Document qaJQEJEpmYbb5YEwQK2depaMq

jr*icd' tfifc'^wlchdtnfom (or 11 \;oka were periuincoty poisoned, wo amputated the rcfenflratmj leg and ;.1 lowed It to regfow wltltoot further administration of k drug. Regeneration wu identical to tjtit ki controls, indi outing that the effeet of homkholinlum Hb#, reversible. To demonstrate that the Initial amputation did not stimulate suh*e<juent regeneration we measured .the growth rate* 10 wooks after the Or* amputation tnd similarly after a reamputaiion. T.w first r.nd ur.: j rates of rogenernt on were iJentier.! in tnCh of two s.iluntmdors studied. We then considered ihnt treatment with hdmlcholinium niij ht inhibit growth be* cause 25 percent of the body weight was lost during treatment. For that study, the hind limb of an untreated salamander was amputated, but no food was given to ihe animal during the ' I0wtck regeneration foriod. Despite a weight Ion of 56 perpont, the rate of limb growth was not different from that in previous controls. The diminished growth rato In treated salamander^ correlated well with (he mlcMscopio findings. Salamander* trooted for 5 week! wlm hcmlcholinium showed only about 30 mitotic figures (n the median sakittol section of the regenerate. However, more than 400 were seen in similar sections from con trols. Colchicine 2 mg/kg) was in jected into both groups 12 hours before the salamanders vere killed so that miiotie figures would accumulate and eoutd bo counted! Evidently hcmicho- llnium did not inhibit regeneration by means of an effect like that of colchi cine, for fur fewer mitotic figures were soon Among treated specimens than among controls. Furthermore, the fact that the mesenchymal cells were not en larged argues againkt the possibility that hcntkho'.wiium blocks cell division at an earlier stage. The drug also prevented the massive dcdUTerontlatioii of muscle tissue normally sm$ hi sections Just proximal to the amputation site. In un treated amputees' the neurilemm&l nuclei Increased lr. number and en larged wit; dedifferentiation of the, nerve Invadlug more proximal parts, hut treated animals showed virtually no nerve changes. Cross and microscopic examinations showed that the vascu- ;.'ity of these regenerating stumps was also strikingly Jininishod. Since the salan aftden won main* tiad in n partially paralyzed or paretic - by treatment [with hemichollnium, a possible that disuse of the stump . --J cocourn for'the observed Jnhlbl- VI liUMVrn, III MIC V*J*W *11*111 IIVIHiMJ l/MI. meat in which tricthylcholinc (400 nig/ ward normal during the period of earJff kg) was administered, tho rate of re differentiation. *, generation was nearly 72 percent of A. yet we have no way of deckling control, despite tho fact that this drug whether treatment with hcmlcholinium paralysed die animal more effectively roturdi growth primarily by blocking than homichollnijm did. Botulinum synthesis of acetylcholine or by func toxin, which also produces paralysis, tioning like curaro (9). Wo have at has been reports. (0) r.ot lo demy re tempted to rulo out the Liter effect by generation of tho salamander limb. Fur simultaneously administering chohne thermore several-changes were observed which eanno; relate to the paralytic effect of drug treatment. Af.sr 5 weeks (10:1 molar ratio) and hcmlcholinium. However, the combination proved toxic. A direct inhihitic.i of mitosis hy hemi- Of UViUiv.c;.l wh . il.o taste buds >',f th.` sal;, r.a slu.v'j regressive and ccgencridivc changes. ciirh.mim has not been ,,xc1uiI:J, but in vi. wof the ability of drug treatment to mimic In the salamander sons# After 19 weeks of treatment the taste features of dysnutonomh. a disease buds disappeared, but t:icy relumed characterised by a cholinergic defect, when treatment was discontinued. Chil hcmicholiniu.n appears lo act as a cho- dren with dysautonomJa not only show linolytic agent. ' a parasympathetic insufficiency with & growth disturbance but also a lack oC ii.^jal taste buws (7), F. Hut A. Smith *>cof Pharmacology and Theft Is much evidence supporting Anesthtiiology, Sew York Medical the hypothesis that acetylcholine is 4 College, Net York 10029 neurotrophic factor (J), but (here ore also compelling reasons against its Rtftnntci uj Nun ready acceptance. Singer (8) reports I. vt. Sinftf. Quart. Ht*. BUt, ST. 1M. (19*2). that microinfusion of atropine into the . 2. ., O. nutlet Afttf O. E. SeHottt. J. t*. Z*>1 rrgtnprjda halts limb i,.owtl;. On tint m, in? (1040). J. P, B. Drxhmen, tyMptnhm vi 0r*-*4 / other hand, infusion of acctylcholioo into a denervated limb Jodi no^. restore tht Ntrrom Q. B. W. WnluoMiwrw nd M. O'Coiuinr, Edt. (Oku.chill, Laadaw, IMS). rt*- iSl-DJ; D. B. U* ichron, Stkntt its regenerative capacity. However, an infusion cannot JupHca.c neural activ US. V.9 (1W.4). 4. .1. D.inct* and A. A. Smith. N. Cmg. J, Mid. . '< 207 (IMS). ity. A more cogent reason for excluding. acetylcholine as tho growth mediator 5. C. Riley, Advait. Min. V, 1ST (l*J7). L D. Orschmsn Md M. Vnjff, fticd in lelriiet Httenreh Bngntl Bulletin t. L. is that the concentration of the sub Outh, Ed, (BrooUJno, MaHachuiflU, IMS), P. H. stance in sensory nerves is negligible compared to that in moior nerves. Vet 7. A. Smith. X Farbnun, J. Dwell, raw# 147, I0af, (IMS). S.M. Sin,:cr, in Dtvtlofilmt C*0 S*Ht`n md the sensory nerve is far moro capable of influencing regeneration than is the Thrtr Cinti*l, D. Rvdnlck. H4. (lUMtA Hew York. 1$f), p. IlS^U). S. R. B. TWci Md V. a. Brook*, ft*. But.- motor, component. On the other hand, regenerating tissue contains concentra 20, MB (1MI). . IQ. F.H. U h fellow of lh Dyiiuionomlt Am tocl* ion it Niw York. tions of acetylcholine whic- are greater 20 July |S7t); mtMd t Odohor 1M0 Poiychlo.-inatoii Duck Hepatitis Vim '..tersetior. 1;h Abstract. Tm-day-old mallard ducklings fed m polychlorinated biphenyl et concenrretfons of 25, 50, and 100 ports per million for 10 days suffered no apparent clinical Intoxication, Five days later these birds were challenged with duck hepatitis virus, and they suffered significantly higher mortality than birds which were not exposed to the polychlorinated biphenyl. Polychlorinated bhhcr.yls (PCB) along with DDE {i,I-d:ch.'cro-2.1-cis(p-chlorophcr./ljeihenc) arc reported to be the most abundar.t of the chlo rinated hydrocarbon pollutants in the global ocor/i em (7). Despite the fact that PCB's have been ^ vide uso inoe 1930 (2), they .emained eco logically inconspicuous until 1964 when a Swedish chemist reported their pres ence ir. tho tissues of pike and other wildlife (2). Since then, their presence has been reported In additional wild life of Europe and North America (/. 4). These compounds are interesting because they ore on important source of interference in the chemical detec tion of DDT and its metabolites (5) and setENce, VOL. ITS MUNS 096662 K*adbo*tlv) are a potential hacard in ' flw environment; on a weight basis l.'CD preparations have been shown to have an tiUudiol-skgraJini potential ghaut live times that of p,/*-Dli>E or techni cal grade DDT (I,l,l-(ff<!h{ro-2,2-bts- (p-mtorophenyDethaneJ (/). Ai part of an investigation of posal- bio Interaction* between o/ganochlorina pollutants and infectious1 diseases, this slpdy was initiuted to determine whether any interaction, antagonistic or synergistic, occurs wi;h PCU anJ duck iwpaiiils virus (DHV) in milliards (Amir piatyrhyncliot). Four separate diets, th *ee with PCR and one without, of a standard duck starter ration were fed |io groups of lO-day-cId mallard duckling*. The three PCB diet* contained Aroclor 125-* (In a corn oil premix) concl.itrat:o:,: cal* cthaicd to be 25, 50, [ .uu , er million (ppm). These dpi* wcit fed to Ave group* o. 25 ducklings each, three principal and two control groups (TaWc 1). 1 All birds were leg-bonded with colorcoded, numbered bands, [weighed, and fed their rt-pcctivc diets1 for 10 days. On day 11 each bird [was weighed again, and every fifth bird from each trcAlment group was killed. Tissues were collected for resldu^ analysis, his tology, end liver weight). During the 10-day feeding trial, no {birds died or became clinically ill, although duck lings In the group given 00 ppm CD were noticeably hypero testable after day 5 as were those ill the 50-ppm group 4 days after treatnent. Body weights of birdi Receiving PCD in their diet wero .`igniftcanlly heavier at tho end of tho 10-day Ceding trial than those fed the diet {without PCB (Table I). Tho heaviest body weigMi occurred in bird* fed th^ highest con- ociOftMioDs of PCB, and{ the tread ap peared to be flnsar. Dttftrettcet la body weighls among treatment means were highly significant (Fw'lO.JO, d.f. 4, IIP) when tested y analysis of covariance. No statfstieat oomparisons between Individual treltmont means were made. A similar analyst of liver weights (Jciermincd as a percontago of Anal body weight) disdojsed highly sig nificant difference* strong treatment means (F s= $.82, d.f. **4, 18). Theso difference* appeared to be within ' groups treated with PC<B rather than between treated and untreated groups (Table I). The sm;.ll sanlpio sites (iV = 5) necessitates caution in interpretation of liver weight data. No ir.,..4.-ci . of differences among (body and hve.' weights are offered at this time. ; occcmoea jv70 Table t. Final mean body and liver weight* oi mailin', ducklings after Oclnp fed PCI) (Arodor 1154, Muin.inip, St. Louis) for 10 gays. Number* in parcnihex. ji.c ^ number ttf bird* tn<d. The liver v "W body weigh** wore adjusted by covariance for a common Martini messt; lit* liver weight was determined ai (K'/venUar of body weight. Tiie ftttaI con trol Ski not receive either PCB or DIIV. Treatment rcu (Ppm) PCll + DIIV PCb + DHV Mil + DHV DHV control Control too 50 25 0 0 UoJy Weight <!'./ ` )*U CJ) 368.0 (25) 352.x , \5) 3,>,' , 3t/... liver wclfht (; 0.04ft (5) 4137(5) .o:vi5) .Q?ft (4) .034 (5) Twelve days after the experiment be gan, birds were placed in cr..<es and transported approximately 5 miles to isolation units whc.u they were main tained ort PCB-freo f.*aj and water for 5 days before u.,..j c.;;tllenged wUh DHV. No birds 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 g-oup 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 intraperitoneally with 1.5 LD.x, (50 percent cfloctive lethal dcae for ducks) of DHV per bird, 'ibc first birds died about *47 hours after inoculation. Mortality was recorded hourly for the remainder of the HOhour experimental period with one ex'ccptiu..; them were no observation* be tween hours 63 through 6V. The onset of mortality In all groups given PCI* plus DHV began at teas' 8 to 16 hours hefor. that In the control group treated .it tho virus only (Fig. I). In addi tion. mortality levels occurring among PCD-phiN-virus treatment groups at the enJ of me experiment were significantly higher at the .01 JjvcI of probability th.. t for ducklings receiving (he virus only (chi square = 7.49. d.f. s 1). There was no significant difference in morality among ducklings receiving the different concentrations of PCU plus vi.-us (chi ><-.,are * 3.79. d.f. = 2). No birds in the entreated group died (Table 2). All birds that died during the experi ment had gross pathognomonic liver lesions, indieating typical duck hepa titis infection (6). No other gross lesions, with the exception of varying amountt of edema within the peri cardium and (horacie cavity of some Tiblc 2 Mortality among mallard duckling* earned by DHV during an 00-hour experimental period. No observation* wera made between 31 to 47 and (3 to 70 boors. Th* Anal control did not receive either PCB or DHV. Tree, mem PCB (Ppm) No. of t'.dihs/ total sample Mortality Percentage Onset (hours) Cctsatio* (hoi 1.. PCB i- DHV too H/l 8 4,4 )H? 75 PCU + DHV so 13/tO 65.0 ' ss 74 PCb + DHV 2$ 7/20 3S.0 31-47 73 DHV eentrol 3/21 U.3 62-70 73 Control 0/20 0.0 Fig. 1. Mortality amor.? nniiard ducklings exposed to both PCB anl .V, DHV only, or neither ar^nt. () MiJ ppm PCB + DHV; (6) 50 ppm PCB + ilV, 25 ppm PCD + DHV; (</) DHV control; and (e) untreated control. Dotted portion of lines prior to 47 hours and between 62 and 70 hours represent periods during which no observations were made. M0NS 096883 ms birds, were observed. Tlscro did not ap DHV control groups would be less than Iwon paid to the role of anc>c `n pear to he any relation between treat- half that in any interaction group on tr.cyme function, its spcc-.'c biochemi pwnt and the preoeftou, dr absence of the basis of chance alone. cal action in vivo has remained un tdcnia. Histological End residue analy This study illustrate' ono of the po- clear (5). A variety of evidence, how sis have not been completed at this tontionnl effee sub.ethal concentra ever, has implicated manganese in mi time. tions of chc; The suggestions that PCD may render often alluded ^Mutants which arc tochondrial funciion. particularly in rarelydocuomxidenateivde. phosphorylation <5, 6). In a host more luicopiiblc to certain types It also empi. .he real diJcrenc. i ,ir laboratoiy, isolated liver i..iloehon- Ct infection* acenlt is the significant (hat cxLl bet finding in this sjiuty. None of the con* ctTcet" conee n;il" and "no- dria fiom manganeso-deficient ralv of pollutants. To showed abnormal oxidative , liosptmry- ccnirntionx of PCD fed resulted in dc* leduhlc chemical intoxication even with the pliyM.'ill stresses of weighing, handling. confinement, and crowding the best of owled^c, other stn ! ies in vivo . j posable efi'cctt f orgnnochlori.e poi..,.uots on the sus ceptibility of a vertebrae hc*t tw a viral l. ;tion which appeared to be di.c to re* di ced oxidative capacity rather than to iiu.. of cou,.iin: *7). \/e nc. v re|Vrt on P;0 ratios (mole* of ;. cnjMiiu during transpoitjilion, transportation it* agent have not been reported. i:ij)hosphn:e forn.jJ relative to gra.n self, or relocation in a different environ* Milton Fiubnd atoms of oxygen consumed) and rate of mem. However, when the stress of an Daniel O. Trainer oxygen uptake in Isolated liver mito- Infectious agent was added, :!csc sub* Department of ''ctcritu -y Science, chor.Jri.i frot.-. mj..isc;ar.cse`i!er.eient and Icih.d co'.ccnlraMor.s appeared ;o in* Alienee the resulting mortality rates, Catwing two* to fourfold increases <14 percent among virus controls versus 35 to 65 percent a nong groups receiving PCD pivn DHV and reduced incuba tion time (Fig. and Table 2). Similar incrcrses in mortality were Obtained with othor organoehlorine compounds in ojur laboratory (7). In those studies 30iduy-old mallard duck lings which had pcen fed sublcth.il con centrations of kp'-DDT or dicldrin exhibited three- to ninefold increases in mortality over that of DHV control (6 percent a.. .-:ijg vims cor. rnis versus 19 to 5'J percent among groups rccciv* ing dicldrin plusj DHV, and 15 to -.0 percent among groups receiving />,/>'DDT plus DHV). It is unlikely that in ail these instances mortality in the University of , '.'.eOi.i.'j,, Madison 53706 Itcftrvncci lad Nglca 1. R. W. RlH'hfoufh, |`. Rlcehc. D. B. PewknM, S. C. Mein an, M, N. Ki.v.n. Suture Us. t9.< (IVMIt. 2. C. A, rending, lnJ. Lug. Chtui. 22, llSO (1920). J. $. Jansen, New Sri. 32. c;: CCM). 4. O. C. 1 Inline*. J. It. Simmon*, J, O'O, Tstinr,, Aolttrc 216. 237 (1967); A. V. Holden and N. Mini)*, iUU., p. r;74; J. It. Kmman. M, C. Ten Noever Oe Uratiw, ;t. II. DV, Uhl. tit, 1126 (1569); V Jcnoi, A. C. Jolin.li, M. OlM>nn, 0. OlU'Ui.i.l, Uil.t. 224. 347 (1969), 5. C. Wltlmnrk, J. An. OO-r. Agr. CS>rm. 50, .009 (1967); L. M. Ki-ynnliit, Out'.. Environ. Conlotu. Toxicol. 4. 121 (1909). 6. I*. P. Levine, in Dtsctie.i of PouUry, H. B. ItcUlcr anti L. It. Srhwaru, Ed, (Iowa Suit Univ. press, Ames, 1963). j. S)l, 7. M. Friend and D. O. Traiiwf, J. Wihlhft Dlt., in press. I. We Uian); llv N' * McGfjw Vildlifo "ounda* lion (or .upp`. . . n-.llardt To* th. S,:p- poricu .it part i>y ilia Paiuxent Wildlife He* vai'ch Cetwr, Bureau o( Sport Fisbertas and W.Jjyfe. 16 July 2070; revised 10 Ct.oixr 1970 u ,'.om pad;u mice, . nd on the fine struc ture of liver tissue as studied with Iho electron microscope. Female mice maintained in a colony originally derived from a four-way cross of inbred strains C57BI/16J, C3H/J, ARK/J. and DDA/2J (.*) were given a purified diet (9) contain ing ] part mangoncso per million (de ficient diet) during pregnancy. Their progeny were maintained on the name diets and wero killed as adults for uso in thoso studies. Hybrid mice of the s.'.mo strain were fed a similar purified diet except that it contained 45 pans of manganese per million (control diet). V.VIid mice {palpo) and :! oir nonp.il id littcrmates (C57B'./IOJ*pa) wart m. iimJr.cd on a stock diet U0). For mitochondrial preparations, four hybrid deficient, four hybrid control, five pnhid, and five nonpnllid mice were decapitated; their livers wero put Live* pndr'a fre::: M\:a:,ai::-t-DoficicRt and Mice: Cuon aud Ul:ra$:rac!u:. into cold 0.25M sucrose, cut into small pieces, and washed three times. Mito chondria were separated as previously escribed (it ). Oxygtn upi..ke was de Abstract. OxUuilve phosphorylation was studied in Isolated liver mitochondria from manganescydeficlcnt mice and in those from a mutant strain, pallid. In mitochondria /rfljm mangonesc*dcficicnt mice, ratios of adenosine triphosphate formed to oxygek consumed were normal, but oxygen uptake was reduced. Elec- termined by fho'polarographlc assay of oxygen after the method of Char.ee and Williams (12), with *hydro\ybul>ratc as the substrate in the reaction medium (Table I). The oxygen electrode tron microscopy of these mitochondria revealed uhrastructural abnormalities Including ehngaiion and reorientation of cristae. bio biochemical or structural abiU/-..iallties welc found in mitochondria from pallid mice. . was according to Packer (IS). We calculated P:0 ratios by determining tho reduction in the amount of added adenosine diphosphate per unit of oxy It number (of animal specie, a causes Congenital ut;.x.a resulting fro a gen consumed. Adenosine diphosphate e.Tect of'dietary . rf.cicncy of impnirca develop;...,-.t of otoliths (3). vrjs measured by absorbance at 260 nuir.;'. .sc during pregnancy is an ir* When .he diet of , regn;;!.t mutant mice nm (Id). The concentration c: protein ` rw...... !l congenital otaxi- in the oil* is supplemented with high amounts of in tho mitochondrial suspension \ras de xp. Vi e atxi.j, characteiaed by ir.ib.ilancc and toss'of body righting re lieves, results frolm abnormal develop ment of the innijr ear, with defective morphogenesis of the oioliths (/, 2). A mutant gene in njdcc, pailid (pa), also mnngahcsc the otoliths nr.d postural be havior of the ofT.sjiri'.-.g are rormrsl. Thus, there is ,, rula.ionship between the g iZ pallid a.-.d r..;.r.^ancsc mc.aJo* lism (A 4). Althojgh considerable attention has termined oy means o* '-e biuret rc.tc* ti. r. (IS). Median lobular hematic tissue w:.s excised from ten mengancsc-dcficicnt hybrid, five control hybrid, and five pallid adult female mice for examina- MONS 096664 SCIENCE, VOL. 170