Document JvvOoJeBErG96yx099w3gNdD6

s 9: RECE1V' DEC 2 9 1882 HASKELL LAB- CLINICAL SIGNS, RADIOLOGY AND TISSUE LEAD DISTRIBUTION OF DOGS ADMINISTERED A MIXTURE OF LEAD CHLORIDE, LEAD BROMIDE AND LEAD SULPHATE ,'aA..H* Ha mir *, N.. D. Su l l iv an *, P. D. HANOspNt, J. S. Wil k in s o n * a n d .. R. B. La v e l l e* .. SUMMARY Eight-month-old dogs maintained on a high-fat-low-calpium diet were administered admixture of teed chloride, lead bromide and lead sulphate for prolonged periods at 4 different dose levels. Dogs on high levels of lead showed marked weight loss and gastrointestinal symptoms followed by death. Two dogs on low lead levels developed . -..neurological signs. Radiological, investigations showed radiopaque particles i:n 27 par centof abdominal radiographs and `lead lines' in the distal radius of 3 dogs. Highest tissue lead ' r-were found in bones followed by liver and kidney, brain and spinal cord. 1- H-V' * *>M -SsB'V- Introduction Lead in petrokis the major source of pollution in the UnitecL.States (Beiiles 1975). During recent years riiuch/attention has been given to the study of chronic,lead, exposure in animals. Most of the expe.rimentaLi'studies have used lead acetate or lead carbonate:!, for producing the toxicity but neither ofr these-substances are significant constituents.of..exhaiist emission of internal combus tion engines using leaded petrol. The purpose, of this paper is to report oil the clinical observations, radiological investigations and tissue distribution of lead in dogs which were exposed for-prolonged periods to a lead salt' mixture .at different dose rates. The lead.salt mixture consisted of lead chlo ride:):, lead.bromide^, and lead sulphate! in 1:1:2 proportions, respectively. These lead compounds in approximately similar proportions are the major byproducts in the exhaust emission of internal combustion engines which utilise leaded petrol (Bloom 1979 personal communication). . , Materials and Methods Animals and Diet Eleven Kelpie-cross dogs of 8 months of age were obtained for the purpose of this experiment. Prior to the commencement of the experiment,, the dogs were vaccinated against canine distemper, canine-hepatitis,, parvovirus infection (using inactivated feline enteritis vaccine) and were dewormed (Pra ziquantel^. and Pyramei Pamoate11). The dogs were then divided into- S',groups of 2 dogs, except group 5 which had * University of Melbourne, Veterinary Clinical Centre, Werribee, Victoria, 3030 * Department of Agriculture, Agricultural Chemistry Laboratories, Melbourne, Victoria. .3000 Ajax Chemicals, Sydney. Mew Soucn V/afes, Bayer Australia Ud. Botany. New .South Wales Pfuer Agricare Pty- Ltd, West Hyde. Ne*.v So.ulh Wales 3 dogs. All the dogs. were maintained on a high-fat-lowcalcium diet which consisted of minced meat scraps and fat. Each dog received 500 g of this diet per day in a stainless steel container. The diet was analysed .and found to contain 3.5% to 45% fat and less than 0.1% calcium' (dry weight, basis). After a period of stabilisation on this diet (7 days), the 5 groups of dogs were administered the lead salt mixture at the dose rate of 0, 5, 13, 30 and 60 mg per kilogram body weight per day (Table 1). The lead.salt mixture was weighed into gelatin capsules and was administered orally. The control group (group 4) was given 2 mg sodium chloride* (containing less than 0.005% lead) per day in gelatiii capsules. The dpgs were weighed once a week and the lead dose for the week was calculated on this weight. Samples Bipod samples (7.5 ml) were drawn from the jugular vein of each dog. 5 ml of this blood was placed in plastic tubescontaining heparin (for lead analysis) and the remainder was placed in EDTA (for routine hematology). Samples were collected daily for the first 3 days and then weekly for the remainder of the experimental period (21 weeks). Routine hematology was carried out using standard laboratory methods. Radiographs were taken of the right foreleg (A-P view) and of the lateral abdomen (which also included the lumbar vertebrae) of each dog prior to the commencement of the experiment. The. radiographs were repeated approximately every 30 days to detect Jead particles in the gastrointestinal tract and to monitor changes in the bones. Radiographs of all the dogs were taken at the same time, except in the case of the dogs that died within the first 30 days in which case the radiographs of the fight foreleg were taken after death. A complete post m.Ortem examination of all dogs was carried out immediately after death. Samples of liver, kidney, right distal radius, fourth lumbar vertebra, cerebrum (frontal lobe) and lumbar spinal cord were frozen for subsequent determmauon of lead content. Representative tissues of most organs including whole.brain were preserved in 10% buffered formalin for histopathological examination. The pathological findings wfii be published elsewhere. Chemical Analysis Ail chemical analyses were performed at the Victorian Department of Agriculture, Division of Agricultural Chemistry 'May aha :Baker Ud.. Dag.enh.am, United Kii Monthly Radiographic Examination of Abdomens of Lead-Administered Dogs Dog No. Dosage No. of NO. Showing X-rays Taken Lead Particles H 176 H 177 H 178 H 179 H 180 H .1.81 H 184 H 185 H 186 5 mg/kg/dav 5 mg/kg/day 15 mg/kg/day 15 mg/kg/day 30 mg/kg/day 30 mg/kg/day 60 mg/kg/day 60 mg/kg/day 60 mg/kg/day 6 3 2 .22 - 1 "-- 2 0 i 0 -* Total {9 dogs) 15 4 (27%) w. Figure S.. Body welgMa (kg) .and blood lead levels (ftg^di) of dogs administered lead at 60 mg. per kilogram body weigh) per day. 3 days. The level then dropped markedly for the next 2 weeks and then gradually increased again (Figures 3 and 5). . . In group 4 (control dogs), the blood lead level increased slightly and remained above 3.5 gg/dl in dog HI82 for approximately 9 weeks (Figure 4). Dog H 18.3 was killed by euthanasia on day 58 since by this time more than half the experimental dogs were dead. Hematology Routine hematological findings indicated a slight drop in the packed cell volume in the dogs fed lead. Early in the course of the experiment, nucleated red blood cells and basophilic stippling were visible in all dogs dosed with lead, but this whs not a regular feature of blood smears from these dogs. No nucleated red blood cells or basophilic stippling were seen in the blood from the control' dogs. Radiology Table .2 shows the radiological findings in the lead administered dogs. Lead particles were detected in the gastrointestinal tract in only 4 abdominal radiographs (27% of the total radiographs of the dogs fed lead). `Lead lines' (radiopaque lines) were detected in the right distal radius of 3 dogs (one each from groups 2, 3 and 5) and were not seen in the lumbar vertebral bodies of any dogs. The `lead lines' were visible in the first series of radiographs which were taken on day 30 of the experiment. At this time they were rather ..faint and could only be appreciated when the radiographs were com pared to the pretreatment films. These lines became progressively denser in subsequent radi ographs (Figure 6B). Radiopaque particles in the alimentary tract or `lead lines' were not seen in the radiographs of the control dogs. Tissue Lead Levels Tissues from the dogs administerd lead showed higher levels of lead than did the tissues of the control dogs (Table 1). Generally the highest lead levels were seen in the distal radius, followed fay the lumbar vertebra, the bones of the calvarium, liver and kidney, cerebrum and spinal cord. The dogs showing `lead lines' on their radiographs had the highest lead levels (over 400 ppm) in the distal radius and dogs that developed neurological signs had high lead levels (over 3 ppm) in the cerebrum. Discussion Anorexia, weight loss, vomiting and diarrhoea as seen in the dogs dosed with lead have been reported in accidental and experimental cases of lead poisoning (Zook et a/ 1969; Stowe et al 1973; Stowe and Vandevclde 1.979; Hamtr 1981). In the low dose group (5 mg/kg), vomiting and diarrhoea were not observed. Thompson (1979) and Mitern,a era/(1980), who used lead carbonate and lead a produce n gastrointe: low dose ; Neurolo of lead in were seen the other note that t gastrointes administra cal signs, poisoning, ment is cc in 63 to 7 H.amir 19;? ditions, unless jfe'TJDU P040007025"Co --- meat The efficacy of our experimental procedure in producing nervous signs thus campares favour ably with'that achieved by previous workers. This suggests that, the simple diet of meat scraps and fat imparted a significant predisposition to lead encephalopathy- However,, our; results also emphasize that the development of the neurological signs is favoured by the use of low levels of lead (5 to 15 mg/kg)-' .-.- - ' A blood lead level of 35 pg/dl is considered as the upper normal limit for dogs (Zook 1978) and levels-of 60 pg/dl or more are diagnostic of lead poisoning (Zook and Carpenter 1977). In our study blood lead levels increased' to above 120 pig/dLia all lead administered dogs. The-increasewas dependent on tile dose leveL_and-the duration of`exposure to lead. This wassalsrpTobserved by Thompson ^:969) and Mitema'ef"3.1 (1980) who used lead crrbonate and lead acetate-respectively to produce lead poisoning in dogs. However, in our study, the rise in blood lead concentration in dogs dosed with lead was steeper and of greater magnitude than was found by-these workers (Figures I, 2., 3 and. 5). This suggests that either the lead salts used were more readily absorbed from gut than are lead acetate-and lead carbonate or that the high-fat-low-calcium diet was condu cive to increased lead absorption- Our view is that the experimental diet played a major role in increasing Bari absorption since- Barttrop and Kboo (1976) have demonstrated-that'diets defi cient in minerals or containing a high proportion of dietary fat increased lead absorption from the gut of laboratory animals by 20 and 7 fold respectively. The slight: increase in the blood lead level of the control dogs between the first and the 13th week of experiment was attributed to the control dogs coming in contact with dogs administered lead during routine experimental procedures (weighing,., radiography and sample collection) since ah extensive search-aimed at detecting any possible sou -ce of contamination during collec tion, storage, handling and analysis of blood _ failed to reveal any source of lead contamination. The assumption that the control dogs acquired lead from the dogs administered lead appears to be valid since after the tenth week of the exper iment, only one dog fed lead remained and .soon after that the biood lead level of the remaining control dog (Hi82) decreased markedly (Figure 4). Radiological investigations of dogs suspected of having lead poisoning for detection of lead particles ip gastrointestinal tract and for detection of `lead lines' in long bones has been recom mended in the standard veterinary texts (Oehme 1975: Buck apd Beskin 1979) and in published work (Zook et ai 1969). Zook et al (1969) however, recommended further investigations on the usefulness of detection of `lead lines' as diagnostic aid in. cases of lead' toxicity. Results of our radiological investigations were not particularly rewarding since only 27 per cent of abdominal radiographs showed' lead particles in the alimentary tract and only 3 dogs (33 per cent of lead-administered dogs) had `lead lines' in the radius. These findings are however com parable to those of Zook ef a/ (i 969) who found radiopaque-, particles in 32 pet cent and `lead lines' in 39 per cent of cases of accidental lead poisonings. In the present experiment, the animals were maintained, on a high-fat-Iow-calcium diet and the lead was administered in a powdered formSince all 3 factors, high dietary fat, low dietary calcium and smairparticle size of lead are known to increase lead absorption (Barttrop and Khoo 1976; Barttrop and Meek- 1979), it is probable that a high proportion of the administered lead was absorbed from the intestines and this may. have contributed to negative radiological find ings in a large proportion of the abdominal radiographs. Acknowledgment We thank Miss S.. Wilkinson for technical assistance. References Bararop, D, and Khoo, H. E. (!976)--Sci. Total Environ. 6: 255. Barttrop, D. and Meek, F. (1979) --Arch. Environ. Health. 34: 2S0. - - --- Beiilcs, R. P. (1975) -- in Toxicology. Ed, Casarett, L. J. and Douil, I. MacMillan Inc, New York, p 477. Buck, W. B. and Hoskin, J. D. (.1979):-- In Canine Medicine Ed. Catcptt, E. i. 4th edn, Vol. 1., American Veterinary Publications. Santa Barbara, California, p 166. Calyery, H. O., Laug, E. P. and Morris, H. j. (.1938) -- Pharmacol, exp. Ther. 64: 364, Hamir, A. N. (1981) -- Vet. Rec. 108: 438. Mitema, E.S., Ochttie, F.S., Penumarthy, L. and Moore, W. E. (1980) --Am. J ret. Res 41: 632. Oehme, F. W. (1975).-- In Textbook of Veterinary interna! Medicine.. Ed. Ettinger, S.,(). Vol. I., Saunders, Philadel phia. p 102. Stowe, H. D., Coyer, R. A., Krieman, M. M., Wilson, M. and Cates, M. (19,73) -- Arch. Pathol. 95: 106. Stowe, H. O. and Vandeveide, M. (1979) -- Neuropathol. exp. Neurol. 38: 463.' Thompson, R, R. (1979) -- In Refresher Course in Clinical Pathology, The Postgraduate Committee in Veterinary Science, University of Sydney, p 4.1. Zook, B. C. (.1978) -- In toxicity of Heavy Metals in the Environment. Part 1. Ed. Oehme, F. W. Dekker, New York, N. Y. p 179, Zook, B. C. and Carpenter J, L. (1977) -- In Current Veterinary Therapy VI, Small Animal Practice. Saunders, Philadelphia, p 128. Zook, ft. C., Carpenter. J, L. and Leeds, E. ft. (1969) --X. Am, v,et. med. Ass. 155: 1329. (Accepted for publication 23 June IRSI) A Blood used freof glosr Many -.. relative'i ratory, c. the relia.therefore ison of s-. in canin(Azostix, method) . and a reft Fifty Moot from 14 nor: I disease (dia>_ haematology taemic dogs these provide 7 and 12 sat\ Immediate: used to deter laced whole t ing to the remaining bit by the other .Serum ure; Unitestf and instructions i natet was the dikite sample.300 photome samples requi necessary wit method was t * 'Ames Cc . E fOeneral Dtagnc BiotOvnamics p3iciochem-Sri-