Predictive Toxicology and the Promise of Embryonic Stem Cells

Predictive toxicology focuses on the evaluation of chemicals that can result in adverse effects on humans. Hundreds of chemicals, namely enzymes, hormones, and neurotransmitters are synthesized endogenously in mammalian organisms and are critical to cellular physiology and homeostasis. Predictive toxicology addresses largely synthetic chemicals that are not produced by target mammalian organisms, or xenobiotics, such as therapeutic small molecules, and environmental toxicants.

Adverse chemical effects on biological systems range from genome-wide nuclei acid mutagenesis, or genotoxicity, to tissue-specific alterations, such as liver failure and cardiac arrhythmia. Importantly, chemical exposure during critical periods of development and/or adulthood may be an underlying cause of several life-threatening and debilitating diseases such as Parkinson's disease,1 autism2 and cancer.3 Strikingly, certain alterations from toxic response can be transmitted through the germ line and may affect as much as three generations from the exposed individual.4'5 The effects of environmental chemicals on subsequent generations is considerable, since inheritable changes are not restricted to a few individuals, but may affect the majority of the offsprings, as suggested by recent findings in epigenetic changes in sperm.5

This discovery urges the development and implementation of robust screening systems to prevent both inheritable and non-heritable abnormalities following human exposure to harmful chemicals.

Animal models have been invaluable for risk assessment of chemical compound safety; however, critical limitations persist for robust prediction of certain toxic outcomes in humans. Toxicity to tissues such as the hematopoetic and gastrointestinal systems has overall similar outcomes between animal models and humans.6 In other organs, such as the liver, where xenobiotic-induced toxicity accounts for approximately 50% of acute liver failure in humans,1 the value of animal models is very limited.8 A favorable prognosis to acute liver failure is conferred upon liver transplantation, which is a challenge in the face of persistent organ shortening. The restriction of animal models to accurately reflect human response is also true for other areas in toxicology.

Abnormal development of human fetuses following in utero chemical exposure may arise despite opposite predictions from animal studies, since the sensitivity of dose exposure can be higher in humans than that detected in animals.9 Indeed, no animal species has been viewed as ideal for developmental toxicity,10 where the majority of studies are conducted on rabbits and rats.

Moreover, decision-making findings from regulatory studies in animals may take up to two years, as indicated by standard carcinogenicity studies in rats.11 In the European Member States, approximately 1 million animals are sacrificed per year for toxicology studies.12 In addition to significant costs and ethical factors associated with the use of experimental animals, the implementation of initiatives such as the REACH program (Registration, Evaluation and Authorization of new and existing Chemicals) by the European Parliament13 strengthens the urgent need of alternative, cost-effective and time-saving models to test for the adverse effects of thousands of chemicals in humans. The goal of REACH is to consolidate full risk assessment of all chemicals marketed at more than one ton within the next 10 to 15 years, which translates into a pressing need for toxicology assays that are more efficient. The opportunity for alternative toxicology models is favored by REACH guidelines, which strongly support the use of in vitro testing for the first phase of chemical analysis. Alternative toxicology models will not replace experimental animals altogether, since certain in vivo systemic interactions that are relevant for toxic outcomes cannot be anticipated in vitro. Nonetheless, alternative in vitro testing may serve as a better predictor for human response in certain cell types and may consequently contribute to refine and reduce the use of animals in research.

In vitro differentiation of embryonic stem (ES) cells provides a renewable source of multiple cell types that are important targets of adverse chemical effects.14'15 The successful isolation and establishment of human embryonic stem cell (hESC, Fig. 1) lines16 enabled striking opportunities for predictive toxicology studies in genetically stable, non-immortalized human cells that can be translated to chemical safety assessments in both embryonic and somatic cell types. Functional neurons17 and cardiomyocytes,18 which are key targets of toxicology effects, have been generated following in vitro differentiation of human embryonic stem cells. hESC-neurons and cardiomyocytes exhibited lineage-specific transcripts

Fig. 1. An undifferentiated colony of H1 human embryonic stem cells grown in the absence of feeder cells.

and structural proteins but most importantly, demonstrated functional phe-notypes with release of neurotransmitters and intercellular conduction of electric stimuli, respectively. The finding that certain human embryonic stem cell-derived cell types exhibit functional properties of in vivo counterparts increases the accuracy in which toxic effects can be detected. Cellular alterations in response to chemical insult can now be identified and interpreted in multiple levels, from changes in gene expression to cellular physiology and metabolism.19,20 In summary, generation of functional differentiated cells from human embryonic stem cells supports systems toxicology studies which provide sensitive detection of chemical safety risks.

The liver is the major organ for metabolism and transformation of xeno-biotics, where hepatocytes, the primary cells of the liver, perform core functions, including metabolism of diverse dietary molecules and chemicals and detoxification of compounds. Therefore, the availability of large numbers of human hepatocytes is a constant critical demand in toxicology and chemical risk assessment.21 Discarded livers which cannot be used for organ transplantation are the limited source for primary human hepatocytes, which in turn exhibit very limited in vitro proliferation ability.22 Initial progress has been achieved to generate hepatocyte-like cells from human embryonic stem cells following extensive research efforts.23,24 Recent studies revealed that differentiation of the endoderm, the liver precursor lineage, evolves from human embryonic stem cells in a more intricate manner than ectoderm and mesoderm lineages.25 For proper differentiation to ensue, an apparent epithelial-to-mesenchymal transition is required.25 This study has also determined in vitro morphogens and culture conditions that favor definitive endoderm, which should facilitate further progress to obtain functional hepatocytes from hESC for toxicity and transplantation applications.

In summary, development of alternative toxicology systems based upon embryonic stem cells have promising opportunities in multiple areas, such as developmental, cardiac and hepatic toxicity. While in vitro assays lack systemic effects that may be involved in toxic response, a humanized model offers a valuable opportunity to replace, reduce and refine (3Rs of animal research) the use of experimental animals. Additionally, the use of human embryonic stem cells to understand signaling pathways involved in toxic response with the translation of these findings to epidemiology and preven-tative medicine may be a shorter term benefit of this technology towards human health in comparison to longer term clinical applications.

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