Genotoxicity Evaluation of the Soybean Isoflavone Genistein in Human Papillary Thyroid Cancer Cells. Study of Its Potential Use in Thyroid Cancer Therapy.

Genistein is one of the several known isoflavonic phytoestrogens found in a number of plants, with soybeans and soy products being the primary food source. The aim of the study is to evaluate if genistein is able to exert antineoplastic action in primary human papillary thyroid cancer (PTC) cells. Thyroid tissues were treated with genistein (1-10-50-100 µM). Cell viability, proliferation, DNA primary damage and chromosomal damage were evaluated. An antiproliferative effect was induced by the highest doses of genistein, and such an effect was synergistically enhanced by the cotreatment with the antineoplastic drug sorafenib. Comet assay did not show any genotoxic effect in terms of primary DNA damage at all the times (4 and 24 h) and tested doses. A reduction of hydrogen peroxide-induced DNA primary damage in primary thyrocytes from PTC cells pretreated with genistein was observed. Data suggest that genistein exerts antineoplastic action, does not induce genotoxic effects while reduces oxidative-induced DNA damage in primary thyrocytes from PTC cells, supporting its possible use in therapeutic intervention.

Both 3,5-diiodo-L-thyronine (T2) and T3 modulate glucose-induced insulin secretion.

3,5-diiodo-L-thyronine (T2), a naturally existing iodothyronine, has biological effects on humans, but no information is available on its action on pancreatic b-cells. We evaluated its effect vs triiodothyronine (T3), on glucose-induced insulin secretion in INS-1e cells, a rat insulinoma line, and on human islets. INS-1e were incubated in the presence/absence of T2 or T3 (0.1 nmol/L-10 µmol/L), and glucose (3.3, 7.5, 11.0, and 20 mmol/L). Insulin release and content (at 11.0 and 20 mmol/L glucose) were significantly (p less than 0.01) stimulated by 1-100 nmol/L T2 and 0.1 nmol/L-1.0 µmol/L T3, and inhibited with higher concentrations of both (1–10 µmol/L T2 and 10 µmol/L T3). Human islets were incubated with 3.3 mmol/L glucose in presence/absence of T3 or T2 (0.1 nmol/L, 0.1 µmol/L, and 1 µmol/L). T2 (0.1 nmol/L-0.1 µmol/L) significantly (p less than0.01) stimulated insulin secretion, while higher concentrations (1 µmol/L) inhibited it. A modest increase in insulin secretion was evidenced with 1 µmol/L T3. In conclusion, T2 and T3 have a direct regulatory role in insulin secretion, depending on their concentrations and the glucose level itself. At concentrations near the physiological range, T2 enhances glucose-induced insulin secretion in both rat b-cells and human islets.

The Comet assay for the evaluation of genotoxic impact in aquatic environments.

This review considers the potential of the Comet assay (or Single Cell Gel Electrophoresis, SCGE) to evaluate the environmental impact of genotoxins in aquatic environments. It focuses on in vivo and in situ studies that have been carried out in various marine and freshwater sentinel species, published in the last 5 years. A large number of the studies reviewed report that the Comet assay is more sensitive when compared with other biomarkers commonly used in genetic ecotoxicology, such as sister chromatid exchanges or micronucleus test. Due to its high sensitivity, the Comet assay is widely influenced by laboratory procedures suggesting that standard protocols are required for both fish and mussel cells. However, there are still a wide variety of personalised Comet procedures evident in the literature reviewed, making comparison between published results often very difficult. Standardization and inter-laboratory calibration of the Comet assay as applied to aquatic species will be required if the Comet assay is to be used routinely by national bodies charged with monitoring water quality.

Cellular responses in the cyprinid Leuciscus cephalus from a contaminated freshwater ecosystem.

The response of wild chubs (Leuciscus cephalus) to chemical pollution was assessed in a metal contaminated river (Cecina River, Italy) through a wide battery of biomarkers which included: Comet assay detecting DNA strand breaks; diffusion assay for apoptosis induction; micronucleus test assessing chromosomal alterations; ethoxyresorufin O-deethylase (EROD) activity for the induction of cytochrome P 4501A; acetylcholinesterase (AChE) activity responsive to pesticide exposure; vitellogenin gene expression in males revealing estrogenic effects. Bioaccumulation of mercury, chromium and polycyclic aromatic hydrocarbons (PAHs) was also determined. Levels of mercury and PAHs were higher in tissues of chubs sampled from the most downstream station, reflecting an anthropogenic pollution of industrial origin. Otherwise, accumulation of Cr was quite similar in fish along the entire course of Cecina River confirming a natural origin due to local geochemical features. Biomarker responses revealed a significant increase of apoptotic cells, DNA stand breaks and micronucleus frequency in chubs from the more impacted sites. A slight EROD induction and AChE inhibition were only seen at the most downstream station demonstrating a limited impact due to PAHs and pesticides. On the other hand, the induction of vitellogenin gene in male chubs was measured in all the sites, suggesting a diffuse estrogenic effect. This study confirmed the utility of large batteries of biomarkers in biomonitoring studies and the suitability of wild chub as bioindicator organism for river basins.

Induction of Th1 chemokine secretion in dermal fibroblasts by vanadium pentoxide.

Vanadium is a soft, silvery­grey metal with a number of different oxidation states. The most common commercial form of vanadium is vanadium pentoxide (V2O5). All vanadium compounds are considered toxic. An increase in skin rashes has been observed in certain vanadium workers, including the development of atopic dermatitis. However, to the best of our knowledge, no prior in vivo or in vitro studies have evaluated the effect of vanadium exposure in human dermal fibroblasts. The present study evaluated the effect of V2O5 on proliferation and chemokine secretion in dermal fibroblasts. The results revealed that V2O5 had no significant effect on the viability or proliferation of fibroblasts, however it was able to induce the secretion of T­helper (Th)1 chemokines from dermal fibroblasts, synergistically increasing the effect of important Th1 cytokines, including interferon­Î³ and tumor necrosis factor­α. Through these processes, V2O5 may lead to the induction and perpetuation of an inflammatory reaction in dermal tissue. The induction and perpetuation of inflammation in the dermis and the variety of involved candidate genes may be at the base of V2O5­induced effects following occupational and environmental exposures. Further studies are necessary to evaluate dermal integrity and manifestations in subjects who are occupationally exposed, or living in polluted areas.

Cellular biomarkers for monitoring estuarine environments: transplanted versus native mussels.

In developed countries, estuarine environments are often subjected to chemical pollution, whose biological impact is profitably evaluated by the use of multi-biomarker approaches on sentinel species. In this paper, we investigate genotoxicity and lysosomal alterations in the Mediterranean mussel (Mytilus galloprovincialis), from the estuary of the River Cecina (Tuscany, Italy), selected as "pilot basin" within the Water Frame Directive (2000/60 European Community). Both native and 1 month transplanted mussels were used in order to compare these two approaches in terms of sensitiveness of specific biomarker responses. Genotoxic effects were evaluated as strand breaks, by single cell gel electrophoresis (or Comet assay), and as chromosomal alterations, by the micronucleus test in gill cells. Lysosomal alterations were assessed by the neutral red retention time (in haemocytes), lipofuscin accumulation and ultrastructure (in digestive cells). Heavy metal bioaccumulation was also analysed. Mussels from the River Cecina showed a general alteration of all the biomarkers investigated, accompanied by an elevation of tissue metal levels. However, some differences in specific responses occurred between transplanted and native mussels. Early biomarkers, such as those based on DNA and lysosomal membrane integrity, were induced at similar degree in native and transplanted mussels; while alterations resulting from cumulative events, as the increase of micronuclei frequency were much more elevated in native specimens (23.1+/-7.6) than in transplanted (9.3+/-4.7) and reference ones (5.8+/-5.2). Similarly, the comparison between lipofuscin accumulation and mean lysosomal diameter in impacted and control sites, gave significant differences exclusively with transplanted mussels. These results suggest that the parallel use of caged and native mussels in environmental biomonitoring can improve the characterization of the study area.

Effects of algal extracts (Polysiphonia fucoides) on rainbow trout (Oncorhynchus mykiss): a biomarker approach.

The genotoxicity of algal extracts (Polysiphonia fucoides) was investigated in erythrocytes of rainbow trout (Oncorhynchus mykiss). Trout were exposed to 0.5% of the algal extract for 7 days. Comet assay (alkaline and neutral versions) and Micronucleus test were used to assess DNA damage, and Diffusion Assay to detect apoptotic cells. EROD activities and oxidative stress parameters in rainbow trout liver were also measured. A significant induction of DNA single strand breaks comparable to the ones induced by the in vivo exposure to 20 mg/kg B[a]P was observed at the end of the treatment, while increases of double strand breaks and apoptotic cells were not observed. The absence of activation of antioxidant responses seems to underline a mechanism of action of the genotoxic algal extract which does not involve oxidative stress.

Does the crystal habit modulate the genotoxic potential of silica particles? A cytogenetic evaluation in human and murine cell lines.

Crystalline silica inhaled from occupational sources has been classified by IARC as carcinogenic to humans; in contrast, for amorphous silica, epidemiological and experimental evidence remains insufficient. The genotoxicity of crystalline silica is still debated because of the inconsistency of experimental results ("variability of silica hazard"), often related to the features of the particle surfaces. We have assessed the role of crystal habit in the genotoxicity of silica powders. Pure quartz (crystalline) and vitreous silica (amorphous), sharing the same surface features, were used in an in vitro study with human pulmonary epithelial (A549) and murine macrophage (RAW264.7) cell lines, representative of occupational and environmental exposures. Genotoxicity was evaluated by the comet and micronucleus assays, and cytotoxicity by the trypan blue method. Cells were treated with silica powders for 4 and 24h. Quartz but not vitreous silica caused cell death and DNA damage in RAW264.7 cells. A549 cells were relatively resistant to both powders. Our results support the view that crystal habit per se plays a pivotal role in modulating the biological responses to silica particles.

Genomic and chromosomal damage in the marine mussel Mytilus galloprovincialis: Effects of the combined exposure to titanium dioxide nanoparticles and cadmium chloride.

Titanium dioxide nanoparticles (TiO2-NPs) continuously released into waters, may cause harmful effects to marine organisms and their potential interaction with conventional toxic contaminants represents a growing concern for biota. We investigated the genotoxic potential of nanosized titanium dioxide (n-TiO2) (100 µg L(-1)) alone and in combination with CdCl2 (100 µg L(-1)) in Mytilus galloprovincialis after 4 days of in vivo exposure. RAPD-PCR technique and Micronucleus test were used to study genotoxicity. The results showed genome template stability (GTS) being markedly reduced after single exposure to n-TiO2 and CdCl2. Otherwise, co-exposure resulted in a milder reduction of GTS. Exposure to n-TiO2 was responsible for a significant increase of micronucleated cell frequency in gill tissue, while no chromosomal damage was observed after CdCl2 exposure as well as after combined exposure to both substances.

n-TiO2 and CdCl2 co-exposure to titanium dioxide nanoparticles and cadmium: Genomic, DNA and chromosomal damage evaluation in the marine fish European sea bass (Dicentrarchus labrax).

Due to the large production and growing use of titanium dioxide nanoparticles (n-TiO2), their release in the marine environment and their potential interaction with existing toxic contaminants represent a growing concern for biota. Different end-points of genotoxicity were investigated in the European sea bass Dicentrarchus labrax exposed to n-TiO2 (1mgL(-1)) either alone and combined with CdCl2 (0.1mgL(-1)) for 7 days. DNA primary damage (comet assay), apoptotic cells (diffusion assay), occurrence of micronuclei and nuclear abnormalities (cytome assay) were assessed in peripheral erythrocytes and genomic stability (random amplified polymorphism DNA-PCR, RAPD assay) in muscle tissue. Results showed that genome template stability was reduced after CdCl2 and n-TiO2 exposure. Exposure to n-TiO2 alone was responsible for chromosomal alteration but ineffective in terms of DNA damage; while the opposite was observed in CdCl2 exposed specimens. Co-exposure apparently prevents the chromosomal damage and leads to a partial recovery of the genome template stability.

Individual sensitivity to cytogenetic effects of 1,2:3,4-diepoxybutane in cultured human lymphocytes: influence of glutathione S-transferase M1, P1 and T1 genotypes.

Although some blood parameters have been suggested to modulate in-vitro induction of sister chromatid exchanges by 1,2:3,4-diepoxybutane (DEB), a metabolite of 1,3-butadiene, the increased sensitivity has largely been assigned to a homozygous deletion of glutathione S-transferase T1 gene (GSTT1 null genotype). However, some DEB-sensitive individuals have been shown to be GSTT1 positive (having at least one undeleted GSTT1 allele). To examine potential causes for this overlap, we evaluated the effect of GSTM1, GSTP1, and GSTT1 genotypes, together with various life-style and blood parameters, on the DEB induction of sister chromatid exchanges and cells with chromosomal aberrations (aberrant cells) in lymphocyte cultures of 115 and 62 human donors, respectively. Our results supported the important role of the GSTT1 genotype in DEB sensitivity; 76% of cultures from GSTT1 null donors but only 4% of those from GSTT1 positive donors were DEB-sensitive, as defined by sister chromatid exchange measurements. The GSTT1 genotype also clearly affected DEB-induced aberrant cells, 92% of GSTT1 null and 8% of GSTT1 positive donors being sensitive to DEB. All individuals showing a high response to DEB in both sister chromatid exchange and aberrant cell analyses were GSTT1 null. Baseline aberrant cell measurements but not sister chromatid exchange measurements were marginally higher among GSTT1 null donors compared with GSTT1 positive donors. GSTM1 and GSTP1 genotypes had no influence on these cytogenetic end-points. Blood transaminases, gamma-glutamyl transferase, urea, creatinine and white blood cell count showed a clear negative association with DEB-induced aberrant cells, whereas wine drinkers had more aberrant cells than non-drinkers. A higher sister chromatid exchange-response to DEB was observed in lymphocytes from women and smokers than from men and non-smokers, respectively. Erythrocyte count correlated negatively with DEB-induced sister chromatid exchanges. Thus, a variety of parameters seemed to modulate the individual DEB-sensitivity together with the GSTT1 genotype. Although the known contributing factors accounted for a considerable part of individual variability in sister chromatid exchanges (59.4%) and aberrant cells (46.7%) in DEB treatment, they did not, however, fully explain the overlap in cytogenetic response between GSTT1 positive and null individuals.

Oxidative damage and cytogenetic analysis in leukocytes of Parkinson's disease patients.

BACKGROUND: Postmortem studies suggest excessive free radical toxicity in the substantia nigra of patients with PD. Increased lipid peroxidation and oxidative DNA damage have been reported in the CNS. Markers of oxidative stress have been identified in the blood of patients with PD. OBJECTIVE: To assess the presence of spontaneous chromosome and primary or oxidative DNA damage in peripheral blood leukocytes of patients with untreated PD. METHODS: Patients with de novo PD (20) and control subjects (16), matched for age, sex, and smoking habits, underwent cytogenetic analysis using the human lymphocyte micronucleus assay coupled with the fluorescence in situ hybridization technique and the Comet assay. RESULTS: Compared with controls, patients with PD showed an increase in the incidence of spontaneous micronuclei (p < 0.001); single strand breaks (p < 0.001); and oxidized purine bases (p < 0.05). Fluorescence in situ hybridization analysis showed micronuclei harboring acentric fragments. CONCLUSIONS: There is chromosomal, primary DNA damage and oxidative DNA damage demonstrable in lymphocytes of patients with untreated PD.

DNA damage and ubiquitinated neuronal inclusions in the substantia nigra and striatum of mice following MDMA (ecstasy).

RATIONALE: 3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative, which is neurotoxic to both serotonin (5HT) and dopamine (DA) nerve terminals. Previous reports, carried out in rodents and non-human primates, demonstrated neurotoxicity to monoamine axon terminals, although no study has analyzed nigral and striatal cell bodies at the sub-cellular level. OBJECTIVE: In this study, we examined intrinsic nigral and striatal cells, and PC12 cell cultures to evaluate whether, in mice, MDMA might affect nigral and striatal cell bodies. METHODS: After administering MDMA, we analyzed effects induced in vivo and in vitro using high-performance liquid chromatography (HPLC) analysis, light- and electron microscopy with immunocytochemistry, and DNA comet assay. RESULTS: We found that MDMA (5 mg/kg x4, 2 h apart), besides a decrease of nigrostriatal DA innervation and 5HT loss, produces neuronal inclusions within nigral and intrinsic striatal neurons consisting of multi-layer ubiquitin-positive whorls extending to the nucleus of the cell. These fine morphological changes are associated with clustering of heat shock protein (HSP)-70 in the nucleus, very close to chromatin filaments. In the same experimental conditions, we could detect oxidation of DNA bases followed by DNA damage. The nature of inclusions was further investigated using PC12 cell cultures. CONCLUSIONS: The present findings lead to re-consideration of the neurotoxic consequences of MDMA administration. In fact, occurrence of ubiquitin-positive neuronal inclusions and DNA damage both in nigral and striatal cells sheds new light into the fine alterations induced by MDMA, also suggesting the involvement of nuclear and cytoplasmic components of the ubiquitin-proteasome pathway in MDMA toxicity.

Validation of single cell gel assay in human leukocytes with 18 reference compounds.

To validate the alkaline single cell gel (SCG) assay as a tool for the detection of DNA damage in human leukocytes, we investigated the in vitro activity of 18 chemicals. Thirteen of these chemicals (pyrene (PY), benzo(a)pyrene (BaP), cyclophosphamide (CP), 4-nitroquinoline-1-oxide (4NQO), bleomycin (BLM), methylmercury chloride (MMC), mitomycin C (MTC), hydrogen peroxide (HP), diepoxybutane (DEB), glutaraldehyde (GA), formaldehyde (FA), griseofulvin (GF), sodium azide (NA)) are genotoxic in at least one cell system, while five compounds (ascorbic acid (AA), glucose (GL), D-mannitol (MAN), O-vanillin (VAN), chlorophyllin (CHL)) are classified as non-genotoxic. In this in vitro SCG assay, PY, BaP and CP were positive with exogeneous metabolic activation (rat S9 mix) while 4NQO, BLM, MMC, MTC, hydrogen peroxide, and diepoxbutane were positive in the absence of metabolic activation. CHL and VAN were unexpectedly found to induce a dose-dependent increase in DNA migration. AA, GL, and MAN were negative in a non-toxic range of doses. GF gave equivocal results, while FA and GA increased DNA migration at low doses and decreased DNA migration at higher doses. This behaviour is consistent with the known DNA damaging and crosslinking properties of these compounds. These data support the sensitivity and specificity of this assay for identifying genotoxic agents.

Herbicide-induced DNA damage in human lymphocytes evaluated by the single-cell gel electrophoresis (SCGE) assay.

The genotoxicity of the herbicides, alachlor, atrazine, maleic hydrazide, paraquat and trifluralin has been evaluated in the single-cell gel electrophoresis (SCGE) assay by using human peripheral blood lymphocytes. All treatments were conducted with and without the presence of an external bioactivation source (S9 mix). The results indicate that all the herbicides tested are able to give positive results by increasing the comet tail length, which would confirm both the genotoxicity of the herbicides and the sensitivity of the assay in front of these chemicals. Alachlor and atrazine give similar results in treatments with and without S9, while when the S9 mix was not used paraquat and trifluralin genotoxicity was higher. On the other hand, although maleic hydrazide genotoxicity was higher when S9 mix was used at normal pH (7.4), our data show that its genotoxicity depends largely on the pH solution, increasing as the pH decreased.

Modulating factors of individual sensitivity to diepoxybutane: sister chromatid exchanges induced in vitro in human lymphocytes.

Spontaneous and diepoxybutane (DEB)-induced sister chromatid exchanges (SCEs) were examined in cultured peripheral lymphocytes (PBL) from 122 healthy donors. SCE-inducing activity under defined experimental conditions and individual sensitivity to genotoxic stress were assessed. SCE means distribution appeared asymmetrical, identifying about 22% of subjects characterized by a 'high-respondent' phenotype with more than 111 SCEs/cell. Confounding factors, such as smoking habit, wine and coffee consumption, work activity and hematological factors, showed a limited capacity to affect individual SCE responsiveness, however hemoglobin and uric acid seemed to antagonize DEB genotoxicity.

Comet assay on children's leukocytes 8 years after the Chernobyl disaster.

DNA damage, mainly single strand breaks, was evaluated by single cell gel electrophoresis, in leukocytes of 36 healthy and 14 thyroid cancer-affected children prior to radio-therapy. The children come from the Gomel region, one of the areas most heavily radio-contaminated by the Chernobyl fallout. In addition, leukocytes were treated with a challenge dose of bleomycin (BLM, 1.5 micrograms/ml), to assess the presence of an adaptive response (AR) potentially resulting from chronic exposure to radionuclides. As controls, 13 children living in Pisa (Italy) were enrolled in the study. Children with thyroid cancer show higher (p < 0.001) DNA damage than healthy ones. No difference was found between healthy children from Gomel and from Pisa. A reduction in the response to BLM was significantly linked to low plasma levels of FT4 hormone (p < 0.0001), to the presence of the tumor (p < 0.002), to being female (p < 0.02), and to a higher 137Cs body burden (p < 0.03).

DNA damage evaluated by alkaline single cell gel electrophoresis (SCGE) in children of Chernobyl, 10 years after the disaster.

Using the alkaline single cell gel electrophoresis (Comet) assay, the extent of DNA damage was evaluated in leukocytes of 43 Belarussian children (16 healthy and 27 affected by thyroid cancer). Thirty-nine healthy children from Pisa (Italy) were enrolled in the study as controls. In addition to basal levels of DNA damage, leukocytes were treated in vitro with bleomycin (BLM), a radiomimetic drug, to evaluate a possible adaptive response in different groups of children. Results with the Comet assay indicated an increased level of DNA damage (P=0.037) in leukocytes of Belarussian children compared to the Italian control group. In addition, within the Belarus group, lower basal levels of DNA damage (P<0.001) were found in children with cancer compared to healthy children. Tumor affected children were living in less radiocontaminated areas (P<0.04) than the healthy children and there was a significant relationship (P=0.03) between the amount of environmental radiocontamination and DNA damage in leukocytes. There were no differences in the sensitivity of leukocytes from different groups of children to BLM, indicating the absence of an adaptive response. The lack of an adaptive response may have been due to the use of noncycling cells and/or the bleomycin dose chosen. Tests for the presence of clastogenic factors (CF) in the blood serum of children showed that 39% of the tumor affected children and 19% of the healthy children in the exposed group were positive as compared to the Italian control group (0%) (Chi-square test, P<0.04). The higher levels of genomic damage in children evaluated 10 years after the Chernobyl disaster could be related to the increased incidence of individuals with CF.

Spontaneous sister chromatid exchange and chromosome aberration frequency in humans: the familial effect.

The possible effects of environmental and genetic factors on spontaneous frequencies of sister chromatid exchanges (SCEs) and cells with chromosome aberrations (CAs) in human lymphocytes were investigated by analysing 177 completed families (mother, father and at least one child). After removing the effects of methodological, biological and life-style factors by the use of multifactor analysis of variance (MANOVA), SCEs and CAs residuals were analysed by simple correlation analysis and principal component analysis. SCEs and CAs inter-familiar variability was higher than that found within families. A significant correlation was found between the average SCE frequencies shared by parents (the so-called 'midpoint parents', or 'midparent') and offspring (linear slope b=0.26+/-0.07, p<0.05), but also between mother and father (b=0.23+/-0.11, p<0.05) suggesting the presence of an effective environmental factor. The midparent-offspring correlation was found to be sustained by the mother-offspring relationship (b=0.28+/-0.08, p<0.05), being the father-offspring correlation not significant (b=0.16+/-0.11, p0.05). Concerning CAs, no statistically significant correlation between parents was found, but the strong relationship between mother and offspring was confirmed (b=0.468+/-0.11, p<0.001). The SCEs correlation between mother vs. offspring disappeared for older offspring (over 23 years old). The obtained findings strongly showed that the genetic make-up is barely detectable in the presence of domestic environment factors which are shown to play the major role in determining the interfamilial variability of SCE and CA in a general population. These results strengthen the suitability of the use of SCEs and CAs analysis in human cytogenetic surveillance for the detection of effective environmental factors.

Evaluation of DNA damage in leukocytes of ex-smokers by single cell gel electrophoresis.

Single cell gel electrophoresis (SCGE), or comet assay, appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. A follow-up study of 90 smokers who ceased smoking was undertaken to determine the possible decrease of DNA damage in their leukocytes. Before beginning the trial, volunteers smoked on average 26.1 +/- 8.4 cigarettes/day. Comet length did not correlate with the number of cigarettes/day or with the condensate tar content. At the end of the study, 28 volunteers had abandoned the trial, 40 volunteers relapsed into smoking at different times, but with a reduced number of cigarettes/day, whereas 22 fully succeeded in smoking cessation. Throughout the 5 sampling times, a great variability of comet length at individual level was found. However, after 1 year of follow-up, comet length means were found to be significantly shorter (p < 0.0001) in those volunteers who completely quit smoking compared to those who relapsed into smoking (27.2 +/- 1.6 vs. 31.9 +/- 5.1 microns, respectively), irrespective of the amount of cigarettes previously smoked. No effect of age or sex was found. Six months later, these results were confirmed by a further study carried out on a reduced sample of volunteers. The present data strongly suggest that, in spite of the great variability observed, 1 year of smoking cessation is associated with a significant reduction of DNA damage in circulating leukocytes.