Very low concentration of cerium dioxide nanoparticles induce DNA damage, but no loss of vitality, in human spermatozoa.

Cerium dioxide nanoparticles (CeO2NP) are widely used for industrial purposes, as in diesel, paint, wood stain and as potential therapeutic applications. The Organization for Economic Cooperation and Development included CeO2NP in the priority list of nanomaterials requiring urgent evaluation. As metal nanoparticles can cross the blood-testis barrier, CeO2NP could interact with spermatozoa. The genotoxicity of CeO2NP was demonstrated in vitro on human cell lines and mouse gametes. However, the effects of CeO2NP on human spermatozoa DNA remain unknown. We showed significant DNA damage induced in vitro by CeO2NP on human spermatozoa using Comet assay. The genotoxicity was inversely proportional to the concentration (0.01 to 10 mg·L-1). TEM showed no internalization of CeO2NP into the spermatozoa. This study shows for the first time that in vitro exposure to very low concentrations of cerium dioxide nanoparticles can induce significant DNA damage in human spermatozoa. These results add new and important insights regarding the reproductive toxicity of priority nanomaterials, which require urgent evaluation.

Radiation protection for surgeons and anesthetists: practices and knowledge before and after training.

The use of radiological activity in the operating room (OR) and a regulatory decrease of the eye lens dose warrant an assessment of how medical staff are protected from radiation. This study aims to evaluate practices and knowledge in radiation protection (RP) for OR doctors before and after training. A descriptive study of surgeons and anesthetists in a French public hospital center was conducted in 2016. An ad hoc questionnaire concerning occupational practices and knowledge about RP was distributed before and one month after RP training. Among 103 doctors attending the training, 90 answered the questionnaire before the training. Results showed a lack of knowledge and good practice in RP. Most of the participants (86.7%) had never been trained in RP and recognized insufficient knowledge. Most of them (92.2%) wore a lead apron, 50.0% a thyroid-shield, 5.6% lead glasses, 53.3% a passive dosimeter and 17.8% an electronic dosimeter. None of them benefitted from collective protective equipment such as a ceiling suspended screen. The questionnaire following the training was completed by only 35 doctors. A comparison before and after training results showed an improvement in knowledge (scores of correct responses: 5.5/16 before and 9.5/16 after training) but not in RP good practices (scores of correct responses: 3.2/7 before and 3.3/7 after training). One training session appears to be insufficient to improve the application of the safety rules when x-rays are used. Communication needs to be improved regarding RP among anesthetists and surgeons, such as training renewal, workstation analysis in OR related to x-ray use and occupational medical follow-up. Otherwise, radiological risks in OR need to be given better consideration, such as radio-induced cataract risk. It is necessary to encourage the use of dosimeters and protective equipment and to strengthen access to lead glasses and collective protective equipment, such as ceiling suspended screens. All these recommendations ensure the received dose is reduced to as low as is reasonably achievable.

[Association between sperm abnormalities and occupational environment among male consulting for couple infertility]. / Association entre anomalies spermatiques et environnement professionnel chez les hommes consultant pour infertilité de couple.

Alteration of sperm parameters related to occupational exposures is the subject of several studies, often on a case-control approach. The study populations usually comprise men consulting in infertility clinics for couple infertility. The objective of this review is to identify, from these case-control studies, the main occupational factors that may be associated with altered sperm parameters. We selected 13 articles in the PubMed database. Participation in these studies varied from 61 to 2619 subjects, with great methodological heterogeneity, particularly in the characterization of exposure. The main occupations that appear significantly associated with a risk of altered sperm parameters are workmen, painters, farmers, welders, plumbers and technicians. When analysis focuses on occupational exposures, a significant result is reported for solvents, heavy metals, heat, vibrations and non-ionizing radiation. None of the selected studies has found a link with exposure to pesticides.

[Toxicity of nanoparticles on reproduction]. / Reprotoxicité des nanoparticules.

Nanoparticles (NPs) are sized between 1 and 100nm. Their size allows new nanoscale properties of particular interest for industrial and scientific purpose. Over the past twenty years, nanotechnology conquered many areas of use (electronic, cosmetic, textile…). While, human is exposed to an increasing number of nanoparticles sources, health impacts and, particularly on reproductive function, remains poorly evaluated. Indeed, traceability of nanoparticles use is lacking and nanotoxicology follows different rules than classical toxicology. This review focuses on the impact of NPs on health and particularly on fertility and addresses potential risks of chronic exposure to NPs on human fertility.

In vivo exposure to benzo(a)pyrene induces significant DNA damage in mouse oocytes and cumulus cells.

STUDY QUESTION: Does in vivo exposure to benzo(a)pyrene (BaP) induce DNA damage in oocytes and cumulus cells (CCs) in mice? SUMMARY ANSWER: Significant increases in DNA strand breaks in oocytes and CCs and in BaP-induced DNA adducts in CCs were detected in exposed mice compared with controls. WHAT IS KNOWN ALREADY: BaP has well-known mutagenic and carcinogenic effects on somatic cells, and is also registered as potential reproductive toxicant by several environmental protection agencies. It has been shown to cause a significant increase in DNA adducts in ovarian tissues; however, to our knowledge, the genotoxic effects of BaP on oocytes and CCs have not been studied to date. STUDY DESIGN, SIZE, DURATION: Female CD1 mice were exposed to BaP via the oral administration of a single dose of 13 mg/kg body weight (bw); matched controls were exposed to the vehicle only (soya oil). A total of 15 groups of 6 mice (exposed or controls) were sacrificed 2, 4, 6, 15 or 22 days after BaP exposure, and after collection of oviducts, the oocyte-CC complexes (COC) were released. PARTICIPANTS/MATERIALS, SETTING, METHODS: The alkaline comet assay was used to quantify the DNA breaks in oocytes and CCs; DNA damage was expressed as the Olive Tail Moment (OTM). Immunofluorescent staining was used to quantify BaP-induced DNA adducts in CCs. Fluorescence was expressed as the average grey value (AGVA; arbitrary units). The differences between the exposed and control groups were assessed using analysis of variance (ANOVA) and the non-parametric Mann-Whitney test. MAIN RESULTS AND THE ROLE OF CHANCE: Higher levels of DNA damage were observed in the oocytes and CCs of BaP-exposed mice than in those of vehicle controls. Significant increases in OTM (mean ± SE) were detected in (i) oocytes from females exposed for 4 (10.5 ± 0.9 versus 3.1 ± 0.4, P < 0.0001) or 6 days before collection (15.6 ± 2.0 versus 3.6 ± 0.9, P < 0.0001) and (ii) CCs from females exposed 2 (6.4 ± 0.6 versus 2.1 ± 0.2, P < 0.0001), 4 (7.8 ± 0.4 versus 2.4 ± 0.1, P < 0.0001) or 6 days before collection (7.3 ± 0.3 versus 3.2 ± 0.5, P < 0.0001) compared with controls. A significant increase in benzo(a)pyrene-7,8-9,10 diol epoxide (BPDE)-DNA adducts and higher AGVA (mean ± SE) scores were observed in CCs from females exposed 2 (6.1 ± 0.3 versus 3.6 ± 0.5, P < 0.0001), 4 (7.5 ± 0.1 versus 3.4 ± 0.1, P < 0.0001) or 6 days before collection (11.6 ± 0.4 versus 3.7 ± 0.1, P < 0.0001) compared with control mice. LIMITATIONS, REASONS FOR CAUTION: Mice were given one treatment via the oral route because this dose and mode of administration have been shown to induce detectable BPDE-DNA adduct levels in mouse organs and sperm cells. Additional data are needed to assess DNA damage in oocytes and CCs after chronic exposure to BaP in vivo. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study examining the in vivo genotoxicity of BaP in oocytes and CCs. We observed significant DNA damage in the oocytes and CCs of mice after acute BaP exposure. BPDE-DNA adducts result directly from BaP metabolism while DNA breaks could result mainly from BPDE-DNA adduct excision and repair and/or through direct genotoxicity from increased reactive oxygen species. These results add new and important insights regarding the recently suggested toxicity of chronic BaP exposure in the ovary. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant (93-CPQ 2012-05) from the DIRRECTE, Provence Alpes Côte d'Azur, France. None of the authors have any conflict of interest to declare.

[Urinary 8-hydroxy-2'-deoxyguanosine: a biomarker of environmental oxidative stress?]. / Dosage de la 8-hydroxy-2'-deoxyguanosine dans les urines: un biomarqueur du stress oxydatif d'origine environnementale?

The oxidative stress plays an important role in certain pathologies, notably in carcinogenesis. Indeed, reactive oxygen species (ROS) can induce a variety of damage to DNA, including oxidized bases which will then be repaired and eliminated in urine. The 8-hydroxy-2'-deoxyguanosine (8-OHdG), the most frequent member of these oxidized bases, can be measured in DNA or in urine by various methods. The urinary measurement was used in several studies among subjects with occupational or environmental exposure. Diverse chemical or physical agents can indeed contribute to the increase of oxidative stress, although the latter has several origins. Our objective is to analyze through these studies the interest, the limits and the implementation of this biomarker. The majority of the studies reveal an increase of the concentration of 8-OHdG in urine in exposed subjects (polycyclic aromatic hydrocarbons, metals, ionizing radiation and other agents) compared with controls or according to exposure levels. The urinary concentration of 8-OHdG is subject to important inter and intra-individual variations. The biomonitoring studies have to take into account diverse confounding factors, which may have conflicting effects. Moreover the results strongly depend on the analytical method used. Thus other investigations are necessary to validate this biomarker and better know its sources of variability, its biological significance, its dose-response relationship and its kinetics after exposure to oxidative stress agents.

Cytogenetic monitoring by use of the micronucleus assay among hospital workers exposed to low doses of ionizing radiation.

The aim of this study was to assess occupationally induced chromosomal damage in a large population of hospital workers exposed to low doses of ionizing radiation. We used the cytokinesis-block micronucleus (CBMN) assay in the peripheral lymphocytes of 132 exposed workers compared with 69 controls matched for gender, age and smoking habits. The CBMN assay was combined with fluorescence in situ hybridization with a human pan-centromeric DNA probe in 32 exposed subjects and 30 controls randomly chosen from the initial populations. Occupational dosimetry records were collected over the last 10-year period and revealed very low exposure levels. The average binucleated micronucleated cell rate (BMCR) was significantly higher in the exposed subjects than in the controls (14.9 per thousand+/-8.1 versus 11.8 per thousand+/-6.5; P=0.011). About one-third of the micronuclei were centromere-negative in the exposed and control groups. BMCR significantly positively correlated with donor age in the exposed population; this correlation was at the border of significance in the control group. In the two groups, BMCR was significantly greater in females than in males, and the significant correlation between age and BMCR was observed in the female population, but not in the male one. No effect of smoking habits emerged. Univariate analysis revealed a possible influence of familial cancer history and diagnostic medical radiation dose (estimated from examinations reported in the questionnaire) on BMCR. Multiple regression analysis, taking into account all the previous confounding factors, showed that only occupational exposure status, gender and age had a significant effect on BMCR. In conclusion, the present study shows that chromosomal damage leading to micronucleated lymphocytes is more frequent in hospital workers exposed to ionizing radiation than in controls, despite the very low levels of exposure.

Exposure to genotoxic agents, host factors, and lifestyle influence the number of centromeric signals in micronuclei: a pooled re-analysis.

We pooled data from three biomonitoring studies using the cytokinesis-block micronucleus assay in peripheral blood lymphocytes in combination with fluorescence in situ hybridization. Centromere-positive micronuclei (C+MN) were classified in two groups: those containing one centromere (C1+MN) and those with two or more (Cx+MN). The three studies evaluated untreated cancer patients, welders, and pathologists/anatomists exposed to formaldehyde. The total number of subjects included in the pooled re-analysis was 113. A higher frequency of C+MN was observed in cancer patients and exposed workers, who showed significant differences from controls in all studies. C1+MN were particularly increased in the group of pathologists/anatomists, who showed a 3.29 times higher frequency than controls (95% CI: 2.04-5.30). A borderline increase in Cx+MN was observed in welders when compared to the corresponding control group (FR: 1.31; 95% CI: 0.99-1.74). An evident effect of gender was found, with significantly increased frequencies of all endpoints measuring aneuploidy in females (C+MN, C1+MN, and Cx+MN). Alcohol consumption had a significant effect on total MN frequency and particularly on C+MN and C1+MN. In conclusion, scoring the number of centromeric signals in the micronucleus assay provides additional information about the mechanism of action of various genotoxic agents, and the role of confounding factors may be more specifically accounted for. Indeed, C+MN could be efficiently used in biomonitoring studies as an independent biomarker of exposure and early biological effect. The use of centromeric signals allows the identification of two further endpoints, representing two alternative pathways of chromosome loss, i.e., impaired chromosome migration, leading to increased C1+MN frequency, and centrosome amplification, possibly leading to Cx+MN with two or more centromeric signals.

Genotoxic risk assessment of pathology and anatomy laboratory workers exposed to formaldehyde by use of personal air sampling and analysis of DNA damage in peripheral lymphocytes.

A study was conducted to evaluate the genotoxic effect of occupational exposure to formaldehyde on pathology and anatomy laboratory workers. The level of exposure to formaldehyde was determined by use of passive air-monitoring badges clipped near the breathing zone of 59 workers for a total sampling time of 15 min or 8 h. To estimate DNA damage, a chemiluminescence microplate assay was performed on 57 workers before and after a 1-day exposure. Assessment of chromosomal damage was carried out by use of the cytokinesis-blocked micronucleus assay (CBMN) in peripheral lymphocytes of 59 exposed subjects in comparison with 37 controls matched for gender, age, and smoking habits. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 18 exposed subjects and 18 control subjects randomized from the initial populations. Mean concentrations of formaldehyde were 2.0 (range <0.1-20.4 ppm) and 0.1 ppm (range <0.1-0.7 ppm) for the sampling times of 15 min and 8 h, respectively. No increase in DNA damage was detected in lymphocytes after a one-workday exposure. However, the frequency of binucleated micronucleated cells was significantly higher in pathologists/anatomists than in controls (16.9‰±9.3 versus 11.1‰±6.0, P=0.001). The frequency of centromeric micronuclei was higher in exposed subjects than in controls (17.3‰±11.5 versus 10.3‰±7.1) but the difference was not significant. The frequency of monocentromeric micronuclei was significantly higher in exposed subjects than in controls (11.0‰±6.2 versus 3.1‰±2.4, P<0.001), while that of the acentromeric micronuclei was similar in exposed subjects and controls (3.7‰±4.2 and 4.1‰±2.7, respectively). The enhanced chromosomal damage (particularly chromosome loss) in peripheral lymphocytes of pathologists/anatomists emphasizes the need to develop safety programs.

A combined analysis of XRCC1, XRCC3, GSTM1 and GSTT1 polymorphisms and centromere content of micronuclei in welders.

The aims of the present study were to assess clastogenic and aneugenic properties of welding fumes using fluorescent in situ hybridization (FISH) with a human pancentromeric DNA probe. The involvement of genetic polymorphisms in DNA repair genes (p.Arg399Gln of XRCC1 and p.Thr241Met of XRCC3) and in detoxification genes (GSTM1 and GSTT1) on the centromere content of micronuclei (MN) was also evaluated. This study included 27 male welders working without any collective protection device and a control group (n = 30). The welders showed significantly higher levels of chromosome/genome damage compared to the controls. The frequencies of MN and centromere-positive MN (C+MN) per 1,000 binucleated cells were significantly higher in the exposed group than in the control group (7.1 per thousand +/- 3.7 versus 4.9 per thousand +/- 1.8; P = 0.012 and 3.5 per thousand +/- 1.8 versus 2.4 per thousand +/- 1.2; P = 0.018, respectively, Mann-Whitney U-test). The centromere-negative MN (C-MN) frequency was higher in the exposed subjects than in the controls (3.6 per thousand +/- 3.4 versus 2.5 per thousand +/- 1.4), but the Mann-Whitney U-test did not yield a significant result. In the total population, the GSTM1 and GSTT1 polymorphisms significantly affected the frequencies of C-MN and C+MN defined by FISH. GSTM1 positive subjects showed an increased C-MN frequency and GSTT1 null subjects showed an elevated C+MN frequency. When GSTM1 and GSTT1 genotypes were included in multiple regression analysis, the effect of the occupational exposure could better be demonstrated; both C+MN and C-MN were significantly increased in the welders. Our results suggest that the combined analysis of genetic polymorphisms and centromeres in MN may improve the sensitivity of the micronucleus assay in detecting genotoxic effects.

Risk assessment of welders using analysis of eight metals by ICP-MS in blood and urine and DNA damage evaluation by the comet and micronucleus assays; influence of XRCC1 and XRCC3 polymorphisms.

The aims of the present study were to assess the occupational risk of welders using analysis of metals in biological fluids, DNA damage evaluation by complementary genotoxic endpoints and the incidence of polymorphisms in DNA repair genes. A biomonitoring study was conducted that included biometrology (blood and urinary concentrations of aluminium, cadmium, chromium, cobalt, lead, manganese, nickel, zinc by ICP-MS), comet and cytokinesis-block micronucleus assays in peripheral lymphocytes and genetic polymorphisms of XRCC1 (p.Arg399Gln) and XRCC3 (p.Thr241Met). This study included 60 male welders divided into two groups: group 1 working without any collective protection device and group 2 equipped with smoke extraction systems. A control group (n = 30) was also included in the study. Higher chromium, lead and nickel blood and urinary concentrations were detected in the two groups of welders compared to controls. Statistically differences between welders of group 1 and group 2 were found for blood concentration of cobalt and urinary concentrations of aluminium, chromium, lead and nickel. The alkaline comet assay revealed that welders had a significant increase of OTMchi2 distribution at the end of a work week compared to the beginning; a significant induction of DNA strand breaks at the end of the week was observed in 20 welders out of 30. The cytokinesis-block micronucleus assay showed that welders of group 1 had a higher frequency of chromosomal damage than controls. The XRCC1 variant allele coding Gln amino acid at position 399 was found to be associated with a higher number of DNA breaks as revealed by the comet assay. Increased metal concentrations in biological fluids, DNA breaks and chromosomal damage in lymphocytes emphasized the need to develop safety programmes for welders.

Cytogenetic monitoring of industrial radiographers using the micronucleus assay.

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years+/-11.2, in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv+/-49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls (10.7 per thousand +/-5.2 versus 6.6 per thousand +/-3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C-MN) in exposed subjects than in controls (8.5 per thousand +/-4.9 versus 2.2 per thousand +/-1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C-MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.

Acentromeric micronuclei are increased in peripheral blood lymphocytes of untreated cancer patients.

Increased micronucleated cell rates, dicentric chromosomes, and other chromosomal damages have been reported in lymphocytes of cancer patients prior to the initiation of chemotherapy, and/or radiotherapy. The cause of these chromosomal damages in these lymphocytes remains unclear. In the present work, we investigated whether these micronuclei mainly reflect structural or numerical chromosomal aberrations by applying the cytokinesis-blocked micronucleus (CBMN) assay in combination with fluorescent in situ hybridization (FISH) of a DNA centromeric probe on blood samples of 10 untreated cancer patients (UCPs), and 10 healthy subjects (HSs). Micronucleated binucleated lymphocyte rate was significantly increased in patients (mean+/-S.D.: 19.0 per thousand +/-14.1 versus 9.2 per thousand +/-4.6 in controls). Trinucleated cytokinesis-blocked cells were not significantly higher in patients than in controls. Acentromeric, centromeric, and multicentromeric micronucleus levels were two-fold higher in patients than in controls, but the difference was significant only with acentromeric micronuclei. The percentage of micronuclei containing one or more centromeres averaged 69.2, and 71.5% in patients, and controls, respectively. The percentage of micronuclei containing several centromeres was 44.7% in patients, and 54.6% in controls. Among centromere-positive micronuclei, the percentage of micronuclei containing several centromeres averaged 59.7% in patients, and 75.4% in controls. These results indicate that genetic instability in peripheral blood lymphocytes of UCPs occurs because of enhanced chromosome breakage. However, a substantial proportion of this genetic instability occurs because of defects in chromosome segregation.

[In vitro study of the antimutagenic activity of alphahederin]. / Etude in vitro de l'activité antimutagène de l'alphahédérine.

Authors have studied the antimutagenic power of alphahederin (a saponin extracted from Hedera helix) versus a clastogenic agent, doxorubicin and an aneugenic agent, carbendazim. We have applied a protocol of incorporation of alphahederin (pretreatment, simultaneous treatment and post treatment) to determine a mechanism of action. According to this protocol, alphahederin induces a significant diminution of the rate of micronuclei wathever the phases of the protocol. These results demonstrate the antimutagenic activity of alphahederin with a mechanism of action, both desmutagenic and bioantimutagenic.

Evaluation of micronucleated lymphocytes, constitutional karyotypes and anti-p53 antibodies in 21 children with various malignancies.

The implication of environmental carcinogens in childhood cancer is still unknown. To assess a possible link between DNA damage and alterations of the tumor suppressor gene p53, blood samples of 21 children with malignancies were examined for the presence of micronuclei in lymphocytes using the cytokinesis blocked micronucleus assay (CBMA). The constitutional karyotypes were analyzed for chromosome abnormalities and the presence of anti-p53 antibodies in blood sera was evaluated by an enzyme-linked immuno sorbent assay (ELISA). A control group of 20 children was also included. The rates of micronucleated cells were 5.1 per thousand+/-3.9 and 2.4 per thousand+/-2.3 for the cancer and control groups, respectively. The difference between the groups were statistically significant (P<0.05 by the Mann-Withney rank sum test). Two children in the cancer group showed extensive chromosome breakage in lymphocytes. The sera of two other children from the cancer group and of one child from the control group contained anti-p53 antibodies. Chromosome breakage and anti-p53 antibodies from the five children were associated with increased micronucleated cell rates. The results of the present study suggest that genotoxic events can occur in the lymphocytes of children with a cancerous state.

Prevalence of self-reported respiratory symptoms in workers exposed to isocyanates.

Until now, no survey had been conducted to assess the prevalence of respiratory symptoms in a large population that had been occupationally exposed to isocyanates, compared with that in a control group. We performed such a survey, using questionnaires administered by occupational physicians. Overall, 1114 workers' questionnaires (585 exposed and 529 control) were analyzed. Exposed workers, primarily painters from small factories, reported significantly (P < 0.05) more wheezing (8.6% vs 3.6%), more breathlessness with wheezing (3.4% vs 0.6%) in the last year, and more rhinitis (33.1% vs 19.1%) than did control workers. A trend for more asthma (2.1% vs 0.8%; P < or = 0.07) was also observed. Furthermore, 16.4%, 16.2%, and 10.6% of exposed workers reported (respectively) cough, rhinitis, and chest tightness when working in contact with isocyanates. We conclude that isocyanate-exposed workers demonstrate significantly higher prevalence rates of rhinitic and asthmatic symptoms than do control subjects.