title: The effects of zinc nanooxide on cellular stress responses of the freshwater mussels Unio tumidus are modulated by elevated temperature and organic pollutants.
abstract: Nanoparticle toxicity is a growing concern in freshwater habitats. However, understanding of the nanoparticle effects on aquatic organisms is impeded by the lack of the studies of the nanoparticles effects in the environmentally relevant context of multiple stress exposures. Zinc oxide nanoparticles (n-ZnO) are widely used metal-based nanoparticles in electronics and personal care products that accumulate in aquatic environments from multiple non-point sources. In this study, we evaluated the effects of n-ZnO in a model organism, a mussel Unio tumidus, and the potential modulation of these effects by common co-occurring environmental stressors. Male U. tumidus were exposed for 14 days to n-ZnO (3.1 μM), Zn(2+) (3.1 μM), Ca-channel blocker nifedipine (Nfd 10 μM), combinations of n-ZnO and Nfd or n-ZnO and thiocarbamate fungicide Tattoo (Ta, 91 μg L(-1)) at 18 °C, and n-ZnO at 25 °C (n-ZnO+t°). Total and metallothionein-bound Zn levels as well as levels of metallothioneins (MT), cellular stress responses and cytotoxicity biomarkers were assessed in the mussels. The key biomarkers that showed differential responses to different single and combined stressors in this study were activities of caspase-3 and lysosomal cathepsin D, as well as protein carbonyl content. At 18 °C, exposures to n-ZnO, organic pollutants and their combinations led to a prominent up-regulation of MT levels (by ∼30%) and oxidative stress response including up-regulation of superoxide dismutase activity, an increase in oxyradical production, and a 2-3-fold decrease in the levels of protein carbonyls in all exposures except nZnO+Ta. Expos ure to n-ZnO in the absence of other stressors also led to a strong (∼7-fold) elevation of cathepsin D activity. Cellular responses to Zn(2+) and n-ZnO were different indicating that n-ZnO was not due exclusively to Zn release. Ca-channel blocker Nfd affected intracellular Zn distribution (reflected in the prominent elevation of Zn-MT levels) and caused reductive stress indicated by elevated levels of reduced glutathione levels and an increase in lactate/pyruvate ratio (reflecting higher NADH/NAD ratio). Elevated temperature (25 °C) abolished most of the typical responses to n-ZnO and induced oxidative injury, DNA fragmentation and caspase-3 mediated apoptosis in n-ZnO-exposed mussels. DNA fragmentation was also induced by exposure to organic toxins (alone and in combination with n-ZnO) but not by n-ZnO alone. These data indicate that n-ZnO toxicity to freshwater organisms is modulated by organic pollutants and enhanced by elevated temperatures.
authors.affiliation: Research Laboratory of Comparative Biochemistry and Molecular Biology, Ternopil National Pedagogical University, 46027, Kryvonosa Str. 2, Ternopil, Ukraine.
authors.forename: Halina
authors.initials: H
authors.lastname: Falfushynska
authors.affiliation: Research Laboratory of Comparative Biochemistry and Molecular Biology, Ternopil National Pedagogical University, 46027, Kryvonosa Str. 2, Ternopil, Ukraine.
authors.forename: Lesya
authors.initials: L
authors.lastname: Gnatyshyna
authors.affiliation: Research Laboratory of Comparative Biochemistry and Molecular Biology, Ternopil National Pedagogical University, 46027, Kryvonosa Str. 2, Ternopil, Ukraine.
authors.forename: Irina
authors.initials: I
authors.lastname: Yurchak
authors.affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, USA. Electronic address: isokolov@uncc.edu.
authors.forename: Inna
authors.initials: I
authors.lastname: Sokolova
authors.affiliation: Research Laboratory of Comparative Biochemistry and Molecular Biology, Ternopil National Pedagogical University, 46027, Kryvonosa Str. 2, Ternopil, Ukraine.
authors.forename: Oksana
authors.initials: O
authors.lastname: Stoliar
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000633:toxicity*; D005618:Fresh Water; D006358:Hot Temperature / Q000009:adverse effects*; D008297:Male; D008668:Metallothionein / Q000378:metabolism; D053758:Nanoparticles / Q000633:toxicity*; D009543:Nifedipine / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D049881:Unio / Q000187:drug effects* / Q000378:metabolism; D015854:Up-Regulation; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D015034:Zinc Oxide / Q000633:toxicity*
chemical_list: D015415:Biomarkers; D002121:Calcium Channel Blockers; D014874:Water Pollutants, Chemical; C021267:zinc thionein; D008668:Metallothionein; D009543:Nifedipine; D015034:Zinc Oxide
keywords: Apoptosis; Metallothionein; Nifedipine; Stressor interactions; Thiocarbamate; Zinc nanooxide