TG), human primary hepatocytes (HPH) from TG, and mouse liver/HepG2 results from the Gene Expression Omnibus (GEO) repository. Gene expression changes for treatments were normalized to controls and analyzed with three methods: 1) gene level for 9071 high expression genes in rat liver, 2) gene set analysis (GSA) using canonical pathways and gene ontology sets, 3) weighted gene co-expression network analysis (WGCNA). Co-expression networks performed better than genes or GSA when comparing treatment effects within rat liver and rat vs. mouse liver. Genes and modules performed similarly at Connectivity Map-style analyses, where success at identifying similar treatments among a collection of reference profiles is the goal. Comparisons between rat liver and RPH, and those between RPH, HPH and HepG2 cells reveal lower concordance for all methods. We observe that the baseline state of untreated cultured cells relative to untreated rat liver shows striking similarity with toxicant-exposed cells in vivo, indicating that gross systems level perturbation in the underlying networks in culture may contribute to the low concordance.

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Transcriptional effects of cell culture in the context of rat liver perturbations achieved with drug treatment.A) Distribution of average absolute eigengene scores for 4182 rat liver drug treatments from DM and TG, and 6 baseline expression comparisons of control samples (shown in red text in the inset). The solid and dashed vertical lines denote the median and 90th percentile values for avg. abs. EG. Rat liver histology sections: B) Unaffected liver. A central vein (asterisk) and small portal triad (arrowhead) are marked. Mild hepatocellular glycogen (recognized by cytoplasmic pallor) is apparent in the periportal region. C) Liver histology 1 day after the last dose of methapyrilene administered at 100 mg/kg for 29 days. Biliary hyperplasia (to the left of the arrows), correlating with increased alkaline phosphatase (ALP) and γ-glutamyltransferase (GGT) activities (S7 Table), and hepatocellular apoptosis/single cell necrosis (arrowheads) are noted. Hepatocytes in the right side of the image are hypertrophied and exhibit prominent anisonucleosis. D) Liver histology 1 day after bortezomib administration at 1 mg/kg. Acute hepatocellular necrosis (left of the arrows) and mild hepatocellular vacuolation (arrowheads) are noted. Images were extracted from the Open TG-GATES website (http://toxico.nibiohn.go.jp/english/).
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pcbi.1004847.g005: Transcriptional effects of cell culture in the context of rat liver perturbations achieved with drug treatment.A) Distribution of average absolute eigengene scores for 4182 rat liver drug treatments from DM and TG, and 6 baseline expression comparisons of control samples (shown in red text in the inset). The solid and dashed vertical lines denote the median and 90th percentile values for avg. abs. EG. Rat liver histology sections: B) Unaffected liver. A central vein (asterisk) and small portal triad (arrowhead) are marked. Mild hepatocellular glycogen (recognized by cytoplasmic pallor) is apparent in the periportal region. C) Liver histology 1 day after the last dose of methapyrilene administered at 100 mg/kg for 29 days. Biliary hyperplasia (to the left of the arrows), correlating with increased alkaline phosphatase (ALP) and γ-glutamyltransferase (GGT) activities (S7 Table), and hepatocellular apoptosis/single cell necrosis (arrowheads) are noted. Hepatocytes in the right side of the image are hypertrophied and exhibit prominent anisonucleosis. D) Liver histology 1 day after bortezomib administration at 1 mg/kg. Acute hepatocellular necrosis (left of the arrows) and mild hepatocellular vacuolation (arrowheads) are noted. Images were extracted from the Open TG-GATES website (http://toxico.nibiohn.go.jp/english/).

Mentions: Given that within-source comparison performed well for all analysis methods, we investigated other sources of variation across experimental models by comparing their baseline states, i.e. basal level of gene expression before perturbation with drug treatment. We find that baseline gene expression in control samples is highly correlated across sources for the same system (TG vs. DM rat liver, TG vs. DM RPH; Tables 2 and S6). We also find higher correlation between expression in liver and primary hepatocytes of the same organism in culture versus liver-to-culture expression comparisons across organisms: i.e., rat liver expression is more correlated with RPH expression, than with mouse or human liver; likewise for human liver vs. HPH and mouse liver vs. MPH. However, viewing differences in relative terms (i.e., treating liver as ‘control’ and culture as the ‘perturbation’) reveals that the transcriptional impact on hepatocytes in going from liver to culture is comparable in magnitude to the effect of administering highly toxic treatments to rat liver causing marked changes in liver morphology (Fig 5). It is noteworthy that the comparison of GEO mouse liver to TG rat liver appears less dramatic on this basis (avg. abs. EG = 1.75) compared to the baseline expression correlation. Thus even in cases where simple gene level correlation suggest that two models are similar, the underlying degree of perturbation reflected by changes in co-regulation behavior of genes, and by analogy the biology associated with those genes, are highly perturbed.


Assessing Concordance of Drug-Induced Transcriptional Response in Rodent Liver and Cultured Hepatocytes.

Sutherland JJ, Jolly RA, Goldstein KM, Stevens JL - PLoS Comput. Biol. (2016)

Transcriptional effects of cell culture in the context of rat liver perturbations achieved with drug treatment.A) Distribution of average absolute eigengene scores for 4182 rat liver drug treatments from DM and TG, and 6 baseline expression comparisons of control samples (shown in red text in the inset). The solid and dashed vertical lines denote the median and 90th percentile values for avg. abs. EG. Rat liver histology sections: B) Unaffected liver. A central vein (asterisk) and small portal triad (arrowhead) are marked. Mild hepatocellular glycogen (recognized by cytoplasmic pallor) is apparent in the periportal region. C) Liver histology 1 day after the last dose of methapyrilene administered at 100 mg/kg for 29 days. Biliary hyperplasia (to the left of the arrows), correlating with increased alkaline phosphatase (ALP) and γ-glutamyltransferase (GGT) activities (S7 Table), and hepatocellular apoptosis/single cell necrosis (arrowheads) are noted. Hepatocytes in the right side of the image are hypertrophied and exhibit prominent anisonucleosis. D) Liver histology 1 day after bortezomib administration at 1 mg/kg. Acute hepatocellular necrosis (left of the arrows) and mild hepatocellular vacuolation (arrowheads) are noted. Images were extracted from the Open TG-GATES website (http://toxico.nibiohn.go.jp/english/).
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pcbi.1004847.g005: Transcriptional effects of cell culture in the context of rat liver perturbations achieved with drug treatment.A) Distribution of average absolute eigengene scores for 4182 rat liver drug treatments from DM and TG, and 6 baseline expression comparisons of control samples (shown in red text in the inset). The solid and dashed vertical lines denote the median and 90th percentile values for avg. abs. EG. Rat liver histology sections: B) Unaffected liver. A central vein (asterisk) and small portal triad (arrowhead) are marked. Mild hepatocellular glycogen (recognized by cytoplasmic pallor) is apparent in the periportal region. C) Liver histology 1 day after the last dose of methapyrilene administered at 100 mg/kg for 29 days. Biliary hyperplasia (to the left of the arrows), correlating with increased alkaline phosphatase (ALP) and γ-glutamyltransferase (GGT) activities (S7 Table), and hepatocellular apoptosis/single cell necrosis (arrowheads) are noted. Hepatocytes in the right side of the image are hypertrophied and exhibit prominent anisonucleosis. D) Liver histology 1 day after bortezomib administration at 1 mg/kg. Acute hepatocellular necrosis (left of the arrows) and mild hepatocellular vacuolation (arrowheads) are noted. Images were extracted from the Open TG-GATES website (http://toxico.nibiohn.go.jp/english/).
Mentions: Given that within-source comparison performed well for all analysis methods, we investigated other sources of variation across experimental models by comparing their baseline states, i.e. basal level of gene expression before perturbation with drug treatment. We find that baseline gene expression in control samples is highly correlated across sources for the same system (TG vs. DM rat liver, TG vs. DM RPH; Tables 2 and S6). We also find higher correlation between expression in liver and primary hepatocytes of the same organism in culture versus liver-to-culture expression comparisons across organisms: i.e., rat liver expression is more correlated with RPH expression, than with mouse or human liver; likewise for human liver vs. HPH and mouse liver vs. MPH. However, viewing differences in relative terms (i.e., treating liver as ‘control’ and culture as the ‘perturbation’) reveals that the transcriptional impact on hepatocytes in going from liver to culture is comparable in magnitude to the effect of administering highly toxic treatments to rat liver causing marked changes in liver morphology (Fig 5). It is noteworthy that the comparison of GEO mouse liver to TG rat liver appears less dramatic on this basis (avg. abs. EG = 1.75) compared to the baseline expression correlation. Thus even in cases where simple gene level correlation suggest that two models are similar, the underlying degree of perturbation reflected by changes in co-regulation behavior of genes, and by analogy the biology associated with those genes, are highly perturbed.

Bottom Line: We examined the concordance of compound-induced transcriptional changes using data from several sources: rat liver and rat primary hepatocytes (RPH) from Drug Matrix (DM) and open TG-GATEs (TG), human primary hepatocytes (HPH) from TG, and mouse liver/HepG2 results from the Gene Expression Omnibus (GEO) repository.Co-expression networks performed better than genes or GSA when comparing treatment effects within rat liver and rat vs. mouse liver.We observe that the baseline state of untreated cultured cells relative to untreated rat liver shows striking similarity with toxicant-exposed cells in vivo, indicating that gross systems level perturbation in the underlying networks in culture may contribute to the low concordance.

View Article: PubMed Central - PubMed

Affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, United States of America.

ABSTRACT
The effect of drugs, disease and other perturbations on mRNA levels are studied using gene expression microarrays or RNA-seq, with the goal of understanding molecular effects arising from the perturbation. Previous comparisons of reproducibility across laboratories have been limited in scale and focused on a single model. The use of model systems, such as cultured primary cells or cancer cell lines, assumes that mechanistic insights derived from the models would have been observed via in vivo studies. We examined the concordance of compound-induced transcriptional changes using data from several sources: rat liver and rat primary hepatocytes (RPH) from Drug Matrix (DM) and open TG-GATEs (TG), human primary hepatocytes (HPH) from TG, and mouse liver/HepG2 results from the Gene Expression Omnibus (GEO) repository. Gene expression changes for treatments were normalized to controls and analyzed with three methods: 1) gene level for 9071 high expression genes in rat liver, 2) gene set analysis (GSA) using canonical pathways and gene ontology sets, 3) weighted gene co-expression network analysis (WGCNA). Co-expression networks performed better than genes or GSA when comparing treatment effects within rat liver and rat vs. mouse liver. Genes and modules performed similarly at Connectivity Map-style analyses, where success at identifying similar treatments among a collection of reference profiles is the goal. Comparisons between rat liver and RPH, and those between RPH, HPH and HepG2 cells reveal lower concordance for all methods. We observe that the baseline state of untreated cultured cells relative to untreated rat liver shows striking similarity with toxicant-exposed cells in vivo, indicating that gross systems level perturbation in the underlying networks in culture may contribute to the low concordance.

Show MeSH
Related in: MedlinePlus