Nutritional factors in relation to heavy metal toxicants.

An increased environmental exposure to various toxic heavy metals such as lead, cadmium, or mercury seems to be a fact of 20th-century life. But relatively little attention has been paid to the possible implications of sucy exposure for the nutritional status of humans and animals. This review summarizes the information available concerning the effect of various nutritional factors in resistance to metal toxicants and the effect of heavy metal toxicity on nutritional status. In particular, the following questions are considered: 1) Are there any examples of heavy metal toxicity that are potentiated by a nutritional deficiency? 2) Is there any evidence that nutritional deficiency can be caused by heavy metal toxicity? 3) Is there any proof that heavy metal toxicity can be decreased by an excess intake of nutrients: 4) Is there any proof that heavy metal toxicity can be increased by an excess intake of nutrients? The discussion is focused primarily on studies with animal models but, wherever possible, implications for human health are pointed out.     

Biokinetic and toxicodynamic modelling and its role in toxicological research and risk assessment

Toxicological risk assessment for chemicals is still mainly based on highly standardised protocols for animal experimentation and exposure assessment. However, developments in our knowledge of general physiology, in chemicobiological interactions and in (computer-supported) modelling, have resulted in a tremendous change in our understanding of the molecular mechanisms underlying the toxicity of chemicals. This permits the development of biologically based models, in which the biokinetics as well as the toxicodynamics of compounds can be described. In this paper, the possibilities are discussed of developing systems in which the systemic (acute and chronic) toxicities of chemicals can be quantified without the heavy reliance on animal experiments. By integrating data derived from different sources, predictions of toxicity can be made. Key elements in this integrated approach are the evaluation of chemical functionalities representing structural alerts for toxic actions, the construction of biokinetic models on the basis of non-animal data (for example, tissue-blood partition coefficients, in vitro biotransformation parameters), tests or batteries of tests for determining basal cytotoxicity, and more-specific tests for evaluating tissue or organ toxicity. It is concluded that this approach is a useful tool for various steps in toxicological hazard and risk assessment, especially for those forms of toxicity for which validated in vitro and other non-animal tests have already been developed.     

An integrated decision-tree testing strategy for repeat dose toxicity with respect to the requirements of the EU REACH legislation

This paper presents some results of a joint research project conducted by FRAME and Liverpool John Moores University, and sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for repeat dose (sub-acute, sub-chronic and chronic) toxicity testing. It reviews the limited number of in silico and in vitro tests available for this endpoint, and outlines new technologies which could be used in the future, e.g. the use of biomarkers and the 'omics' technologies. An integrated testing strategy is proposed, which makes use of as much non-animal data as possible, before any essential in vivo studies are performed. Although none of the non-animal tests are currently undergoing validation, their results could help to reduce the number of animals required for testing for repeat dose toxicity.     

Integrated decision-tree testing strategies for acute systemic toxicity and toxicokinetics with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME conducted a joint research project, sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for acute systemic toxicity and toxicokinetic testing. The paper reviews in vitro tests based on basal cytotoxicity and target organ toxicity, along with QSAR models and expert systems available for this endpoint. The use of PBPK modelling for the prediction of ADME properties is also discussed. These tests are then incorporated into a decision-tree style, integrated testing strategy, which also includes the use of refined in vivo acute toxicity tests, as a last resort. The implementation of the strategy is intended to minimise the use of animals in the testing of acute systemic toxicity and toxicokinetics, whilst satisfying the scientific and logistical demands of the EU REACH legislation.     

Drug delivery and nanoparticles:applications and hazards

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatine and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery. Besides the potential beneficial use also attention is drawn to the questions how we should proceed with the safety evaluation of the nanoparticle formulations for drug delivery. For such testing the lessons learned from particle toxicity as applied in inhalation toxicology may be of use. Although for pharmaceutical use the current requirements seem to be adequate to detect most of the adverse effects of nanoparticle formulations, it can not be expected that all aspects of nanoparticle toxicology will be detected. So, probably additional more specific testing would be needed.     

Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds?

Today reconstructed skin models that simulate human skin, such as Episkin, are widely used for safety or efficacy pre-screening. Moreover, they are of growing interest for regulatory purposes in the framework of alternatives to animal testing. In order to reduce and eventually replace results of in vivo genotoxicity testing with in vitro data, there is a need to develop new complementary biological models and methods with improved ability to predict genotoxic risk. This can be achieved if these new assays do take into account exposure conditions that are more relevant than in the current test systems. In an attempt to meet this challenge, two new applications using a human reconstructed skin model for in vitro genotoxicity assessment are proposed. The skin is the target organ for dermally exposed compounds or environmental stress. Although attempts have been made to develop genotoxicity test procedures in vivo on mouse skin, human reconstructed skin models have not been used for in vitro genotoxicity testing so far, although they present clear advantages over mouse skin for human risk prediction. This paper presents the results of the development of a specific protocol allowing to perform the comet assay, a genotoxicity test procedure, on reconstructed skin. The comet assay was conducted after treatment of Episkin with UV, Lomefloxacin and UV or 4-nitroquinoline-N-oxide (4NQO). Treatment with the sunscreen Mexoryl was able to reduce the extent of comet signal. A second approach to use reconstructed epidermis in genotoxicity assays is also proposed. Indeed, the skin is a biologically active barrier driving the response to exposure to chemical agents and their possible metabolites. A specific co-culture system (Figure 1) using Episkin to perform the regular micronucleus assay is presented. Micronucleus induction in L5178Y cells cultured underneath Episkin was assessed after treatment of the reconstructed epidermis with mitomycin C, cyclophosphamide or apigenin. This second way of using human reconstructed skin for genotoxicity testing aims at improving the relevance of exposure conditions in in vitro genotoxicity assays for dermally applied compounds.     

Long-term in vitro toxicity models: comparisons between a flow-cell bioreactor,a static-cell bioreactor and static cell cultures

In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects. The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse, Integra, Wallisellen, Switzerland) and a static cell bioreactor system (CELLine CL 6-well, Integra), and to compare them with the use of conventional cell culture flasks. A human cell line, Int 407, was exposed to cadmium chloride (CdCl(2); 10-(7-)10-(8)M) for 4 weeks. Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay. In addition, total protein determination and cadmium uptake measurements were performed. The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl(2) concentration. However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl(2). Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl(2) concentration applied and the system used. In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies. It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available.     

Use of in vivo genetic toxicity data for risk assessment

Mutagenicity studies have been used to identify specific agents as potential carconogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1–10 parts per million for man and 0.04–0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risk associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal. In addition, it should allow truly epigenetic or non-genotoxic carcinogens to be distinguished from the genotoxic carcinogens that are not detected by in vitro methods.     

Impact of manganese on and transfer across blood-brain and blood-cerebrospinal fluid barrier in vitro

Manganese occupational and dietary overexposure has been shown to result in specific clinical central nervous system syndromes, which are similar to those observed in Parkinson disease. To date, modes of neurotoxic action of Mn are still to be elucidated but are thought to be strongly related to Mn accumulation in brain and oxidative stress. However, the pathway and the exact process of Mn uptake in the brain are yet not fully understood. Here, two well characterized primary porcine in vitro models of the blood-brain and the blood-cerebrospinal fluid (CSF) barrier were applied to assess the transfer of Mn in the brain while monitoring its effect on the barrier properties. Thus, for the first time effects of MnCl(2) on the integrity of these two barriers as well as Mn transfer across the respective barriers are compared in one study. The data reveal a stronger Mn sensitivity of the in vitro blood-CSF barrier compared with the blood-brain barrier. Very interestingly, the negative effects of Mn on the structural and functional properties of the highly Mn-sensitive blood-CSF barrier were partly reversible after incubation with calcium. In summary, both the observed stronger Mn sensitivity of the in vitro blood-CSF barrier and the observed site-directed, most probably active, Mn transport toward the brain facing compartment, reveal that, in contrast to the general assumption in literature, after oral Mn intake the blood-CSF barrier might be the major route for Mn into the brain.     

Renal proximal tubular cells in suspension or in primary culture as in vitro models to study nephrotoxicity

The kidney forms a frequent target for xenobiotic toxicity. The complex biochemical mechanisms underlying nephrotoxicity are best studied in vitro provided that reliable and relevant in vitro models are available. Since most nephrotoxicants affect primarily the cells of the proximal tubules (PTC), much effort has been directed towards the development of in vitro models of PTC. This review focuses on the preparation of PTC and the use of these cells. Discussed are important criteria such as the viability (survival time) of the cells and the parameters to assess toxicity. Recent studies have shown that isolated PTC in suspension are especially suitable for studies on the biochemical mechanisms of 'acute' nephrotoxicity, whereas PTC in primary culture may be used to investigate mechanisms of nephrotoxic damage at very low concentrations, upon prolonged exposure. PTC cultured on porous filter membranes provide new possibilities to study toxicity in relation to cell and transport polarity. Primary cell cultures of human PTC have been set up. Although a further characterization of these systems is needed, recent data indicate their usefulness.     

Invited Debate/Commentary: Selenium — A Potential Time Bomb or Just Another Contaminant?

Environmental issues related to selenium are complex and not universally understood. This paper provides detailed information regarding selenium and associated aquatic environmental issues, and then introduces and comments on five debate/commentary papers discussing selenium in the context of the title of this paper. Selenium has a complex and not fully understood biogeochemistry in the aquatic environment as well as an unusual mode of toxicity (acute via water column exposure; chronic via food chain exposure). It has the narrowest range between nutritional requirements and toxicity of any essential element, and chronic toxicity is not readily predictable. Selenium contamination of waters or even of tissues does not necessarily indicate a ticking time bomb; there are no generally accepted or universally accepted threshold values for chronic toxicity. Assessing risk must be done site-specifically in a risk assessment framework, focusing on reproductive effects to sensitive exposed fish and waterfowl and on “worst case” hydrologic units. Unless selenium inputs decrease or site-specific biogeochemistry can be shown not to change, continued biological monitoring and testing are required. Provided the necessary investigative and monitoring studies are done, any potential selenium time bombs can be defused.     

The open-access dataset for insilico cardiotoxicity prediction system

Drug cardiotoxicity is one of the main reasons of fatal drug related problem events and the subsequent withdrawals. Therefore, its early assessment is a crucial element of the drug development process. For the drug driven hERG inhibition assessment, which is assumed to be the main reason for toxicity, in vitro techniques are used. Gold standards are based on the Patch Clamp method with the use of various cell models but due to its low throughput, insilico models have become more appreciated. To develop a reliable empirical QSAR model, wide dataset containing a variety of cases has to be available. In this article, a freely available for download, set of data is described. It is based on literature peer-reviewed reports and contains hERG inhibition information expressed as IC50 value for 263 molecules described in 642 records. All studies were done with the use of three cell models (XO, CHO, HEK) and other elements describe the electrophysiological settings of the in vitro study. The above mentioned set was used for the successful development of the predictive models.     

Biostatistical methods for the validation of alternative methods for in vitro toxicity testing

Statistical methods for the validation of toxicological in vitro test assays are developed and applied. Validation is performed either in comparison with in vivo assays or in comparison with other in vitro assays of established validity. Biostatistical methods are presented which are of potential use and benefit for the validation of alternative methods for the risk assessment of chemicals, providing at least an equivalent level of protection through in vitro toxicity testing to that obtained through the use of current in vivo methods. Characteristic indices are developed and determined. Qualitative outcomes are characterised by the rates of false-positive and false-negative predictions, sensitivity and specificity, and predictive values. Quantitative outcomes are characterised by regression coefficients derived from predictive models. The receiver operating characteristics (ROC) technique, applicable when a continuum of cut-off values is considered, is discussed in detail, in relation to its use for statistical modelling and statistical inference. The methods presented are examined for their use for the proof of safety and for toxicity detection and testing. We emphasise that the final validation of toxicity testing is human toxicity, and that the in vivo test itself is only a predictor with an inherent uncertainty. Therefore, the validation of the in vitro test has to account for the vagueness and uncertainty of the "gold standard" in vivo test. We address model selection and model validation, and a four-step scheme is proposed for the conduct of validation studies. Gaps and research needs are formulated to improve the validation of alternative methods for in vitro toxicity testing.     

Human Health Risk Evaluation of ACQ-Treated Wood

Alkaline copper quaternary (ACQ) Type D is one of several compounds currently being used as a chemical preservative to treat wood for prevention of rot and decay. As wood weathers naturally, human exposure to ACQ might occur through dermal contact or incidental ingestion of residues from the wood surface. To understand any potential for health risks from the use of ACQ-treated wood, a health-based evaluation was undertaken on the primary components of ACQ, which are copper, didecyl dimethyl ammonium carbonate, 2-methyl-4-isothiazolin-3-one, and 5-chloro-2-methyl-4-isothiazolin-3-one. For these components, there are no formalized toxicity values in USEPA's Integrated Risk Information System, although extensive toxicity data are available in the scientific literature. Therefore, health-based toxicity benchmarks were derived from a review of existing toxicity data. The exposure assessment was based on methods developed by the Consumer Product Safety Commission in their evaluation of the potential for children's exposure to arsenic from wood treated with chromated copper arsenate. This approach entailed wipe testing the surface of treated wood to determine the amount of chemical that might be removed from the wood, and estimating the amount of chemical that a child might contact via the dermal route or incidental ingestion through hand-to-mouth activities. All calculated exposure estimates were well below toxicity benchmarks.     

Metabolic response to low-level toxicant exposure in a novel renal tubule epithelial cell system

Toxicity testing is vital to protect human health from exposure to toxic chemicals in the environment. Furthermore, combining novel cellular models with molecular profiling technologies, such as metabolomics can add new insight into the molecular basis of toxicity and provide a rich source of biomarkers that are urgently required in a 21st Century approach to toxicology. We have used an NMR-based metabolic profiling approach to characterise for the first time the metabolome of the RPTEC/TERT1 cell line, an immortalised non-tumour human renal epithelial cell line that recapitulates phenotypic characteristics that are absent in other in vitro renal cell models. RPTEC/TERT1 cells were cultured with either the dosing vehicle (DMSO) or with exposure to one of six compounds (nifedipine, potassium bromate, monuron, D-mannitol, ochratoxin A and sodium diclofenac), several of which are known to cause renal effects. Aqueous intracellular and culture media metabolites were profiled by (1)H NMR spectroscopy at 6, 24 and 72 hours of exposure to a low effect dose (IC(10)). We defined the metabolome of the RPTEC/TERT1 cell line and used a principal component analysis approach to derive a panel of key metabolites, which were altered by chemical exposure. By considering only major changes (±1.5 fold change from control) across this metabolite panel we were able to show specific alterations to cellular processes associated with chemical treatment. Our findings suggest that metabolic profiling of RPTEC/TERT1 cells can report on the effect of chemical exposure on multiple cellular pathways at low-level exposure, producing different response profiles for the different compounds tested with a greater number of major metabolic effects observed in the toxin treated cells. Importantly, compounds with established links to chronic renal toxicity produced more diverse and severe perturbations to the cellular metabolome than non-toxic compounds in this model. As these changes can be rationalised with the different pharmacological and toxicity profiles of the chemicals it is suggested that metabolic profiling in the RPTEC/TERT1 model would be useful in investigating the mechanism of action of toxins at a low dose.     

Use of Toxicological Data in Estimating Reference Values for Risk Assessment

A number of programs within the U.S. Environmental Protection Agency (USEPA) currently set less-than-lifetime exposure limits in addition to the chronic reference dose (RfD) and reference concentration (RfC). A review of procedures within the USEPA for setting reference values suggests that less-thanlifetime reference values should be more routinely developed and captured in the USEPA's online IRIS database where chronic RfDs and RfCs, as well as cancer slope factors, are currently available. A review of standard testing study protocols was conducted to determine what data were available for setting acute, short-term, and longer-term reference values, as well as chronic values. This review was done from the point of view of endpoints assessed for specific organ systems (both structural and functional), life stages covered by exposure and outcome, durations of exposure covered and the outcomes evaluated for each, and evaluation of latency to response and/or reversibility of effects. This review revealed a number of data gaps and research needs, including the need for an acute and/or short-term testing protocol that can be used to set acute and shortterm reference values, a strategy for when to conduct more extensive testing based on initial screening data or other information (e.g., chemical class, pharmacokinetics, mode of action), additonal standard testing guidlines protocols to allow more complete assessment of certain organ systems and life stages, development of pharmacokinetic data for different life stages, toxicity related to aging, and latency to response, particularly long-term latency as a result of developmental exposures. The implications of this review are discussed relative to characterizing hazard data for setting reference values, and the potential effects on uncertainty factors and low-dose extrapolation.     

Signaling pathways regulating blood–tissue barriers — Lesson from the testis

Signaling pathways that regulate blood–tissue barriers are important for studying the biology of various blood–tissue barriers. This information, if deciphered and better understood, will provide better therapeutic management of diseases particularly in organs that are sealed by the corresponding blood–tissue barriers from systemic circulation, such as the brain and the testis. These barriers block the access of antibiotics and/or chemotherapeutical agents across the corresponding barriers. Studies in the last decade using the blood–testis barrier (BTB) in rats have demonstrated the presence of several signaling pathways that are crucial to modulate BTB function. Herein, we critically evaluate these findings and provide hypothetical models regarding the underlying mechanisms by which these signaling molecules/pathways modulate BTB dynamics. This information should be carefully evaluated to examine their applicability in other tissue barriers which shall benefit future functional studies in the field. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.