Document V3dEENmv3XEpbnXDnXxwYyYdg

tion ot grovvi;i. However, m one experi th;in normal but which then return n>. 4 treated with hcmichoiinium for 11 ment in which triethylcholine (400 mg/ ward normal during the period of eaiiy vtueVs were permanently po;son?d, we kg) was administered, the rate of re differentiation. amputated the regenerating leg and al generation was nearly 72 percent of As yet we have no way of decuiin* 1 lowed it to regrow without further administration of a drug. Regeneration was identical to that in controls, indi control, despite the fact that this drug paralyzed the animal more effectively than hcmichoiinium did. Botulinum whether treatment with hemicholimun, retards growth primarily by blockin'* synthesis of acetylcholine or by func* cating that the cflect of hemichoUnmm toxin, which also produces paralysis, tioning like curare (9). We have at was reversible. To demonstrate that the has been reported (6) not to delay re tempted to rule out the latter eficct by ' initial amputation slid not stimulate generation of the salamander limb. Fur simultaneously administering choline subsequent regeneration we measured thermore several changes were observed (10:1 molar ratio) and hcmichoiinium. the growth rates 10 weeks after the which cannot relate to the paralytic However, the combination proved toxic. Rm amputation and similarly after a effect of drug treatment. After 5 weeks A direct inhibition of mitosis by hemi- 1 rcumputatioo. The first and second of treatment with bemicholinium the cholinium has not been excluded, but rates of regeneration were identical in taste buds of the salamander showed in view of the ability of drug treatment each of two salamanders studied. We regressive end degenerative changes. to mimic io the salamander some then considered that treatment with After 19 weeks of treatment the taste features of dysautonomia, a disease hemicbolinium might inhibit growth be buds disappeared, but they returned characterized by a cholinergic defect, cause 25 percent of the body weight when treatment was discontinued. Chil hcmichoiinium appears to act as a cho- was lost during treatment. For that dren with dysautonomia not only show linolytic agent. study, the hind limb of an untreated a parasympathetic insufficiency with a salamander was amputated, but no jgrowth disturbance bat also a lack of food was given to the animal during the lingual taste buds (7). F. Hut A. Smith Deportments of Pharmacology and 10-week regeneration period. Despite There is much evidence supporting Anesthesiology, New York Medical a weight loss of 50 percent, the rate of the hypothesis that acetylcholine Is a College, New York 10029 . limb growth was uot different from that neurotrophic factor (5), but there are in previous controls. The diminished growth rate in treated salamanders correlated well with the microscopic findings. Salamanders treated for 5 weeks with bemicholinium 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 hemicholinium did not inhibit regeneration by means of an effect like that of colchi cine, for far fewer mitotic figures were also compelling reasons against its ready acceptance. Sioger (5) reports that microinfusion of atropine into the regenerate halts limb growth. On the other hand, infusion of acetylcholine into a denervated limb 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 the 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 influencing regeneration than is the motor component. On the other hand, regenerating tissue contains concentra RrfmacH mS Nmh - 1. M. Singer, Quart, Her. Biol IT, IS* (lUIt 2. E. O. Bulker and O. E. Schotts, t. tap. ZnI, . SI, 307 (1949). 3. D. B. Dfidmin, Spmpmtlum mm Ormwth mf the Nervous Syttem, O. E. W. Wuhamtimaa and M. O'Connor, Ed*. (Churchill, London. 196k), pp. 231-273; D. B. Drachma*. Stitttee 145. 719 (1964). 4. J. Dancis and A. A. Smith, N. Em$. /. M*4. 374, 307 (19M). 3. C. Riley. Aiemi. MM. 9, 137 (1917). 6. D. Drachma* and M. Sinyer, cited in Nmtm- telenet Research fr#m htiilrrtn 7, L. Guth, Ed. (Brookline, Massachusetts, 1941), p. 33. 7. A. Smith, A. Fatbmao, J. Danri*. Science 147. 1040 (1963). 5. M. Sinyer. in Develeplnt CeU Syitrm, mnt Their Control. D. fturtnick, Ed. (Ronald, Naw York, 1939). p. HJ-133. 9. R. E. Tbits and V. B. Brook*, feO. prme. 24. 369 (I960. 10. F.H. is a fellow of lb* Dysautcnomta Aw Hidation of New York. seen among treated specimens than tions of acetylcholine which are greater 20 July 1970; netted t October 1970 * among controls. Furthermore, the fact that the mesenchymal cells were not en larged argues against the possibility that hcmichoiinium blocks cell division at an Polychlorinated Biphenyl: Interaction with earlier stage. The drug also prevented the ro.iv.ive dedifferentiation of muscle Duck Hepatitis Virus MUNS 084896 tissue normally seen in sections just proximal to the amputation site. In un Abstract. Ten-day-old mallard ducklings fed a polychlorinated biphenyl at treated amputees the neurilemma! concentrations of 25, 50, and 100 parts per million for 10 days suffered no nuclei increased in number and en- apparent clinical Intoxication. Five days later these birds were challenged with Lir;eJ with dedifferentiation of the duck hepatitis virus, and they suffered significantly higher mortality than birds nerve invading more proximal parts, which were not exposed to the polychlorinated biphenyl. be: treated animats showed virtually no rv-.e cV a ~ v CJrosx and microscopic Polychlorinated biphenyls (PCB) a Swedish chemist reported their pres i'\.i.ri:u:ens showed that the vascu- along with DDE n.l*dich1oro*2,2-bis- ence in the tissues of pike and other 1 w -.-.c reccnerating stumps was (p-chlornphenyDethane] are reported wildlife (.?). Since then, their presence ..'o diminished, to be the most abundant of the chlo has been reported in additional wild Vnee :!? v.il.inlanders were main rinated hydrocarbon polliJlants in the life of Europe nnd North America (/. tained in .i p.ini.ilty p.valyred or paretic global ccoNystem (/). Despite the fact 4). These compounds are iutcrcuirv: slate by trevnent w:th hemicbolinium, that PCOs have been in wide use because they are an important source it is jvwrie i: n di.use uf the stump since 1930 (2), they remained eco of interference in the chemic.it ilciec- e-'n'.l .K.vn: :>r ti`c uh'crwd inhibi logically inconspicuous until 1006 when lion of DDI and its metabolites (>) .inj N*cim>c ihcv me it potential h.iz.jrd in the cnviiiMimcnl; on .1 weight lush, PCU piepmations have been sltown to have an cMindtol-ikgmdtng potential about five times iIkiI of />./>'-DDIZ or techni cal grade DDT |1.) tJ-trichloro-2.2-bis- (/>*chlornphcny))cth*nc| </). As part of an investigation of possi ble interactions between organochlorinc pollutanis and infectious diseases, this Study was initiated to determine wltcthcr 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 I0-day*old mallard ducklings. The three PCB diets contained Aroclor 1254 (in a corn oil premix) concentrations cal culated to he 25, 50, and 100 parts per million (ppm). These diets were fed to five groups of 25 ducklings each, three principal and two control groups (Table 1). All birds were leg-banded with color- coded, 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 liver weights. During the 10-day feeding trial, no birds died or became clinically 111, although duck lings In the group given 100 ppm PCB ware noticeably hypercxcitable 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-d*y 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. Differences in body weights among treatment means were ,highly significant (F = 20.30, d.f. =' 4 119) when tested by analysis of cmarianec. No statistical comparisons between individual treatment moans *c>c made. A similar analysis of liver weights (determined as a percentage of final body weight) disclosed highly sig- niluant riitTeicures among treatment me.ms (|- rr 5.S2, d.f. ' 4. IK). These d'hrreiuvs appeared to he within 1*11*111% ucjtvd with IT It r.iiher than between Heated and unhealed gioups O*'!'' 11. The small sample sizes (,V -- S| iw.vs'ii.iti's e,union m tnieipietation *! t -1 w, \o inteipiet.ilion o! .leiiiert.es among body ami liver ,'l's ''O' ottered .it this time. l.tl'le I. 1 in..] mean body anj lucr wcidiis of milliard durklim:* after beini; fed l'CH (AiuMor 1254, Munsaniu. St. Louis) /or U) days. Numbers in parentheses arc the number of Hints used. The liver and body weiplris were atljiiskd hv covariance for a common Marline mean; the liver weight ditcrniimJ as iwtcrnlagc of body weight. Ihc final con trol did not receive eiihti I'CU or DUV. Treatment PCB /ppm) Body Liver weight ' weight <R> <R) PCB 4 DHV 100 381.6 (25) 0.W6 (5) PCB + DHV so 3G8.R (25) .037 (5) PCB 4- DHV 25 352.8 (25) .029 (5) DHV control 0 315.2 (25) .036 (4) Control 0 309.6 (25) .034 (5) 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 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 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 intraperitoneally with 1.5 LD,U (50 percent elective k'.h..! dose for ducka) of DHV jvr b;;2. The first birds died about 47 hours diet inoculation. Mortality was recorded hourly for the remainder cf the hour experimental period wit]) one ex ception; there were 00 observai^ns be tween hours 63 through 69. The or.se! of mortality in all groups given PCB plus DHV began at least S to J6 hcu~s before that in the control group treated with the virus only (Fig. 1). In ade.tion, mortality levels occurring amonr PCB-plus-virus treatment groups at the end of the experiment were significantly higher at the .01 level of piobability than for ducklings receiving the virus only (chi square = 7.49, d.f. = 1). There was no significant difference in mortality among ducklings receiving the different concentrations of PCB plus virus (chi square = 3.79, d.f. = 2). No birds in the untreated group died (Tabic 2). All birds that died during the experi ment had gross pathognomonic liver lesions, indicating typical duck hepa titis infection (6). No other gross lesions, with the exception of varying amounts of edema within the peri cardium and thoracic cavity of some Table 2. Monthly among mallard docUinis caused by DHV during an 10-hour experimental period. No observations were made between 31 to 47 and 62 to 70 hours. The final control did not receive either PCB or DHV. Treatment PCB 4 DHV PCB + DHV PCB 4 DHV DHV control Control PCB (Ppm) too 50 25 No. of deaths/ total sample 8/11 13/20 7/20 3/21 0/20 Mortality Percentage Onset (hours) 44.4 65.0 35.0 14.3 0.0 31-47 55 31-47 62-70 Cessation (hours) 75 75 73 big. 1. Mortality amnnr mallard ducklings exposed lo both f'CH and DHV. DHV only, or neither agent. t./l H'n ppm I'l l'. \ DHV; (M 50 ppm IT'H , DMV. 1, ) ?s ppm ITTl 4. DHV; (,/) DIIV :a,d (, ) mil^alcd mnliol. Dolled p..itioo H lines piior 10 47 hours and between 62 anil 70 hours upie<cnt pciiod-. dm in". wliub no observations vveu* made. birds. were observed. There did not ap pear to be any relation between treat ment ami the presence or absence of edema. Histological and residue analy sis have not been completed at this lime. The suggestions that PCB may render a host more susceptible to certain types of infectious ageots is the significant Coding in this study. None of the con* Generations 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 sublethal concentrations appeared to in fluence the resulting mortality rates, causing two- to fourfold increases (M 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 modality were obtained with other orgauochlorine compounds in our laboratory (7). In those studies 30-day-old mallard duck lings which had been fed sublethal con centrations of p.p'-DDT or dieldrin exhibited three- to ninefold increases In modality over that of DHV controls (6 percent among virus controls versus 19 to 59 percent among groups receiv ing dieldrin plus DHV, and 19 to 40 percent among groups receiving p.p'DDT plus DHV). It is unlikely that In all these instances mortality in the DHV control groups would be less than half that in any interaction group on the basis of chance alone. This study illustrates one of the poleuliuii.il effects of sublethal conevnuations of chemical pollutants which arc often alluded to, but rarely documented. It also emphasizes the real differences that exist between "sublethal" and "noefTect" concentrations of pollutants. To the best of our knowledge, other stud ies in vivo of the possible effects of organochlorine pollutants on the sus ceptibility of a vertebrate host to a viral agent have not been repoded. Milton Friend Daniel O. Trainer Department of Veterinary Science, f/niverjiry of Wisconsin, Madison 53706 Rttimm and Note* 1. R, W. Riiebroufb, P. Rich, D, B. Ptikill, S. G. Herman, M. N. Kirven. Nantr* 220, 1091 (I9<#). 2. C. A. Penning, tnd. Enf. Cfitm. 22, ItSO (1930). 3. S. Jansen, New Set. 32, 612 (1966). 4. D. C. Holme*. J. H. Simmons. J. O'G. Tatton, Nafurt 216, 221 (1967); A. V. Holden and X. Marsde. Ibid., p. 1274; J. H. Koeman, M. C. Tea Notvtr T>a Bnuw, R. 11. DeVt*. ibid. 221, 1126 (1969); S. Jensen. A. G. Johnch. M. Otssos. O. OtteMind. ibid. 32J, 247 (1969). 3. G. Wldmark, /. An. Offic. Agr. Chtm. 50, 1069 (1967); L. M. Reynolds. Bull. iWrai. Cvnum. TonUvl. 4, 12* (1969). 6. F. P. Levine, in Diseases of Poultry, H. E. Belslar and I* H. Schwane, Ed. (Iowa Slaia Utile. Press. Ames, 1965), p. 83ft. 7. M. Friend and D. O. Trainer, J. midllfr DU., in press. 5. W* thank the Max MtOrnw Wildlife Founda tion for supplying mallards (or this study. Sup ported in part by the Patuxent Wildlife Re search Center, Bureau of Sport Fisheries and Wildlife. 16 July 1970; revised 16 October 1970 m Liver Mitochondria from Manganese-Deficient and Pallid Mice: Function and infrastructure Abstract Oxidative phosphorylation was studied in isolated liver mitochondria from manganese-deficient mice 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 wur reduced. Elec tron microscopy of these mitochondria revealed ultrastructural abnormalities including elongation and reorientation of cristae. 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 marcaaese during pregnancy is an ir reversible congenital ataxia in the off>pr:r.g. The ataxia. characterized by im balance and lo\s of body righting re flexes. results from abnormal develop ment of the inner car, with defective morphogenesis of the otoliths (/, 3). A mu:.ml ;vne in mice, pullid (/>n), also causes congenital ataxia resulting from impaired development of otoliths (3). 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. Tims, there is a relationship between the gene pallid and manganese metabo lism (3, -/). Although considerable attention has been paid to the role of manganese hi enzyme function. Us specific biochemk cal action in vivo has remained ww clear (5). A variety of evidence, how ever, has implicated manganese in tochondrial function, particularly is oxidative phosphorylation (5, 6). Is our laboratory, isolated liver mitochorw dria from manganese-deficient raft showed abnormal oxidative phosphory* lation which appeared to be due to rv* duced oxidative capacity rather thas to lack of coupling (7). We now report oo P:0 ratios (moles of adenosine triphosphate formed relative to gram atoms of oxygen consumed) and rate ot oxygen uptake in isolated liver mite* chondria from manganese-deticient and from pallid mice, and on the fine strtis ture of liver tissue as studied with the electron microscope. Female mice maintained in a colony originally derived from a fuur-way cross of inbred strains C57BI/16J C3H/J, ARK/J, and DBA/2J (I) were given a purified diet (9) coatah* ing 1 part manganese per million (de ficient diet) during pregnancy. Thefe progeny were maintained on the sans diets and were killed as adults for uae in these studies. Hybrid mice of th* same strain were fed a similar puriffec diet except that it contained 45 pans or manganese per million (control diet) Pallid mice (pa/pa) and their non pallid littermates (C57BI/10J-pa) wen maintained on a slock diet {10). For mitochondrial preparations, foot hybrid deficient, four hybrid control five pallid, and five nonpnllid mk were decapitated; their livers were pe into cold 0.25M sucrose, cut into smal pieces, and washed three times. Milo chondria were separated as previous! described (//). Oxygen uptake was dr termined by the polarographic assay o oxygen after the method of Chance am Williams {12), with /J-hydroxybutyrab as the substrate in the reaction mediun (Table 1). The oxygen clcctrodi was according to Packer {13). W calculated P:0 ratios by determinim the reduction in the amount of addn adenosine diphosphate per unit of oxy gen consumed. Adenosine riiphosphafr was measured by absorbance at 26< nm (14). The concentration of proteis in the mitochondrial suspension was tie termined by means of the hmict reae tion (15). Median lobular hepatic tissue wa excised from ton m.in^uncse-iL-licicn h\brill, five control Inhrid. and fiv pallid adult tcm.ilo mice tor ex.muiw im Si iimi, Vnl. tH MONS 084898