Transmission of oocyte DNA damage to preimplantation embryos after in vivo mouse exposure to daunorubicin and cytarabine.

International audienceStudy question: Does oocyte DNA damage induced by a previous in vivo mouse exposure to chemotherapy agents is transmissible to preimplantation embryos?Summary answer: DNA damage was observed in preimplantation embryos issued from mice previously exposed to daunorubicin and cytarabine.What is known already: In acute leukemia, the emergency to start a chemotherapydon’t allow a fertility preservation at the time of diagnosis. Some authors have proposed to cryopreserve mature oocytes or embryos after a controlled ovarian stimulation applied shortly after the induction chemotherapy,which is mainly composed by daunorubicin and cytarabine, and reputated to be less gonadotoxic than alkylant agents. We previously observed DNA damage on mouse oocytes issued from antral follicles exposed in vivo to daunorubicin and cytarabine.Little is known about the risk of transmission of oocyte DNA damage to preimplantation embryos after fecundation of oocytes recently exposed to chemotherapy.Study design, size, duration: By three time, two groups of mice (n = 11) were exposed for four days to cytarabine (10 mg/kg IP) or every two days to daunorubicin (1 mg/kg IV). Each group was compared with a negative control group (n = 11) and with a positive control group (n = 11) injected with cyclophosphamide(75 mg/kg IP). Females were mated one week after exposure and preimplantation embryos were collected by flushing the oviducts.Participants/materials, setting, methods: 4 weeks female CD1 mice were mated one week after exposure for studying embryos conceived from oocytes exposed to chemotherapy at late pre-antral stage of follicular development.Cytotoxicity has been assessed by ovulation and fertilization rates and by embryo morphology. DNA embryonic damage was assessed by: (i) alkaline comet assay to quantify the tail DNA (ii) fluorescent immunohistochemical staining in blastomeres to quantify accumulating γH2AX foci.Main results and the role of chance: In mouse, a recent exposure to daunorubicin and cytarabine did not alter the ovarian response to controlled ovarian stimulation with no adverse impact on the fertilization rate and the number of embryo conceived. Ovulation and fertilization rates in mice previouslyexposed to daunorubicin and cytarabine were similar to those in our negative control group. One week after exposure, we observed with the comet assay a significant increase of embryonic DNA damage after exposure to daunorubicin (16.57 ± 1.3, p = 0.0003) and cytarabine (16.46 ± 1.4, p =0.0003) Vs 26.16 ± 2.5 after cyclophosphamide exposure (p < 0.0001) and 7,01 ± 1,1 in negative control group exposed to an injection of sterile saline solution. The analysis γ-H2AX on embryos showed a significant increase of foci corresponding to DNA double-strand breaks, after exposure to daunorubicin (7.97 ± 1.1; p = 0.001), cytarabine (6.47 ± 0.7, p = 0.0039), cyclophosphamide (5.92 ± 0.9; p = 0.0148) compared with negative control group (2,8 ±0,7).Limitations, reasons for caution: Mouse oocyte DNA is not exactly similar to human oocyte DNA, and would be more sensitive to genotoxic effects of chemotherapy agents. After chemotherapy, the kinetic of DNA repair before and after fertilization has to be studied by further assays in exposed oocyte andin embryos.Wider implications of the findings: DNA damage in preimplantation embryos conceived from oocytes exposed to chemotherapy at late pre-antral stage of follicular development lead us to hypothese a transmission of oocyte DNA damage to preimplantation embryo. In acute leukemia, we strongly adviseto not cryopreserve mature oocytes or embryo early after induction chemotherapy.Trial registration number: Experimental protocols and animal handling procedures were reviewed by the French National Ethics Committee on Animal Experimentation (N° 2017033010523688)

Genotoxicity of metal oxide nanomaterials: review of recent data and discussion of possible mechanisms

Nanotechnology has rapidly entered into human society, revolutionized many areas, including technology, medicine and cosmetics. This progress is due to the many valuable and unique properties that nanomaterials possess. In turn, these properties might become an issue of concern when considering potentially uncontrolled release to the environment. The rapid development of new nanomaterials thus raises questions about their impact on the environment and human health. This review focuses on the potential of nanomaterials to cause genotoxicity and summarizes recent genotoxicity studies on metal oxide/silica nanomaterials. Though the number of genotoxicity studies on metal oxide/silica nanomaterials is still limited, this endpoint has recently received more attention for nanomaterials, and the number of related publications has increased. An analysis of these peer reviewed publications over nearly two decades shows that the test most employed to evaluate the genotoxicity of these nanomaterials is the comet assay, followed by micronucleus, Ames and chromosome aberration tests. Based on the data studied, we concluded that in the majority of the publications analysed in this review, the metal oxide (or silica) nanoparticles of the same core chemical composition did not show different genotoxicity study calls (i.e. positive or negative) in the same test, although some results are inconsistent and need to be confirmed by additional experiments. Where the results are conflicting, it may be due to the following reasons: (1) variation in size of the nanoparticles; (2) variations in size distribution; (3) various purities of nanomaterials; (4) variation in surface areas for nanomaterials with the same average size; (5) differences in coatings; (6) differences in crystal structures of the same types of nanomaterials; (7) differences in size of aggregates in solution/media; (8) differences in assays; (9) different concentrations of nanomaterials in assay tests. Indeed, due to the observed inconsistencies in the recent literature and the lack of adherence to appropriate, standardized test methods, reliable genotoxicity assessment of nanomaterials is still challenging

Ultrastructural Interactions and Genotoxicity Assay of Cerium Dioxide Nanoparticles on Mouse Oocytes

Cerium dioxide nanoparticles (CeO2 ENPs) are on the priority list of nanomaterials requiring evaluation. We performed in vitro assays on mature mouse oocytes incubated with CeO2 ENPs to study (1) physicochemical biotransformation of ENPs in culture medium; (2) ultrastructural interactions with follicular cells and oocytes using Transmission Electron Microscopy (TEM); (3) genotoxicity of CeO2 ENPs on follicular cells and oocytes using a comet assay. DNA damage was quantified as Olive Tail Moment. We show that ENPs aggregated, but their crystal structure remained stable in culture medium. TEM showed endocytosis of CeO2 ENP aggregates in follicular cells. In oocytes, CeO2 ENP aggregates were only observed around the zona pellucida (ZP). The comet assay revealed significant DNA damage in follicular cells. In oocytes, the comet assay showed a dose-related increase in DNA damage and a significant increase only at the highest concentrations. DNA damage decreased significantly both in follicular cells and in oocytes when an anti-oxidant agent was added in the culture medium. We hypothesise that at low concentrations of CeO2 ENPs oocytes could be protected against indirect oxidative stress due to a double defence system composed of follicular cells and ZP