ACTIVTox kits are manufactured from a qualified Master Cell Bank (MCB) and Working Cell Banks (WCBs)

The ACTIVTox MCB has been rigorously tested (by an independent CRO) and is free of adventitious agents (table 1).

Table 1. Testing of the Master Cell Bank

Rigorous QA/QC Guarantees Reproducibility

Primary cells are inherently variable because of genetic differences and environmental exposure, while population drift is a continuing problem in cell culture. ACTIVTox cells are genetically defined and are carried for a set number of population doublings out of the MWCB to avoid drift. At the end of each production series, cultures are destroyed and new ones established from the bank.

At each point along the process, cultures are tested for conformance to standards for protein production, glucose consumption and P450 enzyme induction. Release criteria ensure cell quality and assay reproducibility.

ACTIVTox exhibits biochemical features of primary hepatocytes

ACTIVTox cells are similar to primary hepatocytes in a wide variety of characteristics, but without the logistic and cell culture difficulties. Here are summaries; substantially more detailed information is available.

Gluconeogenesis - The synthesis of glucose from less complex molecules is one of the hallmarks of the liver phenotype (2). At least 24 hours post feeding, ACTIVTox cells release glucose when fed with pyruvate and lactate.

Figure 1. Glucose synthesis by ACTIVTox

Testosterone Hydroxylation - Hydroxylation of testosterone to 6-β-hydroxytestosterone by CYP3As is characteristic of human hepatocytes (3). Figure 2 shows the formation of 6-β-hydroxytestosterone by ACTIVTox cells treated with dexamethasone to induce CYP3A.

Figure 2. Formation of 6-β-hydroxy testosterone

Sensitivity to metabolically activated toxins - Some compounds are not toxic until metabolic activation (4). Aflatoxin B1 is such a compound - converted by CYP3A to a highly reactive and hepatotoxic epoxide. ACTIVTox cells were incubated with the aflatoxin B1 for 48 hours in the presence or absence of 10µM ketoconazole, an inhibitor of CYP3As. Figure 3 shows that ACTIVTox cells are sensitive to aflatoxin B1 at similar concentrations as are primary hepatocytes.

Figure 3. Aflatoxin is metabolized to the toxic epoxide by CYP3A

P450 Induction - The enzymes of drug metabolism are often induced by their substrates (5). Some of these inductions, particularly CYP1A induction by aryl hydrocarbons and CYP3A by rifampicin and dexamethasone, are used to assess the liver specific phenotype. For CYP1A induction, cells were incubated with 10µM methylcholanthrene overnight then assayed by the addition of ethoxyresorfuin directly to the cultures. Fluorescence was read one hour later. These enzyme activities are induced appropriately in ACTIVTox cells (figures 4 and 5). For CYP3A induction, cells were incubated with 10µM dexamethasone or 50µM rifampicin for 36 hours, then assayed by the addition of dibenzylfluorescein to the cultures. Fluorescence was read one hour later. The ACTIVTox cells induce appropriately in response to these compounds

Figure 4. Induction of CYP1A in response to 10µM methylcholanthrene

Figure 5. Induction of CYP3A in response to 10µM dexamethasone or 50µM rifampicin

References

1. Kelly, JH (1994) Permanent human hepatocyte cell line and its use in a liver assist device (LAD). US Patent No. 5,290,684.
2. Seifter, S., and England, S. (1994) Energy metabolism, in "The Liver, Biology and Pathobiology", Arias, I.M., et al, Eds., 3rd Edition, Raven Press, NY.
3. Granner, D.K. (1990) Hormones of the Gonads, in "Harper's Biochemistry", Murray, R.K., et al, Eds., 22nd Edition, Appleton and Lange, Connecticut.
4. Park, B.K., et al. (2005) The role of metabolic activation in drug-induced hepatotoxicity. Annu. Rev. Pharmacol. Toxicol. 45, 177 – 202
5. Vessey, D.A. (1989) Metabolism of drugs and toxins by human liver, in "Hepatology", Zakim, D., Boyer, T.D., Eds., 2nd Edition, W.B. Saunders, Pennsylvania.