Chemosphere 268 (2021) 129282 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Endocrine disrupting effects of copper and cadmium in the oocytes of the Antarctic Emerald rockcod Trematomus bernacchii * Chiara Maria Motta a, Palma Simoniello b, , Mariana Di Lorenzo a, Vincenzo Migliaccio c, Raffaele Panzuto a, Emanuela Califano a, Gianfranco Santovito d a Department of Biology, University of Naples Federico II, Naples, Italy b Department of Science and Technology, University of Naples Parthenope, Naples, Italy c Department of Chemistry and Biology “Adolfo Zambelli”, University of Salerno, Fisciano, Italy d Department of Biology, University of Padova, Italy highlights Risks related to metal contamination in Antarctica. Effects of waterborne Cd and Cu on fish oogenesis. Oocyte degeneration and changes in glycan composition. Cd downregulation of alpha and beta estradiol receptors. Cd and Cu reduce fecundity in T. bernacchii by impairing stock recruitment. article info abstract Article history: Antarctica has long been considered a continent free from anthropic interference. Unfortunately, recent Received 5 October 2020 evidence indicate that metal contamination has gone so far and that its effects are still unknown. For this Received in revised form reason, in the present work, the potential endocrine disrupting effect of two highly polluting metals, 28 November 2020 copper and cadmium, were examined in the Antarctic teleost Trematomus bernacchii. After a 10 days Accepted 8 December 2020 waterborne exposure, ovarian metal uptake was determined by atomic absorption; in parallel, classical Available online 18 December 2020 histological approaches were adopted to determine the effects on oocyte morphology, carbohydrate Handling Editor: James Lazorchak composition and presence and localization of progesterone and estrogen receptors. Results show that both metals induce oocyte degeneration in about one third of the previtellogenic oocytes, no matter the Keywords: stage of development. In apparently healthy oocytes, changes in cytoplasm, cortical alveoli and/or Progesterone receptors chorion carbohydrates composition are observed. Cadmium but not copper also induces significant Estrogen receptors changes in the localization of progesterone and beta-estrogen receptors, a result that well correlates with Carbohydrate composition the observed increase in ovarian metals concentrations. In conclusion, the acute modifications detected PAS staining are suggestive of a significantly impaired fecundity and of a marked endocrine disrupting effects of Lectin staining copper and cadmium in this teleost species. Oocyte degeneration © Heavy metals content 2020 Elsevier Ltd. All rights reserved. Ovarian morphology 1. Introduction Once taken up by diet, skin and/or gills (Komjarova and Bury, 2014), metals diffuse in the tissues interfering with conventional The marine environment is spoiled by a multitude of anthro- primary targets such as kidney and liver (Avallone et al., 2017; Koca pogenic pollutants (Di Lorenzo et al., 2020; Motta et al, 2018, 2019; et al., 2008) and with unconventional ones such as, for example, Zizza et al., 2018) including highly toxic metals (Mebane et al., muscles, retina or lateral line (Avallone et al., 2015a, 2015b; 2012; Motta et al., 2016; Rainbow, 2002). Hernandez et al., 2006). Toxic effects are exerted via multiple mechanisms but mainly via a marked prooxidant activity (Craig et al., 2007; Zheng et al., 2016) resulting in damage in protein and lipid metabolism and DNA integrity (Nawaz et al., 2005). * Corresponding author. E-mail address: [email protected] (P. Simoniello). Metal toxicity is also extended to the reproductive organs and https://doi.org/10.1016/j.chemosphere.2020.129282 0045-6535/© 2020 Elsevier Ltd. All rights reserved. C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282 Fig. 1. Cu (A) and Cd (B) accumulation in Trematomus bernacchii ovaries after 10 days exposure to CuCl2 (1.574 mM) or CdCl2 (0.890 mM). Values are media ±standard deviation (n ¼ 10). Significantly different from controls (**, p < 0.01; ***, p < 0.001). embryonic and larval development (Henson and Chedrese, 2004). alterations in carbohydrate composition (Eguchi et al., 2002; In particular, copper (Cu) and cadmium (Cd), are known to interfere Mehinto et al., 2014). In particular, three lectins were used to with fecundity, hatching and larval development in several aquatic highlight the presence and distribution of N-Acetyl-glucosamine species (Capriello et al., 2019; Driessnack et al., 2016). Reported (glcNAc), N-Acetyl-galactosamine (galNAc) and mannose (Man). effects are altered cadherin localization (Prozialeck et al., 2003), Presence and localization of progesterone and alpha and beta cytoskeleton organization (Perrin et al., 2017; Wang and estradiol receptors were also determined being sex hormones Templeton, 1996) and impaired sex hormone binding with re- master regulators of oogenesis also in teleosts (Miura et al., 2007). ceptors (Cao et al., 2019; Nesatyy et al., 2006). Moreover, significant Basic histopathological methods were applied: histology for interference with the expression of many genes, such as those morphology detection (Motta et al., 2005b), histochemistry for coding for ZPR1 zinc finger, metallothionein 1 and vitellogenin, are carbohydrate detection (PAS, Dahlqvist et al., 1965; lectin staining, reported (Craig et al., 2009; Hwang et al., 2000; Gonzalez et al., Motta et al., 2005a) and immunocytochemistry for receptor local- 2006). ization (Motta et al., 2020). Cu and Cd accumulate in the gonads of several teleosts (Karlsson-Norrgren and Runn, 1985; Zhang et al., 2016), including 2. Materials and methods the Antarctic ones, expected to live in an uncontaminated envi- ronment (Beltcheva et al., 2011; Illuminati et al., 2010). This 2.1. Experimental model apparent anomaly is partially explained by the natural occurrence of elevated Cu and Cd concentrations in coastal seawaters (70 ng/L Adult female specimens of Trematomus bernacchii were and 150 ng/L respectively; Westerlund and Ohoman, 1991) and collected in the proximity of Mario Zucchelli Station in Terra Nova surface sediments (from 0.05 to 0.49 mg/g dry mass; Terra Nova Bay, Bay, Antarctica (74420S, 16770E) and kept in aquaria supplied with Bargagli et al., 1996) caused by local volcanism and up-welling aerated seawater at approximately À1 C. After 5-day acclimati- phenomena (Kurtz and Bromwich, 1985). zation, animals were randomly divided in three 180 L aquaria: two Though the presence of metals in the gonads of Antarctic fish is groups of five specimens each were exposed to CuCl or CdCl at the demonstrated, their effects are far less understood. For this reason, 2 2 sub-lethal concentrations of 1.574 mM and 0.890 mM, respectively the aim of the present investigation was to carry out a comparative (Santovito et al., 2003); five unexposed animals were used as evaluation of the endocrine disrupting effects of Cu and Cd on the controls. Exposure occurred under static condition (Avallone et al., oocytes of the Nototheniid Trematomus bernacchii, the Antarctic 2015a; Motta et al., 2016): no metal was added during the experi- Emerald rockcod. T. bernacchii is a teleost widely distributed in mental period so to avoid overlapping the effects due to a daily many areas of Antarctica and for this reason it has often been used renewing. The limited exposure time and the reduced metabolism as a model organism for environmental studies, also in relation to of these fish at À1 C do not favour a significant reduction in the Global Change effects (Garofalo et al., 2019; Todgham and Mandic, concentration of metals, as shown by the analyses carried out on 2020). the water of the tanks before and after the experiment (Santovito To this purpose, adult females were exposed to waterborne et al., 2003). Despite selected exposure concentrations might copper chloride (CuCl ) or cadmium chloride (CdCl ) for 10 days, at 2 2 appear relatively high, they are still environmentally realistic a sub-lethal concentration of 1.574 mM and 0.890 mM, respectively considering the bioaccumulation of metals in the investigated site (Santovito et al., 2003). Ovarian metal uptake was then determined and in polluted areas. After 10 days, specimens were euthanized by atomic absorption (Santovito et al., 2012b). (tricaine methane sulfonate, MS-222; 0.2 g/L) and ovaries were The endocrine disrupting effects of the two metals were deter- quickly excised and stored. mined by using a histopathologic approach since it is reported that Animal treatment and sample collection comply with Research the molecular events triggered by the endocrine disruptors end in regulations concerning activities and environmental protection in an interference with cell and tissue organization (van der Ven et al., Antarctica and with the Protocol on Environmental Protection to 2003). Changes in morphology were determined together with the Antarctic Treaty, Annex II, Art. 3. All the procedures were 2 C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282 Fig. 2. Effects of Cu and Cd on Trematomus bernacchii oocytes morphology. Controls. A) Perinucleolar oocytes (*); Balbiani’s body (Bb), multiple nucleoli (arrow) in nuclei (n). Alveolar oocyte with cortical alveoli (a). B) Early alveolar oocyte with alveoli (a) and multiple nucleoli (arrow). C) Mid alveolar oocyte with alveoli (a) containing nucleoids (arrow). 3