II ’ I l ‘1?!’3.'..,":‘,-‘.)o 1., 9".“ .- ‘. ‘.93¢§1?«IE'\‘-I '-’- THE EFFECTS OF POLYBROMINATED BIPHENYLS ON THE HEMATOLOGY AND PLASMA ERYTHROPOIETIN LEVELS OF THE SINGLE COMB WHITE LEGHORN COCKEREL _ Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY LINDA RAE VAN THIEL 1977 .‘”_-----m,, , _ ~n ollsfm; . ~13?" 2:62 9.3"? [717» 1~~v ("a . . , 1.x... E . , .In , I . . A1»\..A.._‘u. . L’ ‘ ' “' University THE EFFECTS OF POLYBROMINAIED BIPHENYLS ON THE HEMATOLOGY AND PLASMA ERYTHROPOIETIN LEVELS OF THE SINGLE COMB WHITE LEGHORN COCKEREL BY Linda Rae Van Thiel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1977 COI 1111 ha co: ext the lev ABSTRACT THE EFFECTS OF POLYBROMINATED BIPHENYLS ON THE HEMATOLOGY AND PLASMA ERYTHROPOIETIN LEVELS OF THE SINGLE COMB WHITE LEGHORN COCKEREL By Linda Rae Van Thiel Polybrominated biphenyls (PBB), a thermoplastic fire retardant commercially available as Fire Master FF-l, was accidentally incorporated into livestock feed in the State of Michigan. P83 and related compounds, have been found to alter the hematocrits and hemoglobin concentrations of contaminated animals. The purpose of this study was to determine the extent of the hematological disturbance, and to determine whether or not the disturbance was accompanied by a change in plasma erythropoietin (ESP) levels in the single comb white leghorn (SCWL) cockerel. In the first experiment, 27 SCWL cockerels were divided into three equal groups. One group was fed rations containing 150 ppm PBB. The two control groups were fed rations containing 0 ppm PBB; one group was fed ‘gg libitum, the other was pair-fed to the 150 ppm PBB group to eliminate any possible effects due to any decrease in feed consumption. The second experiment was similarly designed using 50 weekrold cockerels. These birds were divided into two equal groups and were fed either 0 or 150 ppm.PBB. Due to limited facilities the 2g libitum control group was pa th th of eliminated in this experiment allowing for maximum numbers of birds in the pair-fed control and the treated groups. The ESF levels were determined by bioassay using Japanese quail. In the first and second experiments, respectively, 50 and 60 adult males were exposed to hypoxic conditions daily for three weeks. This treatment increased their sensitivity to any plasma ESF present in the serum pre— pared from the cockerels fed 0 or 150 ppm PBB. After 4% and 8 weeks of feeding in the first experiment, and after 8 weeks of feeding in the second experiment, statistically significant differences were observed between the control and PBB treated group mean hematocrits and hemoglobin concentrations. Both hematological values were decreased in the treated group versus the control groups. There was also a significant difference between the mean ESF level of the control and treated groups. The plasma ESF level of the treated group was lower than that of the control group. The results of these studies indicated that the decrease in hematological values due to chronic ingestion of P33 was the result of depressed levels of plasma ESF. ACKNOWLEDGMENTS I would like to express my graditude to my major advisor, Dr. Robert K. Ringer, and to the members of my advisory committee, Dr. William L. Frantz and Dr. Lester F. Wolterink for their assistance in my master's program. I would also like to thank.Ms. Ripalda Krasnoborski, Mr. Fredrick Heineman, and Mr. Sulo Hulkonen for their technical assistance, and Dr. H.C. Zindel for the use of research facilities in the Department of Poultry Sciences. I would especially like to thank Ms. Christine Salvaggio and Dr. John H. Fitch for their encouragement, patience, understanding, and, most of all, their friendship during the course of my master's program. TABLE OF LIST OF TABLES . . . . . . . . LIST OF FIGURES . . . . . . . INTRODU m I ON 0 O O O O O O I LITERATURE REVIEW . . . . . . Erythropoietin . . . . . Avian Erythropoietin . . Polyhalogenated Biphenyls 0mm IVES O O O O O O 0 C O 0 MATERIALS AND METHODS . . . . Animals . . . . . . . . . Feeding of SCWL Cockerels Hematological Preparation CONTENTS Erythropoietin.Assay . . . . . . . . . . . . Statistical Analysis . . . . . . . . . . . . RESULTS Mortality . . . . . . . . . . . . Hematocrit . . . . . . . . . . . Hemoglobin Concentration . . . . Erythropoietin Levels . . . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O O I 0 SUMMARY ”PMICES O O O O O O O O O O O O O O O O O O O O I. II. III. IV. V. VI. PBB RATIO" PREPARATION o o o o o o o o o' SAMPLE PAIR FEEDING CALCULATIONS . . . . PREPARATION OF DRABKIN' S REAGENT . DETERMINATION OF HEMOGLOBIN CONCENTRATION SCINTILLATION COUNTING . . . . . ERTTHROCYTE GHOST PREPARATION FLOW CHART BIBLIOM O O O O O O O O O O O O O O C O O O 0 iii iv H wwwu 2O 20 22 22 24 27 28 28 28 33 36 42 49 51 52 56 55 59 60 LIST OF TABLES Quail Breeder QB—72 . . . . . . . . . . . . . . . . . . . . . 21 ChiCk Starter 08-75 0 o o o s o s s o o o o o s o s s o o s o 23 The effect of feeding rations containing 0 and 150 ppm PBB (as Fire Master FF—l) on hematocrit and hemoglobin concentration of SCWL cockerels . . . . . . . . . . . . . . . 29 The effect of injection of either no serum or serum from SCWL cockerels fed 0 and 150 ppm PBB as Fire Master FF-l) on quail erythrocyte thymidine 2-C1 incorporation . . . . . . . 37 iv LIST OF FIGURES Figure 1. The proposed intracellular mechanism.controlling the production of erythropoietin in a renal glomerular epithelial cell 0 O 0 O O O O O O O O O O O O O 0 Factors controlling production of erythropoietin . . The effect of feeding rations of 0 and 150 ppm PBB (as Fire Master FF-l) on the mean hematocrit values of SCWL cockerels . . . . . . . . . . . . . . . . The effect of feeding rations of 0 and 150 ppm PBB (as Fire Master FF-l) on mean hemoglobin concentration Of SM COCkerels 0 O O 0 O O O O O O O O O O O I O O The effect of injection of either no serum or serum prepared from SCWL cockerels fed 0 and 150 ppm PBB (as Fire Master FF-l) on the mean quail erythrocyte thymidine 2-014 incorporation . . . . . . . . . . . . Sample hemoglobin concentration calculation . . . Sample I efficiency calculations . . . . . . . . . Page 32 35 41 54 58 1: 3L INTRODUCTION Polyhalogenated biphenyls have been commercially available since the early 1930's. Polychlorinated biphenyls (PCBs) have been recognized as an environmental contaminant since 1966. Recently, measures have been introduced to reduce the amounts of PCBs released into the environment. Polybrominated biphenyls (PBBs) have been realized as an environmental contaminant in Michigan only within the last few years. PBBs were developed and produced by the Michigan Chemical Company as a fire retardant for thermoplastics under the name of Fire Master FF-l. In May 1973, an unknown number of 50 pound bags, estimated at a total of several hundred pounds, were mistakenly received by the Michigan Farm Bureau. The Farm Bureau routinely mixes a nutritional supplement, magnesium.oxide sold as Nutrimaster, into its feeds. Prior to 1973, the consistency of Fire Master and Nutrimaster were quite different, but due to additional processing of the Fire Master, the compounds looked like identical fine, white powders. In addition, due to a paper shortage in 1973, both compounds were packaged in identical bags, with only the name Iof the compound stenciled across each bag. The mistake in shipment was not realized and the Fire Master FF-l was incorporated into livestock feed. Subsequently, thousands of Michigan beef and dairy cattle, sheep, swine, poultry, and indirectly, humans, were contaminated with unknown amounts of P83. PBBs were not implicated as the source of the contamination until the following year. Gross symptoms of polyhalogenated biphenyl contamination are skin lesions, increased skin pigmentation, edema, loss of appetite, alterations in reproduction, and liver damage. Over a moderate length of time, PCBs cause a noticeable anemia in fowl. It has since been found that PBB intoxication also results in anemia. The purpose of this study was to determine whether or not PBB altered certain hematological values, including hematocrit, hemoglobin concentration and erythropoietin levels in single comb white leghorn cockerels. hu re Va in' or ele It Ilel altI has uriu reSu. LITERATURE REVIEW Erythropoietin For many years it has been known that certain conditions alter the number of circulating erythrocytes. Hypoxia or hyperoxia were thought to influence red cell numbers by the direct action of 02 concentration on the bone marrow. In 1950 Reissman studied parabiotic partially nephrectomized rats and determined that changes in erythrocyte number were due to a humoral factor produced outside the bone marrow. This humoral factor must normally be present, at least intemmittently, for red cell number homeostasis, but until recently the normal concentration was too minute to determine by bioassay. Most erythropoietic studies involve animals with elevated hormone levels induced by chemicals, drugs or environmental changes (Jacobson and Doyle, 1962). Along with elevated plasma erythropoietin (ESF) levels researchers found detectable amounts of ESF in urine, lymph, amniotic fluid and possibly breast milk. It was assumed that such fluids normally contain small amounts of ESP as well (Krantz and Jacobson, 1970). Complete purification of active ESF has not yet been accomplished, although a fraction that is 930,000 times as effective as the original has been attained by extraction on an ion exchange resin at low pH and low NaCl concentration. ESF for purification may be obtained from.the urine or blood of patients and animals with ESF levels elevated as a result of severe anemia or chemical treatment. The highly active CY th Ch Pr: 0r preparation was composed of 71% protein and 292 carbohydrate, but approximately two-thirds of the total was assumed to be impurities. ESF appears to be a protein associated with a glchprotein. Dukes ._§‘_l. (1975) have proposed that the glycoprotein protects the hormone from inactivation and provides for target cell specificity. ESF is relatively stable under normal conditions, retaining its biological activity after boiling for 15 minutes at pH 5.5. Mammalian ESF is destroyed by neuraminidase and such proteolytic enzymes as trypsin, pepsin, 0‘ -and f3 -amylase , 6 -g1ucosidase, cellulase, ribonuclease, carboxypeptidase and lysozyme. Sialic acid is necessary for its activity. Estimates of molecular weight range from 27,000 to 66,000 with an accepted average value of 46,000 (Nakao stflgl., 1975). Early studies attributed the majority of ESF production to the kidneys (Jacobson, 1957). Reissman and Nomura (1962) thought the renal medulla was the source of ESF, while others proposed renal ESF was produced in the renal cortex. In 1965, Fisher and coworkers, using fluorescent antibody techniques and electron microscopy, localized the epithelial cells of the glomerulus as the major source of ESF. However, approximately 102 of ESF circulating is thought to be of extra-renal origin, produced by the liver (Reissman and Nomura, 1962), and possibly spleen, pituitary and/or blood vessels (Fisher gtflgl., 1962). George and coworkers (1975) and Fisher (1975) postulated that 'cyclic-AMP is the intracellular mediator controlling ESF production in the kidney (Figure 1). They proposed that a renal oxygen sensor detects changes in 02 concentration due to any one of many stimuli. They also propose that the sensor may be located in glomerular epithelial cells, or it may be in another region of the kidney which in turn stimulates Figure 1. The proposed intracellular mechanism controlling the production of erythropoietin in a renal glomerular epithelial cell. ESF - Erythropoietic Stimulating Factor REF - Renal Erythropoietic Factor RENAL OXYGEN SENSOR l Adenyl Cyclase I : (activates) ATP ‘1' ; Cyclic-Am /' ./ / (activates) ./ 1’ Inact ive K / Act ive Phosphokinase _§; Phosphokinase ./ 1’ ’(activates) .II’ A Inact ive K Act ive REF i%} REF Renal Plasma REF REF + + Renal Plasma Globulins Globulins II II Renal Plasma ESF ESF V PLASMA ERYTHROPOIETIN F Figure 1. the 035 ini act er) prc orI act The C0! PIC EST uni ste the the the alt "Y flo Or' lev the glomerulus to produce ESF. The primary stimulus acts on the renal oxygen sensor to activate adenyl cyclase. This membrane-bound compound initiates the conversion of intracellular ATP to cycliceAMP. Cyclic-AMP activates a protein kinase which in turn activates a renal erythropoietic factor (REF). A REF was isolated in two separate laboratories; both Hansen (1963) and Kuratowska (1968) reported extremely erythropoietic low activity in their factors. Kuratowska proposed that the less active REF andcx-globulins, of renal or humoral origin, combine to form active ESF. Fisher ££_gl, (1975) found that the action of the renal sensor appeared to be potentiated by prostaglandins. The prostaglandins PGE1 and PGE2 may enhance the renal adenyl cyclase conversion of ATP to cyclic-AMP. Erythropoietin exhibits its effects on the bone marrow to initiate production of red cells. The exact morphological site upon which the ESF acts has not been identified. ESF seems to stimulate an lunidentified stem cell to proliferate and produce erythrocytes. This stem cell, as defined by Gurney (1965), precedes the earliest characterizable erythroblast and differentiates into an erythroblast in the presence of erythropoietin. The primary regulator of ESF production is the 0 concentration at 2 the renal sensory cell. A change in 0 concentration.may result from 2 alterations either in atmospheric 0 pressure, metabolic rate, 2 erythrocyte 0 -carrying capacity, erythrocyte number, or renal blood 2 flow (Figure 2). Blood 02 concentration is directly affected by hypoxia or hyperoxia; the former stimulates ESF production and the latter depresses it. Administration of cobalt is widely used to elevate ESF levels within a few hours. Cobalt has no effect on overall metabolic nowuousuoz use nouuonooum oumoounumuu sound: soon a... a mmwuapaece \ mmm ex mammam A. nowmnmnnnuh 0mm .Ammmv casewomounuhuo mo sowuoavoum mnqaaouuuoo ououoom .N unamum naaonumohaom mammoousurmj. omoouoaa aaaasm No mmsouomug \. I .lv fies—mama \\ \. nommom doumxo_hw Hmamm Amoumasefiumv sm sonata Anson» use woman mo mommanv nouns masonsum.w sass moose as usuuomsu susn o axon: a too can sausages m I m away ~.0 + «.5 Amuv «.0 +.n.0 x N.~ A00 «.0 + ~.0~ +I % Amv ~.o a 0.0 Amv «.0 + 0.0 +I Amy ~.o u 0.e~ anuv 0.0 + n.~m +I “use. «.0 x I a.m~ Ame a.o + m.~n a I «.mm A00 0.0 + ¢.0~ +l Ame a a.o +l fist so N I E «.c + as avafiowrs M I any v.9 + w.mm a I. Aav m.0 + «.mN usoawuonxu mass: 0 osooom unusauonnu mass: 0 uouam usoawuomxm axons we unuwm Asa ooa\mv awooawoaom unuawuommm exam: 0 vacuum usaaauomxu exam: 0 unuam ucoawuomxm names we usage Auv uauooumso: and and one unassumoua mumuoan Houuooo oomluwmm 3380 .593: mm nouoamuom .sasusxooo 030m «0 nowusuunoonoo maaoamoaon one .uwuooumssa so AHImm nouns: swam osv mum and 0n~ one 0 usanawunoo saowumu wmuooou no assume any .m sandy 30 were 8.6% greater than the PBB mean hematocrit. ’The mean hematocrit of the PBB treated group was significantly different from that of both control groups (P- 0.025). Statistically, there was no significant difference between the 2 control groups. The mean hematocrit values were computed from the hematocrits of all surviving birds in each group. At the termination of the same study, after 8 weeks of treatment, the trends were the same as were observed after 48 weeks. The gd_libitum and pair-fed control mean hematocrits increased to 33.81 and 32.8%, respectively. The PBB group mean further decreased to 23.42, nearly 30% lower than the control values. There was no significant difference between the mean hematocrits of the gg_1ibitum and pair-fed control groups. The statistical significant difference between the mean hematocrit values of the control and the PBB-fed groups increased to P- 0.001. Again, the mean hematocrits of the 3 groups were obtained from the values of all surviving birds. The results of the second study were similar to those of the first study. After 8 weeks of feeding, the mean hematocrit of the P88 fed group was 242. The mean value of the pair-fed control group was 32.31. 26! greater than the PBB group mean. There was a significant difference between the means of the 2 groups (P- 0.01). The mean hematocrits were calculated from those of all of the surviving PBB fed cockerels and 21 of the 24 surviving birds in the pair-fed group. The mean hematocrits and their standard errors from both experiments are graphically portrayed in Figure 3. The increase in mean hematocrits of the control groups from 48 to 8 weeks is evident. The decrease in means of the PBB group over the same time is also evident. 31 .moawa Hooauuo> ecu an oouomwamoo ma nouns oumommum on» one mwmonumouma ma oouoofiosa ow macaw you woman no woman: may .maouoxooo 030m «0 cowuouudoocoo canoamoao: some oSu so AdImm momma: swam mmv mum and onfi one 0 mo ocoaumu wswooom mo uoommo one .e ounwwm .n enough 32 as as one H838 nouns: 3:80 .32: we a we a a as a “a one and vow use use use ”:—H .w 0a Amy is ON .IHWL A00 IHL AS 80 :8 80 MI A v , H : 8 a m. m. .Imm. Ls OQ (z) atlaonvman 33 The graph additionally shows that there is little difference between the first and second experiments after 8 weeks of treatment. ggggglgbig_Concentration Table 3 contains the mean hemoglobin concentrations from both experiments. In the nest study, the mean hemoglobin concentration for the PBB treated cockerels was 8.88/100 m1. This value was nearly 112 lower than the 5g libitum and pair-fed control group means, 9.88/100 m1 and 9.98/100 ml, respectively. After 48 weeks, a significant difference was found between the PBB fed group and the 2 control groups (P- 0.025). There was no statistical difference between the means of the control groups. The mean hemoglobin concentrations were determined from the values of all surviving birds in each group. After 8 weeks of the first study, there was a greater difference between the mean hemoglobin concentrations of the PBB treated group and the 2 control groups (P- 0.001). The mean hemoglobin concentrations of the 5d.1ibitum and pair-fed control groups were consistent with the values at 41; weeks to 9.83/100 .1 and 10.13/100 ml, respectively. The mean of the PBB fed group decreased to'7.28/100 ml, 26: lower than the control means. Again, the mean hemoglobin concentrations were determined from the values of all surviving birds within each group. The results of the second experiment were similar to those of the first study at 8 weeks. After 8 weeks of treatment, the mean.hemoglobin concentration of the PBB fed group was 7.48/100 ml, 22! lower than the pair-fed control mean of 9.58/100 ml. The PBB mean hemoglobin concentration was significantly different from the control mean at P- 0.01. The means were derived from the values of the 18 surviving 34 .moswa Hosanna» one he oouoswamoo ma nouns oumooouo any one swoonusouoo ow ooumowoaw ma asouw pom woman 00 Homes: 059 .mHouoxooo 430m 00 mooHo> uHuooumaoa some man no AHIhm nouns: swam mmv mmm and 0m~ one 0 mo moowusu wswooom mo assume 059 .m shaman .e enough 35 as as on 33:8 e315: Houses .323 3. a a as a o .a a we van and one oou and and and and Av n 30 3: A3 33 rHI as E 5 _. A3 *1 a H rml a. o.— (17m 001/8) uonsnuaouoo urqotsoman 36 cockerels in the PBB group and from 21 of the 24 surviving pair-fed cockerels. Figure 4 represents the mean hemoglobin concentrations of the first experiment at 48 and 8 weeks, and the second experiment at 8 weeks. The graph shows that the means remained constant in the 2g libitum and pair- fed control groups from 4% to 8 weeks. In contrast, the means of the PBB treated cockerels decreased over the same time period. Additionally, the graph shows that there was little difference in the mean hemoglobin concentrations of the P88 treated and pair-fed control groups at 8 weeks of treatment between the first and the second experiments. Erythropoietin Levels The mean amounts of thymidine 2-C15 incorporated into quail erythrocytes for various treatment groups are compiled in Table 4. 'Determination of ESF levels at 4k‘weeks was not made in the first study since it was not possible to collect enough blood from the chicks to ' prepare 3 milliliters of serum. In the first study, after 8 weeks of treatment, there was no significant change in the mean amount of thymidine 2-014 incorporated into quail erythrocytes as a result of injection of serum from the cockerels of any of the 3 groups. The mean incorporation, in terms of degradations per minute per 140 mg packed RIC ghosts (DPM), were 3954.5, 3513.0, and 3239.9, respectively for the pg§_1ibitum control, the pair-fed control, and the P33 treated groups. These values indicated that there was no difference in the ESF levels among the 3 groups. The nglibitum.control group mean was determined from 4 of the 7 surviving cockerels in that group. 0f the remaining 3 7 3 8.0 I m “unseat: 39:33.23: one sue—"usage 303qu megs.— uses: Anson» use evade no monsoov mouse muevseum_u easel queue as vouuoeou ounce w I Anav s.hs+m.mwo~ «Ame m.~e-ua.mm~m h I u I Amuv w.no~+s.oeo~ Amy n.sn~+c.smn~ mass: m umuawuonno vacuum Ame o.mo~uo.mamn Ase ~.n«num.smom I «soon a usesquomuo umuwm Aeumogu and vexomm ma ass use zuav souumuomuoosw «~0I~ omwvflahsa amuse amuse abuse mam sea one Houuaou voquamm Houusoo asuanHm v4 amuse oz usuufimumm usuaummus asuom .mouumuonuoomw UIN oaavfiahnu sumoouauhuo Hesse so A~Imm house: when adv mam Bum on“ use 0 mo maoqumu mow mmwuoxooo nsom aouw amuse no sauce on penuae mo moauuofiaw mo avenue oak .e edema 38 survivors, 1 did not provide enough serum to assay. The 0PM of the other 2 survivors were statistically excluded as residuals because their values were extremely high (8510.2 and 7817.7 0PM) when compared to the mean (3954.5 DPM). The pair-fed control mean was calculated from 8 of the 9 surviving birds. One bird lacked a sufficient blood sample to assay. The mean for the PBB fed group was determined from the values of 3 of the 5 birds that survived treatment. The remaining 2 values were not obtained since those quail died after injection with the prepared serum. In the second study, after 8 weeks of treatment, the amounts of thymidine 2-C16 incorporated into the quail erythrocytes, as reflected by the mean number of degradations, were 1397.8 DEM, 1640.7 0PM, and 1028.3 DPM for the quail not injected with any serum, the quail injected with control serum, and the quail injected with PBB serum. These values were all significantly different from one another at P- 0.01. The mean thymidine 2-c1" incorporation in quail not injected with any serum‘was 151 less than that in quail injected with serum from pair-fed control cockerels, and 26! greater than that in quail injected with serum from PBB fed cockerels. There was a 37! difference in the mean thymidine 2-c1“ incorporation in quail injected with serum prepared from pair-fed control cockerels and from.PBB fed cockerels. Serum.from only 19 of the 21 cockerels that survived 8 weeks of treatment was used to determine the mean of the pair-fed cockerels. Sufficient serum.was not obtained from 1 bird, and 1 quail died after injection of the serum. The PBB group mean was determined from the results of all 18 surviving birds. Again, though, 1 value was statistically excluded as a residual. 39 Figure 5 graphically portrays the mean erythrocyte thymidine 2-C14 incorporation in quail injected with the various sera. The values from the first experiment were not directly comparable to those of the second experiment due to differences in the procedures of the 2 studies. In the first experiment, even though the mean DPM decreased from the gg.libitum control group through the PBB treated group, the extent of the maximum range was roughly equal over all 3 groups. In the second experiment, the differences among the 3 groups are evident. 40 .ssawa Hmouuus> he vsusmuaesv ea uouus vusvasum sea was suesnuasuee aw usussavaa mu esouu use mvuan «0 uses:: sea .souusuoeuosau 12"qu anvaaheu suhsounuhus Hausa mesa ssu no amImm usumex suwm may mum see and mas 0 now masusxsos azom souu vsusesun amuse uo abuse on usAuws mo soaussnaa mo uosmms sea .n sumwwh .n 3.8: 41 amuse mum Sues Houusoo lies «oumLJoo luuem see one eouuuu-m sauuAuu ea on new and 3N and and can. I ] Ii 5 a v use I.ocou A3 v $ Any , m I. ..ooo~ Amy J I 4 coon l L L A3 L.83 L . (Had) natavzodaoaut ”Io-z autptlfiqt DISCUSSION Anemia is defined as a decrease in either the ratio of red blood cells to plasma, the size of the red cells, or the amount of hemoglobin contained in the red cells. It is unlikely that the decrease in packed cell volume (hematocrit) and hemoglobin concentration was due to increased plasma volume since similarly treated birds were found to have generalised edema and decreased body weight (Heineman, 1976).1' The decreased hematocrits and hemoglobin concentrations of PBB fed cockerels were also not due to decreased feed intake. There was no difference in hematological values between the g§_libitum and pair-fed control birds in the first study. Because of this it was decided that the g§_libitum control group was no longer a necessary group; thus, the second experiment consisted of only pair-fed control and PBB treated groups. The first and second studies showed a definite decrease in the hematocrits and hemoglobin concentrations of the cockerels fed 150 ppm PBB for 8 weeks as compared to those of the control birds. Thus, PBB administration, at 150 ppm, results in anemia in SCWL cockerels. The mean erythrocyte thymidine 2-014 incorporation data obtained from the first experiment were not extremely accurate due to the small number of birds in each group and the large standard error for each 1' Additional data concerning the effects of PBB and body weight, mortality and food consumption can be found in the M.S. thesis of Fredrick.w. Heineman, M30, 1976. 42 43 group mean. Bach standard error was greatly reduced in the second experiment, so the data were considered more reliable. The amount of thymidine 2-014 incorporated into the quail erythrocytes indicated the relative level of ESF present in each group of serum. The exogenous ESF injected into each quail stimulated its erythropoietic system to produce additional erythrocytes. As the red cells were produced, the thymidine 2-C14 injected was incorporated into the cells. Due to the lack of an avian ESF standard no direct numerical correlation could be made between thymidine 2-c1" DPM and the amount of ESF injected, higher mean DPM indicated greater levels of ESF and lower mean DPM indicated lower levels of ESF. It was assumed that the mean DPM value of the group of quail not injected with any serum represented the basal erythropoietic activity of the hypoxicpolycythemic quail, activity due only to endogenous ESF. Quail erythropoietic activity was increased by the addition of serum prepared from pair-fed control cockerels, presumably due to ESF present in the serum. The amount of ESF present in the serum of the pair-fed control birds reflected the amount of ESF necessary to keep the animals in erythropoietic homeostasis. Erythropoietic activity was decreased in the quail injected with serum from.the PBB fed cockerels. The decrease in erythropoietic activity of the test animals indicated that the ESF level of the PBB birds was lower than that of the birds not fed PBB. In addition, since the erythropoietic activity of the quail injected with serum.from.PBB fed cockerels was lower than the basal quail erythropoietic activity, there was a possibility that a component in the serum inhibited the endogenous quail ESF. Similarly, this component would decrease the activity of the 44 erythropoietic system in the PBB fed cockerels, creating the observed anemia. In general, there are many mechanisms through which anemia may occur. These include the following factors: 1) decreased endogenous hormone levels, 2) altered bone marrow erythroblasts, 3) decreased life span of the red cells leading to increased hemolysis, 4) inability of the renal oxygen sensor to detect changes in blood oxygen, 5) insufficient renal ESF production, 6) increased ESF inhibition, and 7) increased hepatic ESF metabolism. It is unlikely that the first three factors were the primary cause of PBB-induced anemia, singly or in combination with one another. Due to the nature of the erythropoietic negative feedback system, any reduction in the number of red cells stimulates increased ESF formation (Rosssand waldman, 1966). In contrast administration of 150 ppm.PBB resulted in decreased ESF levels. Since endogenous levels of hormones such as androgens, glucocorticoids and thyroxine have been found to be lower with administration of PBB, decreased hormone levels may have an indirect effect upon the erythropoietic system. This mechanism of PCB action has been postulated by Rehfeld ggugl_(l97l) and Platonow and Funnell (1971) although neither assayed for ESF activity. Decreased hormone levels may initiate or contribute to PBB-induced anemia by removing some of the stimuli that result in increased red cell formation during growth. The degree to which these three decreased hormones affect erythropoiesis in PBB-treated animals can be determined by replacing the endogenous hormones with exogenous hormones in the diet. Even though fewer erythroblasts, and the cells into which they differentiate, were observed in bone marrow smears from PBB-treated 45 cockerels (Ringer and Lack, unpublished), this does not necessarily indicate that the erythropoietin responsive stem cells failed to respond to ESF, or that they failed to differentiate into erythroblasts after ESF stimulation. If this had occurred the negative feedback system would have increased ESF production. Rather, the bone marrow observation could additionally indicate absence of plasma ESP, and therefore absence of maturing red cells. This could be determined by injecting large amounts of ESF into the PBB treated animals. If the bone marrow were indeed damaged, there would not be any change in the hematological values in the animals. Finally, the life span of red cells could have been decreased in the PBB treated cockerels. Strik (1973b) found that PBB-fed quail exhibited porphyria and increased ALAS levels. Although porphyric animals often exhibit hemolytic anemia (Merck, 1966), the porphyria is usually a symptom caused by the increased levels of hemoglobin precursors, including~ALAS, rather than the result of the anemia. The actual life span of red cells in PBB- treated animals can be determined by labeling newly-produced red cells with a radioactive isotope, sampling the blood at specific intervals and testing for radioactivity, and calculating the length of time required for the majority of the label to disappear (Rodnan, 1957). The normal life span of fowl erythrocytes is 25 to 30 days (Bell and Freeman, 1971). The radioactive label would disappear before day 25 if PBB increased the rate of red cell destruction and decreased the life span of the average red cell. Since these three mechanisms, if altered, would have increased the plasma concentrations of ESF, it was improbable that the PBB-induced anemia was due primarily to decreased hormone levels, altered bone marrow, and/or decreased red cell life span. 46 While faulty renal oxygen sensors, insufficient renal ESF production, increased BSF inhibition and increased ESF metabolism should all result in decreased plasma ESF levels. neither Iwamoto (1973) nor Norris g£_£l, (1975) found perceptible changes in renal filtration function with either PCB or PBB treatment. In addition, there have not been any reports of damage to the renal glomerulus, the site of renal ESF production (Fisher 35 21., 1965) and possible site of the renal oxygen sensor. Also due to the decrease in cell numbers in the PBB- treated animals, one would expect additional incorporation of hemoglobin into those few cells produced. Strik (1973b) reported that PBB's increased ALAS, a precursor in hemoglobin formation, but the majority of this compound did not seem to be incorporated into red cells, a condition which resulted in porphyria. Also, Iturri EEHSls (1974) showed that the mean corpuscular hemoglobin concentration (HCHC) was not significantly changed after PCB treatment. Rather, there was a trend toward decreased HCEC with administration of increasingly greater amounts of PCB. This indicated that there was a lack of active plasma ESF. Further research is required to determine whether or not decreased plasma ESF levels with administration of 150 ppm PBB were due to failure of the renal oxygen sensor, or the inhibition of BS! synthesis. Inoperative renal oxygen sensors can be detected by exposing the PB!- treated animals to hypo- and hyperoxic environments. If, indeed, the sensors were defective the hematological values of the hypo- and hyperoxic animals would be similar to those of the control animals. Failure of the renal glomerulsr epithelial cells to produce 38! can be determined by measuring the levels of adenyl cyclase, cyclic-AMP and ESP present in the kidneys of PBB-fed animals. Production of BS! in the 47 glomerulus, or lack of it, can also be determined by the use of a fluorescent rsr antibody (Fisher 9; 93,, 1975). According to research done to date, the most probable cause for the PBB-induced anemia was inactivation of large amounts of ESF. The ESF could have been inactivated by an ESF inhibitor or by hepatic degradation. Initially, active ESF may have been displaced from its protective serum binding proteins, as occurs with thyroxine (Batomsky, 1974). This would render the hormone more susceptible to degradation by the liver microsomal enzymes. Many other hormones are destroyed in this manner, among them androgens, glucocorticoids, and thyroxine. Since ESF is degraded in the liver by hepatic microsomal enzymes (Roh and Fischer, 1971) and PHB's induce hepatic microsomal enzymes (Conney, 1967) it follows that PHB's should increase ESF metabolism by increased hepatic microsomal enzymes. This hypothesis can be tested by Boh and Fisher's perfusion techniques. Plasma containing high concentrations of 88! can be perfused through livers of animals pretreated with PBB. In addition, homogenates of liver from animals exposed to PBB can be incubated with plasma containing concentrated ESF. If ESF metabolism were indeed increased with administration of PBB, the plasma from.the perfusion and homogenate experiments would have little erythropoietic activity. The decrease in ESF in the PBB-treated cockerels could have additionally been due to inhibition of plasma ESF by some component. This hypothesis is strongly supported by the limited data from the final experiment. Unfortunately, little is known concerning ESF inhibitors. Dukes g£_£l, (1972, 1975) determined that certain prostaglandins potentiate the erythropoietic activity of ESP and are possibly a cofactor necessary for ESF bioactivity. They proposed that the IS! 48 inhibitor, a protein, has a higher affinity for the prostaglandin cofactor than normal ESF does. Erslev g£_§l, (1971) found an inhibitor that was a lipid. They thought it acted by displacing the prostaglandins normally bound to ESF. This would inactivate the ESP and possibly make it more susceptible to degradation by the liver. The presence of PBB could have increased the amount of inhibitor produced and thus could have decreased erythropoiesis in the cockerels with the resultant anemia. There have not been any reports on the effects of PCBs or PBBs on prostaglandins in living systems. The existence of an BSF inhibitor could be determined by adding active ESF to plasma from PBB-treated animals. Up to a certain point, the preparation would have little or no activity. At that point, sufficient ESF would exist to bind to all of the inhibitor molecules. Past that point, the erythropoietic activity of the preparation would increase as ESP was added. SUMMARY 1. The hematocrits were significantly decreased in the first experiment cockerels fed 150 ppm PBB for 8 weeks relative to the gg_libitum and pair-fed controls. Therefore, the change in hematocrits was directly attributed to PBB. 2. Hemoglobin concentrations were significantly decreased in the first experiment cockerels fed 150 ppm PBB for 8 weeks relative to the‘gg_ libitum and pair-fed controls. Therefore, the change in hemoglobin concentration was directly attributed to PBB. 3. In the second experiment, plasma erythropoietin (ESF) levels were significantly decreased in the cockerels fed 150 ppm.PBB for 8'weeks relative to the pair-fed controls. Therefore, the change in plasma ESF levels was attributed to PBB. 49 APPENDICES APPENDIX I PBB RATION PREPARATION A premix of 12 PBB was prepared by adding 30 g. of pulverized Fire Master FF-l which had been passed through a sieve (U.S. Standard #30) to 2970 g. finely ground, similarily sieved chick starter CS-75 (King Milling Company, Lowell, Michigan). The final 150 ppm PBB ration was prepared in 4 kilogram quantities by combining 60 g. 12 premix with 3940 g. ground, unsieved chick starter 08-75. All ration mixing was done on a Paul G. Abbé', Inc., feed mixer (Little Falls, N.J.), tumbling for 15 minutes in 30 pound capacity feed cans. 50 APPENDIX II SAMPLE PAIR-FEEDING CALCULATIONS Total PBB ration given - GPBB (2500 8) _Total PBB ration not consumed - RPBB (300 g) Total PBB ration consumed - C (2500 g - 300 g - 2200 g) PBB. GPBB- RPBB Number of PBB fed birds - B (22) PBB PBB ration per bird - CPBB/BPBB (2200 g/ 22 . 100 g) The amount of PBB ration consumed per bird every 2 days is equal to the. amount of chick starter that is to be fed to the pair-fed control birds over the next 2 days. Chick starter per bird - PBB ration per bird (100 g - 100 g) Number of pair-fed birds - BPF (24) Total chick starter given - GPF (unknown) G - Chick starter per bird x B (2400 g - 100 g x 24) PP PF 51 APPENDIX III PREPARATION OF DRABKIN'S REAGENT 200 mg 1(3li‘e(CN)6 50 mg KCN 1000 mg NaHC03 1250 mg in 1000 ml distilled water The reagent should be stored, refrigerated in a sealed, dark glass bottle. In this manner it may be usable for 6 months or more. 52 APPENDIX IV DETERMINATION OF HHOGLOBIN C(NCENTRATIW 53 Figure 6. Sample hemoglobin concentration calculation. The line is constructed by plotting the Z absorbance of each standard against its known hemoglobin concentration. 2 Absorbance 0.5 Standard C 0.4 . Standard 8 0.3 ’ 0'25 Absorbance Standard A 0.2 0.1 Calculated Hemoglobin concentration Ask. L ..._.. 4 8 10 12 16 20 Hemoglobin concentration (g/100 ml) Figure 6. APPENDIX V SCINTILLATION COUNTING Reference: Instruction Manual, Beckman Liquid Scintillation Counter LS-lOO-C, Beckman Instruments, Fullerton, Ca. Liquid scintillation counting provides for the determination of radioactivity in samples emitting radiation. The energy emitted by each sample is converted to light energy by the fluorescent compounds (fluors) in the scintillation fluid. The light energy is detected by a photomultiplier tube which is connected to amplifiers and a scalar circuit. The results are expressed as the number of counts of radioactive emissions detected per minute, that is counts per minute (CFM). The counts detected are not always equal to the number of actual emissions of the isotope. This is due to chemical quenching, the absorption of some of the emissions by the chemicals present in the solution. It is important, therefore, to determine the efficiency of the scintillation system and calculate the actual degradations per minute (DPM) of each sample. The efficiency can be determined by detecting the CPM of samples with known DPM values, but with varying degrees of chemical quenching (Quenched C-l4 Standard Set Beckman Instruments, Fullerton, California), and using the following equation: CPM/ 173600 I Efficiency - 199000 DPM x 100 (87.242 - x 100) The Beckman LSlOO-C liquid scintillation counter offers built-in quench ‘55 S6 calibration through an external standard determination. In addition to CPM, the counter printout records external standard values for each sample. For each set of unknowns to be tested a graph is constructed plotting the external standard value of each quenched standard against its calculated efficiency (Figure 7). The efficienty of the unknown. samples is determined from their external standard values using this graph. The actual DFM of each unknown sample can be calculated using the following equation: 1190.5 pp” - CPM/X Efficiency x 100 (1340.6 ' 33.81 x 100) The calculated DPM is more representative of the actual amount of radiation emission of each sample. The major problem in counting whole blood samples is color quenching. The solubilized red cells exhibit a dark red color when in solution with the fluors due to the presence of hemoglobin. Part of the light energy produced by the fluors is absorbed by the solution due to its color, rather than being detected by the photomultiplier tube. This results in less efficient, lowered counts. If the colors of all samples are of unequal intensity, the efficiency of each sample is different. CPM’s are, therefore, unrepresentative of the actual DPM occurring. This can partially be overcome by decolorizing each sample with a few drape of H202, but this only increases the chemical quenching with little change in color of whole blood samples. Quenching can also be corrected by preparing a standard set with constant radioactivity, but varying color intensities. The sample treatment is similar to that followed in correcting chemical quenching. A third method used to compensate for color quenching is to eliminate the source of the color 57 altogether. In this case, the latter method was chosen due to the relative ease of removing the hemoglobin from the red cells. 58 souusasoaso usasusuuue u.e~amsm .h museum vumvasue Heaueuuu cu m— cu N.» n C ‘1 d _ S I nusvnsue Hsnusuuu _ summon . # _ _ i. on _ . _ m essences . . as _ _ m eueeesum _ — it Oh _ a vusossum _ _ cw _ o unmeasum . all. _ uugouuuuum u eouauauuao he as a sausages KauaIOIJJs z APPENDIX VI ERYTHROCYTE GHOST PREPARATION FLOW CHART Heparinized whole blood (centrifuge 2000 X g , 10 minutes) Aspirate serum 4% & Discard fir Packed erythrocytes (wash twice in 1.0% NaCl at 4°C centrifuge 2000 X g, 10 minutes) Discard supernatants