Toxicity testing in the 21st century: a vision and a strategy.

With the release of the landmark report Toxicity Testing in the 21st Century: A Vision and a Strategy, the U.S. National Academy of Sciences, in 2007, precipitated a major change in the way toxicity testing is conducted. It envisions increased efficiency in toxicity testing and decreased animal usage by transitioning from current expensive and lengthy in vivo testing with qualitative endpoints to in vitro toxicity pathway assays on human cells or cell lines using robotic high-throughput screening with mechanistic quantitative parameters. Risk assessment in the exposed human population would focus on avoiding significant perturbations in these toxicity pathways. Computational systems biology models would be implemented to determine the dose-response models of perturbations of pathway function. Extrapolation of in vitro results to in vivo human blood and tissue concentrations would be based on pharmacokinetic models for the given exposure condition. This practice would enhance human relevance of test results, and would cover several test agents, compared to traditional toxicological testing strategies. As all the tools that are necessary to implement the vision are currently available or in an advanced stage of development, the key prerequisites to achieving this paradigm shift are a commitment to change in the scientific community, which could be facilitated by a broad discussion of the vision, and obtaining necessary resources to enhance current knowledge of pathway perturbations and pathway assays in humans and to implement computational systems biology models. Implementation of these strategies would result in a new toxicity testing paradigm firmly based on human biology.

An analysis of the attrition of drug candidates from four major pharmaceutical companies.

The pharmaceutical industry remains under huge pressure to address the high attrition rates in drug development. Attempts to reduce the number of efficacy- and safety-related failures by analysing possible links to the physicochemical properties of small-molecule drug candidates have been inconclusive because of the limited size of data sets from individual companies. Here, we describe the compilation and analysis of combined data on the attrition of drug candidates from AstraZeneca, Eli Lilly and Company, GlaxoSmithKline and Pfizer. The analysis reaffirms that control of physicochemical properties during compound optimization is beneficial in identifying compounds of candidate drug quality and indicates for the first time a link between the physicochemical properties of compounds and clinical failure due to safety issues. The results also suggest that further control of physicochemical properties is unlikely to have a significant effect on attrition rates and that additional work is required to address safety-related failures. Further cross-company collaborations will be crucial to future progress in this area.

Toxicogenomics in risk assessment: an overview of an HESI collaborative research program.

The value of genomic approaches in hypothesis generation is being realized as a tool for understanding toxicity and consequently contributing to an assessment of drug and chemical safety. In 1999 the membership of the International Life Sciences Institute Health and Environmental Sciences Institute formed a committee to develop a collaborative scientific program to address issues, challenges, and opportunities afforded by the emerging field of toxicogenomics. Experts and advisors from academia and government laboratories participate on the committee, along with approximately 30 corporate member organizations from the pharmaceutical, agrochemical, chemical, and consumer products industries. The committee has designed, conducted, and analyzed numerous toxicogenomic experiments within the broad fields of hepatotoxicity, nephrotoxicity, and genotoxicity. The considerable body of data generated by these programs has been instrumental in increasing understanding of sources of biological and technical variability in the alignment of toxicant-induced transcription changes with the accepted mechanism of action of these agents and the challenges in the consistent analysis and sharing of the voluminous data sets generated by these approaches. Recognizing the importance of standardized microarray data formats and public repository databases as the mechanism by which microarray data can be compared and interpreted by the scientific community, the committee has partnered with the European Bioinformatics Institute to develop a database to house the data generated by its collaborative research.

Use of genomics in toxicology and epidemiology: findings and recommendations of a workshop.

The sequencing of the human genome has revolutionized biology and led to an astounding variety of technologies and bioinformatics tools, enabling researchers to study expression of genes, the function of proteins, metabolism, and genetic differences within populations and between individuals. These scientific advances are making an impact in the medical research community and hold great promise for prevention, diagnosis, and treatment of diseases. This developing field also holds great promise for improving the scientific basis for understanding the potential impacts of chemicals on health and the environment. A workshop sponsored by the International Council of Chemical Associations was held to review the state of the science in the application of genomics technologies in toxicology and epidemiology. Further, consideration was given to the ethical, legal, and regulatory issues and their influence on the direction and application of genomics technologies to environmental health research. Four overarching themes emerged from the workshop: Genomics technologies should be used within a framework of toxicology and epidemiology principles and applied in a context that can be used in risk assessment; effective application of these technologies to epidemiology will require suitable biologic samples from large and diverse population groups at the relevant period of exposure; ethical, legal, and social perspectives require involvement of all stakeholder communities; and a unified research agenda for genomics technologies as applied to toxicology, epidemiology, and risk assessment is urgently needed for the regulatory and scientific communities to realize the potential power and benefits of these new technologies.

Downregulation of lactoferrin by PPARalpha ligands: role in perturbation of hepatocyte proliferation and apoptosis.

PPARalpha (peroxisome proliferator activated receptor alpha) is a transcription factor that mediates the rodent liver tumorigenic responses to peroxisome proliferators via regulation of genes that remain to be identified. Using microarray gene expression profiling of mRNA from wild type versus PPARalpha null mice, we detected a 3- to 7-fold downregulation of hepatic lactoferrin (LF) in response to the PP, diethylhexylphthalate (DEHP; 1150 mg/kg). Northern blot analyses confirmed a significant downregulation of LF mRNA by DEHP in wild type mouse liver. Since LF has been reported to repress tumor necrosis factor-alpha (TNF-alpha), LF downregulation by PPs may permit TNF-alpha levels to rise, enhancing hepatocyte survival and proliferation. To test this hypothesis, we asked if exogenous LF could prevent the perturbation of hepatocyte growth by PPs but not by TNF-alpha. In vitro, the PPs monoethylhexylphthalate (MEHP; 500 microM, the active metabolite of DEHP) and another PP, nafenopin (50 microM) or exogenous TNF-alpha (5000 U/ml) induced hepatocyte proliferation and suppressed apoptosis. LF (200 microM) blocked the growth but not the peroxisome proliferation response to PPs but could not block the growth response to TNF-alpha. Immunocytochemistry using specific antibodies to LF but also to transferrin (TF), a related gene previously shown to contain a PP response element (PPRE), demonstrated that both LF and TF are expressed in murine liver. Furthermore, both were downregulated by DEHP in both wild type and PPARalpha null mouse liver. These data suggest that the regulation of iron binding proteins by PPARalpha ligands plays a role in PP-mediated liver growth, but not in peroxisome proliferation.

Custom cDNA microarrays; technologies and applications.

Technologies designed to characterise genes and their products on a discovery scale are now having an impact on many areas of biology, including toxicology. A number of platforms exist which measure changes in expression of potentially thousands of genes simultaneously. These approaches, when applied to toxicology, are termed 'toxicogenomics' and promise to greatly facilitate mechanism-based research on toxicant action with the longer term possibility of assisting in the identification of potential toxicity issues earlier in the development of new pharmaceutical, agrochemical and chemical products. An example of such a platform developed in our laboratory is ToxBlot II, a custom microarray containing cDNAs representing 12564 human genes chosen on the basis of their potential relevance to a broad range of toxicities. ToxBlot II can assist in characterising many outcomes including processes as diverse as immune system response, receptor biology, signal transduction, protein modification, membrane transport, growth and development, metabolism, oxidative stress and regulation of the cell cytoskeleton. Furthermore, ToxBlot II allows the simultaneous expression profiling of genes representing entire cellular pathways, facilitating a very detailed investigation of potential mechanisms of toxicity. Our laboratory is applying this and other custom microarrays to many areas of toxicology, including endocrine disruption, receptor biology, stress response and the effect of toxicants on immune function. Such approaches can be particularly valuable when used in conjunction with 'functional genomics' such as transgenic or knockout models.

A high content screening assay for identifying lysosomotropic compounds.

Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable. Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP>2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity.