In Vitro Co-Exposure to CeO2 Nanomaterials from Diesel Engine Exhaust and Benzo(a)Pyrene Induces Additive DNA Damage in Sperm and Cumulus Cells but Not in Oocytes

Abstract

Benzo(a)pyrene (BaP) is a recognized reprotoxic compound and the most widely investigated polycyclic aromatic hydrocarbon in ambient air; it is widespread by the incomplete combustion of fossil fuels along with cerium dioxide nanomaterials (CeO₂ NMs), which are used in nano-based diesel additives to decrease the emission of toxic compounds and to increase fuel economy. The toxicity of CeO₂ NMs on reproductive organs and cells has also been shown. However, the effect of the combined interactions of BaP and CeO₂ NMs on reproduction has not been investigated. Herein, human and rat gametes were exposed in vitro to combusted CeO₂ NMs or BaP or CeO₂ NMs and BaP in combination. CeO₂ NMs were burned at 850 °C prior to mimicking their release after combustion in a diesel engine. We demonstrated significantly higher amounts of DNA damage after exposure to combusted CeO₂ NMs (1 µg·L^-1) or BaP (1.13 µmol·L^-1) in all cell types considered compared to unexposed cells. Co-exposure to the CeO₂ NMs-BaP mixture induced additive DNA damage in sperm and cumulus cells, whereas no additive effect was observed in rat oocytes. This result could be related to the structural protection of the oocyte by cumulus cells and to the oocyte's efficient system to repair DNA damage compared to that of cumulus and sperm cells.

Keywords: additivity; cocktail; genotoxicity; germ cells; nanomaterials; polycyclic aromatic hydrocarbons.

Figure 1

Evaluation of DNA damage using the comet assay following in vitro exposure of human (a) and rat sperm (b) to NMs+BaP. Tested concentrations: Negative control = Figure 1. S9 mix, 1% DMSO), NMs: aged CeO₂ NMs at 1 µg·L⁻¹; BaP: BaP at 1.13 µmol·L⁻¹; NMs+BaP: aged CeO₂ NMs at 1 µg·L⁻¹ previously incubated with 1.13 µmol·L⁻¹ BaP. p < 0.05, for differences compared versus *: negative control (NEG); §: vs. NMs, £: vs. BaP.

Figure 2

Evaluation of DNA damage using the comet assay following in vitro exposure of rat cumulus cells (a) and oocytes (b) to NMs+BaP. Tested concentrations: negative control = Ferticult^® medium, IC = intern control (Ferticult^® 1% S9 mix, 1% DMSO), NMs: aged CeO₂ NMs at 1 µg·L⁻¹; BaP: BaP at 1.13 µmol·L⁻¹; NMs+BaP: aged CeO₂ NMs at 1 µg·L⁻¹ previously incubated with 1.13 µmol·L⁻¹ BaP. p < 0.05, for differences compared versus *: negative control (NEG); §: vs. NMs, £: vs. BaP.