Current status and future challenges of genotoxicity OECD Test Guidelines for nanomaterials: a workshop report.

Genotoxicity testing for nanomaterials remains challenging as standard testing approaches require some adaptation, and further development of nano-specific OECD Test Guidelines (TGs) and Guidance Documents (GDs) are needed. However, the field of genotoxicology continues to progress and new approach methodologies (NAMs) are being developed that could provide relevant information on the range of mechanisms of genotoxic action that may be imparted by nanomaterials. There is a recognition of the need for implementation of new and/or adapted OECD TGs, new OECD GDs, and utilization of NAMs within a genotoxicity testing framework for nanomaterials. As such, the requirements to apply new experimental approaches and data for genotoxicity assessment of nanomaterials in a regulatory context is neither clear, nor used in practice. Thus, an international workshop with representatives from regulatory agencies, industry, government, and academic scientists was convened to discuss these issues. The expert discussion highlighted the current deficiencies that exist in standard testing approaches within exposure regimes, insufficient physicochemical characterization, lack of demonstration of cell or tissue uptake and internalization, and limitations in the coverage of genotoxic modes of action. Regarding the latter aspect, a consensus was reached on the importance of using NAMs to support the genotoxicity assessment of nanomaterials. Also highlighted was the need for close engagement between scientists and regulators to (i) provide clarity on the regulatory needs, (ii) improve the acceptance and use of NAM-generated data, and (iii) define how NAMs may be used as part of weight of evidence approaches for use in regulatory risk assessments.

Functional analysis of TLK2 variants and their proximal interactomes implicates impaired kinase activity and chromatin maintenance defects in their pathogenesis.

INTRODUCTION: The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with 'Mental Retardation Autosomal Dominant 57' (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies. METHODS: We re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity. RESULTS: We identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage. CONCLUSION: Our study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging.

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

A harmonized and standardized in vitro approach produces reliable results on silver nanoparticles toxicity in different cell lines.

Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated by comet and micronucleus assays, while oxidative DNA damage by modified comet assay and 8-oxodG/8-oxodA detection. We also investigated immunotoxicity and immunomodulation by cytokine release and NO production in RAW 264.7 and MH-S cells, with or without lipopolysaccharide (LPS) stimulus. Transmission electron microscope (TEM) analysis was used to analyze cellular uptake of AgNPs. Our results indicate different values of AgNPs hydrodynamic diameter depending on the medium, some genotoxic effect just on BEAS-2B and no or slight effects on function of RAW 264.7 and MH-S in absence or presence of LPS stimulus. This study highlights the relevance of using optimized protocols and multiple endpoints to analyze the potential toxicity of AgNPs and to obtain reliable and comparable results.

An integrated approach for chemical water quality assessment of an urban river stretch through Effect-Based Methods and emerging pollutants analysis with a focus on genotoxicity.

The impact of emerging chemical pollutants, on both status and functionality of aquatic ecosystems is worldwide recognized as a relevant issue of concern that should be assessed and managed by researchers, policymakers, and all relevant stakeholders. In Europe, the Reach Regulation has registered more than 100.000 chemical substances daily released in the environment. Furthermore, the effects related to the mixture of substances present in aquatic ecosystems may not be predictable on the basis of chemical analyses alone. This evidence, coupled with the dramatic effects of climate changes on water resources through water scarcity and flooding, makes urgent the application of innovative, fast and reliable monitoring methods. In this context, Effect-Based Methods (EBMs) have been applied in the urban stretch of the Tiber River (Central Italy) with the aim of understanding if detrimental pressures affect aquatic environmental health. In particular, different eco-genotoxicological assays have been used in order to detect genotoxic activity of chemicals present in the river, concurrently characterized by chemical analysis. Teratogenicity and embryo-toxicity have been studied in order to cover additional endpoints. The EBMs have highlighted the presence of diffuse chemical pollution and ecotoxicological effects in the three sampling stations, genotoxicological effects have been also detected through the use of different tests and organisms. The chemical analyses confirmed that in the aquatic ecosystems there is a diffuse presence, even at low concentrations, of emerging contaminants such as pharmaceuticals, not routinely monitored pesticides, personal care products, PFAS. The results of this study can help to identify an appropriate battery of EBMs for future studies and the application of more appropriate measures in order to monitor, mitigate or eliminate chemical contamination and remediate its adverse/detrimental effects on the ecosystem health.

Critical issues in genotoxicity assessment of TiO2 nanoparticles by human peripheral blood mononuclear cells.

In the last years, a number of in vitro studies have been performed to assess the genotoxic activity of titanium dioxide (TiO2 ). To resolve the contradictory results, in this study, we investigated the genotoxic activity of commercial TiO2 nanoparticles (NPs) and microparticles of different forms (anatase, rutile and mix of both). We evaluated micronucleus formation in stimulated lymphocytes, as well as DNA strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine in peripheral blood mononuclear cells (PBMCs), a mixed population of lymphocytes and monocytes. Different responses to TiO2 exposure were obtained depending on the assay. Both TiO2 NPs and microparticles and all the crystalline forms elicited a significant increase in 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in the whole PBMC population, without a concurrent increase of micronuclei in proliferating lymphocytes. The distribution of DNA damage in PBMCs, detected by the comet assay, that measures DNA damage at level of single cells, indicated the presence of a more susceptible cell subpopulation. The measurement of side scatter signals by flow cytometry highlighted the preferential physical interaction of TiO2 particles with monocytes that also displayed higher reactive oxygen species generation, providing a mechanistic explanation for the different responses observed in genotoxicity assays with PBMCs and lymphocytes. This study confirmed the suitability of human PBMCs as multi-cell model to investigate NP-induced DNA damage, but suggested some caution in the use of stimulated lymphocytes for the assessment of NP clastogenicity.

Effects of cigarette smoking on circulating leukocytes and plasma cytokines in monozygotic twins.

BACKGROUND: Despite the well-documented role of cigarette smoke in the development of chronic obstructive pulmonary disease (COPD), lung cancer and cardiovascular disease, biomarkers for screening or monitoring disease progression and outcome remain elusive, particularly for COPD and lung cancer. Inflammatory cells and mediators are likely to be involved in the disease processes, but their importance is still poorly understood. The purpose of this study was to investigate early changes in immunological markers associated with smoking in healthy monozygotic twins without a detectable disease discordant for smoking, thereby minimising data variability due to genetic background. METHODS: Twenty-two monozygotic twin pairs, aged 31.5±6.3 years, entered the study. One of each twin pair was a smoker and the other a non-smoker. None of the subjects reported any diseases or clinically defined respiratory symptoms or airflow limitation. Each subject donated blood samples for determination of total leukocytes and subpopulations, lymphocyte subpopulation plus pro-inflammatory mediators (interleukin-8, tumour necrosis factor-α, soluble tumour necrosis factor-α receptors and C-reactive protein). RESULTS: We observed a significant increase in the number of circulating leukocytes and neutrophils in smokers compared to non-smokers. Smokers also had significantly higher numbers of B cells and CD4+ T cells, plus an increased CD4/CD8 ratio. The numbers of NK cells were statistically significant lower in smokers compared to non-smokers. CONCLUSIONS: While the prognostic significance of these changes is uncertain, results suggest that smoking is associated with immune changes, independent of genetic background and environmental conditions.

Re-evaluation of silicon dioxide (E 551) as a food additive in foods for infants below 16 weeks of age and follow-up of its re-evaluation as a food additive for uses in foods for all population groups.

The present opinion is the follow-up of the conclusions and recommendations of the Scientific Opinion on the re-evaluation of silicon dioxide (E 551) as a food additive relevant to the safety assessment for all age groups. In addition, the risk assessment of silicon dioxide (E 551) for its use in food for infants below 16 weeks of age is performed. Based on the newly available information on the characterisation of the SAS used as E 551 and following the principles of the 2021 EFSA Guidance on Particle-TR, the conventional safety assessment has been complemented with nano-specific considerations. Given the uncertainties resulting from the limitations of the database and in the absence of genotoxicity concern, the Panel considered that it is not appropriate to derive an acceptable daily intake (ADI) but applied the margin of exposure (MOE) approach for the risk assessment. The Panel concluded that the MOE should be at least 36 for not raising a safety concern. The calculated MOEs considering the dietary exposure estimates for all population groups using the refined non-brand loyal scenario, estimated at the time of the 2018 re-evaluation, were all above 36. The Panel concluded that E 551 does not raise a safety concern in all population groups at the reported uses and use levels. The use of E 551 in food for infants below 16 weeks of age in FC 13.1.1 and FC 13.1.5.1 does not raise a safety concern at the current exposure levels. The Panel also concluded that the technical data provided support an amendment of the specifications for E 551 laid down in Commission Regulation (EU) No 231/2012. The paucity of toxicological studies with proper dispersion protocol (with the exception of the genotoxicity studies) creates uncertainty in the present assessment of the potential toxicological effects related to the exposure to E 551 nanosize aggregates.

DNA damage response in monozygotic twins discordant for smoking habits.

Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

Pyrogenic synthetic amorphous silica (NM-203): Genotoxicity in rats following sub-chronic oral exposure.

The genotoxicity of nano-structured synthetic amorphous silica (SAS), a common food additive, was investigated in vivo in rats. A 90-day oral toxicity study was performed according to OECD test guideline 408 and the genotoxicity of pyrogenic SAS nanomaterial NM-203 was assessed in several organs, using complementary tests. Adult Sprague-Dawley rats of both sexes were treated orally for 90 days with 0, 2, 5, 10, 20, or 50 mg SAS/kg bw per day. Dose levels were selected to approximate expected human dietary exposures to SAS. DNA strand breaks were evaluated by the comet assay in blood, bone marrow, liver, and spleen according to OECD test guideline 489; mutations induced in bone marrow precursors of erythrocytes were assessed by the Pig-a assay and chromosome/ genome damage by the micronucleus assay in blood (OECD test guideline 474) and colon. No treatment-related increases of gene (Pig-a) or chromosome/genome (micronucleus) mutations were detected in the blood. The percentage of micronucleated cells was not increased in the colon of treated rats. Among the organs analyzed by the comet assay, the spleen was the only target showing a weak but biologically relevant genotoxic effect.

Cross-sectional study of biomarkers of exposure and biological effect on monozygotic twins discordant for smoking.

BACKGROUND: The aim of this study was to investigate the possible correlation between smoking status and biomarkers of exposure (BoE) and biological effect (BoBE) in monozygotic twins discordant for smoking status (smoker and non-smoker pairs). By eliminating potential genetic variability in this manner, a clearer pattern of the effects of lifestyle and environmental exposures should become apparent. METHODS: This was a cross-sectional study on monozygotic healthy twins (44 subjects, 26 males and 18 females) with a mean age 31.5 years. BoE to cigarette smoke and BoBE were measured in body fluids (24 h urine and blood) after medical pre-screening. RESULTS: All BoE were significantly higher in the smoker twins. Among BoBE, 11-dehydrothromboxane B(2) (11-dehydro TBX), 2,3-dinorthromboxane B(2) (2,3-dinor TBX), 8-epi-prostaglandin F2α (8-epiPGF), hydroxyproline (OH-P), fibrinogen, white blood cell (WBC), neutrophil and lymphocyte counts and heart rate were statistically significantly increased in the smoker compared to the non-smoker twins. Moreover, statistically significant correlations between neutrophil count and 11-dehydro TBX (r=0.32), WBC and 8-epiPGF (r=0.33), OH-P and 8-epiPGF (r=0.49) and heart rate and fibrinogen (r=0.46) were observed. CONCLUSIONS: The study results confirmed the reliability of the BoE for the evaluation of smoking status. Moreover, a subset of the BoBE, reported as being associated with inflammatory conditions and early stages of vascular disorders, has emerged as showing a consistent relationship with smoking status from the present and the previous studies. By using monozygotic twin pairs, genetic variability has been excluded as a possible source of variability in this study. These results should assist in the interpretation of other population studies using these biomarkers.

FAIRification of nanosafety data to improve applicability of (Q)SAR approaches: A case study on in vitro Comet assay genotoxicity data.

(Quantitative) structure-activity relationship ([Q]SAR) methodologies are widely applied to predict the (eco)toxicological effects of chemicals, and their use is envisaged in different regulatory frameworks for filling data gaps of untested substances. However, their application to the risk assessment of nanomaterials is still limited, also due to the scarcity of large and curated experimental datasets. Despite a great amount of nanosafety data having been produced over the last decade in international collaborative initiatives, their interpretation, integration and reuse has been hampered by several obstacles, such as poorly described (meta)data, non-standard terminology, lack of harmonized reporting formats and criteria. Recently, the FAIR (Findable, Accessible, Interoperable, and Reusable) principles have been established to guide the scientific community in good data management and stewardship. The EU H2020 Gov4Nano project, together with other international projects and initiatives, is addressing the challenge of improving nanosafety data FAIRness, for maximizing their availability, understanding, exchange and ultimately their reuse. These efforts are largely supported by the creation of a common Nanosafety Data Interface, which connects a row of project-specific databases applying the eNanoMapper data model. A wide variety of experimental data relating to characterization and effects of nanomaterials are stored in the database; however, the methods, protocols and parameters driving their generation are not fully mature. This article reports the progress of an ongoing case study in the Gov4nano project on the reuse of in vitro Comet genotoxicity data, focusing on the issues and challenges encountered in their FAIRification through the eNanoMapper data model. The case study is part of an iterative process in which the FAIRification of data supports the understanding of the phenomena underlying their generation and, ultimately, improves their reusability.

Towards FAIR nanosafety data.

Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology.

The hCOMET project: International database comparison of results with the comet assay in human biomonitoring. Baseline frequency of DNA damage and effect of main confounders.

The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.

Telomerase activity, telomere length and hTERT DNA methylation in peripheral blood mononuclear cells from monozygotic twins with discordant smoking habits.

Increased telomerase expression has been implicated in the pathogenesis of lung cancer and, since the primary cause of lung cancer is smoking, an association between telomerase reactivation and tobacco smoke has been proposed. In this work an investigation has been performed to assess the relationship between tobacco smoke exposure and telomerase activity (TA) in peripheral blood mononuclear cells of healthy smokers. The methylation status of the catalytic subunit of telomerase hTERT was concurrently investigated to assess the possible association between epigenetic modifications of hTERT and TA. Besides, the association between smoke and telomere length (TL) has been evaluated. Healthy monozygotic twins with discordant smoking habits were selected as study population to minimize inter-individual differences because of demographic characteristics and genetic heterogeneity. Statistically significant higher values of TA and TL were observed in smokers compared to nonsmoker co-twins. The multivariate analysis of data showed, besides smoking habits (P = 0.02), an influence of gender (P = 0.006) and BMI (P = 0.001) on TA and a borderline effect of gender (P = 0.05) on TL. DNA methylation analysis, focused on 100 CpG sites mapping in hTERT, highlighted nine CpG sites differentially methylated in smokers. When co-twins were contrasted, selecting as variables the intra-twin difference in TA and hTERT DNA methylation, a statistically significant inverse correlation (P = 0.003) was observed between TA and DNA methylation at the cg05521538 site. In conclusion, these results indicate an association of tobacco smoke with TA and TL and suggest a possible association between smoke-induced epigenetic effects and TA in healthy smokers. Environ. Mol. Mutagen. 58:551-559, 2017. © 2017 Wiley Periodicals, Inc.

[Evaluation of the cytotoxicity of cigarette smoke condensate by using different solvents]. / Valutazione della citotossicità del condensato di fumo di sigaretta utilizzando diversi solventi.

The use of short term in vitro tests is an important step in the evaluation of the mechanism of action of cigarette smoke condensate (CSC). This study evaluated the effects of both CSC and solvents on Swiss 3T3 murine fibroblast cells. Results show that after 24 hours of treatment, CSC exerts a dose-dependent cytotoxic activity, both in methanol and dimethyl sulfoxide (DMSO), through mechanisms involving cell death and alterations in cell proliferation and metabolism. On the contrary, after 1 hour of treatment, a cytotoxic effect is seen only with CSC in DMSO. In cell cultures devoid of CSC, similar results have been obtained after 24 hours with both types of treatment.

Different mechanisms are involved in oxidative DNA damage and genotoxicity induction by ZnO and TiO2 nanoparticles in human colon carcinoma cells.

In this work we investigated the genotoxicity of zinc oxide and titanium dioxide nanoparticles (ZnO NPs; TiO2 NPs) induced by oxidative stress on human colon carcinoma cells (Caco-2 cells). We measured free radical production in acellular conditions by Electron Paramagnetic Resonance technique and genotoxicity by micronucleus and Comet assays. Oxidative DNA damage was assessed by modified Comet assay and by measuring 8-oxodG steady state levels. The repair kinetics of DNA oxidation as well as the expression levels of hOGG1 were also analyzed. Even if both NPs were able to produce ROS in acellular conditions and to increase 8-oxodG levels in Caco-2 cells, only ZnO NPs resulted genotoxic inducing micronuclei and DNA damage. Furthermore, Caco-2 cells exposed to ZnO NPs were not able to repair the oxidative DNA damage that was efficiently repaired after TiO2 NPs treatment, through OGG1 involvement. These results indicate that the high oxidant environment caused by ZnO NPs in our cellular model can induce DNA damage and affect the repair pathways.

Biomonitoring of primary aluminium industry workers: detection of micronuclei and repairable DNA lesions by alkaline SCGE.

The genetic effects of occupational exposure to low polycyclic aromatic hydrocarbon (PAH) concentrations were investigated in primary aluminium industry workers. The study subjects were employed in a plant that uses pre-baked anode cells, and has relatively low PAH contamination. Forty-two male workers belonging to different job categories (anode fabrication, baking, rodding, electrolysis, maintenance), together with 16 male local residents with no occupational exposure to PAHs were selected for the analysis of micronuclei and DNA lesions in peripheral lymphocytes. The incidence of micronuclei determined in 1000 cytokinesis-blocked cells in each subject was not significantly different between workers and controls (8.5+/-5.4 per thousand versus 9.7+/-4.9 per thousand, respectively), nor between smokers and non-smokers (8.3+/-5.8 per thousand versus 9.2+/-5.1 per thousand), but was significantly (P<0.05) related to the subjects' age. Also the analysis of DNA damage in unstimulated and mitogen-stimulated lymphocytes by single cell gel electrophoresis (SCGE) did not show significant differences between the studied groups (average tail moment values were 0.53+/-0.53 and 0.49+/-0.45 microm in exposed subjects and controls, respectively). However, when lymphocytes were cultured in the presence of cytosine arabinoside (Ara-C, 1 microg/ml for 16h) the SCGE analysis revealed a significant (P=0.018) difference in tail moment values between aluminium workers and controls (1.73+/-1.05 microm versus 0.93+/-0.88 microm, respectively). This difference may highlight an excess of relatively stable DNA lesions, that do not affect strand integrity, and are expressed as intermediates of excision repair in stimulated cells, when gap refilling is inhibited by cytosine arabinoside (Ara-C).

Assessment of individual sensitivity to ionizing radiation and DNA repair efficiency in a healthy population.

Inter-individual variation in response to exposure to carcinogens has been ascribed to differences in carcinogen metabolism as well as to variability in DNA repair capacity (DRC). In order to investigate the role of inherited and acquired factors on individual variation in DNA repair capacity, a mutagen sensitivity assay was carried out on 31 healthy subjects. Fresh blood samples were irradiated with gamma-rays (2Gy) and the kinetics of DNA repair in leukocytes assessed by the comet assay 0, 15, and 30 min after irradiation. Whole blood cultures were set up to detect spontaneous and induced structural chromosomal aberrations in lymphocytes 48 h after irradiation. The results obtained were evaluated with respect to age, gender, smoking habits, occupational exposure to chemicals and metabolic genotype (NQO1, GSTM1 and GSTT1) of the study subjects. A higher frequency of radiation-induced aberrations was observed in GSTM1-positive individuals compared with GSTM1-null subjects (P=0.025), as well as in non-smokers compared with heavy smokers (P=0.05). Similar results were obtained by measuring residual DNA damage (RD) shortly after irradiation by means of the comet assay, with non-smokers showing a higher amount of RD compared with smokers (P=0.016). Moreover, a significant correlation (P=0.008) was observed between the amount of RD and the frequency of chromosome breaks after irradiation. The results of this pilot study suggest a modulator effect of smoking habits and GSTM1 genotype on the individual DNA repair capacity, possibly related to the higher expression of enzymes involved in the repair of oxidative DNA damage in heavy smokers and GSTM1-null subjects.

A review of in vitro methods to assess the biological activity of tobacco smoke with the aim of reducing the toxicity of smoke.

In the last few years tobacco companies have been developing several research strategies in order to reduce the risks associated with smoking. These strategies include, for example, the refining of alternative cigarette designs that reduce the amount of hazardous chemicals in the mainstream smoke by introducing modified filters, and/or reducing the amount of biologically significant ingredients in tobacco-burning cigarettes. In the last few decades numerous studies have been published to assess the biological activity of tobacco smoke using in vivo and in vitro test systems. In this scenario a general scientific consensus on how to measure and characterize the risk associated with cigarette smoke is still lacking. Short-term in vitro assays, which are widely accepted by regulatory agencies around the world, are useful tools to evaluate both the biological activity and the progress towards a reduction of tobacco smoke toxicity. These assays could be mainly applied to evaluate cytotoxicity and genotoxicity properties on whole cigarette smoke as well as condensates or fractions of whole smoke. Cytotoxicity induction can be measured as cellular viability and growth rates using different end-points. Otherwise, the target of genotoxicity studies is the DNA molecule. For genotoxicity evaluation, the end-points and cell systems should be chosen from those that are relevant and appropriate as clinical surrogate markers. In this respect, the occurrence of early biological effect markers, such as mutational or clastogenic events (point mutations, frameshifts, micronuclei, SCE, DNA adducts) in bacterial and mammalian cells should be studied in a tiered approach following the guidelines of regulatory agencies. The choice of criteria shall be matter of discussion.