Popliteal lymph node assay: facts and perspectives

The popliteal lymph node assay (PLNA) derives from the hypothesis that some supposedly immune-mediated adverse effects induced by certain pharmaceuticals involve a mechanism resembling a graft-versus-host reaction. The injection of many but not all of these compounds into the footpad of mice or rats produces an increase in the weight and/or cellularity of the popliteal lymph node in the treated limb (direct PLNA). Some of the compounds known to cause these adverse effects in humans, however, failed to induce a positive PLNA response, leading to refinements of the technique to include pretreatment with enzyme inducers, depletion of CD4(+) T cells or additional endpoints such as histological examination, lymphocyte subset analysis and cytokine fingerprinting. Alternative approaches have been used to improve further the predictability of the assay. In the secondary PLNA, the test compound is injected twice in order to illicit a greater secondary response, thus suggesting a memory-specific T cell response. In the adoptive PLNA, popliteal lymph node cells from treated mice are injected into the footpad of naive mice; a marked response to a subsequent footpad challenge demonstrates the involvement of T cells. Finally, the reporter antigens TNP-Ficoll and TNP-ovalbumin are used to differentiate compounds that induce responses involving neo-antigen help or co-stimulatory signals (modified PLNA). The PLNA is increasingly considered as a tool for detection of the potential to induce both sensitization and autoimmune reactions. A major current limitation is validation. A small inter-laboratory validation study of the direct PLNA found consistent results. No such study has been performed using an alternative protocol. Other issues include selection of the optimal protocol for an improved prediction of sensitization vs autoimmunity, and the elimination of false-positive responses due to primary irritation. Finally, a better understanding of underlying mechanisms is essential to determine the most relevant endpoints. The confusion resulting from use of the PLNA to predict autoimmune-like reactions as well as sensitization should be clarified. Interestingly, most drugs that were positive in the direct PLNA are also known to cause drug hypersensitivity syndrome in treated patients. This observation is expected to open new avenues of research.     

The popliteal lymph node assay: a tool for predicting drug allergies

A considerable number of drugs is able to induce systemic hypersensitivity in man. Systemic hypersensitivity can be drug- or autoantigen-specific, but in either case a complex of immunological processes and predisposing factors are involved and it is rarely if ever noticed in standard toxicity testing. The popliteal lymph node assay (PLNA) is regarded a suitable test to screen for the immunostimulating ability of a chemical, which may indicate its immunosensitizing potential. The most simple, primary PLNA measures popliteal lymph node hyperplasia after subcutaneous injection of a chemical into the footpad of the hindpaw of a mouse or rat. In order to assess the involvement of T cells, and hence immunosensitizating potential of a chemical, anamnestic immune reactions to a chemical or its metabolite can be measured in previously exposed (and sensitized) animals or in naive animals that received an adoptive transfer of syngeneic T cells from previously exposed animals. In the recently introduced modified PLNA, defined reporter antigens TNP-OVA (T cell-dependent antigen) and TNP-Ficoll (T cell-independent antigen) are used to distinguish between sensitizing and non-sensitizing (IgG1-response or not to TNP-Ficoll, respectively) and between mere inflammatory and complete innocent (no IgG1-response to TNP-Ficoll and an IgG1-response or not to TNP-OVA, respectively) drugs. Results with about 130 compounds (drugs and environmental pollutants) with the various types of the PLNA show a good correlation with documented immunostimulating (both autoimmunogenic and allergic) potential and no false negative chemicals were detected if metabolism was considered. The PLNA awaits further validation before this test can be recommended as a tool for prediction of drug allergy.     

Immunomodulatory effects of tetrachlorobenzoquinone,a reactive metabolite of hexachlorobenzene

Hexachlorobenzene (HCB) is an environmental pollutant that causes autoimmune-like effects in humans and rats. It is not completely clear whether T cells are involved and, if so, how they are stimulated after oral exposure to HCB. HCB as a rather inert chemical is not likely to bind covalently to macromolecules. The oxidative metabolite of HCB, tetrachlorobenzoquinone (TCBQ), which is in a redox equilibrium with tetrachlorohydroquinone (TCHQ), can bind to macromolecules, hence may form hapten-carrier complexes in vivo. We have assessed in the reporter antigen-popliteal lymph node assay whether HCB or TCHQ and TCBQ are able to induce a 2,4,6-trinitrophenyl (TNP) specific IgG1 response to the T cell-independent antigen TNP-Ficoll, which is indicative of neoantigen specific T cell help. To this end, these compounds and silica were injected into the footpad of Balb/c mice. Silica was included as an inert model compound, which causes autoimmune-like effects by activating macrophages. Seven days later, cell number and TNP specific antibody-secreting cells (ASC) in the popliteal lymph node (PLN) were determined. Furthermore, a secondary PLNA was performed to find out if TCHQ was capable of eliciting a memory response. Silica, TCHQ, and TCBQ, but not HCB, increased PLN cellularity and the number of IgM-producing ASC by ELISPOT. Both oxidative metabolites were able to induce the formation of germinal centers as assessed by immunohistochemistry and an IgG1 response to TNP-Ficoll. In the secondary PLNA, only mice primed with TCHQ and challenged with TCHQ together with TNP-Ficoll showed a significant increase in TNP specific IgG1 ASC. Present data show that TCHQ and TCBQ are capable of inducing neoantigen specific T cell help and that TCHQ can induce a compound specific memory response.     

Interferon-alpha2b secretion by adenovirus-mediated gene delivery in rat, rabbit, and chimpanzee results in similar pharmacokinetic profiles

Gene delivery, with subsequent protein synthesis and secretion, in vivo has been proposed as an alternative way to deliver a therapeutic protein to the systemic circulation. Interferon-alpha (IFN) protein is effective in the treatment of viral and malignant diseases but has short serum half-life that requires frequent administration. An E1 region-deleted adenovirus vector encoding human IFN-alpha2b gene driven by the cytomegalovirus immediate early promoter (rAd-IFN) was generated to assess the serum concentration-time profiles of expressed IFN protein in animal models. Intravenous administration of rAd-IFN, normalized for body weight, resulted in dose-dependent serum IFN concentrations that persisted 8-40 days with similar concentration-time profiles in rats and rabbits. We sought to determine if serum concentration-time profiles in the rat and rabbit animal models would be predictive for a larger animal and would therefore be relevant models for potential dosing of human patients. Two chimpanzees (approximately 70 kg) dosed with rAd-IFN by intravenous administration normalized to body weight achieved serum IFN concentration-time profiles similar to those observed in rats and rabbits. The role of the immune response in limiting the persistence of transgene expression was highlighted by the persistence of serum IFN concentrations for over 200 days in beige/SCID immunodeficient mice. These studies suggest that serum concentration of secreted transgene products after gene delivery in small animal models may be highly predictive for larger species and will help define dosing strategies in human patients.     

神经发育毒性动物实验替代方法研究进展

胚胎早期暴露于某些工业化学物中,即使是很小剂量,也可导致胚胎脑损伤,引起神经发育性疾病和亚临床脑功能不良。虽然化学物基于动物毒性实验的安全性评价是较可靠的,但这种方法耗时长、成本高,而且不符合目前减少实验动物使用的趋势,因此神经发育毒性(DNT)实验的替代模型逐步引起重视。为建立和完善快速、经济又可高通量筛选受试物的替代方法,本文分别介绍了体外细胞模型和非哺乳动物模型的优势、现阶段应用以及所面临的挑战。这些替代法虽不能完全取代包括哺乳动物在内的体内实验,但它们在区分化合物和识别DNT机制方面将发挥巨大的作用。     

Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA–PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP–OVA or TNP–Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.     

Comparative dermal toxicity of dicyclohexylcarbodiimide and diisopropylcarbodiimide in rodents

Dicyclohexylcarbodiimide (DCC) and Diisopropylcarbodiimide (DIC) are two representative chemicals in the carbodiimide class of chemicals used in industry as stabilizing agents. There is a potential of dermal exposure to these agents in chemical, pharmaceutical and recombinant DNA industries. The National Toxicology Program conducted a number of animal studies to characterize toxicity and carcinogenicity of DIC and DCC. Dermal administration of DCC and DIC in F344/N rats and B6C3F1 mice for 90-days induced skin irritation at the site of application in a dose-dependent manner. Microscopically, dose-dependent increases in epidermal hyperplasia and chronic inflammation were observed. We further evaluated the effects of dermal exposure of DCC and DIC in p53 haploinsufficient and Tg.AC mouse models. Results revealed the skin as the primary target of DCC and DIC exposure as indicated by dose - dependent skin lesions (hyperplasia, inflammation and necrosis). DCC induced squamous cell papillomas in Tg.AC mice but did not induce any neoplastic lesions in p53 haploinsufficient mice. Dermal application of DIC did not induce any neoplastic lesions in Tg.AC mice and p53 haploinsufficient mice. Based on these studies, it was predicted that DIC would be negative and DCC positive for carcinogenic activity in the traditional two-year bioassay. In the subsequent studies, the carcinogenic potential of DIC only in F344 rats and B6C3F1 mice in a traditional 2-year chronic carcinogenicity bioassay was evaluated by the dermal route. Findings revealed the skin as the major target organ of toxicity in both sexes in rats and in male mice. There were no neoplastic lesions observed in rats or mice with the administration of DIC. In rats, there were clinical signs of toxicity in the highest dose-group which included ataxia, excitability, impaired gait, low muscle tone, abnormal breathing, lethargy, and seizures. This was accompanied by non-neoplastic lesions in the brain and lung only at the highest dose level. In conclusion, both DIC and DCC are dermal toxicants. DIC did not have any carcinogenic activity in transgenic mouse models or in the traditional NTP two-year carcinogenicity studies in F344 rats and B6C3F1 mice. DCC was positive in the Tg.AC mouse model and likely to be carcinogenic in the 2-year bioassay as well.     

Comparative analysis of predictive models for nongenotoxic hepatocarcinogenicity using both toxicogenomics and quantitative structure-activity relationships

The primary testing strategy to identify nongenotoxic carcinogens largely relies on the 2-year rodent bioassay, which is time-consuming and labor-intensive. There is an increasing effort to develop alternative approaches to prioritize the chemicals for, supplement, or even replace the cancer bioassay. In silico approaches based on quantitative structure-activity relationships (QSAR) are rapid and inexpensive and thus have been investigated for such purposes. A slightly more expensive approach based on short-term animal studies with toxicogenomics (TGx) represents another attractive option for this application. Thus, the primary questions are how much better predictive performance using short-term TGx models can be achieved compared to that of QSAR models, and what length of exposure is sufficient for high quality prediction based on TGx. In this study, we developed predictive models for rodent liver carcinogenicity using gene expression data generated from short-term animal models at different time points and QSAR. The study was focused on the prediction of nongenotoxic carcinogenicity since the genotoxic chemicals can be inexpensively removed from further development using various in vitro assays individually or in combination. We identified 62 chemicals whose hepatocarcinogenic potential was available from the National Center for Toxicological Research liver cancer database (NCTRlcdb). The gene expression profiles of liver tissue obtained from rats treated with these chemicals at different time points (1 day, 3 days, and 5 days) are available from the Gene Expression Omnibus (GEO) database. Both TGx and QSAR models were developed on the basis of the same set of chemicals using the same modeling approach, a nearest-centroid method with a minimum redundancy and maximum relevancy-based feature selection with performance assessed using compound-based 5-fold cross-validation. We found that the TGx models outperformed QSAR in every aspect of modeling. For example, the TGx models' predictive accuracy (0.77, 0.77, and 0.82 for the 1-day, 3-day, and 5-day models, respectively) was much higher for an independent validation set than that of a QSAR model (0.55). Permutation tests confirmed the statistical significance of the model's prediction performance. The study concluded that a short-term 5-day TGx animal model holds the potential to predict nongenotoxic hepatocarcinogenicity.     

Development of quantitative structure-activity relationship (QSAR) models to predict the carcinogenic potency of chemicals. II. Using oral slope factor as a measure of carcinogenic potency

The overall risk associated with exposure to a chemical is determined by combining quantitative estimates of exposure to the chemical with their known health effects. For chemicals that cause carcinogenicity, oral slope factors (OSFs) and inhalation unit risks are used to quantitatively estimate the carcinogenic potency or the risk associated with exposure to the chemical by oral or inhalation route, respectively. Frequently, there is a lack of animal or human studies in the literature to determine OSFs. This study aims to circumvent this problem by developing quantitative structure-activity relationship (QSAR) models to predict the OSFs of chemicals. The OSFs of 70 chemicals based on male/female human, rat, and mouse bioassay data were obtained from the United States Environmental Protection Agency's Integrated Risk Information System (IRIS) database. A global QSAR model that considered all 70 chemicals as well as species and/or sex-specific QSARs were developed in this study. Study results indicate that the species and sex-specific QSARs (r(2)>0.8, q(2)>0.7) had a better predictive abilities than the global QSAR developed using data from all species and sexes (r(2)=0.77, q(2)=0.73). The QSARs developed in this study were externally validated, and demonstrated reasonable predictive abilities.     

Exposure to chronic intermittent nicotine vapor induces nicotine dependence

Animal models of drug exposure are important tools for the study of the neurobiological mechanisms of nicotine dependence and as preclinical models for medication development. There are few non-invasive animal models of nicotine exposure and currently there is no known animal model of second-hand exposure to nicotine. We hypothesized that chronic administration of nicotine vapors would produce blood levels of nicotine in rodents that are clinically relevant to those observed in human smoking and that rodents exposed to nicotine vapors would develop dependence to nicotine. We developed a system that vaporizes nicotine in the air in a stable, reliable and consistent manner. Intermittent exposure to nicotine vapor (0.2 mg/m³) for 8 or 14 h per day for 7 days produced a concentration of nicotine in the blood of 22 ng/mL. Sixteen hours after removal from nicotine vapors, rats showed significant somatic withdrawal signs precipitated by mecamylamine (1.5 mg/kg). These results provide a new rodent model of nicotine dependence using vapor administration that produces consistent levels of nicotine in the blood that are relevant for both heavy smoking and second-hand smoking, using a non-invasive technique that mimics the intermittent aspect and route of administration in humans.     

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization.     

Latex allergy in the workplace.

While less than 1% of the general population is sensitized to latex, the U.S. Occupational Safety and Health Administration estimates that 8-12% of health-care workers are sensitized. The major source of workplace exposure is powdered natural rubber latex (NRL) gloves. NRL is harvested from HEVEA: brasiliensis trees and ammoniated to prevent coagulation resulting in the hydrolysis of the latex proteins. Prior to use in manufacturing, the latex is formulated by the addition of multiple chemicals. Thus, human exposure is to a mixture of residual chemicals and hydrolyzed latex peptides. Clinical manifestations include irritant contact dermatitis, allergic contact dermatitis (type IV), and type I immediate hypersensitivity response. Type I (IgE-mediated) NRL allergy includes contact urticaria, systemic urticaria, angioedema, rhinitis, conjunctivitis, bronchospasm, and anaphylaxis. Taking an accurate history, including questions on atopic status, food allergy, and possible reactions to latex devices makes diagnosis of type-I latex allergy possible. To confirm a diagnosis, either in vivo skin prick testing (SPT) or in vitro assays for latex-specific IgE are performed. While the SPT is regarded as a primary confirmatory test for IgE-mediated disease, the absence of a U.S. Food and Drug Administration-licensed HEVEA: brasiliensis latex extract has restricted its use in diagnosis. Serological tests have, therefore, become critically important as alternative diagnostic tests. Three manufacturers currently have FDA clearance for in vitro tests, to detect NRL-specific IgE. The commercially available assays may disagree on the antibody status of an individual serum, which may be due to the assay's detecting anti-NRL IgEs to different allergenic NRL proteins. Sensitized individuals produce specific IgE antibody to at least 10 potent HEVEA: allergens, Hev b 1-Hev b 10, each of which differs in its structure, size, and net charge. The relative content and ratios of Hevs in the final allergen preparation most probably could effect diagnostic accuracy. The Hev proteins have been cloned and expressed as recombinant proteins. Sequencing demonstrates both unique epitopes and sequences commonly found in other plant proteins. Sequence homology helps to explain the cross reactivity to a variety of foods experienced by latex allergic individuals. The development of recombinant allergens provides reagents that should improve the diagnostic accuracy of tests for latex allergy. Although clinical and exposure data have been gathered on the factors affecting response in latex-allergic individuals, less is known regarding the development of sensitization. Coupled with in vitro dermal penetration studies, murine models have been established to investigate the route of exposure in the development of latex sensitization. Time-course and dose-response studies have shown subcutaneous, intratracheal, or topical administrations of non-ammoniated latex proteins to induce IgE production. Both in vitro penetration and in vivo studies highlight the importance of skin condition in the development of latex allergy, with enhanced penetration and earlier onset of IgE production seen with experimentally abraded skin. The diagnosis of latex allergy is complicated by these variables, which in turn hinder the development of intervention strategies. Further epidemiological assessment is needed to more explicitly define the scope, trends, and demographics of latex allergy. Diagnostic accuracy can be improved through greater knowledge of proteins involved in the development of latex allergy, and better documentation of the presently available diagnostic tests. In vivo and in vitro models can elucidate mechanisms of sensitization and provide an understanding of the role of the exposure route in latex allergy-associated diseases. Together, these efforts can lead to intervention strategies for reducing latex allergy in the workplace.     

Application of the popliteal lymph node assay in immunotoxicity testing: complementation of the direct popliteal lymph node assay with flow cytometric analyses

The popliteal lymph node assay (PLNA) has been proposed to measure the immunosensitizing potential of chemicals. The direct PLNA detects an immunomodulating effect but does not give insight into the mode of action of the chemical under test. Modifications of this test have been proposed, but they are difficult to perform in routine toxicity testing and require many animals. In the present investigation the direct PLNA was extended with the flow cytometric determinations of: (a) lymphoblasts; (b) the phenotyping of lymphoid subpopulations; (c) the determination of expression of proliferation/activation markers CD25, CD69 and CD62L/CD44 and (d) the analysis of intracellular cytokines interferon gamma, interleukin 2 and interleukin 4. Streptozotocin, hydrazine, HgCl2 and trinitrobenzene sulfonic acid were used as model chemicals. The different mode of action of these substances was well documented by the techniques applied. As the proposed flow cytometric methods can easily be performed and do not require additional test animals this complementation of the direct PLNA seems a promising approach in immunotoxicity testing.     

Toxicologic evidence of developmental neurotoxicity of environmental chemicals

Developmental neurotoxicity constitutes effects occurring in the offspring primarily as a result of exposure of the mother during pregnancy and lactation. To exert their effect, these chemicals or their metabolites must pass the placenta and/or the blood-brain barrier. In experimental animals, exposure to neurotoxic chemicals during critical periods of brain development has induced permanent functional disturbances in the CNS. Although available data suggest that proper animal models exist, only few chemicals have been tested. Neurotoxicity testing is not required by national authorities for classification of chemicals. Epidemiological evidence is very limited, but severe irreversible effects have been observed in humans following in utero exposures to a few known developmental neurotoxicants. The large number of chemicals with a potential for developmental neurotoxicity in humans stresses the importance of generating basic kinetic data on these chemicals based on relevant experimental models. First of all, data are needed on their ability to pass the placenta and the developing blood-brain barrier, to accumulate, and to be metabolized in the placenta and/or the fetus. These kinetic data will be essential in establishing a scientifically based hazard evaluation and risk assessment.     

Alternative approaches to the identification and characterization of chemical allergens.

Chemical allergy can take a variety of forms, those of greatest importance in an occupational setting being skin sensitization resulting in allergic contact dermatitis and sensitization of the respiratory tract associated with asthma and other symptoms. In both cases there is a need for predictive test methods that allow the accurate identification of sensitizing chemicals. Well characterized methods are available for skin sensitization testing, and although to date no tests for respiratory sensitization have been formally validated, progress has been made in defining suitable animal models. In recent years there have been significant advances in our understanding of the cellular and molecular mechanisms through which allergic sensitization to chemicals is induced and regulated. Such progress provides us now with new opportunities to consider alternative approaches to sensitization testing, including the design of in vitro test methods. The greatest investment has been in exploring novel methods for the identification of contact sensitizers and it is upon this aspect of chemical allergy that this article is focused. Described here are some of the general requirements of in vitro test methods for skin sensitization, and progress that has been made in developing suitable approaches with particular emphasis on the utility of dendritic cell culture systems.     

Use of computer-assisted prediction of toxic effects of chemical substances

The current revision of the European policy for the evaluation of chemicals (REACH) has lead to a controversy with regard to the need of additional animal safety testing. To avoid increases in animal testing but also to save time and resources, alternative in silico or in vitro tests for the assessment of toxic effects of chemicals are advocated. The draft of the original document issued in 29th October 2003 by the European Commission foresees the use of alternative methods but does not give further specification on which methods should be used. Computer-assisted prediction models, so-called predictive tools, besides in vitro models, will likely play an essential role in the proposed repertoire of "alternative methods". The current discussion has urged the Advisory Committee of the German Toxicology Society to present its position on the use of predictive tools in toxicology. Acceptable prediction models already exist for those toxicological endpoints which are based on well-understood mechanism, such as mutagenicity and skin sensitization, whereas mechanistically more complex endpoints such as acute, chronic or organ toxicities currently cannot be satisfactorily predicted. A potential strategy to assess such complex toxicities will lie in their dissection into models for the different steps or pathways leading to the final endpoint. Integration of these models should result in a higher predictivity. Despite these limitations, computer-assisted prediction tools already today play a complementary role for the assessment of chemicals for which no data is available or for which toxicological testing is impractical due to the lack of availability of sufficient compounds for testing. Furthermore, predictive tools offer support in the screening and the subsequent prioritization of compound for further toxicological testing, as expected within the scope of the European REACH program. This program will also lead to the collection of high-quality data which will broaden the database for further (Q)SAR approaches and will in turn increase the predictivity of predictive tools.     

Chronic bioassays: relevance to quantitative risk assessment of carcinogens

Conventional carcinogenic potency estimates for chemicals have been compared across rodent species. The high correlations previously demonstrated between maximum likelihood estimates (MLE) for chemicals identified as positive carcinogens in both rats and mice (r = 0.83, n = 83) are shown to occur also for chemicals that were negative in both species (r = 0.85, n = 51), for chemicals causing tumors in rats but not mice (r = 0.55, n = 15), and for those causing tumors in mice but not rats (r = 0.68, n = 25). Corresponding upper-bound estimates of carcinogenic potency are also highly correlated across rodent species. The correlations arise from (i) the strong interspecies correlation between maximum doses tested in chronic bioassays, (ii) the small group sizes utilized, and (iii) the narrow range of doses typically tested. These factors constrain conventional ML and upper-bound potency estimates to lie very close to the inverse maximum doses tested, irrespective of the bioassay's qualitative outcomes. The potency estimates are thus artifacts of experimental design and would seem to provide little information on the actual human cancer risks from chemical exposure. Risk assessment models can reveal true potency differences across species, but they must incorporate relevant mechanistic information before quantitative risk extrapolation from rodents to humans is scientifically defensible.     

Xenobiotic metabolism in human skin and 3D human skin reconstructs: a review

In this review, we discuss and compare studies of xenobiotic metabolism in both human skin and 3D human skin reconstructs. In comparison to the liver, the skin is a less studied organ in terms of characterising metabolic capability. While the skin forms the major protective barrier to environmental chemical exposure, it is also a potential target organ for adverse health effects. Occupational, accidental or intended-use exposure to toxic chemicals could result in acute or delayed injury to the skin (e.g. inflammation, allergy, cancer). Skin metabolism may play a role in the manifestation or amelioration of adverse effects via the topical route. Today, we have robust testing strategies to assess the potential for local skin toxicity of chemical exposure. Such methods (e.g. the local lymph node assay for assessing skin sensitisation; skin painting carcinogenicity studies) incorporate skin metabolism implicitly in the in vivo model system used. In light of recent European legislation (i.e. 7(th) Amendment to the Cosmetics Directive and Registration Evaluation and Authorisation of existing Chemicals (REACH)), non-animal approaches will be required to reduce and replace animal experiments for chemical risk assessment. It is expected that new models and approaches will need to account for skin metabolism explicitly, as the mechanisms of adverse effects in the skin are deconvoluted. 3D skin models have been proposed as a tool to use in new in vitro alternative approaches. In order to be able to use 3D skin models in this context, we need to understand their metabolic competency in relation to xenobiotic biotransformation and whether functional activity is representative of that seen in human skin.     

Untargeted metabolomics of neuronal cell culture: A model system for the toxicity testing of insecticide chemical exposure

Toxicity testing is essential for the protection of human health from exposure to toxic environmental chemicals. As traditional toxicity testing is carried out using animal models, mammalian cell culture models are becoming an increasingly attractive alternative to animal testing. Combining the use of mammalian cell culture models with screening‐style molecular profiling technologies, such as metabolomics, can uncover previously unknown biochemical bases of toxicity. We have used a mass spectrometry‐based untargeted metabolomics approach to characterize for the first time the changes in the metabolome of the B50 cell line, an immortalised rat neuronal cell line, following acute exposure to two known neurotoxic chemicals that are common environmental contaminants; the pyrethroid insecticide permethrin and the organophosphate insecticide malathion. B50 cells were exposed to either the dosing vehicle (methanol) or an acute dose of either permethrin or malathion for 6 and 24 hours. Intracellular metabolites were profiled by gas chromatography–mass spectrometry. Using principal components analysis, we selected the key metabolites whose abundance was altered by chemical exposure. By considering the major fold changes in abundance (>2.0 or <0.5 from control) across these metabolites, we were able to elucidate important cellular events associated with toxic exposure including disrupted energy metabolism and attempted protective mechanisms from excitotoxicity. Our findings illustrate the ability of mammalian cell culture metabolomics to detect finer metabolic effects of acute exposure to known toxic chemicals, and validate the need for further development of this process in the application of trace‐level dose and chronic toxicity studies, and toxicity testing of unknown chemicals.