Isolation, cloning, and tissue distribution and functional analysis of ShP-glycoprotein in the freshwater crab Sinopotamon henanense exposed to Cd and Cd-QDs.

P-glycoprotein (Pgp), a member of ATP binding cassette (ABC) transporter family, can extrude toxic substances out of cells by mediating multi-xenobiotic resistance (MXR) in aquatic organisms, however, its regulation and association with MXR are still unclear. In this work, the genetic information of Pgp in freshwater crab Sinopotamon henanense (ShPgp) was revealed for the first time. ShPgp with a total of 4488 bp was cloned and analyzed, which includes 4044 bp open reading frame, 353 bp 3' untranslated region, and 91 bp 5' untranslated region. The recombinant ShPGP were expressed in Saccharomyces cerevisiae and taken for SDS-PAGE and western blot analysis. ShPGP was widely expressed in the midgut, hepatopancreas, testis, ovary, gill, hemocytes, accessory gonad and myocardium of the crabs studied. The images of immunohistochemistry indicated that ShPgp was mainly distributed in the cytoplasm and cell membrane. When the crabs were exposed to cadmium or cadmium containing quantum dots (Cd-QDs), not only the relative expression of ShPgp mRNA and the protein produced were enhanced, but also the MXR activity and ATP contents. The relative expression of target genes related to energy metabolism, detoxification and apoptosis was also determined in the carbs exposed to Cd or Cd-QDs. The results showed that bcl-2 was significantly down-regulated, while other genes were up-regulated except PPAR (not affected). However, when the Shpgp in treated crabs was interfering by knockdown technique, their apoptosis and the expression of proteolytic enzyme genes and transcription factors MTF1 and HSF1 were also elevated, while the expression of apoptosis inhibiting and fat metabolism genes were compromised. Based on the observation, we concluded that MTF1 and HSF1 were involved in gene transcription regulation of mt and MXR, respectively, while PPAR had limited regulatory effect on those genes in S. henanense. NF-κB may play a negligible role in the process of apoptosis in testes induced by cadmium or Cd-QDs. However, the detail information regarding Pgp involvement in SOD or MT, and its association with apoptosis during xenobiotics insults remain to be explored.

Detoxification and recovery after cadmium exposure in the freshwater crab Sinopotamon henanense.

Cadmium (Cd) is a common pollutant in the aquatic environment, which puts the health and safety of aquatic organisms and humans at risk. In the present study, the freshwater crab Sinopotamon henanense was exposed to Cd (0, 50, 100, and 500 µg·L-1) for 14 d (0-14th d), followed by 21 d (14-35th d) of depuration. The changes in Cd bioaccumulation, microstructure, biomacromolecules (polysaccharides, neutral lipids, DNA and total proteins), and biochemical parameters (SOD, CAT, GR, TrxR, MDA and AChE) in the gills and hepatopancreas were tested. The injured microstructure, activated antioxidant system, increased MDA, and inhibited AChE of the gills and hepatopancreas responded with progressive bioaccumulation of Cd. Meanwhile, the polysaccharides and neutral lipids in the hepatopancreas reduced and DNA synthesis enhanced. During depuration, more than 58.80 ± 8.53% and 13.84 ± 12.11% of Cd was excreted from the gills and hepatopancreas, respectively. Recovery of microstructure and biomacromolecules as well as alleviated oxidative damage and neurotoxicity were also found in these two organs. Additionally, based on PCA, Ihis, GR and MDA were identified as the optimal biomarkers indicating the health status of crabs. In conclusion, S. henanense could resist Cd stress through antioxidant defence and self-detoxification.

Reproductive toxicity of quantum dots on gonads of the fresh water crab Sinopotamon henanense.

Since nano-quantum dots (QDs) are increasingly used as fluorescent dyes in biomedical sciences, the possibility of QDs contaminating aquatic environments is generally increasing. There is concern about potential toxicity of QDs. However, their risks in the aquatic environment are not entirely understood. In this study, the freshwater crab Sinopotamon henanense was exposed to cadmium telluride (CdTe) QDs by intraperitoneal injection to detect the reproductive toxicity of QDs (1/32, 1/16 and 1/4 LD50; Crab was exposed for 1, 3, 5, and 7 days). After CdTe QD exposure, no significant effect was detected on the body weight and gonadosomatic index. Additionally, morphological observations showed tissue vacuolation in the testis, and inflammatory cell infiltration in the ovary. The submicroscopic structure showed that exposure to CdTe QDs damaged the organelles and cell structures of the gonads of S. henanense. Among the adverse effects, pathological changes in the nuclear membrane, mitochondria and lysosomes were particularly significant. Antioxidant enzymes responded differently to different doses of QDs. The 0.5-mg/kg dose induced superoxide dismutase activity in the testes. And in the 1-mg/kg and 4-mg/kg dose QD exposure test, the testis responded by activating glutathione peroxidase and inducing reduced glutathione and overconsuming glutathione peroxidase. Respectively, the ovaries responded by overconsuming superoxide dismutase and glutathione peroxidase and reduced glutathione. Thus, we conclude that the gonads of S. henanense were injured by CdTe QD, and male are better indicators of the toxicity of QDs than female crabs according to greater alterations in tissue structure and antioxidant enzyme in the analyses.