3D InSight™ Investigative Toxicology | 3D In Vitro Liver Safety Services

Fast and reliable identification of mechanisms of toxicology based on the market-leading human 3D Liver Safety platform

Industry-ready 3D In Vitro Investigative Toxicology

InSphero’s Investigative Toxicology services offer a comprehensive set of solutions to decipher the mechanism of toxicity of a substance. Profiling and hypothesis generation are supported by state-of-the art ‘omics technology, while a comprehensive portfolio of biomarker analysis and classical toxicology mechanistic assays can test a broad set of toxic reactions. InSphero’s team supports you in identifying potential hepatotoxic substances and understanding their mechanisms of toxicity to guide your risk assessment with confidence within weeks.

Why Investigative Toxicology with InSphero?

We help toxicologists, worldwide, to understand the mechanism of action of liver toxicity through an in-depth analysis based on the advanced 3D InSight™ Human Liver Microtissues.
An icon demonstrating the market-leading 3D in vitro liver toxicology solutions by InSphero

Market-leading 3D in vitro biology you can rely on

Finding answers to complex questions requires a model faithfully modeling liver biology. InSphero’s spheroid system containing all liver cells, both parenchymal and non-parenchymal, delivers.

We seamlessly go from hazard identification to investigative toxicology

By using our 7 or 14-day DILI service to identify an issue, you can proceed to an investigative study in no time.

An icon demonstrating InSphero's 3D in vitro human liver model which enables fast and efficient safety assessment projects

Fast turnaround to accelerate your projects

Our streamlined, automated, and 3D-optimized workflow guarantees reliable data in record time.
An icon showcasing the 3D in vitro liver safety platform trusted by pharmaceutical companies worldwide. 3D in vitro DILI testing used by the top ten pharmaceutical companies.

Trusted by pharmaceutical companies worldwide

Your team will appreciate safety data that has been proven by thousands of compound tests.

Your Success is Our Mission

Schedule a consultation with our experts

3D In Vitro Investigative Toxicology Assays

Cytochrome P450 enzymes play a central role in the metabolism of xenobiotics to facilitate their excretion. However, this metabolic activity may produce toxic reactive species [1,2].

Aflatoxin B1, a mycotoxin produced by A. flavus and A. parasiticus, is an example of a substance that forms toxic reactive metabolites when metabolized by CYP1A2 and CYP3A4 into the reactive aflatoxin B1 exo-8,9-epoxide [4].

InSphero’s standard “CYP450 causality” assay investigates the role of cytochrome P450 enzymes in the toxicity to a test item by comparing its cytotoxicity in 3D InSight™ Liver microtissues co-treated with and without 1-Aminobenzotriazole, a broad-spectrum cytochrome P450 inhibitor [3].

As shown in Figure 1, aflatoxin B1 is less cytotoxic to 3D InSight™ Liver microtissues cotreated with 1-Aminobenzotriazole, thereby demonstrating the deleterious role of cytochrome P450s in the response to aflatoxin B1.

Figure 1: InSphero´s standard “CYP450 causality” assay with Aflatoxin B1 - 3D InSight™ Liver microtissues are treated for 7 days with 4 concentrations of a test item, with or without a fixed concentration of 1-Aminobenzotriazole. The ATP content of the microtissues is measured on day 7, while the leakage of cytosolic lactate dehydrogenase into the cell culture supernatant is assessed at three different timepoints on day 2, 5 and 7. A change in the cytotoxicity of the test item suggests CYP450 activity is involved in its toxicity.

Resources

[1] Iversen, Ditte B et al. “Drug metabolism and drug transport of the 100 most prescribed oral drugs.” Basic & clinical pharmacology & toxicology vol. 131,5 (2022): 311-324. doi:10.1111/bcpt.13780

[2] Mahajan, Piyush et al. “Drug reactive metabolite-induced hepatotoxicity: a comprehensive review.” Toxicology mechanisms and methods vol. 34,6 (2024): 607-627. doi:10.1080/15376516.2024.2332613

[3] de Montellano, Paul R Ortiz. “1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology.” Medicinal chemistry vol. 8,3 (2018): 038. doi:10.4172/2161-0444.1000495

[4] Eaton, D L, and E P Gallagher. “Mechanisms of aflatoxin carcinogenesis.” Annual review of pharmacology and toxicology vol. 34 (1994): 135-72. doi:10.1146/annurev.pa.34.040194.001031

The inhibition of mitochondrial oxidative phosphorylation by drugs can lead to adverse liver effects [1]. In its most severe form, this inhibition results in lactic acidosis, as pyruvate is converted into lactate for cellular excretion rather than being converted into acetyl-CoA to undergo oxidation for energy production in the citric acid cycle.

Phenformin, a hepatotoxic drug from the biguanide class [2, 3, 4], is an example of a drug that inhibits mitochondrial oxidative phosphorylation, and this mechanism is likely to play a key role in its potential for hepatotoxicity [5].

InSphero´s standard “Mitochondrial function” assay investigates whether a test item inhibits mitochondrial oxidative phosphorylation by monitoring its effect on lactate production.

As shown in Figure 1, phenformin induces a dose-dependent increase in lactate production in 3D InSight™ Liver microtissues, thereby demonstrating the inhibition of mitochondrial respiration by phenformin.

Figure 1: InSphero’s standard “Mitochondrial function” assay with Phenformin
3D InSight™ Liver microtissues are treated for 7 days with 4 concentrations of a test item and the secretion of lactate into the cell culture supernatant is assessed at three different timepoints on day 2, 5 and 7. An increase in lactate secretion by 3D InSight™ Liver microtissues suggests the test item inhibits of mitochondrial oxidative phosphorylation.

Resources

[1] Mihajlovic, Milos, and Mathieu Vinken. “Mitochondria as the Target of Hepatotoxicity and Drug-Induced Liver Injury: Molecular Mechanisms and Detection Methods.” International journal of molecular sciences vol. 23,6 3315. 18 Mar. 2022, doi:10.3390/ijms23063315

[2] Kwong, S C, and J Brubacher. “Phenformin and lactic acidosis: a case report and review.” The Journal of emergency medicine vol. 16,6 (1998): 881-6. doi:10.1016/s0736-4679(98)00103-6

[3] McGuinness, M E, and R L Talbert. “Phenformin-induced lactic acidosis: a forgotten adverse drug reaction.” The Annals of pharmacotherapy vol. 27,10 (1993): 1183-7. doi:10.1177/106002809302701004

[4] YOUNG, J M, and L P ARMANINO. “LACTIC ACIDOSIS AND PHENFORMIN INTOXICATION. REPORT OF TWO CASES WITH REVIEW OF LITERATURE.” California medicine vol. 103,3 (1965): 198-202.

[5] Bridges, Hannah R et al. “Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria.” The Biochemical journal vol. 462,3 (2014): 475-87. doi:10.1042/BJ20140620

Bile acid transporters play a key role in maintaining bile acid homeostasis, and their inhibition by drugs contributes to the onset of intracellular cholestasis.

Bosentan a hepatotoxic endothelin receptor antagonist, is an example that inhibits bile acid transporters, and this inhibition is likely to play a key role in its potential for hepatotoxicity [2,3].

InSphero´s standard “Canalicular Secretion Inhibition” assay investigates whether a test item inhibits bile acid transporters by monitoring the flow of the fluorescein-labelled bile acid analog cholyl-Lys-Fluorescein (CLF) into the canalicular network of 3D InSight™ Liver microtissues [4].

As shown in Figure 1, Bosentan leads to a dose-dependent decrease in the CLF signal in the canalicular network of 3D InSight™ Liver microtissues, thereby demonstrating the cholestasis potential of Bosentan.

Figure 1: InSphero’s standard “Canalicular Secretion Inhibition” assay with Bosentan
3D InSight™ Liver microtissues are treated for 7 days with 4 concentrations of a test item, and CLF signal in the canalicular network is assessed on day 7 by confocal imaging. A decrease in CLF signal in the canalicular network suggests the test item inhibits bile acid transporters.

Resources

[1] Saran, Chitra, and Kim L R Brouwer. “Hepatic Bile Acid Transporters and Drug-induced Hepatotoxicity.” Toxicologic pathology vol. 51,7-8 (2023): 405-413. doi:10.1177/01926233231212255

[2] Fattinger, K et al. “The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions.” Clinical pharmacology and therapeutics vol. 69,4 (2001): 223-31. doi:10.1067/mcp.2001.114667

[3] Oorts, Marlies et al. “Bosentan Alters Endo- and Exogenous Bile Salt Disposition in Sandwich-Cultured Human Hepatocytes.” The Journal of pharmacology and experimental therapeutics vol. 379,1 (2021): 20-32. doi:10.1124/jpet.121.000695

[4] de Waart, Dirk R et al. “Hepatic transport mechanisms of cholyl-L-lysyl-fluorescein.” The Journal of pharmacology and experimental therapeutics vol. 334,1 (2010): 78-86. doi:10.1124/jpet.110.166991

Hepatic steatosis refers to the excessive accumulation of triglyceride in hepatocytes and can be an adverse effect of drugs [1].

Amiodarone, an antiarrhythmic drug, is an example of a drug that inhibits mitochondrial β-oxidation, and this inhibition is likely to play a key role in its potential to induce hepatic steatosis [2-5]

InSphero’s standard “Steatosis Induction” assay investigates whether a test item induces hepatic steatosis by assessing the triglyceride content of 3D InSight™ Liver microtissues

As shown on Figure 1, amiodarone induces a dose-dependent increase in the triglyceride content of 3D InSight™ Liver microtissues, thereby demonstrating its potential to induce hepatic steatosis.

Figure 1: InSphero´s standard “Steatosis induction” assay with Amiodarone
 3D InSight™ Liver microtissues are treated for 7 days with 4 concentrations of a test item, and the triglyceride content of the microtissues is assessed on day 7. An increase in triglyceride content of the 3D InSight™ Liver microtissues suggests that a test item induces hepatic steatosis

Resources

[1] Satapathy, Sanjaya K et al. “Drug-induced fatty liver disease: An overview of pathogenesis and management.” Annals of hepatology vol. 14,6 (2015): 789-806. doi:10.5604/16652681.1171749

[2] LiverTox: Clinical and Research Information on Drug-Induced Liver Injury [Internet]. Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases; 2012-. Amiodarone. [Updated 2016 Mar 1]

[3] Anthérieu, Sébastien et al. “Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in HepaRG cells.” Hepatology (Baltimore, Md.) vol. 53,6 (2011): 1895-905. doi:10.1002/hep.24290

[4] Hubel, Einav et al. “Repetitive amiodarone administration causes liver damage via adipose tissue ER stress-dependent lipolysis, leading to hepatotoxic free fatty acid accumulation.” American journal of physiology. Gastrointestinal and liver physiology vol. 321,3 (2021): G298-G307. doi:10.1152/ajpgi.00458.2020

[5] Begriche, Karima et al. “Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver.” Journal of hepatology vol. 54,4 (2011): 773-94. doi:10.1016/j.jhep.2010.11.006

The liver's metabolism of xenobiotics can produce toxic reactive species, including reactive xenobiotic metabolites and free radicals, which can interact with cellular components to form nonfunctional adducts [1-3]. If the formation of these reactive species exceeds the liver's ability to detoxify them through the antioxidant response, it may lead to oxidative stress and, ultimately, liver injury [4]. Glutathione is an endogenous organic compound that plays a central role in the cellular antioxidant response by harmlessly neutralizing reactive species, thereby protecting essential cellular components from detrimental alterations.

Acetaminophen, a non-opioid analgesic and antipyretic agent, is an example of a drug that forms toxic reactive metabolites detoxified by cellular glutathione [5, 6].

InSphero’s standard “Glutathione causality” assay investigates the role of glutathione in the detoxification of a test item by comparing its cytotoxicity in 3D InSight™ Liver microtissues co-treated with or without L-buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis.

As shown in Figure 1, acetaminophen is more cytotoxic to 3D InSight™ Liver microtissues co-treated BSO, thereby demonstrating the protective role of glutathione in the detoxification of acetaminophen.

Figure 1: InSphero´s standard “Glutathione causality” assay with Acetaminophen
3D InSight™ Liver microtissues are treated for 7 days with 4 concentrations of a test item, with or without a fixed concentration of BSO. The ATP content of the microtissues is measured on day 7, while the leakage of cytosolic lactate dehydrogenase into the cell culture supernatant is assessed at three different timepoints on day 2, 5 and 7. A change in the cytotoxicity of the test item suggests glutathione is involved in its toxicity.

Resources

[1] Baillie, T.A. Drug–protein adducts: past, present, and future. Med Chem Res 29, 1093–1104 (2020). doi.org/10.1007/s00044-020-02567-8

[2] Attia, Sabry M. “Deleterious effects of reactive metabolites.” Oxidative medicine and cellular longevity vol. 3,4 (2010): 238-53. doi:10.4161/oxim.3.4.13246

[3] Donato, MaríaTeresa, and Laia Tolosa. “High-Content Screening for the Detection of Drug-Induced Oxidative Stress in Liver Cells.” Antioxidants (Basel, Switzerland) vol. 10,1 106. 13 Jan. 2021, doi:10.3390/antiox10010106

[4] Villanueva-Paz, Marina et al. “Oxidative Stress in Drug-Induced Liver Injury (DILI): From Mechanisms to Biomarkers for Use in Clinical Practice.” Antioxidants (Basel, Switzerland) vol. 10,3 390. 5 Mar. 2021, doi:10.3390/antiox10030390

[5] Mirochnitchenko, O et al. “Acetaminophen toxicity. Opposite effects of two forms of glutathione peroxidase.” The Journal of biological chemistry vol. 274,15 (1999): 10349-55. doi:10.1074/jbc.274.15.10349

[6] Torres, Sandra et al. “Endoplasmic Reticulum Stress-Induced Upregulation of STARD1 Promotes Acetaminophen-Induced Acute Liver Failure.” Gastroenterology vol. 157,2 (2019): 552-568. doi:10.1053/j.gastro.2019.04.023

Inflammation may contribute to drug-induced liver injury through two main mechanisms: either by increasing the liver's sensitivity to a particular drug treatment, or conversely, a drug or its metabolites can initiate an inflammatory response, ultimately leading to hepatocellular necrosis [2].

Trovafloxacin, a broad-spectrum antibiotic withdrawn due to hepatotoxicity, is an example of a drug whose toxicity is exacerbated by otherwise benign inflammation [3].

InSphero’s standard "Inflammation Causality" assay investigates whether the toxicity of a test item is potentiated by inflammation by comparing its cytotoxicity in 3D InSight™ Liver microtissues co-treated with or without lipopolysaccharide (LPS), a potent inducer of the inflammatory response. [4]

As shown in figure 1, trovafloxacin is more cytotoxic to 3D InSight™ Liver microtissues co-treated with LPS, thereby demonstrating the exacerbating role of the inflammatory response in trovafloxacin toxicity.

Figure 1: InSphero´s standard “Inflammation causality” assay with Trovafloxacin
3D InSight™ Liver microtissues are treated for 7 days with 4 concentrations of a test item, with or without a fixed concentration of LPS. The ATP content of the microtissues is measured on day 7, while the leakage of cytosolic lactate dehydrogenase into the cell culture supernatant is assessed at three different timepoints on day 2, 5 and 7. An increase in the cytotoxicity of the test item suggests a synergy between the inflammatory response and the toxicity of the test item.

Resources

[1] Nguyen-Lefebvre, Anh Thu, and Anatolij Horuzsko. “Kupffer Cell Metabolism and Function.” Journal of enzymology and metabolism vol. 1,1 (2015): 101.

[2] Role of Inflammation in Drug-Induced Liver Injury -Chapter 10, Robert A. Roth, Patricia E. Ganey,Drug-Induced Liver Disease (Third Edition),Academic Press, 2013, Pages 157-173, ISBN 9780123878175, doi.org/10.1016/B978-0-12-387817-5.00010-8

[3] Shaw, Patrick J et al. “Lipopolysaccharide and trovafloxacin coexposure in mice causes idiosyncrasy-like liver injury dependent on tumor necrosis factor-alpha.” Toxicological sciences : an official journal of the Society of Toxicology vol. 100,1 (2007): 259-66. doi:10.1093/toxsci/kfm218

[4] Bilzer, Manfred et al. “Role of Kupffer cells in host defense and liver disease.” Liver international : official journal of the International Association for the Study of the Liver vol. 26,10 (2006): 1175-86. doi:10.1111/j.1478-3231.2006.01342.x

Our Investigative Toxicology Process

Expert project planning

Our 3D in vitro liver safety experts develop a concise plan to identify the underlying mechanism of toxicity.

Executing the action plan

We know your request is urgent. Our team will execute the action plan swiftly and update you on the progress.

Expert review by experienced liver toxicology scientists

We know your request is urgent. Our team will execute the action plan swiftly and update you on the progress.

Impress with a comprehensive report

We know your request is urgent. Our team will execute the action plan swiftly and update you on the progress.

Featured resources

A Novel Framework for Teasing Out Mechanisms of DILI
3D InSight™ Liver Platform - Investigative Toxicology

Related products

3D InSight Human Liver -Assay-ready liver spheroids
3D InSight™ Human Liver Microtissues
Akura™ 3D SureKit
Scroll to Top

Your Success is Our Mission - Get in Touch

Fill in form below to contact our 3D in vitro experts

Sign up for our Newsletter

Get the latest news on 3D in vitro research

View resource

Fill in the form below to view this resource