Tricyclic antipsychotics promote adipogenic gene expression to potentiate preadipocyte differentiation in vitro.

Antipsychotic-induced weight gain is a well-established but poorly understood clinical phenomenon. New mechanistic insights into how antipsychotics modulate adipose physiology are sorely needed, in hopes of either devising a therapeutic intervention to ameliorate weight gain or contributing to improved design of future agents. In this study, we have hypothesized that the weight gain-associated tricyclic antipsychotics clozapine and chlorpromazine directly impact adipose tissue by potentiating adipogenic differentiation of preadipocytes. Utilizing a well-established in vitro model system (3T3-L1 preadipocyte cell line), we demonstrate that, when applied specifically during induction of adipogenic differentiation, both clozapine and chlorpromazine significantly potentiate in vitro adipogenesis, observed as morphological changes and increased intracellular lipid accumulation. These persistent effects, observed at endpoints well after the end of antipsychotic exposure, are accompanied by increased transcript- and protein-level expression of the mature adipocyte marker perilipin-1, as indicated by RT-qPCR and Western blotting, but not by further upregulation of pro-adipogenic transcription factors versus positive controls. Our findings point to a possible physiological mechanism of antipsychotic-induced hyperplasia, with potentiated expression of mature adipocyte markers enhancing the differentiation and maturation of preadipocytes.

Assessment of the genotoxic and carcinogenic risks of p-nitrophenol when it is present as an impurity in a drug product.

According to the 2008 US FDA (draft) and 2006 EMEA guidance documents for genotoxic impurities, an impurity that is positive in an in vitro genotoxicity study, in the absence of in vivo genotoxicity or carcinogenicity data, should be treated as genotoxic and typically controlled to 1.5 microg/day for chronic use. For p-nitrophenol (PNP), existing study results (i.e., positive in vitro clastogenicity in mammalian cells, no information on its in vivo genotoxicity, and negative with respect to carcinogenicity in a dermal mouse study with no confirmation of systemic exposure) indicated that it should be considered genotoxic and exposure as a drug impurity limited. Therefore, to more completely characterize the genotoxic potential of PNP (consistent with the guidance documents), in vivo mouse micronucleus and dermal pharmacokinetic bridging studies were conducted. In the micronucleus study, PNP was negative, demonstrating that the reported in vitro clastogenicity is not present in vivo. In the pharmacokinetic study, PNP was well absorbed dermally, validating the negative dermal carcinogenicity assessment. These results indicate that PNP should be considered a non-genotoxic impurity and, as a drug impurity, a threshold limit of 4 mg/day would be set (per ICH Q3C). This threshold limit is higher than the EPA reference dose (listed in the 2006 Edition of the Drinking Water Standards and Health Advisories), so if present at such levels, the specification limits for PNP should be determined on a case-by-case basis, based on risk-benefit.

Predictive models for carcinogenicity and mutagenicity: frameworks, state-of-the-art, and perspectives.

Mutagenicity and carcinogenicity are endpoints of major environmental and regulatory concern. These endpoints are also important targets for development of alternative methods for screening and prediction due to the large number of chemicals of potential concern and the tremendous cost (in time, money, animals) of rodent carcinogenicity bioassays. Both mutagenicity and carcinogenicity involve complex, cellular processes that are only partially understood. Advances in technologies and generation of new data will permit a much deeper understanding. In silico methods for predicting mutagenicity and rodent carcinogenicity based on chemical structural features, along with current mutagenicity and carcinogenicity data sets, have performed well for local prediction (i.e., within specific chemical classes), but are less successful for global prediction (i.e., for a broad range of chemicals). The predictivity of in silico methods can be improved by improving the quality of the data base and endpoints used for modelling. In particular, in vitro assays for clastogenicity need to be improved to reduce false positives (relative to rodent carcinogenicity) and to detect compounds that do not interact directly with DNA or have epigenetic activities. New assays emerging to complement or replace some of the standard assays include Vitotox, GreenScreenGC, and RadarScreen. The needs of industry and regulators to assess thousands of compounds necessitate the development of high-throughput assays combined with innovative data-mining and in silico methods. Various initiatives in this regard have begun, including CAESAR, OSIRIS, CHEMOMENTUM, CHEMPREDICT, OpenTox, EPAA, and ToxCast. In silico methods can be used for priority setting, mechanistic studies, and to estimate potency. Ultimately, such efforts should lead to improvements in application of in silico methods for predicting carcinogenicity to assist industry and regulators and to enhance protection of public health.

Regulatory aspects of genotoxicity testing: from hazard identification to risk assessment.

This symposium focused on the use of tests for chromosomal damage, and other genotoxicity measures, for detection of potentially harmful chemicals. The speakers discussed the information that has been gained over the last three decades about the use of "short-term tests" for genotoxicity in cultured cells and in animals (mainly rodents), and the ongoing debates about the rational use of data from such experimental systems in trying to extrapolate to an understanding of potential human risk. The overall theme was that the field of regulatory toxicology currently is over-reliant on qualitative outcomes of in vitro hazard-screening tests, generally conducted at the maximum achievable exposures, and needs a more realistic approach that incorporates in vivo exposure levels and dose-response information.

Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. I. Theoretical aspects.

A decrease in the cytokinesis-block proliferation index (CBPI) or replication index (RI) is routinely used to determine cytotoxicity of a test compound and therefore the choice of its appropriate test concentration for the in vitro micronucleus (MN) test conducted in the presence of cytochalasin B. As a number of laboratories prefer to conduct the in vitro MN test in the absence of cytochalasin B, it is important that selected test concentrations, based on cytotoxicity, should be similar to what they would have been if cytochalasin B had been used, and should be relevant of a true cytotoxicity. By using models to analyse the dynamics of the cell cultures with and without cytochalasin B we have compared different methods for evaluation of cytotoxicity, and demonstrate that relative decrease in population doubling or relative increase in cell counts are the most appropriate measures of cytotoxicity to compare with reduction in CBPI or RI.

In vitro approaches to develop weight of evidence (WoE) and mode of action (MoA) discussions with positive in vitro genotoxicity results.

A recent analysis by Kirkland et al. [Kirkland, D., Aardema, M., Henderson, L. and Müller, L. (2005) Evaluation of the ability of a battery of 3 in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat. Res. 584, 1-256] demonstrated an extremely high false positive rate for in vitro genotoxicity tests when compared with carcinogenicity in rodents. In many industries, decisions have to be made on the safety of new substances, and health risk to humans, without rodent carcinogenicity data being available. In such cases, the usual way to determine whether a positive in vitro genotoxicity result is relevant (i.e. indicates a hazard) for humans is to develop weight of evidence (WoE) or mode of action (MoA) arguments. These are based partly on further in vitro investigations, but usually rely heavily on tests for genotoxicity in one or more in vivo assays. However, for certain product types in the European Union, the use of animals for genotoxicity testing (as well as for other endpoints) will be prohibited within the next few years. Many different examples have been described that indicate DNA damage and genotoxic responses in vitro can arise through non-relevant in vitro events that are a result of the test systems and conditions used. The majority of these non-relevant in vitro events can be grouped under a category of 'overload of normal physiology' that would not be expected to occur in exposed humans. However, obtaining evidence in support of such MoAs is not easy, particularly for those industries prohibited from carrying out in vivo testing. It will become necessary to focus on in vitro studies to provide evidence of non-DNA, threshold or in vitro-specific processes and to discuss the potential for such genotoxic effects to occur in exposed humans. Toward this end, we surveyed the published literature for in vitro approaches that may be followed to determine whether a genotoxic effect observed in vitro will occur in humans. Unfortunately, many of the approaches we found are based on only a few published examples and validated approaches with consensus recommendations often do not exist. This analysis highlights the urgent need for developing consensus approaches that do not rely on animal studies for dealing with in vitro genotoxins.

Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing.

This report summarizes the proceedings of the September 9-10, 2005 meeting of the Expert Working Group on Hazard Identification and Risk Assessment in Relation to In Vitro Testing, part of an initiative on genetic toxicology. The objective of the Working Group was to develop recommendations for interpretation of results from tests commonly included in regulatory genetic toxicology test batteries, and to propose an appropriate strategy for follow-up testing when positive in vitro results were obtained in these assays. The Group noted the high frequency of positive in vitro findings in the genotoxicity test batteries with agents found not to be carcinogenic and thought not to pose a carcinogenic health hazard to humans. The Group agreed that a set of consensus principles for appropriate interpretation and follow-up testing when initial in vitro tests are positive was needed. Current differences in emphasis and policy among different regulatory agencies were recognized as a basis of this need. Using a consensus process among a balanced group of recognized international authorities from industry, government, and academia, it was agreed that a strategy based on these principles should include guidance on: (1) interpretation of initial results in the "core" test battery; (2) criteria for determining when follow-up testing is needed; (3) criteria for selecting appropriate follow-up tests; (4) definition of when the evidence is sufficient to define the mode of action and the relevance to human exposure; and (5) definition of approaches to evaluate the degree of health risk under conditions of exposure of the species of concern (generally the human). A framework for addressing these issues was discussed, and a general "decision tree" was developed that included criteria for assessing the need for further testing, selecting appropriate follow-up tests, and determining a sufficient weight of evidence to attribute a level of risk and stop testing. The discussion included case studies based on actual test results that illustrated common situations encountered, and consensus opinions were developed based on group analysis of these cases. The Working Group defined circumstances in which the pattern and magnitude of positive results was such that there was very low or no concern (e.g., non-reproducible or marginal responses), and no further testing would be needed. This included a discussion of the importance of the use of historical control data. The criteria for determining when follow-up testing is needed included factors, such as evidence of reproducibility, level of cytotoxicity at which an increased DNA damage or mutation frequency is observed, relationship of results to the historical control range of values, and total weight of evidence across assays. When the initial battery is negative, further testing might be required based on information from the published literature, structure activity considerations, or the potential for significant human metabolites not generated in the test systems. Additional testing might also be needed retrospectively when increase in tumors or evidence of pre-neoplastic change is seen. When follow-up testing is needed, it should be based on knowledge about the mode of action, based on reports in the literature or learned from the nature of the responses observed in the initial tests. The initial findings, and available information about the biochemical and pharmacological nature of the agent, are generally sufficient to conclude that the responses observed are consistent with certain molecular mechanisms and inconsistent with others. Follow-up tests should be sensitive to the types of genetic damage known to be capable of inducing the response observed initially. It was recognized that genotoxic events might arise from processes other than direct reactivity with DNA, that these mechanisms may have a non-linear, or threshold, dose-response relationship, and that in such cases it may be possible to determine an exposure level below which there is negligible concern about an effect due to human exposures. When a test result is clearly positive, consideration of relevance to human health includes whether other assays for the same endpoint support the results observed, whether the mode or mechanism of action is relevant to the human, and - most importantly - whether the effect observed is likely to occur in vivo at concentrations expected as a result of human exposure. Although general principles were agreed upon, time did not permit the development of recommendations for the selection of specific tests beyond those commonly employed in initial test batteries.

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes.

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.

Chromosomal aberration frequencies in patients with thalassaemia major undergoing therapy with deferiprone and deferoxamine in a comparative crossover study.

Measurements of chromosomal aberrations were made in 10 thalassaemia major patients treated long-term with deferiprone (at least 5 years) and compared with an equal number of patients matched for age, sex and iron overload, treated long-term with deferoxamine. Two blood samples were collected from each patient, 7 and 20 days after a transfusion episode, and the frequency of chromosomal aberrations (gaps, breaks and exchanges) in the patients' circulating lymphocytes analysed in both samples using standard cytogenetic staining techniques. The frequency of reciprocal translocations was also analysed using fluorescence in situ hybridization. Relatively low frequencies of cells with stable and unstable aberrations were seen at both sampling times in all patients, with no statistically significant differences between sexes. Chromosomal aberrations were less frequent in patients treated long-term with deferiprone than in patients treated with deferoxamine, although the difference did not reach statistical significance. After the second blood sample had been collected, all patients had their iron chelation therapy switched to the other chelator. Patients treated long-term with deferiprone had their therapy switched to deferoxamine and patients treated long-term with deferoxamine had their therapy switched to deferiprone. After the switch, two further blood samples were collected 7 and 20 days after transfusion for each of the next two transfusion cycles in all patients. Analysis of the post-switch samples also revealed a slightly higher frequency of chromosomal aberrations during therapy with deferoxamine than with deferiprone at all time points. A small, but statistically significant, increase in cells with aberrations was observed at the first post-switch assessment in the group of patients whose therapy was switched from deferiprone to deferoxamine, whereas the switch from deferoxamine to deferiprone was associated with a decrease in the frequency of chromosomal aberrations. The results of the study demonstrate that, in a clinical setting, deferiprone has no greater clastogenic activity than that of deferoxamine.

Evaluation of thresholds for benomyl- and carbendazim-induced aneuploidy in cultured human lymphocytes using fluorescence in situ hybridization.

Threshold mechanisms of activity for mutagenic agents have been debated for some time, especially for those substances which induce aneuploidy by inhibiting mitotic spindle function. No observed effect levels (NOELs) or "practical thresholds" have been demonstrated for several aneugens both in vitro and in vivo generally by either counting chromosomes in metaphase preparations or by observing micronuclei. Recently, fluorescence in situ hybridization (FISH) has proven to be a sensitive and useful technique for the assessment of aneuploidy at low concentrations. Using binucleate human lymphocytes coupled with FISH, we have been able to characterize a threshold mechanism of action for two spindle inhibitors, benomyl and its active metabolite, carbendazim. Test chemicals were added 24 h following culture initiation. After a further 20 h, cytochalasin B was added, and cells were harvested 28 h later (72 h post initiation). The distribution of chromosomes between the nuclei of binucleate cells was evaluated by fluorescence microscopy for the simultaneous detection of centromeres labeled with FITC (green) or Cy-3 (red). Six human chromosomes were investigated in pairs (1 and 8, 11 and 18, and X and 17). Abnormalities were classified as chromosome loss (including centromeric positive micronuclei), chromosome gain, non-disjunction, or polyploidy. Dose-response data were generated over a range of closely spaced concentrations at 100 ng/ml intervals. The threshold, defined as the lowest "effect" concentration using statistical methods, was determined for each chromosome. Non-disjunction proved to be the most sensitive endpoint for the detection of aneuploidy occurring at higher frequencies and lower concentrations. Results for the six chromosomes demonstrated similar dose-response data which included a series of concentrations with no statistically significant increase above background, followed by a second range of higher concentrations with a statistically significant, concentration-dependent increase. Nearly equimolar threshold concentrations were determined for benomyl- and carbendazim-induced non-disjunction.

Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds.

Despite recent improvements in genotoxicity protocols, we have observed an increase in the occurrence of positive results, particularly in chromosomal aberration tests in vitro, yet very few of these are accompanied by positive responses in vivo. Thus, the positive results may not be biologically relevant either for rodents or humans in vivo, but how should we determine "biological relevance"? Chemicals that produce thresholded dose-responses may well not pose a genotoxic risk at low (relevant to human) exposures, but thresholds should not just be "seen"; there must be an explanation and understanding of the underlying mechanism. In addition to extremes of pH, ionic strength and osmolality, as have been identified previously, such mechanisms include indirect genotoxicity resulting from interaction with non-DNA targets, chemicals/metabolites which are inherently genotoxic but which, at low concentrations, are effectively conjugated and unable to form adducts, and production of specific metabolites under in vitro conditions that are not formed in rodents or humans in vivo. If such thresholded mechanisms can be identified at exposures which are well in excess of expected human exposure, then there may be a strong argument that the positive results are not biologically relevant.