Elsevier

Aquatic Toxicology

Volume 213, August 2019, 105220
Aquatic Toxicology

Tamoxifen affects the histology and hepatopancreatic lipid metabolism of swimming crab Portunus trituberculatus

https://doi.org/10.1016/j.aquatox.2019.06.003Get rights and content

Highlights

Abstract

Tamoxifen (TAM) is an antiestrogenic agent and can enter the aquatic environment in wastewater. It has been reported that TAM can induce hepatic steatosis in vertebrates, however, the effects of TAM exposure on lipid metabolism of hepatopancreas in crustaceans remains unclear. In this study, four TAM concentrations (0, 6.7, 13.4 and 20 μg g−1 crab body weight) were injected into the swimming-leg of swimming crabs Portunus trituberculatus, as a means of evaluating the effects of TAM on the expression levels of lipid metabolism-related genes, lipid composition, and hepatopancreas histology. The results showed that the mRNA levels of three lipogenic related genes (diacylglycerol acyltransferase 1 (DGAT1), acetyl-CoA carboxylase (ACC) and fatty acyl desaturase (FAD)) decreased significantly in the 6.7 μg g−1 and 20 μg g−1 TAM treatments compare to the control. The mRNA levels of fatty acid synthase (FAS) decreased significantly in a dose-dependent manner as TAM concentration increased. The mRNA levels of two lipid catabolism-related genes (acyl-CoA oxidase (ACOX) and fatty acid transport protein (FATP)) were down-regulated among the three TAM treatments, while the enzyme activity and mRNA level of carnitine palmitoyltransferase I (CPT-I) was up-regulated by TAM treatments. Compared to the control, the lowest levels of total lipids and phospholipids were detected in the 6.7 μg g−1 TAM treatment, while the 20 μg g−1 TAM treatment had the lowest free fatty acids concentration. The 6.7 μg g−1 TAM treatment had the lowest percentages of 16:1n-7, 18:1n-9, 18:1n-7 and total monounsaturated fatty acids (∑MUFA), whilst simultaneously recording the highest percentages of 18:2n-6 and 20:2n-6 in this treatment. Moreover, histological observations indicated that TAM caused the walls of the hepatopancreatic tubules to become brittle, with a concurrent increase in the number of blister-like cells. These results suggest that TAM damages the hepatopancreas and leads to a reduction in hepatopancreatic lipid deposition in P. trituberculatus.

Introduction

Environmental endocrine disrupting compounds (EDCs) are chemicals that can either directly or indirectly affect the normal synthesis, transport, metabolism, and secretion of endogenous hormones (Segner et al., 2006). Among EDCs, pharmaceuticals are becoming increasingly important as potential environmental contaminants due to their widespread presence in aquatic ecosystems (Fent et al., 2006a,b). However, the ecotoxicological potential of pharmaceutical residues remains scarcely researched (Fent et al., 2006a,b; Laville et al., 2004).

Tamoxifen, [Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphenyl-1-butene (TAM), is a known selective estrogen receptor modulator (SERM) which, in the last four decades, has been widely used in breast cancer therapy (Jordan, 2003; Poirot and Jordan, 2011). However, it has been reported to cause steatosis (fatty liver) in 43% of recipients (Nishino et al., 2003), thought to manifest as a result of impaired fatty acid β-oxidation (Egawa et al., 2003; Lelliott et al., 2005). Following administration, TAM is known to enter the aquatic environment through municipal sewage effluents (Ashton et al., 2004; Hilton and Thomas, 2003). In a study of the environmental occurrence of TAM in the UK, concentrations in sewage effluents were found to range from 0.146 to 0.369 g L−1 (Fent et al., 2006a,b). As such, an understanding of the ecotoxicological impacts of TAM to aquatic organism is important. To date, the reproductive, biochemical and lethal effects of tamoxifen in some aquatic animals have been reported (Sun et al., 2007; Pagano et al., 2001; Kawahara and Yamashita, 2000). However, in this regard, no information is yet available on the effects of TAM on lipid metabolism.

Lipids play vital roles in cell structure and energy supply for physiological processes, including growth, health and reproduction (Pasquevich et al., 2011), whilst acting as important precursors of steroidal hormones, such as estrogen and progesterone (Warren et al., 2002; Janer and Porte, 2007). It is well known that in crustaceans the hepatopancreas is the central organ for lipid metabolism, including digestion, storage, catabolism and anabolism of lipids (Vogt et al., 1989; Vogt, 1994; Cheng et al., 2000). Crustacean’ hepatopancreas performs the all the functions which are typically done by the adipose tissues and the liver of vertebrates (Vogt, 1994; Wen et al., 2001). Therefore, the hepatopancreas is the logical target organ for studying lipid changes in crustaceans. Furthermore, crustaceans are one of the most ubiquitous groups of invertebrates, inhabiting all types of aquatic habitats. Resultantly, they have been chosen as test species for evaluating EDC effects in numerous studies (DeFur et al., 1999; Oberdörster and Cheek, 2000; Depledge and Billinghurst, 1999).

The swimming crab, Portunus trituberculatus, is an important marine-culture species that is widely distributed in the coastal waters of East Asia, including Korea, Japan, Philippines and China (Hamasaki et al., 2006). In this research, this crab species was used as an experimental organism since its hepatopancreas is not only the center of lipid metabolism, but also an important detoxification organ (Wang et al., 2014; Wu et al., 2014; Li et al., 2015). Therefore, it is proposed that P. trituberculatus, in addition to being an important cultured species, could also be used as a model species to understand the potential toxicity mechanisms and lipid metabolism effects of marine crustaceans exposed to TAM.

As such, the objective of the present study was to comprehensively evaluate the effects of exogenous tamoxifen on the expression levels of lipid metabolism-related genes, lipid composition and histology of hepatopancreas in P. trituberculatus. To the authors’ knowledge, this is the first study reporting the effect of TAM on lipid metabolism in a crustacean species. The results are envisaged to also provide valuable information to further understand the toxic effects on the lipid metabolism in crustaceans.

Section snippets

Animals and culture conditions

The experiment was conducted at the Qidong scientific research base of Shanghai Fisheries Research Institute, Qidong county, Jiangsu Province, China. The experimental crabs, obtained from an outdoor aquaculture pond of the Qidong scientific research base, were reared in an indoor recirculating water system. After 7 days of acclimation in indoor culture conditions, healthy, intact and active females with body weight of 175 ± 25 g and that had just finished their puberty molt, were selected for

Results

In present study, TAM injection significantly decreased gonadosomatic index of crab compared to the control, while no significant effects on crab survival rate or hepatosomatic index were observed. These results have been published in our previous article (Lu et al., 2018).

Discussion

It is known that the molecular mechanism by which TAM can prevent and treat breast cancer is that it forms a relatively stable complex with the estrogen receptors (ER), thereby reducing the number of receptors available for subsequent estradiol binding (Jordan, 2003; Kitano et al., 2007). Additionally, previous studies showed that TAM also has estrogen agonistic properties, which depend on the species and tissues (Osborne et al., 1996). Owing to its multiple actions, TAM is typically referred

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

This study was funded by the two projects (No. 41276158 and No. 41606169) from the Natural Science Foundation of China. Analysis costs were partially supported a research and extension project (No. 2016-1-18) from Shanghai Agriculture Committee and a special research project (No. SZ-LYG2017019) for North Jiangsu area from Science and Technology Department of Jiangsu Province and. Infrastructure costs were partially supported by the research project (No. A1-2801-18-1003) for high level

References (65)

  • J. Kerner et al.

    Fatty acid import into mitochondria

    Biochim. Biophys. Acta

    (2000)
  • R. Koskela et al.

    The influence of prostaglandin E2 and the steroid hormones, 17α-hydroxyprogesterone and 17β-estradiol on moulting and ovarian development in the tiger prawn, Penaeus esculentus Haswell, 1879 (Crustacea: Decapoda)

    Comp. Biochem. Physiol. A: Mol. Integr. Physiol.

    (1992)
  • N. Laville et al.

    Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes

    Toxicology

    (2004)
  • S. Li et al.

    Characterization, mRNA expression and regulation of Δ6 fatty acyl desaturase (FADS2) by dietary n-3 long chain polyunsaturated fatty acid (LC-PUFA) levels in grouper larvae (Epinephelus coioides)

    Aquaculture

    (2014)
  • K. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method

    Methods

    (2001)
  • R. Loizzi et al.

    Lipolytic sites in crayfish hepatopancreas and correlation with fine structure

    Comp. Biochem. Physiol. B: Biochem. Mol. Biol.

    (1971)
  • T. Miura et al.

    Estradiol-17β stimulates the renewal of spermatogonial stem cells in males

    Biochem. Biophys. Res. Commun.

    (1999)
  • W. Morrison et al.

    Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron trifluoride-methanol

    J. Lipid Res.

    (1964)
  • T. Oosthuyse et al.

    Oestrogen’s regulation of fat metabolism during exercise and gender specific effects

    Curr. Opin. Pharmacol.

    (2012)
  • G. Pagano et al.

    The role of oxidative stress in developmental and reproductive toxicity of tamoxifen

    Life Sci.

    (2001)
  • M. Pasquevich et al.

    Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade)

    Comp. Biochem. Physiol. B: Biochem. Mol. Biol.

    (2011)
  • J. Schaffer et al.

    Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein

    Cell

    (1994)
  • L. Sun et al.

    Tamoxifen effects on the early life stages and reproduction of Japanese medaka (Oryzias latipes)

    Environ. Toxicol. Pharmacol.

    (2007)
  • X. Wen et al.

    Variation in lipid composition of Chinese mitten-handed crab, Eriocheir sinensis during ovarian maturation

    Comp. Biochem. Physiol. B: Biochem. Mol. Biol.

    (2001)
  • C. Yen et al.

    DGAT enzymes and triacylglycerol biosynthesis

    J. Lipid Res.

    (2008)
  • G. Almudena et al.

    Molecular and functional characterization and expression analysis of a Δ6 fatty acyl desaturase cDNA of European Sea Bass (Dicentrarchus labrax L.)

    Aquaculture

    (2009)
  • P. Carthew et al.

    Tamoxifen induces endometrial and vaginal cancer in rats in the absence of endometrial hyperplasia

    Carcinogenesis

    (2000)
  • Y. Cheng et al.

    The lipid accumulation during the stages of the ovarian fast maturation and their effect on the spawning of Eriocheir sinensis

    J. Fish. China

    (2000)
  • L. Cole et al.

    Tamoxifen induces triacylglycerol accumulation in the mouse liver by activation of fatty acid synthesis

    Hepatology

    (2010)
  • P. DeFur et al.

    Endocrine Disruption in Invertebrates: Endocrinology, Testing and Assessment

    (1999)
  • T. Egawa et al.

    Pitavastatin ameliorates severe hepatic steatosis in aromatase‐deficient (Ar−/−) mice

    Lipids

    (2003)
  • K. Fent et al.

    Ecotoxicology of human pharmaceuticals

    Aquat. Toxicol.

    (2006)
  • Cited by (13)

    • The effects of overwintering temperature on the survival of female adult mud crab, Scylla paramamosain, under recirculating aquaculture systems as examined by histological analysis of the hepatopancreas and expression of apoptosis-related genes

      2023, Aquaculture
      Citation Excerpt :

      Before the experiment, the initial water temperature (15–16 °C) was cooled or heated at a rate of 2 °C per day to reach the target temperature. Analysis of the hepatopancreas histology was conducted as described by Liu et al. (2019b). Briefly, a small portion of hepatopancreas from each sampled crab was fixed in fixative (10% neutral buffered formalin) for 24 h (two crabs/replicate, a total of 6/treatment).

    • Transcriptome analysis of hepatopancreas in penaeus monodon under acute low pH stress

      2022, Fish and Shellfish Immunology
      Citation Excerpt :

      The induction of CA in freshwater crayfish was enhanced by low pH stress [32], cytoplasmic CA in Pacific oyster was significantly increased after the reduction of seawater pH [33], and the expression of CA in Portunus trituberculatus was significantly enhanced under low pH conditions [34], and in this study, CA expression was an upregulated gene, indicating that penaeus monodon is exposed to low pH stress, the organism is involved in immune response and regulation of acid-base balance through enhanced CA catalytic activity.diacylglycerol acyltransferase catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates, DGAT gene family members include DGAT1 and DGAT2 [35,36]. DGAT1 and DGAT2 regulate lipid metabolism (ko00561) in liver tissue during exposure to environmental factors stress [37–39] and induce oxidative stress and inflammatory responses [40,41]. TIM is a key reaction in the metabolic pathway that catalyzes the isomerization of glyceraldehyde 3- pHospHate to dihydroxyacetone pHospHate [42,43], and TIM expression levels were found to be significantly upregulated in the organism when subjected to stress by biotic factors (including bacteria, viruses, parasites, etc.) or abiotic factors (including pH, temperature, etc.) in both plants and animals [44–48].

    • EDCs trigger immune-neurotransmitter related gene expression, and cause histological damage in sensitive mud crab Macrophthalmus japonicus gills and hepatopancreas

      2022, Fish and Shellfish Immunology
      Citation Excerpt :

      In the present study, vacuolization and an expanded necrotic area were observed in the hepatopancreas after exposure to BPA and DEHP (Fig. 6I – P). Severe histological alterations were observed in the hepatopancreas, including the disruption of the basement membrane and the accumulation of hemocytes, in a concentration-dependent manner, consistent with the results of previous studies [53,54]. Epithelial cell damage and structural changes in the hepatopancreas disrupted the normal functions of the liver.

    • Dietary zinc levels affects lipid and fatty acid metabolism in hepatopancreas of mud crab (Scylla paramamosain)

      2021, Aquaculture
      Citation Excerpt :

      Triglyceride (TG) and cholesterol (CHO) contents in hepatopancreas supernatant and hemolymph of crab were measured by commercial assay kits (Nanjing Jiancheng Co., Nanjing, China) according to corresponding protocols. Determinations of the hepatopancreas histology was processed as described by Liu et al. (2019) with some modifications. Briefly, hepatopancreas tissues were gradated dehydrated in gradient concentrations of ethanol, embedded in paraffin, sliced into sections of 4 μm, stained with haematoxylin and eosin (H&E), then images were obtained by a microscope (Olympus, DP72) and further measure the diameter, area and number of hepatopancreatic R cell by ImageJ.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text