Impaired behaviour, learning and memory, in adult mice neonatally exposed to hexabromocyclododecane (HBCDD)
Introduction
Brominated flame-retardants (BFRs) are a new, diverse group of global environmental pollutants (de Boer et al., 1998, de Wit, 2002). The BFRs most frequently used are hexabromocyclododecane (HBCDD) and the polybrominated diphenyl ethers (PBDEs). These chemicals are added to variuos materials, such as electronic equipment, paints and textiles, to prevent them from catching fire. HBCDD is also widely used in the building industry as a thermal insulator additive in polystyrene foam. Neither HBCDD nor the PBDEs bind covalently to the polymer product to which they are added, and can thus leach into the environment throughout the lifetime of the product (Hutzinger et al., 1976, Hutzinger and Thoma, 1987).
Hexabromocyclododecane, C12H18Br6, has a lipophilicity value of log Kow 5.6. This can be compared to the PBDEs having a log Kow ranging between 4.28 and 9.9, with higher values for the more highly brominated congeners. The physical and chemical properties of HBCDD are similar to those of PBDEs and other persistent organic pollutants, including polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT), which are known to be persistent and bioaccumulative (de Wit, 2002, Sellström et al., 1998, Tomy et al., 2004).
In contrast to the more classical persistent organic pollutants like DDT and PCBs, the BFRs are steadily increasing in our environment. In Sweden, PBDEs were found to increase exponentially in mother's milk from 1972 to 1992 (Meironyté and Norén, 1999, Norén and Meironyté, 2000) and in U.S. the levels of PBDEs in sewage sludge have been found to exceed the PCB levels (Hale et al., 2001, Hale et al., 2003). Less is known about HBCDD, though it is reportedly present in the aquatic food web, and in different fish species (Sellström et al., 1998, Tomy et al., 2004, Gerecke et al., 2003, Remberger et al., 2004). A recent report indicates also that HBCDD is present in eggs of peregrine falcons (Lindberg et al., 2004).
Exposure to low doses of persistent environmental agents, e.g. PCBs and DDT, during neonatal brain development in mice, has been shown to induce irreversible disruption in adult brain function (Eriksson, 1997, Eriksson et al., 2001b). In recent studies, we have shown that neonatal exposure to three different PBDEs, PBDE 47, PBDE 99 and PBDE 153, on postnatal day 10, induced persistent brain dysfunction in adult male NMRI mice (Eriksson et al., 2001a, Eriksson et al., 2002, Viberg et al., 2002, Viberg et al., 2003a). The effects observed were: altered spontaneous motor behaviour, including reduced habituation capabilities; impaired learning and memory faculties; and changes in the cholinergic transmitter system. The behavioural defects were seen to worsen with increasing age.
The persistent effects are induced during a defined critical period in the neonatal development of the mouse brain, namely around postnatal day 10 (Eriksson et al., 1992, Eriksson et al., 2000, Ahlbom et al., 1995, Eriksson, 1998), which coincides with the period of rapid brain growth—the ‘brain growth spurt’ (BGS) (Davison and Dobbing, 1968). This is when the brain undergoes several fundamental changes, such as dendritic and axonal outgrowth and the establishment of neural connections (Davison and Dobbing, 1968, Kolb and Whishaw, 1989). During the BGS, animals also acquire many new motor and sensory abilities (Bolles and Woods, 1964) and their spontaneous behaviour peaks (Campbell et al., 1969). The BGS is associated with numerous biochemical changes that transform the feto-neonatal brain into that of the mature adult (Coyle and Yamamura, 1976, Fiedler et al., 1987). In mammals, the timing of the BGS in terms of onset and duration varies from one species to another. In the human, it begins during the third trimester of pregnancy and continues throughout the first 2 years of life. In rodents, however, the BGS is neonatal, spanning the first 3–4 weeks of postnatal life and reaching its peak around day 10.
There is a lack of knowledge regarding the toxic and – especially the developmental toxic – effects of HBCDD. Some studies have found a low acute toxicity in mammals and also low repeated dose toxicity with LOAEL around 100 mg/kg body weight (for reference, see Darnerud, 2003). One developmental toxicity study in rats exposed to HBCDD on days 0–20 of gestation reported a NOAEL of 75 mg/kg body weight/day (for reference, see Darnerud, 2003). However, a recent in vitro study by Mariussen and Fonnum (2003) showed that HBCDD can have an effect on the plasma membrane uptake of neurotransmitters such as dopamine, glutamine and GABA in rat brain synaptosomes at concentrations similar to those for PCBs. They also showed that HBCDD can inhibit the vesicular uptake of dopamine similar to that of PBDEs.
In view of the lack of in vivo data on developmental neurotoxicity and the reported similarities in in vitro neurotoxicity between HBCDD and the PCBS and PBDEs, the present study was undertaken to investigate the developmental neurotoxic effects of HBCDD on the developing brain in neonatal mice, when administered during the critical window of BGS. This was achieved by measuring (a) spontaneous behaviour, and (b) spatial learning and relearning ability.
Section snippets
Chemicals and animals
Pregnant NMRI mice, purchased from B&K, Sollentuna, Sweden, were housed individually in plastic cages in a room with an ambient temperature of 22 °C and a 12-h light/12-h dark cycle. The animals were supplied with standardized pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. The size of the litters was adjusted to 10–12 mice, within the first 48 h after birth, by killing excess pups. The litters contained pups of both sexes during the neonatal period and no separation with
Results
There were no clinical signs of dysfunction in the treated mice throughout the experimental period, nor were there any significant deviations in body weight gain in the HBCDD-treated mice, compared with the vehicle-treated mice.
Discussion
The present study has shown that neonatal exposure to HBCDD (0.9 mg HBCDD and 13.5 mg HBCDD/kg body weight) on postnatal day 10 can cause persistent aberrations in spontaneous behaviour in the adult animals, when 3 months old. Furthermore, neonatal exposure to HBCDD also affected learning and memory functions in adult animals. These defects were dose–response related.
The spontaneous motor behaviour data showed a dose–response related disruption of habituation in mice neonatally exposed to HBCDD.
Acknowledgements
Financial support was provided by the Swedish Research Council for Environmental, Agricultural Sciences and Spatial Planning, Foundation for Strategic Environment Research and the Swedish Environmental Protection Board.
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