Proteomic analysis and biochemical alterations in marine mussel gills after exposure to the organophosphate flame retardant TDCPP

Highlights

• TDCPP was quickly bioaccumulated and eliminated by mussels soft tissues.

• TDCPP inhibited AChE activity and caused differential regulation of few gill proteins.

• Gill proteome was more responsive to time than to chemical exposure itself.

Abstract

Organophosphate flame retardants (OPFRs) are (re-)emergent environmental pollutants increasingly being used because of the restriction of other flame retardants. The chlorinated OPFR, tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is among those of highest environmental concern, but its potential effects in the marine environment have rarely been investigated. We exposed a widely used sentinel marine mussel species, Mytilus galloprovincialis, to 10 μg L⁻¹ of TDCPP during 28 days and studied: (i) the kinetics of bioaccumulation and elimination of the compound, (ii) the effect on two molecular biomarkers, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities, and (iii) proteomic alterations in the gills, following an isobaric labeling quantitative shotgun proteomic approach, at two exposure times (7 and 28 days). Uptake and elimination of TDCPP by mussels were very fast, and the bioconcentration factor of this compound in mussels was 147 L kg_ww^-1, confirming that this compound is not very bioaccumulative, as predicted by its chemical properties. GST activity was not affected by TDCPP exposure, but AChE activity was inhibited by TDCPP at both 7 and 28 days of exposure. Proteomic analysis revealed subtle effects of TDCPP in mussel gills, since few proteins (less than 2 % of the analysed proteome) were significantly affected by TDCPP, and effect sizes were low. The most relevant effects detected were the up-regulation of epimerase family protein SDR39U1, an enzyme that could be involved in detoxification processes, at both exposure times, and the down-regulation of receptor-type tyrosine-protein phosphatase N2-like (PTPRN2) after 7 days of exposure, which is involved in neurotransmitter secretion and might be related to the neurotoxicity described for this compound. Exposure time rather than TDCPP exposure was the most important driver of protein abundance changes, with 33 % of the proteome being affected by this factor, suggesting that stress caused by laboratory conditions could be an important confounding factor that needs to be controlled in similar ecotoxicology studies. Proteomic data are available via ProteomeXchange with identifier PXD019720.

Introduction

Flame retardants are widely used in several consumer and industrial products like textile, polyvinyl chloride plastics, cellulose, coatings, polyester resins, polyurethane foams, etc. These compounds are designed to prevent combustion and to delay the spread of fire after ignition, and can reach the environment by volatilization, leaching and abrasion during the entire lifespan of the products to which they are added (Betts, 2008; Van der Veen and de Boer, 2012). Several compounds that have been used as flame retardants since the 60 s, such as PCBs or PBDEs, have been banned or restricted afterwards because of their persistence in the environment, and their bioaccumulative and toxic effects (Beiras, 2018). Alternative compounds are being used in substitution, such as organophosphate flame retardants (OPFRs), which are re-emerging as environmental pollutants in recent years (Wei et al., 2015; Pantelaki and Voutsa, 2019). Within existing OPFRs, chlorinated ones, such as tris(2-chloroethyl)phosphate (TCEP), tris(chloropropyl)phosphate (TCPP) and tris(1,3-dichloro-2-propyl)phosphate (TDCPP), are within the most frequently used, but are also the ones of highest environmental concern, due to their persistence and reported toxicological effects, such as neurotoxicity and carcinogenicity (Reemtsma et al., 2008; Van der Veen and de Boer, 2012).

Within them, TDCPP was considered of highest priority for aquatic life risk assessment by the European Union (EU, 2008), it is persistent and has been detected in rivers, coastal waters and marine biota (Aznar-Alemany et al., 2018; Pantelaki and Voutsa, 2019). Toxicological studies with mammals and cell lines have pointed towards its carcinogenicity and neurotoxicity (WHO, 1998; ATSDR, 2009; Dishaw et al., 2011). Despite its potential effects in the aquatic environment are less known, studies with zebrafish have shown developmental and endocrine alterations upon exposure to this compound (McGee et al., 2012; Liu et al., 2013; Wang et al., 2015a,b).

Blue mussels (Mytilus sp.) are frequently used in marine pollution monitoring (Sericano, 2000; Beyer et al., 2017). Because they are sessile, widespread, easy to sample, and filter-feeders that bioaccumulate pollutants in their tissues, they are a preferred biomonitoring species not only for the measurement of pollutants in their tissues, but also for the evaluation of biomarkers of exposure or effect, as early warning tools for the detection of deleterious effects of pollutants in the marine environment (Beliaeff and Burgeot, 2002; Vidal-Liñán et al., 2010; Davies and Vethaak, 2012). In the last years, the development of proteomic tools has allowed the study of regulation level of hundreds or even thousands of proteins in mussel tissues upon exposure to contaminants, which has opened the door to the discovery of new biomarkers and to increase knowledge about the molecular mechanisms of toxicity of contaminants (Campos et al., 2012; Gouveia et al., 2019). Proteomic studies of the effects of pollutants are usually performed at a fixed exposure time (e.g. Apraiz et al., 2006; Duroudier et al., 2019). However, the exposure time needed for a contaminant to elicit a response is not always known a priori, and even the effects of the contaminant can be different depending on the exposure time (Oliveira et al., 2016). Laboratory experiments with mussels have revealed that protein abundance was more affected by exposure time (including control organisms) than by exposure to a pollutant (Oliveira et al., 2016). However, the specific proteomic changes caused by exposure time, and their possible confounding effects on the outcomes of this type of experiments have not yet been evaluated.

As mussels pump water for feeding and breathing purposes, their gills represent the primary organ in contact with pollutants, and are a sensitive tissue for detecting toxicological effects, as previously demonstrated with the use of molecular biomarkers (Vidal-Liñán et al., 2010; Vidal-Liñán and Bellas, 2013; Vidal-Liñán et al., 2015a).

The objective of this work was to study the bioaccumulation of TDCPP and the effects in the gill proteome produced by this re-emerging pollutant in the marine mussel Mytilus galloprovincialis, at two different exposure times, 7 and 28 days, to test for short vs. long term exposure, and also to test for the effects of exposure time itself on mussel proteome. Likewise, this study focused on traditional biomarkers assessed in gills of exposed mussels to detect the early onset of adverse effects, including a neurotoxic response as acetylcholinesterase (AChE) and a Phase II detoxification enzyme: glutathione S-transferase (GST).