The early pronucleus stage of the mouse zygote has been characterised in vitro as radiosensitive, due to a high rate of induction of chromosome-type chromosome abnormalities (CA). We have investigated the repair of irradiation induced double strand DNA breaks in vivo by γH2AX foci and first cleavage metaphase analysis. Breaks were induced in sperm and in the early zygote stages comprising sperm chromatin remodelling and early pronucleus expansion. Moreover, the role of PARP1 in the formation and repair of spontaneous and radiation-induced double strand breaks in the zygote was evaluated by comparing observations in C57BL/6J and PARP1 genetically ablated females. The results confirmed in vivo that the rate of chromosome aberration induction by X-rays was approximately 3-fold higher in the zygote than in mouse lymphocytes. This finding was related to a diminished efficiency of double strand break signalling, as shown by a lower rate of γH2AX radiation-induced foci compared to that measured in most other somatic cell types. The spontaneous frequency of CA in PARP1 depleted zygotes was slightly but significantly higher than in wild type zygotes. Also, these zygotes showed some impairment of the radiation-induced DNA Damage Response when exposed closer to the start of S-phase, revealed by a higher number of γH2AX foci and a longer cell cycle delay. The rate of chromosome aberrations, however, was not elevated over that of wild type zygotes, possibly thanks to backup repair pathways and/or selection mechanisms against damaged cells. When comparing with the literature data on irradiation induced CA in mouse zygotes in vitro, the levels of induction were strikingly similar as was the frequency of misrepair of double strand breaks (γH2AX foci). This result can be reassuring for in vitro human gamete and embryo handling, because it shows that culture conditions do not significantly affect double strand DNA break repair.

Bisphenol A (BPA) is a synthetic monomer widely used to polymerize polycarbonate plastics and resins. It is shown in vitro to interfere with microtubules, producing aberations in mitotic and meiotic spindles. An increase of meiotic abnormalities in untreated female mice from an experimental colony was temporally correlated with the accidental release of BPA from polycarbonate cages and bottles damaged by inadvertent treatment with harsh alkaline detergents [P.A. Hunt, K.E. Koehler, M. Susiarjo, C.A. Hodges, A. Ilagan, R.C. Voigt, S. Thomas, B.F. Thomas, T.J. Hassold, Bisphenol A exposure causes meiotic aneuploidy in the female mouse, Curr. Biol. 13 (2003) 546-553].In the present study, potential aneugenic effects of BPA on mouse male and female germ cells and bone marrow cells have been evaluated after acute, sub-chronic or chronic in vivo exposure. Female mice were orally treated with a single BPA dose, with 7 daily administrations or exposed for 7 weeks to BPA in drinking water. No significant induction of hyperploidy or polyploidy was observed in oocytes and zygotes at any treatment condition. The only detectable effect was a significant increase of metaphase 11 oocytes with prematurely separated chromatids after chronic exposure; this effect, however, had no irreversible consequence upon the fidelity of chromosome segregation during the second meiotic division, as demonstrated by the normal chromosome constitution of zygotes under the same exposure condition.With male mice, no delay of meiotic divisions was found after six daily oral doses of BPA with the BrdU assay. Similarly, no induction of hyperploidy and polyploidy was shown in epydidimal sperm hybrized with probes for chromosomes 8, X and Y, 22 days after six daily oral BPA doses. Finally, two daily oral BPA doses did not induce any increase of micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow. In conclusion, our results do not add evidence to the suspected aneugenic activity of BPA and suggest that other factors or co-factors should be considered to explain the unexpected burst of meiotic abnormalities previously attributed to accidental BPA exposure. (c) 2007 Elsevier B.V. All rights reserved.

Aneuploidy tests are important in evaluating genetic hazards especially when chemical exposures are suspected to affect the fidelity of chromosome segregation in oocytes and embryos. In the current study, a newly established method, mouse preantral follicle culture, was employed to grow oocytes in vitro within follicles. The sensitivity of in vitro grown follicle enclosed oocytes was compared with oocytes maturing in vivo in the ovary. In both the cases, oocytes were exposed to the cytostatic chemical, nocodazole, from the time of hormonally stimulated resumption of meiosis. The in vivo study revealed a significant decrease in the number of ovulated mouse oocytes and an increase in meiosis I-arrested and hyperploid metaphase II oocytes at a single i.p. dose of 70 mg/kg body weight of nocodazole. A significant increase was also observed in the number of meiosis I-arrested and hyperploid mouse oocytes from preantral follicle culture, when they were cultured in the presence of >or=30 nM nocodazole during the final stages of maturation. This concentration is slightly lower than that previously shown to induce nondisjunction in denuded mouse oocytes or in cultured human lymphocytes. The higher sensitivity of the in vitro matured oocytes from preantral follicle culture than that of denuded oocytes may be related to a synergistic adverse influence of nocodazole on the oocyte, on somatic cell integrity and on cell-cell communication, which possibly also affects ovulation in vivo. When expressed in molarity relative to the mouse weight, the effective dose of the acute exposure in vivo is 3-4 orders of magnitude higher than the lowest effective concentration employed continuously in vitro. Reduced bioavailability of nocodazole to the target cells due to its poor water solubility may contribute to this difference. Preantral follicle culture can be helpful in analysing mechanisms in chemically induced aneuploidy in mammalian oogenesis, and in predicting the consequences of chemical exposures in vivo.

The genotoxic effects associated with automobile painting were analyzed using a panel of biomarkers. Chromosomal aberrations (CAs), sister chromatid exchange (SCE), and micronuclei were evaluated in 25 car painters (12 smokers, 13 nonsmokers) working in different automobile paint‐shops in Italy and in 37 control subjects. The controls were healthy blood donors (14 smokers, 23 non‐smokers) that were matched with the experimental population for gender and age. Air samples were analyzed regularly at the work places, and elevated concentrations of benzene and toluene were detected consistently. The exposed group had higher frequencies of CAs (both chromosome‐ and chromatid‐type), micronuclei, and SCE (P < 0.5 – P < 0.001). Furthermore, exposed and control subjects were also genotyped for GSTM1 and GSTT1 polymorphism. No significant associations were detected between the biomarker responses and either the GSTM1 or GSTT1 genotype of the subjects, but the small sample size does not allow definite conclusions on the relationship between the genetic polymorphism and the biomarkers. The results indicate that automobile painters have increased levels of clastogenic and possible aneugenic damage and that smoking may be a confounding factor for the responses. Environ. Mol. Mutagen., 2005. © 2005 Wiley‐Liss, Inc.

Melphalan is an alkylating substance used as a therapeutic agent; its mutagenicity is related to its ability to produce monoadducts and to form DNA cross-links. The alkaline comet assay is a useful test for the detection of DNA lesions. However, cross-links are not easily detected under standard conditions. Recently, modifications to the test have been introduced to measure cross-links by evaluating the reduction in induced DNA migration.In this work, the standard comet assay and an assay modified by prolonging the electrophoresis time have been applied to evaluate DNA lesions induced by single, 4 or 26 weekly oral administrations of melphalan to p53+/− knockout and to isotype parental mice. Cells were analysed from the liver, bone marrow, peripheral blood and the distal intestine. Moreover, a further protocol in which the presence of cross-links was inferred by the reduction in X-ray-induced DNA migration was applied to bone marrow cells and the sensitivity of the different methods was compared.The majority of groups examined by the standard protocol showed no difference compared to controls, while the modified protocol (prolonged electrophoresis time) could detect a retarded DNA migration in cells from all the organs analysed with the exception of bone marrow cells.Only the protocol based on X-ray in vitro irradiation showed the presence of melphalan-induced cross-links in bone marrow cells exposed to 2mg/kg for 4 weeks, demonstrating that this was the most sensitive approach for detecting this type of lesion.DNA lesions were evident in all the organs analysed. However, results suggest that the kinetics of cross-link repair could be different in bone marrow cells compared to other organs tested. After comparison between genotype-matched treated and control groups, a significant effect was shown more frequently in p53+/− than in wild type groups.

A cytogenetic monitoring study was carried out on a group of workers in clinical analysis laboratories to investigate the risk of occupational exposure to chronic low levels of chemicals.Thirty-four clinical laboratories have been involved in the study. In these laboratories, toxicants and analytical procedures utilized have been characterized. The individual occupational exposure of workers was assessed by use of a questionnaire concerning the chemical substances utilized. About 300 different chemicals have been identified. Cytogenetic analyses (chromosomal aberration and micronucleus tests) were carried out on a strictly selected group of 50 workers enrolled from these laboratories and compared to 53 controls (healthy blood donors) matched for gender and age. The exposed group shows a significantly higher frequency of genetic damage than the control group. Both chromatid and chromosome aberration frequencies in workers appear significantly higher than in controls. Similarly, comparison between micronucleated cells rates of exposed and unexposed groups show significantly higher frequencies of binucleated cells with micronucleus (BNMN) and of total micronuclei (MN tot) in workers than in controls.

The Semipalatinsk region (Kazakhstan Republic) has been affected by extensive radioactive contamination due to more than 450 nuclear tests of which almost 100 were exploded in the atmosphere. The present results refer to cytogenetic assessments in a study cohort of the population of Dolon, a settlement located on the NE boundary of the nuclear weapon test site, which was exposed to elevated doses of ionising radiation primarily due to the first Soviet nuclear test in 1949. Conventional cytogenetic analyses were carried out on 21 blood samples from individuals (more than 50 years old) living in Dolon since the very beginning of nuclear testing. A matched control group included 20 individuals living in non-contaminated areas. Higher frequencies of chromosome aberrations were found in the Dolon cohort compared to the control group, even though they remain within the range of the background levels reported for large normal human population studies on elderly individuals.

Lung fibroblasts from BD-exposed mice have been analysed for the occurrence of micronuclei. Primary cultures set up 24h after the end of exposure were treated with cytochalasin B and micronuclei scored in binucleate cells. A three-fold statistically significant increase of micronucleated cells was detected after exposure to 500ppm, the lowest tested concentration. A linear dose effect relationship was observed between 500 and 1300ppm. Immunofluorescent staining of kinetochore proteins was applied to distinguish between acentric micronuclei produced by chromosome breaks and micronuclei containing a centromeric region, most likely induced by chromosome loss. A statistically significant increase of both types of MN in 1300ppm-exposed females and a significant increase in centromeric MN in 500ppm-exposed males were detected. These data demonstrate that an intermediate of BD metabolism with a potential for clastogenic and aneugenic effects is active in lung cells after inhalation exposure. These effects can play a role in the initiation and promotion of BD-induced lung tumours.

Method description and initial results of a study to assess risks to health sector workers and environment due to chemical agents used and waste products generated in diagnostic clinical chemical laboratories, and image diagnostic testing. A survey was conducted of the methods and agents used and their toxicological classification, the number of workers exposed and an analytical profile of the waste produced. The assessment of risk to workers was based on cytogenetic tests (chromosome aberrations and micronuclei); the assessment of environmental risk from waste disposal was based on tests on plant systems.

The genotoxic effects of benzene in lung cells of mice exposed to single acute doses by inhalation have been estimated by cytogenetic analysis of micronuclei in primary cultures of lung fibroblasts. Mice were nose-only exposed to 1000 p.p.m. for 30 or 60 min or to 3500 p.p.m. for 30 min and sacrificed 24 h after the end of exposure. Lung fibroblasts were cultured attached to coverslips for 72 h, the last 48 h in the presence of 0.75 microgram/ml cytochalasin B. Micronuclei were scored in binucleate cells. The mechanism(s) of micronucleus induction was characterized by immunofluorescent staining of kinetochore proteins (CREST staining), which allowed micronuclei due to chromosome loss (kinetochore-positive) to be distinguished from those produced by chromosome breakage (kinetochore-negative). Three- and 4-fold statistically significant increases in total micronucleus frequencies were observed in all benzene-exposed mice with respect to unexposed controls. The effect was neither concentration nor time dependent. This is compatible with a plateau dose-effect relationship for the effects on bone marrow, which is explained by saturation of metabolism. Both chromosome loss and chromosome breakage appear to contribute to micronucleus formation, suggesting that in addition to chromosome rearrangements, aneuploidy may be a relevant early genotoxic event associated with benzene carcinogenicity. Under the same treatment conditions no micronucleus induction could be shown in spleen lymphocytes, suggesting that with very short benzene exposures cells at the first contact site with local metabolizing capacity have a higher probability of genetic alterations potentially leading to neoplasia.

Reproductive effects of 1,3 butadiene inhalation have been evaluated in male mice by reduction of post-meiotic germ cells, alteration of sperm chromatin structure and transmission of chromosome aberrations to one-cell embryos. Animals were exposed for 5 consecutive days for 6 h per day to butadiene concentrations of 130, 500 or 1300 ppm. The testicular fraction of post-meiotic germ cells was measured by flow cytometric analysis on the basis of their DNA content. Round spermatids were discriminated from mature, elongated spermatids by their different degree of chromatin condensation. Butadiene-induced cytotoxic effects on differentiating spermatogonia were shown by a concentration-dependent decrease of round spermatids occurring 21 days after chemical exposure, confirmed by a similar decrease of elongated spermatids measured in testes sampled 7 days later. Statistically significant effects were seen already at 130 ppm. An incomplete repopulation of the elongated spermatid compartment observed 35 days after exposure to 1300 ppm suggested that, at the highest concentration tested, butadiene toxicity extended to stem cells. Alterations of sperm chromatin were revealed by its increased sensitivity to acidic denaturation in situ. The percentage of abnormal sperm was significantly increased after butadiene exposure of differentiating spermatogonia and spermatocytes. This suggested the induction of persistent effects interfering with chromatin remodelling during spermiogenesis. Chromosome-type structural aberrations were significantly elevated in first-cleavage embryos conceived by males mated during the first and second week after the end of exposure. The lowest effective tested concentration was 500 ppm, the same reported for dominant lethal induction under identical exposure conditions. As in the dominant lethal assay, the effect of this dose was confined to exposed sperm, while both sperm and late spermatids were affected by the inhalation of 1300 ppm. A quantitative comparison between the effects induced by intraperitoneal injections of diepoxybutane or butadiene inhalations suggested that other reactive intermediates, in addition to diepoxybutane, might contribute to mediate butadiene-induced reproductive toxicity.

A study was conducted on the genotoxicity of butadiene diepoxide (DEB) in mouse oocytes. Superovulated female mice were injected intraperitoneally with DEB and mated with untreated males. Oocyte exposure occurred approximately 1.5 days before ovulation. DEB doses ranged between 26 and 52 mg/kg. Chromosome aberrations were scored in C-banded metaphases of one-cell embryos. The percentage of mated females, the average number of zygotes harvested per female, the frequencies of unfertilized oocytes and developmentally delayed zygotes did not reveal any overt sign of chemical toxicity which hindered the propensity of animals to mate or affected the ovulation, fertilization, or cell cycle progression of treated oocytes. A dose-dependent induction of chromosome aberrations was observed which was best fitted by a linear-quadratic equation. Half of all the aberrations transmitted by DEB-treated oocytes were chromatid-type breaks or exchanges. Among chromosome-type aberrations, double fragments for exceeded chromosome exchanges. This spectrum of structural aberrations differed markedly from what was previously observed in one-cell embryos conceived by DEB-treated sperm, where 97% were chromosome-type aberrations and 40% were dicentrics or translocations. This difference suggests that chromosome damage in one-cell embryos can be fixed by different mutagenic pathways influenced by the targeted gamete and its specific chromatin configuration. After exposure to the same dose, oocytes transmitted to one-cell embryos between 4 and 8 times fewer aberrations than DEB-treated sperm. While the rate of aberration induction suggests that female germ cells may be less at risk than mature sperm, especially at low-dose levels, the higher threshold for reproductive toxicity observed in female than in male mice may justify inclusion of data on female germ cell mutagenicity in the genetic risk assessment of butadiene exposure.