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- v , " *V > ' ` *1 in1 ^ VW^I> I li I'llKir1, aVW.y CARDIOVASCULAR AND HEMATOLOGICAL PARAMETERS AFFECTED BY FEEDING VARIOUS POLYCHLORINATED BIPHENYLS TO THE SINGLE COMB WHITE LEGHORN COCKEREL S. J. ITURRI, E. A. COGGER, and R. K. RINGER Department of Poultry Science Michigan State University Bast Lansing, Michigan 48823 The effect of dietary PCBs (50-200 ppm) on some cardiovascular and hemato logical parameters in the SCWL cockerel was investigated. Heart rate was signifi cantly reduced by PCB 1242 and 1254 at > 100 ppm and was not altered by PCB 1221 or 1260 at 150 ppm. A significant decrease in arterial blood pH was observed with PCB 1254 at 150 ppm. Mean blood pressure was unaffected by any treatment used. Hemoglobin concentration, HCT, and total erythrocyte count were found to be significantly decreased by PCBs 1242 and 1254 at > 50 ppm. These same parameters were not affected by PCB 1221 or 1260 at any level used. It was con cluded that the anemia observed was due solely to a decrease in total erythrocyte concentration. The possibility that these changes may be due to a decrease in erythropoiesis is discussed. Pericardial fluid volume was increased with PCBs 1242 and 1 254; however, there was no correlation between the magnitude of the brady cardia and the amount of pericardial fluid. Introduction Many biological changes have been reported in different animal species after ingestion of polychlorinated biphenyls (PCBs), McCune er al. (1962) observed that various levels of Aroclor 1242 produced labored respiration, hydropericardium, enlarged heart, abdominal edema, and a decrease in body weight in chickens. Koeman el al (1969) reported hydro pericardium in Japanese quail at 1000 ppm of dietary PCB (60% chlorinated). In addition to the above symptoms, Rehfeld el al. (1971) observed internal hemorrhaging, depression of secondary sexual characteristics, and an increase in liver weight as percentage of body weight when PCB (48% chlorinated) was fed to one-day-old chickens. Flick el al (1965) reported that hemoglobin, hematocrit, and blood glucose values were reduced after feed ing Aroclor 1242 to White Leghorn cockerels. Little is known concerning the cardio vascular and hematological changes following exposure to various levels of PCB. The following study was undertaken to determine the effects of PCB on some cardiovascular parameters of the White Lc- horn cockerel. Michigan Agricultural Experiment Station Journal Article 6411. Archive* of Environmental Contamination and Toxicology, Vol. 2, No, 2, 1974. 1974 by Springer--Verlag Ntw York Inc. 130 DSW 025519 " ^ ,'** t i \ * * , ` , r : ': v( ,*- ^ `; '; s ' V .'' 1 ` " *~i. , >*> ' >;***'' * * , .-.WW ----------------------------------- -------------------- `-------------------- ' ' ' ' STLCOPCB4009474 u^Jrir* mm n**t'*Af m aiiWh Parameters Affected by PCBs in the SCWL Cockerel 131 Material and methods Diets containing different levels of PCBs were prepared by adding to a chick starter ration the respective PCB (Aroclor 1221, 1242, 1254, or 1260; Monsanto Chemical Co., St. Louis. Mo.) (Table I). The PCBs were dissolved in acetone and blended into a premix to facilitate a final even distribution. The acetone was evaporated and the premix blended into the basal ration. An equal amount of acetone-treated premix was added to the basal ration for the control groups. The chicks were reared in starting batteries with raised wire floors, weighed, individually wing banded, and randomly distributed into groups of 12 birds. Feed and water were available ad libitum. Weekly feed consumption was measured. All parameters studied were measured for each bird. Systemic arterial blood pressure and heart rate were recorded on a Grass polygraph (Model 7) through a cannulated carotid artery' using a Statham pressure transducer (Model P23). Mean blood pressure was calcu lated according to Sturkie (1965): mean blood pressure * 3/8 pulse pressure + diastolic blood pressure. Blood samples for analysis were taken in heparinized syringes from the cannulated carotid artery. Packed erythrocyte volume (HCT) was determined by collect ing two samples in heparinized capillary tubes. The tubes were centrifuged at 11,500 rpm for live minutes in an International microhematocrit centrifuge. Total erythrocyte counts were made by diluting blood 1:200 with Wiseman's solution in dilution pipettes and counting in a hemocytometer (Exp. 1) or by diluting 20X of blood 1:50,000 with Isoton solution for counting on a Coulter Counter (Model B) (Exp. 2). The mean of two de terminations was used as an observation. Table I. Chick Starter Ration Ingredient Pounds per ton of feed Yellow com, fine ground Ground oats Wheat middlings Alfalfa meal, dehydrated, 17% protein Meat and bone scraps, 50% protein Solvent process soybean meal, 45% protein Fish meal, Vitaproil, 55% protein Dried whey Ground limestone ' Dicalcium phosphate (24% Ca, 18.5% P) Salt, iodized Vitamin trace mineral premix Coccidiostat 1060 100 100 80 50 500 40 40 10 10 6 5 + 2001 + V--' '.K . \ ' ,>!; ...c-- A ?. ,= , :* * - -v ' f" f' /'K.J'/"; A".'-' "i - ` V tC .: . STLCOPCB40 13; S. J. Iturri et al. .Mean corpuscular volume (MCV) was determined by dividing the packed cell volume by the total erythrocyte count (Wintrobe 1961). Volumes were converted to cubic microns (ji3) using appropriate conversion factors. Hemoglobin was determined using the modified Newcomer acid hematin method (Denington and Lucas 1955). Standard curves were made using a prediction equation de termined by linear regression using four hemin standards (5, 7.5, 10, and 15 X 10-3 mg.'ml). These hemin standards corresponded to 5.05, 7.58, 10.11, and 15.16 gm of hemoglobin per 100 ml of blood, respectively, as determined from a commercial hemo globin standard (Hycel Hemoglobin control; Hycel, Inc., Houston, Texas). Mean corpus cular hemoglobin concentration (MCHC) was calculated by dividing hemoglobin concen tration by HCT. Blood samples for pH measurement (Coming, Mode! 12 pH meter and Beckman ihermomatic constant temperature block), in experiment 2 were obtained from the cannulated carotid artery. Hydropericardium volume was determined by inserting a 20-gauge needle attached to a 2.5-ml syringe into the pericardium and withdrawing the fluid measured to the nearest 0.1 ml. Data were analyzed by either analysis of variance or row by column chi-square analy sis. Parameters showing significant effects by analysis of variance were further tested by the new multiple range procedure of Duncan (1955). Experiment 1. In trial 1, rations containing 25, 50, and 100 ppm of Aroclor 1242 were fed to one-day-old pure strain Single Comb White Leghorn (SCWL) cockerels main tained on our research farm. In a second trial the same Aroclor was added to rations to give 50, 100, and 200 ppm. All groups were terminated at eight weeks except in trial 2, when the group fed 200 ppm was sacrificed at four weeks due to high mortality. A corresponding number of controls were sacrificed at this time. . Experiment 2. Rations containing 50, 100, and 150 ppm of Aroclor 1221, 1254, and 1260 were given to one-day-old commercial (DeKalb strain) SCWL cockerels. The experi ment was terminated at nine weeks of age. Results and discussion The effects on heart rate and mean blood pressure of experiments 1 and 2 are shown in Tables II and III. A significant decrease in heart rate was observed when Aroclor 1242 was present in the diet at 100 or 200 ppm, and when Aroclor 1254 was added in the diet at 100 or 150 ppm. No effect on heart rate was observed for Aroclors 1221 and 1260 at the levels fed. There was no change in mean blood pressure with any PCB at the levels fed. It is known that cholinesterase activity is decreased with lowered pH (Hestrin 1950); hence in experiment 2 arterial blood pH was measured in an attempt to correlate this parameter with the possible change in heart rate as observed with Aroclor 1242 feeding. Using this fact, Emerson et ol. (1964) postulated that the bradycardia observed in dogs treated acutely with endrin was the result of increased acetylcholine activity. Our results were inconclu sive because arterial blood pH was lowered only when the cockerels were fed 150 ppm (Aroclor 1254) whereas bradycardia was also observed at 100 ppm with no change in arterial blood pH (Table III). DSW 025521 r-: 5k. STLCOPCB4009476 14 t.- Table II. The Effect of Feeding Rations Containing Aroclor 1242 on Heart Rate and Mean Blood Pressure ofSOVL Cockerels Parameter Heart rate (beats/min) (weeks) 8 (trial 1) 8 (trial 2) 4 (trial 2) Mean blood pressure (mm Hg) 8 (trial I) 8 (trial 2) 4 (trial 2) feeding period Dietary levels (ppm) 0 25 50 100 200 (12) 398 8.8^b (12)371 10.9mn (10) 388 8.9X " (6)436 13.2X - (11)361 + 10.9,, (12)351 12.5xy - (7)349 + 15. S,, (10)328 15.3y - (6) 326 23.ly (12) 137 4.2 (10)133+ 3.7 (6) 133 7.9 (12)142+ 2.7 ~ ~ (II) 142 4.6 (12) 136 6.9 '- (7)149+ 7.3 (10) 128 4.8 - (6)126+ 8.6 a(No. of birds) mean standard error. bMean having different subscripts are significantly different; m,n (P<0.05); x, y (P<0.01). <C%L *C<T* C"n0O <o/> 5: noro U) t,; K f- K r-; A; S,/ ;;i i`, V. Jr "rC.iV''' *r i- o t/5 O fv :* -h. Uu>l v IV rv .1. -1'V^* w.' >:- Iv . . A* ; * : tv '* V.' STLCOPCB4009477 STLCOPCB4009478 ..... Parameters Affected by PCBs in the SCWL Cockerel 135 Tire effects of Aroclor 1242 on HCT, hemoglobin concentration, total erythrocyte concentration, MCV, and MCHC are presented in Tables IV and V. It is evident that HCT, hemoglobin concentration, and total erythrocyte concentration were significantly re duced by Aroclor 1242. Tables VI and VII show the effect of Aroclors 1221,1254, and 1260 on the same parameters. Aroclor 1254 at levels as low as 50 ppm in the diet caused a significant decrease in HCT, hemoglobin concentration, and total erythrocyte concen tration, whereas neither Aroclor 1221 nor 1260 had any effect on these param eters at the dietary levels fed. MCV and MCHC were not significantly altered. Similar changes in HCT and hemoglobin concentration have been reported by Flick etal. (1965) as early as one week of age after feeding Aroclor 1242 at 200 and 400 ppm in the diet. They believed that these changes were transient since recovery was observed at three weeks of age. However, the experiment was terminated at that time and no further ob servations were made. Rehfeld et al. (1972a, b) observed decreases in hemoglobin con centration and HCT wiien feeding Aroclor 1248 at levels as low as 20 ppm and 30 ppm for four and one-half weeks. These changes were reversible when the PCB was withdrawn after two and one-half weeks. The anemia observed when feeding PCBs to cockerels could be due to a decrease in total concentration of erythrocytes, a decrease in erythrocyte size, an increase in plasma volume, a decrease in mean corpuscular hemoblobin concentration, or any combination of the above. An increase in plasma volume is improbable since loss in body weight and edema formation was observed with Aroclor 1242 at 50 ppm and Aroclor 1254 at 100 ppm. Edema formation has been reported by other investigators when feeding various PCBs (McCune et at. 1962, Flick et al. 1965, De Vos and Koeman 1970, Rehfeld et at. 1971). Since there were no significant differences in MCV and MCHC, it was concluded that the normochromic normocytic anemia was due solely to a decrease in total erythro cyte concentration. This change may be explained by a decrease in erythropoiesis which is partially controlled by sex steroids. A marked decrease in comb size, indicating re duced testosterone secretion, was observed in birds fed Aroclors 1242 and 1254 although it was not quantitated. This observation is in agreement with other workers (Rehfeld et al. 1971, Platonow and Funnell 1971). Several investigators have reported that testos terone increases total erythrocyte concentration in chickens (Domm et al. 1943, Domm and Taber 1946, Tanaka and Rosenberg 1955) and in Japanese quail (Nirmalan and Robinson 1972). Also, Fried ef al. (1966), Alexanian et al. (1967), and Malgor and Fisher (1970) have suggested that testosterone has an effect on erythropoiesis by stimu lating in vitro production of erythropoietin in mammalian species. PCB may decrease red cell formation through enhanced hepatic catabolism of testos terone (Nowicki and Norman 1972). Another possibility is that PCB's may directly affect erythropoiesis or erythropoietin production. This is supported by the work of Bilman et al. (1972) who demonstrated that some PCBs have estrogenic-like activity in rats. It has been suggested that estrogens depress erythropoietin precursor formation. There was a significant relationship between dietary level of PCB's and hydroperi cardium (Table VIII); however, there was no correlation within individual treatments between the magnitude of tljc bradycardia and the amount of pericardial fluid. y "V u-' i-V-' ; ; fT-:-' ,v ... - "I r 'r* 'i i 4; 4 .*: . ' v 1 I '>( :.V-' ib i-S;,*;>' .... .J,r;.V. / "s *+ -t STLCOPCB4009480 S' K* v- A ' w' rs%1 :'Vy '/. V; ' . .* .V' 1- o tx/>: oM U1 U1 NU)l Cu>h Table IV. 77ie Effect of Feeding Rations Containing Aroclor 1242 (Trial 1} on the Packed Cell Volume, Hemoglobin Concentration, Total Erythrocyte Count, Mean Corpuscular Volume, and Mean Corpuscular Hemoglobin Concentration of SCWL Cockerels Parameter Length of feeding period 0 Dietary levels (ppm) 25 50 100 Packed cell volume (%) Hemoglobin cone. (gm/100 ml) Total erythrocyte count (millions/mm3) Mean corpuscular volume (m3) Mean corpuscular hemoglobin cone. (gm/100 ml) 8 wks 8 wks 8 wks 8 wks 8 wks (12)27.4 ,83jb (12)25.8 ,59xy (10)23.3 ,86y (7) 19.8 + 31* (12) 9.24 ,3I6X (12) 8.39 .180, (11) 7.59 + ,205y (7) 6.29 13S, (12) 2.77 ,134X (11) 2.53+ ,12IX (10) 2.56 ,096x (7) 2.00 + . 1 68y (11)96.1 2.46 (10)99.2 3.09 '(9)93.9 +3.10 (7)99.7 2.51 (12)33.8 .53 (12)32.6 .53 (10)32.7 + .68 (7)31.8 .34 "(No. of birds) mean standard error b Means having different subscripts are significantly different; x, y,z (P < 0.01). m v -v hv* if.v- '-.v 'i" * 1 *: 'T 1 </;; ' r- ; V ` ->:V fif-'C'JfCff. '--v cf :V'V ^ ^ Vr:^m. |v v::-y ' Parameters A ffe c te d by PCBs in the SCWL Cockerel Table Vi The Effect of Feeding Rations Containing Aroclor 1242 {Trial 2} on the Packed Cell Volume, Hemoglobin Concentration, Total Erythrocyte Count, Mean Corpuscular Volume, and Mem Corpuscular Hemoglobin Concentration of SCWL Cockerels Parameter Length of feeding period 0 Dietary levels (ppm) 50 100 200 Packed ceil volume (%) Hemoglobin cone. (gm/100 ml) Total erythrocyte count (miilions/mm3) Mean corpuscular volume ip3) Mean corpuscular hemoglobin cone. (gm/100 ml) 8 wks 4 wks 8 wks 4 wks 8 wks 4 wks 8 wks 4 wks 8 wks 4 wks (10)23.4 +0.74^b (6)25.1 1.40 (12)20.8 l.S6mn (10)17.3 1.53,, (10) 8.28 .351* (6) 7.51 .270 (12) 7.47 + ,354*y (10) 5.86 .638y (10) 2.64 ,089m (12) 2.24 ,170mn (10) 1.91 .192,, (6) 2.61+ .053 (10)88.8 +3.83 (6)96.0 3.92 (12)93.8 1.81 (10)92.3 2.56 (10)35.2 0.68 (6)30.1 0.95 (11)34.3 0.62 (10)33.8 0.95 (6)20.6 2.69 (6) 6.24 + 1.025 (6) 2.15 .300 (6)96.8 2.07 (5)31.2 0.37 a(No. of birds) mean standard error. bMean having different subscripts are significantly different; m, n (P<0.05); x,y (P<0.01). 137 STLCOPCB4009481 ..I.v v.-HVtvv :<> ,<,, A,..' ' ":-V ' 'v-*: O' :' -.y'**:'V.?::' .'.-..V, V/'V-VV'.. * - . - .U . * - V i..,, .4 " O00J S. J. Itu rri et ai. Table VI. 77ie Effect of Feeding Rations Containing Aroclors 1221, 1254, and 1260 on the Packed Erythrocyte Volume, Total Erythrocyte Count, and Hemoglobin Concentration of SCWL Cockerels Parameter Aroclor Packed erythrocyte volume (%) 1221 1254 1260 Total erythrocyte 1221 count (millions/mm3) 1254 1260 Hemoglobin cone. (gm/100 ml) 1221 1254 1260 0 (11)29.5 .45* (12)29.5 .44$ (12)27.6 .96 (11) 2.25 .061 (12) 2.16 .074m (11) 2.08 .096 (11) 9.87 .156 (12) 10.04+ .182, (11) 9.31+.404 Dietary levels (ppm) 50 100 (11)27.4 1.46 (10)23.3 1.36y (12)28.8 .79 (11) 2.04+ .096 (10) 1.84 .119n (12) 2.16+ .077 (H) 9.04 .565 (11) 7.91 ,416y (12) 9.84 .239 (10)29.5 .57 (8)20.4 2.36y (12)27.4 .89 (10) 2.22 .103 (7) 1.77 ,168n (12) 1.96 .063 (10) 9.91 .256 (8) 6.97 + ,906y (12) 9.32 .255 150 (11)26.7 .91 (7)18.0 1.86y (22) 26.6 .61 (11) 2.12 .157 (6) 1.48 . .118,, (12) 1.96 .063 01) 9.03 + .293 (7) 6.10+ .728y 02) 9.02+ .240 KNo. of birds) mean standard error. bMean having different subscripts are significantly different; m, n (P<0.05); x, y (P< 0.01). Parameters Affected by PCBs in the SCWL Cockerel Table VII. The Effect of Feeding Rations Containing Aroclors 1221, 1254, and 1260 on Mean Corpuscular Volume, and Mean Corpuscular Hemoglobin Concentration of SCWL Cockerels Parameter Mean corpuscular volume Ox3) Aroclor 1221 1254 1260 0 (11) 131.4+1.97* (12) 137.8 + 4.44 (11) 131.9 4.72 Dietary levels (ppm) 50 100 (11) 133.9 + 3.42 (8) 134.1 + 5.16 (12) 134.8 5.21 (11) 131.9 + 4.72 (7) 127.7 3.28 (11) 138.6 3.09 150 , (12) 134.8 5.21 (6) 130.8 + 3.78 (11) 134.7 2.43 Mean corpuscular hemoglobin cone. (gm/100 ml) 1221 1254 1260 (11) 33.4 0.30 (12) 34.0 0.39 (12) 33.7 + 0.63 (11) 32.8 0.46 (9) 33.9 0.40 (12) 34.3 0.44 (10) 33.5 0.33 (8) 33.5 -.96 (12) 34.2 0.47 (11) 33.9 0.43 (7) 33.6+1.10 (12) 33.9 0.27 *(No. of birds) mean standard error. 139 .v- , - t-v W ;V. -\ A ' f> I :.'V S. J. Itu rri et at. DSW 0 2 5 5 2 9 Arodor 1221 1242 1242 1254 1260 Table VIH. Percentage of Chickens Exhibiting Hydropericardium0 when Fed Various PCB's from One Day of Age Treatment period (weeks)*5 9 0 o%(UF 25 - Dietary level (ppm) 50 0%(11) 100 0%(10) 150 0%(11) 8 (trial 1) 0% (12) 0% (12) 20%(10) 62% (8) - 8 (trial 2) 0%(10) - 25% (12) 80% (10) - 9 0% (12) - 8% (11) 50% (8) 57% (7) 9 0% (12) - 0% (12) 9% (12) 8% (12) X3 NS PC.001 PC.001 PC.05 NS aHydropericardium taken as > 1.0 ml pericardial fluid. b Average at sacrifice. c(No. of birds). o to*i***>&4<v ,, '> Parameters Affected by PCBs in the SCWL Cockerel 141 Acknowledgments The authors wish (o acknowledge the technical assistance of Mrs. S. Asher and Mr. M. Dickens. References Alcxanian, R., W. K. Vaughn, and M. W. Ruchelman: Erythropoietin excretion in man following androgens. J. Lab. Clin. Med. 70, 777 (1967). Bitman, J., H. C. Cecil, and S. J. Harris; Biological effects of polychlorinated biphenyls in rats and quail. Environ. With. Perspectives 1, 145(1972). Denington, E. M.t and A. M. Lucas: Blood technics for chickens. Poultry Sci. 34, 360 (1955). De Vos, J. G,, and J, H. Koeman: Comparative toxicologic study with polychlorinated biphenyls in chickens with special reference to porphyria, edema formation, liver necrosis and tissue residues. Toxicol. Appl. Pharmacol. 17,656 (1970). Domm, L. V., E. Taber, and D. E. Davis: Comparison of erythrocyte numbers in normal and hormone-treated Brown Leghorn fowl. Proc. Soc. Exp. Biol. Med. 52, 49 (1943). Domm, L. V. and E. Taber: Endocrine factors controlling erythrocyte concentration in the blood of the domestic fowl. Physiol. Zoo!. 19, 258 (1946). Duncan, D. B.: Multiple range and multiple F tests. Biometrics 11,1 (1955). Emerson, Jr., T. E., C. M. Brake and L. B. Hinshaw: Cardiovascular effects of the insecti cide endrin. Can. J. Physiol. Pharmacol. 42, 41 (1964). Flick, D. F., R. G. O'Dell, and V. A. Childs: Studies of the chick edema disease. 3, Simi larity of symptoms produced by feeding chlorinated biphenyl. Poultry Sci. 44, 160 (1965). Fried, \V., D. Marver, R. D. Lange, and C. W. Gurney: Studies on the erythropoietic stimulating factor in the plasma of mice after receiving testosterone. J. Lab. Clin. Med. 68, 947 (1966). Hestrin, S.: Acylation reactions mediated by purified acetylcholine esterase 11. Biochem. Biophys. Acta 4, 310 (1950). Koeman, J. H., M. C. Ten Noever De Bratiw, and R. IT. Dc Vos: Chlorinated biphenyls in fish, mussels and birds from the river Rhine and the Netherlands coastal area. Nature 221, 1126 (1969). Malgor, L. A., and J. W. Fisher: Effects of testosterone on erythropoietin production in isolated perfused kidneys. Am. J. Physiol. 218, 1732 (1970). McCune, E. L., J. E. Savage, and L. B. O'Dell: Hydropericardium and ascites in chicks fed a chlorinated hydrocarbon. Poultry Sci. 41. 295 (1962). f i j ! osw 025530 STLCO 142 S. J. Iturri et al. Nirmalan, G. P., and G. A. Robinson: Hematology of Japanese quail treated with exo genous stilbestrol dipropionate and testosterone propionate. Poultry Sci. 51, 920 (1972). Nowicki, H. G., and A. W. Norman: Polychlorinated biphenyls -- induced increase in hepatic metabolism of testosterone, estradiol-17/5 and 4-androstene-3, 17 dione. (Abstract). Amer. Chem. Soc. Nat. Meet., 163rd, Boston (1972). Platonow, N. S. and H. S. Funnell: Anli-androgenic-like effect of polychlorinated bi phenyls in cockerels. Vet. Record 88, 109 (1971). Rehfeld, B. M., R. L. Bradley, Jr., and M. L. Sunde: Toxicity studies on polychlorinated biphenyls in the chick. 1. Toxicity and symptoms. Poultry Sci. 50, 1090 (1971). Rehfeld, B. M., R. L. Bradley, Jr., and M. L. Sunde: Toxicity studies on polychlorinated biphenyls in the chick. 2. Biochemical effects and accumulations. Poultry Sci. 51, 436 (1972a). Rehfeld, B. M., R. L. Bradley, Jr., and M. L. Sunde: The effect of polychlorinated bi phenyls on chicks. 3. Recovery. Poultry Sci. 51, 488 (1972b). Sturkie, P. D.: Avian Physiology, 2nd. ed. Ithaca, New York: Cornell University Press (1965). Tanaka, T,, and M. M. Rosenberg: Effect of testosterone and dienestrol diacetate on hemoglobin levels of cockerels and capons. Poultry Sci. 34, 1429 (1955). Wintrobe, M. M.: Clinical Hematology, 5th ed., Philadelphia: Lea and Fibiger (1961). Manuscript received June 11, 1973; accepted September 21, 1973. >... Jd DSW 025531 STLCOPCB4009486