Document 1QMwm6XBwYoNmkk3aV3enByJo

.- llilnw 4U4U4*****uW* tr-qted* w^thv hemkhdBnfijm for 11 weoks were pbhnancnfy poisoned, wo amputated live regenerating leg and al lowed it to regrow witliout further administration of a drug. Regeneration was identical to that in controls, indi cating that the effect of hcmicholinium 'skrtis reversible. To demonstrate that the initial amputation did not stimulate subsequent regeneration we measured the growth rates 10 weeks after the first amputation and similarly after a rcamputat ion. The first and s-c-.: J rates of regeneration were identical in each of two salamanders studied. We then considered that treatment with hcmicholinium might inhibit grow th 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 the animal during titc ` 10-Wbck regeneration foriod. Despite a weight loss of 50 percent, the rate of limb growth was not different from that in previous controls. The diminished growth rate in treated salamanders correlated well with the microscopic findings. S;.lnnmmlcn> treated for 5 weeks witn hcmicholinium showed only about 30 mitotic figures in the median sagittal 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 were killed so that mitotic figures would accumulate and could be counted. Evidently hcmiciio- linium did not inhibit regeneration by means of an effect like that of colchi cine, for far fewer mitotic figures were seen among treated specimens than among controls. Furthermore, the fact that the mesenchymal cells were not en larged argues against the possibility that hcmicholinium blocks ceil division at an earlier stage. The drug also prevented the massive dedifferentiation of muscle tissue normally seen fak sections just proximal to the amputation site. In un treated amputees the neurilemmal nuclei increased ir. number and en larged wifi dedifferentiation of the nerve invading more proximal parts, but treated animals showed virtually no nerve changes. Gross and microscopic examinations showed that the vascu larity of these regenerating stumps was also strikingly diminished. Since the salamanders wen main- aiir.ed in a partially paralyzed Or paretic c by treatment with bemicholinium, -i possible that disuso of the stump _id account for the observed inhibi- i:-,4 UVSJ4 Ui. ^UWiU. jriUVTWVVf lit WHO CA|Ui- man iKJijjiui uui witivu uiwn ictuiq ui* meat in which tricthyieholinc (400 nig/ ward normal during the period of ariy kg) was administered, the rate of re differentiation. *, generation was nearly 72 percent of As yet we have no way of deciding control, despite the fact that this drug whether treatment with hemichnlinium paralyzed the animal more effectively retards growth primarily by blocking than hcmichoUnijm did. Botulinum synthesis of acctyichoiinc or by func toxin, which also produces paralysis, tioning like curare (9). We have at has been reporter (O') r.ot to dciay re tempted to rule out the latter effect by generation of the salamander limb. Fur simultaneously administering choline thermore several-changes were observed (10:1 molar ratio) and hemichnlinium. which cannot. relate to titc paralytic However, the combination proved toxic. effect of drug treatment. Af.cr 5 weeks A direct inhibition of mitosis by hemi- Of treatment \vi... re:.' lei.auv; the cnc'.inium has not been excluded, but taste buds of the sal;. v.a:v.. showed in view of the ability of drug treatment regressive and regenerative changes. to mimic in the salamander some After 19 weeks of treatment the taste features of dysautonomia, a disease buds disappeared, but they returned characterized by a cholinergic defect, when treatment was discontinued. Chil hcmicholinium appears to act as a cho- dren with dysautonomia not only show linolytic agent. ' a parasympathetic insufficiency with a F. Hui growth disturbance but also a lack of A. Smith iii.^jal taste bui.s (7). Dehorn..cuts of Pharmacology and Theft is much evidence supporting Anesthesiology, New York Medical the hypothesis that acetylcholine is n College, New York 10029 neurotrophic factor (3), but there are also compelling reasons against its References and Notes ready acceptance. Singer (5) reports that microinfusion of atropir.c into the regenerate hulls limb growth. Gn the 1. M. Singer, Quart. Rev. Biol. 27, 169 (1952). 2. P,. G. Butler nnd O. E. Schotic, J. Exp, ZooL es, an? (iw), 3. D. B. Drachitun, Symposium on Groiw'i of other hand, infusion of acetylcholine into a denervated limb dodt ne^rcstore - the Nervous Sysicm, G. E. W. \Volstcnhomc and M. O'Connor, Eds. (Churchill, London, 3968), pp. 251-273; D. 3. Drachman, Science its regenerative capacity. However, an infusion cannot duplicate neural activ US, 7!9 (1964). 4. .1. D.nncix and A. A. Smith, N, Eng, 1. Med. . /4, 207 (1966). ity. A more cogent reason for excluding, acetylcholine as the growth mediator 5. C. Riley, Advau. Pediat. 9, 157 (1957). 6. D. Drachman and M. Singer, cited in Neurc* science Research Progress Bulletin 7. L. is that the concentration of the sub stance in sensory nerves is negligible compared to that in motor nerve?. Yet Guth, Ed. (Brookline, Massachusetts, 1968), P. 35. . 7. A. Smith, A. Farbman, J. Dands, Science 147, 1040 (1965). 8. M. Singer, in Developing Cell Systems mtd the sensory nerve is far more capable of influencing regeneration than is the Their Control, D. Rydnick, Ed. (Ronald, New York, 1959), p. 115-133. 9. R. E. Tfcks and V. B. Brooks, Fed. Proc. motor, component. On the other hand, regenerating tissue contains concentra 20, 569 (1961). . 10. F.H. is a fellow of the Dysautonomia As sociation at New York. tions of acetylcholine which are greater 20 July 1970; rerisad 1 October 1970 Polychlorinated Zi^hcnyl: I..ferection wi-h Dock Hepatitis Virus Abstract. Ten-day-old mallard ducklings fed a polychlorinated biphenyl at concentrations of 25, 50, and 100 parts 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 biphenyls (PCB) along with DDE [i,l-d:ch.cro-2,l-fcij- (p-chlorophcr.y!) etr.r.nc] arc reported to be the most abundant of the chlo rinated hydrocarbon pollutants in the global ccosys.cm (I). Despite the fact that PCB's have been vide use since 1930 (2), they .emained eco logically inconspicuous until 1966 when a Swedish chemist reported their pres ence ir. the tissues of pike and other wildlife (3). Since then, their presence has been reported in additional wild life of Europe and North America (I, 4). Thesc compounds arc interesting because they are an important source of interference in the chemical detec tion otDDT md it* metabolites (5) and DSW 332422 SCIENCE. VOX.. 170 STLCOPCB4078998 tOdlbs*c*they arc a potential hazard in The environment; on a weight basis i'CB preparations have been shown to have an estradiol-degrading potential nbout live times that of p.p'-DDE or techni cal grade DDT [1,1,1 -trichloro-2,2-bis- (/>^blorophcnyl)ethane] (/). As part of an investigation of possi ble interactions between orgnnochlorinc pollutants and infectious diseases, this study was initiated to determine whether any interaction, antagonistic or synergistic, occurs wi;h PCD and duck hepatitis virus (DHV) in mallards (A mu platyrliynehos). Four separate diets, three with PCB and One without, of a standard duck starter ralion were fed to groups of 10-day-old mallard ducklings. The three PCB diets contained Arocior 1254 (in a corn oil premix) concentrations cal cinated to be --5, 50, ...lb . \j\j /....'is , or million (ppm). These diets went fed to five groups 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 11 each bird was weighed again, and every fifth bird from each treatment group was killed. Tissues were collected for residue analysis, his tology, and iiver weights. During the 10-d*y feeding trial, no birds died or became clinically ill, although duck lings in the group given 100 ppm 1 CB were noticeably hyperexcitable after day 3 as were those in the 50-ppm group 4 days 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 (TaWc 1). The heaviest body weights occurred in birds fed the highest con centrations of PCB, and the trend ap peared to be linear. Diftitrencee in body weights among treatment means were highly significant (Fa 20.30, d.f. = 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 difference* among treatment means (F ~ 5.82, d.f. = 4, 18). These differences appeared to be within groups treated wnh PCB rather than between treated and untreated groups (Table 1). The small sample sizes (N = 5) necessitates caution in interpretation of liver weight data. No ir._/.-ct of differences among (body and live.weights are offered at this time. id DECEMDER 1970 Table I. Final mean body and liver weight? off mallard, ducklings after being fed PCI! (Aroctor 1154, Mimsnnip, St. Louis) for 10 days. Numbers in parenthev, arc - number of bird* u-ed. The liver end body weights were adjusted by covariance for a common starting mean; die liver weight was determined us percentage of body weight. The final con trol did not receive either l'CB or DIIV. LD.v, (50 percent effective lethal dose for ducks) of DHV per bird. The first birds died about *47 hours after inoculation. Mortality was recorded hourly for the remainder of the X0hour experimental period with one cx'ceptio..; there were no observations be Treatment 1`CU (PPin) PCI! 4- DIIV PCT. + DHV PC'U + DHV DHV control Control too 50 25 0 0 U,)d y weight (gi ` Liver weight (t) 3*1.6 (25) 368.* (25) 352.8 ,'75) 3; 30'X,. ,e > 0.046 (5) .037 (5) .029 (5) .036 (4) .034 (51 tween hours 63 through 69. The onset of mortality in all groups given PC'B plus DHV began at least 8 to 16 hours ' before that in the control group treated .h the virus only (Fig. 1). In addi tion, mortality levels occurring among PCB-plus-virus treatment groups at the end of ,.ic experiment were significantly higher at the .01 level of probability Twelve days after the experiment be tha.i for ducklings receiving the virus gan, birds were placed in cr-ics and only (chi square = 7.49, d.f. = 1). transported approximately 5 miles to There was no significant difference in isolation units where they were main mor.ality among ducklings receiving the tained on PCB-free feed and water different concentrations of PCB plus for 3 days before Owing challenged vi,(eh: scaare = 3.79, d.f. = 2). No with DHV. No birds died or became birds in iho untreated group died clinically ill during the interim period (Table 2). between PCB feeding and virus chal All birds that died during the experi lenge although two birds were lost from ment had gross pathognomonic liver the 100-pprr. group due to accidents. lesions, indicating typical duck hepa On day 15 of the experiment all but titis infection (6). No other gross the untreated controls (birds not re lesions, with the exception of varying ceiving either PCB or DHV') were in amounts of edema within the peri oculated intraperitoncally with 1.5 cardium and thoracic cavity of some Table 2. Mortality among mallard ducklings caused by DHV durins an 80-hour experimental period. No observations were made between 51 to 47 and 62 to 70 hours. The final control did not roceiv* either PCB or DHV. Treatment PCB (ppm) No. of cd-iihs/ tola: sample Mortality Percentage Onsel (hours) Cessation (hours, PCB 4- DHV PCB + DHV 100 8/18 44.4 . 31-47 . 75 50 13/20 65.0 7 5J 76 PCli + DHV 25 7/20 35.0 31-47 75 DHV control 3/21 14.3 62-70 73 Control 0/20 0.0 Time (hours) Fig. 1. Mortality among mallard ducklings exposed to both PCB ami T .V, DHV only, or neither agent. (<; IGd ppm PCB -j- DHV; (6) 50 ppm PCB -j- CiiV; <c) 25 ppm PCB -J- DHV;.(tl) DHV control: and (e) untreated control. Dotted portion of lines prior'to 47 hours and between 62 and 70 .hour*-"-----* ----------during which no observations were made. . DSW 3__3_24,,2,,3,, 1315 I j 1 f STLCOPCB4078999 . rI 1 ! I j i ) i 1 \ i4 birds, were observed. Tltcrc did not ap- DHV control groups would be less than been paid to the role of manganese in penr to be any relation between treat- half that in any interaction group on enzyme function, its specific biochemi n>cnl and the presence dr absence of the basis of chance alone. cal action in vivo has remained un edema. Histological and residue analy This study illustrates or.c of the po- clear (5). A variety of evidence, how sis have not been completed at this tcntional effee ' suo.ethat concentra ever, has implicated manganese in mi time. The suggestions that PCB may render a 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 tectable chemical intoxication even with the physical stresses of weighing, handling, confinement, and crowding during transportation, transportation it self, or relocation in a different environ ment. However, when the stress of an infectious agent was added, these subleth:.! concentrations appeared to in fluence the resulting mortality rates, causing two- to fourfold increases (14 percent among virus controls versus 35 to 65 percent among groups receiving PCB plus DHV) and reduced incuba tion time (Fig. 1 and Table 2). Similar increases in mortality were obtained with other orgnnochlorine compounds in our laboratory (7). In those studies 30-day-old mallard duck lings which had been fed sublcthnl con centrations of p,p'-DDT or dicldrin exhibited three- to ninefold increases in mortality over that of-DHV controls (6 percent a., .nig virus cor. rols versus 19 to 59 percent among groups receiv ing dicldrin pius DHV, and T9 to -rO percent among groups receiving p,p'DDT plus DHV). It is unlikely that in all these instances mortality in the tions of chc: pollutants which arc tochondrial function, particularly in often alluded rarelydocouxmideantitveod.'phosphorylation (5, 6). In It also empi. die real diiTorenci our laboratory, isolated liver mitochon that cxi. t bei "sjoletual" and "no- dria from manganese-deficient rats cITcci" conce of pollutants. To showed abnormal oxidative . hosphory- the best of , owlc.lge, other stud l.-.tion which appeared to be One to re ies irt vivo . j possible effects f duced oxidative capacity rather than organochlorn.e poi.^.ants on the sus to urn.. of co.t,.ling 17). \/e r.o.v report ceptibility of a vcrtebr.uc host to a viral on P:0 ratios (moles of adenosine agent have not been reported. triphosphate formed relative to gram Milton Friend atoms of oxygen consumed) and rale of Daniel O. T.tainer oxygen uptake in isolated liver mito Department oj Veterinary Science, chondria from manganese-deficient and University oj . ".'_co;.ab., from padiu mice, t.nd on the fine struc Madison 53706 ture of liver tissue as studied with the electron microscope. Reference* and Notca Female mice maintained in a colony 1. R. W. Rlscb rough, 1*. Ricchc. D. B. Pcnk.ill, S. G. Kerman, M. N. Kirven, Nusi.re 220, 109.S (1968). 2. C. A. Penning, ir.d. Lug. Client. 22, 1180 (1930). 3. S. Jansen, New Sci. 32, C.2 (1966). 4. D. C. Holmes, J. H. Simmons, J. O'G. Tatton, Nefurs 216. 227 (1967); A. V. Holden and K. Mantle, ibid.. p. 1274; J, H. Kocman, M. C. Tun Noever Dc Jlrauw, R. H. DeVes, ibid. 221, 1126 (1969); S. jciis.m, A. G. Johncls, M. Olsson. G. Oiterlind, ibid. 224. 247 (1969). 5. G. Wulmark, J. Ass. OQ:>c. Agr. C7a*/n. 50, ;069 (1967); L. M. Reynolds, Bull. Environ, Contain. Toxicol. 4. 128 (1969). 6. P. P. Levine, in Diseases of Poultry, H. E. Bcistcr and L. II. Schwario, Ed. (Iowa Slate Univ. Press, Ames, 1965), p. S3R. 7. M. Friend and D. O. Trainer, J. Wildlife Dis in press. 8. We iliank the M x XTeGraw Wildlife Founda tion Xor -.uppi.*r...Hards .'or this st^oy. Sup- pone- .n pari by the Paiuxenl Wildlife Re search Center, Bureau of Sport Fisheries and w;;jiiCc. 1C July 1970; revised 16 Cc.ober 1970 w originally derived from a four-way cross of inbred strains C57B1/IGJ, C3H/J, ARK/J, and DBA/2J (S) were given a purified diet (9) contain ing I part manganese per million (de ficient diet) during pregnancy. Their progeny were maintained on the same diets and were killed as adults for use in these studies. Hybrid mice of the same strain were fed a similar purified diet except that it contained 45 parts of manganese per million (control diet). TT.kid mice (pa/pa) and their nonpahid littermates (C57B1/lOJ-pa) were maintained on a stock diet (10). For mitochondrial preparations, four hybrid deficient, four hybrid control, five pailid, and five nonpallid mice were decapitated; their livers were put into cold 0.25M sucrose, cut into smr.il Liver Mitochondria fix::: coc-Dcficiezit and PsIlIJ Mice: F;:r.cIcn and UlirasiracfTi : pieces, and washed three times. Mito chondria were separated as previously described (If). Oxygen uptake was de termined by fhe'polarographic assay of Abstract. Oxidative phosphorylation was studied in isolated liver mitochondria oxygen after the method of Chance and from manganese-deficient mice and in those from a mutant strain, pallid. In Williams (12), with /J-hydroxybutyrate mitochondria from manganese-deficient mice, ratios of adenosine triphosphate as the substrate in the reaction medium formed to oxygen consumed were normal, but oxygen uptake was reduced. Elec (Table 1). The oxygen electrode tron microscopy of these mitochondria revealed ultrastructural abnormalities was according to Packer (13). We including elongation and reorientation of cristae. No biochemical or structural calculated PtO ratios by determining abnor.nalities were found in mitochondria from pallid mice. . the reduction in the amount of added adenosine diphosphate per unit of oxy 1 r. number o[ animal specie.. a causes congenital ataxia resulting from gen consumed. Adenosine diphosphate si. ....g c.Tect of dietary .. :fic:cncy of impaired develop:---of otoliths (3). was measured by absorbance at 260 ivuu-.g. _^e during pregnancy is an ir- When .he diet of regnant mutant mice nm (14). The concentration g: protein rc'.-..con genual ataxL in the off- is supplemented with high amounts of in the mitochondrial suspension was de sp."'itT -h e ataxia, characterized by im manganese the otoliths ar.d postural be termined oy means ot ike biuret reac balance and loss of body righting re havior of the offspring are r. nrmal. tion (75). flexes, results from abnormal develop Thus, there is - role. Task ip between Median lobular hematic tissue was ment of the inner car, with defective the gene pallid and rnar.^ancsc metabo excised from ten mangancse-dcTicicnt morphogenesis of the otoliths (/, 2). A lism (A 4). hybrid, five control hybrid, and five mutant gene in mice, pallid (pa), also Although considerable attention has pallid adult female mice for examina- 1316 SCIENCE, VOL. 170 DSW 332424 STLCOPCB4079000