Preservation, induction or incorporation of metabolism into the in vitro cellular system − Views to current opportunities and limitations
Highlights
► Metabolic capability is needed in in vitro systems for improving in vivo relevance. ► Bioreactors produce metabolites for target exposure. ► Preservation by appropriate culture conditions enhance metabolic competence. ► Incorporation by genomic techniques restore metabolism in otherwise deficient cells. ► Stem cell-derived hepatocytes will guarantee unlimited supply of competent cells.
Introduction
Toxicokinetic (TK) characteristics of chemicals are determined by passive and active factors, and among active factors, metabolism (xenobiotic-metabolizing enzymes) and transport (uptake and efflux transporters) are the most important. The expression and activity of the enzymes and transporters is regulated, for example, by ligand-activated nuclear receptors in the organs of importance for TK. If these processes were to be measured in in vitro cellular systems, it is of utmost importance to ensure that they are actually functional and preferably at the similar level as in vivo (see e.g. Coecke et al., 2006, Adler et al., 2011). Fig. 1 demonstrates the central role of metabolism affecting the concentration of a substance at the active site (receptor) and thus determining to a large extent the eventual toxicodynamic consequences. Thus the preservation, induction and/or incorporation of relevant metabolic capacity are one of the challenges, which have to be solved before truly reliable toxicity testing systems can be achieved.
Section snippets
Why it is important to have metabolism in the cellular system for toxicity testing
From the test system point of view, metabolic competence may become of importance for determining local extra or intracellular concentrations of the studied substance and its active or inactive metabolites. It is obvious that efficient metabolism may decrease the concentration of the substance to the point that no effect could be revealed. On the other hand, lack of metabolism may be detrimental to the performance of the test if metabolic activation is a prerequisite for the effect.
One of the
Toxicologically important organs and activities for metabolism
Critical organs for toxicologically important xenobiotic metabolism and active transporters are the portals to the body (lung, skin, gastrointestinal tract), internal organs for metabolism (liver) and excretion (liver and kidneys) and specialized barriers such as the placenta regulating fetal exposure or blood–brain barrier for CNS drug action and toxicity.
One of the crucial considerations here is the adequacy of the liver as a master organ for metabolism studies. Hepatotoxicity has been one of
Examples of commonly used in vitro systems
Some representative in vitro systems currently in use are presented in Table 1. For comparison, the table starts with some non-cellular systems, which are regularly used especially in drug development. Many of these in vitro non-cell ex vivo systems such as liver microsomes or homogenates rather consistently represent the original tissue and can be used in the initial studies on the characterization and quantitation of metabolic profiles and activities. However, such systems are not suitable as
Preservation, induction and/or incorporation of metabolism into the cellular systems
There are several potential techniques to incorporate metabolism into cellular systems, which are collected into Table 2.
Extra-hepatic tissues and incorporation of metabolism
Xenobiotic metabolism enzymes display tissue specific-expression and regulation (e.g. Baron et al., 2008), which is of importance for several reasons. First of all, cell-specific profile of drug metabolizing enzymes should be studied so that an appropriate in vitro cell model could be sought. Table 3 contains some examples of the expression of drug-metabolizing enzymes in extrahepatic tissues. These data are required as a necessary framework for the development of metabolically competent
How to get the information about metabolism without actual incorporation
Chemically reactive metabolites pose a major challenge especially in drug development, as they have the potential to alter biological function and initiate serious adverse drug reactions. These include hepatotoxicity, skin rashes, agranulocytosis, and aplastic anemia, in drug-treated patients. Pharmaceutical companies employ today several different strategies to evaluate reactive metabolites. At early stages of drug development, molecular modeling (in silico) methods can fairly reliably predict
The integration of in vitro data into human risk assessment
Ultimately, the information derived from in vitro and in silico studies described above has to be used for risk assessment. Thousands of articles have been published on the in vitro metabolism and interactions of particular chemicals using human and animal-derived in vitro models. However, the main challenge is how to translate the in vitro measured characteristics into the information useful for the risk assessment process.
For risk assessment, when only in vitro data is available, a number of
Conclusions
A necessary condition to develop realistic and reliable experimental in vitro systems is their early characterization regarding metabolism and other important kinetic processes (e.g. transporters), because the thorough characterization is the prerequisite for the validation and use of cellular systems for pharmacological and toxicological purposes. There are currently a number of experimental possibilities, although also many limitations, to develop cells capable of metabolism and possessing
Conflict of interest
None declared.
Acknowledgments
Original research of the authors has been support by respective universities and by grants from The Academy of Finland, TEKES State Granting Agency for Research and Innovation, and by intellectual and travel support from EU COST and FW programmes.
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