Electromagnetic exposure of scaffold-free three-dimensional cell culture systems

In recent years, a number of in vitro studies have reported on the possible athermal effects of electromagnetic exposure on biological tissue. Typically, this kind of study is performed on monolayers of primary cells or cell lines. However, two‐dimensional cell layer systems lack physiological relevance since cells in vivo are organized in a three‐dimensional (3D) architecture. In monolayer studies, cell‐cell and cell‐ECM interactions obviously differ from live tissue and scale‐ups of experimental results to in vivo systems should be considered carefully. To overcome this problem, we used a scaffold‐free 3D cell culture system, suitable for the exploration of electrophysiological effects due to electromagnetic fields (EMF) at 900 MHz. Dissociated cardiac myocytes were reaggregated into cellular spheres by constant rotation, and non‐invasive extracellular recordings of these so‐called spheroids were performed with microelectrode arrays (MEA). In this study, 3D cell culture systems were exposed to pulsed EMFs in a stripline setup. We found that inhomogeneities in the EMF due to electrodes and conducting lines of the MEA chip had only a minor influence on the field distribution in the spheroid if the exposure parameters were chosen carefully. Bioelectromagnetics 32:351–359, 2011. © 2011 Wiley‐Liss, Inc.     

Review of the in vivo genotoxicity tests performed with styrene

Results from new genotoxicity tests in laboratory animals have necessitated a comprehensive re-evaluation of the mutagenic potential of styrene in vivo. Available data suggest that styrene, after being metabolized to styrene oxide, is weakly positive in indicator tests detecting DNA adducts, DNA strand breaks and sister chromatid exchanges (SCEs). There is no convincing evidence of styrene clastogenicity in experimental animals when the quality of the studies and the plausibility of the test results are considered. Equivocal results were obtained after exposure to high doses causing lethality. A recently published in vivo micronucleus test (MNT) in bone marrow cells of mice conforming to the current OECD guideline was clearly negative. Consequently, our evaluation of the published genotoxicity data comes to the conclusion that styrene at high doses can induce genotoxic effects in indicator tests. These DNA effects depend upon the exposure levels of the target cells, the metabolic activation to styrene oxide and the efficiency of detoxification. Mutagenic effects of styrene can only be expected under extreme exposure conditions if styrene oxide is not efficiently detoxified and primary DNA lesions are not completely repaired. However, there is no clear evidence that styrene induces mutagenic/clastogenic effects in vivo when tested under appropriate test conditions.     

Review of the genotoxicity of styrene in humans

Styrene (CAS No. 100-42-5) is an important industrial chemical for which positive results have been reported in in vitro and in vivo genotoxicity assays. Styrene-exposed workers have been studied extensively over two decades for the induction of various types of genotoxic effects. The outcomes of these studies have been conflicting, and where positive responses have been reported, it has proved difficult to demonstrate clear relationships between levels of damage reported and exposure levels. In this review, we have assessed studies addressing mutagenicity (chromosome aberrations, micronuclei and gene mutations) and other endpoints (sister chromatid exchanges, DNA breaks and DNA adducts) using criteria derived from the IPCS guidelines for the conduct of human biomonitoring studies. Based on the re-evaluated outcomes, the data are not convincing that styrene induces gene mutations. The evidence for induction of clastogenicity in occupationally exposed workers is less clear, with a predominant lack of induction of micronuclei in different studies, but conflicting responses in chromosome aberration assays. The results of numerous studies on sister chromatid exchanges do not provide evidence of a clear positive response, despite these being induced in animals exposed to styrene at high concentrations. However, there is evidence that both DNA adducts and DNA single strand breaks are induced in styrene workers. These types of damage are considered indicative of exposure of the target cells and interaction with cellular DNA but do not necessarily result in heritable changes. There is evidence that the metabolism of styrene in humans is affected by genetic polymorphisms of metabolizing genes and that these polymorphisms affect the outcome of in vitro mutagenicity studies on styrene. Therefore, studies that have addressed the potential of this factor to affect in vivo responses were considered. To date, there are no consistent relationships between genetic polymorphisms and induction of genotoxicity by styrene in humans, but further work is warranted on larger samples. The analyses of individual studies, together with a consideration of dose-response relationships and the lack of a common profile of positive responses for the various endpoints in different studies, provide no clear evidence that styrene exposure in workers results in detectable levels of mutagenic damage. However, evidence of exposure to genotoxic metabolites is demonstrated by the formation of DNA adducts and strand breaks.     

Inflammatory and genotoxic effects of diesel particles in vitro and in vivo

Recent studies have identified an indirect genotoxicity pathway involving inflammation as one of the mechanisms underlying the carcinogenic effects of air pollution/diesel exhaust particles (DEP). We investigated the short-term effects of DEP on markers of inflammation and genotoxicity in vitro and in vivo. DEP induced an increase in the mRNA level of pro-inflammatory cytokines and a higher level of DNA strand breaks in the human lung epithelial cell line A549 in vitro. For the in vivo study, mice were exposed by inhalation to 20 or 80 mg/m3 DEP either as a single 90-min exposure or as four repeated 90-min exposures (5 or 20 mg/m3) and the effects in broncho-alveolar lavage (BAL) cells and/or lung tissue were characterized. Inhalation of DEP induced a dose-dependent inflammatory response with infiltration of macrophages and neutrophils and elevated gene expression of IL-6 in the lungs of mice. The inflammatory response was accompanied by DNA strand breaks in BAL cells and oxidative DNA damage and increased levels of bulky DNA adducts in lung tissue, the latter indicative of direct genotoxicity. The effect of a large single dose of DEP was more pronounced and sustained on IL-6 expression and oxidative DNA damage in the lung tissue than the effect of the same dose administered over four days, whereas the reverse pattern was seen in BAL cells. Our results suggest that the effects of DEP depend on the rate of delivery of the particle dose. The mutation frequency (MF), after DEP exposure, was determined using the transgenic Muta Mouse and a similar exposure regimen. No increase was observed in MF in lung tissue 28-days after exposure. In conclusion, short-term exposure to DEP resulted in DNA strand breaks in BAL cells, oxidative DNA damage and DNA adducts in lungs; and suggested that DNA damage in part is a consequence of inflammatory processes. The response was not associated with increased MF, indicating that the host defence mechanisms were sufficient to counteract the adverse effects of inflammation. Thus, there may be thresholds for the inflammation-associated genotoxic effects of DEP inhalation.     

The comet assay as an indicator test for germ cell genotoxicity

The in vivo comet assay is a well-established genotoxicity test. It is currently mainly performed with somatic cells from different organs to detect a genotoxic activity of potential carcinogens. It is regarded as a useful test for follow-up testing of positive or equivocal in vitro test results and for the evaluation of local genotoxicity. However, the comet assay also has the potential to detect germ cell genotoxicity and may be used for demonstrating the ability of a substance or its metabolite(s) to directly interact with the genetic material of gonadal and/or germ cells. Such results are important for the classification of germ cell mutagens, e.g. in the context of the "Globally Harmonized System of Classification and Labelling of Chemicals" (GHS). This review summarizes and discusses available information on the use of the comet assay with germ cells and cells from the gonads in genetic toxicology. The literature contains results from in vitro studies, ex vivo studies and in vivo studies. With regard to the assessment of germ cell genotoxicity, only in vivo studies are relevant but the other kind of studies provided important information on various aspects of the methodology. Many comet assay studies with human sperm have been performed in the context of male infertility and assisted fertilization. The results of these studies are not reviewed in detail here but various aspects of the assay modifications used are discussed. Measuring DNA effects by the comet assay in sperm requires additional steps for chromatin decondensation. Many different modifications of the alkaline and the neutral comet assay are in use but a standard protocol has not been established yet. High and variable background levels of DNA effects were reported and there is still need for standardization and validation of the comet assay with sperm. Some human biomonitoring studies with human sperm were published, but it seems to be premature to use these data for hazard identification and classification of chemicals. In contrast, the standard alkaline in vivo comet assay can easily be adapted to investigations with cells from reproductive organs. Tests with cells from the gonads (testis and ovary) seem to be most appropriate and a promising tool for demonstrating that a test compound reaches the gonads and is able to interact with the genetic material of germ cells. However, studies to standardize and validate these methods are necessary before the comet assay can be usefully applied in risk assessment of germ cell mutagens.     

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.     

How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop

Workshop participants agreed that genotoxicity tests in mammalian cells in vitro produce a remarkably high and unacceptable occurrence of irrelevant positive results (e.g. when compared with rodent carcinogenicity). As reported in several recent reviews, the rate of irrelevant positives (i.e. low specificity) for some studies using in vitro methods (when compared to this "gold standard") means that an increased number of test articles are subjected to additional in vivo genotoxicity testing, in many cases before, e.g. the efficacy (in the case of pharmaceuticals) of the compound has been evaluated. If in vitro tests were more predictive for in vivo genotoxicity and carcinogenicity (i.e. fewer false positives) then there would be a significant reduction in the number of animals used. Beyond animal (or human) carcinogenicity as the "gold standard", it is acknowledged that genotoxicity tests provide much information about cellular behaviour, cell division processes and cellular fate to a (geno)toxic insult. Since the disease impact of these effects is seldom known, and a verification of relevant toxicity is normally also the subject of (sub)chronic animal studies, the prediction of in vivo relevant results from in vitro genotoxicity tests is also important for aspects that may not have a direct impact on carcinogenesis as the ultimate endpoint of concern. In order to address the high rate of in vitro false positive results, a 2-day workshop was held at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy in April 2006. More than 20 genotoxicity experts from academia, government and industry were invited to review data from the currently available cell systems, to discuss whether there exist cells and test systems that have a reduced tendency to false positive results, to review potential modifications to existing protocols and cell systems that might result in improved specificity, and to review the performance of some new test systems that show promise of improved specificity without sacrificing sensitivity. It was concluded that better guidance on the likely mechanisms resulting in positive results that are not biologically relevant for human health, and how to obtain evidence for those mechanisms, is needed both for practitioners and regulatory reviewers. Participants discussed the fact that cell lines commonly used for genotoxicity testing have a number of deficiencies that may contribute to the high false positive rate. These include, amongst others, lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g. Aroclor-induced rat S9), impaired p53 function and altered DNA repair capability. The high concentrations of test chemicals (i.e. 10 mM or 5000 microg/ml, unless precluded by solubility or excessive toxicity) and the high levels of cytotoxicity currently required in mammalian cell genotoxicity tests were discussed as further potential sources of false positive results. Even if the goal is to detect carcinogens with short in vitro tests under more or less acute conditions, it does not seem logical to exceed the capabilities of cellular metabolic turnover, activation and defence processes. The concept of "promiscuous activation" was discussed. For numerous mutagens, the decisive in vivo enzymes are missing in vitro. However, if the substrate concentration is increased sufficiently, some other enzymes (that are unimportant in vivo) may take over the activation-leading to the same or a different active metabolite. Since we often do not use the right enzyme systems for positive controls in vitro, we have to rely on their promiscuous activation, i.e. to use excessive concentrations to get an empirical correlation between genotoxicity and carcinogenicity. A thorough review of published and industry data is urgently needed to determine whether the currently required limit concentration of 10mM or 5000 microg/ml, and high levels of cytotoxicity, are necessary for the detection of in vivo genotoxins and DNA-reactive, mutagenic carcinogens. In addition, various measures of cytotoxicity are currently allowable under OECD test guidelines, but there are few comparative data on whether different measures would result in different maximum concentrations for testing. A detailed comparison of cytotoxicity assessment strategies is needed. An assessment of whether test endpoints can be selected that are not intrinsically associated with cytotoxicity, and therefore are less susceptible to artefacts produced by cytotoxicity, should also be undertaken. There was agreement amongst the workshop participants that cell systems which are p53 and DNA-repair proficient, and have defined Phase 1 and Phase 2 metabolism, covering a broad set of enzyme forms, and used within the context of appropriately set limits of concentration and cytotoxicity, offer the best hope for reduced false positives. Whilst there is some evidence that human lymphocytes are less susceptible to false positives than the current rodent cell lines, other cell systems based on HepG2, TK6 and MCL-5 cells, as well as 3D skin models based on primary human keratinocytes also show some promise. Other human cell lines such as HepaRG, and human stem cells (the target for carcinogenicity) have not been used for genotoxicity investigations and should be considered for evaluation. Genetic engineering is also a valuable tool to incorporate missing enzyme systems into target cells. A collaborative research programme is needed to identify, further develop and evaluate new cell systems with appropriate sensitivity but improved specificity. In order to review current data for selection of appropriate top concentrations, measures and levels of cytotoxicity, metabolism, and to be able to improve existing or validate new assay systems, the participants called for the establishment of an expert group to identify the in vivo genotoxins and DNA-reactive, mutagenic carcinogens that we expect our in vitro genotoxicity assays to detect as well as the non-genotoxins and non-carcinogens we expect them not to detect.     

Genotoxicity of inhaled nanosized TiO(2) in mice

In vitro studies have suggested that nanosized titanium dioxide (TiO(2)) is genotoxic. The significance of these findings with respect to in vivo effects is unclear, as few in vivo studies on TiO(2) genotoxicity exist. Recently, nanosized TiO(2) administered in drinking water was reported to increase, e.g., micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs) and DNA damage in leukocytes. Induction of micronuclei in mouse PCEs was earlier also described for pigment-grade TiO(2) administered intraperitoneally. The apparent systemic genotoxic effects have been suggested to reflect secondary genotoxicity of TiO(2) due to inflammation. However, a recent study suggested that induction of DNA damage in mouse bronchoalveolar lavage (BAL) cells after intratracheal instillation of nanosized or fine TiO(2) is independent of inflammation. We examined here, if inhalation of freshly generated nanosized TiO(2) (74% anatase, 26% brookite; 5 days, 4 h/day) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5 mg/m(3) could induce genotoxic effects in C57BL/6J mice locally in the lungs or systematically in peripheral PCEs. DNA damage was assessed by the comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. MN were analyzed by acridine orange staining in blood PCEs collected 48 h after the last exposure. A dose-dependent deposition of Ti in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to nanosized TiO(2) anatase. Our inhalation exposure resulted in much lower systemic TiO(2) doses than the previous oral and intraperitoneal treatments, and lung epithelial cells probably received considerably less TiO(2) than BAL cells in the earlier intratracheal study.     

DEHP: genotoxicity and potential carcinogenic mechanisms-a review

Di(ethylhexyl) phthalate (DEHP) is a manufactured chemical commonly added to plastics: it is a ubiquitous environmental contaminant to which humans are exposed through multiple routes. DEHP is a rodent carcinogen with an extensive data base on genotoxicity and related effects spanning several decades. Although DEHP has been reported to be negative in most non-mammalian in vitro mutation assays, most studies were performed under conditions of concurrent cytotoxicity, precipitation, or irrelevant metabolic activation. However, a number of in vitro rodent tissue assays have reported DEHP to be positive for effects on chromosomes, spindle, and mitosis. A robust database shows that DEHP increases transformation and inhibits apoptosis in Syrian hamster embryo cells. In a transgenic mouse assay, in vivo DEHP exposure increased the mutation frequency only in the liver, which is the target organ for cancer. In vitro exposure of human cells or tissues to DEHP induced DNA damage; altered mitotic rate, apoptosis, and cell proliferation; increased proliferation, tumor mobility, and invasiveness of tumor cell lines; and activated a number of nuclear receptors. DEHP has been shown to be an agonist for CAR2, a novel constitutive androstane receptor occurring only in humans. Environmental exposures of humans to DEHP have been associated with DNA damage. After taking into account study context and relevant issues affecting interpretation, in vitro studies reported that a similar DEHP concentration range induced both mutagenic and non-mutagenic effects in human tissues and, using a much more limited rodent database, transformation of embryonic rodent tissues. The human and rodent data suggest that DEHP induces cancer through multiple molecular signals, including DNA damage. The analyses presented here may provide guidance for similar data sets used in structure-activity relationships, computational-toxicology extrapolations, and attempts to extrapolate in vitro results to predict in vivo effects for hazard characterization.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper No. 5. Genotoxicity tests as predictors of carcinogens: an analysis

Differences between the results of numerical validation studies comparing in vitro and in vivo genotoxicity tests with the rodent cancer bioassay are leading to the perception that short-term tests predict carcinogenicity only with uncertainty. Consideration of factors such as the pharmacokinetic distribution of chemicals, the systems available for metabolic activation and detoxification, the ability of the active metabolite to move from the site of production to the target DNA, and the potential for expression of the induced lesions, strongly suggests that the disparate sensitivity of the different test systems is a major reason why numerical validation is not more successful. Furthermore, genotoxicity tests should be expected to detect only a subset of carcinogens, namely genotoxic carcinogens, rather than those carcinogens that appear to act by non-genetic mechanisms. Instead of relying primarily on short-term in vitro genotoxicity tests to predict carcinogenic activity, these tests should be used in a manner that emphasizes the accurate determination of mutagenicity or clastogenicity. It must then be determined whether the mutagenic activity is further expressed as carcinogenicity in the appropriate studies using test animals. The prospects for quantitative extrapolation of in vitro or in vivo genotoxicity test results to carcinogenicity requires a much more precise understanding of the critical molecular events in both processes.     

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.     

Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice

The genotoxicity of an acetone extract of locally collected airborne particles was evaluated both in vitro and in vivo using the sister-chromatid exchange (SCE) assay in mice. At the highest concentration (5.36 mg/5 ml culture), the extract caused approximately a 3-fold increase in SCEs over controls in mouse bone marrow and spleen primary cells in vitro. However, the same airborne particle extract did not induce a significant increase in the SCE level over controls in vivo in mouse bone marrow and spleen cells when administered intraperitoneally or through oral gavage. This indicates that bone marrow and spleen primary cell cultures can be used in in vitro genotoxicity studies of complex mixtures, and that the genotoxicity of airborne particles detected in the in vitro system cannot always be detected in vivo with the same cell types. In addition, the same acetone extract of airborne particles caused dose-related his+ revertants in the strain TA98 of Salmonella typhimurium, both with and without S9 activation. The significant finding of this study is that the in vitro genotoxicity results of airborne particle extract may not be very meaningful in an in vivo situation.     

Human biological relevance and the use of threshold-arguments in regulatory genotoxicity assessment: experience with pharmaceuticals

Issues of biological relevance and thresholds for genotoxicity are discussed here based upon the background of experience with the submissions for the approval of new pharmaceuticals to the German regulatory authority over the period between 1990 and 1997. This experience shows that out of the genotoxicity test systems which are required according to existing guidelines in the European Union (EU), the in vitro tests for chromosomal aberrations (CA) and the mouse lymphoma tk assays (MLA) yield a rate of positives that is about four-fold higher than that of other genotoxicity tests. A detailed analysis of chemical and pharmacological classes of compounds and their effects in these systems reveals that in addition to direct DNA reactivity several mechanisms of indirect genotoxicity such as nucleoside analogue incorporation into DNA, interaction with microtubule assembly, topoisomerase inhibition and high levels of cytotoxicity are relevant. New pharmaceuticals, for which the latter mechanisms apply, often display threshold-like characteristics in their genotoxic effects in vitro or even in vivo in experimental animals. This casts doubt upon the relevance of positive in vitro test results for such compounds. However, the discussion of examples shows that it may not be easy to demonstrate the exact thresholded mechanism of genotoxicity in a given case. In particular, the demonstration of a coincidence of genotoxicity and high levels of cytotoxicity, which seems to be a major factor for biologically non-relevant in vitro positive new pharmaceuticals, usually requires quite extensive testing. Hence, for new pharmaceuticals it is practice to provide in addition to in vitro results that may be thresholded a wealth of information from in vivo studies on genotoxicity, carcinogenicity, metabolism, pharmacokinetics, etc. the results of which help in assessing the biological relevance of in vitro positives. The regulatory acknowledgement of biologically non-relevant, thresholded mechanisms of (in vitro) genotoxicity in addition to those that are considered relevant for human risk ensures a better understanding of test results and is needed for the credibility of genotoxicity testing practice in general.     

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.     

Assessment of the mutagenic potential of arecoline in gpt delta transgenic mice

Until recently, knowledge about the genotoxicity of roxarsone in vitro or in vivo was limited. This study assessed the genotoxicity of roxarsone in an in vitro system. Roxarsone was tested for potential genotoxicity on V79 cells by a Comet assay and a micronucleus (MN) test, exposing the cells to roxarsone (1-500 μM) and to sodium arsenite (NaAsO?, 20 μM) solutions for 3-48 h. Roxarsone was found to be cytotoxic when assessed with a commercial cell counting kit (CCK-8) used to evaluate cell viability, and moderately genotoxic in the Comet assay and micronucleus test used to assess DNA damage. The Comet metrics (percentages TDNA, TL, TM) increased significantly in a time- and concentration-dependent manner in roxarsone-treated samples compared with PBS controls (P<0.05), while the data from samples treated with 20 μM NaAsO? were comparable to those from 500 μM roxarsone-treated samples. The MN frequency of V79 cells treated with roxarsone was higher than that in the negative control but lower than the frequency in cells treated with 20 μM NaAsO?. A dose- and time-dependent response in MN induction was observed at 10, 50, 100 and 500 μM doses of roxarsone after 12-48 h exposure time. The DNA damage in V79 cells treated with 500 μM roxarsone was similar to cells exposed to 20 μM NaAsO?. The uptake of cells was correlated with the DNA damage caused by roxarsone. This investigation depicts the genotoxic potentials of roxarsone to V79 cells, which could lead to further advanced studies on the genotoxicity of roxarsone.     

Electromagnetic fields and epidemiology: An overview inspired by the fourth course at the International School of Bioelectromagnetics

The fourth course at the International School of Bioelectromagnetics addressed various aspects of the epidemiology of exposure to electromagnetic fields (EMF). In this overview, inspired by the lectures and the discussions among participants, we summarize current knowledge on exposure to EMF and disease risk, with emphasis on studies of use of mobile phones and brain tumours and exposure to power lines and childhood leukaemia. Sources of bias and error hamper straightforward conclusions in some areas and, in order to move forward, improvements in study design and exposure assessment are necessary. The scientific evidence available to date on possible long‐term effects from exposure to ELF and RF fields is not strong enough to revise current protection limits based on the known acute effects of such exposures. Precautionary measures may be considered to reduce ELF exposure of children or exposure to RF during mobile phone use, keeping in mind that it is unclear whether they involve any preventive benefit. Possible health effects from mobile phone use in adults and in children should be investigated further by prospective epidemiological studies with improved exposure assessment and brain tumour incidence rates should be monitored. Further studies on the relation between childhood leukaemia and ELF magnetic fields would be worthwhile if they focus on heavily exposed groups and attempt to minimize possible selection bias. In conclusion, epidemiological studies conducted with appropriate diligence can play a key role in finding the answers. Bioelectromagnetics 30:511–524, 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of toxicological monitoring markers using proteomic analysis

In our study, we chose three different concentrations of FA (0, 5, and 10 ppm), and cytotoxic (lipid peroxidation and protein oxidation) and genotoxic assays (DNA damage) were carried out on plasma, blood, and liver cells of rats subjected to FA-inhalation treatment. The profiles of plasma protein changes determined using 2-DE analysis were also evaluated to identify potential toxicological monitoring markers in FA-exposed rats. Concern was raised that our genotoxic analyses did not follow previously published research data and that the results of our rat plasma proteomic studies were difficult to interpret because we did not directly determine the plasma concentration of FA. However, we had already determined the concentration of FA using HPLC in an exposure chamber to monitor FA inhalation concentrations. We suggest that our experimental design was suitable to determine the FA effects on rat using an inhalation chamber system. For the similarity of genotoxic effects in lymphocytes and liver cells, we chose to present our data on the general cytological toxic effects on lipid peroxidation and protein oxidation which revealed a similarity between plasma and liver cells of FA-exposed rats. We have shown strong correlations between genotoxicity and lipid peroxidation, and lipid peroxidation is known to mediate DNA damage in many in vitro, and in vivo studies. We are well aware of the 'implausibility' of leukemia induction by FA, but for precisely this reason, we feel the need for further study to prove the systemic genotoxic effects of FA.     

A genotoxicity study on N-acryloyl-N′-phenylpiperazine: implications of aneugenic effects in risk evaluations

Further to a previous genotoxicity study, we analyzed sister-chromatid exchange (SCE) and DNA-repair induction (V79 and EUE cells in vitro) and DNA damage (rat liver in vivo) with regard to N-acryloyl-N-phenylpiperazine (AcrNPP), a chemical proposed for biomaterial polymerization which contains an aromatic tertiary amino function in a piperazine cycle. This chemical induced SCEs in a dose-dependent fashion (up to ≈ 3.7 times the control value), while it was negative for DNA-repair induction and weakly yeat significantly positive for in vivo DNA damage (maximum increase ≈ 1.4 times the control value). Taken together with our previous genotoxicity data on AcrNPP and structurally related compounds, the present results confirm that aneuploidy is a possible major effect of aromatic tertiary amines. As regards exposure to aneugenic agents, considerations on cancer risk evaluation are presented.     

Genetic susceptibility, biomarker respones, and cancer

A large number of studies have reported associations between polymorphisms of xenobiotic-metabolizing enzymes (XMEs) and various cancers. However, the carcinogenic exposures behind such findings have usually been unclear. Information on susceptibility to specific carcinogens could better be obtained by examining situations where the exposure and the endpoint studied are nearer in time, i.e., by studying biomarkers of carcinogen exposure and early (genotoxic) effect in exposed humans. For example, analyses of DNA adducts and cytogenetic endpoints have indicated an increased susceptibility of glutathione S-transferase M1 (GSTM1) null genotype to genotoxicity of tobacco smoking, supporting the view that the associations of the GSTM1 null genotype with bladder and lung cancer are partly related to smoking. In vitro genotoxicity studies with human cells offer an experimental tool that can be used, within the limits of the cell systems, to predict individual sensitivity and genotype-carcinogen interactions. In vitro sensitivity to the genotoxicity of 1,2:3,4-diepoxybutane, an epoxide metabolite of 1,3-butadiene has clearly been shown to depend on GSTT1 genotype, which has also been implicated to modify, along with GSTM1 genotype, the in vitro genotoxicity of 1,2-epoxy-3-butene, another epoxide metabolite of 1,3-butadiene. These genotypes appear to modulate the excretion of 1,3-butadiene-specific mercapturic acids, and influence genotoxicity biomarker levels in 1,3-butadiene-exposed workers. The excretion of specific mercapturic acids (PHEMA) in workers exposed to styrene has clearly been shown to depend on GSTM1 genotype, and GSTT1 genotype seems to modulate the excretion of one PHEMA diastereoisomer. These genotypes have also been implicated to modulate the in vitro genotoxicity of styrene. In general, the genetic polymorphisms potentially important for biomarker response largely depend on the exposing agent, biological material examined, and ethnicity of the population under study. Individual exposure level may vary a lot, and a reliable estimate of the exposure is essential for correct interpretation of genotype-exposure interaction. Besides XME polymorphisms, any polymorphisms that affect cellular response to DNA damage could, in principle, modify individual sensitivity to genotoxins. For instance, those concerning DNA repair proteins are presently being studied by many laboratories.     

In Vitro and In Vivo Magnetic Resonance Tracking of Sinerem-Labeled Human Umbilical Mesenchymal Stromal Cell-Derived Schwann Cells

Tracking of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles-labeled embryonic stem cells, neural stem cells, or adult mesenchymal stem cells in vitro and in vivo by using magnetic resonance (MR) imaging have been reported. However, whether the transdifferentiated cells can be effectively labeled by USPIO has not yet been investigated. The requirement for nerve donor material evokes additional morbidity and inability to generate a sufficiently large number of cells in a short time to hamper the clinic application of Schwann cells (SCs) transplantation. These limitations may be avoided if SCs can be generated from clinically accessible sources, such as bone marrow and umbilical cord. However, a reliable means of inducing the selective differentiation of human mesenchymal stromal cells isolated from the umbilical cord (HUMSCs) into SCs in vitro has not yet been established. In this study, we induce HUMSCs into Schwann-like cells in terms of morphology, phenotype, and function by an improved protocol basing on our previous studies. Furthermore, HUMSCs-derived SCs are labeled efficiently in vitro with ultrasmall superparamagnetic iron oxide contrast agent (USPIO) Sinerem and poly-l-lysine (PLL) without affecting morphology, cell cycle, proliferation, and differentiation ability of the labeled cells between the concentration of 200 to 800 μg/ml. Importantly, when grafted into the intact cerebral cortex and striatum, the survival and migration of these Sinerem-labeled cells were observed using MRI. Our study suggest the effective concentration field for Sinerem use in tracking transdifferentiated HUMSCs, and Sinerem labeling transdifferentiated HUMSCs is feasible, efficient, and safe for MRI tracing following grafting into nervous system.