Document pe3aamroy6eJwJwRmJRMyR17

i'o .Lvnnuio whether salamanders f^.ticd wiih bemicholimum for '.Vefci-wen; pcrnwnci-.tly poisoned, wg amputated the regenerating teg and al^ .\1 it to regrow without further , ',;r;iinn of a drug. Regeneration ,s i.icntical in that in controls, indi- .. that (lie clTcct of Insmicholintum v .a jcvcrsihlk'. To demonstrate that the in. ;.il amputation did not stimulate Mii'sci|iicni ugcnoratioo we measured die growth rate* 10 weeks after the first amputation and similarly after a r*a -.ipuiation. The first and second rates of regeneration were identical in cacti of two salamandeh studied. We then considered that treatment with hcmicholinium might inhibit growth be cause 25 percent of the body weight was lost during treatment Few that study, the hind limb of an untreated salamander waa amputated, but no food waa given to the animal during the I0week regeneration period. Despite a weight lost of 50 percent the rate of limb growth waa not different from that in previous controls. The diminished growth rate in treated salamanders correlated well with tho microscopic findings. Salamanders treated for 5 weeks with bemlchoUninm showed only about 30 mitotic figures iu the median sagittal section of the regenerate. However, more than 400 were seen in similar sections from con trols. Colchicine (2 mg/kg) waa in jected into both groups 12 hours before the salamanders were killed so that mitotic figures would accumulate and could be counted. Evidently hstnieho- linium did not inhibit regeneration by means of an effect like that of colchi cine. f. r far fewer mitotic figure! were seen among treated specimens then among controls. Furthermore, the feet that the mesenchymal cells were not en larged argues against the possibility that hemiehoUnium blocks oeQ division at an earlier stage. The drag also prevented the massive dedlffereatlerton of muscle tissue normally seen In seotioee )ust proximal to the amputation site. In ua- ueated ampttteee the neurflemmal nuclsi increased io number and em larged with dedifferentiation of tho nerve invading more proximal ports, but treated animals showed virtually no nerve changes. Gross and microscopic examinations showed that the vascu larity of those regenerating stumps waa also strikingly diminished. " Slum the salamanders were main tained in a partially paralysed or paretle ststo by treatment with hemiehoUnium, it it possible that disuse of tho stump could aceouot for the observed inhiM- Pc6- Li* tion of growth. However, in one experi, than normal but which ilvn rcHun ioment in which triethylchoiine (400 mg/ *~>'rd normal during ihc ; r <-d u, curly kg) was administered, the rate of re ^.iTcrcntiation. generation was nearly 72 percent of As yet we have no wny of Ucvi-'in-' control, despite the fact that this drug whether treatment with hcmlchnii.i; nn paralyzed the animal more effectively retards growth primarily by blockm; than hcmicholinium did. Botulinum synthesis of acetylcholine nr by func toxin, which also produces paralysis, tioning like curnre (9). Wc have ,i. has been reported (6) not to delay re tempted to rule out the Liter effect by generation of the salamander limb. Fur. simultaneously administering choline thermore several changes were observed (10:1 molar ratio) and homicholinium. which cannot relate to the paralytio However, the combination proved toxic. effect of drug treatment After 5 weeks A direct inhibition of mitosis by hemi- of treatment with hemicbolinium the cholinium has not been but taste buds of the salamander showed in view of the ability of drug treatment regressive and degenerative changes. to mimic in tho salamander some After 19 weeks of treatment the taste features of dyuutooomia, a disease buds disappeared, but they returned characterised by a cholinergic defect, when treatment was discontinued. Chil hemiehoUnium appears to act as a chodren with dyiautonomia not only show Unolytic agent. a parasympathetic insufficiency with a F. Hm growth disturbance but alio a lack of A. Smith Ungual taste buds (7). Departments of Pharmacology unj There is much evidence supporting Anesthesiology, New York Medical the hypothesis that aeetylchoUne Is a College, New York 10029 neurotrophic factor (J), but there are also compelling reasons against Its ready acceptance. Singer (&) reporta that mkroinfusioo of atropine Into the regenerate halts limb growth. On the other hand, infusion of acetylcholine into a denervated Until does not restore its regenerative capacity. However, an infusion cannot duplicate neural activ ity. A more cogent reason for excluding acetylcholine as the growth mediator is that tho concentration of the sub stance in sensory nerves is negligible compared to that in motor nerves. Yet the sensory nerve Is far more capable of infiuapdag regeneration than is tho motor component On the other hand, regenerating tiseue contains concentre- !. M. SIMM, Omft, Sea, Stmt, S7, 14V 0932). a. a. o. eter we o. a. swim. /. zmi. SS. 307 (1949), ). O. B. DnwkfttM. Srareeehm an Grewih at *A Ntrtemt Syttmt, O. B. W. WMoconhomo and M. 0`CMoer, Eda (CfamttB. toad*. 1941), pp. Ul-tni D. a. DracHiwait. Scianaa 14f, 719 (1944). 4. i. Panda and A. A. tafck, ft. But. J. Mad. 374, 307 (194*). J. C. Biter, Adeem. Pedlar. 9. 137 fi->57V 4. D. DradMnan Mid M. Slnsr. died In Xeutn- irtww Swiff* Proanst .Inll.im 7. L Orth, Id. (Breoklloa. p. . \9(A). 7. A. Smith. A. Pirbwn. J. o.ui.i . Scisitea 147, 1040 (1949). t M. Sinjor, hi Deaetaptms Call Syttunt I Thatr Cannot, O. Bmrtkk. 84. (KonaM. Nav York, 1999). p. 119-13). 9. R. B. TMaa aod V. B. awnhi. rad, Hoe. --, 949 (1941). 10. ML la a totem at tkn D9mmmw-- Am mhdn at N*w Ywk. tiona of aeetylchoUne which are greater 30 Jets ttm fadaad I OaaaSar 1974 33wV" Polychlorinated Biphenyk Intenctioa witfe Hack Hqpdtb Vina Abstract Ten-day~old mallard duckling* fed a polychlorinated biphenyl at concentrations of 23, 30, and 100 parte per mUUan for 10 dope suffered no apparent clinical Intoxication, Five days later thorn birds ware challenged with duck hepatitis virus, and they suffered stgriificantty higher mortality than birds which were not exposed to the polychlorinated, biphenyl. Polychlorinated biphenyls (PGB) along with DDB (M-dtehloio-24-bJo(p-chlorophanyl) ethane) are reported^ to be the most abundant of the chlorinoted hydrocarbon pollutants In tho global ecosystem (i). Despite the fact that PCB's have bean in wide use tinoe 1930 (2), they remained eco logically inconspicuous until 1964 when a Swedish chemist reported their prev enoe in the tin*-- of pike sod m'icr wildlife (J). Since then, their presence baa been reported in additional wild life of Europe and North America (I. 4h The-- compounds ire interesting becau-- they are an important sour-- of interference in the chemical detec tion of DDT sod iu metabolites (<) and 1)14 SCIENCE, VOL 170 HONS 0330 Id i'* ill.**, ,irv poit jiitiil hn/.uril in , , itummnoiU; on .t weight baxi* 'U '(vi;.nn',h hove been shown to . .0 . n . . ./i.ivlcgi.ulmg potential about :i'c mnes liiat of p.p'-DDE or technie;ii grade DDT lU,Urichloro-2,2-Ws{fi-chlv>rophenyl)clhane] (J). As part of an investigation of possi ble interactions between organochkmne pollutants and infectious diseases, this study was initiated to determine whether any interaction, antagonistic or synergistic, occurs with PCB and duck hepatitis virus (DHV) in mallards (Anas platyrhynchos). Four separate diets, three with PCB and one without, of a standard duck starter ration were fed to groups of 10-diiy-old mallard ducklings. The three PCB diets containod Aroclor 1254 (in a corn oil premix) concentrations cal culated lo be 25, 50, and 100 parts per million (ppm). Thcso diets were fed to live groups of 25 ducklings each, three principal and two control groups (Table 1). All birds were leg-banded with colorcoded, numbered bands, weighed, and fed their respective diets for 10 days. On day II each bird was weighed again, and every fifth bird from each treatment group was killed. Tissues were collected for residue analysis, his tology, and liver weights. During the 10-day feeding trial, no birds died or became clinically ill, although duck lings in the group given 100 ppm PCB were noticeably hyperexeitabio after day 3 as were those in the 50>ppm group 4 dtys after treatment. Body weights of birds receiving PCB in their diet were significantly heavier at the end of the 10-day feeding trial than those fed the diet without PCB (Table 1). The heaviest body weights occurred in birds fed the highest con centrations of PCB, and the trend ap peared to be linear. Difference! in body weights among treatment means were highly significant (F=s 20.30, ds 4, 119) when tested by analysis of covariance. No statistical comparisons between individual treatment means were made. A similar analysis of liver. weights (determined as a percentage of final body weight) disclosed highly sig nificant differences among treatment means (Fas5.82, d.f. = 4, 18). These differences appeared to be within groups treated with PCB rather than between treated and untreated groups (Table 1). The smell sample siaes (Nos 5) necessitates caution in interpretation of liver weight data. No interpretation of differences among body and liver weights are offered at this time. I Milk' I. HimI Mh iin htHlv xit<| |j\ci w. ii-liis of niallnnl itucUmas nftur l>cin* fid l*< 'll (Aroulur 1234, Motunnio, .St. Louis) for 10 days. Numbers in parentheses am the number of Unla used. The liver and body weifMa were adjusted by covariance for a common tuning meant the liver weight wee determined m percents!* of body weight. The final controt did not receive either PCB or DHV. Treatment PCD (PPm) PCB + DHV PCB + DHV PCB + DHV DHV control Control 100 50 25 0 0 Body weight (s) 3SI.6 <2S) 361.1 (23) 352.1 (25) 315.2 (23) 309.6 (23) Liver weight (g) 0.046 (5) .037 (3) .029 (3) .036 (4) .034 (3) Twelve days after the experiment be gan, birds were placed in crates and transported approximately 5 miles to isolation units where they were main tained on PCB-free feed and water for 3 days before being challenged with DHV. No bird* died or became clinically ill during the interim period between PCB feeding and virus chal lenge although two birds were lost from the 100-ppm group due to accidents. On day 15 of the experiment all but the untreated controls (birds not re ceiving either PCB or DHV) were in oculated intreperitoneally with 1.5 I I > < `>0 |h'!\ i ill ll> i....... i or ducks) ol III IV tT i.,m , .rst birds died about 47 houis .ihcr inoculation. Mortality was recorded hourly for the remainder of the HOhour experimental period with one ex ception: them wero no observations be tween hours 63 through 69. The onset of mortality in all groups given PCB plus DHV began at least 8 to 16 hours before that in the control group treated with the virua only (Fig. I). In addi tion, mortality levels occurring among PCB-plus-virus treatment groups at the end of the experiment were significantly higher at the .01 level of probability than for ducklings receiving the virua only (chi square =: 7.49, d.f. = n There was no significant difference ... mortality among ducklings rccc:v.,ij .. different concentrations of PCI* plus virua (chi square = 3.79, d.(. = 2). No birds in the untreated group died (Table 2). All birds that died during the experi ment had gross pathognomonic hv.r lesions, indicating typical duck :;a.titis infection (6). No other jruss lesions, with the exception of varying amounts of edema within the peri cardium and thoracic cavity of miroc Table 2. Mortality amoaf aaltaid dockiinii caused by DHV during m SO-hour experimental period. No observations ware mad# between 31 to 47 and 62 to 70 hours. The final control did not receive sttber PCB or DHV. Mortality Treatment (PPM) No. of deaths/ total cample , Percentage Onset (hours) PCB + DHV PCB + DHV PCB 4- DHV DHV control Control . too 90 25 S/tl 44.4 31-47 13/20 65.0 35 7/20 35.0 31-47 3/21 14.3 62-70 0/20 0.0 CeuaiHut (hours) 73 76 73 73 Pig. 1. Mortality among mallard ducklings exposed to both PCB end n:;v. only, or neither agent (a) 100 ppm PCB 4- DHV; (P) 50 ppm PCB 4- DHV .. ppm PCB + DHV; (d) DHV control; end () untreated control. Dotij p.--< lines prior to 47 hours end between 62 and 70 hours repvcMot periods umm, no observations were made. IS OgCBMggR W0 M0NS 083073 birds, were observed. There did not ap pear to be any relation between treat* men! and the presence or absence of edema. Histological and residue analy sis have not been completed at (his lime. Thu suggestions that PCB may render .1 host more susceptible to certain types of infectious agents is the significant finding in this study. None of the con* centrations of PCB fed resulted in de lectable chemical intoxication even with the physical stresses of weighing, handling. confinement, and crowding x'.'.M-Ing irampovutinn, transportation itscir. or relocation in a different environ ment. However, when the stress of an inicciunts agent was added, these sub- lethal concentrations appeared to inilocnco the resulting mortality rates, causing two* to fourfold increases (14 percent among virus controls versus 35 to 65 percent among groups receiving PCB plus OHV) and reduced incuba tion time (Fig. 1 and Table 2). Similar Increases in mortality were obtained with other organochlorine compounds in our laboratory (7). In those studies 30-day-o)d mallard duck lings which had bean fed sublethal con centrations of p.p'-DDT or dieldrin exhibited three- to ninefold ineresess in mortality over that of DHV controls (6 percent among virus controls versus 19 to S9 percent among groups receiv ing dieldrin plus DHV, and 19 to 40 percent among groups receiving p,pfDDT plus DHV). It l> unlikely that in ail these instances mortality la the DHV control groups would be less than half that in any interaction group on the bssia of chance alone. This study illustrates one of the po* tcntional effects of sublethal concentra tions of chemical pollutants which are often alluded to, but rarely documented. It also emphasizes the real differences that exist between "sublethe!" and "no effect" concentrations of pollutants. To the best of our knowledge, other stud ies in vivo of the possible effects of orgnnochlorine pollutants on the sus ceptibility of s vertebrate host to a viral agent have not been reported. Milton Friend Daniel O. Tiuinbk Department of Veterinary Science, University of Wisconsin, Madison 53706 1. R. W. RlwfcwmS, I*. RtoclM. O. 1. hiklll, S. O. Herman, M. N. Klrmt, Nmere SM, toee (iMt). X C. A. tonifift In*. Inf. Chtm. U, UN (lWf>. i. S. Jaown, New Sri. , *12 (IMS). A, D. C. Holms, I. H. Stsmont, I. O'O. Triton, Wrier* SIS, 2*1 <tM7); A. V. HeWra u4 X. Monies, MS., 9. 12741 1. H. Kostnim, M. C. Ton Noovor Do Bmow, K. K. DsVss, DM. SSI, ms (IMS); S. ianasn, A. O. Joritrit, M. OlMen, O. OttOfUne. DM. US. 247 (IMS). S. O. Wienork, J. At* Ogle. Aer. Ckem. SO, IOCS (1M7); L. M. RfljrnoMl, MM forimn. Coofton. Tetteei. 4, 12S (IM9). , t. 9. Lovtno, in DUhm of fritftrv, H. 1. Mttor anS L. H. Schwarts, Be. (tow* Siam Urir. 9nm. Am, IMS), 9. SIS. 7. M. Frtane me D. O. TMasr, /. WMUte DU. %. VfttSL Da Mai MsOfri* WtttiU* Fowri*. IS My |S7S| mane IS OrtoSar tf70 Livwr Mitochondria from Minpmwr Deficient and Pallid Micei Functioa and Ultraatructore Abstract Oxidative phosphorylation was studied in Isolated liver mitochondria from manganese^defkient mica and In those from a mutant strain, pallid. In mitochondria from manganese deficient mice, ratios of adenosine triphosphate formed to oxygen consumed were normal, but oxygen uptake was reduced, fire men microscopy of these mitochondria revealed ultrastructural abnormalities including elongmion and reorientation of cristaa. No biochemical or structural abnormalities were found In mitochondria from pallid mice. In a number of animal species a striking effect of dietary deficiency of mangnneM during pregnancy is an ir reversible congenital ataxia in the off spring. The ataxia, characterized by im balance and low of body righting re flexes, results from abnormal develop ment of the inner ear, with defective morphogenesis of the otoliths (/, 2). A mutant gene in mice, pallid (pa), also causes congenital ataxia resulting from impaired development of otoliths (J). When the diet of pregnant mutant mice is supplemented with high amounts of manganese the otoliths and postural be havior of the offspring are normal Thus, there is a relationship between the gene pallid and manganese metabo lism (7, 4). Although considerable attention has been paid to the role of manganese in enzyme function, its specific btochami. cal action in vivo has remained un clear (5). A variety of evidence, how ever, has implicated manganese in mi tochondrial function, particularly in oxidative phosphorylation (5. 6). in our laboratory, isolated liver mitochor,. drla from manganese-dcficinu r .* showed abnormal oxidative plios;^. . lation which appeared to be due io re duced oxidative capacity rather ilmn to lack of coupling (7). We now report on P:0 ratios (moles of adenosine triphosphate formed relative to gram atoms of oxygen consumed) and rate of oxygen uptake in isolated liver mito chondria from manguncsc-dcflcicnt ,imi from pallid mice, and on the fine struc ture of liver tissue as studied with (no electron microscope. Female mice maintained in a colony originally derived from a four-wny cross of inbred strains C57B1/IGJ, C3H/J, ARK/J, and D8A/2J (8) were given a purified diet (9) contain ing 1 part manganaaa per million (de ficient diet) during pregnancy. Their progeny were maintained on the same diets and ware killed as adults for use in these studies. Hybrid mice of the same strain ware fed a similar purified diet except that it contained 43 parts of manganese per million (control diet). Pallid mice (pa/pa) and their nonpallid littermates (C57BI/10J-pe) were maintained on a stock diet (JO). For mitochoodria! preparations, four hybrid deficient, four hybrid control, five pallid, and five nonpallid mica were decapitated; their liven were put into cold 0.25M sucrose, cut into small pieces, and washed three times. Mito chondria were separated as previously described (//). Oxygen uptake was de termined by the polarographic assay of oxygen after the method of Chance and Williams (12), with /3-hydroxybutyrate as the substrate in the reaction medium (Table 1). The oxygen electrode was according to Packer (13). We calculated P:0 ratio* by determining the reduction in the amount of added adenosine diphosphate per unit of o\>gen consumed. Adenosine cipbosphnuwas measured by absorbance at 260 nm (14). The concentration of protein in the mitochondrial suspension was de termined by means of the biuret reac tion (15). Median lobular hepatic tissue was excised from ten manganese-deficient hybrid, five control hybrid, and five pallid adult female mice for examina- mt SCI8NCS, VOL. 110 MQNS Uti307A