A frequent misinterpretation in current research on liver fibrosis: the vessel in the center of CCl4-induced pseudolobules is a portal vein

Hammad, Seddik; Braeuning, Albert; Meyer, Christoph; Mohamed, F.; Hengstler, Jan G.; Dooley, Steven

doi:10.1007/s00204-017-2040-8

Cited by 20 publications

(12 citation statements)

References 16 publications

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“…An explanation may be that hepatocytes exhibit gene expression alterations that increase with the length of the cultivation period, leading to a decrease in the hepatocyte differentiation status (Godoy et al 2015;Zellmer et al 2010;Rowe et al 2013). Moreover, hepatotoxic effects caused by repeated doses in vivo often require the involvement of cell types other than hepatocytes, such as stellate cells (Hammad et al 2014(Hammad et al , 2017, endothelial cells (Hoehme et al 2010;Ghallab et al 2016Ghallab et al , 2018, and cholangiocytes (Jansen et al 2017;Vartak et al 2016;Ghallab et al 2018), which are not represented adequately in currently available in vitro methods. If, however, hepatocytes are the primary target of a hepatotoxic compound, it seems that this type of toxicity can be adequately identified by a single compound exposure for 48 h, at least for the compound set tested in the present study.…”

Section: Discussionmentioning

confidence: 99%

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

et al. 2019

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Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (C max) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC 10) yielded better metrics than higher toxicity thresholds (EC 50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of C max were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC 10 and C max as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

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Section: Discussionmentioning

confidence: 99%

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

et al. 2019

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“…However, it should be considered that an in vitro system may respond differently to repeated or chronic exposure than an organ in vivo. A suitable example of this is liver cirrhosis where repeated exposure to hepatotoxic compounds leads to hepatocyte death followed by stellate cell activation and secretion of extracellular matrix (Hammad et al 2017;Jansen et al 2017). However, since the currently available in vitro systems do not recapitulate Fig.…”

Section: Discussionmentioning

confidence: 99%

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes

Albrecht

Edlund

et al. 2018

Arch Toxicol

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Primary human hepatocytes (PHHs) remain the gold standard for in vitro testing in the field of pharmacology and toxicology. One crucial parameter influencing the results of in vitro tests is the incubation period with test compounds. It has been suggested that longer incubation periods may be critical for the prediction of repeated dose toxicity. However, a study that systematically analyzes the relationship between incubation period and cytotoxicity in PHHs is not available. To close this gap, 30 compounds were tested in a concentration-dependent manner for cytotoxicity in cultivated cryopreserved PHHs (three donors per compound) for 1, 2 and 7 days. The median of the EC 50 values of all compounds decreased 1.78-fold on day 2 compared to day 1, and 1.89-fold on day 7 compared to day 1. Median values of EC 50 ratios of all compounds at day 2 and day 7 were close to one but for individual compounds the ratio increased up to almost six. Strong correlations were obtained for EC 50 on day 1 and day 7 (R = 0.985; 95% CI 0.960-0.994), day 1 and day 2 (R = 0.964; 95% CI 0.910-0.986), as well as day 2 and day 7 (R = 0.981; 95% CI 0.955-0.992). However, compound specific differences also occurred. Whereas, for example, busulfan showed a relatively strong increase on day 7 compared to day 1, cytotoxicity of acetaminophen did not increase during longer incubation periods. To validate the observed correlations, a publicly available data set, containing data on the cytotoxicity of human hepatocytes cultivated as spheroids for incubation periods of 5 and 14 days, was analyzed. A high correlation coefficient of EC 50 values at day 5 and day 14 was obtained (R = 0.894; 95% CI 0.798-0.945). In conclusion, the median cytotoxicity of the test compounds increased between 1 and 2 days of incubation, with no or only a minimal further increase until day 7. It remains to be studied whether the different results obtained for some individual compounds after longer exposure periods would correspond better to human-repeated dose toxicity. Keywords Hepatotoxicity • Primary human hepatocyte • Incubation period • Cell-titer-blue Abbreviations APAP Acetaminophen ASP Aspirin BOS Bosentan BPR Buspirone Xiaolong Gu and Wiebke Albrecht shared first authorship.

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“…This serves to detoxify xenobiotics before they are drained into the central vein [2,11]. However, for compounds metabolically activated by CYPs, such as CCl 4 or acetaminophen, the zonated expression causes a pericentral pattern of necrosis [12,13]. A zonated metabolic pattern is also known for ammonia metabolism [14].…”

Section: Introductionmentioning

confidence: 99%

Influence of Liver Fibrosis on Lobular Zonation

Ghallab

Myllys

Holland

et al. 2019

Cells

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Little is known about how liver fibrosis influences lobular zonation. To address this question, we used three mouse models of liver fibrosis, repeated CCl4 administration for 2, 6 and 12 months to induce pericentral damage, as well as bile duct ligation (21 days) and mdr2−/− mice to study periportal fibrosis. Analyses were performed by RNA-sequencing, immunostaining of zonated proteins and image analysis. RNA-sequencing demonstrated a significant enrichment of pericentral genes among genes downregulated by CCl4; vice versa, periportal genes were enriched among the upregulated genes. Immunostaining showed an almost complete loss of pericentral proteins, such as cytochrome P450 enzymes and glutamine synthetase, while periportal proteins, such as arginase 1 and CPS1 became expressed also in pericentral hepatocytes. This pattern of fibrosis-associated ‘periportalization’ was consistently observed in all three mouse models and led to complete resistance to hepatotoxic doses of acetaminophen (200 mg/kg). Characterization of the expression response identified the inflammatory pathways TGFβ, NFκB, TNFα, and transcription factors NFKb1, Stat1, Hif1a, Trp53, and Atf1 among those activated, while estrogen-associated pathways, Hnf4a and Hnf1a, were decreased. In conclusion, liver fibrosis leads to strong alterations of lobular zonation, where the pericentral region adopts periportal features. Beside adverse consequences, periportalization supports adaptation to repeated doses of hepatotoxic compounds.

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A frequent misinterpretation in current research on liver fibrosis: the vessel in the center of CCl4-induced pseudolobules is a portal vein

Cited by 20 publications

References 16 publications

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes

Influence of Liver Fibrosis on Lobular Zonation

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