Charcterization of biotransformation systems in human cells

Efforts are continuously being made to improve test procedures so that predictions of toxicity can be made earlier and more accurately. Understanding mechanisms of toxicity is an important part of improving safety assessment, but for this, simplified methods and models to screen for mechanisms associated with toxicity are required.

Glutathione (GSH) is important in protecting cells from oxidants and electrophiles, and the availability of GSH in the nucleus would be highly advantageous in the protection of DNA. A novel immunocytochemical method for visualization of subcellular compartmentalization of GSH was described. Confocal microscopy of stained A549 cells showed that the GSH levels of the nuclear and cytosolic compartments are close to equilibrium, and that the highest levels of cellular GSH are associated with mitochondria. In addition, the method described provided visualization of the compartmentalization of protein-glutathione mixed disulphides formed in A549 cells exposed to diamide. The method described allow for studies of the organization of intracellular GSH in intact cells, and may potentially offer means to monitor the regiospecificity in the GSH levels and the cellular redox state after exposure to xenobiotics. The liver is a frequent target of xenobiotic-induced toxicity and it is of great importance to assess potential hepatotoxicity.

In order to evaluate the use of hepatocyte-like cells derived from human embryonic stem cells (hESC) as a model system for metabolism and xenobiotic-induced hepatotoxicity, the expression of biotransformation enzymes was evaluated. The protein expression of glutathione transferases (GSTs) in hepatocyte-like cells was studied and results show high levels of GSTA1-1, whereas GSTP1-1 is not present, and this was in accordance to the results in primary human hepatocytes. In addition, GST activity was detected in hepatocyte-like cells at levels comparable to human hepatocytes. The mRNA and protein expression of several important cytochrome P450s (CYPs) in hepatocyte-like cells was also investigated.

CYP mRNA expression and CYP1A2 and 3A/7 protein expression was detected in hepatocyte-like cells, but at low levels compared to human hepatocytes. The expression of other liver-related genes, such as UDP-glucuronosyltransferases (UGTs), drug transporters and transcription factors were also studied using low density arrays. The mRNAs for a variety of CYPs and liver-related factors as well as CYP1A2 and CYP3A4/7 protein were shown to be inducible. The hepatic phenotype of hepatocyte-like cells was demonstrated by characteristic hepatic morphology, expression of several hepatic markers and transcription factors as well as glycogen storage. Taken together the results indicate that the hepatocyte-like cells have the potential of being used as a model system for studying hepatotoxicity, although further differentiation is needed before they have a fully mature hepatic phenotype.

Just like hESC, human adult stem cells may be a source of differentiated cells for safety assessment, but they may also be an in vivo target for xenobiotics and toxicity. To evaluate the biotransformation capacity in human adult stem cells from breast and liver tissue, the expression of GSTs and CYPs was evaluated. The results show a clear difference in the expression pattern of these enzymes between adult stem cells from both tissues, and their differentiated counterparts. This indicates that these stem cells would respond differently to exposure of xenobiotics and that they are not suitable as a model system for safety assessment without differentiation of the cells to more closely resemble the target cell type.

List of scientific papers

I. Soderdahl T, Enoksson M, Lundberg M, Holmgren A, Ottersen OP, Orrenius S, Bolcsfoldi G, Cotgreave IA (2003). Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells. FASEB J. 17(1): 124-6.
https://doi.org/10.1096/fj.02-0259fje

II. Soderdahl T, Kuppers-Munther B, Heins N, Edsbagge J, Bjorquist P, Cotgreave I, Jernstrom B (1970). Glutathione transferases in hepatocyte-like cells derived from human embryonic stem cells. Toxicol In Vitro. 21(5): 929-37.
https://pubmed.ncbi.nlm.nih.gov/17346923

III. Soderdahl T, Ek M, Kuppers-Munther B, Bjorquist P, Edsbagge J, Cotgreave I, Jernstrom B, Andersson TB, Ingelman-Sundberg M, Johansson I (2007). Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells. Biochem Pharmacol. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/17568565