In Vitro Evaluation of Human Xenobiotic Toxicity: Scientific Concepts and the Novel Integrated Discrete Multiple Cell Co-culture (IdMOC) Technology

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Organ Toxicity

In Vitro Evaluation of Human Xenobiotic Toxicity: Scientific Concepts and the Novel Integrated Discrete Multiple Cell Co-culture (IdMOC) Technology

Albert P. Li, Advanced Pharmaceutical Sciences Inc./In Vitro ADMET Laboratories Inc.

Published: December 6, 2007

About the Author(s)
Dr. Albert P. Li obtained his Ph.D. degree in Biomedical Sciences from the University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences, with Dr. Abraham Hsie of Oak Ridge National Laboratories. He is currently Co-Founder, President and Chief Executive Officer of In Vitro ADMET Laboratories LLC (IVAL, Columbia, Maryland), Advanced Pharmaceutical Sciences Inc. (APSciences, Columbia, Maryland), and BRiVAL Inc. (Vancouver, British Columbia). Dr. Li’s companies provide in vitro toxicity testing service (IVAL), in vitro drug metabolism service (BRiVAL), and in vitro cell-based products (APSciences). Dr. Li was previously President and CEO of Phase I Molecular Toxicology Inc. (Santa Fe, New Mexico), Chief Scientific Officer of In Vitro Technologies Inc. (Baltimore, Maryland), Director and Research Professor, Surgical Research Laboratories, St. Louis University Medical School (St. Louis, Missouri), Senior Research Fellow, Monsanto Company (St. Louis, Missouri), Cellular and Genetic Toxicology Group Leader, Lovelace Inhalation Research Institute (Albuquerque, New Mexico), and Research Scientist and Assistant Professor, Cancer Research and Treatment Center, University of New Mexico (Albuquerque, New Mexico).

Dr. Li is one of the pioneers in the development and applications of human-based in vitro experimental systems in the evaluation of human drug properties. He was instrumental in the now generally accepted approach of the use of in vitro human liver models in the evaluation of drug metabolism and drug-drug interactions. His latest contributions include the optimization of cryopreservation procedures for human hepatocytes and, most notably, the invention of the Integrated Discrete Multiple Organ Co-culture (IdMOC) system which models the whole animal/human, allowing the evaluation of multiple organ toxicity while allowing multiple organ interactions as occur in vivo. The IdMOC system has been allowed a US patent in 2007. Dr. Li has published over 150 peer-reviewed papers, edited 5 books and 5 journal issues, and authored three US patents.  He serves as editor and editorial board members on various journals in toxicology and drug metabolism.

Albert P. Li, Ph.D.
Advanced Pharmaceutical Sciences Inc. and In Vitro ADMET Laboratories Inc.
9221 Rumsey Road Suite 8
Columbia, Maryland 21045
E-mail: lialbert@invitroadmet.com

Species-differences in xenobiotic toxicity is a well-documented phenomenon. This occurrence of species-species differences in xenobiotic toxicity illustrates the major deficiency of the routine practice of xenobiotic safety evaluation, namely, human-specific effects cannot be detected with non-human animals.

There are two major contributing factors to species-differences in xenobiotic toxicity:

  1. Species-differences in drug metabolism.
  2. Species-differences in target organ sensitivity.
Species-species differences in xenobiotic metabolism

Xenobiotic metabolism is in general a protective mechanism against toxic substances that can enter an organism. In general, toxic substances are modified by xenobiotic metabolism to neutralize their toxicity and to facilitate their excretion. Variations in drug metabolizing enzymes are evolved among the various animal species, presumably due to the specific exposure of each species towards environmental toxicants.

Species-differences in metabolite formation is believed to be one of the major contributors to species-differences in xenobiotic toxicity for the following reasons:

  1. A toxic metabolite that is formed in humans but not in nonhuman animals would lead to underestimation of the toxicity of the parent xenobiotic.
  2. A toxic metabolite that is formed in nonhuman animals but not in humans would lead to the overestimation of the toxicity of the parent xenobiotic.
Species-species differences in target cell sensitivity towards toxicants

Besides differences in metabolism, species-differences in xenobiotic toxicity can also be due to differences in the sensitivity of the cells in the target organs towards to the toxicants in question.

The following are well-defined examples of this phenomenon:

  1. Peroxisome proliferation receptor ligands: Antilipidemic agents such as the fibrates are agonists of PPARalpha, leading to the upregulation of lipid metabolism pathways. These agents have been found be nongenotoxic hepatocarcinogens in rodents but generally not believed to be carcinogenic in primates, including humans.
  2. Ah-receptor ligands: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxic effects of halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and other structurally diverse ligands. Species differences in toxicity of these ligands, for instance, TCDD-dioxin and polybrominated biphenol (PBB), are well-established.
  3. PXR-ligands: Pregnane X receptor (PXR) is an orphan nuclear receptor that regulates the expression of genes encoding drug-metabolizing enzymes and transporters. Activation of PXR leads to up-regulation of drug metabolizing enzymes including cytochromes P450 isoforms and the transporter Pgp. Although the consequence is mainly in drug-drug interactions, there is an apparent correlation between enzyme induction potential and hepatotoxicity for drugs that are known to cause idiosyncratic toxicity. Human-rodent differences in PXR activation is well-established. For instance, rifampin has a higher affinity for human than for rodent PXR ligand, while pregnenolone 16alpha-carbonitrile (PCN), has a higher affinity for rodent? PXR than human PXR, and with CYP3A induction potential reflecting these differences.
  4. Anticancer agents: Species-species differences in target cell sensitivity have been illustrated with direct-acting cytotoxicants evaluated in vitro using metabolically incompetent cells such as bone-marrow cells.
In vitro toxicity assays as an approach to define human-specific xenobiotic toxicity

Human-specific xenobiotic toxicity, whether as a result of drug metabolism or target cell sensitivity, by definition cannot be accurately evaluated with nonhuman animal models. Short of experimentation in humans in vivo, toxicity testing in vitro using experimental systems with relevant human specific properties represents the only practical preclinical approach to derive human-specific information for the accurate prediction of human xenobiotic toxicity.

It is important to note that the in vitro experimental systems used should have the following components that are critical to human-specific xenobiotic toxicity as discussed earlier:

  1. Human xenobiotic metabolism.
  2. Human target cells.

The use of dedifferentiated cell lines such as transformed or immortalized mouse or human fibroblasts would not be useful, as neither of the above critical properties is present. The use of human-organ derived primary cells as monogenic culture (single cell type cultures) will only allow the evaluation of the effect of xenobiotic on a specific cell type which may or may not possess significant human xenobiotic metabolism pathways.

One cell system that represents the target cells and has human metabolism capacity is human hepatocytes. This cell system has the following advantages:

  1. Human xenobiotic metabolism: Fresh isolates or cryopreserved fresh isolates of human hepatocytes are known to contain most, if not all, of the in vivo hepatic xenobiotic metabolism capacity.
  2. Human target cells: The hepatocytes are the cells in the human liver that are damaged by hepatotoxicants, leading ultimately to liver failure.

A commonly used cell line, the HepG2 cell line, however, does not have these properties, and therefore would not represent a relevant in vitro model for the investigation of hepatotoxicity:

  1. Human xenobiotic metabolism: HepG2 cells only retain a minimal capacity (estimated to be less than 1%) of the normal hepatic xenobiotic metabolism.
  2. Human target cells: HepG2 cells are derived from a human adenocarcinoma of the liver, not the parenchymal cells which are the in vivo target of hepatotoxicants.
It is to be emphasized that the use of cell lines without human metabolism and not representative of in vivo target cells will not have any advantage over the use of laboratory animals in the evaluation of xenobiotic toxicity.

Besides hepatocytes, primary cells cultured from various organs such as kidney, nervous system, heart, skeletal muscle, vascular endothelium, lung, and blood/bone marrow cells have also been applied towards the evaluation of xenobiotic toxicity. The various primary cell systems used may be representative of the appropriate in vivo target cells for their respective organs. However, the use of these cells alone would lack an important in vivo determinant of xenobiotic toxicity, namely, hepatic metabolism.

Integrated Discrete Multiple Organ Co-culture (IdMOC)

The IdMOC is a novel technology developed in our laboratory as an in vitro experimental system for the evaluation of human xenobiotic metabolism, distribution, and toxicity. It is based on the concept that in the human body, there are multiple organs that are physically separated but are interconnected by the systemic circulation. The systemic circulation allows multiple organ interactions. An example of multiple organ interaction is the metabolism of a toxicant by the liver, with the resulting metabolites entering the systemic circulation, leading to the exposure of distal, nonhepatic organs to these metabolites, resulting in toxicity in these distal organs.

The schematic presentation of the scientific concept and configuration of IdMOC is presented in Fig. 1, with a photograph of an IdMOC plate shown on Fig. 2. The IdMOC uses a wells-in a well concept. Cells from individual organs are cultured separately in each of the inner wells. The inner wells are then interconnected by filling the outer well with an overlying medium to cover all inner wells. A xenobiotic introduced into the overlying medium will interact with the multiple cell types in each of the inner wells, and will be exposed to the metabolites collectively generated by the cells. The IdMOC system therefore satisfy our requirements for human metabolism (via the use of human hepatocytes as one of the cell types to provide hepatic xenobiotic metabolism), and human target cells (via the use of cell types from different organs to allow evaluation of organ-specific toxicity). An advantage of the use of IdMOC over the conventional mixed-cell type cultures is that after treatment, the cells from each well can be evaluated for cytotoxicity, thereby allowing the evaluation of cell-type specific effects after co-culturing that is extremely difficult with mixed-cell type co-cultures.

Discussion

Demonstration of advantages over conventional approaches using laboratory animals is key to the acceptance and universal applications of alternative methods. A major advantage of the use of in vitro methods is that human-specific information can be obtained with human-based in vitro experimental systems. The same information, by definition, cannot be obtained with nonhuman laboratory animals. This advantage, namely, that in vitro systems can provide human-specific information, has led to the present universal acceptance of human-based in vitro hepatic experimental systems such as human hepatocytes and human liver microsomes in the evaluation of drug metabolism and drug-drug interactions. It is to be noted that U. S. Food and Drug Administration (FDA) now accepts data from human in vitro hepatic systems alone, and considers in vivo animal tests as inappropriate, for the definition of drug-drug interaction potential of human pharmaceuticals.

In vitro toxicity has always been highly criticized, mainly due to the lack of factors that are believed to be key to the manifestation of in vivo toxicity. We thereby embarked on the development of in vitro approaches incorporating these key in vivo factors. Our research has led to the development of the IdMOC technology.

The IdMOC system has the simplicity and practicality of in vitro system, but with the incorporation of the following key components that are crucial to the manifestation of in vivo toxicity:

  1. Hepatic metabolism via the use of hepatocytes.
  2. Hepatic and nonhepatic target cells via the co-culturing of hepatocytes with key cell types from major organs susceptible to toxicant effects.
  3. Multiple organ interactions via an overlying medium.
  4. Discrete cultures allowing evaluation of effects of a toxicant on a specific cell type.

The IdMOC is a simple experimental system to be applied without the need for specialized laboratory equipments. Virtually all assays developed for cells in culture can be applied to the IdMOC, including higher-throughput approaches. IdMOC has the potential to be a valuable, universally applicable in vitro system for the evaluation of xenobiotic properties including metabolism, distribution, and toxicity.


Fig. 1. Schematic presentation of the principles of the Integrated Discrete Multiple Organ Co-culture (IdMOC) experimental system. The human body is envisioned as consisting of multiple organs which are physically discrete but are interconnected by blood (top figure). This concept is applied in the development of IdMOC (bottom) consisting of multiple inner wells, with cells from each organ cultured individually in the inner wells, and an overlying medium overfilling the inner wells. The IdMOC therefore have multiple organs that are physically discrete but interconnected by an overlying medium akin to a human in vivo.

Fig. 2. A photograph of an IdMOC plate with 6 inner wells per outer well, and with 16 outer wells per plate. The separation of the inner wells from each other is illustrated by the colored fluids on the left half of the IdMOC plate. The use of an overlying medium to integrate the inner wells in each outer well is illustrated on the right half of the plate. This IdMOC plate therefore has 16 independent units for experimentation.
©2007 Albert Li

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