The use of an in vitro approach to assess marine invertebrate carboxylesterase responses to chemicals of environmental concern
Introduction
Marine ecosystems are the final sink for many land-based chemicals and this raises concerns on the toxicological consequences that man-made compounds may impose on aquatic wildlife and aquaculture species (Nilsen et al., 2019; Tornero and Hanke, 2016). In addition to the long list of classical chemical pollutants, there is a broad range of new drugs of environmental concern which have been more recently introduced into aquatic systems. Despite removal efforts of waste-water plants, technological limitations, accidental discharges and the high concentration and recalcitrant properties of some of these chemicals, make these substances reach marine systems (Sanchez-Avila et al., 2009). Among the latter, pharmaceuticals and personal care products (PPCPs) stand out, as they are increasingly used in a growing and aging population worldwide (Fent et al., 2006; Gaw et al., 2014). Consequently, there is increasing concern over the toxicological implications of PPCPs exposures in aquatic species (Corcoran et al., 2010; Dussault et al., 2008; Fabbri and Franzellitti, 2016), including some particular drugs such as statins or lipid regulators (Santos et al., 2016). Plastics, and more particularly the additives included in their manufacturing process to improve some of their properties, are another group of chemicals of increasing environmental concern (Avio et al., 2017; Hermabessiere et al., 2017). Among them, brominated flame retardants (BFRs) had largely been used worldwide as plastic additives and reported to persist in aquatic environments (de Wit, 2002). Due to their widespread use and lipophilic nature, they are ubiquitously present in sewage sludge and even in marine sea-food in Europe (Aznar-Alemany et al., 2017; Gorga et al., 2013). Among them, tetrabromobisphenol A (TBBPA) and other bisphenol A derivatives can pose a threat to aquatic fauna because of its endocrine disrupting character and oxidative stress condition (Abdallah, 2016; Liu et al., 2018; Perez-Albadalejo et al., 2020). Organophosphorus-based flame retardants such as Tris(1-chloropropan-2-yl) phosphate (TCP) and triphenyl phosphate (TPP) have been introduced in the market to replace BFRs, as they have been reported to be less toxic (van der Veen and de Boer, 2012). However, there is recent evidence of their occurrence in high trophic level marine fauna, being able to cross the blood-brain barrier and accumulate in the brain (Sala et al., 2019). Another type of commonly-used plasticizers are phthalates, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), di-isobutyl phthalate (DiBP), di-n-butyl phthalate (DnBP), benzylbutyl phthalate (BzBP) and di(2-ethylhexyl) phthalate (DEHP). These compounds have been detected in Mediterranean waters (Paluselli et al., 2018) and are known to be accumulated by zooplankton, posing a critical problem given their link position between primary producers and the upper levels of the marine trophic web (Schmidt et al., 2018). Recent evidences in marine shellfish species, including wild and farmed mussels confirm that plasticizers bioaccumulate in animal tissues: TCPP and phthalates may be found at about 20 ng/g d.w. and may reach up to 600 ng/g d.w. in the case of the more lipophilic DEHP (Castro-Jimenez and Ratola, 2020). Other plastic additives such as TCS, and more importantly its metabolite methyl-TCS, are also of concern due to their higher bioaccumulation potential in shellfish (Azzouz et al., 2019). Flame retardants, including TBBPA, as well as their metabolites have also been reported to bioccumulate in benthic and pelagic biota of the marine web (Choo et al., 2019).
Carboxylesterases (CEs) are members of the superfamily of α/β hydrolases involved in hydrolysis of endogenous compounds and detoxification of many chemicals of the ester, amide, thioester bonds and carbamates (Sogorb and Vilanova, 2002; Satoh and Hosokawa, 2006; Wheelock et al., 2008). In humans, two CE isoforms have been described to display a particular organ distribution and substrate preference: hCE1, mainly located in the liver and preferring substrates with a small alcohol or amine group and a large acyl moiety; and hCE2, located in intestine and extra-hepatic organs and showing preference for substrates with a large alcohol or amine and a small acyl moiety (Fukami and Yokoi, 2012; Hosokawa, 2008; Laizure et al., 2013). A large number of in vitro inhibition studies of this family of enzymes have been carried out and have been recognized as a useful tool for the evaluation of drug-drug interactions and drug development in pharmacology (Fukami et al., 2010; Imai et al., 2006; Takahashi et al., 2009; Xu et al., 2013). An adaptation of the former in vitro approach, currently used in mammalian systems, was here applied to reveal the potential interaction of drugs of environmental concern in hepatic CEs. In vitro experimentation has a number of limitations, including the fact that they use higher concentrations than those considered as environmentally realistic. Furthermore, they may not reflect the cell compartment interactions given that analyses are carried out under the lack of the metabolic and homeostasis processes which take place in vivo. Nevertheless, this approach was here adopted to conduct a first screening of a proxy of the potential interactions occurring in vivo, and aiming to set the bases for further environmental studies. A selection was made of representative marine invertebrates of three phyla that constitute a large proportion of Mediterranean shellfish. Recent studies on marine bivalves have demonstrated the in vivo ability of CEs to metabolise drugs such as the retroviral Tamiflu® (Dallarés et al., 2019) and to respond to the presence of pesticides in the environment (Dallarés et al., 2018). The CEs in vitro sensitivity to pesticides, pharmaceutical drugs and plastic additives has also been reported in a large number of aquatic species (Nos et al., 2020; Ribalta et al., 2015; Solé and Sanchez-Hernandez, 2015, 2018). Gastropods CEs have also been revealed to be sensitive to pesticides but studies are largely based on freshwater species (Bianco et al., 2014; Laguerre et al., 2009; Otero and Kristoff, 2016). Marine gastropods such as Bolinus brandaris are considered as good sentinels of endocrine disrupting chemical pollution (e.g. organotin compounds, inducing imposex), a long-lasting concern in the Mediterranean (Anastasiou et al., 2016; Solé et al., 1998). CEs in marine crustaceans have also been targeted as biomarkers of chemical exposure (Antó et al., 2009; Solé et al., 2009; Varó et al., 2019). In the later, the juvenile hormone methyl farnesoate is metabolised by a particular type of CE. Since this hormone is involved in key physiological events, including moulting, metamorphosis and reproduction (Homola and Chang, 1997; Lee et al., 2011), any interactions of xenobiotics on CE hydrolytic activity are of physiological concern.
The aim of this study was to apply an in vitro approach, currently used in mammalian studies, to reveal interactions of toxic chemicals of environmental concern on hepatic CEs of selected marine shellfish invertebrates. For that, commercial recombinant human CEs were included in the in vitro exposures to validate the methodological approach. The selected species are representative elements of the marine food chain with a significant commercial interest NW Mediterranean region. Any drug interactions with CE activities which may affect shellfish physiological performance, drug metabolism capacity and bioaccumulation to ultimately infer on the consequences to wildlife and humans as shellfish consumers.
Section snippets
Sample collection
The present study considered three bivalves (the mussel Mytilus galloprovincialis, the cockle Cerastoderma edule and the razor shell Solen marginatus), the muricid gastropod Bolinus brandaris, the portunid crab Macropipus tuberculatus and the pandalid shrimp Plesionika martia. Representatives of all these species were collected in July 2019 from traditional fishing grounds of the NW Mediterranean (Spain) and immediately transported in cold conditions to the laboratory where they were dissected.
Substrate preference and baseline CE activities in selected marine invertebrates
In Table 1 CE baseline activities in the hepatic organ are detailed for the six marine invertebrate species and substrates assayed. Regarding the bivalves, CE substrate preference (i.e. measures displaying higher hydrolysis rates) in the mussel and the cockle followed a similar, and statistically significant, trend: pNPA < pNPB < αNA < αNB (mussels: χ2(2) = 26.040, p < 0.001; cockles: χ2(2) = 26.760, p < 0.001). For razor shell (S. marginatus), however, hydrolysis rates with αNA were the
Conclusions
The in vitro approach used in this study, adopted from mammalian studies, confirmed the selectivity of certain drugs to modulate particular CE isoforms in humans, and confirmed its methodological suitability to be applied to other biological groups. Despite a lower responsiveness when using S9 fractions of hepatic organs of marine invertebrates we were still able to identify CE-related measures and the most sensitive substrate for each species. The substrate pNPA was confirmed as the most
CRediT authorship contribution statement
Montserrat Solé: Conceptualization, Investigation, Funding acquisition, Writing - original draft. Rosa Freitas: Formal analysis, Writing - review & editing. Georgina Rivera-Ingraham: Formal analysis, Writing - review & editing.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
This work was financed by Spanish Ministry of Economy, Industry and Competitivity (ref CGL2016-76332-R MINECO/FEDER, UE). SAP-ICATMAR is acknowledged for providing biological samples. Two of us (M.S and R.F) are member of the CYTED network RIESCOS ref. 419RT0578.
References (82)
- et al.
Microplastics in commercial molluscs from the lagoon of Bizerte (Northern Tunisia)
Mar. Pollut. Bull.
(2019) - et al.
Imposex and organotin compounds in ports of the Mediterranean and the Atlantic: is the story over?
Sci. Total Environ.
(2016) - et al.
Characterisation of integrated stress biomarkers in two deep-sea crustaceans, Aristeus antennatus and Nephrops norvegicus, from the NW fishing grounds of the Mediterranean sea
Ecotoxicol. Environ. Saf.
(2009) - et al.
Risk of triclosan based on avoidance by the shrimp Palaemon varians in a heterogeneous contamination scenario: How sensitive is this approach?
Chemosphere
(2019) - et al.
Plastics and microplastics in the oceans: from emerging pollutants to emerged threat
Mar. Environ. Res.
(2017) - et al.
Occurrence of halogenated flame retardants in commercial seafood species available in European markets
Food Chem. Toxicol.
(2017) - et al.
A multi-residue method for GC-MS determination of selected endocrine disrupting chemicals in fish and seafood from European and North African markets
Environ. Res.
(2019) - et al.
Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases
Aquat. Toxicol.
(2013) - et al.
Resistance in cholinesterase activity after an acute and subchronic exposure to azinphos-methyl in the freshwater gastropod Biomphalaria straminea
Ecotox. Environ. Safe.
(2014) - et al.
First evidence of ingested plastics by a high commercial shrimp species (Plesionika narval) in the eastern Mediterranean
Mar. Pollut. Bull.
(2018)
Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding
Anal. Biochem.
Spatial occurrence and effects of microplastic ingestion on the deep-water shrimp Aristeus antennatus
Mar. Pollut. Bull.
Species and habitat-dependent accumulation and biomagnification of brominated flame retardants and PBDE metabolites
J. Hazard. Mater.
An overview of brominated flame retardants in the environment
Chemosphere
Ecotoxicology of human pharmaceuticals
Aquat. Toxicol.
The emerging role of human esterases
Drug Metab. Pharmacokinet.
Determination of PBDEs, HBB, PBEB, DBDPE, HBCD, TBBPA and related compounds in sewage sludge from Catalonia (Spain)
Sci. Total Environ.
Occurrence and effects of plastic additives on marine environments and organisms: a review
Chemosphere
Microplastic contamination and pollutant levels in mussels and cockles collected along the channel coasts
Environ. Pollut.
Distribution and regulation of esterases that hydrolyze methyl farnesoate in Homarus americanus and other crustaceans
Gen. Comp. Endocr.
Bioconcentration of triclosan and methyl-triclosan in marine mussels (Mytilus galloprovincialis) under laboratory conditions and in metropolitan waters of Gulf St Vincent, South Australia
Mar. Pollut. Bull.
B-type esterases in the snail Xeropicta derbentina: an enzymological analysis to evaluate their use as biomarkers of pesticide exposure
Environ. Pollut.
Two juvenile hormone esterase-like carboxylesterase cDNAs from a Pandalus shrimp (Pandalopsis japonica): cloning, tissue expression, and effects of eyestalk ablation
Comp. Biochem. Physiol. B
Transformation/degradation of tetrabromobisphenol A and its derivatives: a review of the metabolism and metabolites
Environ. Pollut.
Population-structure and exploitation of bolinus-brandaris (mollusca, gastropoda) off the catalan coast (northwestern mediterranean)
Fish. Res.
An ultraviolet spectrophotometric assay for α-naphthyl acetate and α-naphthyl butyrate esterases
Anal. Biochem.
A multi-biomarker approach to assess effects of Triclosan in the clam Ruditapes philippinarum
Mar. Environ. Res.
In vitro and in vivo studies of cholinesterases and carboxylesterases in Planorbarius corneus exposed to a phosphorodithioate insecticide: finding the most sensitive combination of enzymes, substrates, tissues and recovery capacity
Aquat. Toxicol.
Comparative study of hydrolytic metabolism of dimethyl phthalate, dibutyl phthalate and di(2-ethylhexyl) phthalate by microsomes of various rat tissues
Food Chem. Toxicol.
Distribution of phthalates in Marseille Bay (NW Mediterranean Sea)
Sci. Total Environ.
The effect of temperature on Triclosan and Lead exposed mussels
Comp. Biochem. Physiol., B
Microplastic contents from maricultured and natural mussels
Mar. Pollut. Bull.
Hepatic biotransformation and antioxidant enzyme activities in Mediterranean fish from different habitat depths
Sci. Total Environ.
Different effects of subchronic exposure to low concentrations of the organophosphate insecticide chlorpyrifos in a freshwater gastropod
Ecotoxicol. Environ. Saf.
First determination of high levels of organophosphorus flame retardants and plasticizers in dolphins from Southern European waters
Environ. Res.
Determination and occurrence of phthalates, alkylphenols, bisphenol A, PBDEs, PCBs and PAHs in an industrial sewage grid discharging to a municipal wastewater treatment plant
Sci. Total Environ.
Statins: an undesirable class of aquatic contaminants?
Aquat. Toxicol.
Structure, function and regulation of carboxylesterases
Chem. Biol. Interact.
Effects of water warming and acidification on bioconcentration, metabolization and depuration of pharmaceuticals and endocrine disrupting compounds in marine mussels (&ITMytilus galloprovincialis&IT)
Environ. Pollut.
Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis
Toxicol. Lett.
An in vitro screening with emerging contaminants reveals inhibition of carboxylesterase activity in aquatic organisms
Aquat. Toxicol.
Cited by (13)
Functional roles and localization of hydrolases in the Japanese mitten crab Eriocheir japonica
2024, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular BiologyIn vitro screening of imidazolium and pyrrolidinium based ionic liquids toxicity on subcellular fractions of the Mediterranean mussel Mytilus galloprovincialis
2023, Environmental Toxicology and PharmacologySubcellular tissue-specific responses of Mytilus galloprovincialis to fluoroquinolone antibiotics
2023, Environmental Toxicology and PharmacologyB-esterase measurements and other blood related biomarkers in loggerhead sea turtles (Caretta caretta) as indicators of health status
2023, Science of the Total EnvironmentThe influence of ecological factors in the modulation of pollution biomarkers of two small pelagic marine fish
2023, Marine Pollution Bulletin