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
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 H and OH, 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.
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2022, Science of the Total EnvironmentCitation Excerpt :The potential grey literature (e.g. conference proceedings and listed references) was also checked. A total of 56 studies were retained in this study (Abe et al., 2021; Arukwe et al., 2018; Breitholtz and Wollenberger, 2003; Chan and Chan, 2012; Cheng et al., 2017; Chou et al., 2010; Dong et al., 2018; Feng et al., 2013a, 2013b, 2014; Folle et al., 2021; Gagné et al., 2017; Giraudo et al., 2015; Han et al., 2017; He et al., 2015; Hu et al., 2015; Huang et al., 2017; Jian et al., 2017; Jiang et al., 2017, 2018, 2021; Kang et al., 2016; Key et al., 2008; Lee et al., 2012; Li et al., 2015, 2018, 2019, 2020a, 2020b, 2020c; Liu et al., 2013, 2019; Nakari and Huhtala, 2010; Oliveri et al., 2015; Parolini and Binelli, 2012; Ronisz et al., 2004; Sha et al., 2015; Sun et al., 2016; Torres et al., 2013; Usenko et al., 2016; Wang et al., 2015b, 2015c, 2015d, 2019, 2021; Wu et al., 2016; Xia et al., 2021; Xu et al., 2015; Yang et al., 2013, 2015, 2021; Zeng et al., 2018; Zhang et al., 2016, 2021a, 2021b; Zhao et al., 2021). The inclusion criteria were: (i) peer-reviewed research paper; (ii) the exposed chemicals were OFRs; (iii) the tested species were aquatic animals; (iv) at least one OFRs-treated group presented the hormetic effect; (v) the determination of hormesis was that the stimulatory response to at least one dose below the no-observed-adverse-effect-level (NOAEL) was 10% or more different from the control (i.e. ≥ 110% of the control response), or less than 10%, but with statistical difference (Calabrese and Blain, 2011; Agathokleous et al., 2019b).