1 15 2 https://www.nal.usda.gov/exhibits/speccoll/files/original/d5ceb7a3bd6c83844d6909cc862117d5.pdf a526bd953e096b5ff17252ddc385d02f PDF Text Text Item D Number °2414 Author Vecchi, A. Corporate Author Report/Article TldO Typescript: Effect of Acute Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on Humoral Antibody Production in Mice Journal/Book Title Year 000 ° Month/Day Color Number of Images D 14 Descripton Notes Friday, October 05, 2001 Page 2414 of 2422 �ALVIN L. YOUNG, Mc.]ir, USAF Consultant, Environs; uai Sciences EFFECT OF ACUTE EXPOSURE TO 2,3.7,8-TETRA-CHLORODIBENZO-p-DIOXIN (TCDD) ON HUMORAL ANTIBODY PRODUCTION IN. MICE* A. VECCHI, A. MANTOVANI, M. SIRONI, W. LUINI, M. CAIRO and S. GARATTINI Istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea, 62 - 20157 MILAN, Italy. Supported by Regione Lombardia, Milan, Italy. Special Research Program on TCDD. Proofs should be sent to : Dr. Annunciata VECCHI Istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea, 62 - 20157 MILAN, Italy. �1 S U M M A R Y The effect of single dose of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDDj 1.2, 6 or 30 pg/kg i.p.) on primary humoral antibody production was studied in young adult C57B1/6J mice. TCDD profoundly suppressed the primary response to thymus-dependent (sheep erythrocytes) and independent (Type III pneumococcal polysaccharide) antigens. The inhibitory effect of TCDD was still detectable 42 days after treatment. In contrast, under these experimental conditions, in vitro lymphoproliferative responses to Concanavallin A and bacterial lypopolysaccharides and the ability to mediate graft versus host reaction were not significant affected per unit number of lymphoid cells. �2 - I N T R O D U C T I O N 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an extremely toxic compound found as a contaminant of some chlorinated phenols used as herbicides. Previous work (1) on the lymphoid system of rats, mice, and guinea pigs revealed that TCDD causes profound atrophy of the thymus and thymusdependent areas of lymphoid organs. Suppression of cell-mediated immunity and increased susceptibility to bacterial infections have also been described (1,2,3). The most dramatic immunosuppressive effects were seen after animals were exposed to TCDD in utero , or in young adults animals after chronic treatment. Although the effect of TCDD on humoral antibody production has not been adequately tested, it has been generally assumed that the chemical does not affect humoral responses (1,4,5). The present study, prompted by the pollution with TCDD of Seveso,a densely populated area near Milan (6,7) was made to evaluate the effects of single doses of TCDD on primary humoral antibody production against T-dependent and T-independent antigens in young adult mice, and on cellmediated reactivities. �3 - MATERIALS AND METHODS Mice - C57B1/6 and (C57B1/6 X DBA/2)F1 male mice obtained from Charles River, Calco, Italy, were used at 6-8 weeks of age. TCDD - TCDD (1.2 ,6 and 30 pg/kg) obtained from Kor Isotopes, Cambridge, Mass, was given as a single i.p. injection in a volume of 0.25 ml of acetone corn-oil (1:6 v:v). Control mice received the vehicle alone. The TCDD doses used were well toletared by the animals (5,8,9). G.V.H. assay - Graft versus host (G.V.H.) reaction was assayed by the popliteal lymph node weight gain assay (10). 5 X 10 splenocytes from C57B1/6 treated mice were injected s.c. into the right hind foot-pad of (C57B1/6 X DBA/2)F, mice. An equal number of formol inactivated cells was injected controlaterally. Seven days later both popliteal lymph nodes were removed and weighed. The ratio of the weight of the right to the left lymphnode (lymphnodal index L.I.) was taken as a measure of GVH reaction. Four mice per group were tested individually, injecting the splenocytes in 4 recipients mice per donor animal. Mitogen stimulation - The splenocyte response to Concanavallin A (Con A) (Calbiochem., San Diego, California, USA) and E. coli lipopolysaccharide B (IPS, Difco Laboratories, Detroit, Mich., USA) was evaluated essentially 5 by the method described by Kirchner (11). Briefly, 5-10 splenocytes were cultured in triplicate in RPMI 1640-10% FCS (Gibco, Bio Cult, Glasgow, Scotland) with graded mitogen doses in 0.2 ml microtiter plates (No. 76-013-05 Linbro Scientific, Hamden, Conn. USA) and incubated at 37°C in a humidified 3 atmosphere with 5% COp. Forty-eight hours later 1 uCi H-thymidine (5Ci/mmole, Amersham Radiochemical Center, Amersham, England) was added to each sample and cultures were incubated for a further 16 h. Samples were collected on a filter with an automatic harvester (Titertek, Skatron, Norway) and radioactivity was counted in a liquid scintillation spectrometer. Four mice per experimental group were inidividually tested. �4- Response to sheep erythrocytes and type III pneumococcal polysaccharide A Mice (6 per experimental group) were injected i.p. with 4 x 10 sheep erythrocytes (SRBC) 7, 14, 21 or 42 days after TCDD treatment and spleen hemolytic plaque forming cells (PFC) were counted by the technique of Jerne and Nordin (12) 5 days later. For type III pneumococcal polysaccharide (SIII, a gift from Dr. P.O. Baker, N.I.A.I.D., Bethesda, Md), the optimally immunizing dose of 0.5 pg was injected i.p. 18 days after TCDD to 7 mice per experimental group and specific anti-SIII PFC were measured 5 days later as described elsewhere (13). Statistical analysis - Results are presented as mean +_ S.E. of 4-7 mice per experimental group. Statistical significance was evaluated by Dunnett's test. R E S U L T S Table 1 shows the effect of TCDD on primary humoral antibody production assessed by injecting the antigen (SRBC) 7 to 42 days after treatment. Exposure to TCDD resulted in a marked, dose-dependent reduction of the PFC count both per unit number of lymphoid cells (PFC/10 cells) and per organ (PFC/spleen). Impairment of the primary humoral response to the thymusdependent antigen SRBC was seen throughout the 42 days observation period, although by the end partial recovery was starting. In view of the reported selective toxicity of TCDD for the thymus and thymus-dependent areas of lymphoid organs (1,5), it was of interest to investigate the effect of this chemical on the response to the thymusindependent antigen SIII. As shown in Table 2, 18 days after treatment the humoral response to SIII was reduced to approximately 50% of the control value. �5- The effect of TCDD on cell-mediated reactivity has been extensively investigated, but some what different experimental conditions, including schedule of treatment and animal species, have been employed in previous studies (1,2,3,8). Therefore in a series of experiments GVH reactivity and the lymphoproliferative responses to ConA and IPS were measured at times (day 7-14) when humoral antibody production was greatly reduced. As illustrated in Tables 3, 4 and 5, no significant impairment of these cellmediated reactions was observed on a per cell basis. However, as previously repeatedly demonstrated (1,2,5), significantly reduced (30-50%) numbers of spleen cells were recovered from mice given 6 or 30 jug/kg TCDD. Therefore the total cell-mediated reactivity per organ was presumably decreased by this chemical. D I S C U S S I O N The results presented here show that single doses of TCDD markedly suppress primary humoral antibody production against thymus-dependent (SRBC) and independent (SIII) antigens in 6-8 week-old mice. In contrast, under these experimental conditions, the in vitro blastogenic responses to ConA and IPS and the GVH reactivity were not significantly affected on a per cell basis, although the smaller number of spleen cells recovered from mice given TCDD (2,5) presumably results in impairment of the total capacity per organ to mediate these reactions. Suppression of the PFC response to SRBC induced by TCDD was long lasting, a dose of 6 ^ig/kg still inhibiting the response by more than 75 percent 42 days after treatment. The effect of TCDD on humoral antibody production has not been adequately investigated (1,8), but it has been frequently assumed that this chemical spares humoral responses (5,14), although Vos et al. (8) reported a significant decrease injifp, and U-globulin in mice given non-toxic doses of TCDD. �6- It has been recenlty shown that in utero and neonatal exposure of rats to TCDD selectively suppresses the response to T cell mitogens without affecting antibody levels (4,14). The different animal species (mice versus rats) or the different time of exposure in relation to the ontogenesis of the immune system might at least partially account for the apparent discrepancy between previous data and the results presented here . In this context it is noteworthy that the effect of repeated doses of TCDD on G.V.H. reaction in adult mice (1) was not confirmed by the same authors (2) in subsequent studies and that non-toxic doses of TCDD had no effect on PHA splenocyte responsiveness. In view of the repeated selectivity of TCDD for the thymus and for thymus-dependent areas of lymphoid organs the marked inhibition we observed of the response to the thymus-independent antigen SIII is somewhat surprising. The hypocellularity of the bone marrow of TCDD-treated mice (5) might represent a cellular basis for the impairment of thymus-independent humoral responses reported here. Mice exposed to TCDD show reduced resistance to bacterial infections (3). Inhibition of humoral antibody production might contribute to the TCDDinduced impairment of host defense mechanisms against infections. ACKNOWLEDGEMENTS We wish to thank Mr. R. Motta and Mr. L. Vaghi for their helpful technical assistance in the animal house. gb/ �7 - TABLE 1 EFFECT OF TCDD ON PRIMARY HUMORAL RESPONSE TO SRBC Day after treatment TCDD (fig/kg ) PFC/106 splenocytes - 240) - 114)+* PFC/spleen 204 94 29 11 (173 ( 77 ( 21 ( 9 - 41)** 12)** ' 6 30 266 154 79 30 (236 (114 ( 52 ( 19 - 306) 208) * 121) * 48)** 33,871 19,600 9,064 3,381 21 ^ 0 ' 6 30 509 202 119 58 (452 (173 ( 96 ( 40 - 572) 237)** 147)** 85)** 88,583 (82,041 - 95,647) 30,609 (26,046 - 35,971)** 42 0 1.2 6 30 1,028(938 -1,127) 458(367 - 572)* 152(115 - 201)** 128( 98 - 167)** 83,086 (73,215 - 94,287) 51,844 (45,153 - 59,562) 16,795 (11,865 - 23,774)** 12,226 (10,247 - 14,586)** 7 1 0 ' 6 1 2 30 0 14 14 Results + P<0.05 ** P<0.01 ] 2 ] 2 27,334 (24,106 - 30,990) 10,514 ( 8,698 - 12,708)** 3,723 ( 2,666 - 5,199)** 915 ( 765 - 1,095)** (30,844 (13,869 ( 5,967 ( 2,065 - 37,194) 27,700)* 13,768)** 5,536)**- 14,230 (11,476 - 17,643)** 5,890 ( 4,274 - 8,117)** are the meansjH (1: S.E. )after logarithmic transformation of the data �8TABLE 2 EFFECT OF TCDD ON PRIMARY RESPONSE TO SI 11 Day after TCDD treatment PFC/106 splenocytes (jug/kg ) 18 0 127 PFC/spleen 19,561 (108 - 150) 1.2 30 (15,746 - 24,300) 60* ( 49 - 74) 11,339 ( 9,441 - 13,619) 69* ( 58 - 83) 8,159* ( 6,751 - 10,130) Results are the means +_ (1 S.E. ) after logarithmic transformation of the data. * p<0.05 �9 TABLE 3 EFFECT OF TCDD ON THE MITOGENIC RESPONSE TO ConA Day after jrnn (ug/kg ) Con A fyg/sample) ,, 0.1 0.8 1.6 3.2 0 2,304+ (1,209) 9,209 (3,271) 59,400 (15,267) 28,593 ( 7,465) 3,404 (2,445) 1.2 1,142 (587) 8,222 (1,318) 52,654 (18,434) 23,549 ( 9,280) 4,404 (1,663) 6 5,148 (2,478) 10,416 (2,875) 72,484 (26,386) 76,140 (31,561) 10,477 (7,866) 30 4,991 ( 166) 3,721 ( 671) 92,710 (15,319) 121,340* (15,020) 44,589* (14,087) 0 5,929 (981) 19,619 (1,789) 331,256 (58,585) 306,293 (62,486) 68,693 (21,406) 1.2 4,430 (1,340) 18,624 (7,072) 258,133 (60,329) 249,430 (67,545) 91,642 (33,658) 6 9,012 (1,582) 29,054 (10,111) 295,477 (55,390) 291,792 (47,099) 122,720 (30,622) 30 5,129 (613) 10,415 ( 2,771) 309,645 (61,953) 368,125 (52,192) 103,837 (27,788) 7 14 CPM = counts per minute (+ 1 S.E.) p<0.05 �10 - TABLE 4 EFFECT OF TCDD ON THE MITOGENIC RESPONSE TO LPS Day after treatment TCDD (P9/kg) r LPS (^ig/sample) o 0.05 5 120 0 10,620+ (1,428) 15,185 (2,104) 43,069 (2,542) 1,478 (283) 30 12,380 (3,451) 13,706 (3,085) 36,230 (10,231) 3,189 (833) 0 6,287 (1,396) 15,853 (3,270) 21,077 ( 5,315) 1,738 (281) 30 10,541 (3,060) 20,736 (4,078) 23,861 ( 2,987) 1,693 (341) 7 14 i CPM = counts per minute (+ 1 S.E.) �11 TABLE 5 EFFECT OF TCDD ON 6.V.H. REACTION Day after + 0 30 2.50 +_ 0.31 2.07 + 0.10 0 2.31 + 0.05 30 2.48 + 0.26 14 Lymphnodal index. �12 - REFERENCES 1 J.G.Vos, J.A. Moore and J.G. Zinkl, Effect of 2,3,7,8tetrachlorodibenzo~p-dioxin on the immune system of laboratory animals, Environ. Health Perspect., 5 (1973) 2 149. J.G. Vos and J.A. Moore, Suppression of cellular immunity in rats and mice by maternal treatment with 2,3»7»8tetrachlorodibenzo-p-dioxin, Int. Arch. Allergy Appl. Immunol., ^7 097Z0 777. 3 J.E. Thigpen, R.E. Faith, E.E. McCormell and J.A. Moore, Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7»8-tetrachlorodibenzo-p-dioxin, Infect. Immun., 12 (1975) 1319. 4 J.A. Moore and R.E. Faith, Immunologic response and factors affecting its assessment, Environ. Health Perspect., 18 (1976) 125. 5 E.E.McConnell,J.A.Moore, J.K. Haseman and M.¥. Harris, The comparative toxicity of chlorinated dibenao-p— dioxin isomers in mice and guinea pigs, Toxicol. Appl. Pharmacol,, 44 (1978) 335. 6 A. Hay, Toxic cloud over Seveso, Nature (London), 262 (1976) 636. 7 S. Garattini, TCDD poisoning at Seveso, Biomedicine, 26 (1977) 28. 8 J.G. Vos, J.A. Moore and J.G. Zinkl, Toxicity of 2,3,7,8tetrachlorodibenzo-p~dioxin (TCDD) in C57B1/6 mice, Toxicol. Appl. Pharmacol., 29 (197^) 229. 9 R.E. Kouri, T.H. Rucle, R. Joglekar, P.M. Dansette, D.M. JeriJig, S.A. Atlas, I.S. Owens and D.¥. Nebert, 2,3»7»8-tetrachlorodibenzo-p-dioxin as cocarcinogen causing 3-niothylcolanthroneinitiated subctitaneous tumors in mice genetically "nonresponsive" at ah locus, Cancer Res., 38 (1978) 2777. �13 - 10 W.L. Ford, ¥. Burr and M. Simonsen, A lymph node weight assay for the graft-versus-host activity of rat lyniphoid cells, Transplantation, 10 (19?0) 258. 11 H. Kirchner, H.T. lioldon and R.B. Herberman, Splenic suppressor macrophages induced in mice by injection of Corynebacteriurn parvum, J. Immunol., 115 (1975) 1212. 12 N.K. Jerne and A.A.Nordin, Plaque formation in agar by single antibody-producing cells, Science, 140 13 A. Vecchi, A. Mantovani, A. Tagliabue (19^3) ^05. and F. Spreafico, A charactei'ization of the ianmunosuppressive activity of adriamycin and daunoraycin on humoral antibody production and tumor allograft rejection, Cancer Res., 36 (1976) 1222. 14 R.E. Faith, M.I. Luster and J.A. Moore, Chemical separation of helper coll function and delayed hypersensitivity responses, Cell. Immunol., 40 (19?8) 275. � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 095 Folder The folder containing the original item. 2414 Series The series number of the original item. Series IV Subseries IV Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Vecchi, A. A. Mantovani M. Sironi W. Luini M. Cairo S. Garattini Title A name given to the resource Typescript: Effect of Acute Exposure to 2,3,7,8-Tetra-chlorodibenzo-p-dioxin (TCDD) on Humoral Antibody Production in Mice Subject The topic of the resource animal testing Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 106 Folder The folder containing the original item. 2924 Series The series number of the original item. Series V Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Vecchi, A. A. Mantovani M. Sironi W. Luini M. Cairo S. Garattini Source A related resource from which the described resource is derived Chemico-Biological Interactions Date A point or period of time associated with an event in the lifecycle of the resource 1980 Title A name given to the resource Effect of Acute Exposure to 2,3,7,8-Tetra-chlorodibenzo-p-dioxin on Humoral Antibody Production in Mice