Document YGeL3dXeDzmLnR5ZdN6BjJeXO

MAxstfc. \P-- AUC1 1978 e^3 r S . I.tsr n il with Polychlorinated Biphenyl and Hcxachlorobenzcnc Induced Humoral Immunosuppression L. 1>. Loose. K. A. Pittman, K.-F. Bcnitz and J. B. Silkworth institute of Comparative and Human Toxicology Albany Medical College Albany, New York 12208 INTRODUCTION The influence of pesticides and other environmental chemicals on immune re sponses has received scant attention. Immunosuppression by chemicals of en vironmental importance is a developing concern in chronic toxicity assessment. Also, chemicals that are persistent in the environment arc of particular interest because of their possible accumulation in animal tissues and subsequent magnifica tion in the food ehain (4. 23, 28). Indeed, most feared and least understood arc the possible effects of the chemicals on cell regulatory processes which Ciolherg (13) has termed "the three geneses" -- carcinogenesis, teratogenesis and immunofenesis (13). .Several environmental contaminants have been reported to be synergistic with infectious agents. Polychlorinated biphenyls (PCB's) (8), arscnicals (12), cobalt sulphate (II) and sulphur dioxide (3) increased the mortality of animals infected with viral agents. Lead nitrate (14) and 2,3.7.8-tctrachlorodibcnzo-p-dioxin (T('I)D) (24) enhanced the mortality of mice to Salmonella $p. challenge. Evidence indicating an impaired host resistance to protozoan or rickettsial infections is presently unavailable. The enhanced susceptibility to bacterial and viral challenge in animals exposed to environmental chemicals may be related to a suppression in inlihndy (Ah) formation previously reported in animals administered either lead, cadmium, mercury (17), PCB's (18), or DDT (30, 31). or to an impaired cellmediated immunity (V. 10, 28, 29) in organo-chlorinc treated animals. Two environmental chemicals of importance which have not been extensively investigated with regard to their impact on immune responsiveness are heMchiorobenzcnc (HCB) and PCB's Polychlorinated biphenyls arc extremely viable compounds, are not hydrolyzed by water and have high thermal stability. They have a low acute toxicity and are poorly metabolized, particularly those with more than 4 chlorine atoms on the phenyl rings. Hcxachlorobenzcnc (CCI) is used tgricullimilly as a fungicide and industrially as a chemical intermediate in organic vvnthcsis. 23.3 HONS 001112 254 i oust it m,. The increased mortality observed in ducks fed a diet containing 25,50 or KKlppm of Aroclor 1254 for 10 days and subsequently challenged with duck hepatitis virus <g), coupled with (he demonstrated reduction in neutralizing Ah tilers to pscudoi abies virus in rabbits administered 3(H) mg ofeither PCU 1221, 1242. or 1254 once a week for 4 weeks (Ik), suggests an immunosuppressive action of P('H\ Therefore, the present studies were conducted to evaluate the influence of dictar> administration of PCB's (Aroclor 1242) and HCB on Ab production and immunoglobulin concentrations. Concurrent analysis of tissue levels of the chemicals anil hisiopathology of select lymphoid tissues and reticuloendothelial oigans were utilized to relate tissue concentration and morphology to functionality. ; . ! \ i T t : * of I i. . MATERIALS AND MUTHODS Animals- Male Balb/c mice (18 to 20 g) were used throughout, hood and watcr were provided ml libitum. Test Chemicals. The polychlorinated biphenyl Aroclor 1242 (Monsanto) and HCB were mixed in the diet at a concentration of 167 ppm. Hexachloioben/cne (Kastman practical grade) was first passed through activated charcoal and then 2X crystallized from boiling benzene. Control animals received the normal Wayne mouse diet. Diet analyses were conducted to confirm the correct concentration of Aroclor 1242 and MC'B in the food. /immmn/ogtV Parameters. Six weeks after being placed on diets containing tf7 ppm of cither PCB 1242 or HCB. all mice, including control animals, received an i.v. injection of0.1 ml of 10% sheep erythrocytes (SRBC). The primary A b response was evaluated hy the splenic direct plaque-forming cell (PKC) method as described by Jerne and Nordin (16). The plaques formed in this technique have been suggested to represent l9,Sor lgM Ab producing cells (3. 20). The primary PKC response measured on days 4. 5. 6 and 7 following immunization. Liver, lung, spleen, mesenteric lymph nodes and thymus tissue samples were also evaluated at this time for histopathologic changes and for concentrations of PCI) 1242 and HCB. Setum lg(n. IgA and lgM concentrations were quantitated by radial immunodiffusion (RID) using commercially prepared (Meloy Laboratories) RID plates for mouse immunoglobulins. A secondary i.v. challenge of 0.1 ml of 109? SRBC was adminis tered 2 weeks following the primary immunization. Direct splenic PK* formation, serum immunoglobulin (IgCii. lgM and IgA) concentrations, hisiopathology of liver, lung, spleen, thymus and mesenteric lymph nodes and tissue levels of PCD 1242 and HCB were evaluated on days 3. 4. 5 and 6 following the secondan immunization. To determine the effect oft he PCB 1242 and HCB on Ab memory cells, a separate group of animals was immunized with 0.1 ml of 10% SRBC 2 weeks prior to hemp placed on the diets containing either PCB or HCB and then challenged with SR IK* ft weeks later while still on their respective diets. The parameters measured were the same as previously described. Analysis of fICI) and Aroclor 1242. Measured samples of spleen, thymus, livvi. lung and serum were placed into separate glass vials. Two ml of acetonitrile undone ml of 2% sodium sulfate were added to each sample. The samples were allowed a* stand overnight, shaken and were extracted with one 5 ml and two 2 ml portions nl hexane. The hexane extracts were evaporated over a stream of nitrogen to a volume MONS 001U3 CHI:MK`AU.Y INDUCED HUMORAL IMMUNOSUPPRESSION 255 of I lo 2 ml and then passed through a microcolumn fitted with a glass wool plugand containing l,t> g of anhydrous sodium sulfate above 1.6 g of PR grade Florisil (100/120 mesh. Sigma Chemical Co.). The column was prewetted with 10 m) of hexane and the extracts were eluted from the column with 10 ml of hexane. The clunte was brought lo an appropriate volume with hexane for gas-liquid chromato graphic (Gt.O analysis. Scrum samples of'0.03 lo 0.10 ml were pipetted into glass extraction tubes and 2 ml of 2% sodium sulfate were added. The samples were extracted with three 2 ml aliquots of hexane and evaporated to an appropriate volume by drying over a stream of nitrogen. Known amounts of HCH or Aroclor 1242 were added to control tissue or serum samples and analyzed simultaneously with experimental samples and direct stan dards. A Hewlett-Packard gas chromatograph (Model 57I3A), equipped with a B:,Ni electron capture detector held at 300 Cand containing a 6' x Vi"(0.25 mm l.D.) glass column filled with 3%OV-l on Chromsorb Q. 100/120 mesh, was maintained ul an oven temperature of 2 IOC for analysis of Aroclor 1242. A carrier gas of 5% methane in argon was used at a flow rate of 45 m!/min. The GLC conditions for HC13 analysis required the use of the above glass column filled with 1.5% OV-17/1.95% QF-1 on Chromsorb W HP. 80/100 mesh, maintained at an oven temperature of 190 C. Two glilcvsofcaeh sample were used for the analysis of cither Aroclor 1242 or HCJ3. A regression line was calculated from peak urea of Aroclor 1242 x attenuation or peak height of HCH x attenuation vs. concentration of Aroclor 1242 or of HCD for extraction standards, and the resulting slopes and intercepts were used to deter mine the concentrations of the compounds in the tissue and serum samples. Peak urea for Aroclor 1242 was determined using a Sargent-Welchdual pen recorder and integrator. The percent extraction, determined by comparing the slopes of the regression lines for extracted and direct standards, was found to range between 90 * ami ItXW. liistopathnlouy -- General. Mice were ancsthcsizcd with ether and killed by txssmgumation. The peritoneal and thoracic cavities were opened and their con tents examined in .situ. The spleen, mesenteric lymph nodes, liver, thymus and lungs were removed in the stated order. The spleens were cut longitudinally and one blf was used for immunologic studies and the other half was fixed foY histology. Two representative samples of liver were fixed in 10% buffered formaldehyde. The lungs were injected with a small amount of formaldehyde lo expand the pulmonary parenchyma to its normal size; all other organs were fixed vn hloe. The tissue rumples were embedded in paraffin and cut at 6 gm. Hcmatoxylin-cosin was used as igenera) stain for all organs. A parallel set of lung sections was stained according to the method of Cason (2) for the demonstration of type II pncumocytcs. , Spleen. Facb hematoxylin-stained section was projected on a sheet of millimeter I paper at 40X magnification. The outline of the entire section and the areas of while pulp weiv drawn in. resulting in a topographical map (>r every spleen section. Ptonimetcr values expressed in mm* were obtained for the entire area of the section indforall follicles (which were individually numbered at the time). The total area of follicles was then expressed in percent of total area of the spleen section. The composition of the follicles which contained both small and large lymphocytes, or Urge lymphocytes alone, was determined by a point sampling method (I) at a 256 LOOSB I.T AL. magnification of 400X. Each follicle was represented by 100 test points and 10 follicles were chosen from each section, resulting in a total of 1.000 lest points for each spleen. The results were expressed as percent of total individual follicular volume. These 10 values were then averaged to give a percent volume figure for the entire section. Increases in number of pyknoscs and amounts of nuclein debus m splenic follicles were graded as: normal, minimal, slight, moderate and marked. Thymus und Mesenteric Lymph Nodes. Hematoxylimeosin stained sections were examined microscopically and the presence or absence of some morphologic details was notedwithoutfurther quantification. l.ivcr, Hematoxyiin-cosin stained sections were used to quantify microscopic changes, using thefollowing scores: normal,minimal, slight and moderate. Luntis. Hemaloxylm-eosin stained slides and parallel sections stained with C'ason's method (2) were used to enumerate microscopic changes, using the scores: norma), minute, slight (few) and moderate (some), the terms in parentheses being used to describe numbers, the terms outside the parentheses indicating degrees of change. Siulisncui HA'alualum. Data arc presented as the mean standard error with significance, as determined by the Student t test, denoted by the lettered superscript. RESULTS The dietary administration of 167 ppm of Aroclor 1242 or HCB to male Balb/e mice for 6 weeks resulted in a > 2x reduction in peak primary (T) splenic Ah PK` response to SRHC antigen (Ag) (Table 1). The peak PEC response occurred on day 5 following Ag injection in PCB, HCB and control mice, suggesting that there was no delay in response lime. Also, the total yield of mononuclear spleen cells in PCB or HCB treated mice was unaltered from control values (Table l), A concomitant reduction in scrum lgGi, IgA and IgM was demonstrated in PCB and IK'B treated mice throughout the measured response period. Although all the immunoglobulin concentrations measured were decreased, the most consistently and significantly reduced was IgA. Polychlorinated biphenyl and HCB treated mice had scrum IgA levels which were consistently 40 to 80 mg/di lower than control values throughout the I" response, Scrum IgA levels were more profoundly reduced in HCB treated mice than in those animals which received PCB 1242 (Table 2). Lung, thymus and spleen weights in the PCB and HCB treated mice were comparable to control values. A significant increase (111 to 160% of control values) in relative liver weight in mice receiving PCB or HCB was attributed to ecntrilohu lar and pericentral hcpatocytc hypertrophy (Table 3). Histopathologic examination of the lung, spleen, thymus, or mesenteric lymph nodes did not reveal any histologic alterations. Morphometric analysis of the spleens from PCB or HCB treated mice did not indicate any changes from control values in the number, size or cellular composition of the germinal follicles. Mice maintained on the diets containing 167 ppm of PCB 1242 or HCB and injected with a secondary challenge of SRBC 2 weeks following the primary immunization manifested a significant reduction in the secondary splenic Ab PFC response. Control mice elicited a maximal response of 133 15 PFC/10* spleen cells while PCB treated mice had 52 4 PFC/KE spleen cells and HCB treated mice had 79 15 PFC/IO" spleen cells. Since there was no difference in the yield of total j . j \ j ) i ( t j * I I : . j j ) HONS 001115 TABLE I KfTcct of I'C'H 1242 oi HC'H on Splenic Anti-SRBC Primary (!> Response in Day 4 Day 5 Day 6 [>,(> 7 l cjfcplecn XMP Control 2.9t 0.38 PCB 2.7# - 0.31 HCB 2.16 0.26 Control 3.14 i 0.75 PCB 2.60 0.21 HCB 2.77 0.4) Control 1.75 i 0.43 PCB 2.05 i 0.29 HCB 1.64 i 0.30 Control 2.36 i 0 23 PC 1 OK < 0 11 IKK 1 7S I'r PI-'(`/Spleen 7.435 1.159 5.787 * 520 6.538 t 706 65.980 9.812 26.6Sf 2MMT R.33# 4.612 3.021 2.214 4.3K4 958 4.476 984 1 LORI 1.278 2.74?- *. 14V 426 K17 I'l l /ID* Spleen cells 25 22 .30 233 99r IKT 55 21" 31 48 Dr *2 3 i2 33 1! It. 13 - .3 K S 2 4 The primary duect splenic PH' lo the SRBC Ag was evaluated in mice fed 167 ppm of PCD 1242 or HCH for 6 weeks. at which time all mice received a primary i v mlmmiMnritim of O.t vc of 10% SRDC. The mice were maintained on (heir respective diets during the response period. ''|)iitH ate expressed as ineiin standard error with significance levels indicated as h) p < 0.0A; c) p ; (I 02; dip < 0.01; ftp < 0 002; ftp * 0 OM-,n 7 m# in all groups. mons oom6 TABLE 2 Scuim Immunoglobulin Concentrations in Mice Fed PCH or HCH and Immunised (1) with SKUC* Day 4 Day 5 Day 6 Day 7 Control ITU HCH Control ITB HCH Control 111) HCH Control IVH IK H IgCn mg/dl 300 * 13 280 - 40 260 2 24 490 * 35 29<r 2 31 360" 2 38 510 2 63 190" i 16 340 2 12 540 * 30 400 69 \;u 7' IgA mg/dl 170 i 12 130 20 hkt ?3 190 13 I4(r i5 i i<r t2 340 2 41 14(T 280" 190 i 3 i 18 2 1 1 i in4* 4 IgM mg/dl 430 e 3.' 410 2. 72 390 i 15 410 i 27 360 i. 21 330 i 12 380 i 63 I2(f* 27 2W 2 14 370 i 18 Mtr - 17 ' Mallei male mice were fed Atoclor (ITH) 1242 or HCH in their diel at a concentration of 167 ppm for 6 weeks at which lime they raeivcd .i pnm.n v immuniwilum ti.v.) of 0.1 it of HXtf SKHC. Scrum immunoglobulins were determined on days 0,6 and 7 by KID. Data ate exptessed as mean 2 standaid error: b) p < 0.05; c) p< 0.02; d) p < 0.01; c) p < 0.002; f) p < 0.001; n = 6 to H in all gioups TABLH3 FfTcct of ITH and HCB on Body and Organ Weight* in Mice Fed fTB and Mr |> wi )< ,i . . HONS 001117 TAHI R 3 l-.ffvd of K'll and HCB on Body and Orpin Weights in Mice Fed PCD and HCB and Immunized tl") with SKBC*'6 Day 4 [Jay 5 Day 6 Day 7 Control IM'B MCI! Control PCB HCB Control f'CH HCB Coniiol K'H H( H Body Wi. (I) Kelaiivr I ivci Wi. % 32 1 0.35 5.K9 0 22 31.9 - 0 5| 601 * 0.09 28.9*' 30.5 * 0.71 . 0.69 6.76* 5.50 i 0.12 * 0.18 31.8 ' 0.99 30.9 29.3 - 0.46 0.72 6.10* 7.12* 5.45 0.18 t 0.22 2 0.21 72. y 0 .37 27 8 29 I - 0.85 7 0.68 6 32", 8.74a 5.37 i 0.21 0.83 i 0.45 31 5" '061 Mi (i (1 M 6 17 7 76r ' o.i5 :: Relative Spleen Wt. *7f 0.758 0.708 0.03 . 0.01 0.807 0.716 a 0.05 - 0.04 0.609 0.06 0.717 t 0.02 0.671 0.07 0.524 0 699 0.679 - 0.02 - 003 * 0.05 0A24 (1 <77 (I 01 - (MM Kcliilive Thymus Wi % 0.107 0.142'1 0.01 * 0.01 0 126 0.128 0.01 - 0.01 0.124 0.153 0.143 - 0.01 0.01 i O.Ot 0 110 .: 0.01 0112 0.155 0 01 * 0.01 0.119 (MIX 001 0 01 Relative I tint: Wt. '.f 0.522 0.532 0.02 - 0.03 0 496 0.562 - 0.01 1 0.03 0.506 0.514 0.710 i 0.01 + 0.01 - 0.02 0616 0.686 0635 rt 0.01 - 0.04 t 0.01 0,629 06'h 0 02 - 0 01 Aindoi 1242 and HCB weic fed in llic did at a concentration of 167 ppm for6 weeks, at which time all mice received a primary immunization li.v ) withO | it ot Mint SHIU .Oigan weights, histopathology. ehemien) analysis and measurements of immune parameters were conducted on day s4. 5.6 anti 7 following intimim/ation. ''Outa aie espicsscit as mean * Mandate! error with significance levels indicated as 6) p < 0.05; c) p 0.02, d) p < 0.01; et p 0.002; f) p - 0.001; n 5 i k in all poops. HONS ooms TAB! F. 4 l-fleci of Aroclor 1242 or HCU on Splenic Anii-SKRC Secondary (II") Response in Mice*'* Day 1 Control PCH Day 4 non Control pen Day 5 HCB Control l*CB Day 6 HCB Control PCB HCIt Crlls/Splern XI0` I'lC/SpImi 3.1' r- 0.31 40.737 - 4.120 3.09 ?. 0.16 2.94 i 0.27 3.01 t 0.39 2.52 l 0.20 3.40 2. 0.81 1.72 0.14 11,724' 22.034' 26,590 14.926 19.152 11.859 - 855 i 3.022 - 4.299 4.946 i 9.210 2 1.566 2.00 * 0.32 2.12 i 0.28 1.69 4 0.26 1.82 ? 0 34 1 60 - 0 09 3.175* 4 493 3,494* 6,710 7,478 5.047 4 659 - 1.147 ? 1.613 i.w; *ir vnr Spleen Cells 111 57r 79*' 91 52** 4T 70 17- 17* 45 44 10 ' IS ? 4 ? 15 10 i 13 1 10 5 9 i 2 4 3 .4 10 * 8 9 The sccnmlniy duett splenic PIC to the SKMC Ag whs evaluated 2 weeks after primary immunization. At this time all mice received it second challenge ol 0.1 cc of 10% NKIU*. The mice wete maintained on lheir diets of 167 ppm of PCB or HCB during the response period. ''Diilanic ckpiesscd as mean -> standard error with significance levels indicated as h) p < 0.05; c) p < 0.02; d) p < 0 111; c) p < 0.002; f) |> <> (KM. n 7 log in all groups. \ HONS oqiii-9 TABLE 5 Scium Immunoglobulin Concentrations in Mice bed PCI) and MCI) and Immunized (11) with SRUC* * Day 3 Day 4 Day 3 1 >;ti 6 Control 1*0) HCD Coutiol P('B HCB Control It'll IK'll Control it n lit It ie<: > mg/dl .120 36 300 .? 24 490 i 81 380 fel 460 * 34 310 ' 42 440 t 47 46(1 -- 82 S40 68 340 i 10 2ur 19 'll) 9H IgA tmg/illl 140 140 110 160 190 130 190 17(1 I40b 180 170 i or *7 3 t 3 11 i 19 l 13 i 20 -- 13 5 3 14 It 6 IgM Img/ill) 330 n 340 i. 23 300 rt 23 380 - 17 370 i 18 4'0 s 43 400 t 33 370 i 26 30(P 20 330 *. 14 HO too * 28 33 *Sonim immunoglobulin concentrations wcie determined by KIP in mice challenged with SRRC 2 weeks following then lv immunization Ml mice u eie nuimtnmrd on thcii diets containing 167 ppm of PCM or lid) during the secondary response period. 'Paluaioccpirssed ns mean *. stand** d citoi with levels of significance indicated as: b)p < 0.03; c)p < 0.02;d) p < 0.01;c)p " 0.002,ftp 0 0(11; n 7 In H in all pimps. M0NS 001120 TABLE 6 Effect of PCH and HCB on Rody nm< Organ Weighs in Mice during the Secondary Response to SRUC, h Day 3 Day 4 Day 5 nit> {, Body Wt. <g> Relative I.ivci Wt. % Kel.dtve Spleen Wt. 4 Control 29.9 : 0.52 5.41 : 0.28 0.724 * 0.06 pen HCB Control 30.8 * 0.4# 28.5 29.5 2 0.84 t 0.55 6. MK ? 0.22 861' 5.54 l 0.22 i 0.64 0.638 0.70V 0.536 7 0.02 ? 0.03 0.06 pen HCB Control 31.2 i 0.71 28.6 30.I - 0.86 i 0 56 6.80' i 0.17 10.42' .4.3.3 2 0.23 i 0.30 0.692 0.754** 0.569 i. 0.05 x 0 0.4 * 0 03 K'H HCH Control 32.0" i 061 .30.9 29.8 ? 0.12 0.33 7.CM' t 0.15 9.4 3' 5 35 ?. 0.22 ? OKI 0.583 0,69| ' 0.644 i. 0.04 a 0 03 a <UH 1*1 U l II 30 4 ?6 7" ' 0.76 on? 6 6V 8 S81 U 12 114' 0 377 0 MS 005 0IH Keliitivc riiymus Wt. u Keliil.ve l ung Wi. ; 0.110 0.01 0.6% ? 0.04 0.112 0.105 0.086 0.01 * 0.01 *. 0.01 0.7W 0 76# 0.59.3 ? 0.05 7 p.03 0.01 0086 0.088 0 085 ? 0.01 0.01 1 0.01 0.576 0.679 0.662 0.01 a 0.04 i 0 06 0.t>90 0 076 i 0.01 ?. 0.01 0.082 t*m 0.604 0650 0 636 * 0.02 - 0.08 ? 0 03 0.090 o w: 0.01 0 III 0 6S l 0 674 ' 0 04 0(U AukIim 1242 ami IK'lt were fed in the did at a concentration oft 67 ppm for 6 weeks, at which time all mice received j pmnaty immunization of 0 K i ni UV'i SKUCTwo weeks later all mice received a secondary challenge of0.1 cc oft WA SR1H'. Body and organ weights, hisiopathology. chemical analym s amt mciistticments of immune pmameters were determined on days 3.4.5 and 6 following 11 challenge. "Diifaair expressed ns mean r* standard error with significance levels indicated as: hi p < 0.05;e| p < 0.02.d)p * 0.01. r) p 0.002:1) p 0 001. ii s t,,x in all gioups. HONS 001121 TABLE 7 KITcct of PCB or HCH on the Secondary (IP) Splenic Anti-SRBC Response ii Mice Pnmrd wiili .SKC Prior to Diclary Administration of ihc Chemicals* '' Day 4 Day 6 Day K Day II Cnitliol PC U HCB Control PCB HCB Control PCB HCB Control It'll 1*1 M < Ylls/Splern xio* 2.1H * 0. IK 2.7.1 - 0.16 2.8ft 0.1' 1.7' 2 0.1.' 1.62 t 0.11 2.21 i 0.28 I .'4 - 0.1.' 1.4ft - 0.16 1.58 i 0.14 1 68 0 11 2 14 2 (HI O.ftH o .'4 PH/Spleen PIC/KP Spleen Cells 4.06' * .'7' 17 -4 2.910 - M2 12 ?1 I.72K' 11' ft" 1 '.KHft 6K2 16 -' 1.718* 1% Iff it 2.108* 6.418 4.'9 .1 1.179 12* i. 1 41 i ft 2.461" i 710 2.uv t 480 IK" IV s *2 9/2 . 11' 1 2.092 * I.4K0 ft 2 , ,,, ,, '2 *Mice woic imnmniml will) 0 I cc ol MYA SKHC 2 weeks prior to being placed on diets containing 167 ppm of Ml oi HCH. After 6 weeks on the ilu-i.o y icjlinirn, the mnr lereivcd h sccondaiy challenge of SRBC. Responses were evaluated on days 4. 6. 8 and 11. ''Dalnaierspiessedas mean slandaid enor with levels ofsignificance indicated as: blp < 0.05. Op 0.02:d)p < 0.01: c) p 0.002. Op * 0 (tot, it ' to 8 in all gtoii|>s. MONS 001122 table s Srrum Immunoglobulin Concentrations During Secondary Response to SRBC in Mice IVimrd with SRBC Prior to Being Placed on the Dietary Regimen Containing PCH or HCB*" Day 4 Day 6 Day K Day 11 Control pen HCB Control PCB HCB Control PCH HCB Control W It HCH lg(i< (mg/dl) IgA (mg'dl) 380 a 20 140 ft. 7 360 ?. 15 noa2 450 a 30 460 ft 14 1 Iff 150 +3 - 5 460 ft 45 I3CT ft 8 490 a 90 480 ft. 31 IMF i4 130 i6 420 ft 31 110" a4 470 ft 61 480 ft 35 1 Iff' ft 2 130 ft 7 410 ? 41 130 *4 600 7S Po 4 IgM (mg/dl) 4(K) ft 19 34(1" ft. 7 350" a7 300 i 13 290 i 10 290 ft 13 390 l 37 300" ft 13 290" ft 25 320 ft IV 280 - 24 1?0 14 Mice wcie imtmmircd with 0.1 cc of 10% SRIK' 2 weeks prior to being placed on diets containing l(>7 ppm of PC'Bor UCH. Aftci 6 weeks on tin- ilut.uv regimen, the mice tcccivcd a secondary challenge of SRIK". Responses were evaluated on days 4. 6. 8 and II. "Dataaier spicssed as mean ft standaid enoi with levels of significance indicated as: hip < 0.05; c)p < 0.02; d) p <. 0 01; c) p . 0.002. f) p 0 (Mil. u s 8 in all gitnips oou23 HONS TABLE 9 KfTccl of PC1J 1342 or HCH on Body and Organ Weights During the Secondary Response in Mice Primed with SKHC Prior to Dietary Administration of {*011 or HCH* b Day 4 Day 6 Day 8 Day I \ Hotly Wt. (Hi Relative 1 ivet Wt. % Control 51.9 a 0.3 3 6. OH 0.37 PCI* 30.0" i 0 73 HU* Control 30.4 29.8 i 0.64 * 071 6 27 - 0. It* 7.46' 0.21 5.54 0.12 PCU HCH Control 30.1 A 1.00 29.1 30.0 i 1.44 - 0.73 6. .31" 7.43' 5.19 a 0.27 0.24 *0.17 PCB HCH Coni ml 30.3 0.57 29 0 29 - 0.87 1 0.68 6.14' a 0.12 7.58" 4.93 - 0.77 * 0.05 KH 31 4 083 6 1P * 0 24 IK H 30 1 0 os 7 24' o t: Relative Spleen Wt. '/> Kelmive Thymus Wt. W- 0.559 * 0 03 0.1 87 0.05 0.750" 0 595 0.578 0.04 i 0.03 t 0.04 0.1100.154 0.129 < 0 03 A 0.01 * 0.01 0.453 0.680 0.560 i 0.02 1 0.04 0.04 0.136 0.149h 0.125 A 0.01 A 0.01 * 0.01 0.500 0.SS9 (MM A 0.04 0.02 * 0.01 0.137 0.137 0 141 . 0.01 - 0.01 A 0.01 0 461 - (MM 0 123 - (Mil (1 S'|| o o: 0 120 (1 01 Ketativc Lung Wt. W 0. 471 003 0.526 0.505 0.646 0.05 - 0.01 i 0.03 0.695 0.702 0.656 1 0.07 0.06 i 0.06 0.649 0 609 0 768 a 0.05 0.03 * 0.02 0.02(MMK- o Kin 0 (8. Mice wcio timmmt/.cd witUU.I ccof lGW SRUC 2 weeks prior to being placed on diets containing 167 ppmofPCll ot HCH. Aftci 0 weeks on the diet.nv regimen, (lie mice icccived a secondary challenge of SKBC, Responses were evaluated on days 4, 6. 8 and 11. 'Data are expressed as mcah a standard error with levels ofsignificance indicated as b) p < 0.05; c)p 0.02; dip *. 0.01 ;o)p 0.002; Dp 0 001.n s i. K in all groups. M0NS 001124 TABU- 10 I'.lcilliKi riipUiK- (ins ( hiomalopiaphie Analysis of Arnclor 1242 and HCH m Tissues of Mice Ped Diets Comaimn* If? Pfm of WH or M( It-1 ...................... "................ Tissue Conccnlration (jig/g) Set inn Livi Limp Spleen '1 Innim Day Wit HCH pen HCB Jt'H HCB PCB HCB pen Hi It 0 84 12 0 2.8 56 7.0 269 2.2 47 7 1 15? - 0 03 20 0.35 9.2 1.9 36 2 0.33 2 2.8 * i i 36 S 1.0 5U 4.4 80 5.8 243 3 0 '2 4 5 1811 - 0 1? s 3-0 * 0.53 i 7.5 0.90 i' 25. 3 0.38 l 6.2 0'*0 ?' 1.0 * 0.17 12.0 3.4 87 6.4 214' 1.4 0.21 2 7.4 2 0.68 1 14 .3 6 - 0.11 5) 3.6 7l 047 171 * 79 0.95 - O K. 9.6 2 0.8 4.3 0.43 75 * .5.2 7.6 3 1.6 157 - 15 4.4 0.58 52 2 4.3 V9 10 IM - K7 (ilOUp ? II" 1 s 7 0.63 2 0.13 0.51 2 0.04 0.57 0.01 0.46 - 0.02 0.70 2 0.11 M ? 0. 35 15 2 1.6 11 - 0.64 14 2. 1.4 -- 6.9 3 0.59 52 : 0.75 6.0 2 0.48 7.8 i 0.82 4.0 2. 0.65 99 3 12 m 2 9.1 94 2 9.0 99 4.7 -- 5.4 i 0.53 12 2.3 6.5 2 0.58 7.1 i 1.2 5.7 2.8 265 21 320 72 217 2. 54 208 2. 37 - 2.8 2 074 17 0. 30 1.5 3 0.22 2.6 * 0.40 3.0 ? 0.53 * 54 i 7.5 60 3 6.0 69 ? 4.6 77 -. 8.5 - 6? - 1.4 90 * 14 10 * 11 91 i: 79 * 03(1 14' 224 54 7: ' 15 l?6 - Ml Day 4 0.86 8.8 2.6 60 9.1 247 3 0 41 i on 0.73 * 0.23 ?. 3.2 i 3.2 2 44 0 50 2 80 0 84 1.3 j 0.14 9.7 i 0.20 3.2 2. 0.38 67 2 5.4 9.6 1.4 183 2 16 36 2 39 50 2 4.8 3 7 1 17 0 3S 11 K 1.5 n 4.0 63 7.8 198 3.3 67 7 t ?44 * o.:? 2 0.84 i 0.45 8.7 2. 2.3 2 30 i 0.24 5.5 2 1 0 ?n 0.80 s 0.30 11 2 0.41 2.9 2 0.32 82 7.7 8.7 1.2 250 2 42 3.0 2. 0.79 66 2. 6.9 H4 * l2 I'M) 3? ,, ... , ....... 1 IMVnl in nna i y immimiM.iim it") of SKHC fiUinwmf fc weeks dietary administration of If7 ppm >l Wit i IK It ............? M" i< Ht.S.tl i .> SHIM OOll-*5 HONS I I HI'.MICM l Y INDUCED MUMORM. IMMUNOSUPPRUSSJON 267 xplccn cells obtained from control versus PCB or HCB treated animals and since KMhthe control group and experimental groups had the greatest PFC response on day 3 following II" challenge, either an alteration in the splenic Ab producing cell population and/or an impaired cell function may be suggested (Table 4). No consistent significant alterations, as compared to control values, in serum IgGi or IgM concentrations in the PCB or HCB treated mice, were demonstrated during the 11" response to SRBC. However, a significant decrease (72 to 79% of control values) in IgA was measured in HCB treated mice on days 3. 5 and 6 during the secondary response period; day 4 IgA concentration was decreased but was not Miilislically significant. IgA levels in PCB treated mice were comparable to controls (Table 5), Hepatomegaly, attributed to a ccntrilobular and pericentral hcpatocyte hyper trophy, wjts a consistent finding in the PCB and HCB treated mice during the II" response period. Control liver weights were unaltered. Significant splenomegaly (21 to 41% of control values) was observed in HCB treated mice on days 4 and 5 following the 11" SRBC administration. However, this change was not reflected in the splenic germinal follicle number, size or cellular composition. Thymus and lung eights and histology were unaltered from control tissue (Table 6). To ascertain the influence of the environmental chemicals on memory cell func tion, one group of mice was immunized (1) with SRBC 2 weeks prior to being placed on the diets containing PCB 1242 or HCB. Six weeks following dietary administra tion of the chemicals, a II" challenge of SRBC was administered. Control animals manifested a direct splenic Ab response of 41 6 PFC/10* spleen cells, whereas FCTltrcatcd mice had only I8 5 PFC/IO spleen cellsand HCB animals had I3 2 PI:(7I0K cells. Maximal response in all groups was achieved on day 8 following the 11 immunization. The yield of spleen cells in the environmental chemical-treated mice was unaltered from control values (Table 7). A consistent and significant decrease of 20 to 40 mg/d! from control IgA concentration was demonstrated on days4. band 8 in mice receiving PCB or HCB. IgA values returned to normal by day 10. IgCn and IgM levels in the PCB and HCB treated animals were unaltered from control levels (Table K). A significant increase in liver weight in PCB and HCB treated mice (117 to 149% of control values, respectively) was determined to be a result of hypertrophy of ccntrilobular ami pericentral hcpatocyles. Thymus, liver, lung and spleen weights ind hislopathology were unaltered from control tissues (Table 9). l-lcction-capture gas chromatographic analysis of PCB 1242 and HCB in scrum and tissues revealed: 1) an approximate steady state condition occurred by the 6 week period. i.e., no significant increase in tissue chemical levels occurred between 6nd 8.5 weeks in lung, thymus, spleen or scrum; however, liver content of PCB and HCB did increased) liver, spleen and thymus manifested an approximate 20:1 concentration of JK'B/PCB while the lung HCB/PCB ratio was *"38:1; and 3) maximal tissue uptake of PCB occurred equally in the thymus and lung while the principle tissue site of HCB localization was the lung, with the second largest deposition being present in the thymus (Table 10). DISCUSSION Clinically suhloxic levels ofPOH 1242 or HCB were profoundly immunosupprcs\ivc when fed to mice for A weeks prior to and during the Ag challenge (Tables 1 and oou*6 HONS B*S! 26K LOOSE UT AL. 4). Noth the 1 and II" splenic Ph'C responses to SRBC were impaired in the environmental chcmical-trcatcd mice. Animals immunized 2 weeks prior to being placed on the diets containing either the PCB or HCB and challenged with a IP SRBC injection 6 weeks after the dietary administration of the chemicals man ifested a significantly depressed secondary response, suggesting cithei that Ah memory cell function was impaired or that there was an alteration in cellular cooperation (Table 7). Peak Ab formation occurred on the same day in experimental animals as in control mice, indicating that there was no delay in Ab formation. Also. since the total number of mononuclear spleen cells obtained from the PCB or HCB treated mice was essentially the same as the number obtained from controls, the decreased PKC response in the treated mice may be dm* to an impaired cell function and/or an alteration in the composition of the spleen eel) population, i.e,. a change in the macrophage. T and B lymphocyte ratios. A PCB induced immunosuppression was also noted by Roller and Thigpen (18). who demonstrated a significant 50 to 59% reduction in neutralizing Ab titers to pscudorabies vims in rabbits adminislered 300 mg of either PCB 1221. 1242, or 1254 once a week for 14 weeks; PCB 1242 treated rabbits had the lowest Ab titers. Vos and van Dricl-Grooienhuis (27) previously observed a decrease in cellular immunity and humoral Ah formation in guinea pigs exposed to a 60% solution of PCB 1260. Street and Sharma (23) did not delect any change in hemolysin oi hemagglutinin titers in rabbits fed 3.7 to 170 ppm of PCB 1254 for 4 weeks. However, since Ab titers were measured only at 10 and 17 days post*)" injection, it is possible a change in peak titers could have been missed. An interaction between organochlorines and infectious diseases was determined by Priend and Trainer (8), who demonstrated a significant (p < 0.01) increase, as compared to control values, in mortality of ducklings fed 25. 50 or 100 ppm of Aroclor 1254 for 10 days prior to i.p. inoculation of duck hepatitis virus. A 2 to 4.V increase in mortality rates over control (duck hepatitis virus alone) mortality was observed in the PCB plus virus group. There was no significant difference in mortality among ducklings receiving the different concentrations of PCB 1254 plus virus. A .3 u>OX increase in mortality rates, ovcrcontrol (duck hepatitis virus alone) values, was manifested in ducklings receiving p.p'-DDT or dicldrin and inoculated with duck hepatitis virus. The suppressed host resistance to the duck hepatitis virus may be associated with the suppressed immune response demonstrated in the present study and also observed by Vos and van l>ricl-Grootenhiiis (27) and by Roller and Thigpen (18). The reduction in primary direct splenic PI*C formation, a measurement primarily of IgM Ab producing cells (3, 20), was associated with a significant decrease in scrum IgM on days 6 and 7 following immunization in PCB and HCB treated mice. IgM concentration was reduced from control values by approximately 23% in HCU treated mice, whereas the IgM levels in PCB treated mice were reduced 69% and I(>% on days 6 and 7. respectively. Scrum IgCii was significantly reduced. 41% and 63%. from control values on days 5 and 6, respectively, in PCB treated mice; a slight, but significant decrease of 27% was observed on day 5 in the HCB treated animals. The most consistent alteration in immunoglobulins was a decrease in serum IgA in the HCB treated animals during both the l" and U" immunization periods. A mean decrease of 43% below control concentrations was measured on days 4 to 7 in the HCB-treated animals during the 1" response and a mean decrease of approximately 23% during both 11" responses, i.e.. in animals which received ihi- 5 { I ' , , 1 j ) * { 1 I t j j I , * ; I I * l HOI** CHI.MJCAl l.Y INDUCED HUMORAL IMMUNOSUPPRESSION 269 I* prior lo being placed on the diets and in animals which received the 1 while on thetr respective diets. Polychlorinated biphenyl-treated mice also manifested a lignificanl decrease in scnmi IgA of approximately 35% during the 1 response and a llight, but significant, decrease of 16% below control values, in animals primed prior to dietary administration of (he PCI* (Tables 2.5 and 8). It is significant to note that Shigematsucr/. (22) reported that IgG in the sera of 203 victims of the rice oil poisoning incident (Yusho disease) was increased by 23.8%. whereas IgM was decreased by 18.6% and IgA by 28.5%. Intensive histopathologic examination of the lung, thymus, mesenteric lymph nodes and spleen did not reveal any (issue alterations different from controls. However, a significant and consistent hepatomegaly, attributed to a eendilobular and pericentral hcpatocyic hypertrophy, was observed. This type of hepatic re sponse inconsistent with that seen following exposure toorganochlorines. Detailed morphometric analysis of the spleens from PCB or HCB treated mice throughout the experimental period did not indicate any alteration from control values in the germinal follicle numbers, size or cellular composition. In guinea pigs fed 10 or 50 ppm of PCB 1260 for 8 weeks Vos and De Roij (26) also noted an absence of any histomorphologie changes in the spleen, thymus, cervical and mesenteric lymph nodes. Similarly. Kollcr and Thigpen (18) did not detect any pathologic changes in the spleen, thymus or lymph nodes in rabbits fed 300 mg of PCB 1221, PCB 1242 or PCIl 1254 by gavage once a week lor 14 weeks. However, in rabbitsfed PCB 1254 in their diet at doses ranging from 3.7 to 170 ppm for 4 weeks. Street and Sharma (23) suggested that alterations in lymphoid organ histology were the most sensitive indicators of immunosuppression, i.c.. treatment with PCB 1254 resulted in a decreased count of plasma cells in the popliteal lymph nodes, reduction of splenic germinal centers and cortical atrophy of the thymus. Tissue localization of the PCB 1242 and HCB in the liver, lung, spleen and thymus is of 2*fold importance. First, the Jiver. lung and spleen contain the principle populations of fixed tissue macrophages which have been suggested to act as initiators of the immune response (6, 7. 25) and to be essential in thymocytic mitogcncsis (19. 2 J); second, (he thymus is a primary lymphoid organ in addition to SRIK' being a thymic or T dependent Ag. It is interesting to speculate that the approximate 20:1 ratio in tissue concentration of HCB and PCB is correlative with flic* increased immunosuppressive effects of HCB as compared to PCB. The ques tion remains that, in the absence of histopathologic changes, excepting the heputocytc hypertrophy, and with the demonstrated immunosuppression,, is the tissue concentration of PCB and HCB in lymphoid and reticuloendothelial organs induc ing a functional but not morphologic alteration and is the functional change a more sensitive indicator of toxicity than a pathologic alteration? ACKNOWLEDGMENTS Supported by National Institute of Environmental Health Sciences Research Gram No. 2POI1-.S00226-10ami hy a ininl program between the Gesellschnn ftir Strahlcn und Umweltforschurtg mhH. Munich, Germany and (lie Insliuile of Comparative and Human Toxicology, Albany Medical College. Albany, New York. HONS 00U-2S 270 I.OOSfi f:T At.. ) \ ki:ki;ki:n ci:s 27. V,... nu\li 1. BcniU. K -P. and Dumhach. G. Quantification ofdrug-induced morphologic change* with Hcnnig'l 2H Vos. poml uml line-sampling methods using Zeiss integrating eyepieces I and ll.Orun Rex. 14, 92V, (V64 i/i, 1 2. Cason, J. H. A rapid one-step Mallory-Hcidcnhain slain for connective lixsuc. Slain Tn hnol. 25. 29 Vos 225. WSO. 'sill. I .V Dicsser. 1). W. arul Wort is. H. H. Use of amiglohtilm set um to deled celts producing antibody with low hemolytic eflictency. Nature 208. 85V. 1965. Ml W.,. 4. ( iccgovich, C. P, Relationship of pesticides to immune responses, /tv/, t'nu 32. 2010. 197.7 V Fairchild. Ci. A.. 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