Development in in vitro toxicology

There are three principal pressures driving the development of in vitro toxicology: (1) the need for more efficient testing systems to cope with the large number of xenobiotics currently being developed; (2) public pressure to reduce animal experimentation; and (3) a need for a better understanding of the mechanisms of toxicity. Within this, in vitro toxicology is focused on local, systemic, and target-organ toxicity. It is becoming increasingly apparent that a step or decision-tree approach using input of a variety of experimental data (physicochemical properties, biokinetics, cytotoxicity) provides the most efficient system for predicting toxicity. Examples of the use of in vitro toxicity systems for prediction of systemic toxicity and target-organ (liver) toxicity are presented.     

Emerging approaches in predictive toxicology

Predictive toxicology plays an important role in the assessment of toxicity of chemicals and the drug development process. While there are several well‐established in vitro and in vivo assays that are suitable for predictive toxicology, recent advances in high‐throughput analytical technologies and model systems are expected to have a major impact on the field of predictive toxicology. This commentary provides an overview of the state of the current science and a brief discussion on future perspectives for the field of predictive toxicology for human toxicity. Computational models for predictive toxicology, needs for further refinement and obstacles to expand computational models to include additional classes of chemical compounds are highlighted. Functional and comparative genomics approaches in predictive toxicology are discussed with an emphasis on successful utilization of recently developed model systems for high‐throughput analysis. The advantages of three‐dimensional model systems and stem cells and their use in predictive toxicology testing are also described. Environ. Mol. Mutagen. 55:679–688, 2014. © 2014 Wiley Periodicals, Inc.     

Genetic toxicology in the 21st century: reflections and future directions

A symposium at the 40th anniversary of the Environmental Mutagen Society, held from October 24–28, 2009 in St. Louis, MO, surveyed the current status and future directions of genetic toxicology. This article summarizes the presentations and provides a perspective on the future. An abbreviated history is presented, highlighting the current standard battery of genotoxicity assays and persistent challenges. Application of computational toxicology to safety testing within a regulatory setting is discussed as a means for reducing the need for animal testing and human clinical trials, and current approaches and applications of in silico genotoxicity screening approaches across the pharmaceutical industry were surveyed and are reported here. The expanded use of toxicogenomics to illuminate mechanisms and bridge genotoxicity and carcinogenicity, and new public efforts to use high‐throughput screening technologies to address lack of toxicity evaluation for the backlog of thousands of industrial chemicals in the environment are detailed. The Tox21 project involves coordinated efforts of four U.S. Government regulatory/research entities to use new and innovative assays to characterize key steps in toxicity pathways, including genotoxic and nongenotoxic mechanisms for carcinogenesis. Progress to date, highlighting preliminary test results from the National Toxicology Program is summarized. Finally, an overview is presented of ToxCast?, a related research program of the U.S. Environmental Protection Agency, using a broad array of high throughput and high content technologies for toxicity profiling of environmental chemicals, and computational toxicology modeling. Progress and challenges, including the pressing need to incorporate metabolic activation capability, are summarized. Environ. Mol. Mutagen., 2011. © 2011 Wiley‐Liss, Inc.     

磁性纳米介质的毒理学研究进展

磁性纳米介质在医药等诸多领域有广泛的应用价值,如肿瘤磁感应热疗、影像诊断、药物载体等。而磁性介质的生物相容性是其在医学领域中应用的前提。本文主要对磁流体、磁性脂质体这类磁性纳米介质的毒理学研究进展磁性纳米介质体内、体外毒性评价实验,相关评价指标的研究进展及磁性介质毒理学研究中存在的问题进行综述。     

Prevalidation of in vitro continuous flow exposure systems as alternatives to in vivo inhalation safety evaluation experimentations: Outcome from MAAPHRI-PCRD5 research program

Diesel engine emission aerosol-induced toxicity patterns were compared using both in vitro (organotypic cultures of lung tissue) and in vivo experimentations mimicking the inhalation situation with continuous aerosol flow exposure designs. Using liquid media resuspended diesel particles, we show that toxic response pattern is influenced by the presence of tensioactive agent in the medium which alter particle-borne pollutant bioavailability. Using continuous aerosol exposure in vitro, we show that with high sulfur fuel (300ppm) in the absence of oxidation catalysis, particulate matter was the main toxic component triggering DNA damage and systemic inflammation, while a very limited oxidant stress was evidenced. In contrast, with ultra-low sulfur fuel in the presence of strong diesel oxidation catalysis, the specific role of particulate matter is no longer evidenced and the gas phase then becomes the major component triggering strong oxidant stress, increased NO(2) being the most probable trigger. In vivo, plasma tumor necrosis factor alpha (TNFalpha), lung superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activity levels varied in agreement with in vitro observations. Diesel emission treatment with oxycat provokes a marked systemic oxidant stress. Again NO(2) proved to account for a major part of these impacts. In conclusion, similar anti-oxidant responses were observed in in vitro and in vivo experiments after diesel emission aerosol continuous flow exposures. The lung slice organotypic culture model-exposed complex aerosol appears to be a very valuable alternative to in vivo inhalation toxicology experimentations in rodents.     

A 3-D organoid kidney culture model engineered for high-throughput nephrotoxicity assays

Cell-cell and cell-matrix interactions control cell phenotypes and functions in vivo. Maintaining these interactions in vitro is essential to both produce and retain cultured cell fidelity to normal phenotype and function in the context of drug efficacy and toxicity screening. Two-dimensional (2-D) cultures on culture plastics rarely recapitulate any of these desired conditions. Three dimensional (3-D) culture systems provide a critical junction between traditional, yet often irrelevant, in vitro cell cultures and more accurate, yet costly, in vivo models. This study describes development of an organoid-derived 3-D culture of kidney proximal tubules (PTs) that maintains native cellular interactions in tissue context, regulating phenotypic stability of primary cells in vitro for up to 6 weeks. Furthermore, unlike immortalized cells on plastic, these 3-D organoid kidney cultures provide a more physiologically-relevant response to nephrotoxic agent exposure, with production of toxicity biomarkers found in vivo. This biomimetic primary kidney model has broad applicability to high-throughput drug and biomarker nephrotoxicity screening, as well as more mechanistic drug toxicology, pharmacology, and metabolism studies.     

Adjuvant arthritis 50 years on: the impact of the 1956 article by C. M. Pearson, ‘Development of arthritis,periarthritis and periostitis in rats given adjuvants’

Summary. The Science Citation Index (Web of Science) has now accumulated over 700 citations to this report published in Proc Soc exp Biol Med 1956; 91: 95–101, including several in 2006. This memoire is a tribute to its author for revealing the opportunities for so much subsequent research in experimental pharmacology and toxicology. For half a century, the adjuvant disease in rats has enormously aided research on drugs to control arthritis and other chronic inflammatory disorders. The adjuvant also triggers many systemic responses beyond the articular tissues. So we are given simultaneously a window to explore the converse phenomenon: namely, how a chronic disease can affect drug efficacy and toxicity. This phenomenon has been variously described as patho-pharmacodynamics, conditional pharmacology/toxicology and ‘a right nuisance’ ! Nevertheless it has enormous heuristic value for clinical therapeutics. Received 19 July 2006; returned for revision 16 October 2006; accepted by M. Parnham 19 October 2006     

Opportunities to integrate new approaches in genetic toxicology: An ILSI‐HESI workshop report

Genetic toxicity tests currently used to identify and characterize potential human mutagens and carcinogens rely on measurements of primary DNA damage, gene mutation, and chromosome damage in vitro and in rodents. The International Life Sciences Institute Health and Environmental Sciences Institute (ILSI‐HESI) Committee on the Relevance and Follow‐up of Positive Results in In Vitro Genetic Toxicity Testing held an April 2012 Workshop in Washington, DC, to consider the impact of new understanding of biology and new technologies on the identification and characterization of genotoxic substances, and to identify new approaches to inform more accurate human risk assessment for genetic and carcinogenic effects. Workshop organizers and speakers were from industry, academe, and government. The Workshop focused on biological effects and technologies that would potentially yield the most useful information for evaluating human risk of genetic damage. Also addressed was the impact that improved understanding of biology and availability of new techniques might have on genetic toxicology practices. Workshop topics included (1) alternative experimental models to improve genetic toxicity testing, (2) Biomarkers of epigenetic changes and their applicability to genetic toxicology, and (3) new technologies and approaches. The ability of these new tests and technologies to be developed into tests to identify and characterize genotoxic agents; to serve as a bridge between in vitro and in vivo rodent, or preferably human, data; or to be used to provide dose response information for quantitative risk assessment was also addressed. A summary of the workshop and links to the scientific presentations are provided. Environ. Mol. Mutagen. 56:277–285, 2015. © 2014 Wiley Periodicals, Inc.     

Endocrine toxicology: A Review of the application of endocrinology in experimental toxicology

In the past endocrine toxicology has not been a common subject in routine toxicity studies. However, since the endocrine system is an important integrating system of the body, controlling the major physiological functions, it is important to investigate the mechanism of action of exogenous compounds in endocrine target organs or hormonal target cells. The following procedure is suggested to detect effects on the endocrine system in routine toxicity experiments: (1) determination of the weight of endocrine organs and histology as screening parameters; (2) determination of circulating hormones in combination with morphological or immunocytochemical methods: (3) specific function tests and in vitro methods to determine dysfunction of specific endocrine organs or cells. That the use of such an approach has provided insight into the mechanism of action of chemical compounds will be demonstrated by results of endocrine toxicity studies with the antibiotic compound sulphadimidine, interfering with thyroid hormone synthesis as a secondary mechanism leading to thyroid tumour formation, the androgenic compound trenbolone acetate, used for growth promotion, for which the disturbance of the gonadal function formed the basis for the establishment of the no-observed-hormonal-effect level, the antibacterial compound furazolidone, suspected of having an oestrogenic activity which was hypothesized as the underlying mechanism for the observed mammary tumour formation, and the antimicrobial agent carbadox, used as feed additive for pigs, for which the interference with adrenal function, resulted in a severe disturbance of the water and salt balance in target animals.Originally presented at ECCP 93.     

Endocrine toxicology: A Review of the application of endocrinology in experimental toxicology

In the past endocrine toxicology has not been a common subject in routine toxicity studies. However, since the endocrine system is an important integrating system of the body, controlling the major physiological functions, it is important to investigate the mechanism of action of exogenous compounds in endocrine target organs or hormonal target cells. The following procedure is suggested to detect effects on the endocrine system in routine toxicity experiments: (1) determination of the weight of endocrine organs and histology as screening parameters; (2) determination of circulating hormones in combination with morphological or immunocytochemical methods: (3) specific function tests and in vitro methods to determine dysfunction of specific endocrine organs or cells. That the use of such an approach has provided insight into the mechanism of action of chemical compounds will be demonstrated by results of endocrine toxicity studies with the antibiotic compound sulphadimidine, interfering with thyroid hormone synthesis as a secondary mechanism leading to thyroid tumour formation, the androgenic compound trenbolone acetate, used for growth promotion, for which the disturbance of the gonadal function formed the basis for the establishment of the no-observed-hormonal-effect level, the antibacterial compound furazolidone, suspected of having an oestrogenic activity which was hypothesized as the underlying mechanism for the observed mammary tumour formation, and the antimicrobial agent carbadox, used as feed additive for pigs, for which the interference with adrenal function, resulted in a severe disturbance of the water and salt balance in target animals. Originally presented at ECCP 93.     

The applications of buckminsterfullerene C60 and derivatives in orthopaedic research

Buckminsterfullerene C₆₀ and derivatives have been extensively explored in biomedical research due to their unique structure and unparalleled physicochemical properties. C₆₀ is characterized as a “free radical sponge” with an anti-oxidant efficacy several hundred-fold higher than conventional anti-oxidants. Also, the C₆₀ core has a strong electron-attracting ability and numerous functional compounds with widely different properties can be added to this fullerene cage. This review focused on the applications of C₆₀ and derivatives in orthopaedic research, such as the treatment of cartilage degeneration, bone destruction, intervertebral disc degeneration (IVDD), vertebral bone marrow disorder, radiculopathy, etc., as well as their toxicity in vitro and in vivo. We suggest that C₆₀ and derivatives, especially the C₆₀ cores coupled with functional groups presenting new biological and pharmacological activities, are advantageous in orthopaedic research and will be promising in clinical performance for musculoskeletal disorders treatment; however, the pharmacokinetics and toxicology of these agents as local/systemic administration need to be carefully determined.     

Immunotoxicology: an overview

It is now known that chemicals and drugs may induce selective toxicity which may alter the interactions between immunocompetent cells, especially if the toxicity occurs during proliferation and differentiation. Hence, a flexible panel of sensitive in vivo and in vitro assays has been developed and validated to assess the immunotoxicity or immunopharmacology of suspect agents in rodents. The combined use of such sequential analysis methods with host resistance assays can effectively define immunomodulation following exposure to xenobiotics. Methods development, refinement and validation will be an ongoing requirement because of our rapidly expanding knowledge of the cell biology of the immune system. Classic studies of the comparative preclinical toxicology of several immunosuppressive drugs have substantiated species similarities and have contributed significantly to the development of predictive rodent models for extrapolation to humans. Studies of immunopharmacology and immunotoxicity of cyclosporin A, for example, produced both the desired pharmacology and the undesired toxicity at similar doses in both rodents and humans. When species differences are observed during toxicology studies they are most probably due to differences in absorption, disposition, metabolism, excretion, or delivered dose at the target tissue, rather than major species differences in cellular targets or cell physiology. This assumption is the basis for using rodent species to predict the toxicity of chemicals and drugs under development.(ABSTRACT TRUNCATED AT 250 WORDS)     

复合水凝胶人工角膜裙边支架材料的生物相容性评价

背景：β-磷酸三钙/聚乙烯醇复合水凝胶具有高含水量、良好柔软性优点,有利于成纤细胞生长及胶原沉积,适用于做人工角膜裙边支架材料。 目的：评价β-磷酸三钙/聚乙烯醇复合水凝胶人工角膜裙边支架材料的生物相容性。 方法：①迟发型超敏反应：在豚鼠脊柱两侧皮内由头到尾分别注射A液(完全弗氏佐剂与生理盐水等体积混合液)、B液(β-磷酸三钙/聚乙烯醇复合水凝胶浸提液或生理盐水或2-巯基苯并噻唑)、C液(A液与B液等体积混合稳定性乳化剂),并进行局部诱导及激发。②急性全身性毒性实验：由昆明小鼠尾静脉分别注射生理盐水与β-磷酸三钙/聚乙烯醇复合水凝胶浸提液。③体外细胞毒性实验：分别以β-磷酸三 钙/聚乙烯醇复合水凝胶浸提液、细胞培养液及含苯酚的细胞培养液培养MRC-5细胞。④皮内反应：在兔脊柱皮内分别注射β-磷酸三钙/聚乙烯醇复合水凝胶生理盐水(或芝麻油)浸提液、生理盐水及芝麻油。 结果与结论：β-磷酸三钙/聚乙烯醇复合水凝胶人工角膜裙边支架材料的迟发型超敏反应分级为0-1级,急性全身毒性实验结果为正常无症状,体外细胞毒性均为0级或1级,皮内反应为极轻微。表明β-磷酸三钙/聚乙烯醇复合水凝胶人工角膜裙边支架材料生物相容性指标达到生物植入医用材料的要求。     

How do we compare dose to cells in vitro with dose to live animals and humans? Some experiences with inhaled substances

The inhalation route provides closer contact between the ambient environment and living cells than the other major routes-of-entry to the body. In addition, the ambient air transporting exogenous agents to the close proximity of living cells can maintain the reactivity of such agents until they deposit in the extracellular lining layer of the lungs at micrometer distances from the airway epithelium. While toxicity may often occur following the systemic distribution of exogenous substances via the respiratory tract, it is the situation when toxicity occurs at the airway portal-of-entry that pose a particular challenge in the mapping of the dosimetry. In such case, the volume of distribution of solutes before they may enter the airway cells is very small. Therefore exposures of the airway epithelium are more variable and can be much higher than exposures of viable cells of the skin or the gastro-intestinal tract at similar concentrations in the ambient environment. Especially with aerosol exposures, local concentrations around deposited particles can be exceedingly high. To simulate these exposures in a cell culture is a difficult task. However, in order to bridge the in vivo-in vitro gap two methods can be used: (1) Specially designed in vitro systems to better mimic the physiology/morphology of cells residing in the respiratory tract. (2) Mathematical models to analyse the toxicokinetics of the in vitro systems and extrapolate to the corresponding in vivo situation. Both strategies will be exemplified and discussed.     

Reticuloendothelial system function and histamine release in shock and trauma: Relationship to microcirculation

Summary The material reviewed, and presented, here lends credence to the concept that the severity or course of the shock syndrome can be evaluated, quantitatively, at a tissue level by assessing RES phagocytic function. In general, the available data indicate that RE cell stimulants can adapt animals (and probably man) to the insults of circulatory shock and trauma; such substances could have important value in pretreating patients scheduled for massive surgery. The fact that a number of biologically active materials with vasotropic, and RE cell depressant, effects appear in the tissues and blood in shock, particularly when the organism becomes refractory to therapy, suggests that the final functional deterioration of the cardiovascular system may be due to the specific action of one or more of these biologically active materials; such a contender is, without doubt, histamine.Histamine has all the attributes of a typical shock-toxin. Evidence is presented that histamine can be a potent splanchnic (shock target-organ) arteriolar (microcirculatory) dilator even in physiologic (circulating) concentrations. Concentrations of histamine found in plasma of shocked animals and human subjects would produce extremely potent splanchnic vasodilator actions at the microcirculatory level. Evidence is also presented to indicate that microvessels can synthesize and release free, pharmacologically-active histamine.Endogenous release of histamine (e.g., with compound 48/80) produces dose-dependent and lethal shock-like anaphylactic actions; such release also produces, dose-dependently, RES phagocytic depression. Repeated administration of the histamine releaser, compound 48/80, results in almost a 400% enhancement of RES phagocytic function and cross-tolerance to lethal doses of whole-body trauma. Such results raise the possibility that the RES plays a pivotal role in the circulatory manifestations of compound 48/80 and anaphylactic-type (histamine release) shock syndromes.Evidence is presented to indicate that H₁-receptor antihistamines can ameliorate circulatory shock (and trauma) and prevent RES phagocytic depression, whereas H₂-receptor antihistamines do the reverse. Direct in situ microscopy revealed that the former types of histamine receptor blockers prevent tissue ischemia, whereas H₂-receptor blockers exacerbate tissue ischemia in circulatory shock. Histamineinduced vasodilatation via H₂-receptors may thus be a beneficial effect in circulatory shock and trauma; one must think seriously about the potential value of antihistamines as adjuvant drugs in the treatment of low-flow states and as preoperative medication.Collectively, the data reviewed herein could be taken as strong support for a pivotal role for the release (and possible synthesis) of free, pharmacologically-active histamine in shock.Supported by Research Grants H1 18002, HL 18015 and DA 02339 from the U.S.P.H.S.     

Ankle-brachial index in HIV infection

Roche's protease inhibitor nelfinavir mesylate (Viracept^®) produced between March 2007-June 2007 was found to contain elevated levels of ethyl methanesulfonate (EMS), a known mutagen (alkylator) – leading to a global recall of the drug. EMS levels in a daily dose (2,500 mg Viracept/day) were predicted not to exceed a dose of ~2.75 mg/day (~0.055 mg/kg/day based on 50 kg patient). As existing toxicology data on EMS did not permit an adequate patient risk assessment, a comprehensive animal toxicology evaluation of EMS was conducted. General toxicity of EMS was investigated in rats over 28 days. Two studies for DNA damage were performed in mice; chromosomal damage was assessed using a micronucleus assay and gene mutations were detected using the MutaMouse transgenic model. In addition, experiments designed to extrapolate animal exposure to humans were undertaken. A general toxicity study showed that the toxicity of EMS occurred only at doses ≥ 60 mg/kg/day, which is far above that received by patients. Studies for chromosomal damage and mutations in mice demonstrated a clear threshold effect with EMS at 25 mg/kg/day, under chronic dosing conditions. Exposure analysis (C_max) demonstrated that ~370-fold higher levels of EMS than that ingested by patients, are needed to saturate known, highly conserved, error-free, mammalian DNA repair mechanisms for alkylation. In summary, animal studies suggested that patients who took nelfinavir mesylate with elevated levels of EMS are at no increased risk for carcinogenicity or teratogenicity over their background risk, since mutations are prerequisites for such downstream events. These findings are potentially relevant to >40 marketed drugs that are mesylate salts.     

Monoclonal antibody first dose cytokine release syndromes-mechanisms and prediction

Acute cytokine release syndromes are associated with some therapeutic antibodies in man, leading to a spectrum of clinical signs from nausea, chills and fever to more serious dose limiting hypotension and tachycardia. When anticipated this syndrome is typically manageable, however this adverse reaction recently became headline news when a massive and unexpected cytokine release syndrome occurred within a few hours of dosing six healthy volunteers with a therapeutic antibody, putting their lives at risk due to multiple organ failure. Preclinical studies did not predict this adverse event, emphasising the need to compare the relative potency of the product in man and the chosen toxicology species, so that additional margins of safety can be applied when conducting first in man (FIM) studies if there is uncertainty over the predictability of the toxicology species. In vitro human PBMC and whole blood cultures may be useful for predicting cytokine release. However since cytokine release arises through at least two distinct mechanisms, it should be emphasised that the utility of these in vitro methods needs to be established for each antibody product.