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REVIEW' OF LEAD STUDIES IN ANIMALS CARRIED OUT AT HASKELL LABORATORY - TWO-YEAR FEEDING STUDY AND RESPONSE TO HEMORRHAGE STUDY Alex Azar, M.D*, Henry J. Troehimowicz, D.Sc., Mary E. Maxfield, Ph.D. E. I. du Pont de Nemours and Company Haskell Laboratory Elkton Road Newark, Delaware 19711 N40702 SUMMARY A brief review Of two animal studies carried out at Haskell Laboratory is given. The first describes a two-year feeding study during which rats and dogs were fed diets to which was added 0, 10, 50, 100 and 500 ppm lead as lead acetate. Rats were also fed levels of 1000 and 2000 ppm Pb, Various clinical, biochemical, histopathologic and reproductive studies were done on the animals. In the second study, dogs with markedly depressed ALA-dehydrase activity were severely hemorrhaged. Their recovery curves of hemoglobin, red cell count and hematocrit were compared to those obtained on hemorrhaged control dogs. DUP050057949 REVIEW OF LEAD STUDIES IN ANIMALS CARRIED OUT AT HASKELL LABORATORY - TWO-YEAR FEEDING STUDY AND RESPONSE TO HEMORRHAGE STUDY INTRODUCTION Today, I should like to swimnarize two animal studies carried out at Haskell Laboratory to provide additional information on the toxicology of lead. Both studies have been presented in more detail elsewhere (I?2) and are only summarized here. It has generally been agreed that in man, thp^iet is the major source \f d intake; however, long-term lea ceding /studies in animals have onljKreeently been/tSarried out .er> orted that 5 ppm l^ad\cetate in/the drinking i mice an increased mortality arwL decr^tsed longevity, subseque: cation(4), however, theseVWvestigators reported that the initial cudy publishe/aSln 1964 contained/25 rather tjai and the the animals arere chromium-deficient. When ; epeated the sti sing non-^rondxim^eficient rats dr: containing 2Vj?pra Pb, the mortality and lcnagevitv>were not and fund to be non-tumdrigenic. M?re\r^ently, the; vestigators reported that ppm Pb in the dmoking water significant^ on the indices of reproduction o^jnice and. Jessup (6) fried out oheof the most comprehensive toxicologic studies of le^cfl. ever undertaken, which include^a 22-month feedip study using/xats, dogs and(m6nk<^s, a three^generation reprobation study using rats, a teratology siu^y in rsrobits, a rat an&smozikey behavior^ study, a one-year newborn rat ..Carcinogenesis study, tissue enzyme/studies, electron microscopy Mid radiotracer studies. Ingestion of l(fjpPm Pb produced no demonstrate adverse effep^s; 50 ppm resulted in minimal equivocal effects; and 100 anaSlOOO pp^L produced histologic but not functional changes in the kidney or\rSts. Ten ppm Pb was equivalent to approximately 100 times the quantity of lead ingested by an average adult man. METHODS In order to provi< .tion on the effects of long- term ingestion of lead diets containing lead acetate for two years. The concentration of lead added was 0, 10, 50, 100 and 500 ppm. There were 50 male and 50 female rats per dose level and 100 male and 100 female control rats receiving the basal laboratory diet. Four male.^and ,/ppr,female beagle dogs were used at each dose level. After the'^myrols'Ln effect for several months, a second two-year feeding study was initiated in rats to provide dietary lead levels of 0, 1000 and 2000 ppm added lead. In the latter study, only 20 male and 20 female rats were used per dose level. DUP050057950 . (During animafl-s wars by a irecogaf of Pepnsy] vi Periodic tlx atomic at> so; hematocrit, xirea| nitrpgi to gLobulin Routine apid enzyme de'lt excretion o urine were -2 C/. -y ^ *Cf 7tik> IAM&- ;a^ \A ttor nTA&f f/ was dpne jon^j js/ty <HkXc 't y> e the two study s lar to that described by Oser and OgerlXl rats fed'O, 10^.50, 100; 1000 and 2000 ppm lead* / .n. on KESULTS There were no significant eriects on the appearance, behavior, weight gain, mortality or neurologic examination of dogs receiving as much as 500 ppm Fb in their diet. The clinical appearance and behavior of the rats receiving as much as 2000 ppm Pb was normal; however, the rate of weight gain was depressed in both male and rats ingesting diets containing 1000 and 2000 ppm Pb. J % 1 iaLiaBb--w, .... "Br^ievcl^was" &6TZ .Vrft -i * i V* <?>**<:>, : ffi./ t Jy n^7 i^dney t^Srs were seen in male rats at the 500 ppm Pb level and above. These were not seen in the r^, y xb&itc. females until the 2000 ppm Fb level. Most of the tumors were adenomas derived from the tubular epithelium. There were no pathologic changes, seen in the rats fed up to and including 100 ppm lead in their diet. With the dogs, there were no pathologic effects of dietary lead on any of the organ systems in the females. A slight degree of cytomegaly was found in the proximal convoluted tubule of the kidney in two of four male dogs fed 500 ppm Pb. ifobte-'II* shows the effect of dietary lead on the hematopoietic system. Hemoglobin and hematocrit were depressed only in the rats receiving 1000 ppm Fb or more. Stippled cells were increased at the 10 ppm level in the rat and were no ^jLc^eased in the dog until the diet contained 500 ppm Fb. A ^^fOTroOTC^decrease in ALAI) activity was seen at a dietary Pb concentration of 50 ppm in the rats and 100 ppm in the dogs. This decrease, however, was not associated with any increased urinary excretion of its substrate BALA until a concentration of 500 ppm Pb was present in the diet. These findings are consistent with the functional enzyme reserve concept seen in many biological systems. (-) No significant abnormalities were found in the other clinical chemistries examined. i DUP050057951 -3- Figure 1 shows that the blood, f d concentration of dogs rose rapidly during the first four to six months and, thereafter, appeared to plateau. Similar findings were found in the rats and with urine lead. The apparent lag time between continued ingestion of lead and subsequent rise in blood Fb may be a factor in the finding that pediatric lead poisoning cases occur more frequently in the summer months as opposed to the winter season when children would more likely be exposed to paint containing lead, v9) A high degree of correlation was found between blood Fb concentration and concentrations of lead found in the kidney, bone, liver and brain of both rats and dogs. Figure 2 shows the high degree of correlation between blood Fb and kidney Fb concentration in dogs. Thus, blood Fb concentration appears to be a good index of tissue Fb deposition as well as exposure to Fb, The three-generation, six-litter, reproduction study shoved that there was no effect of dietary lead at concentrations of 0, 10, 50, 100, 1000 and 2000 ppm Fb on the number of pregnancies, number of pups born alive, fertility index, gestation index, viability index or lactation index. At 1000 and 2000 ppm dietary Pb, however, the average weight of weanling rats was slightly decreased and histologic examination of-eMr-orgau sys the 21-day old weanling rats (F3B) showed * histologic changes in the kidney comparable to thog^seen in adult rats receiving 500 npm or more Fb ijjj, their ' k -C f (tat A-n/ (% iMT The average concentration of lead in the blood, urine, kidney, bone and hna&Kr of the rats and dogs after ingesting lead for 24 months is shown in Table 111, As the amount of lead in the diet was increased, the lead content of the tissues increased. -Tnir-H" The blood and urine. Fb concentration of dogs receiving 10 ppm Fb or more was significantly greater than corresponding control values. Similar findings occurred in the rat. if i' iftt 1,7?t U t/lfi n tf, 't&* (Hf T&i# -t&jM ^ -p - 1 A , ft Xc,r/-*- iA* ^ " A* -t~/ 4, jt dUU+*A W ft A H - 4c yA-t Ml* tulyy'y * y `tr/7,, , ft't ft ` (Cl&xA&U^ 4^ ' `^ ^ . <2Hu" svft T tfo t e l l "Ah o s e w h o h a v e a n e e d t o k n o w DUP050057952 -h- Similarly, adult humans generally begin to show clinical signs of.lead poisoning at blood Fb values ahpveJSO |ig/lOO gm. In this a dietary Fb level of 500 ppm pfeSwed- a blood Pb concentration of about 80 iag/100 ml which was associated with an increase in DAIA excretion. At this level and higher, increased mortality and renal changes were'fooserved in the rats. The latter may be attributable to the very nigh concentration of lead found in the kidney of the rat compared to man and the dog Thus, when the results of this animal study are examined in terms of blood and tissue lead concentrations rather than on a basis of dose level, the findings are similar to those reported in humans. HEMORRHAGE STUDY ~hc/4t<J.K ALA-dehydrase is one of the enzymes involved in hemoglobin systhesis. Our findings and those of others(8) suggest that despite a depression of its activity by lead, the body has sufficient enzyme reserve to meet its day to day needs. However, it could be argued that the body might not be able to cope with a stressful situation involving the loss of large quantities of blood. In order to investigate this situation, the following experiment was carried out. Thirty-six dogs were divfcE^^ groups of 12, with each containing six males and six females. One group served as a control; the second group received 100 ppm Pb as lead acetate in their diet and the third group was given 500 ppm Pb. After the animals were on this diet for 31 weeks, the dietary lead concentration of the high level group was doubled to 1000 ppm because the enzyme activity was not as low as desired. the ALA- dehydrase activity q severely depressed WhojaHiotegtrablff, thn The group of dogs given 100 ppm Pb in the showed a %$ reduction in ALA-dehydrase activity. At the end oirjpo weeks, the' blood concentration of the dogs given highest level of lead averaged 60~80 jig Pb/lOO gm blood. OjFbehavioro^ pi a approximately one-half of the dogs circulating blood volume was removed from the jugular vein in a two-stage operation .using a modified Whipple (l) procedure, ^^esthoala was^niot necessary, hemorrltagew--- \ DUP050057953 -5A 30 to 40 percent reduction in hemoglobin concentration, red cotmfcand hematocrit ratio occurred as a result of the hemorrhage. sEoyrs the recovery curve for hemoglobin concentration, Bigorous statistical treatment of the data demonstrated that the recovery curves of the hemoglobin concentration, red cell count and hematocrit ratio were not effected by the presence of lead. This study clearly demonstrates that despite severe depression of AIA-dehydrase activity, the ability of the blood-forming mechanisms to manufacture hemoglobin and red clood cells was not measurably affected even when required to junction at accelerated rates, it is reasonable to conclude that while ALAI) is essential to the synthesis of hemoglobin, the amount of enzyme needed for this function is but a small fraction of that normally present. DUP050057954 REFERENCES 1. Trochimowicz, H. J., J. R. Barnes, H. Sherman, E. F. Stula and N. W. Henry: The Effect of Prolonged Feeding of Low Levels of Lead to Laboratory Animals. Presented at the American Industrial Hygiene Association Meeting, San Francisco, California, May 1972. Paper will be submitted to the American Industrial Hygiene Journal. 2. Maxfield, M. E., G. J. Stopps, J. R, Barnes, R. D. Snee and A. Azar: Effect of Lead on Blood Regeneration Following Acute Hemorrhage in Dogs. American Industrial Hygiene Association Journal (May 1972). 3. Schroeder, H. A., J. J. Balassa and W. H. Vintom; Chromium, Lead, Cadmium, Nickel and Titanium in Mice: Effect on Mortality, Tumors and Tissue Levels. Journal of Nutrition, 8^: 239-250 . (July 1964). 4. Schroeder, H. A., M. MLtchener and A. P. Nason: Zirconium, Niobium, Antimony, Vanadium and Lead in Rats: Life-Term Studies. Journal of Nutrition, 100:99-68 (1970). ,. , 5. Schroeder, H. A. and M. Mitchener: Toxic Effects of Trace Metals on the Reproduction of Mice and Rats. Archives of. Environmental Health, 23:102-106 (1971) ` 6. Jessup, D. C.: The Chronic Toxicity of Lead, American Petroleum Institute Medical Research Report No. EA7102. The Clearinghouse House for Federal Scientific and Technical Information, National Technical Information Services, 5285 Port Royal Road, Springfield, Virginia 22151 (April 1971). 7. Oser, B. L. and M. Oser: Nutritional Studies on Rats on Diets Containing High Levels of Partial Ester Emulsifiers, Journal of Nutrition, 60:4, pp. 489-505 (December 1956). ' ;: , 8. Lead, Airborne Lead in Perspective, National Academy of Sciehces,';;,';'',':!'.:,'' 2101 Constitution Avenue, N.W., Washington, D. C* 204l8 (1972). 9. Chisolm, J. J., Jr.:, Lead Poisoning,'Scientific American, 224:15-23!'- ; 10, Whipple, 6, H., C. W. Hooper and F. S. Robschert: Blood Regeneration Following Simple Anemia* I, Mix Diet Reaction, American Journal of Physiology, 52.(2): 151 (September 1920). XI. Barry, P.Sal. and Mossman, D. B.: Lead Concentrations in Human Tissues. Brit. J. Industr. Hed. 22:339-351 (1970). 12. Goldwater, L. J. and Hoover, A. W,: An International Study of "Norma 1,? Levels of Lead in Blood and Urine. Arch. Environ. Health 15:60-63, (1967). DUP050057955 TABLE X MORTALITY AND 1aixm t u mo r s nI RATS FED LEAD ACETATE FOR TWO YEARS Dietary Fba^ (ppm) 5 IS 62 l4l 548 No. of Rats of Each Sex 100 50 50 50 50 4> Mortality^ ^ Male Female 57 34 56 >0 56 28 56 28 52 36 Kidney Tumors Male Female 00 00 00 00 10 0 5 1150 2102 20 50 35 0 0 20 50 50 50 o 20 So 35 80 35 a) Measured concentration of pb in diet. vi rove vtwu# ao.ea or were Bacrincea, in extremis. TABLE II EFFECT OF DIETARY Pb ON HEMOPOIETIC SYSTEM (Mean 95% C. L.)> Dietary Pb Hemoglobin Hematocrit Stippled Cells ------CeepI J 00 (cells/50 mg) . 5 Rat 15.39 (2) (Dog) (16.23) 42.9 (44.1) 0.05 (0.00) ALAD (units/ml rbc^ 22.0 (23.4) DALA (mg 70 0.42 <0,26) 18 (16) 62 (57) 141 <155) 548 (576) 15.27 <16.04) 15.42 (16.12) 15.32 (16.27) 15.03 <15.64) 42.2 (43.1) 42.3 (43.6) 42.1 <44,1) 42.1 (42.4) 0.18* <0.0i) 0,33 (0.00) 0.50 <0.04) 2.11 (0.23)* 22.2 <21.8) 14.4* (17,8) 9.3 (10.6) 3.6 (3.9) 0.43 (0.27) 0.42 (0.29) 0.44 (0.27) 0.97* (0.57) * 95% C, L. 0.24 (0.57) 0.7 <1.8) 0.14 <0.11) 2.2 ' (A.J) 6.53 (0.08) 3 15.72 45.2 0.04 17.3 0.49* 1130 14.73* 43.1* 4.27 2.4* 2.09* 2102 14,37 41.1 7.57 1.6 2.38 95% C. L. 0.29 0.8 0.63 0.7 0,21 a) C, L. = Confidence Limits. * lowest*.level at which significant difference from control occurred (P < . 05) TABES III AVERAGE TISSUE LEAD CONCENTRATION IN RATS AND DOGS FED DIETS CONTAINING LEAD ACETATE Dietary Pb 5 Bat (2) Bog XB (16) 62 (57) l4l(155) 548 (576) Blood Pb* . (uR$) 12.7 (15.8) 11.0 (16.6)* 18.5* (31.5) 35.2 Xfe.5) 77.8 (75.8) Urine Pb^ 19 42 59* 62* 79 103 148 156 451 325 Kidney Pba Liver Pb* (ug/g) (ug/g) 0.40 (0.22) 0.31 (0,41)* 0.18 (0.30) 0.16 (0.66)* 0.S5* (0.74) 0.31* (1.57) 2.31 (1.16) 13.20 (2.91) 4 o.6o (2.26) 1.87 (7.09) Brain pb* 0.14 (0,05) 0.19* (0.06) 0,28 (0.98)* 0.38 (0.12) 1.06 (0.55) Bone Fba 9.19 (4.88) 9.00 (6.19) 17.50* (6.36) 26.75 (10.37) 91.40 (17.35)* 3 3130 2102 Human-^>^ Mean Range 16.4 98.6* 98.4 <: 17 .15-40 44 1046* 1281 0.17 13.37* 11.60 0.13 1.87* 2.86 o.ii 0.88* 1.48 1.87 280,54 409.05 < 35 20-65 0.52 .Cl-2.20 6.84 .03-3.13 0.13 19.04 .01-. 78 . .21-49.5 a value at 24 months; b average over 2-year period; * - lowest level at which significant ( p < *05) difference from control occurred. gpp !s * DUP050057956 FIGURE 1 TWO YEAR LEAP FEEDING STUDY (DOGS) REGRESSION < KIDNEY Pb on 8L00D Pb f i l FIGURE 2 TWO YEAR LEAD FEEDING STUDY IN DOGS 500ppm i & 100ppm SOppm 10 ppm Oppm fcV t j*?; jL? i DUP050057957 FIGURE 3 BLOOD ALA DEHYDRASE (CROUP AVERAGES) 34 I I Pb 0 ppm -- O MALES i 2T Pb IOO ppm A 3JX Pb 500--1000ppm n~--g| .!I J26* ACTIVITY E OOl. o q i i I UNITS PER m l R B C -l 14 X 10 ^ ^ --------- "--a-" 4. .10- 16- ........ 22- 28. PRE TREATMENT WEEK - 34. FIGURE 4 RECOVERY FOLLOWING HEMORRHAGE KEMOGLOBIN CONCENTRATION CROUP AVERAGE ONE STANDARD DEVIATION X Pb 0 ppm # 31 Pb tOO ppm A HI Pb 500-r-(000 ppm MALES I 4001 40 -ZMm PERCENT OF PRE-HEMORRHAGE AVERAGE 20 I| I 0 *0. 20. 25 30. .35 40. . .45 OAY AFTER END OF HEMORRHAGE it t}' ! b I i r "i-- fy. i4 DUP050057958