Document xza36rqqw6ERjBeO7aqYK9dz1

TWO YEAR FEEDING STUDIES ON LEAD Medical Research Project 787 HEMATOLOGIC AND BIOCHEMICAL RESPONSES OF DOGS TO LEAD ACETATE IN THEIR DIET PROCEDURE: Samples of blood and urine were collected three times before the test began, after one, two and three months and then every three months for the remainder of the two years. Blood was taken by venipuncture for the hematologic and biochemical measurements. A 24-hour urine specimen was collected on the day preceeding or following the blood sample. Two dogs, one male and one female, were sacrificed at the end of one year. All but one female survived the second year. At the end of the test period, the remaining animals were sacrificed and tissues were taken for lead analysis. METHODS: The hematologic examination of the blood, made on a portion of the sample collected, was the same as described for the rats. The urinalysis consisted of a measure of the specific gravity, a semi-quantitative measure of urobilinogen^ and a test for acetone^ and bilirubin3 as well as the routine analyses described for the rats. Urine glutamic-pyruvic transaminase, however, was not measured. The biochemical measurements made on the blood included a measure of: glucose using Boehinger reagent^; urea nitrogen by the method of Karr5; cholesterol by the method of Pearson, Stine and McGarack^; alkaline phosphatase by a modification of the method of Bessey, Lowry and Brock using Boehinger reagent?; glutamic-pyruvic transaminase by the method of Wroblewski and LaDueS. 1. Watson, C.J., Am. J. Clin. Path., _6:458 (1936). 2. Acetestf, Ames Co., Elkhart, Indiana. 3. Ictotesd, Ames Co., Elkhart, Indiana. 4. C.F. Boehringer and Soehne GmbH, Mannheim. 5. Karr, W.G., Manual of Clin. Biochem., Stephenson Bros., Phila (1940). 6. Pearson, S., S. Stein and T.H. McGarack, Anal, Chem,, 25:881 (1953). 7. Bessey, O.A,, O.H. Lowry and M.S. Brock, J. Biol. Chem., 164:321 (1946). 8. Wroblewski, F. and J. S. LaDue, Proc. Soc. Expt'l. Biol. Med., 91:569 (1956). N36836 -2- METHODS (Cont'd.): Delta-aminolevulinic dehydrase was also measured after 6, 9, 12, 15, 18 and 24 months using the method of Bonsignore, e_t al. 9. Prothrombin time was measured using the Lab-Tek Prothrombin systemlO and the total protein with the buiret reagentH. Albumin globulin ratios were determined after electrophoretic separation of the proteins using the dye elution technique*2. Samples of blood were taken for lead analysis and stored in plastic bottles at -4 C until analyzed. Urine and feces samples were collected as described previously for rats. Tissues from individual animals were collected at the terminal sacrifice and kept frozen until analyzed. Lead was determined by atomic absorption spectrophotometry following the same procedure described for the samples from rats. A simple correlation coefficient was calculated for the response vs. lead in the diet for each of the quantitative measurements made on the blood, urine, feces or tissue. The coefficient of regression with time was also calculated for these responses. An analysis of variance was made on the data for those responses that showed a significant correlation with the lead level of the diet and a "t" test of the difference between means was used to determine the effect level. RESULTS: The average value found for each response measured during the pre exposure period and for the test period, when lead was added to the diet, are listed in Table IV. The simple correlation coefficient for the response vs. lead content of the diet also appears in this table. The group averages at the various dietary levels of lead are listed in Table V for males and in Table VI for the females. Averages for each sampling interval during the two-year period are found in the appendix. The correlation coefficient indicated that there was a very significant relationship between the lead in the diet and the delta-aminolevulinic acid in the urine, the aminolevulinic dehydrase activity of the blood, and the lead content of the urine, feces and blood. A similar, but much less definite relationship appeared to exist for several other responses that are of interest because of their association with lead intoxication. 9. Bonsignore, D., P. Calissano, and C. Cortasegud, Bui. Hygiene 40:1337 (1965). 10. Lab-Tek Instrument Co., Westmont, 111. 11. Reiner, M., Standard Methods of Clinical Chemistry, Vol. I, Academic Press, New York (1953). 12. Gelman Instrument Co., Ann Arbor, Michigan, Manual 51199-A (1963). DUP050308536 -3- RESULTS (Cont'd.): A. delta-Aminolevulinic Dehydrase: The enzyme, delta-aminolevulinic dehydrase (ALD) is involved in the early stages of heme synthesis. Two molecules of aminolevulinic acid are condensed to form porphobilinogen by the catalytic action of this enzyme in the biological system. Lead has been shown to inhibit ALD activity both in vivo and in vitro, and its activity has been reported to be one of the most sensitive indications of an exposure to lead. The simple correlation coefficient indicated that the decrease in ALD activity with increasing levels of lead in the diet were definitely related. The "t" test of the difference between means showed that the addition of 50 ppm or more of lead to the diet of the dogs resulted in a significant decrease in ALD activity in the blood. B. delta-Aminolevulinic Acid: Although the increase in the concentration of delta-aminolevulinic acid (DALA) in the urine with increasing levels of lead in the diet was not very marked at the lower lead supplements, the correlation coefficient indicated a significant relationship. The DALA in the urine of dogs receiving up to 100 ppm of added lead in their diet, however, did not differ significantly from the dogs receiving no added lead, A "t" test of the difference between / means showed that only the dogs receiving 500 ppm of added lead had elevated [ levels of DALA in their urine. .................... ........ C. Other Changes in the Blood: A small, but nevertheless, significant relationship between the lead in the diet and the hemoglobin concentration and hematocrit was also indicated by the simple correlation coefficient. There was no marked difference between the average value observed for any group for these responses. The mean value for all dogs during the two-year period was actually somewhat higher than the average value measured during the pre-test period. In view of the demonstrated effect on heme synthesis, however, the results might be considered to suggest the beginning of a trend toward abnormal at the highest lead supplement to the diet. Less significant, but noteworthy, was the slight decrease in alkaline phosphatase activity that showed some correlation with the lead added to the diet, for such a response also occurred in rats fed these lead supplements. The transaminase activity and dietary lead showed a similar relationship and suggests the effect may have resulted from simple heavy metal inhibition of enzyme activity rather than reflecting an effect on a tissue at the levels of lead that were added to the diet. DUP050308537 -4- RESULTS (Gont'd.): D. Lead in Blood: During the pre-test period and for the first two months of the test, it was not possible to detect or measure lead in the blood with the sample available. After that time, however, the analytical procedure was modified to increase its sensitivity and lead was measured in all subsequent samples. The correlation coefficient showed a very marked relationship between the lead concentration in the blood and the lead in the diet. At the various levels of lead added to the diet in this study, the males usually had higher levels of lead in the blood than the females. The length of time that lead was fed also influenced the lead level in the blood for there was a trend toward higher values as the experiment progressed. The difference between the mean blood lead level of the group receiving no added lead in the diet and the mean of the group receiving a 10 ppm lead supplement was significant (sexes tested separately). The mean blood-lead con centration for the control group (males and females) was 0.8 ^ig/10 ml + 0.6 ^ig/10 ml. Xf the upper range of a normal population is taken to be plus two, standard deviations from the mean, only 24% of the measurements in the group fed 10 ppm of added lead were outside this range. The chance of detecting an exposure to 10 ppm of lead in the diet by measuring blood lead would have been one time in four. Even at the highest lead supplement (500 ppm) nearly 9% of the values were in the ''normal'' range (i.e. less than +2 S.D. from the control mean). For the control population itself, approximately 3% of the values were above this "normal" range. The control group mean of 0.8 jig/10 ml is somewhat less than half the value 1.7 ug/10 ml + 1.1 pg/10 ml (17 + 11 ^ig/100 ml) reported by Goldwater and Hoover13 for "normal'' blood levels in humans. The dogs receiving the 10 ppm lead supplement more closely approximated this value. A level of 8 ^ug/10 ml (80 ug/100 ml) according to Haeger-Aronson1^ has usually been given as the upper normal limit of the concentration of lead in the blood of humans. Values exceeding this range were seen only in the dogs receiving the highest lead supple ment approximately 20% of the time. E. Lead in Urine and Feces: j There was a very significant correlation between the level of lead in `the urine and feces and the level of lead in the diet. Most of the lead entering the human body by ingestion is eliminated by way of the intestines without being absorbed into the blood stream!3. Since this is probably true for other mammalian species as well, only the urinary lead data, representing the absorbed lead, was analyzed statistically. 13. Goldwater, L. J. and A. W. Hoover, Arch. Environ. Health, _15:64 (1967). 14. Haeger-Aronson, B., Second. J. Clin. & Lab. Invest. _12: Supplement 47 (1966). DUP050308538 -5- RESULTS (Cont'd.): The amount of lead eliminated by the kidneys did not change significantly as the study progressed nor did one sex have, consistently, higher or lower urinary lead levels than the other. The difference between the mean level of urinary lead of the group receiving the 10 ppm lead supplement and the control group with no added dietary lead was significant. The mean urine .lead concentration of the control group (male plus female) was 0.41 Ug/10 ml + 0.26 ^ig/ml. Only 13% of the urine lead concentrations in the group' fed the 10 ppm supplement were outside the normal range, if plus two standard deviations from the control mean is used to establish the upper limit of normal. The chance of detecting an exposure to an increase of 10 ppm of lead in the diet by measuring urinary lead would he about 1 time in 8. At the highest dietary lead supplement somewhat less than 2% of the urine lead concentrations were within the normal range established by the controls. The control group mean of 0.41 jig/10 ml is about 25% higher than the value 0.33 y-g/10 ml (33 ug/L) reported by Goldwater and Hoover for "normal" lead levels in human urine. The upper normal limit of human urinary lead given by most authors according to Haeger-Aronson is 0.6 - 0.8 ug/10 ml (6.0 - 8.0 ug/100 ml). The mean for the group, males and females, receiving the 10 ppm lead supplement was in this range and 40% of the individual analyses for this group gave results which were in excess of 0.6^ig/10 ml. F. Lead in Tissues: ~The results of the lead analyses of the various tissues collected at the termination of the study are summarized in Table VII. Since the number of samples at each level of dietary lead was quite small all of the statistical techniques could not be used to analyze the data. A simple correlation coefficient indicated that for all but the muscle there was a significant relationship between the lead content of the tissue and the lead in the diet. For those tissues whose lead content was related to the lead in the diet, the highest concentration was found in the bone and the lowest in the brain. There was, in general, a similar relative increase in tissue lead with increasing amounts of lead in the diet indicated by the slope for the transformed data, log x on log y, although the rate of increase was somewhat greater for the liver and somewhat lower for the bone than for the other tissues. (Figure 4) COMMENTS: The significant responses in rats and dogs fed lead in their diet are summarized in Table VIII for comparison. The average lead level in the diet of the rats during the two-year C-- period was higher than in the diet of the dogs. Some of this difference resulted from variation in the lead content of the basal diet and some undoubtedly resulted from errors in the mixing, sampling and analyses for lead. The average fecal U<uL content was also higher in the rats than in the dogs. DUP050308539 6- - COMMENTS (Cont'd.): The urine lead concentration, expressed both as ^ug/10 ml and as a per cent of the lead ingested was higher in the dogs than in the rats. The significance of this observation is not certain. It may represent a species difference in the absorption of dietary lead, a difference in the route of elimination, a difference in the retention and storage of lead or a combination of these and other factors. The biochemical evidence for an effect of added lead in the diet, an increase in urinary DALA reflecting an interference with heme synthesis and an increase in stippled cells reflecting a greater incidence of defective erythrocytes would suggest that the rat is the more sensitive species. Higher concentrations of lead were found in the bone, kidney and brain of the rat than the dog. In the blood and liver higher concentrations of lead were found in the dog than the rat. Because of the association of bone with erythrocyte production, hence indirectly with heme synthesis, the greater accumulation of lead in the bone of rats may account for the species difference in sensitivity to ingested lead. Aside from possible or probable species difference in absorption, transport, accumulation or elimination of dietary lead, there were important differences in the treatment of each species that may have more bearing on the apparent difference in sensitivity. The rats began receiving the dietary lead when they were weaned and continued to receive it for -essentially their lifetime. The dogs were young adults when lead was added to their diet and were fed for a portion of their lifetime. As a consequence of this, the rats received a higher relative dose--grams lead/grams body weight when they were young and perhaps more vulnerable than they did when they were mature. For the dogs there was little change in the relative dose throughout the feeding period. The level of lead in the tissues and in the blood, urine and feces was directly related to the lead content of the diet. An increase in the lead content of any of these may be considered, per se, an effect or response. In the blood and excretia, such an effect was discernible even at the lowest dietary lead supplement. A response observed in both rats and dogs that can be considered an adverse effect in that it is evidence for an interference with a necessary biochemical process was found for the synthesis of heme. This occurred only at the highest dietary level of lead. A similar kind of effect was also found at this level of lead for erythrocyte development in rats. JRB:gdc September 6, 1968 DUP050308540 TABLE 4 SIMPLE CORRELATION COEFFICIENT OF RESPONSE TO LEAD IN THE DIET TWO YEAR FEEDING STUDY WITH DOGS CO *o0P !--<u! 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Vm> o1 O o o O oo o rrO--~Nli stf o oCM vO sCMt IN fi--N4. rCCMM- coo <CC3OM- ot O o O o O ' O ot o 1 sP CcOo CPON CO --i cdo- oo o H40<JU5 00 it} O 4p0OJ a p-n mm CO o m Ct-PO ON a\ CO O CO co CM mo in Vr-Ol on t-P CinO r-1 o CCMO ON <t o o o o COCMM oo ON CO CM ON Mt* ON 00 rP CM m r-l m Ir~DH <CfM* i<--nt CM m -4- o o co CO O o O o NVCoOOM mON Sot CCOM Mi- l-H vjv0O0 tC-Mi r-l OCMN CHM COO4 rH oin rC-M*l r-l vVOO O CO rC--Ml VO Ir^-Sl m co 00 CoO *frNnp. \i0n CN U0 t-i CCr-OO4 r-l or-ol ON vO t--[ irnP o* Pr-'Sl vC+M rU-0l <*t rmC--Ml N i rP CO C1--Ol o Jo--1 r-l CrmNO. 1UrP-34 rl VcOo -4 CM vrO-l CM CO 1t 54 54 w cod g oo 400J3 oop xu: l>-is W rP s oO1-p C5 W) rl XoM-4) i o m COg3s. COrOP S'? txl V 4uoO- 4CcJO> CtOo3 wcg<td> 0<a1) 4 T4<>Ju 00 & CJ> W CO lr-r*ll 4COpJl . 0CJ FI t*--asr>tl *">05 03 rl X0PopJ0<13 F5 0005 or> <11 3-S ool~t >5 XsS cn 03 54 I1----COl1 a> *CO u fvj 4<J1) XoosCJsU xCr:lU *0OroH3i 03 03 0CU rl oOo0i^x0ooC3iUj o rP 03 OCHU 4>-11 T? 40-1 roc0aPd) 4WS FA < F5 w s om 03 ri---4l o0 nr0-dl '4araJl.. CO to Xi "sC +Mv. 03 W) E U XS <-CSMf^. --1 H 0 4r-4l >0 03 o 6 0u3 A "CSM <OrX3*Jv orl 0rl r-l M0 r-0C > 00rl o 4ol--1l rl o CocOu 4>J, rr--coH-dll 0E) O E > i<rJUl PoJ-M4 r0O>-l adH I '-a 40-13 * rl 0 3 0o<d) 54 6C E to0oN o i--0i o 0 4pt00rJ>il0 E5 c0d DU c>o 0 r0-l> 4p<Ju 03 r--0l 0 Xi o 00<f10s fA0 x40o<0<<C:3J3dudUN 0 arl rt-dl r-l < 40PJ3 00 /HNn vo/ 003 0irdl isd CtdJ Hp o003 0> fl H 0rl 0o3 Xul o IuP rP oo1--{ l> s 4c0rJll POP4 r4--tdJ4 O H rc0p4 rO3 t H0 No. 0rl rrO3p < P xt0ss M XOr3l <1 -Orl r0rll r0>P O *0rl <01 '^d rP i--l 0 or--l -S. 0 NorPs. w CO UO < M X 00url 03s 0O<3u nods r0p 0> fP P3 0 0rl 0rl *00-4 'Ocidu c03d DUP050308541 Q w p <0 o O ja P P o HI f&tf 8 <&3 o 1-1 OS3 MP OO K mH HS3 C3 Mg OW <5 BQ C<O <w 3 pi r^. vo o r- o CM vO uo o o o n CO m -d- VO rH rH vO CM CM O CM co m ft * * t tn vD r> CO CM o vO m v0 o o O O CM rH rH CM CM p -d" rH m CM CO m oo m > p tn M W pH w }H 61 p H <N H w to MH CO 3 CM CM CO C\ CM o CO CO CM o ON CO CM o uo vO MO CM rH CM o CO CO oooo rH <1* i--1 in CM rH CM CO oO ft uo rH o ON ON <t* rH CO o rH O rH rH CO m CO O -d- in CO in o -d- CO CM ft * m m co in in CM o ON r*- <F o o O o r-i -a- rH m CM CM CM O uo rH VO in vO rH CO tn oft rH H pH M t--1 vO H iH 00 x co rH m r-* rH CO X CM CO pH m vD CO vO -t CM o VO o m o o o o rH Hi* rH m CM iH CM O co x. i--I vO co ON r>. CM co co CM rH uo CM CO CM t--i rH ON t rH uo UO vO CO --i tn rH CO rH o (d- ON oo rH U0 rH co pH UO pH ON CM CM uo rH CM rH pH o r*^ rH co vO o rH X. ft rH <f CO CO o vO rH ON VQ CM so rH ^ rH rH UO m ON MT CO ON VO ft rH CO o CO vO o ON uo CM vO rH CM CM rH m ON r^. CM o i--1 ON J--1 w ftmF rH in rH m m VO ON rH CM m o m CM * * * O ON COft CM ftuo O rH CO in VO CO <F CO CM o VO i--l Ht o rH o o ON rH ON X rH rH ON rH <t ON vO rH ON U0 rH m CM rH X. rH CM rH vO rH rH CM m CO rH ON CM O r*. uo O vO o n rH CM vO vO VO VO CO VO rH uo * .O CM * CM uo -d- CM S) o CO Hi- CO CM o VQ rH o -H o o d- o rH vO o O 03 ON CO ON o O CO vO CM 1--l Fl rH CO 1 CO vO ftCM o n o m CO ON <F rH O o rH vO CM * m uo CO vO rH m o ON ON CO o o o o rH <1* m CM rH CO Om * ON rH m o Mf o CM CO CM rH in o VO -d- ft rH x- uo CM rH U0 in UO oo uo rH ON rH i-^ rH rH i--H 1 o n ON o n GN 00 oo CO CO CO 00 OO CO CO 00 O o O o o ON O ON ON ON o O o 2 uo in in m m uo uo m m m m uo in m vO V0 vO V0 vO m VO UO uo U0 VD vO CM l CO B^S a- co 'D s vO o rH X Ss o o COB3 \ Sdo 5*S O pH X 43J) Q> d g C<DO d idr>l CO s-s ^2 5^ CO opq L$r oe o m>S rH O 03 CO . 03 s-e --1 C) rH rH pq rH 4GJ> d 4J o O .on pH oUo 60 4J iJ O d X S <0 CO CO CO Vi CO 4Jd) H .du rd M CD rH CD *rH w 4J o X M O o oCu oO. u J u X r> 0 oo rn, 04 -U O d oo od d d CD U] a <i) oi X P, O 0) <D CJ O 03 rH rH 0) *ri orl d u 0) 0 rH CU H <D O- o cu X o03 H dU W S3 p 2! S3 g P w M < S3 CO n * rH CuO o ja <F XCM rH s 4XJ rH p oCO rHmC -d- o *iHd P r3H> > S CM <D (1) d rH gu 60 O 3 rH o 4g-> s a rH CO O O rH ^ s p > rH rH d <j UH co rH 1 <F <D rH rH o j d CM d O Q 4J 4J srH CD ti0>u a oCO O rH JH <D P 'O 60 rH rH CD CduO c6 sd 4CJO *H o rdt H ti o o s-s rH 60 s g-2 6j : "***, Ea. d rl o a < <D o rH CO drl ti (!) a i--rHJ d uVi rH tu CoO u J2 S3 >d a XM CO 111 p rH Ph p dat g rl o6 H ID `rH US p* CO d dN b d rH rH > c d H <D <D *> rH rQ rH d o6C 60 rH O O o d6 O +J rH rH <D oJH u o 2 H to \ u a> u v. s 6 d rt0) rH u Ph 5< u 03 S3 w rH 1 03 rH P- C:j d 0 d) rH E d 4Jd d O d rH CJ d u U) rd rt od M aj O 4- rO r--1 0J <D--1 rH e-f r--H IH p O rH H no <3 a o KJ H < Pn DUP050308542 On vO vO O CO n CO CO O 0 CM *4- CM O m ON CM in rH CM O O in O 00 rH CO VD CO vO 00 CO > ** 4 * * in in n O 4 CO CM 0 0 rH <f 0 O O O rH 00 0 CM rH 4- rH in CO rH co vD rH rH CO O 4 ON VQ 0 ON 4 VO CO 4 00 rH CM CM O m CM 4 CO n- 00 in > Pm cn ON co vO 0 OO m vO CO CM vO CM 0 * n * O 4 CM * <t- rH \o rH CO Q M W rH in in co 4 CM rH O CO vo 4' O O O 0 CO rH CM 00 rH rH O 4 4 ON m rH CM ON M W r*H <r rH in CO uo ON rH CM vO rH CM rH h Pm m VO rH co g 0 TO O }H 0 61 *^v. w 0 rH O 4co CO CO 0 VO 1-1 h H 3; r^. M Wm 4 in in rH CM O CO vO CO vO co 00 CM 4 < 0 CO CM 4 4 0 rH *0 > H 1-1 m VO 4 4 CO CM 0 O CO 4 0 0 O 0 m rH CM co rH rH VO CO 4 m vO rH ON O M rH 4 rH n CO 0 CO rH rH n rH CM CM pt| co c 4 rH rH rH Pm TO O JM 6t |Y| rt CO P>^ 00 CM CO CM ON CO r> ON M M rH vO CM O CO ON CM CM m rH rH t --1 vD 4 CM 0 O ON rH CM 4 ON O CO hH 1--1 1 cotro m VO CM CO CM CM 0 ON O 4 O rH O 0 co rH O CO rH rH CO CO VO in rH ON ON corH <r rH m CM rH in rH rH m rH m rH vtnOHino4*\Ocoirv<ocvi tn^mcO^NONOO'd-OHOO r4 <j* H tT| CM vD CM o vO rl H H C\ CM CO 4- 00 CO co 4 o cOn H00M in h cmo o h 4O VO 00 CM CM P CM CO co CO [/jcniNOMnovoocoovvovoco co vO uo in c m OHfOCOC^MOCOfOHOOO I CO O CM ON CO CM CO 00 vO VO rH CM pH *4* CM iM-H HinC<J tioonf'rsNN(<iri CO O n* o ON m t-i in co n o n vo h <f* IM tnvDCOUOCMMfOCMvOCOOOOO o% h in n* rH *4* rH in CO oo o *rH t--m1 rh> in in 1--1 <0 in h t--o{ CO CM o o o o o >o n o ^^o n o \o n o n o n o n 0000 vo vo vo vo in m m in m m m m m m vO vO vO vO on m o 0000000 vo VO vo VO vo vo VO CM CO *d d *t H H o CO o O TnO 4J MH < o TO <c o o oTO TO vO o to cu 8s 4 CM H w C5 CTOO CO Oo rH co ^HO \m rH 5> Jh iH . JS rH u 03 00 * > s `rH TO rH Xo H )H X CO <1) co CO ^ CO 03 nw ^ CO - CO 4 r1--H1 OpQ r--Hl TO P3 TO 6.2 TOV CO EU 4 CM to s tO O rTO fX CO *rH P* t o* 4 TO 60 *H S O'* 3 HrH H >P TO o M co a> . rl n (5 ts <0 60 60 rH TO O TO4J a o H rH o 4HJ U o CTOO Hh Hh M to <U U 4-> JG PP 4J > 0, P to O rH ** .CJ -r4 cn P< -u r'HH rTHO T3 -0U) O TTPO u jTO rtHo -OU OO OOO Jh ^ o o o TO to ,2 o 0 CU *H TO rH TO Cu Et o O to u to w tC (gU to W oTO to P u0 S3 W3TO 5*5 H>TO s SD*H P>> Wo OJ S O (C0O pq CU rH Uto o3 <q ps CU Url co 30 CO pTI* H l 4 TO TO CM 4J 4J O rH 60 P W O fU4 'OG> S 'rH rTHO 4 iPh CJ 1 'rH 62 t *v o 1hJ O 4-1 rH ^TuO COUm ^OTO TO TOSE U co TO co & CO 'rH o TO rH` E O TO rH TO TO to 4-) 4J & TO TO O 4J P a p o H Jh t o. -d a -o O H < Pm -d DUP050308543 lTABLE AVERAGE VALUE FOUND FOR THE LEAD CONTENT OF TISSUES FROM DOGS FED LEAD ACETATE FOR TWO YEARS DUP050308544 &TABLE SIGNIFICANT RESPONSES IN RATS AND DOGS FED LEAD ACETATE FOR TWO YEARS '''C a lc u la te d fro m yvg/day and 24 h o u r u r in e volum e DUP050308545 FIGURE h Increase in the. level of lead in dog tissue with increasing levels of lead in the diet DUPO 503 08546