The liver is rich in metabolic activity, which may create toxic metabolites from otherwise safe drugs. Broadly, the metabolism of substances by the liver is classified in two main phases:
- Phase I: Oxidation, Reduction, Hydrolysis
- Phase II: Conjugation with molecules such as glutathione or glucuronic acid
Cytochrome P450 enzymes form a superfamily of enzymes that metabolize numerous drugs in the liver. The CYP450 causality assay tests the role of cytochrome P450 enzymes in the toxicity of a substance by comparing its cytotoxicity in the presence of in the absence of 1-Aminobenzotriazole, a broad-spectrum inhibitor of cytochrome P450 enzymes extensively used to study drug metabolism.
For example, aflatoxin B1 is metabolized by cytochrome P450 enzymes 1A2 and 3A4 into a mutagen epoxide metabolite.
As shown in Figure 1, 1-Aminobenzotriazole decreases aflatoxin B1 cytotoxicity in human liver microtissues, demonstrating the deleterious role of cytochrome P450 enzymes in the response to aflatoxin B1.
Figure 1: Liver microtissues were treated for 3 days with increasing concentrations of aflatoxin B1 in the presence or absence a fixed concentration of 1-Aminobenzotriazole (ABT1). Cellular ATP was measured on day 3.
In InSphero’s standard CYP450 causality assay, 4 concentrations of a test item are applied on liver spheroids for 7 days in the presence or absence of a fixed concentration of 1-Aminobenzotriazole. The ATP content of the liver microtissues is determined on day 7 at the end of the assay, whereas LDH activity leakage from the cytosol into the cell culture supernatant is determined on three different time points on days 2, 5 and 7.
A decrease in the cytotoxicity of the test item shows cytochrome P450 enzymes contribute to the cytotoxicity of the test item. Conversely, an increase in the cytotoxicity of the test item shows cytochrome P450 enzymes detoxify the test item.
