HSP90 and pCREB alterations are linked to mancozeb-dependent behavioral and neurodegenerative effects in a marine teleost

Highlights

• Fish exposed to mancozeb exhibited an evident latency to reach T-maze arms.

• Mancozeb caused immobility and reduction of explorative attitudes.

• Fish exposed to mancozeb showed anxiogenic performances in the Light/Dark apparatus.

• The brain of fish exposed to mancozeb supplied pCREB plus HSP90 mRNA up-regulations.

• Some brain areas of fish exposed to mancozeb revealed an evident neurodegeneration.

Abstract

The pesticide mancozeb (mz) is recognized as a potent inducer of oxidative stress due to its ability to catalyze the production of reactive oxygen species plus inhibiting mitochondrial respiration thus becoming an environmental risk for neurodegenerative diseases. Despite numerous toxicological studies on mz have been directed to mammals, attention on marine fish is still lacking. Thus, it was our intention to evaluate neurobehavioral activities of ornate wrasses (Thalassoma pavo) exposed to 0.2 mg/l of mz after a preliminary screening test (0.07–0.3 mg/l). Treated fish exhibited an evident (p < 0.001) latency to reach T-maze arms (> 1000%) while exploratory attitudes (total arm entries) diminished (− 50%; p < 0.05) versus controls during spontaneous exploration tests. Moreover, they showed evident enhancements (+ 111%) of immobility in the cylinder test. Contextually, strong (− 88%; p < 0.01) reductions of permanence in light zone of the Light/Dark apparatus along with diminished crossings (− 65%) were also detected. Conversely, wrasses displayed evident enhancements (160%) of risk assessment consisting of fast entries in the dark side of this apparatus. From a molecular point of view, a notable activation (p < 0.005) of the brain transcription factor pCREB occurred during mz-exposure. Similarly, in situ hybridization supplied increased HSP90 mRNAs in most brain areas such as the lateral part of the dorsal telencephalon (Dl; + 68%) and valvula of the cerebellum (VCe; + 35%) that also revealed evident argyrophilic signals. Overall, these first indications suggest a possible protective role of the early biomarkers pCREB and HSP90 against fish toxicity.

Introduction

Water pollution represents a serious threat to aquatic organisms and especially fish. Pesticide runoff from agricultural lands are one of the main sources of water contamination, which induces dangerous disturbances to the different physiological facets of fish such as swimming and feeding behaviors (Ullah et al., 2014). Pesticides may easily enter in the fish body through gills, skin and via the food-chain thereby reaching target organs like the brain (Atamaniuk et al., 2014). At this regard, deleterious effects of pesticides are often related to neuronal dysfunctions in both mammals (Lee et al., 2015) and fish (Renick et al., 2016). Consequently the dithiocarbamate fungicides, widely used for preserving different agricultural yields, are considered hazardous toxicants (Rath et al., 2011) due to the alteration of key enzymes such as α-carbonic anhydrase thus accounting for the failure of pH homeostasis, respiration and electrolyte secretion (Kolayli et al., 2011). Among dithiocarbamates, mancozeb (manganese (Mn)/zinc (Zn)-ethylene-bis-dithiocarbamate; mz) is composed of different sub-compounds (Mn, Zn plus ethylene thiourea) that together account for multiple toxic mechanisms operating simultaneously during exposure to this fungicide (Geissen et al., 2010). For this reason, we purposely decided to focus on the effects of the integral compound, which causes wide neuronal damages (Harrison Brody et al., 2013). Indeed, the presence of this molecular complex permits it to catalyze the production of reactive oxygen species (ROS) as well as inhibiting mitochondrial respiration at the brain level (Todt et al., 2016) thus proposing it as an environmental risk for neurodegenerative diseases such as Parkinson's (Pezzoli and Cereda, 2013). At present, indications are beginning to consider mz as an important endocrine disruptor (Thienpont et al., 2011), an oxidative stressor of gills and blood (Kubrak et al., 2012), plus being an inductor of oxidative damage to lipids and proteins in brain, liver and kidney of fish (Atamaniuk et al., 2014).

Based on the above considerations, together with the lack of toxicological studies on marine fish contaminated by mz, the present study aimed to investigate neuronal and behavioral effects of this fungicide on the marine teleost ornate wrasse (Thalassoma pavo) exposed to 0.2 mg/l of mz. This concentration was chosen on the basis of a preliminary screening test of sublethal concentrations (0.07, 0.14, 0.2, 0.3 mg/l) handled in our laboratory. These doses are in line with those used in other studies (Jarrard et al., 2004) along with environmentally relevant concentrations detected in waterbodies near agricultural fields (Shenoy et al., 2009) and in view of the recommended mz concentrations being a thousand times higher for crop treatment (Atamaniuk et al., 2014). Despite the frequent application of mz throughout the world, indications regarding its environmentally relevant concentrations in seawater are still lacking, perhaps because this fungicide is often considered a compound with a low toxicity even if recent evidences are beginning to indicate a high risk imposed to fish due to water contamination (Marques et al., 2016). In any case, it is considered a marine pollutant as reported by the Pesticide Properties Database of the University of Hertfordshire (http://sitem.herts.ac.uk/aeru/ppdb/en/Reports/424.htm) and it is known that the runoff of mz from the river or soil near coasts can easily reach the sea especially since Italy is geographically surrounded by the Mediterranean sea.

Hence, the different behavioral parameters (Light/Dark Test, Spontaneous Exploration Test in a T-maze and Cylinder Test) along with specific molecular markers were investigated during cellular impairments following exposure to this fungicide. For the present work, the phosphorylated and active form of CREB (cAMP response element-binding protein, pCREB) was preferred since it is an important transcription linker between a number of neurotransmitters and downstream gene expression thus favoring neuronal survival and proliferation (Dworkin et al., 2007) together with antianxiety-like conditions (Chakravarty et al., 2013). At the same time, HSP90 was also evaluated due to its well-known role on environmental stress (Wang et al., 2016, Zizza et al., 2016) in which they mitigate deleterious effects of protein misfolding in a similar manner to neurodegenerative diseases (Marino Gammazza et al., 2016). Indications deriving from the present study may provide novel bearings concerning the activation of protective mechanisms against mz-dependent toxicity on fish neurobiological activities.