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Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis

https://doi.org/10.1016/j.etap.2020.103379Get rights and content

Highlights

  • The toxicity of the dinoflagellate Coolia malayensis crude extract was studied.

  • Mitochondrial toxicity assessment and cell-based assays were performed.

  • Coolia malayensis crude extract was found to be toxic to mitochondrial bioenergetics.

  • Toxicity in mitochondria also elicited HepG2 and H9c2(2−1) cell mass decrease.

  • Coolia malayensis toxicity at sub-cellular and cellular levels were demonstrated.

Abstract

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨm) generation and the fluctuations of ΔΨm associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2−1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨm and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca2+-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.

Introduction

Some marine dinoflagellate species produce a variety of potent and persistent toxins that may cause harm to aquatic life and human health (Anderson, 2009). Haemolytic activity, hypothermia and respiratory failure in mice are associated with some Coolia species (Holmes et al., 1995; Pagliara and Caroppo, 2012; Rhodes et al., 2014), as well as lethality and abnormal development and behaviour in brine shrimp and sea urchin (Leung et al., 2017). Also, yessotoxin-like compounds produced by C. monotis exerted effects on zebrafish embryo development (Lewis et al., 2018).

An undescribed mono-sulfated compound was found in C. malayensis (Rhodes et al., 2014), and five other possible toxic compounds were reported (Wakeman et al., 2015). However, comparing with other toxic dinoflagellate species, knowledge of the toxicity of C. malayensis was still lacking. Thus, the present study aimed to address critical knowledge gaps regarding the effects of C. malayensis at subcellular and cellular levels through mitochondrial toxicity assessment and cell-based assays.

Mitochondria are involved in the production of the ATP required for cellular viability and functionality (Lehninger, 1964), which is synthesized by the coupling of two interdependent processes: the mitochondrial electron transport system, which generates an electrochemical potential across the mitochondrial inner membrane (ΔΨm), and ATP synthase, which phosphorylates ADP using the energy of ΔΨm (Will and Dykens, 2014). Therefore, the effect of C. malayensis toxicity was studied by following mitochondrial ΔΨm fluctuations associated with oxidative phosphorylation in rat liver mitochondria energized with the substrates glutamate/malate (respiratory chain complex I-linked substrates) and succinate (respiratory chain complex II-linked substrate). Since mitochondrial maximal ability to accumulate Ca2+ is an indicator of mitochondrial permeability transition (MPT) susceptibility (De Marchi et al., 2014), the collapse of ΔΨm associated with Ca2+ additions in the presence of C. malayensis was also studied. In addition, the toxicity of C. malayensis was determined in human liver HepG2 and rat cardiomyoblast H9c2(2−1) cells.

Section snippets

Material and methods

The C. malayensis strain UNR-02 used in the present study came from a monoclonal culture maintained at the Collection of Marine Microalgae of the Federal University of the State of Rio de Janeiro. Algal cells in the exponential growth phase were harvested and lyophilised (1.16 × 107 cells). Then, intracellular compounds were extracted in 500 μL of dimethyl sulfoxide (DMSO, ≥99 %, Sigma-Aldrich D2650).

The animal experimental protocol used was previously approved by the local Institutional Animal

Results and discussion

The effect of C. malayensis DMSO crude extract was studied at the subcellular level by following mitochondrial ΔΨm fluctuations associated with oxidative phosphorylation in mitochondria energized with the substrates glutamate/malate (Fig. 1A) and succinate (Fig. 1B). Before extract addition, mitochondria developed a ΔΨm between 212 and 220 mV (negative inside). Incubation with increasing concentrations of 2500, 5000, 10,000 and 20,000 algal cells mL−1 resulted in a progressive decrease of the

CRediT authorship contribution statement

Ana T. Varela: Formal analysis, Investigation, Writing - original draft, Visualization. Raquel A.F. Neves: Formal analysis, Investigation, Visualization. Silvia M. Nascimento: Methodology, Investigation, Resources, Writing - review & editing, Funding acquisition. Paulo J. Oliveira: Methodology, Validation, Resources, Writing - review & editing. Miguel A. Pardal: Funding acquisition. Elsa T. Rodrigues: Conceptualization, Validation, Writing - original draft, Supervision, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The Portuguese Foundation for Science and Technology supported this work through the fellowship SFRH/BPD/116152/2016, the FishFree Project PTDC/AAG-TEC/4966/2014 and the Centre for Functional Ecology Strategic Project UID/BIA/04004/2013.

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