A. Botta

Assessment of the genotoxicity of three cryoprotectants used for human oocyte vitrification: Dimethyl sulfoxide, ethylene glycol and propylene glycol

Vitrification requires high concentrations of cryoprotectants that may induce long-term toxic effects on cells. The aim of this study was to evaluate the possible genotoxicity of three cryoprotectants extensively used for oocyte vitrification: dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PROH). For this purpose, a Chinese Hamster Ovary cell line (CHO), commonly used in genetic toxicology, was selected as an in vitro biological model to assess both the induction of DNA strand-breaks as identifiable by the alkaline comet assay and the persistence of chromosomal damages (micronuclei) as analyzed by the micronucleus assay. Results showed that DMSO was not genotoxic. EG did not exert direct genotoxic activity, however EG exhibited significant genotoxic and clastogenic activities in the presence of an external cytochrome-based P450 oxidation system (S9 Mix). PrOH produced in vitro DNA-damage leading to chromosome mutations in the presence and absence of the S9 Mix. These results showed that high concentrations of EG and PrOH could induce in vitro chromosomal damage in eukaryotic cells.

Evaluation of the genotoxic activity of paclitaxel by the in vitro micronucleus test in combination with fluorescent in situ hybridization of a DNA centromeric probe and the alkaline single cell gel electrophoresis technique (comet assay) in human T‐lymphocytes

Paclitaxel is a recent chemotherapeutic agent that inhibits tubulin depolymerization in tumoral cells. Despite its increasing use against various human cancers, the genotoxicity of paclitaxel has never been studied on normal human cells. The in vitro genotoxic effects of the drug were evaluated with two complementary mutagenesis tests on human T‐lymphocytes: (1) the cytokinesis‐blocked micronuclei assay (CBMN) in combination with fluorescent in situ hybridization (FISH) of nonspecific centromeric probes and (2) the comet assay performed in three ways: on stimulated lymphocytes as in the CBMN, and on freshly isolated lymphocytes at both 4 and 37°C. A slight cytotoxicity of 2.5 to 10 nM paclitaxel was found in the CBMN and a significant increase in the binucleated micronucleated cell rates was observed, with a concentration‐dependent manner. In the FISH analysis, more than 85% of the micronuclei (MN) were centromere positive, and a ratio of 72.2 to 78.6% of these MN contained more than one centromere. Moreover, at 10 nM of paclitaxel, 35.6% of the cells are multimicronucleated lymphocytes. Unexpectedly, paclitaxel induced single‐strand breaks on proliferating lymphocytes at 5 and 7.5 nM but not in resting cells, even at 5 to 15 μM. These in vitro results showed that (1) paclitaxel does not present any direct DNA action in resting cells, (2) DNA damage detected in stimulated lymphocytes may be linked either to a high frequency of cells in the S‐phase cell cycle or to a direct DNA damaging effect on replicating cells, and (3) paclitaxel is a strong in vitro aneugenic drug on human normal cells, at clinically relevant concentrations. Environ. Mol. Mutagen. 34:269–278, 1999

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.

Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells.

OBJECTIVE To develop and validate an efficient comet assay on mouse oocytes without depellucidation. DESIGN In vitro experiments using a murine model. SETTING Biogenotoxicology research laboratory in Aix-Marseille II University, France. ANIMAL(S) CD1 prepubescent female mice. INTERVENTION(S) DNA lesions in oocytes were evaluated by the alkaline comet assay. After oocyte retrieval, we first studied the effect of zona pellucida (ZP) on comet morphology. For this study, we applied the comet assay to mature oocytes with and without ZP after exposure to simulated sunlight irradiation (SSI) compared with negative controls. Next, nondepellucidated mouse oocytes were exposed to three well-known genotoxic agents (SSI, methylmethanesulfonate [MMS], and hydrogen peroxide [H(2)O(2)]) and compared with negative controls. Images of oocytes were analyzed with Komet software. MAIN OUTCOME MEASURE(S) DNA damages were quantified and expressed as olive tail moment (OTM), defined as the product of the tail length and the fraction of total DNA in the tail. OTMχ(2) were calculated from OTM; they corresponded to the degrees of freedom (n) of each OTM distribution obtained from at least 50 oocytes. OTMχ(2) is an indicator of DNA lesions. The test was considered positive and statistically significant when OTMχ(2) increased in oocytes compared with the medium-only control cells. RESULT(S) There was no difference in comet aspect between oocyte groups with and without ZP. The three genotoxic agents significantly increased DNA damages as compared with the control groups. The OTMχ(2) values were (mean ± SD): 2.1 ± 0.07, 7.73 ± 0.35, 3.35 ± 0.15, and 12.4 ± 0.51 in control, SSI, MMS, and H(2)O(2) groups, respectively. CONCLUSION(S) Comet assay on non depellucidated mouse oocytes is a rapid and easy test. This assay would be useful to assess the genotoxicity on female germ cells of chemicals, drugs, or environmental pollutants and the efficiency of antioxidant molecules.

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.

In smokers, swim-up and discontinuous gradient centrifugation recover spermatozoa with equally lower amounts of DNA damage than spermatozoa obtained from neat semen

We compared the abilities of two commonly used semen preparation techniques to decrease the amount of benzo(a)pyrene-diol-epoxide (BPDE)-DNA adducts in the spermatozoa of ten smokers. Semen processing by swim-up or discontinuous gradient centrifugation recovered spermatozoa showing an equally significantly lower amount of BPDE-DNA adducts than in unprepared spermatozoa from neat semen.

Risque génotoxique et exposition au formaldéhyde en laboratoire d'anatomo-pathologie : métrologie atmosphérique et biogénotoxicologie

Resume Objectif Le but de l’etude est d’evaluer les effets genotoxiques associes a l’exposition au formaldehyde aupres de sujets travaillant dans des laboratoires hospitaliers d’anatomo-pathologie. Methodes L’exposition au formaldehyde a ete mesuree au moyen de badges de prelevements passifs portes a proximite des voies respiratoires de 59 travailleurs pendant 15 minutes et 8 heures. L’evaluation des mutations chromosomiques a ete realisee a l’aide du test des micronoyaux avec blocage de la cytodierese au niveau des lymphocytes de 59 exposes et de 37 temoins apparies pour l’âge, le sexe et le tabagisme. Le test des micronoyaux a ete associe a l’hybridation in situ fluorescente d’une sonde pancentromerique chez 18 exposes et 18 temoins tires au sort dans les populations initiales. Resultats et discussion Les concentrations moyennes en formaldehyde etaient de l’ordre de 2,0 ppm (0,1 a 20,1 ppm) et de 0,1 ppm (0,1 a 0,7 ppm) pour des temps de prelevements respectivement de 15 minutes et de 8 heures. Les taux de lymphocytes binuclees micronuclees etaient plus eleves chez les sujets exposes que chez les temoins (16,9 ‰ ± 9,3 versus 11,1 ‰ ± 6,0 ; p = 0.001). Les taux de micronoyaux centriques etaient plus eleves chez les sujets exposes que chez les temoins (17,3 ‰ ± 11,5 versus 10,3 ‰ ± 7,1) mais la difference n’etait pas significative. Les taux de micronoyaux acentriques etaient similaires entre les deux populations exposee et temoin. Conclusion L’elevation des dommages chromosomiques, lies principalement a la perte de chromosomes entiers, dans les lymphocytes des sujets travaillant dans des laboratoires d’anatomo-pathologie souligne la necessite de mettre en œuvre des programmes de prevention appropries.