Elsevier

Aquatic Toxicology

Volume 69, Issue 3, 25 August 2004, Pages 229-245
Aquatic Toxicology

Effects of the brominated flame retardants hexabromocyclododecane (HBCDD), and tetrabromobisphenol A (TBBPA), on hepatic enzymes and other biomarkers in juvenile rainbow trout and feral eelpout

https://doi.org/10.1016/j.aquatox.2004.05.007Get rights and content

Abstract

Brominated flame retardants (BFRs) leak out in the environment, including the aquatic one. Despite this, sublethal effects of these chemicals are poorly investigated in fish. In this study, a screening of selected biomarkers in juvenile rainbow trout (Oncorhynchus mykiss) and feral eelpout (Zoarces viviparus) was performed after exposure to hexabromocyclododecane (HBCDD) and tetrabromobisphenol A (TBBPA).

Rainbow trout was injected intraperitoneally (i.p.) with HBCDD or TBBPA. Two out of four short-term experiments with HBCDD showed an increase in the activity of catalase. A 40% increase in liver somatic index (LSI) could be observed after 28 days. HBCDD did also seem to have an inhibitory effect on CYP1A’s activity (ethoxyresorufin-O-deethylase (EROD)). A putative peroxisome proliferating activity of the compound was investigated without giving a definite answer. HBCDD did not seem to be estrogenic or genotoxic. TBBPA increased the activity of glutathione reductase (GR) after 4, 14 and 28 days in rainbow trout suggesting a possible role of this compound in inducing oxidative stress. The compound did not seem to be estrogenic. TBBPA seemed to compete with the artificial substrate ethoxyresorufin in vitro, during the EROD assay.

In eelpout, only one 5 days in vivo experiment was performed. Neither of the compounds gave rise to any effect in this fish.

This was the first screening of sublethal effects of the two chemicals in fish, using high doses. Our results indicate that there is a need for further studies of long-term, low-dose effects of these two widely used flame retardants.

Introduction

Brominated flame retardants (BFRs) are added to polymeric materials in order to interfere with the combustion process in different ways, e.g. by emission of brominated species into the gas phase. The species will destroy the radicals Hradical dot and OHradical dot, which are produced in the early stage of the fire (Luijk and Govers, 1992).

Flame retardants can be either reactive (i.e. covalently bound to the polymer) or additive. Electronic and electric devices stand for about 70% of the consumption of BFRs. Model calculations have shown that these compounds by large are spread to the environment by volatilisation, from production, use and from waste disposal sites, but as they often are both persistent/semi-persistent and lipophilic they also accumulate in sediment or living organisms (Sellström et al., 1998).

Tetrabromobisphenol A (TBBPA) (Fig. 1) has the largest production volume among brominated flame retardants in the world (over 120,000 tons annually or 30% of all BFRs) (WHO/ICPS, 1995). The primary application is in epoxy polymers used in circuit boards and in enclosing material in electrical components. TBBPA is used mostly as a reactive retardant. During incomplete combustion such as spontaneous fires at waste disposal sites, polymers containing TBBPA can generate polybrominated dibenzofurans (PBDFs) and polybrominated dibenzodioxins (PBDDs) (Luijk and Govers, 1992).

TBBPA has a very high acute toxicity to algae, the water flea Daphnia magna, mysid shrimp Mysidopsis bahia and fish (KemI, 16/95, 1995).

In an in vitro study (Brouwer, 1998), TBBPA bound strongly to human transthyretine, a thyroxine binding and transporting protein. TBBPA could thus act as a competitor to thyroxine and disturb the endocrine system in the organism. This binding was, however, not seen in vivo (Meerts et al., 1999).

There are few data about the levels of TBBPA in biota: the compound was e.g. found in human blood plasma in low ng/g lipid weight range (de Wit, 2002).

Hexabromocyclododecane (HBCDD) (Fig. 2) is used as an additive flame retardant for thermoplastic polymers. Its principal use is in expanded polystyrene foams used for insulation in building industry, but it is also used in textile coatings such as furniture and car interior textiles, polyvinylchloride wire/cable, latex and even in isolation put beneath road surfaces, although it does not fulfill any purpose there (Bernes, 1998, de Wit, 2000, National Academy of Sciences, 2000). In 1998, the consumption of HBCDD was 14% of the total consumption of brominated flame retardants in Western Europe (Miljøstyrelsen, 494, 1999), and the world total production numbers probably goes beyond 3000 tons/year (Bergman, 1999; in Swedish). HBCDD was further reported in both sediment and fish in the Swedish river Viskan, close to several textile industries (Sellström et al., 1998).

HBCDD has been found in eggs of guillemot (Uria aalge) (∼100 ng/g lipid weight) in the Baltic. Guillemot feeds on pelagic fish, e.g. sprats (Sprattus sprattus) and herring (Clupea harengus) (Lundstedt-Enkel et al., 2001). In another study HBCDD has been found in herring (4.9–9.8 ng/g fat in Bothnian Bay and northern Baltic Proper, ∼32 ng/g fat in southern Baltic Proper) (Nylund et al., 2001). The 10-fold higher levels of HBCDD in guillemot eggs than in herring seem to indicate that HBCDD biomagnifies. The levels of HBCDD in guillemot eggs were also found to increase in a temporal study performed between 1969 and 1997 (Kierkegaard et al., 1999).

HBCDD was negative in Ames test (KemI 16/95, 1995), but caused a statistically significant, dose-dependent increase in recombination frequency in cultured mammalian cell lines SPD8 and Sp5, an activity that can provoke cancer (Helleday et al., 1999).

We have measured the effects of HBCDD and TBBPA in vivo on several biomarkers in rainbow trout (Oncorhynchus mykiss), a fresh water fish with well-known physiology, commonly used in laboratory studies. We also measured the effects in vivo on eelpout (Zoarces viviparus), a marine fish. The eelpout is an abundant and relatively stationary viviparous fish, used in Swedish and German biomonitoring programs. The biomarkers employed in this study were: changes in the activities of the enzyme CYP1A (measured as ethoxyresorufin-O-deethylase or EROD), glutathione-S-transferase (GST), and the antioxidant enzymes glutathione reductase (GR) and catalase, induction of vitellogenin (VTG) in male fish, formation of DNA adducts, and finally liver somatic index (LSI).

EROD activity is induced by planar molecules, such as polycyclic aromatic hydrocarbons (PAHs), planar polychlorinated biphenyls (planar PCBs) and dioxins (including the very potent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) through a cytosolic receptor—the aryl hydrocarbon receptor or AHR (e.g. Landers and Bunce, 1991, Xiao et al., 1995, Hahn, 1998, Waxman, 1999). EROD activity is an established biomarker in fish (e.g. Haux and Forlin, 1988, Forlin et al., 1995, Whyte et al., 2000, Oost et al., 2003).

GST conjugates electrophilic substrates with the tripeptide glutathione, making them more water-soluble and easier to excrete in urine or bile. Examples include PAH-epoxides produced during CYP-mediated metabolism of PAHs.

Oxidative stress is a condition when the defenses of an organism can no longer get rid of all the undesired radicals and other reactive oxygen species (ROS), which can result in damage to lipids, proteins and DNA (Timbrell, 1991, Stephensen et al., 2002). The cytosolic enzyme GR and mainly peroxisomal enzyme catalase are important part of that defense. The formation of ROS may be enhanced by xenobiotics, e.g. through induction of cytochrome P450 system, Fenton reaction involving free metal ions, or through uptake of lipophilic xenobiotics into membranes resulting in disturbance of electron flow between components of the cytochrome P450 system (Lemaire and Livingstone, 1993).

VTG is a yolk-precursor produced in the liver of mature female fish in response to the 17β-estradiol (Mommsen and Walsh, 1988). It is exported into the blood and absorbed by the developing oocytes. Foreign substances mimicking endogenous estradiol, such as alkylphenolic compounds, synthetic estrogen (e.g. from contraceptive pills) and some pesticides can induce an increase of vitellogenin blood plasma levels in males and juveniles as well as in females. Induction of vitellogenin in male and juvenile fish, measured in blood plasma thus becomes a biomarker for foreign, estrogenic chemicals (Sumpter and Jobling, 1995, Sumpter et al., 1996, Larsson et al., 1999).

Some substances, especially electrophilic ones may bind to DNA forming DNA-adducts. This can lead to DNA-damage, mutations and ultimately cancer. Measuring the formation of DNA-adducts is used as a biomarker for the genotoxicity of a compound (Walker et al., 1996).

In addition LSI (liver weight as percentage of whole body weight (bw)) was measured. An enlargement of the liver is often seen in fish living in areas polluted with stable organic substances. This enlargement could be a result of increased deposit of fat and glycogen and/or increased protein synthesis in the hepatocytes. This is possibly caused by pollutant-induced disturbances of metabolism and/or induction of biotransformation enzymes by the pollutants (Andersson et al., 1988).

Section snippets

Aim

The data concerning the sublethal effects of TBBPA and HBCDD on fish are very limited. The aim of this study was to perform basic screening of such effects on the selected biomarkers in juvenile rainbow trout and feral eelpout, fish species used in laboratory and for biomonitoring of the aquatic environment.

Chemicals

HBCDD (Broomchemie V.B., Terneuzen, The Netherlands) was generously provided by Åke Bergman at the University of Stockholm. TBBPA (Aldrich, Milwaukee, USA) was a gift from Ulrika Örn, also at the University of Stockholm. 1-Chloro-2,4-dinitrobenzene (CDNB), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), 7-ethoxyresorufin, oxidised glutathione (GSSG), reduced glutathione (GSH), reduced nicotinamide adenin dinucleotide phosphate (NADPH) and 2,4-dichlorophenoxyacetic acid (2,4-D) were bought from

In vivo experiments with rainbow trout given TBBPA

Due to the large number of doses, not all injections were made on the same day. In order to present more clear charts, we decided to show the responses as percentages of control levels (Fig. 3, Fig. 4, Fig. 5, Fig. 6).

Rainbow trout and TBBPA

TBBPA seemed to give rise to two effects: GR activity was induced by the 100 mg/kg-dose after 4, 14 and 28 days and also in the combination experiment with BNF after 4 days (but it was reduced after 1 day) in vivo and the compound inhibited the EROD activity in vitro. Recently, induction of GR activity was indicated to be one of the most sensitive biomarker for oxidative stress in fish in laboratory studies (Stephensen et al., 2002). This could point to a possible oxidative stress-inducing

Final remarks

TBBPA seemed to induce oxidative stress in vivo in rainbow trout. It inhibited EROD activity in vitro, possibly through competition with the ethoxyresorufin, which means that it may interfere with metabolism of CYP1A substrates. HBCDD caused an increase in LSI after 28 days in rainbow trout and it also strongly inhibited EROD activity after that time. It seemed to inhibit CYP1A protein (measured in the 5 days experiment only). The reason for this is unknown. There is also a possibility that

Acknowledgements

This study was generously supported by grants from the Swedish Foundation for Strategic Environmental Research (MISTRA), Helge Axson Johnson Foundation, The Royal Swedish Academy of Sciences, Swedish Council for Work Life Research, Swedish Environmental Protection Agency and The Swedish National Chemicals Inspectorate. We would also like to thank Gunilla Ericson and Margareta Adolfson-Erici for the analysis of the DNA-adducts and bile, respectively.

References (64)

  • L. Förlin et al.

    Biochemical and physiological effects in fish exposed to bleached kraft mill effluents

    Ecotoxicol. Environ. Safety

    (1995)
  • C. Garcia-Alfonso et al.

    Regulation of horse-liver glutathione reductase

    Int. J. Biochem.

    (1993)
  • W.H. Habig et al.

    Glutathione S-transferases. The first step in mercapturic acid formation

    J. Biol. Chem.

    (1974)
  • M.E. Hahn

    The aryl hydrocarbon receptor. A comparative perspective

    Comp. Biochem. Physiol.

    (1998)
  • T. Helleday et al.

    Brominated flame retardants induce intragenic recombination in mammalian cells

    Mutat. Res.

    (1999)
  • M. Hermes-Lima et al.

    Quatification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation

    Free Radic. Biol. Med.

    (1995)
  • S. Jobling et al.

    Comparative responses of molluscs and fish to environmental estrogens and estrogenic effluent

    Aquat. Toxicol.

    (2004)
  • D.G.J. Larsson et al.

    Ethinylestradiol: an undesired fish contraceptive?

    Aquat. Toxicol.

    (1999)
  • C. Lindholst et al.

    Estrogenic response of bisphenol A in rainbow trout (Oncorhynchus mykiss)

    Aquat. Toxicol.

    (2000)
  • O.H. Lowry et al.

    Protein measurements with the Folin reagent

    J. Biol. Chem.

    (1951)
  • R. Luijk et al.

    The formation of polybrominated dibenzo-p-dioxins (PBDDs) and dibenzofurans (PBDFs) during pyrolysis of polymer blends containing brominated flame retardants

    Chemosphere

    (1992)
  • D. Ronisz et al.

    Interaction of isosafrole, beta-naphthoflavone and other CYP1A inducers in liver of rainbow trout (Oncorhynchus mykiss) and eelpout (Zoarces viviparus)

    Comp. Biochem. Physiol.

    (1998)
  • A.E.C.M. Simpson

    The cytochrome P450 (CYP4) family

    Gen. Pharm.

    (1997)
  • E. Stephensen et al.

    Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver

    Comp. Biochem. Physiol.

    (2002)
  • K. Van den Belt et al.

    Comparison of vitellogenin responses in zebrafish and rainbow trout following exposure to environmental estrogens

    Ecotoxicol. Environ. Safety

    (2003)
  • J.P. Vanden Heuvel

    Peroxisome proliferator-activated receptors: a critical link among fatty acids, gene expression and carcinogenesis

    J. Nutr.

    (1999)
  • D.J. Waxman

    P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR

    Arch. Biochem. Biophys.

    (1999)
  • J.-H. Yang et al.

    Induction of peroxisome proliferation in rainbow trout exposed to ciprofibrate

    Toxicol. Appl. Pharmacol.

    (1990)
  • Aebi, H., 1984. Catalase. In: Bergmeyer, H.U. (Ed.), Methods in Enzymatic Analysis, vol. II. Academic Press, New York,...
  • T. Andersson et al.

    Physiological disturbances in fish living in coastal water polluted with bleached kraft pulp mill effluents

    Can. J. Fish. Aquat. Sci.

    (1988)
  • Bergman, Å., 1999. Brominated Flame Retardants (BFR)-A proposal for further studies within the program “A New Strategy...
  • Bernes, C., 1998. Organic Environmental Toxins (Organiska miljögifter), Monitor 16. Published by the Swedish...
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