Operationalization of a multidimensional sex/gender concept for quantitative environmental health research and implementation in the KORA study: Results of the collaborative research project INGER.

Background: In environmental health research, sex and gender are not yet adequately considered. There is a need to improve data collection in population-based environmental health studies by comprehensively surveying sex/gender-related aspects according to gender theoretical concepts. Thus, within the joint project INGER we developed a multidimensional sex/gender concept which we aimed to operationalize and to test the operationalization for feasibility. Methods: In an iterative process, we created questionnaire modules which quantitatively captured the requirements of the INGER sex/gender concept. We deployed it in the KORA cohort (Cooperative Health Research in the Region of Augsburg, Germany) in 2019 and evaluated response and missing rates. Results: The individual sex/gender self-concept was surveyed via a two-step approach that asked for sex assigned at birth and the current sex/gender identity. Additionally, we used existing tools to query internalized sex/gender roles and externalized sex/gender expressions. Adapted to the KORA population, we asked for discrimination experiences and care and household activities contributing to explain structural sex/gender relations. Further intersectionality-related social categories (e.g., socio-economic position), lifestyle and psychosocial factors were covered through data available in KORA. We could not identify appropriate tools to assess the true biological sex, sexual orientation and ethnic/cultural identity, which have yet to be developed or improved. The response-rate was 71%, the evaluation of 3,743 questionnaires showed a low missing rate. Prevalence of marginalized groups regarding sex/gender identity and definable by experiences of discrimination was very low. Conclusion: We have shown how the multidimensional INGER sex/gender concept can be operationalized according to an European and North American understanding of sex/gender for use in quantitative research. The questionnaire modules proved feasible in an epidemiologic cohort study. Being a balancing act between theoretical concepts and its quantitative implementation our operationalization paves the way for an adequate consideration of sex/gender in environmental health research.

Urinary cotinine and exposure to passive smoke in children and adolescents in Germany - Human biomonitoring results of the German Environmental Survey 2014-2017 (GerES V).

Passive smoking is a preventable and significant cause of many serious health problems, with children being particularly at risk. In the fifth German Environmental Survey (GerES V), conducted from 2014 to 2017, information reflecting the extent of passive smoke exposure in children and adolescents was collected by interview-based questionnaires and human biomonitoring (HBM) analyses of cotinine in urine from 2260 participants, aged 3-17 years. Based on these population-representative data, we describe current passive smoke exposure stratified by different subgroups and identify specific exposure determinants using multivariate logistic regression. The questionnaire data revealed that 42% of children and adolescents lived with at least one smoker in the household. Quantifiable concentrations of cotinine could be detected in 56% of the participants. The overall median concentration of cotinine was 0.2 µg/L, with children and adolescents of low socioeconomic status found to be a group particularly affected by passive smoke with higher cotinine concentrations (median = 1.2 µg/L). In the multiple analysis, the most significant predictor of cotinine levels derived from the questionnaire was passive smoking at home (odds ratio (OR) 13.07 [95CI: 4.65, 36.70]). However, parental smoking and passive smoking among friends and relatives could also be identified as independent factors influencing elevated cotinine levels. The comparison between the previous cycle GerES IV (2003-2006) on 3-14-year-olds and GerES V shows that tobacco smoke exposure of children decreased significantly. This decrease is likely an effect of extensive non-smoker protection laws being enforced 2007-2008 on federal and state level. This is reflected by a halving of urinary cotinine concentrations. Nevertheless, our results indicate that passive smoke is still a relevant source of harmful pollutants for many children and adolescents in Germany, and thus support the need for further efforts to reduce passive smoke exposure, especially in the private environment.

Lysmeral Exposure in Children and Adolescences Participating in the German Environmental Survey (2012-2015): Integrating Sex/Gender into Analysis.

Comprehensive consideration of the biological and social diversities of sex and gender as well as their interdependencies is mostly missing in human biomonitoring (HBM) studies. Using the INGER sex/gender concept as theoretical background, we analyzed differences in exposure to lysmeral, a compound commonly found as a fragrance in cosmetics, personal care, and household products, in 2294 children and adolescents in Germany using decision tree, regression, and mediation analysis. The variables "sex assigned at birth" and "age", as well as well as use of personal care products and fabric conditioner proved to have the highest explanatory value. Mediating effects of behaviour associated with societal gender expectations were observed, as the use of cosmetics correlated highly with lysmeral metabolites concentrations in girls between 6 and 17 years, with the strongest effect in adolescents between 14 and 17 years old. In the youngest age group (3-5 years) boys showed higher concentration of the metabolite tert-butylbenzoic acid (TBBA) compared to girls of the same age but only if TBBA urine concentrations were normalized on creatinine. Our study offers the first retrospective sex/gender assessment of HBM data. It demonstrates the possibilities to rethink and broaden sex/gender analysis in existing HBM-studies and highlights the need for inclusion of new sex/gender concepts in the design of new studies.

Formaldehyde, aliphatic aldehydes (C2 -C11 ), furfural, and benzaldehyde in the residential indoor air of children and adolescents during the German Environmental Survey 2014-2017 (GerES V).

Indoor air concentrations of formaldehyde, furfural, benzaldehyde, and 11 aliphatic aldehydes (C2 -C11 ) were measured in residences of 639 participants in the German Environmental Survey for Children and Adolescents 2014-2017 (GerES V). Sampling was conducted using passive samplers over periods of approximately seven days for each participant. The most abundant compounds were formaldehyde and hexanal with median concentrations of 24.9 µg m-3 and 10.9 µg m-3 , respectively. Formaldehyde concentrations exceeded the Guide Value I recommended by the German Committee on Indoor Guide Values (Ausschuss für Innenraumrichtwerte - AIR) (0.10 mg m-3 ) for 0.3% of the participating residences. The sum of aliphatic n-aldehydes between C4 (butanal) and C11 (undecanal) exceeded their Guide Value (0.10 mg m-3 ) for 2.0% of the residences. The geometric mean concentrations of most aldehydes were lower than in the earlier GerES IV (2003-2006) study. Formaldehyde and hexanal concentrations, however, were comparable in both studies and showed no significant difference. Indoor aldehyde concentrations did not exhibit significant correlations with factors collected in questionnaires, such as the age of the participants, their socio-economic status, the location of the residence (former East/West Germany), migration background, tobacco exposure, and the type of furniture used. The validity of the passive sampler measurements was verified against active sampling techniques in a test chamber experiment.

Integrating Sex/Gender into Environmental Health Research: Development of a Conceptual Framework.

There is a growing awareness about the need to comprehensively integrate sex and gender into health research in order to enhance the validity and significance of research results. An in-depth consideration of differential exposures and vulnerability is lacking, especially within environmental risk assessment. Thus, the interdisciplinary team of the collaborative research project INGER (integrating gender into environmental health research) aimed to develop a multidimensional sex/gender concept as a theoretically grounded starting point for the operationalization of sex and gender in quantitative (environmental) health research. The iterative development process was based on gender theoretical and health science approaches and was inspired by previously published concepts or models of sex- and gender-related dimensions. The INGER sex/gender concept fulfills the four theoretically established prerequisites for comprehensively investigating sex and gender aspects in population health research: multidimensionality, variety, embodiment, and intersectionality. The theoretical foundation of INGER's multidimensional sex/gender concept will be laid out, as well as recent sex/gender conceptualization developments in health sciences. In conclusion, by building upon the latest state of research of several disciplines, the conceptual framework will significantly contribute to integrating gender theoretical concepts into (environmental) health research, improving the validity of research and, thus, supporting the promotion of health equity in the long term.

Substitutes mimic the exposure behaviour of REACH regulated phthalates - A review of the German HBM system on the example of plasticizers.

The population is constantly exposed to potentially harmful substances present in the environment, including inter alia food and drinking water, consumer products, and indoor air. Human biomonitoring (HBM) is a valuable tool to determine the integral, internal exposure of the general population, including vulnerable subgroups, to provide the basis for risk assessment and policy advice. The German HBM system comprises of five pillars: (1) the development of suitable analytical methods for new substances of concern, (2) cross-sectional population-representative German Environmental Surveys (GerES), (3) time trend analyses using archived samples from the Environmental Specimen Bank (ESB), (4) the derivation of health-based guidance values as a risk assessment tool, and (5) transfer of data into the European cooperation network HBM4EU. The goal of this paper is to present the complementary elements of the German HBM system and to show its strengths and limitations on the example of plasticizers. Plasticizers have been identified by EU services and HBM4EU partners as priority substances for chemical policy at EU level. Using the complementary elements of the German HBM system, the internal exposure to classical phthalates and novel alternative plasticizers can be reliably monitored. It is shown that market changes, due to regulation of certain phthalates and the rise of substitutes, are rapidly reflected in the internal exposure of the population. It was shown that exposure to DEHP, DiBP, DnBP, and BBzP decreased considerably, whereas exposure to the novel substitutes such as DPHP, DEHTP, and Hexamoll®DINCH has increased significantly. While health-based guidance values for several phthalates (esp. DnBP, DiBP, DEHP) were exceeded quite often at the turn of the millennium, exceedances today have become rarer. Still, also the latest GerES reveals the ubiquitous and concurrent exposures to many plasticizers. Of concern is that the youngest children showed the highest exposures to most of the investigated plasticizers and in some cases their levels of DiBP and DnBP still exceeded health-based guidance values. Over the last years, mixture exposures are increasingly recognized as relevant, especially if the toxicological modes of action are similar. This is supported by a cumulative risk assessment for four endocrine active phthalates which confirms the still concerning cumulative exposure in many young children. Given the adverse health effects of some phthalates and the limited toxicological knowledge of substitutes, exposure reduction and surveillance are needed on German and EU-level. Substitutes need to be monitored, to intervene if exposures are threatening to exceed acceptable levels, or if new toxicological data question their appropriateness. It is strongly recommended to reconsider the use of plastics and plasticizers.

Risk assessment for irritating chemicals - Derivation of extrapolation factors.

Irritation of the eyes and the upper respiratory tract are important endpoints for setting guide values for chemicals. To optimize the use of the often-limited data, we analysed controlled human exposure studies (CHS) with 1-4 h inhalation of the test substance, repeated dose inhalation studies in rodents, and Alarie-Tests and derived extrapolation factors (EF) for exposure duration, inter- and intraspecies differences. For the endpoint irritating effects in the respiratory tract in rodents, geometric mean (GM) values of 1.9 were obtained for the EF for subacute→subchronic (n = 16), 2.1 for subchronic→chronic (n = 40), and 2.9 for subacute→chronic (n = 10) extrapolation. Based on these data we suggest an EF of 2 for subchronic→chronic and of 4 for subacute→chronic extrapolation. In CHS, exposure concentration determines the effects rather than exposure duration. Slight reversible effects during 4 h exposure indicate that an EF of 1 can be considered for assessing chronic exposures. To assess species extrapolation, 10 chemicals were identified with both, reliable rat inhalation studies and CHS. The GM of the ratio between the No Observed Adverse Effect Concentration (NOAEC) in rats and humans was 2.3 and increased to 3.6 when expanding the dataset to all available EF (n = 25). Based on these analyses, an EF of 3 is suggested to extrapolate from a NOAEC in a chronic rat study to a NOAEC in a CHS. The analysis of EFs for the extrapolation from a 50% decrease in respiratory frequency in the Alarie test in mice (RD50) to a NOAEC in a CHS resulted in a GM of 40, for both, the reliable (n = 11) and the overall dataset (n = 19). We propose to use the RD50 from the Alarie test for setting guide values and to use 40 as EF. Efs for intraspecies differences in the human population must account for susceptible persons, most importantly for persons with chemical intolerance (CI), who show subjective signs of irritation at low concentrations. The limited data available do not justify to deviate from an EF of 10 - 20 as currently used in different regulatory settings.

[Integration of sex/gender into environmental health research. Results of the interdisciplinary research network Sex/Gender-Environment-Health (GeUmGe-NET)]. / Integration von Geschlecht in die Forschung zu umweltbezogener Gesundheit. Ergebnisse des interdisziplinären Forschungsnetzwerks Geschlecht ­ Umwelt ­ Gesundheit (GeUmGe-NET).

The comprehensive consideration of sex/gender in health research is essential to increase relevance and validity of research results. Contrary to other areas of health research, there is no systematic summary of the current state of research on the significance of sex/gender in environmental health. Within the interdisciplinary research network Sex/Gender-Environment-Health (GeUmGe-NET) the current state of integration of sex/gender aspects or, respectively, gender theoretical concepts into research was systematically assessed within selected topics of the research areas environmental toxicology, environmental medicine, environmental epidemiology and public health research on environment and health. Knowledge gaps and research needs were identified in all research areas. Furthermore, the potential for methodological advancements by using gender theoretical concepts was depicted. A dialogue between biomedical research, public health research, and gender studies was started with the research network GeUmGe-NET. This dialogue has to be continued particularly regarding a common testing of methodological innovations in data collection and data analysis. Insights of this interdisciplinary research are relevant for practice areas such as environmental health protection, health promotion, environmental justice, and environmental health monitoring.

[Indoor air pollution-current fields of action]. / Schadstoffe im Innenraum ­ aktuelle Handlungsfelder.

People in Central Europe spend most of their time in private dwellings, offices, education centres or other public buildings. In these indoor places, they are exposed to a variety of gaseous or particulate pollutants that potentially exert adverse health effects. This work compiles current fields of action that are discussed in the public, among expert panels, and in the scientific community. These address ventilation in buildings, the impact of building product emissions and particulate matter sources on indoor air quality, the detection and prevention of mould as well as the assessment of indoor air quality using guidance values and the determination of the internal exposure by human biomonitoring. Indoor air quality appears as a dynamic field of action that has become more complex due to the interaction between new chemicals introduced into the indoor environment through a variety of products and the observed reduction of ventilation rates. Implications for human health have thus become less predictable.

Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of N7-ETE-guanine DNA adducts, the excision rate of CEES-induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD(+) levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES-induced short-term cytotoxicity 24h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi-functional mustards such as SM and HN2. In conclusion, we demonstrate that PARylation plays a functional role in mustard-induced cellular stress response with substance-specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM-related pathologies and to sensitize cancer cells for mustard-based chemotherapy, potential long-term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy.

Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions.

Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy. PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the molecular mechanism of PARP1 activation and its functional consequences after mustard treatment in general. Such a study is presented in an accompanying article (Mangerich et al., 2016).

Antibiotics and sweeteners in the aquatic environment: biodegradability, formation of phototransformation products, and in vitro toxicity.

In the present study, in vitro toxicity as well as biopersistence and photopersistence of four artificial sweeteners (acesulfame, cyclamate, saccharine, and sucralose) and five antibiotics (levofloxacin, lincomycin, linezolid, marbofloxacin, and sarafloxacin) and of their phototransformation products (PTPs) were investigated. Furthermore, antibiotic activity was evaluated after UV irradiation and after exposure to inocula of a sewage treatment plant. The study reveals that most of the tested compounds and their PTPs were neither readily nor inherently biodegradable in the Organisation for Economic Co-operation and Development (OECD)-biodegradability tests. The study further demonstrates that PTPs are formed upon irradiation with an Hg lamp (UV light) and, to a lesser extent, upon irradiation with a Xe lamp (mimics sunlight). Comparing the nonirradiated with the corresponding irradiated solutions, a higher chronic toxicity against bacteria was found for the irradiated solutions of linezolid. Neither cytotoxicity nor genotoxicity was found in human cervical (HeLa) and liver (Hep-G2) cells for any of the investigated compounds or their PTPs. Antimicrobial activity of the tested fluoroquinolones was reduced after UV treatment, but it was not reduced after a 28-day exposure to inocula of a sewage treatment plant. This comparative study shows that PTPs can be formed as a result of UV treatment. The study further demonstrated that UV irradiation can be effective in reducing the antimicrobial activity of antibiotics, and consequently may help to reduce antimicrobial resistance in wastewaters. Nevertheless, the study also highlights that some PTPs may exhibit a higher ecotoxicity than the respective parent compounds. Consequently, UV treatment does not transform all micropollutants into harmless compounds and may not be a large-scale effluent treatment option.

High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay.

DNA interstrand crosslinks (ICL) are induced both by several cytotoxic anti-cancer drugs as well as by the chemical warfare agent sulphur mustard (SM). Although measurement of ICL formation could be used in risk assessment or provide valuable predictive information on the response of malignant cells to crosslinking chemotherapeutic agents, respectively, it is currently not applied due to lack of appropriate standardized methodology. Here we describe a fast and convenient procedure for detection of ICL in human peripheral blood mononuclear cells (PBMC) as high-throughput method, termed 'reverse FADU assay'. This assay detects ICL based on the prevention of time-dependent alkaline unwinding of double-stranded DNA in a cell lysate that starts from single or double strand breaks. We have successfully established and optimized the reverse FADU assay by using human PBMC exposed to the model compounds mitomycin C, melphalan and SM. Our fully automated assay version is faster than currently used methods and possesses similar sensitivity. It operates in a 96-well format, thus allowing parallel analysis of multiple samples. Furthermore, we describe optimized protocols for sample preparation, with sample volume minimized to 100µl of blood, storage and shipment conditions. We conclude that the reverse FADU assay is an attractive candidate method for monitoring DNA damage induced by DNA crosslinking agents.

Induction of DNA strand breaks by dental composite components compared to X-ray exposure in human gingival fibroblasts.

The toxicity of dental composites has been attributed to the release of residual monomers from polymerized resin-based composites due to degradation processes or incomplete polymerization. Some of these eluted substances have a genotoxic potential. We tested the hypothesis that realistic concentrations (and/or worst case concentrations/situations) of bisphenol-A-glycidyldimethacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) found in elution experiments can cause DNA strand breaks in human gingival fibroblasts (HGF). Such DNA damage was compared with that resulting from ionizing radiation coming from natural sources, dental radiography or tumor therapy. TEGDMA, HEMA and MMA did not induce DNA strand breaks at concentrations of up to 10 mM. About 24 h after incubation with 0.25 mM BisGMA, significantly more DNA strand breaks were found in HGF compared to controls. DNA strand breaks caused by 0.25 mM BisGMA, correspond to DNA strand breakage caused by irradiation with 4 Gy, only used in the high single-dose irradiation tumor therapy. But 0.25 mM BisGMA is more than 100-fold higher than that concentration found in worst case calculations. Therefore, our data did not support our hypothesis.

Role of poly(ADP-ribose) polymerase in sulfur mustard toxicity.

Sulfur mustard (SM) is a chemical warfare agent leading to severe blistering of skin and mucosal surfaces, and as a long-term effect, to an increased risk for malignancies. At the molecular level, SM acts as a bifunctional alkylating agent, leading to DNA mono-adducts and di-adducts. This review is focussed on the role of poly(ADP-ribosyl)ation in the cell and tissue responses to SM-induced damage and potential role of inhibitors of poly(ADP-ribosyl)ation as therapeutic agents for SM injury.

Loss of ATM sensitizes against O6-methylguanine triggered apoptosis, SCEs and chromosomal aberrations.

A critical pre-cytotoxic and -apoptotic DNA lesion induced by methylating carcinogens and chemotherapeutic drugs is O6-methylguanine (O6MeG). The mechanism by which O6MeG causes cell death via apoptosis is only partially understood. The current model ascribes a role to DNA replication and mismatch repair, which converts O6MeG into a critical distal lesion (presumably a DNA double-strand break) that is finally responsible for genotoxicity and apoptosis. Here we analysed whether the PI3-like kinase ATM is involved in this process. ATM is a major player in recognizing and signaling DNA breaks, but most reports are limited to ionizing radiation. Comparing mouse ATM knockout fibroblasts (ATM-/-) with the corresponding wild-type (ATM+/+) we show that ATM-/- cells are hypersensitive to the cytotoxic and apoptosis-inducing effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Inhibition of O6-methylguanine-DNA methyltransferase (MGMT) activity by O6-benzylguanine enhanced cell killing whereas the increase of MGMT activity by transfection with an expression vector provoked MNNG resistance. This was more pronounced in ATM-/- than in ATM+/+ cells, suggesting that O6MeG is responsible, at least in part, for increased MNNG sensitivity of ATM-/- cells. Cytogenetic studies showed that MNNG-induced sister-chromatid exchange frequencies were the same in ATM-/- and ATM+/+ cells in the first mitoses following treatment, but higher in ATM-/- cells than in the wild-type in the second post-treatment mitoses, when MGMT was depleted. Also, a significant higher frequency of MNNG-induced chromosomal aberrations was observed in ATM-/- than in ATM+/+ cells when analysed at a late recovery time, which is consistent with O6MeG being the inducing lesion. In summary, we conclude that ATM is not only involved in resistance to ionizing radiation but also to methylating agents, playing a role in the repair of secondary DNA damage generated from O6MeG lesions. The data also show that ATM is not required for activating the apoptotic pathway in response to O6MeG since ATM-/- cells are able to undergo apoptosis with high frequency.