Effects of di-(2-ethylhexyl) phthalate on Transcriptional Expression of Cellular Protection-Related HSP60 and HSP67B2 Genes in the Mud Crab Macrophthalmus japonicus

Kiyun Park,Ihn-Sil Kwak,Won-Seok Kim

doi:10.3390/app10082766

Abstract

Di-2-ethylhexyl phthalate (DEHP) has attracted attention as an emerging dominant phthalate contaminant in marine sediments. Macrophthalmus japonicus, an intertidal mud crab, is capable of tolerating variations in water temperature and sudden exposure to toxic substances. To evaluate the potential effects of DEHP toxicity on cellular protection, we characterized the partial open reading frames of the stress-related heat shock protein 60 (HSP60) and small heat shock protein 67B2 (HSP67B2) genes of M. japonicus and further investigated the molecular effects on their expression levels after exposure to DEHP. Putative HSP60 and small HSP67B2 proteins had conserved HSP-family protein sequences with different C-terminus motifs. Phylogenetic analysis indicated that M. japonicus HSP60 (Mj-HSP60) and M. Japonicus HSP67B2 (Mj-HSP67B2) clustered closely with Eriocheir sinensis HSP60 and Penaeus vannamei HSP67B2, respectively. The tissue distribution of Heat shock proteins (HSPs) was the highest in the gonad for Mj-HSP60 and in the hepatopancreas for Mj-HSP67B2. The expression of Mj-HSP60 Messenger Ribonucleic Acid (mRNA) increased significantly at day 1 after exposure to all doses of DEHP, and then decreased in a dose-dependent and exposure time-dependent manner in the gills and hepatopancreas. Mj-HSP67B2 transcripts were significantly upregulated in both tissues at all doses of DEHP and at all exposure times. These results suggest that cellular immune protection could be disrupted by DEHP toxicity through transcriptional changes to HSPs in crustaceans. Small and large HSPs might be differentially involved in responses against environmental stressors and in detoxification in M. japonicus crabs.

Highlights

Artificial chemical additives have come to the fore as one of the main environmental pollution triggers
We found that Mj-heat shock protein 60 (HSP60) included a chaperonin-like super family main domain, whereas a RHOD superfamily motif was detected in Mj-heat shock protein 67B2 (HSP67B2) (Figure 2A)
We carried out pairwise alignment of Mj-HSP67B2 using EMBOSS alignment (EMBL-EBI, Cambridgeshire, UK) [35] with sequences identified in BLAST searches

Summary

Introduction

Artificial chemical additives have come to the fore as one of the main environmental pollution triggers. Plasticizers, which assign flexibility and durability to plastic, have been heavily utilized, owing to the widespread application of plastic products. As the most common plasticizer, di-2-ethylhexyl phthalate (DEHP) has contributed to the manufacture of flexible products from solid plastics such as polyvinyl chloride [1]. Owing to its widespread use, DEHP is ubiquitously released into the aquatic environment [2,3]. A recent study showed that the main source of DEHP is emissions from household sewage and sludge disposal activities [2]. DEHP is detected at high levels in all sediment samples taken from coastal bays, indicating ubiquitous contamination of the marine environment [3].

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Results

Conclusion