Understanding Of Toxic Mechanisms Research Articles

Anticancer Potential of the S-Heterocyclic Ring Containing Drugs and its Bioactivation to Reactive Metabolites.

Sulfur-containing heterocyclic derivatives have been disclosed for binding with a wide range of cancer-specific protein targets. Various interesting derivatives of sulfur-containing heterocyclics such as benzothiazole, thiazole, thiophene, thiazolidinedione, benzothiophene, and phenothiazine, etc have been shown to inhibit diverse signaling pathways implicated in cancer. Significant progress has also been made in molecular targeted therapy against specific enzymes such as kinase receptors due to potential binding interactions inside the ATP pocket. Sulfur-containing heterocyclic ring metal complexes i. e., benzothiazole, thiazole, thiophene, benzothiophene and phenothiazines are among the most promising active anticancer compounds. However, sulfur heteroaromatic rings, particularly thiophene, are of high structural alert due to their metabolism to reactive metabolites. The mere presence of a structural alert itself does not determine compound toxicity therefore, this review focuses on some specific findings that shed light on factors influencing the toxicity. In the current review, synthetic strategies of introducing the sulfur core ring in the synthesized derivatives are discussed with their structure-activity relationships to enhance our understanding of toxicity mechanisms and develop safer therapeutic options. The sulfur-containing marketed anticancer drugs included in this review direct the synthesis of novel compounds and will help in the development of potent, safer sulfur-based anticancer drugs in near future.

Defining the in vivo mechanism of air pollutant toxicity using murine stress response biomarkers

Air pollution can cause a wide range of serious human diseases. For the informed instigation of interventions which prevent these outcomes there is an urgent need to develop robust in vivo biomarkers which provide insights into mechanisms of toxicity and relate pollutants to specific adverse outcomes. We exemplify for a first time the application of in vivo stress response reporters in establishing mechanisms of air pollution toxicity and the application of this knowledge in epidemiological studies. We first demonstrated the utility of reporter mice to understand toxicity mechanisms of air pollutants using diesel exhaust particles compounds. We observed that nitro-PAHs induced Hmox1 and CYP1a1 reporters in a time- and dose-dependent, cell- and tissue-specific manner. Using in vivo genetic and pharmacological approaches we confirmed that the NRF2 pathway mediated this Hmox1-reporter induction stress reporter activity. We then correlated the activation of stress-reporter models (oxidative stress/inflammation, DNA damage and Ah receptor -AhR- activity) with responses in primary human nasal cells exposed to chemicals present in particulate matter (PM; PM2.5-SRM2975, PM10-SRM1648b) or fresh roadside PM10. To exemplify their use in clinical studies, Pneumococcal adhesion was assessed in exposed primary human nasal epithelial cells (HPNEpC). The combined use of HPNEpC and in vivo reporters demonstrated that London roadside PM10 particles induced pneumococcal infection in HPNEpC mediated by oxidative stress responses. The combined use of in vivo reporter models with human data thus provides a robust approach to define the relationship between air pollutant exposure and health risks. Moreover, these models can be used in epidemiological studies to hazard ranking environmental pollutants by considering the complexity of mechanisms of toxicity. These data will facilitate the relationship between toxic potential and the level of pollutant exposure in populations to be established and potentially extremely valuable tools for intervention studies for disease prevention.

Deriving time-concordant event cascades from gene expression data: A case study for Drug-Induced Liver Injury (DILI).

Adverse event pathogenesis is often a complex process which compromises multiple events ranging from the molecular to the phenotypic level. In toxicology, Adverse Outcome Pathways (AOPs) aim to formalize this as temporal sequences of events, in which event relationships should be supported by causal evidence according to the tailored Bradford-Hill criteria. One of the criteria is whether events are consistently observed in a certain temporal order and, in this work, we study this time concordance using the concept of "first activation" as data-driven means to generate hypotheses on potentially causal mechanisms. As a case study, we analysed liver data from repeat-dose studies in rats from the TG-GATEs database which comprises measurements across eight timepoints, ranging from 3 hours to 4 weeks post-treatment. We identified time-concordant gene expression-derived events preceding adverse histopathology, which serves as surrogate readout for Drug-Induced Liver Injury (DILI). We find known mechanisms in DILI to be time-concordant, and show further that significance, frequency and log fold change (logFC) of differential expression are metrics which can additionally prioritize events although not necessary to be mechanistically relevant. Moreover, we used the temporal order of transcription factor (TF) expression and regulon activity to identify transcriptionally regulated TFs and subsequently combined this with prior knowledge on functional interactions to derive detailed gene-regulatory mechanisms, such as reduced Hnf4a activity leading to decreased expression and activity of Cebpa. At the same time, also potentially novel events are identified such as Sox13 which is highly significantly time-concordant and shows sustained activation over time. Overall, we demonstrate how time-resolved transcriptomics can derive and support mechanistic hypotheses by quantifying time concordance and how this can be combined with prior causal knowledge, with the aim of both understanding mechanisms of toxicity, as well as potential applications to the AOP framework. We make our results available in the form of a Shiny app (https://anikaliu.shinyapps.io/dili_cascades), which allows users to query events of interest in more detail.

Graphene oxide nano-bio interaction induces inhibition of spermatogenesis and disturbance of fatty acid metabolism in the nematode Caenorhabditis elegans

Graphene oxide (GO) has the potential for wide applications, which necessitates an intensive investigation of its potential hazard on human and environmental health. Even if previous studies show reproductive toxicity in the nematode Caenorhabditis elegans, the mechanisms of reproductive toxicity by GO are poorly understood. To understand the underlying mechanisms of GO-induced reproductive toxicity, we investigated the interaction between GO and C. elegans using Raman spectroscopy, sperm counts produced by spermatogenesis, progeny and analyzed the fatty acid metabolism using molecular techniques. GO-characteristic Raman spectral bands measured throughout C. elegans, brood size and Hoecst staining of dissected gonads clearly showed GO accumulation in the reproductive organs, reduced progeny and low sperm counts, which are possibly direct results of the reproductive toxicity from GO exposure. Interestingly, reduced fatty acid metabolites, such as stearic, oleic, palmitoleic, and palmitic acids, were found with GO exposure. We found that GO increased intestinal fat accumulation in wild type N2, fat-5(tm420), and fat-7(wa36) mutants, whereas it decreased fat storage in the fat-6(tm331) and nhr-49(nr2041) mutants. GO exposure affected C. elegans fat accumulation and consumption, which was possibly regulated by daf-16 and nhr-80 gene activity. Also, GO exposure suppressed the survival of long-lived fat-5(tm420) mutants, whereas it increased the survival of short-lived nhr-49(nr2041) mutants. Hence, our studies collectively indicated that GO accumulation in reproductive organs, suppression of spermatogenesis, and the alteration of fatty acid metabolism play critical roles in understanding mechanisms of toxicity in C. elegans.

A systematic review of metabolomics biomarkers for Bisphenol A exposure.

Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl) propane, a common industrial chemical which has extremely huge production worldwide, is ubiquitous in the environment. Human have high risk of exposing to BPA and the health problems caused by BPA exposure have aroused public concern. However, the biomarkers for BPA exposure are lacking. As a rapidly developing subject, metabolomics has accumulated a large amount of valuable data in various fields. The secondary application of published metabolomics data could be a very promising field for generating novel biomarkers whilst further understanding of toxicity mechanisms. To summarize the published literature on the use of metabolomics as a tool to study BPA exposure and provide a systematic perspectives of current research on biomarkers screening of BPA exposure. We conducted a systematic search of MEDLINE (PubMed) up to the end of June 25, 2017 with the key term combinations of 'metabolomics', 'metabonomics', 'mass spectrometry', 'nuclear magnetic spectroscopy', 'metabolic profiling' and 'amino acid profile' combined with 'BPA exposure'. Additional articles were identified through searching the reference lists from included studies. This systematic review included 15 articles. Intermediates of glycolysis, Krebs cycle, β oxidation of long chain fatty acids, pentose phosphate pathway, nucleoside metabolism, branched chain amino acid metabolism, aromatic amino acids metabolism, sulfur-containing amino acids metabolism were significantly changed after BPA exposure, suggesting BPA had a highly complex toxic effects on organism which was consistent with existing studies. The biomarkers most consistently associated with BPA exposure were lactate and choline. Existing metabolomics studies of BPA exposure present heterogeneous findings regarding metabolite profile characteristics. We need more evidence from target metabolomics and epidemiological studies to further examine the reliability of these biomarkers which link to low, environmentally relevant, exposure of BPA in human body.

Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives

Mitochondria are critical cellular organelles for energy generation and are now also recognized as playing important roles in cellular signaling. Their central role in energy metabolism, as well as their high abundance in hepatocytes, make them important targets for drug-induced hepatotoxicity. This review summarizes the current mechanistic understanding of the role of mitochondria in drug-induced hepatotoxicity caused by acetaminophen, diclofenac, anti-tuberculosis drugs such as rifampin and isoniazid, anti-epileptic drugs such as valproic acid and constituents of herbal supplements such as pyrrolizidine alkaloids. The utilization of circulating mitochondrial-specific biomarkers in understanding mechanisms of toxicity in humans will also be examined. In summary, it is well-established that mitochondria are central to acetaminophen-induced cell death. However, the most promising areas for clinically useful therapeutic interventions after acetaminophen toxicity may involve the promotion of adaptive responses and repair processes including mitophagy and mitochondrial biogenesis, In contrast, the limited understanding of the role of mitochondria in various aspects of hepatotoxicity by most other drugs and herbs requires more detailed mechanistic investigations in both animals and humans. Development of clinically relevant animal models and more translational studies using mechanistic biomarkers are critical for progress in this area. Relevance for patients:This review focuses on the role of mitochondrial dysfunction in liver injury mechanisms of clinically important drugs like acetaminophen, diclofenac, rifampicin, isoniazid, amiodarone and others. A better understanding ofthe mechanisms in animal models and their translation to patients will be critical for the identification of new therapeutic targets.

An in vitro metabolomics approach to identify hepatotoxicity biomarkers in human L02 liver cells treated with pekinenal, a natural compound.

An in vitro cell metabolomics study was performed on human L02 liver cells to investigate the toxic biomarkers of pekinenal from the herb Euphorbia pekinensis Rupr. Pekinenal significantly induced L02 cell damage, which was characterised by necrosis and apoptosis. Metabolomics combined with data pattern recognition showed that pekinenal significantly altered the profiles of more than 1299 endogenous metabolites with variable importance in the projection (VIP) > 1. Further, screening correlation coefficients between the intensities of all metabolites and the extent of L02 cell damage (MTT) identified 12 biomarker hits: ten were downregulated and two were upregulated. Among these hits, LysoPC(18:1(9Z)/(11Z)), PC(22:0/15:0) and PC(20:1(11Z)/14:1(9Z)) were disordered, implying the initiation of inflammation and cell damage. Several fatty acids (FAs) (3-hydroxytetradecanedioic acid, pivaloylcarnitine and eicosapentaenoyl ethanolamide) decreased due to fatty acid oxidation. Dihydroceramide and Cer(d18:0/14:0) were also altered and are associated with apoptosis. Additional examination of the levels of intracellular reactive oxygen species (ROS) and two eicosanoids (PGE2, PGF2α) in the cell supernatant confirmed the fatty acid oxidation and arachidonic acid metabolism pathways, respectively. In summary, cell metabolomics is a highly efficient approach for identifying toxic biomarkers and helping understand toxicity mechanisms and predict herb-induced liver injury.

Short- and long-term neurological and psychiatric sequelae of developmental exposure to antiepileptic and anesthetic drugs.

Phenobarbital has been in clinical use for over a century; phenytoin for more than three-quarters of a century, and volatile anesthetics, such as isoflurane and halothane for more than half a century. Despite the long history of use of these compounds for the treatment of epilepsy, and for anesthesia, their effects on brain development are still relatively poorly understood. For this reason, the use of neuroactive drugs during gestation and infancy remains one of the thorniest issues in clinical neurology and neonatology. I had the privilege of co-editing this research topic with my mentor, colleague, and friend, Dr. Karen Gale. Dr. Gale passed away in August of 2014. She and I saw this research topic as an opportunity to highlight the state of the field, and in particular, underscore important future research directions. Together, these articles do exactly that. In this research topic, the current state of the field is reviewed from pre-clinical and clinical perspectives by Turski and Ikonomidou (1) and Gedzelman and Meador (2). These reviews highlight converging results from animal and human studies that underscore the profound impact of these drugs can have on the developing brain. Complementing these reviews, Rochelle Caplan’s translational review focuses on the role of antiepileptic drugs in psychopathology seen in pediatric epilepsy (3). From a translational perspective, the review by Khanna and colleagues (4), and the opinion piece by Pressler and Auvin (5) highlight the importance of neuronal chloride homeostasis as a key regulator of neuronal activity during brain development and as a critical mediator of the efficacy of the first-line therapies for neonatal seizures (i.e., phenobarbital). With respect to anesthesia, the review from Jevtovic-Todorovic and colleagues (6) approaches the problem of anesthesia-induced damage to the developing brain from a highly mechanistic perspective. In particular, they highlight the role of sub cellular organelles, such as mitochondia, in the pathophysiology of neonatal anesthesia exposure. Furthermore, the review by Cheng Wang outlines research approaches and models that have been employed in pre-clinical pediatric neurotoxicology experiments (7). Finally, the research article by Murphy and Baxter highlights sex-dependent effects of neonatal anesthesia exposure on long-term cognitive outcomes (8). In the decade and a half since the first characterization of apoptotic neurodegeneration in the developing rat brain after exposure to these drugs (9–11), this field has made considerable advances in understanding mechanisms of toxicity and characterizing the types of lasting injury that occur after early drug exposure. Indeed, the importance of this research was highlighted in the 2014 NINDS Benchmarks for Epilepsy Research, which lists as a goal, “[To] identify the impact of pharmacological treatment of the epilepsies on fetal and neonatal development. Develop strategies to control seizures in pregnancy without causing harm to either the mother or child” (12). There are, however, many questions that remain to be addressed. To what degree do the mechanisms of developmental neurotoxicity overlap and differ between classes of drugs (e.g., anesthesia vs. antiepileptic drugs). How can pre-clinical research influence clinical practice with respect to these drugs? What therapeutic strategies and adjunct treatments can be used to minimize/avoid developmental neurotoxicity? How does sex modulate risk for developmental neurotoxicity? What features and types of toxicity will be revealed by studies in non-human primate models? It is my sincere hope that 10 years from now, we will have made considerable progress in addressing these issues.

130IN - Gastrointestinal Toxicity in Oncology: Evolutionary Science

ABSTRACT Proper practice of Medical Oncology requires an understanding of the science of regimen-related toxicity (RRT); and as treatment regimens evolve, so too does that science. Mouth ulcers, pain, diarrhea and constipation are among the common side effects of chemotherapy and hinder our ability to give adequate, perhaps curative, doses. As we increase our understanding of toxicity mechanism, so we slowly improve its prevention and treatment. However, this must not come at the cost of tumour-response, thus requiring understanding of the interaction between mechanism of action and of toxicity. Animal models have become increasingly sophisticated, and are used to screen for interventions that reduce toxicity without affecting tumour response. The introduction of the targeted anti-cancer (TAT) agents has further complicated the picture, with GI toxicity being a major, and not always predicted effect. The mechanism of toxicity is often the same as the mechanism of action, making successful management difficult. Each new class of TAT has a new mechanism, epidemiology and presentation of toxicity, so we constantly need to update our understanding of the science. A mouth ulcer caused by methotrexate is not the same as one caused by an mTOR inhibitor. There has been a corresponding evolution in the doctor-patient relationship surrounding toxicity, as we have moved from the paternalistic relationship to a partnership, with Patient Reported Outcomes (PROs) increasingly important. Clinicians will usually rate a given toxicity less severely than will the patient; the message being that we need to listen to the patient. Risk prediction is receiving more attention, with Oncologists now talking about response prediction and risk prediction as two sides of the same coin, aiming to offer treatment to non-toxic responders, treatment with toxicity interventions to toxic responders, and completely avoid ineffective treatment for non-responders. We have moved from the simplistic risk prediction of age, sex, comorbidity and drugs, to complex gene and SNP analysis using modern bioinformatics. However, given the common lag between drug development and successful toxicity interventions, toxicity specialists should be involved earlier in drug development. The science will continue to evolve as long as cancer treatment evolves. Disclosure D. Keefe: Prof Keefe works with many companies in the development of agents in the field of GI toxicity. These include: Helsinn, Entera Health, Pfizer, GSK and Merck.

(Q)SAR Modeling and Safety Assessment in Regulatory Review

The ability to predict clinical safety based on chemical structures is becoming an increasingly important part of regulatory decision making. (Quantitative) structure-activity relationship ((Q)SAR) models are currently used to evaluate late-arising safety concerns and possible nonclinical effects of a drug and its related compounds when adequate safety data are absent or equivocal. Regulatory use will likely increase with the standardization of analytical approaches, more complete and reliable data collection methods, and a better understanding of toxicity mechanisms.

Toxicity of Chlorine-Produced Oxidants to Arabian Killifish Embryos for Acute Developmental Stages Exposures

Background: Fish embryos are excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible acute and chronic effects. Therefore, we present the use of Arabian killifish (Aphanius dispar) embryos, an indigenous species to the Arabian Gulf, to study the effect of chlorine-produced oxidants to marine organisms in the Qatari coastal area. Objective: The objective of this study is to develop chlorine toxicity data for the marine Fish Embryo Toxicity (mFET) test. The test is designed as a means to replace or refine the use of marine juvenile and/or adult fish in standard approaches evaluating toxicity of chemicals and effluents. Methods: Embryos were collected from a breeding stock of sexually mature Arabian killifish. Testing was initiated as soon as possible after fertilization of the eggs with exposure to aqueous concentrations of calcium hypochlorite (0.10 – 12.3 mg/l) for up to 240 hours. The investigated endpoints included; coagulated eggs, somite development, heartbeat, tail detachment, hatchability and post hatch mortality. Results: The results showed a developmental stage-dependent response to chlorine. During earlier developmental stages, chlorine had reduced effects on the embryos and the survival rate and hatchability were high, even at relatively high concentrations. In later developmental stages (pre to post hatch, eleutheroembryo), the embryos were significantly more sensitive to chlorine than in the early stages of development. The chorion, (membrane surrounding the egg), is believed to provide a barrier against chlorine in early stages of embryo development. Conclusions: Taking into account the findings presented here, Killifish embryos exhibit the ability to be an indicator organism for environmental risk assessments of the Qatari coastal area. Benefits include, animal alternative, ease of fish breeding, clarity of the embryos, reduced sample size, reduced waste generation and shorter study duration.

The Role of Drug Metabolism in Drug Discovery and Development

Drug metabolism and pharmacokinetic studies have essential roles to play in all stages of the research and development process, ideally being involved from drug discovery through to post-marketing surveillance, because pharmacokinetic variability is the cause of major problems in drug development. The origins of these problems have been termed “pharmacokinetic defects” and include, among others, poor absorption, very short or very long half-life, enzyme inhibition and high first-pass effect. Traditionally, the significance of many of these has been realized only after extensive clinical development. Failure to take these into consideration can result in expensive delay or failure during development and make an approved drug vulnerable in the marketplace. The thoughtful inclusion of new feedback loops offers the prospect of improved decision-making at various stages of drug development. These should be based on quality metabolic and pharmacokinetic data and exploit the opportunities which the new biology offers to “humanize” drug development by predicting human metabolic pathways, anticipating kinetic variability in human populations and understanding mechanisms of toxicity. Such improved decision making should contribute to enhanced time- and cost-efficiency of development and ultimately lead to safer, more easily used drugs.

Mapping Progress in Toxicology Research by the Content of the Best Papers Published in Society of Toxicology Journals: A Synopsis of the Best Paper Awardees (1974-2011)

In 1973, an anonymous donor contributed funds in honor of Dr Frank Blood for an award that would bring recognition to the best paper published in the Society of Toxicology journal. As time was needed to establish criteria for the award and review papers, the first award was bestowed in 1974. At its inception, responsibility for selecting the best paper was assigned to the Awards Committee, but over time, this role was transferred to the Board of Publications. Members are encouraged to nominate papers for consideration, but the Editorial offices have also played an active role in selecting papers for consideration. In general, articles selected as the best paper are recognized because they contain novel, new data, represent significant progress in understanding mechanisms of toxicity and/or advance the protection of human and environmental health. A total of 51 papers have been awarded best paper honors in Toxicology and Applied Pharmacology (specifically designated as the Frank R. Blood awardee), Fundamental and Applied Toxicology, and Toxicological Sciences since 1974. The complete list of the papers selected as the best manuscripts published in Society of Toxicology journals is presented in Table 1. It is not possible to provide a full account of the scope and significance of each manuscript recognized as the best paper since 1974. Rather, the purpose of this article is to highlight the content of these papers as they reflect on fundamental tenets in toxicology. Additionally, this collection of papers also serves to illustrate important advances in toxicologic research. Although the papers are diverse in content, several general themes emerged while reviewing the collection of best papers, and these themes served to organize the content, scope, and significance of the articles. The general themes by which the papers are discussed in this article include the following:

Highlights of the XII International Congress on Toxicology, 19 – 23 July 2010, Barcelona, Spain

Importance of the field: There are few true international meetings dedicated to covering multiple areas of toxicology. The XII International Congress of Toxicology (IUTOX) held from 19 to 23 July 2010 in Barcelona, Spain is one such meeting. The IUTOX is important as its emphasis is on chemical safety and integrating approaches and alternative possibilities to protecting public health. The meeting was an important forum with professional interactions in different subspecialties of toxicology addressing these current topics.Areas covered in this review: The basis of toxic effects including mechanistic, effects testing, monitoring and alternative methods are covered in this meeting highlights article. Coverage of industry, clinical toxicologists, environmentalists, regulators and technological developers is provided.What the reader will gain: Insight into the coverage of topics discussed at the XII IUTOX meeting.Take home message: Current topics in toxicology with international impact are presently centered on new testing strategies, biomarkers and understanding mechanisms of toxicity to help address the safety and risk of substances relevant to public health.

RESEARCH ISSUES AND NITIATIVES: Agreement Could Speed Reductions in Animal Use

A Memorandum of Cooperation signed by representatives of the agencies responsible for evaluating and recommending alternative toxicity testing methods in the United States, the European Union, Japan, and Canada represents a major step foward in the use of improved alternatives to animal testing. The agreement will enhance international cooperation and coordination in the validation of alternative test methods, the execution of independent peer-review meetings and reports, and the development of harmonized test method recommendations for regulatory consideration. “Early and consistent cooperation in these three critical areas will greatly enhance the likelihood of international acceptance of alternative test methods determined to be useful for safety evaluations,” says William Stokes, director of the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and executive director of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). “Most importantly, there will be international discussion and agreement on the design of validation studies, including test method protocols and chemical selection, before studies are initiated.” Linda Birnbaum, director of the National Toxicology Program and the NIEHS, signed the agreement 27 April 2009 on behalf of NICEATM, along with representatives for the European Centre for the Validation of Alternative Methods, the Japanese Centre for the Validation of Alternative Methods, and the Environmental Health Science and Research Bureau of Health Canada. “It’s a big step to have agreement between the Europeans, the Japanese, the Canadians, and us in terms of approaching the evaluation of alternative test methods,” says Birnbaum. “There will be more sharing of information because of the agreement, and hopefully that will lead to more rapid decision making.” That’s not to say that enhanced international cooperation and leveraging of resources will guarantee acceptance of new test methods. “Because each regulatory agency has different legislative authority within their own country, I don’t foresee this as being an automatic, across-the-board acceptance,” says Jodie Kulpa-Eddy, vice chair of ICCVAM and a senior staff veterinarian with the Animal and Plant Health Inspection Service of the U.S. Department of Agriculture. “Each group is going to have to take a look at the data and determine whether or not a test is going to be applicable under their umbrella.” Still, speedier and more coordinated consideration of new test methods is expected to increase the likelihood that methods will be accepted by individual regulatory agencies. The benefits, says Stokes, will include a reduction in the use of animals for product safety testing and improved public health with the availability of more predictive toxicity tests achieved through advances in understanding of toxic mechanisms and incorporation of new technologies. He adds that the initiative has received “very positive support and feedback” from all stakeholders, including the animal welfare community and industry. Many companies with a global focus stand to benefit from the international cooperation. “Instead of having to validate test methodologies multiple times, you can now potentially do it once and have it applied to multiple markets,” says Syed Ahmed Mustafa, director of sales and marketing at CeeTox, Inc., an in vitro safety testing company in Michigan. “This will enable multinational companies to only have to do one test to cover all of their operating regions.” According to Birnbaum, this agreement and other recent advances in the field have brought about substantial progress in the development of alternative test methods. “It took a long time to get to this point; there was a great deal of evaluation that needed to be done,” she says. “And now we’re at the point where I think things are beginning to move much more rapidly.”

Systems biology and functional genomics approaches for the identification of cellular responses to drug toxicity

Extensive growth in the field of molecular biology in recent decades has led to the development of new and powerful experimental and computational tools that enable the analysis of complex biological responses to chemical exposure on both a functional and structural genetic level. The ability to profile global responses on a transcriptional level has become a valuable resource in the science of toxicology and attempts are now being made to further understand toxicity mechanisms by incorporating metabolomics and proteomics approaches. In addition, recent progress in understanding the extent of the genetic diversity within and between species allows us to take a fresh look at research on genetic polymorphisms that may influence an individual's susceptibility to toxicity. Whereas new technologies have the potential to make a sizeable impact on our understanding of the mechanisms of toxicity, considerable challenges remain to be addressed, especially with regard to the regulatory acceptance and successful integration of omics data. This review highlights recent advancements in the application of functional and structural genomics techniques to chemical hazard identification and characterization, and to the understanding of the interindividual differences in susceptibility to adverse drug reactions.

The zebrafish embryo model in environmental risk assessment—applications beyond acute toxicity testing

The use of fish embryos is not regulated by current legislations on animal welfare and is therefore considered as a refinement, if not replacement of animal experiments. Fish embryos represent an attractive model for environmental risk assessment of chemicals since they offer the possibility to perform small-scale, high-throughput analyses. Beyond their application for determining the acute toxicity, fish embryos are also excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible adverse and long-term effects. Therefore, we have reviewed the scientific literature in order to indicate alternative applications of the fish embryo model with focus on embryos of the zebrafish. The analysis of the mode of action is important for the risk assessment of environmental chemicals and can assist in indicating adverse and long-term effects. Toxicogenomics present a promising approach to unravel the potential mechanisms. Therefore, we present examples of the use of zebrafish embryos to study the effect of chemicals on gene and protein patterns, and the potential implications of differential expression for toxicity. The possible application of other methods, such as kinase arrays or metabolomic profiling, is also highlighted. Furthermore, we show examples of toxicokinetic studies (bioconcentration, ABC transporters) and discuss limitations that might be caused by the potential barrier function of the chorion. Finally, we demonstrate that biomarkers of endocrine disruption, immune modulation, genotoxicity or chronic toxicity could be used as indicators or predictors of sub-acute and long-term effects. The zebrafish embryo represents a model with an impressive range of possible applications in environmental sciences. Particularly, the adaptation of molecular, system-wide approaches from biomedical research is likely to extend its use in ecotoxicology. Challenges for future research are (1) the identification of further suitable molecular markers as indicators of the mode of action, (2) the establishment of strong links between (molecular) effects in short-term assays in embryos and long-term (toxic) effects on individuals, (3) the definition of limitations of the model and (4) the development of tests that can be used for regulatory purposes.

Toward an integrative systems toxicology

10.1517/14622416.5.8.1163 © Assessing toxicity is among the most ancient and important of human endeavors. Long before the taming of fire or the advent of tool use, humankind was confronted with a most basic problem of existence: what to ingest. Today, toxicology impacts every facet of our lives – from the food, supplements and therapeutics that we consume, to the consumer products that we utilize and, ultimately, the environment in which we live. Toxicity must, therefore, be considered not only as a problem for basic science, but in the context of safety and its accompanying regulatory imperatives. In its most basic form, toxicity testing can be distilled down to two concepts: exposure and assessment. A model system – typically a laboratory animal – is exposed to a test article, and then examined to determine whether any adverse effects have occurred. Stated differently, a biological system is perturbed and the resulting phenotype is characterized. Phenotype is conventionally defined as the visible properties of an organism resulting from gene–environment interactions. Detailed phenotypic characterization in the form of histopathology has dominated the field of toxicology and provided a strong basis for understanding specific mechanisms of toxicity and, more generally, the effects of chemical exposure and its relationship to disease. Importantly, the depth with which the effects of chemical exposure can now be described has been fundamentally enhanced through the introduction of comprehensive profiling technologies. Toxicogenomics, in its most narrow sense, represents a synthesis of toxicology and genomics in an effort to understand mechanisms of toxicity through the use of genomic information, such as gene expression modulation captured using microarrays [1]. An advantage of this approach is that a large number of attributes – in this case, transcripts – can be used as descriptors of the biological system under scrutiny. In principle, the potential for accurately differentiating between closely related systems and the outcomes induced in these systems could be enhanced by enlarging the potential descriptor space. Thus, a profile, or signature, of gene expression can be used to classify mechanisms of toxicity [2]. But genomics represents only a piece of the puzzle, and alternative data streams, such as proteomics [3] and metabolomics [4], can also be used to provide highcontent system descriptors. Proponents of each technique can point to technological advantages of their chosen method, as well as state their case as to the comparative relevance of each data type for understanding mechanisms of toxicity. Clearly, no single method can provide a truly comprehensive view of the state of a biological system, nor can any single method adequately describe the interplay between genes, proteins and metabolites in a way that reflects the dynamics of the system. The goal of using molecular profiling techniques in the context of toxicology should be to move the science beyond the mere classification of known compounds and test articles and into the realm of real understanding and knowledge about the specific mechanisms, whereby chemical and environmental agents effect adverse outcomes. For this, we must capture data from multiple sources and provide a holistic framework for their integration. Toxicogenomics must, therefore, expand beyond the narrow scope of its own definition to encompass multiple, high-content data streams in a manner that accounts for the interdependencies inherent between the elements that comprise a system. In short, we must advance toxicology toward systems biology. Systems biology has been described in many ways, but the essential elements of the original definition remain the most relevant: systematically perturbing a biological system, monitoring the behavior of the elements that make up a system (e.g., genes, proteins, and metabolites), integrating these data, and describing the structure of their interrelationships using mathematical models in an effort to account for emergent properties of the system [5]. The perceived promise of a systems approach to biological problems has led to a great deal of interest and activity in the pharmaceutical, biotechnology and educational sectors. In particular, enhancing our understanding of safety and efficacy issues as a prelude to personalized medicine has galvanized previously disparate efforts. Significant progress has been made in developing a suite of tools for perturbing biological systems by changing their

Application of toxicogenomics to toxicology: basic concepts in the analysis of microarray data.

Toxicology and the practice of pathology are rapidly evolving in the postgenomic era. Observable treatment related changes have been the hallmark of toxicology studies. Toxicogenomics is a powerful new tool that may show gene and protein changes earlier and at treatment levels below the limits of detection of traditional measures of toxicity. It may also aid in the understanding of toxic mechanisms. It is important to remember that it is only a tool and will provide meaningful results only when properly applied. As is often the case with new experimental tools, the initial utilization is driven more by the technology than application to problem solving. Toxicogenomics is interdisciplinary in nature including at a minimum, pathology, toxicology, and genomics. Most studies will require the input from the disciplines of toxicology, pathology, molecular biology, bioinformatics, biochemistry, and others depending on the types of questions being asked.

Advances in understanding mechanisms of toxicity and implications for risk assessment

Knowledge of mechanism of action of a toxicant can greatly improve the accuracy of risk estimation by replacing with data the many default assumptions of risk assessment. Results from studies on comparative pharmacokinetics, metabolism, cell biology, and molecular biology have been successfully applied to problems of interspecies extrapolation, interindividual differences in susceptibility, and the relevance of high-dose findings for low-dose risk estimation. Examples are provided. Extremely rapid progress in understanding the molecular control of embryonic pattern formation and organogenesis has the potential to significantly improve the accuracy of risk assessment, especially by providing a sounder basis for characterizing interspecies differences, individual susceptibility, and multifactorial (gene-environment) etiologies of abnormal development. However, it will be necessary to quantitate toxicant-induced changes at the molecular level and to determine the level of change needed to perturb higher levels of biological organization at which adverse effects are manifested. It will also be important for risk assessment methodology to evolve so that it can better and more routinely accommodate mechanistic information. There is great potential for the recent and coming advances in knowledge of the molecular and cellular basis of abnormal development to be applied to risk assessment. Consideration should be given to shifting some of the resources now allocated to hazard screening to investigating the mechanisms of chemically induced abnormal development.