Document Yrk6Q20ymd2VLw1715z4aOG0E

Reduction of Pentobarbital-induced Sleeping Times in PCIMrented Cottontail Rabbits by It. L. 7.t>*i\ Jn.p 0. T. Sandliis, and H. L. Kiuwi'.vtmck Department uf fisheries and Wildlife Sciences Virginia I'olytei hnic Institute and State Unit ersity Blacksburg, I 'a. 24061 Many scientists have expressed concern about the *ffcrts environmental pollutants may have on wild animal populations, although few studies have demonstrated that small levels of contaminants can have marked influences on the physiology of wild aaimnallan species. Polychlorinated biphenyls (PCBs) are well known pollutants whose significance within various ecosystems It just beginning to be understood. One property of PCBs, as with other chlorinated hydrocarbons, Is their ability to stimulate hepatic microsomal enzymes. Ingestion of 10 ppm PCB for 28 daya by domestic rabbits significantly increased the activity of microsomal enzymes in liver homogenates (VlLLENEl/VE et al. 1971a). Wistar rats fed 20 ppra of the PCB Arocior 1254, for 30-350 days exhibited shorter sleeping times, indirectly Indicating induced hepatic enzyme activity (VILLENEUVE et al. 1972). Barbitutteinduced sleeping time is a commonly employed technique used for indirectly measuring hepatic microsomal enzyme induction (PUYEAR and PAULSON 1972, SANDERS et el. 1973, VILLENEUVE et al. 1972). We have employed this method to demonstrate hepatic microsomal enzyme Induction in a captive wild mamnal, the cottontail rabbit (Svlvllaitus florldanus). fed a ration containing 10 ppm Arocior 1254 for ten to eleven weeks. METHODS Wild cottontail rabbits were llve-trepped during December, 1972, and feeding of the experimental diet began on January 22, 1973. The experimental design was a 2 x 2 factorial arrangement with two non-PCB-treated groups receiving e conwerel.il rabbit ration (Purina Rabbit Chow, 167. protein) ad llblturn or 75% ad libitum. The two remaining groups (PCB-treAtod) received the same two levels of the ration treated with Arocior 1254 . The PCB was dissolved in acetone and sprayed onto tha chow while mixing it on a large feed mixer. The a libitum ration contained 10 mg Arocior 1254 per kg of chow (10 ppm). The restricted rstton was adjusted in order to provide the same total PCB Intake ss the ad libitum group. Therefore, 13.3 mg Arocior 1254 was added to each kg of chow. The rabbits were maintained on these diets for 12 weeks. During the tenth week^all males (17) were injected with sodi um pentobarbital (Nembutal ) at a level of 45 mg/kg body weight *Aroclor 1254 generously supplied by Monsanto Company, St. Louis Mo., 63166. (Lot No. KB-01-604). \ ol. !3, Kv. & S 1171 Jy S|ir swy viwjwywp,i sia ^{i^rnmnnsr^iysy1 O8105A HONS ''i ': I (measured to the nearest 100 gn). All females (49) were injected the following week with 40 mg/kg. Sleeping times were measured s the interval from Injection of the barbltuate to the regaining of the righting ability. RESULTS Rabbits treated with PCB at a level of L0 ppm, exhibited significantly shorter periods of sleep (Tables 1 and 2). Sleep ing times were shorter both in PCB-treated males (P<.01) and PCBtreated females (P<,05). Level of nutrition had no significant effect on sleeping tlsws in either sex and thaxe wara no algnlflesnt interactions of PCB and dietary level. Table 1 Id Sleeping times for swila cottontail rabbits*. Treatment Diet No. of Animals Sleeping Times (minutes + S.E.M.) PCBb (Aroclor 1254) Ad libitum 75% Ad lib. 4 5 116.3 + 12.2' 118.0 + 16.9 Non-PCB Ad libitum 75% Ad lib. 4 3 162.8 + 21.8' 231.0 + 30.2 KK a Sodium pentobarbital (Nembutal ) lnjectad at 45 mg/kg body waight. b Significantly different from the non-PCB treated group (P<.01). e,d Meant bearing different superscripts are significantly dif ferent from eech other at the 5% Level (Duncen's Multiple Range Test) Table 2 Sleeping tines for female cottontail rabbits*. Treatment Diet No. of Animals Sleeping Times (minutes + S.E.M.) d PCB1* Ad libitum 10 on (Aroclor 1254) 75% Ad lib. 13 161.9 + 16.5C<1 129.7 + 11.4C Non-PCB Ad libitum 75% Ad lib. 10 11 157.8 + 17.7'd 174.1+ 8.9 1 Sodium pentobarbital (Nembutal ) Injected at 40 mg/kg body weight. b Significantly different from the non-PCB treated groups (P<.05). c,d Means bearing different superscripts ere significantly different from each other at the 57. level (Duncan's Multiple Range Test) ........... * -7'- ',7:*v;. > *<:- > VA i m HONS 081855 Tli# lengths of time for rabbits to succumb to the sedative effects of the pentobarbital were quite variable. However, the differences were not related to treatment. Five females, two from the ad libitum control group, one from the restricted con* trol, and two from the ad libitum PCB group, failed to aleep. One male from the ad libitum PCB group alao failed to sleep. These lx animals were not Included In the calculations. DISCUSSION Although some information has been published concerning the ability of PCBs to Induce hepatic microsomal enzymes In laboratory animals or birds, little Information is available pertaining to wild sunmnals. These results suggest thst the cottontail rabbit, like the domestic rabbit, responds to relatively low levels of these compounds. Further studies are necessary to determine If enzyme induction is related to lowered reproductive function. VILLENBUVE at al.(1971a) have reported Increased carboxyletters## activity in liver homogenates taken from pregnant female labora tory rabbits fed with 10 ppm Aroclor 1254 during the 2S days of gestation. Carboxylesterase activity was used as an indicator of microsomal enzyme activity. Aniline hydroxylase and amlnupyrlne N-demethylaae, both drug-metabolising enzymes, were also significantly induced. These same workers havs reported placental transfer of PCBs and have shown PCBs to be fetopathlc at doses of 12.5 and 50 mg/kg/day (GRANT at al. 1971, VILLENEUVE et al. 1971b). However, ICR strain albino mice exhibited no apparent enzyme induction at dietary levels of 62.5 ppm Aroclor 1254. Levels of 250 and 1000 ppm were necessary to Induce liver enzyaws, demonstrating species-specific sensitivity to the PCB (SANDERS et al. 1973). Although level of nutrition had no significant effect on sleeping tlsws as shown by analysis of variance, there was a trend when the means ware analysed by Duncan's multiple range test for non-PCB restricted animals to sleep longer. DIXON et el. (I960) have shown that starvation decreases detoxication of druga; however RATO (1967) and WEATHERH0L12 and WEBB (1971) have reported an increase in ensyme Induction due to starvation or pro tein restriction. The fact that we have demonstrated Induction of liver enzyme systems In a wild animal is In Itself, not surprising, but the fact Chat such induction was caused by a relatively low level of PCB ingestion should certainly stimulate further study of the effects this Increased activity may have on reproduction and the animals' physiology. SUMMARY Pentobarbital sleeping time was used to examine the effects of Aroclor 1254 on hepatic microsomal enzyme activity in cotton tail rabbits. Ten ppm fed for 10-11 weeks significantly rsduced sleeping times in both sexes. Restriction of food intake had no conslatant effect on sleeping times. $20 Ml ... V ,:r.. i- HQNS 081856 '-",,$ ,.v.,Ji.^-*:1.'.,``,j v'-' %?; - *';* w'M:.. .,: ;,}-v.." .' v ;:/:^i-:^, V`*`-'Vvv' ;' *.> VVr ';!<:'* i`->v-"'-l; V '':"/.:' ;** ' ,.',v-,sV'.^^,/.*.:..,-* t...';'V'x' .$: v5;vov'' f.wAi'd'i ,f v *ati fia i.*.!.V .<,,? i -^wtvf. ' DIXON, R.L., R.W. SHULTICE, J.K. FOUTS: Proc. Soc. Exp. Biol. Med 103. 333 (1960). CHANT, D.L., D.C. VILLENEUVE, K.A. MCCULLY, W.E.J. PHILLIPS: Environ. Physio. 1, 61 (1971). NATO, A.: Uiochera. Phanacol. 16, 671 (1967). TWEAK, R.L. and G.D. PAULSON: Toxicol. Appl. Pharmacol. 22, 621 (1972). SANDERS, O.T., R.L. ZKPP, R.L; KIRKPATRICK: Submitted for publi cation (1973). V1LLKNEUVK, D.C., D.L. CRANT, W.E.J. PHILLIPS, M.L. CLARK, D.J. CLEGG: Bull. Environ. Concata. Toxicol. ' 120 (1971a). V1LLKNKUVK, D.C., D.L. GRANT, K. KHERA, D.J. CLEGG, H. BAER, W.E.J. PHILLIPS: Environ Phytlo. 1, 67 (1971b). VILLKNKUVE, D.C., D.L. GRANT, W.E.J. PHILLIPS: Bull. Environ. Contaou Toxicol. 2, 264 (1972). WPATHEKHOLTZ, W.M. and R.B. WEBB: J. NuCr. 101. 9 (1971). \ . ., hktv'WtZi ::ht.XX-Zi bo. *:"*$ 'f' viJT. SsP'y- ?n*pa i* ;? ' 'V v I- o;. - 1 - MONS O01B57