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An important goal of neurotoxicological research is to provide relevant and accurate risk assessment of environmental and pharmacological agents for populations and individuals. Owing to the challenges of human subject research and the real possibility of species specific toxicological responses, neuronal lineages derived from human embryonic stem cells (hESCs) and human neuronal precursors have been offered as a potential solution for validation of neurotoxicological data from model organism systems in humans. More recently, with the advent of induced pluripotent stem cell (iPSC) technology, there is now the possibility of personalized toxicological risk assessment, the ability to predict individual susceptibility to specific environmental agents, by this approach. This critical advance is widely expected to facilitate analysis of cellular physiological pathways in the context of human neurons and the underlying genetic factors that lead to disease. Thus this technology opens the opportunity, for the first time, to characterize the physiological, toxicological, pharmacological and molecular properties of living human neurons with identical genetic determinants as human patients. Furthermore, armed with a complete clinical history of the patients, human iPSC (hiPSC) studies can theoretically compare patients and at risk groups with distinct sensitivities to particular environmental agents, divergent clinical outcomes, differing co-morbidities, and so forth. Thus iPSCs and neuronal lineages derived from them may reflect the unique genetic blueprint of the individuals from which they are generated. Indeed, iPSC technology has the potential to revolutionize scientific approaches to human health. However, before this overarching goal can be reached a number of technical and theoretical challenges must be overcome. This review seeks to provide a realistic assessment of hiPSC technology and its application to risk assessment and mechanistic studies in the area of neurotoxicology. We seek to identify, prioritize, and detail the primary hurdles that need to be overcome if personalized toxicological risk assessment using patient-derived iPSCs is to succeed.
Copyright © 2012 Elsevier Inc. All rights reserved.
The field of cytochrome P450 (P450) research has developed considerably over the past 20 years, and many important papers on the roles of P450s in chemical toxicology have appeared in Chemical Research in Toxicology. Today, our basic understanding of many of the human P450s is relatively well-established, in terms of the details of the individual genes, sequences, and basic catalytic mechanisms. Crystal structures of several of the major human P450s are now in hand. The animal P450s are still important in the context of metabolism and safety testing. Many well-defined examples exist for roles of P450s in decreasing the adverse effects of drugs through biotransformation, and an equally interesting field of investigation is the bioactivation of chemicals, including drugs. Unresolved problems include the characterization of the minor "orphan" P450s, ligand cooperativity and kinetic complexity of several P450s, the prediction of metabolism, the overall contribution of bioactivation to drug idiosyncratic problems, the extrapolation of animal test results to humans in drug development, and the contribution of genetic variation in human P450s to cancer incidence.
Of the 57 human cytochromes P450 (P450) and 58 pseudogenes discovered to date, (http://drnelson.utmem.edu/CytochromeP450.html ), 1/4 still remain "orphans" in the sense that their function, expression sites, and regulation are still largely not elucidated. The post-human genome-sequencing project era has presented the research community with novel challenges. Despite many insights gathered about gene location and genetic variations in our human genome, we still lack important knowledge about these novel P450 enzymes and their functions in endogenous and exogenous metabolism, as well as their possible roles in the metabolism of toxicants and carcinogens. Our own list of such orphans currently consists of 13 members: P450 2A7, 2S1, 2U1, 2W1, 3A43, 4A22, 4F11, 4F22, 4V2, 4X1, 4Z1, 20A1, and 27C1. Some of the orphans, e.g. P450s 2W1 and 2U1, already have putative assigned functions in arachidonic acid metabolism and may activate carcinogens. However, at this point, for the majority of them more knowledge is available about their genes and single nucleotide polymorphisms than of their biological functions. It is noteworthy that most P450 orphans express high interspecies sequence conservation and have orthologs in rodents (e.g. CYP4X1/Cyp4x1, CYP4V2/Cyp4v3). This review summarizes recent knowledge about the P450 orphans and questions remaining about their specific roles in human metabolism.
The biochemical facets of toxicology have always had a major role in providing insight into mechanisms. Some of the history of the development of this area is summarized, including metabolism, enzymology, and the chemistry of reactive intermediates. Knowledge in these fields has had a major impact in the areas of drug metabolism and safety assessment, which are both critical steps in the development of pharmaceuticals and the rational use of commodity chemicals. The science of toxicology has developed considerably with input from other disciplines and today is poised to emerge as a predictive science with even more dramatic impact. The challenges ahead are considerable but there is renewed excitement in the potential of the field. As in the past, further advances in the field of toxicology will require the input of knowledge from many disciplines.
Herbert Remmer was a pioneer in the study of the phenomenon of enzyme induction. He was also a leading figure, if not the foremost, in the development of the discipline of toxicology in Germany during his tenure as Professor of Toxicology at the University of Tübingen. Included here are some brief thoughts about where toxicology came from, where it is today, and what the future is. Toxicology is at a crossroads today, at an interface between basic science and applied projects. From its past as a descriptive discipline, it has incorporated a medley of concepts and technology from basic science. The usefulness of many approaches is now being evaluated. The hope is that toxicology will be able to be much more predictive in the future; a great need exists in the pharmaceutical industry. The shape of academic toxicology is also changing.
Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity.