Stefano Magni

Environmentally relevant concentrations of galaxolide (HHCB) and tonalide (AHTN) induced oxidative and genetic damage in Dreissena polymorpha

Synthetic musk compounds (SMCs) are extensively used as fragrances in several personal care products and have been recognized as emerging aquatic pollutants. Among SMCs, galaxolide (HHCB) and tonalide (AHTN) are extensively used and have been measured in aquatic ecosystems worldwide. However, their potential risk to organisms remains largely unknown. The aim of this study was to investigate whether 21-day exposures to HHCB and AHTN concentrations frequently measured in aquatic ecosystems can induce oxidative and genetic damage in Dreissena polymorpha. The lipid peroxidation (LPO) and protein carbonyl content (PCC) were measured as oxidative stress indexes, while the DNA precipitation assay and the micronucleus test (MN test) were applied to investigate genetic injuries. HHCB induced significant increases in LPO and PCC levels, while AHTN enhanced only protein carbonylation. Moreover, significant increases in DNA strand breaks were caused by exposure to the highest concentrations of HHCB and AHTN tested in the present study, but no fixed genetic damage was observed.

Amphetamine exposure imbalanced antioxidant activity in the bivalve Dreissena polymorpha causing oxidative and genetic damage.

Illicit drugs have been recognized as emerging aquatic pollutants due to their presence in aquatic ecosystems up to µg/L level. Among these, the synthetic psycho-stimulant drug amphetamine (AMPH) is commonly found in both surface and wastewaters worldwide. Even though the environmental occurrence of AMPH is well-known, the information on its toxicity towards non-target freshwater organisms is completely lacking. This study investigated the imbalance of the oxidative status and both oxidative and genetic damage induced by a 14-day exposure to two concentrations (500 ng/L and 5000 ng/L) of AMPH on the freshwater bivalve Dreissena polymorpha by the application of a biomarker suite. We investigated the activity of antioxidant enzymes (SOD, CAT and GPx), the phase II detoxifying enzyme GST, the lipid peroxidation level (LPO) and protein carbonyl content (PCC), as well as primary (Single Cell Gel Electrophoresis assay) and fixed (DNA diffusion assay and Micronucleus test) genetic damage. Our results showed that a current realistic AMPH concentration (500 ng/L) did neither cause notable imbalances in enzymatic activities, nor oxidative and genetic damage to cellular macromolecules. In contrast, the bell-shaped trend of antioxidants showed at the highest tested concentration (5000 ng/L) suggested an overproduction of reactive oxygen species, leading to oxidative damage, as confirmed by the significant increase of protein carbonylation and DNA fragmentation.

Multi-biomarker investigation to assess toxicity induced by two antidepressants on Dreissena polymorpha

Antidepressants are one of the main pharmaceutical classes detected in the aquatic environment. Nevertheless, there is a dearth of information regarding their potential adverse effects on non-target organisms. Thus, the aim of this study was the evaluation of sub-lethal effects on the freshwater mussel Dreissena polymorpha of two antidepressants commonly found in the aquatic environment, namely Fluoxetine (FLX) and Citalopram (CT). D. polymorpha specimens were exposed to FLX and CT alone and to their mixture (MIX) at the environmental concentration of 500ng/L for 14days. We evaluated the sub-lethal effects in the mussel soft tissues by means of a biomarker suite: the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and the activity of the phase II detoxifying enzyme glutathione-S-transferase (GST). The oxidative damage was evaluated by lipid peroxidation (LPO) and protein carbonylation (PCC), while genetic damage was tested on D. polymorpha hemocytes by Single Cell Gel Electrophoresis (SCGE) assay, DNA diffusion assay and micronucleus test (MN test). Finally, the functionality of the ABC transporter P-glycoprotein (P-gp) was measured in D. polymorpha gills. Our results highlight that CT, MIX and to a lesser extent FLX, caused a significant alteration of the oxidative status of bivalves, accompanied by a significant reduction of P-gp efflux activity. However, only FLX induced a slight, but significant, increase in apoptotic and necrotic cell frequencies. Considering the variability in biomarker response and to perform a toxicity comparison of tested molecules, we integrated each endpoint into the Biomarker Response Index (BRI). The data integration showed that 500ng/L of FLX, CT and their MIX have the same toxicity on bivalves.

Sublethal effects induced by morphine to the freshwater biological model Dreissena polymorpha

Opioids are considered as emerging contaminants in aquatic ecosystems, mainly due to their large illicit consume worldwide. Morphine (MOR) is the main opiate and it was commonly found at measurable concentrations in freshwaters. Even though its occurrence is well documented, just limited information is available regarding its hazard to nontarget organisms. The aim of this study was of the evaluation of sublethal effects induced by MOR to the freshwater bivalve Dreissena polymorpha. We exposed mussels to two MOR concentrations (0.05 µg/L and 0.5 µg/L) for 14 days and we investigated the sublethal effects by a suite of biomarkers. The Neutral Red Retention Assay (NRRA) was used as a test of cytotoxicity, while the oxidative stress was evaluated by the activity of antioxidant and detoxifying enzymes, namely catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione‐S‐transferase (GST), and by measuring the levels of lipid peroxidation (LPO) and protein carbonylation (PCC). The genetic damage was assessed by the Single Cell Gel Electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test). Finally, the filtration rate of D. polymorpha was evaluated in order to investigate possible physiological effects. Both tested concentrations reduced the lysosome membrane stability of bivalves, but only the highest MOR concentration induced significant changes in the activity of antioxidant enzymes (SOD, CAT, and GPx) and increase in lipid peroxidation levels. Slight increase in primary DNA fragmentation was noticed, while no fixed genetic damage and alterations of the filtering rate were found.

Carbon nanopowder acts as a Trojan-horse for benzo(α)pyrene in Danio rerio embryos

Abstract Carbon-based nanoparticles (CBNs) are largely distributed worldwide due to fossil fuel combustion and their presence in many consumer products. In addition to their proven toxicological effects in several biological models, attention in recent years has focussed on the role played by CBNs as Trojan-horse carriers for adsorbed environmental pollutants. This role has not been conclusively determined to date because CBNs can decrease the bioavailability of contaminants or represent an additional source of intake. Herein, we evaluated the intake, transport and distribution of one of the carbon-based powders, the so-called carbon nanopowder (CNPW), and benzo(α)pyrene, when administered alone and in co-exposure to Danio rerio embryos. Data obtained by means of advanced microscopic techniques illustrated that the “particle-specific” effect induced a modification in the accumulation of benzo(α)pyrene, which is forced to follow the distribution of the physical pollutant instead of its natural bioaccumulation. The combined results from functional proteomics and gene transcription analysis highlighted the different biochemical pathways involved in the action of the two different contaminants administered alone and when bound together. In particular, we observed a clear change in several proteins involved in the homeostatic response to hypoxia only after exposure to the CNPW or co-exposure to the mixture, whereas exposure to benzo(α)pyrene alone mainly modified structural proteins. The entire dataset suggested a Trojan-horse mechanism involved in the biological impacts on Danio rerio embryos especially due to different bioaccumulation pathways and cellular targets.

Ultrastructural and biochemical characterization of mechanically adaptable collagenous structures in the edible sea urchin Paracentrotus lividus.

The viscoelastic properties of vertebrate connective tissues rarely undergo significant changes within physiological timescales, the only major exception being the reversible destiffening of the mammalian uterine cervix at the end of pregnancy. In contrast to this, the connective tissues of echinoderms (sea urchins, starfish, sea cucumbers, etc.) can switch reversibly between stiff and compliant conditions in timescales of around a second to minutes. Elucidation of the molecular mechanism underlying such mutability has implications for the zoological, ecological and evolutionary field. Important information could also arise for veterinary and biomedical sciences, particularly regarding the pathological plasticization or stiffening of connective tissue structures. In the present investigation we analyzed aspects of the ultrastructure and biochemistry in two representative models, the compass depressor ligament and the peristomial membrane of the edible sea urchin Paracentrotus lividus, compared in three different mechanical states. The results provide further evidence that the mechanical adaptability of echinoderm connective tissues does not necessarily imply changes in the collagen fibrils themselves. The higher glycosaminoglycan (GAG) content registered in the peristomial membrane with respect to the compass depressor ligament suggests a diverse role of these molecules in the two mutable collagenous tissues. The possible involvement of GAG in the mutability phenomenon will need further clarification. During the shift from a compliant to a standard condition, significant changes in GAG content were detected only in the compass depressor ligament. Similarities in terms of ultrastructure (collagen fibrillar assembling) and biochemistry (two alpha chains) were found between the two models and mammalian collagen. Nevertheless, differences in collagen immunoreactivity, alpha chain migration on SDS-PAGE and BLAST alignment highlighted the uniqueness of sea urchin collagen with respect to mammalian collagen.

Adsorption of B(α)P on carbon nanopowder affects accumulation and toxicity in zebrafish (Danio rerio) embryos

The increasing use of nanomaterials raises several concerns regarding their potential risk for the environment and human health. In particular, the aquatic ecosystems appear highly susceptible. In this context, we investigated the interplay between carbon nanopowder (CNPW) and the common pollutant benzo(α)pyrene (B(α)P) in zebrafish embryos. CNPW was contaminated with B(α)P, and showed significant adsorption towards the hydrocarbon. Embryos were then exposed to CNPW (50 mg L−1) or B(α)P (0.2, 6, 20 μg L−1) alone, or to the CNPW doped with the three B(α)P concentrations. We demonstrated that CNPW helps B(α)P uptake by zebrafish embryos and we also demonstrated that the interaction between CNPW and the hydrocarbon affects the stress response pathways of the organism, so eliciting the toxic effect. In particular, the modulation of genes related to the cellular stress response (cyp1a, hsp70, sod1, sod2) and the measurement of oxidative stress enzyme activities allowed us to identify critical molecular events modulated by the pollutants alone and in co-exposure. Finally, to evaluate the toxic effects due to CNPW interactions with B(α)P, we analyzed biomarkers of cyto-genotoxicity. No significant genotoxicity was induced by B(α)P and CNPW alone, but the co-exposure led to an increase of cytotoxicity, and a higher incidence of necrotic and apoptotic cells. Altogether our data show that nanomaterials, even if they are not toxic per se, could help to enhance the toxicity of common pollutants.

Removal of enteric viruses and Escherichia coli from municipal treated effluent by zebra mussels

Dreissena polymorpha is a widespread filter-feeder species, resistant to a broad range of environmental conditions and different types of pollutants,which has recently colonized Italian freshwaters. Although widely used to monitor pollution in freshwater environments, this species is also an important food source for some fish and water birds. It can also be used to concentrate or remove particulate organic matter to interrupt avian-to-human transmission of pollutants and control health risks for animals and humans. In this study, the accumulation/inactivation in D. polymorpha of human health-related spiked enteric viruses was described. The removal of endogenous Escherichia coli, the classical indicator of fecal contamination,was tested as well.Our preliminary lab-scale results demonstrate that zebra mussels can reduce significantly poliovirus titer after 24 h and rotavirus titer after 8 h. E. coli counts were also reduced in the presence of zebra mussels by about 1.5 log after 4 h and nearly completely after 24 h. The fate of the two enteric viruses after concentration by zebra mussels was also investigated after mechanical disruption of the tissues. To our knowledge, the accumulation from water and inactivation of human health-related enteric viruses by zebra mussels has never been reported.

Genotoxic effects induced by the exposure to an environmental mixture of illicit drugs to the zebra mussel

Despite the growing interest on the presence of illicit drugs in freshwater ecosystems, just recently the attention has been focused on their potential toxicity towards non-target aquatic species. However, these studies largely neglected the effects induced by exposure to complex mixtures of illicit drugs, which could be different compared to those caused by single psychoactive molecules. This study was aimed at investigating the genetic damage induced by a 14-day exposure to a realistic mixture of the most common illicit drugs found in surface waters worldwide (cocaine, benzoylecgonine, amphetamine, morphine and 3,4-methylenedioxymethamphetamine) on the zebra mussel (Dreissena polymorpha). The mixture caused a significant increase of DNA fragmentation and triggered the apoptotic process and micronuclei formation in zebra mussel hemocytes, pointing out its potential genotoxicity towards this bivalve species.

Increase in cannabis use may indirectly affect the health status of a freshwater species

Cannabis is the most used illicit drug worldwide and in some countries a new regulatory policy makes it legal under some restrictions. This situation could lead to a substantial increase in environmental levels of the cannabis active principle (Δ-9-tetrahydrocannabinol [Δ-9-THC]) and its main metabolite, 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH). Although previous studies have highlighted the toxicity of Δ-9-THC, the adverse effects of THC-COOH on aquatic organisms is completely unknown, even though such effects could be more significant because the environmental concentrations of THC-COOH are higher than those of the parent compound. The present study aimed to assess oxidative and genetic damage to the zebra mussel (Dreissena polymorpha) because of 14-d exposures to 3 THC-COOH concentrations, mimicking a current environmental situation (100 ng/L), as well as exposure to 2 possible worst-case scenarios (500 ng/L and 1000 ng/L), because of the potential increase in THC-COOH in surface waters. Variations in the activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) were measured, as well as levels of lipid peroxidation and protein carbonyl content. Genetic injuries were investigated by single-cell gel electrophoresis assay, DNA diffusion assay, and the micronucleus test. A significant imbalance in antioxidant defense enzymes was noted in response to the 3 tested concentrations, whereas oxidative damage was noted only at the higher one. Moreover, an increase in DNA fragmentation in zebra mussel hemocytes, but no fixed genetic damage, was found. Although the results showed that THC-COOH toxicity was lower than that of Δ-9-THC, the increase in cannabis use might increase its levels in freshwaters, enhancing its hazard to bivalves and likely to the whole aquatic community. Environ Toxicol Chem 2017;36:472-479.