Zebrafish as a correlative and predictive model for assessing biomaterial nanotoxicity

The lack of correlative and predictive models to assess acute and chronic toxicities limits the rapid pre-clinical development of new therapeutics. This barrier is due in part to the exponential growth of nanotechnology and nanotherapeutics, coupled with the lack of rigorous and robust screening assays and putative standards. It is a fairly simple and cost-effective process to initially screen the toxicity of a nanomaterial by using invitro cell cultures; unfortunately it is nearly impossible to imitate a complimentary invivo system. Small mammalian models are the most common method used to assess possible toxicities and biodistribution of nanomaterials in humans. Alternatively, Daniorerio, commonly known as zebrafish, are proving to be a quick, cheap, and facile model to conservatively assess toxicity of nanomaterials.     

Zebrafish as model organisms for studying drug-induced liver injury

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review highlights the strengths and weaknesses of using zebrafish as a high-throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different from that of the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways to those in humans; they possess a wide range of cytochrome P450 enzymes that enable metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de-ethylation. Following exposure to a range of hepatotoxic drugs, the zebrafish liver develops histological patterns of injury comparable to those of mammalian liver, and biomarkers for liver injury can be quantified in the zebrafish circulation. The zebrafish immune system is similar to that of mammals, but the zebrafish inflammatory response to DILI is not yet defined. In order to quantify DILI in zebrafish, a wide variety of methods can be used, including visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring of histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers.     

Zebrafish as a model vertebrate for investigating chemical toxicity.

Zebrafish (Danio rerio) has been a prominent model vertebrate in a variety of biological disciplines. Substantial information gathered from developmental and genetic research, together with near-completion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model. Although still in its infancy, there is a clear potential for zebrafish to provide valuable new insights into chemical toxicity, drug discovery, and human disease using recent advances in forward and reverse genetic techniques coupled with large-scale, high-throughput screening. Here we present an overview of the rapidly increasing use of zebrafish in toxicology. Advantages of the zebrafish both in identifying endpoints of toxicity and in elucidating mechanisms of toxicity are highlighted.     

抗肿瘤新药研究的新型模式生物斑马鱼

斑马鱼（Danio rerio），是一种用于研究脊椎动物胚胎学和发育遗传学的模式生物。近年来，利用斑马鱼来建立人类疾病研究模型已成为新的研究亮点。笔者就斑马鱼作为一种抗肿瘤新药研究模式生物所具有的优势、应用研究及其前景做一综述。     

Zebrafish assays for drug toxicity screening

Zebrafish are vertebrate organisms that are of growing interest for preclinical drug discovery applications. Zebrafish embryos develop most of the major organ systems present in mammals, including the cardiovascular, nervous and digestive systems, in < 1 week. Additional characteristics that make them advantageous for compound screening are their small size, transparency and ability to absorb compounds through the water. Furthermore, gene function analysis with antisense technology is now routine procedure. Thus, it is relatively simple to assess whether compounds or gene knockdowns cause toxic effects in zebrafish. Assays are being developed to exploit the unique characteristics of zebrafish for pharmacological toxicology. This review discusses assays that may be used to assess in vivo toxicity and provides examples of compounds known to be toxic to humans that have been demonstrated to function similarly in zebrafish.     

利用斑马鱼建立成瘾性药物对运动机能及认知功能影响的评价模型

目的利用斑马鱼这一新型模式动物,建立斑马鱼自发活动及学习记忆模型,并考察成瘾性药物对模型的影响。方法本实验以吗啡及甲基苯丙胺作为受试药物,利用斑马鱼行为视频跟踪分析系统自动记录动物行为参数,在自发活动模型主要测试运动路程、穿越次数及在顶部停留时间等行为,而进入营养富集区(EC区)的潜伏时间与总停留时间则作为T迷宫模型建立的主要指标,分别考察斑马鱼在运动机能及认知功能方面的行为变化。结果与正常组相比,6min内吗啡预处理组中斑马鱼运动路程、垂直穿越次数及在上部的停留时间均明显增加(P<0.05),甲基苯丙胺预处理组的垂直穿越次数及在上部的停留时间有明显的下调(P<0.01,P<0.01),提示吗啡短时暴露使斑马鱼活动性增强,甲基苯丙胺急性处理使得斑马鱼处于焦虑状态;在T迷宫测试中,正常的斑马鱼随着训练次数增加,与首次测试相比进入营养富集区(EC区)的时间有下降趋势,在EC区的停留时间明显增加(P<0.05),而与正常组相比,吗啡重复处理组中斑马鱼在EC区的停留时间无变化,而甲基苯丙胺重复处理组斑马鱼的空间学习记忆能力明显受到影响。结论成瘾性药物影响斑马鱼游泳行为与认知功能,且不同药物产生特征性影响,表明所建立模型的可靠性,可为药物引发的神经精神障碍及损伤提供快速有效的技术平台。     

A zebrafish phenotypic assay for assessing drug-induced hepatotoxicity

IntroductionNumerous studies have confirmed that zebrafish and mammalian toxicity profiles are strikingly similar and the transparency of larval zebrafish permits direct in vivo assessment of drug toxicity including hepatotoxicity in zebrafish.MethodsHepatotoxicity of 6 known mammalian hepatotoxic drugs (acetaminophen [APAP], aspirin, tetracycline HCl, sodium valproate, cyclophosphamide and erythromycin) and 2 non-hepatotoxic compounds (sucrose and biotin) were quantitatively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration, changes in liver size and yolk sac retention. Zebrafish liver degeneration was originally screened visually, quantified using an image-based morphometric analysis and confirmed by histopathology.ResultsAll the tested mammalian hepatotoxic drugs induced liver degeneration, reduced liver size and delayed yolk sac absorption in larval zebrafish, whereas the non-hepatotoxic compounds did not have observable adverse effect on zebrafish liver. The overall prediction success rate for hepatotoxic drugs and non-hepatotoxic compounds in zebrafish was 100% (8/8) as compared with mammalian results, suggesting that hepatotoxic drugs in mammals also caused similar hepatotoxicity in zebrafish.DiscussionLarval zebrafish phenotypic assay is a highly predictive animal model for rapidly in vivo assessment of compound hepatotoxicity. This convenient, reproducible animal model saves time and money for drug discovery and can serve as an intermediate step between cell-based evaluation and conventional animal testing of hepatotoxicity.     

Evaluation of toxicity of Personal Care Products (PCPs) in freshwaters: Zebrafish as a model

Personal care products (PCPs) are part of the large and growing family of emerging contaminants (ECs). Many daily products such as sunscreens, toothpaste, make-up products, perfume, and others, fall under this definition, and their use is increasing exponentially. Furthermore, the degradation of some components of these products is limited. Indeed, they are able to easily reach and accumulate in aquatic systems, representing a new class of contaminants. Moreover, due to their chemical properties, they can interfere at different biological levels, and for this reason, they need to be thoroughly investigated. We have reviewed the literature on PCPs, with a special focus on the adverse effects on the freshwater zebrafish (Danio rerio). The aim of this work is to provide a careful assessment of the toxicity of these compounds, in order to raise awareness for more conscious and responsible use.     

Zebrafish as a neurotoxicological model

At a time when common regulatory pathways are being identified in several different species and genomics is beginning to allow comparisons of genes, how they are arranged on chromosomes and how they are regulated, zebrafish has emerged as a valuable and complementary vertebrate model. Some of the characteristics that prove of value are described and illustrated. Fluorescent transgenic lines of zebrafish embryos are presented for time-line studies with neurotoxicants. While genetic knockout technology has yet to be developed for the model, the anti-sense, morpholino approach allows for knockdown of expression of genes for the 3 day, embryonic period. This can provide for phenocopies of mutant genes for those genes essential to embryonic development or it can provide for a limited inhibition of gene expression that allows subsequent development of the fish. With the zebrafish genomic sequencing effort, microarray technology is now developing for the model system. These resources and technologies allow one to challenge the system with toxicants, and to view the immediate effects of the toxicants with transgenic embryos that fluoresce in part or all of the nervous system. Behavioral and learning protocols have been developed for the organism so that early exposures can be assayed for effects upon adult fish. Microarray technology should allow for one to identify specific genes and pathways affected by a neurotoxicant. In the future, these approaches should provide a working protocol for exploring molecular mechanisms of neurotoxicants. This type of complementary approach should then allow for more efficient examination and testing of mechanisms in mammalian models.     

A high-throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays

Mitochondrial toxicity is a major reason for safety-related compound attrition and post-market drug withdrawals, highlighting the necessity for higher-throughput screens that can identify this mechanism of toxicity during the early stages of drug discovery. Here, we present the validation of a 384-well dual parameter plate-based assay capable of measuring oxygen consumption and extracellular acidification in intact cells simultaneously. The assay showed good reproducibility and robustness and is suitable for use with both suspension cells and adherent cells. To determine if the assay provides additional value in detecting mitochondrial toxicity over existing platforms, 200 commercially available drugs were tested in the assay using HL60 suspension cells as well as in two conventional mitochondrial toxicity assays: an oxygen consumption assay that uses isolated mitochondria and a cell-based assay that uses HepG2 cells grown in glucose and galactose media. The combination of the dual parameter assay and the isolated mitochondrial oxygen consumption assay identified more compounds that caused mitochondrial impairment than any other combination of the three assays or each of the three assays on its own. Furthermore, novel information was obtained from the dual parameter assay on drugs not previously reported to cause mitochondrial impairment.     

Elucidating mechanisms of drug-induced toxicity

The early and high-throughput application of assays for non-genetic toxicity is of great interest to the pharmaceutical industry, although few systems have been validated as being of good predictive value. New technologies could enable toxicity to be studied in the context of systems biology. An important factor to be considered is the metabolism of drugs to reactive intermediates. Chemical reactions of these with cell and tissue nucleophiles are relatively well understood, but predicting how biological modifications will affect signalling and regulatory networks remains a challenge. Some of these pathways could be useful as sentinels for toxicity. This article will cover some examples of drug toxicity and the prospects for future technology development.     

Autophagy in drug-induced liver toxicity

Liver injury resulting from exposure to drugs and chemicals is a major health problem. Autophagy is an important factor in a wide range of diseases, such as cancer, liver disease, muscular disorder, neurodegeneration, pathogen infection, and aging, and emerging evidence indicates that autophagy makes a substantial contribution to the pathogenesis of drug- and chemical-induced liver toxicity. In this review, we summarize current knowledge on autophagy triggered by toxicants/toxins, the protective role of autophagy in liver toxicity, and the underlying molecular mechanisms. We also highlight experimental approaches for studying autophagy.     

An integrated addition and interaction model for assessing toxicity of chemical mixtures.

The high propensity for simultaneous exposure to multiple environmental chemicals necessitates the development and use of models that provide insight into the toxicity of chemical mixtures. In this study, we developed a mathematical model that combines concepts of concentration addition, response addition, and toxicokinetic chemical interaction to assess toxicity of chemical mixtures. A ternary mixture of acetylcholinesterase inhibiting organophosphates (malathion and parathion) and the P450 inhibitor piperonyl butoxide was used to model toxicity. Concentration-response curves were generated for individual chemicals as well as for mixtures of the chemicals using acute toxicity tests with Daphnia magna. The toxicity of binary combinations of malathion and parathion adhered to the principles of concentration addition. The contribution of piperonyl butoxide to mixture toxicity was integrated using a model for response addition. Piperonyl butoxide also modified the toxicity of the organophosphates by inhibiting their metabolic activation. The antagonistic effects of piperonyl butoxide towards the organophosphates were quantified as coefficients of interactions (K-functions) and incorporated into the mixture model. Finally, toxicity of the ternary mixture was modeled at 30 different mixture formulations using three additive models that assumed no interaction (concentration addition, response addition, and integrated addition) and using the integrated addition and interaction (IAI) model. Toxicity of the 30 mixtures was then experimentally determined and compared to model results. Only the IAI model accurately predicted the toxicity of the mixtures. The IAI model holds promise as a means for assessing hazard of complex chemical mixtures.     

Role of maternal toxicity in assessing developmental toxicity in animals: a discussion.

The belief that any drug or chemical, when administered at a high enough dose, can be expected to produce fetal malformations is not consistent with the facts. However, the stress associated with maternally toxic doses can be expected to result in associated, often transient, fetal abnormalities that may not be the result of deviant organogenesis. Sometimes the toxicity toward the pregnant animal, including her embryos/fetuses since they are hardly in a sanctuary, is severe enough to result in resorption of the embryo or abortion of the fetus. Thus, it is possible that the embryolethality and other indications of developmental toxicity, produced by some drugs and chemicals, may be the result of a mechanism(s) other than selective toxicity toward the embryo. Also, some test materials have been shown to affect maternal homeostasis, thereby disrupting support to the embryo, without causing significant overt toxicity to the embryo or dam; e.g., the endocrine system of the dam is altered. Routine testing has thus far revealed a relatively limited number of true teratogens, although a large number of drugs and chemicals have resulted in fetal effects such as developmental variations when administered at doses that approach lethal levels. Such effects on the fetus should be expected when the maternal animals are stressed by the high dosages usually employed. A better understanding of the etiology and biological relevance of the embryo/fetal deviations often seen in developmental toxicology studies might help to avoid the sometimes unjustified withholding of potentially useful drugs and chemicals from the marketplace.     

Complement activation-related pseudoallergy: a new class of drug-induced acute immune toxicity

A major goal in modern pharmacotechnology is to increase the therapeutic index of drugs by using nanoparticulate vehicle systems in order to ensure slow release or targeted delivery of drugs. With all great benefits, however, these innovative therapies can carry a risk for acute immune toxicity manifested in hypersensitivity reactions (HSRs) that do not involve IgE but arises as a consequence of activation of the complement (C) system. These anaphylactoid reactions can be distinguished within the Type I category of HSRs as "C activation-related pseudoallergy" (CARPA). Drugs and agents causing CARPA include radiocontrast media (RCM), liposomal drugs (Doxil, Ambisome and DaunoXome) and micellar solvents containing amphiphilic lipids (e.g., Cremophor EL, the vehicle of Taxol). These agents activate C through both the classical and the alternative pathways, giving rise to C3a and C5a anaphylatoxins that trigger mast cells and basophils for secretory response that underlies HSRs. Pigs provide a useful model for liposome-induced CARPA as minute amounts of reactogenic lipomes cause C activation with consequent dramatic cardiovascular and laboratory abnormalities that mimic some of the human symptoms. Consistent with the causal role of C activation in liposome-induced HSRs, a recent clinical study demonstrated correlation between the formation of C terminal complex (SC5b-9) in blood and the presence of HSRs in patients treated with liposomal doxorubicin (Doxil). Overall, the CARPA concept may help in the prediction, prevention and treatment of the acute immune toxicity of numerous state-of-the-art drugs.     

Zebrafish as a genomics research model

The zebrafish (Danio rerio) is a recent addition to the genomic scientists' repertoire of vertebrate animal model systems. Unlike simple invertebrates such as the fly or the nematode, this teleost maintains the biological and genomic complexity found in higher vertebrates. Furthermore, the zebrafish has many advantageous technical and genomic properties that open the door to experimental approaches not practical using more classical models. The zebrafish genome can be functionally accessed using both forward and reverse genetics based approaches. A notable recent addition to the zebrafish genomics toolbox is the development of morpholino-based antisense gene inhibition for sequence-based 'knockdown' screening. This method offers the opportunity to examine the role of significant subsets of the vertebrate genome for specific gene function in vivo. The zebrafish embryo can rapidly provide critical information for drug target discovery purposes when examined with an emphasis on clinically-relevant biological processes. Finally, the advent of chemical genetics in zebrafish suggests that, in addition to the identification and understanding of drug targets and their biology, this system will be a powerful tool in the direct development of novel pharmaceuticals in the near future.