Tozicology, 49 (1988) 43-48 Elsevier Scientific Publishers Ireland Ltd.

EFFECTS OF 3,3 ' ,4 ,4 ' -TETRACHLOROBIPHENYL IN T H E B R A I N OF T H E N E O N A T A L MOUSE

PER ERIKSSON

Department of Zoophysioiogy, Box 560, S-751 2~ Uppsala {Sweden}

SUMMARY

To study the retention of TCB in the brain of the immature mouse and its effects on the muscarinic cholinergic receptors in the cerebral cortex and hippocampus, [14C]TCB and TCB were administered to 10-day-old mice as a single peroral dose of 1.5 MBq/kg body wt and 0.41 mg or 41 mg/kg body wt, respectively. The mice were killed 24 h or 7 days after treatment. The amount of radioactivity in the whole brain decreased by 78% between 24 h and 7 days after administration. The density of muscarinic receptors was measured using the muscarinic antagonist [SH]QNB. A significant decrease (16°/o) in specific [3H]QNB binding was observed in the hippocampus 7 days after treatment. Despite the relatively rapid elimination of TCB from the immature mouse brain, a single low dose of TCB given during the period of rapid development of the cholinergic system appeared to affect the muscarinic cholinergic receptors in the immature mice.

K e y words: Muscarinic receptors; Brain; Chlorinated hydrocarbons; TCB; Mouse

INTRODUCTION

Polychlorinated biphenyls (PCBs) consists of a large group of chlorinated biphenyls that are widespread and persistent environmental contaminants. PCBs are bioconcentrated in the food-chain and are readily accumulated in mammals. Behavioural deviations and changes in brain transmitter metabolism have been reported from mammals exposed to commercial mixtures of PCBs [1--4]. One of the most toxic PCB congeners is 3,3',4,4'- tetrachlorobiphenyl (TCB) [5]. Neurobehavioural and neuropathological changes as well as changes in the level of dopamine and density of dopamine receptors have been observed in mice exposed to TCB in utero [6--8].

Recent studies have shown that another PCB congener, 2,2',4,4',5,5'-

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hexachlorobiphenyl (6-CB), and other chlorinated hydrocarbons, such as DDT and a chlorinated paraffin (polychlorohexadecane, PCHD), are retained in the mouse brain to a greater extent when given to pre-weanling mice at the age of 10 days [9,10]. This point in time coincides with a rapid development of the cholinergic system in the mouse brain [11--13]. It has also been seen that chlorinated hydrocarbons such as DDT and PCHD when given at low doses to the 10-day-old mouse cause changes in the variables of the cholinergic system as the muscarinic receptors and sodium-dependent choline uptake in the brain of the immature mouse [14,15].

The aim of the present s tudy was to compare the retention of TCB in the 10-day~ld mouse brain to that of the previously mentioned chlorinated hydrocarbons and to determine whether TCB affects the muscarinic cholinergic receptors in the immature mouse brain.

MATERIALS AND METHODS

The animals used were offspring of both sexes from pregnant NMRI-mice purchased from ALAB, Sweden. Li t ter size was adjusted within 24 h so that each litter contained 6 animals in the retention study and 8 - 1 0 animals in the muscarinic receptor study. Each adult female and her litter were kept in a plastic cage in a room at 22°C and subjected to a 12/12-h light/dark cycle. The animals were supplied with standardized pellet food (Ewos, Sweden) and tap water ad libitum.

Unlabelled 3,3',4,4'-tetrachlorobiphenyl (TCB) purity /> 980/0) and 14C- labelled TCB (spec. act. 259 GBq/mol, purity >t 950/0) were gifts from Dr /~ke Bergman, Section of Organic Chemistry, Wallenberg Laboratory, University of Stockholm, Sweden. The unlabelled TCB was dissolved in a mixture of egg lecithin (Merck) and peanut oil (Oleum arachidis) (1:10, w/w) and thereaf ter sonicated with water to yield a 200/0 (w/w) fat emulsion vehicle [15] containing 4.1 or 0.041 mg TCB/ml, by a method similar to that described by BrunstrSm and 0 r b e r g [16]. [14C]TCB was dispersed by gentle warming (<50°C) and ultrasonic t rea tment in a fat emulsion (Intralipid ® 200 mg/ml, Vitrum, Sweden) so as to contain about 0.11 MBq/ml.

Retention study Three to five mice at the age of 3 and 10 days received a single

administration of 1.5 MBq [14C]TCB/kg body wt per os (corresponding to 1.7 mg/kg body wt) via a PVC-tube. One mouse in each litter was used as a background control. The mice were killed by decapitation 24 h or 7 days after t rea tment and the brain (sectioned just behind the cerebellum) was removed and prepared for liquid scintillation counting (LSC) [9]. Radioactivity was measured in a Chicago Mark II LCS-system and corrected for quenching [9]. The data is presented as the total amount of radioactivity found in the brain, as per mille of the dose administered. No at tempt was made to differentiate between the radioactivity of the original substance and its metabolites.

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Muscarinic receptor binding study Ten,lay-old mice received a single administration of 0.41 or 41 mg TCB/kg

body wt per os via a PVC-tube. Mice used as controls received in the same manner 10 ml/kg body wt of the 20% fat emulsion vehicle. The animals were killed by decapitation 24 h or 7 days after the administration and the brains were removed and dissected on an ice-cold glass plate into the cerebral cortex and hippocampus [17]. A crude synaptosomal fraction (P2) (with a protein content of about 2 mg/ml) was prepared and kept frozen ( -20° C ) until the receptor assay was performed [15]. The protein content of the P2 fraction was determined by the Lowry method [18]. The muscarinic receptor assay was performed by m~asuring [SH]QNB (spec. act. 1.59 TBq/mmol; 0.2 mM, Amersham International plc, U.K.) specifically bound in the P2 fraction [15,19].

Statistics The statistical evaluation (Student's t-test) in the retention study was

made by comparing the amount of radioactivity 24 h after administration with that after 7 days. The t-values were corrected for small samples [20]. In the receptor s tudy the statistical difference between the vehicle-treated mice and the TCB-treated mice was assessed with Student 's t-test.

RESULTS AND DISCUSSION

The amount of radioactivity in the brain 24 h and 7 days after the administration of [14C]TCB to 3- and 10-day-old mice is shown in Fig. 1. The amount of radioactivity found 24 h after administration was about the same magnitude (2--50/00) as that earlier found for DDT, 6-CB and PCHD [10]. However, in both age categories there was a significant decline in retained radioactivity between 24 h and 7 days after administration. There was no

m

I~= 24 hours

(~ = 7 days

3 iO ABE

(days at time of administration}

Fig. 1. Radioactivity levels (%o of total radioactivity administered) in the mouse brain 24 h and 7 days after oral administration of 1.5 MBq[I~C]TCB/kg body wt. The height of the bars represents the mean ± S.D. for 3 to 5 animals. The statistical difference between 24 h and 7 days is indicated by asterisks: ***P ~ 0.001.

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pronounced retention of radioactivity in mice receiving [14C]TCB on the tenth postnatal day, as previously reported for DDT, 6-CB and PCHD [10]. The elimination of radioactivity from the brain in the 3- and 10-day-old mice were similar. The retention ratio, 7 days over 24 h, in the 3- and 10-day-old mice was 0.28 and 0.22, respectively. However, this does not exclude that the concentration and retention of TCB can differ between different areas of the brain. Seegal et al. [3] noticed changes in the concentration of PCBs in different brain areas depending on time following a single peroral dose of commercial mixtures of PCBs given to adult rats.

Despite the comparatively low retention of TCB in the immature mouse brain, the muscarinic cholinergic receptors appeared to be affected. There was a significant decrease in the amount of specific [3H]QNB binding sites in the hippocampus 7 days after a single peroral dose of 0.41 or 41 mg TCB/kg body wt (Fig. 2). No significant change was seen 24 h after t reatment . In the cerebral cortex no significant change was noticed either 24 h or 7 days after t reatment . The increase in [SH]QNB binding sites in both the hippocampus and cerebral cortex in control animals between 11 and 17 days is in agreement with previous investigations [11, 15].

Regional differences in response within the brain, as shown in the present study, have also been observed in adult animals exposed to PCBs [3,4]. Seegal et al., [3,4] noticed region specific changes in the concentration of norepinephrine and dopamine in the brain of adult rats exposed to high doses of commercial mixtures of PCBs. Despite the 100-fold differences between the 2 doses of TCB used in the present study there was no significant dose-related change in [3H]QNB binding sites in the hippocampus.

iO00

° 800

~ 600

. ~ ~ 400

200

0

HIPPOCAMPUS

24 hours

Con- TCB TCB tro] Ai mg 4i mg (7) {B) (7)

days

Con- TCB TCB trol .41 mg 41 mg (6) (iO) {7)

Fig. 2. [SH]QNB binding (pmol/g protein) in the hippocampus 24 h and 7 days after oral administrat ion of 0.41 mg or 41 mg TCB or 10 ml of the vehicle/kg body wt to 10-day-old mice. The brain tissues from 11- and 17-day-old mice were pooled from 3 and 2 animals, respectively. The height of the bars represents means _+ S.D. of the specific binding of [SH]QNB, which was carried out as previously described [15]. The statistical difference from the control was estimated by Student ' s t-test and is indicated by asterisks: *P ~ 0.02, **P ~ 0.01.

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This might be due to tha t the immatu re mouse brain is not biochemically complete ly developed and tha t in the growing animal the uptake and dis tr ibut ion and/or red i s t r ibu t ion of a subs tance might be dependen t on the amount tha t is given.

Most s tudies of the neuro toxic effects of PCBs on mammals have been pe r fo rmed by adminis te r ing PCB e i ther by gavage to p regnan t animals [ 6 - 8], via a cer t i f ied PCB-contaminated diet t h roughou t the ges ta t ion and lactat ion period [1,2], or d i rec t ly to adult animals [3,4]. The neurochemical and neurobehavioura l s tudies were t h e r e a f t e r pe r fo rmed on weaned animals. According to expe r imen t s by Vodicnik and Lech [21,22] and Vodicnik [23], the major i ty of the PCB congeners , regard less of the i r planari ty, are e l iminated via the milk in lacta t ing mice. In expe r imen t s where ra t s were fed a diet adu l te ra ted with cert i f ied amounts of a commercial mix tu re of PCBs, TCB was not de tec ted in the feed, bu t was found in the milk from the lactat ing ra ts and in the brain of the i r offspr ing [24]. The p re sen t s tudy shows tha t a single low dose of TCB given to pre-weanl ing mice affects the muscarinic cholinergic r ecep to r s dur ing a period of rapid deve lopment of the cholinergic sys tem. The cholinergic sys tem is involved in many behavioural phenomena such as memory and learning, neurological syndromes , audition, vision and aggression [25]. There fo re , it seems likely tha t the neurotoxic effects seen in PCB- and especially TCB-exposed animals can be caused by multiple changes within the neurons of the brain.

The p resen t resul t s f u r t he r suppor t previous s tudies [14,15] tha t have shown the cholinergic sys tem of the immatu re mouse brain to be sensi t ive to low doses of cer ta in chlor inated hydrocarbons known to be envi ronmenta l contaminants .

ACKNOWLEDGEMENTS

Dr. Ake Bergman is acknowledged for the synthes is of unlabelled and ~4C- labelled 3,3 ' ,4,4 '- tetrachlorobiphenyl. This work was financially suppor ted by grants f rom the National Swedish Env i ron m en t Pro tec t ion Board.

REFERENCES

1 J.E. Storm, J.L. Hart and R.F. Smith, Behavior of mice after pre- and postnatal exposure to Arochlor 1254. Neurobehav. Toxicol. Teratol., 3 (1981) 5.

2 R.E. Bowman and M.P. Heironimus, Hypoactivity in adolescent monkeys perinatally exposed to PCBs and hyperactive as juveniles. Neurobehav. Toxicol. Teratol., 3 {1981) 15.

3 R.F. Seegal, B. Bush and K.O. Brosch, Polychlorinated biphenyls induce regional changes in brain norepinephrine concentration in adult rats. Neurotoxicology, 6 {1985) 13.

4 R.F. Seegal, K.0. Brosch and B. Bush, Polychlorinated biphenyls produce regional alterations of dopamine metabolism in rat brain. Toxicol. Lett., 30 (1986) 197.

5 S. Safe, Polychlorinated biphenyls {PCBs) and polybrominated biphenyls (PBBs): Biochemistry, toxicology, and mechanism of action. CRC Crit. Rev. Toxicol., 13 {1984) 319.

6 S.M. Chou, T. Miike, W.M. Payne and G.J. Davis, Neuropathology of "spinning syndrome" induced by prenatal intoxication with a PCB in mice. Ann. N.Y. Acad. Sci., 320 (1979) 373.

7 H.A. Tilson, G.J. Davis, J.A. McLachlan and G.W. Lucier, The effects of polychlorinated

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biphenyls given prenatally on the neurobehavioral development of mice. Environ. Res., 18 (1979) 466.

8 A.K. Agrawal, H.A. Tilson and S.C. Bondy, 3,4,3',4'-tetrachlorobiphenyl given to mice prenatally produces long-term decrease in striatal dopamine and receptor binding sites in the caudata nucleus. Toxicol. Lett., 7 (1981) 417.

9 P. Eriksson, Age-dependent retention of [I~C]DDT in the brain of the postnatal mouse. Toxicol. Lett., 22 (1984) 323.

10 P. Eriksson and P.O. Darnerud, Distribution and retention of some chlorinated hydrocarbons and a phthalate in the mouse brain during the pre~weaning period. Toxicology, 37 (1985) 189.

11 Y. Falkeborn, C. Larsson, A. Nordberg and P. Slanina, A comparison of the regional ontogenesis of nicotine- and muscarine-like binding sites in mouse brain. Int. J. Dev. Neurochem., 1 (1983) 187.

12 C.C. H6hman and F.F. Ebner, Development of cholinergic markers in mouse forebrain. I. Choline acetyltransferase enzyme activity and acetylcholinesterase histochemistry. Dev. Brain Res., 23 (1985) 225.

13 C.C. H6hmann, C.C. Pert and F.F. Ebner, Development of cholinergic markers in mouse forebrain. II. Muscarinic receptor binding in cortex. Dev. Brain Res., 23 (1985) 243.

14 P. Eriksson, Y. Falkeborn, A. Nordberg and P. Slanina, Effects of DDT on muscarine- and nicotine-like binding sites in CNS of immature and adult mice. Toxicol. Lett., 22 (1984) 329.

15 P. Eriksson and A. Nordberg, The Effects of DDT, DDOH-Palmitic acid, and a chlorinated paraffin on muscarinic receptors and the sodium-dependent choline uptake in the central nervous system of immature mice. Toxicol. Appl. Pharmacol., 85 (1986) 121.

16 B. Brunstr6m and J. (3rberg, A method for studying embryotoxicity of lipophilic substances experimentally introduced into hens' eggs. Ambio, 11 (1982) 209.

17 J. Glowinski and L.L. Iversen, Regional studies of catecholamines in the rat brain. I. The disposition of [aH]norepinephrine, [SH]dopamine and [SH]dopa in various regions of the brain. J. Neurochem., 13 (1966) 655.

18 0.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265.

19 H.I. Yamamura and S.H. Snyder, Muscarinic cholinergic binding in rat brain. Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 1725.

20 J.B. de V. Weir, Standardized t. Nature, 185 (1960) 558. 21 M.J. Vodicnik and J.J. Lech, The transfer of 2,4,5,2',4',5'-hexachlorobiphenyl to fetuses and

nursing offspring. I. Disposition in pregnant and lactating mice and accumulation in young. Toxicol. Appl. Pharmacol., 54 (1980) 293.

22 M.J. Vodicnik and J.J. Lech, The transfer of 3,4,5,3',4',5'-hexachlorobiphenyl from mothers to fetuses and nursing offspring. The Toxicologist, 2 (1982) 136.

23 M.J. Vodicnik, The effect of pregnancy and lactation on the disposition of [2,4,2',4'- l~C]tetrachlorobiphenyl in the mouse. Fundam. Appl. Pharmacol., 6 (1986) 53.

24 W. Shain, S.R. 0vermann, L.R. Wilson, J. Kostas and B. Bush, A congener analysis of polychlorinated biphenyls accumulating in rat pups after perinatal exposure. Arch. Environ. Contam. Toxicol., 15 (1986) 687.

25 A.G. Karczmar, Cholinergic influences on behavior, in P.G. Waser (Ed.), Cholinergic Mechanisms, Raven Press, New York, 1975, pp. 501- 529.

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