1 15 7 https://www.nal.usda.gov/exhibits/speccoll/files/original/eb3a0f653bc6c863caddf5bc819dbdbc.pdf d0a6a8d81b69aefc041ddd6b323097b2 PDF Text Text Item D Number 02331 Author Rizzardini, M. Corporate Author Report/ArticleTltlO Typescript: Toxicological Evaluation of Urban Waste Incinerator Emissions Journal/Book Title Year 1982 Month/Day November Color D Number of Images 6 DBSCriptOn NOtBS Chemosphere, submitted November '82 Monday, September 24,2001 Page 2331 of 2337 �Chemosphere Submitted, Novemberr82 TOXICOLOGICAL EVALUATION OF URBAN WASTE INCINERATOR . ' - ., ; EMISSIONS :.M« Rizzardini, M. Romano, F. Tursi, M. Salmona, A. Vecchi, M. Sironi, F. Gizzi, E, Benfenati, S. Garattini and R. Fanelli Istituto di Ricerche Farmacologiche "Marie Negri" Via Eritrea 62, 20157 MILAN, Italy ft.EGIONE L O M B A R ' DI A • . • Ufficso Specials di Sevesp' • Servizio Documentazibns Seie Via S. Carlo, 4 . - , 20030 SEVESO (Ml) INTRODUCTION •'"_". .The- incineration of solid urban waste raises- serious problems related to the presence of highly toxic micropollutants in the emissions of incinerating plants. In particular polychlorinated dibenzo-p-dioxins(PCDD) and polychlorinated dibenzofurans (PCDF) mixtures have been found in these emissions (1-5). Assessment of the toxicological risk associated with these mixtures is a major, still unsolved problem. In fact only a few of the 75 PCDD isomers and 135 PCDr isomers have been studied as regards toxicological properties (6,7), and even scarcer are the data on the toxicological effects of their mixtures, most of them artificially built by joining some individual isomers (8-13). In a first attempt to study the potential toxicological properties of a PCDD/PCDF mixture released from an urban incinerator, we carried out an experiment to characterize its immunodepressive and enzyme-inducing properties. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDi) has been shown to. be a very potent immunosuppressant in animals (14), single doses of 1 jig/kg depressing antibody production for more than 40 days ( 5 . Little information is 1) available on the immunosuppressive effects of 2,3,7,8-tetrachlorodibenzofuran (TCDF) in laboratory animals ( 6 . .Very recently TCDF was compared with TCDD for its toxicity on the 1) immune system of mice ( 7 . It is generally believed that these two compounds induce 1) similar toxic effects (18) when 10-30 times more TCDF is given. We reported (17) that the humoral response is more sensitive than cell-mediated reactions (Graft versus Host reactionmitogenic responsiveness) to single TCDF exposure and that the immunosuppression induced by TCDF recovers faster than that induced by TCDD, antibody induction being totally restored by day 40. We thus chose to investigate the effects of extracts from an incinerator on humoral antibody production, using heterologous erythrocytes as antigen. An artificial mixture containing known amounts of TCDD and TCDF was prepared as reference control, to evaluate the interaction of the two toxic agents or their possible synergism. TCDD is among the most potent mixed-function oxidase (MFO) inducing agents yet demonstrated in mammalian liver. Low dose studies in the rat gave an ED5Q of TCDD induction .of benzo(oO pyrene-hydroxylase of 0.63 ug/kg and the smallest dose of TCDD which significantly increased enzyme activity was 0.002 ug/kg (19). In mice (C57B1/6J) an £050 of 0.29 ug/kg TCDD for hepatic AHH induction was estimated (20). �4' * TCDF is closely related to TCDD in structure and acute toxicity ( 8 . Its bio1) logical activity (ED5Q for AHH induction) is almost equivalent to TCDD in the chick embryo (21). In mice (C57B1/6J) and ED50 of 7 ug/kg TCDF was found (20) (AHH induction). An important observation was that there is an excellent correlation between AHH induction and the toxic potency of certain PCDD and PCDF isomers ( 2 . Despite the large 2) amount of information about the single compounds none is yet available on the effect of a mixture of TCDD and TCDF on these parameters (P-450 content and hepatic MFO enzyme system). It was therefore deemed interesting to investigate the inducing properties of a mixture of the two compounds and of extracts from an urban waste incinerator, containing complex mixtures of PCDD and PCDF. " - ' - - - . . / - ' .; MATERIALS Animals ' • , - . , • ' " . • • C57B1/6J male mice where obtaine from Charles River, Calco. (Italy) and used when 10 weeks old* Chemicals ' ' . . • All chemicals involved in the extraction, purification and analysis of the PCDD and PCDF micture have been reported ( ) TCDF was a kind gift from Dr. C. Rappa, University 5. of Umea, Sweden. ' • " ' ' • "^ ' EXPERIMENTAL The PCDD and PCDF mixture was drawn from a modern municipal incinerator in Italy, equipped with electrostatic precipitators. Gas condensate collection and the methods of extraction with n-hexane and of purification were as previously described ( ) An LKB 2091 5. GC-MS equipped with an LKB 2130 computer was used for GC-MS analysis, in the reported conditions (5). An imal_t r eatment After analysis, the sample (mixture 1) was dissolved in 10 ml of acetone-corn oil (1:6 v/v) and given as a single i.p. injection to mice (7-8 per group) as such or diluted 10 times (mixture 1/10), at a dose of 10 ml/kg. TCDD (1.2 ug/kg), TCDF (10 ug/kg) and the mixture of TCDD and TCDF (respectively 1.2 ug/kg and 10 ug/kg) were dissolved and given as described for mixture 1. '~~~' groduction assay_ Q 4- 10° sheep red blood cells were injected i.p. 7 days after treatment with the different toxic agents and plaque forming cells (PFC) in the spleen were counted 5 days later by the Yeme's technique (23). �1 statistical analysis -r- _ - Results are presentad as means +_ s.e.; the statistical significance was evaluated by Duncan's test* Enzyme indue tion_ass ay Mice were killed by cervical dislocation and livers were immediately removed, rinsed in saline and blotted dry. The tissues were homogenized with an Ultra-Turrax apparatus in 0.05M phosphate buffer pH 7.4 (1:4 v/v) and centrifuged at 9000xg for 20 min in a refrigerated centrifuge. The supernatant fractions were stored at -70°C until used for cy to chrome P-450, 7-ECD and protein assay. Cytochrome P-450 was determined according to Omura and Sato (24) . 7-Ethoxycoumarin 0-deethylase activity was assayed according to Greenlee and Poland. ( 5 . Proteins were measured according to Lowry et al. (26). 2) ' ' • ' * . - . . , RESULTS The amounts of individual classes of PCDD and PCDF employed for the experiment are listed in Table 1. They correspond approximately to the amount of micropollutants •released in about . . seconds or the amount obtained from about 0.5 kg of waste ( 7 . 05 2) Table 1 - PCDD-PCDF dose administered (ng/kg) , corresponding to mixture 1 PCDD PCDF a Tetra Penta Hexa Hepta Octa a 3688 (17.5) 4816 (22.9) 7152 (34.0) 4332 (20.6) 1064 (5.0) 8800 (35.3) 7696 (30.9) 4540 (18.2) 3004 (12.1) 880 (3.5) Total 21052 . 24920 In brackets the percentage of the individual classes of isomers. Effect_on_antibody_groduction The effect of an artificial mixture of TCDD and TCDF at a ratio of ' about 1:8 was first investigated. As shown in Fig. 1, TCDD inhibited antibody production by 80% while TCDF at the dose used was not depressive. However, when the two toxic agents were administered together in the same solution, the degree of inhibition (50%) was significantly lower than that induced by TCDD alone (80%) when results are expressed either as PFC/10° splenocytes or as PFC/spleen. Thus, the presence of TCDF can modify the immunosuppressive capacity of TCDD. The simultaneous administration of 3.688 ;ug/kg tetrachlorodibenzodioxin and 8.8 jug/kg tetrachlorodibenzofuran (mixture 1) or of a dose 10 times lower (mix I/ 1C) did not significantly modify the immune response, antibody production being only 15% inhibited. �EFFECT OF SIMULTANEOUS ADMINISTRATION OF TCOD AND TCDF AND OF MIXTURES FROM INCINERATOR ON ANTIBODY PRODUCTION. PFC/10 6 splenocytes r? RFC /spleen 100- o cc H~ z o u u. 50O ** TCDD TCDF TCDO TCDF Mix. Mix. 1/10 1 TCOO TCOF TCDD TCOF Mix. Mix. 1/10 1 •FIG. 1 TCDD (1.2 ug/kg), TCDF (10 ug/kg) and extracts from an incinerator were dissolved in acetone:corn oil (1:6 v/v) and given i.p. on day -7. 4-108 SRBC were given i.p. on day 0 and the test was done 5 days later. Mix 1 = extract containing 3.7 and 8.8 ug/kg of tetrachloro-dibenzodioxin and -dibenzofuran respectively; see Table 1. • Mix. 1/10 » dose one-tenth Mix 1 Effect_on_CYtochrome_P-450_content_and_on_7-etox2Coumarin-0-deeth2lase activity Twelve days after a single dose of 1.2 pg/kg TCDD, cytochrome P-450 content was still about 3 times higher than, in control mice. As expected, there was a shift of 2 nm in.-the peak absorption maximum (from 450 nm to 448 nm; see,Table 2). This shift was also observed when TCDD was administered simultaneously with TCDF. No effect on maximum peak absorption was observed with TCDF alone. In this respect, no clear-cut response was seen when incinerator extracts were given. Parallel to cytochrome P-450 induction, a ten-fold increase was observed in the activity of 7-etoxycoumarin-O-deethylase. TCDF at 10 ug/kg had no inducing effect. When the same doses of chlorinated compounds were given in combination, an induction pattern similar to that caused by TCDD alone was observed; however the content of cytochrome P450 was only doubled in comparison to controls and was significantly lower (p^O.05) than in mice treated with TCDD alone. In the same treatment groups the activity of 7-ECD was 8 times higher than in control mice, but somewhat lower (1179 pmol/min/mg prot. versus 1315) than in TCDD treated animals. The effect of an extract from urban incinerator smoke containing 3.3 and 8.8 ,ug/kg of tetrachloro-dibenzodioxins and dibenzofurans was then evaluated: cytochrome P-450 content �», significantly raised and this inductive effect was more evident on 7-ECD activity. — ten times lower dose of incinerator extract had no effect on cytochrome P-450 or 7-ECD, ' Table 2 - Effect of simultaneous administration of TCDD, TCDF and incinerator mixtures on cytochrome P-450 levels and 7-ethoxycoumarin-O-deethylase activity. Treatment Cytochrome P-450 or P-448a (nmol/mg prot) Vehicle 0.63 +:0.028b ( ) 6c 2,3,7,8 TCDD 2,3,7,8 TCDF 1.87 0.75 1.57 0.97 0.82 TCDD +• TCDF Mix 1 Mix 1/10 + 0.07** (6) £0.04- (6) +_ 0.18**+(5) ^ 0.06* (5) -t- 0.017 ' 6 () 7-Ethoxycoumarin-O-deethylase (pmol/min/mg prot) 143 + 6 (5) 1315 +_ 62 (6)" 173 1179 352 234 ^ 15 (6) +_ 98 ( ) 5' +_ 40 ( ) 6' + 16 (6) +•* p^O.Ol from control group (Duncan's test) *- p^O.05 + pjgO.05 from mice treated with TCDD alone (Duncan's test) 3 . Cytochrome P-450 indicates the microsomal co-binding pigment found in control mice, b Mean + S.E. c In. parentheses the number of observations. For treatment schedule refer to Fig. 1 I CONCLUSIONS Our preliminary results indicate that microsomal enzyme induction is a prompt, sensitive biological parameter of exposure to PCDD and PCDF, even when these compounds are in mixtures whose composition is not completely known. The observation that the administration of a dose of TCDF inactive per se simultaneously with an active dose of TCDD reduced the immunosuppfessive and enzyme inducing capacity of TCDD suggest a competitive effect at the receptor level. From this finding with a given TCDD:TCDF ratio, however, it* is impossible to extrapolate the effects of different ratios or of higher active doses of TCDF. These unexpected biological effects illustrate the complexity of problems to be tackled in order to improve the prediction of toxic effects of mixtures of chemicals released simultaneously in the environment. For the time being, statements on the harmlessness to humans of urban waste incinerator emissions appear untimely (28). / ACKNOWLEDGEMENTS The authors express their gratitude to M. Lodi and R. Tagliaferri (ECOLAB s.r.l. Coop., Vignate, Milano, Italy) for sampling and to Dr. C. Rappe (Department of Organic Chemistry, University of Umea, Sweden), for .supplying the TCDF standard. �'•-.. ^1. K..;.01ie, P.L.. Vermeulen and 0..Hutzinger, Chemosphere, £, 455-459 (1977).* t 2. H.R. Buser, H-P. Bosshardt and C. Rappe, Chemosphere, 7_, 165-172 (1978). 3. G.A. Eiceman, R.E. Clement and F..W. Karasek, Anal. Chem., 51, 2343-2350 (1979). f j 4. J.W.A. Lustenhouwer, K. Olie and 0. Hutzinger, Chemosphere, 9, 501-522 (1980). . ' i c ~~ 5. F. Gizzi, R. Reginato, E. Benfenati and R. Fanelli, Chemosphere, 11, 577-583 (1982). [ 6. M.P. Esposito, T.O. Tiernan and F.E. Dryden, "Dioxins" tJ.S. EPA ; Cincinnati, Ohio (1980). 7. J.A. Goldstein. Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodioxins and Related Products, Elsevier, Amsterdam, R.D. Kimbrough (ed.), 151-190 (1980). ; 8. M. Nishizumi, Toxicol. Appl. Pharmacol., 45, 209-212 (1978). —*—* 9. S.. Oishi, M. Morita and H. Fukuda., Toxicol. Appl. Pharmacol., 43, 13-22 (1978). i i ] 10. H. Bauer, K.H. Schulz and U.Spiegelberg,Arch.iGewerbepathol.Gewerbehyg. 1_8_,538-555( 1961). ! 11. 5. Kawano and. K. Hiraga,, Jpn. J. Pharmacol.,, 28, 305-315 (1978). "" ' 12. S^ Oishi and K. Hiraga, Food Cosmet. Toxicol., 16» 47-48 (1978). 13. K.D. Courtney, Bull. Environ. Contain. Toxicol., 16, 674-681 (1976). 14. J.G. Vos, R.E. Faith and M.I. Luster. Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodioxins and Related Products, Elsevier, Amsterdam, R.D. Kimbrough (ed.), 241-266 (1980). 15* A. Vecchi, A. Mantovani, M., Sironi, W. Luini, M. Cairo and S. Garattini, Chem. Biol. Interact., 20, 337-342 (1980). 16. M.I. Luster, R.E. Faith and L.D. Lawson, Drug Chem. Toxicol., 2_, 49-60 (1979). 17. A. Vecchi, M. Sironi, M.A. Canegrati and S. Garattini, Symposium on Chlorinated Dioxins and Dibenzofurans in the Total Environment, Ann Arbor, Science Publishers, L.H. Keith, G. Chondhary and C. Rappe (eds.), 1983, in press. 18. E.E. McConnell and J.A. Moore, Ann. N.Y. Acad. Sci., 320, 138-150 (1979). 19. K.T. Kitchin and J.S. Woods, Toxicol. Appl. Pharmacol., 47, 537-546 (1979). S'. *"" """ 20. A. Poland, E. Glover, M. DeCamp, C.M. Giandomenico and A.S. Kende, Science, 194, 627-630 (1976). 21. A. Poland, E. Glover and A.S. Kende, J. Biol. Chem., 251, 4936-4946 (1976). 22. A. Poland, W.F. Greenlee and A.S. Kende, Ann. N.Y. Acad. Sci., 320, 214-230 (1979). 23. N.K. Jerne and A.A. Nordin, Science, 140, 405 (1963). 24. T. Omura and R. Sato,'J. Biol. Chem., 239, 2370-2378 (1964). 25. W.F. Greenlee and A. Poland, J. Pharmacol. Exp. Ther., 205, 596-605 (1978). 26. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193, 265-275 (1951). 27. E. Benfenati, F. Gizzi, R. Reginato, R. Fanelli, M. Lodi and R. Tagliaferri, Chemosphere, in press. i / "28. U.S. Environmental Protection Agency, Environmental News, Nov.19 (1981). _, } � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 093 Folder The folder containing the original item. 2331 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 Rizzardini, M. M. Romano F. Tursi M. Salmona A. Vecchi M. Sironi F. Gizzi E. Benfenati S. Garattini R. Fanelli Date A point or period of time associated with an event in the lifecycle of the resource 1982-11-01 Title A name given to the resource Typescript: Toxicological Evaluation of Urban Waste Incinerator Emissions Subject The topic of the resource incineration dioxin animal testing enzyme induction 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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 https://www.nal.usda.gov/exhibits/speccoll/files/original/295b865ea83d8d0648321427d1677b2e.pdf c5a95ff97af14fe6c9583566c4565ae8 PDF Text Text Item D Number °2415 Author Mantovani, A. Corporate Author Report/Article TltlO Typescript: Effect of 2,3,7,8-Tetrachlorodibenzo-pdioxin on Macrophage and Natural Killer Cell-Mediated Cytotoxicity in Mice Journal/Book Title Year 000 ° Month/Dey Color Number of Images D 17 Descrlpton Notes Friday, October 05, 2001 Page 2415 of 2422 �ALViN L Y::V:'~ ••' EFFECT OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN ON MACROPHAGE AND NATURAL KILLER CELL-MEDIATED CYTOTOXICITY IN MICE. A. Mantovani, A. Vecchi, W. Luini, M. Sironi, G.P. Candiani, F. Spreafico and S. Garattini. Istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea, 62 - 20157 MILANO, Italy Key words: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); macrophages; NK cells. �2- INTRODUCTION 2,3,7,8 Tetrachloro-dibenzo-p-dioxin (TCDD) is one of the most toxic chemicals known, occurring as a contaminant in the production of chlorinated phenols or compounds synthesized from them. One of the major toxic effects of TCDD in rodents is thymic atrophy (12, 15-17). Concomitantly, TCDD-treated animals show impaired cell-mediated immune reactivity (2, 15, 16) and decreased resistance to bacterial infection (14). The effect of TCDD on humoral antibody production has not been thoroughly investigated (2,15) but it appears so far that humoral responses are relatively spared by this agent (2,14-17). The present investigation, prompted by the severe pollution with TCDD of a densily populated area (Seveso) near Milan (4,18), was designed to elucidate TCDD's effects on natural host defense mechanisms involving natural killer (NK) cells and macrophages. Both these mechanisms are currently taken to represent effective lines of resistance against infection and neoplasia (5,13). MATERIALS AND METHODS Mice. Male C57B1/6 J mice (6 to 8 weeks old), obtained from Charles River Breeding Lab., Calco, Italy were employed throughout. TCDD. TCDD (1,2, 6 and 30 mg/kg), obtained from Kor Isotopes, Cambridge, Mass., was injected i.p. in a volume of 0.25 ml of an acetone-corn oil mixture (1:6 v:v). Control mice were given the vehicle alone. Target cells. The YAC-1 lymphoma, mKSATU5 (TUS) kidney line and SL2 lymphoma were obtained through the courtesy of Dr. R. Kiessling (Karolinska Institute, Stockholm, Sweden), Dr. J. Dean (Litton Bionetics, Kensington, Md., USA) and Dr. R.Evans (Chester Beatty Research Institute, Sutton, Surrey, England) respectively. The tumor lines were maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (growth medium). �3- Macrophages. Peritoneal exudate cells (PEC) were obtained by washing the peritoneal cavity with 4 ml of basal medium Eagle (BME) (8,9). After centrifugation and resuspension in BME, differential cell counts were made with Turk's solution and macrophages were seeded in 1 ml BME in the wells of Costar Trays (3524 Costar, Cambridge, Mass.) for cytostasis experiments (10 macfophages/well) or in 0.3 ml BME in flat bottomed 6.4 mm culture wells (3596, Costar, Cambridge, Mass.) for cytolysis 5 experiments (5x104, 10 or 2x106 macrophages/well). After 2 h of incubation at 37°C, the wells were thoroughly washed with medium to remove non-adherent cells. More than 90% of the adherent cells were macrophages as assessed by marphology and phagocytosis of latex particles. Macrophage-mediated cytolytic activity. The cytolytic activity of macroO "3 phages was evaluated in terms of J H-thymidine (H-TdR) release from prelabelled TU5 target cells, as recently described (10). Briefly, non confluent TU5 cultures in 25 cm 2 tissue culture flasks (Costar, Cambridge, Mass., USA) were incubated overnight with 5 ml of medium containing •3 0.5 uCi/ml of H-TdR (6 Ci/mmole, Amersham Radiochemical Centre, Amersham, England). After exposure to 1 ml trypsin-EDTA for 5 min at 37°C, the cells were washed twice with 50 ml of growth medium. Target cells (10 /0.3 ml) were incubated for 48 h with a range of attacker to target cell (A:T) ratios in flat bottomed 0.4 mm culture wells (3596, Costar, Cambridge, Mass., USA). At the end of the incubation, 0.1 ml of the supernatant was harvested and counted in a liquid scintillation spectrometer. Total incorporated radioactivity was calculated from tumor cells incubated with 1% sodium dodecyl sulfate (SDS) in water. Percentage isotope release was calculated as A/B x 100, where A is the isotope released in test samples and B is the SDS releasable radioactivity. Specific lysis was determined by subtracting the spontaneous release of tumor cells incubated in the absence of effectors, which under these conditions did not exceed 25% of the total incorporated radioactivity. A semi log plot of the specific cytotoxicity values versus the number of effector cells per sample was obtained and the number of cells required to give 20% specific lysis was arbitrarily defined as one �4- lytic unit (LU^g). This approach permitted a quantitative estimate of the total cytotoxic capacity per peritoneal cavity. Macrophage-mediated cytostatic activity. Macrophage-mediated cytostatic activity was evaluated using SL2 lymphoma cells as targets as previously described (8,9). Briefly, SL2 lymphoma cells (5x10 in 1 ml growth median) were seeded on macrophage monolayers (approximately 1x10 cells) in the wells of Costar trays (3524, Costar, Cambridge, Mass.). Tumor growth was checked daily under an inverted microscope. After 48 h of incubation, lymphoma cells were vigorously resuspended with a Pasteur pipette, transferred to plastic tubes and washed. After resuspension in 1 ml growth medium, SL2 lymphoma cells were pulsed with 0.5 uCi H-TdR (specific activity 1.9 Ci/mmoles Schwarz Mann, Orangeburg, N.Y.). A Percentage inhibition of 3H-TdR uptake was calculated as (1- - ) x 100, where A is the isotope uptake in the presence of macrophages and B is the isotope uptake by tumor cells alone. Spleen NK activity. NK activity was evaluated using splenocytes as effectors and 51 Cr-labelled YAC-1 lymphoma cells as targets as previously described (11). Briefly, 5xl0 4 » 51 Cr-labelled YAC-1 lymphoma cells were cultivated for 12 h with different numbers of splenocytes, the resulting A:T ratios ranging from 10:1 to 50:1. The percentage of specific cytotoxicity was calculated as /"(release with effector cells-spontaneous release)/ total releasable radioactivity/ x 100. Spontaneous release in the absence of effector cells was 0.6-1.5% per hour of incubation and total releasable radioactivity, assessed by osmotic lysis of target cells, was 75% of total isotope incorporated. The total cytotoxic capacity per spleen was calculated in terms of LU 33 as described above for macrophage-mediated lysis. Statistical analysis. Five mice per experimental group were employed throughout and data obtained with 2-3 replicates per A:T ratio were analysed by Dunnett's test. Results are presented as mean + S.E. �5 - RESULTS Table I shows the effect of TCDD on the number and cellular composition of PEC obtained from C57B1/6 J mice. Stimulation of the peritoneal cavity with acetone-oil, employed as a vehicle in these studies, resulted in doubling of the number of PEC seven days after treatment, the number of nucleated cells slowly declining thereafter to reach normal values on day 47. Seven days after vehicle administration, greater percentage (34%) of polymorphs was observed than in unstimulated mice (1%). The polymorphonuclear infiltrate had returned to normal values by day 14. TCDD (1.2-30 ,ug/kg) had no effect on the cellular composition of PEC throughout the observation period (47 days). On the other hand, TCDDtreated mice showed a marked reduction ( ^ 75% at the dose of 30 >jg/kg) in total nucleated cell numbers on day 14 and 26 after treatment. No significant difference from control values was detected 47 days after treatment with TCDD. The effect of TCDD on macrophage-mediated cytolytic and cytostatic activity was then investigated. Macrophage-mediated cytolysis was studied using TU5 target cells, which have been shown to be susceptible to the natural cytotoxic capacity of human and murine mononuclear phagocytes (10, Tagliabue et al., in preparation). Under these experimental conditions, TCDD did not significantly modify the spontaneous cytocidal activity of murine f)eritoneal macrophages tested at A:T ratios from 5:1 to 20:1 (Table II). However, when the total number of recovered LU20/mouse was calculated, TCDD-treated PEC showed a lower cytotoxic potential than controls on day 14 and 26, LU20 values of TCDD-treated macrophages being 6 and 2 compared to 14 and 8 for controls. Results 3 presented in Table II were obtained in a 48 h H-TdR release assay, but findings were similar when the incubation time was prolonged to 72 h (results not shown). In a series of experiments the fucntional capacity of macrophages was evaluated by measuring their response to the activating stimulus endotoxin (13). As shown in Table III, in the presence of endotoxin macrophages showed increased cytocidal activity on tumor cells and treatment with �6 - TCDD did not alter their capacity to respond to this activating stimulus. The relationship between the cytostatic and cytolytic effects of macrophages on tumor cells is still not clear (10). Therefore it was considered of interest to evaluate the cytostatic activity of endotoxintreated macrophages after administration of TCDD. As shown in Table IV, the growth inhibitory capacity of TCDD-treated macrophages, in terms of 3 inhibition of H-TdR uptake by SL2 lymphoma cells, was the same as controls. In an effort to elucidate better the interaction of TCDD with natural cell-mediated host defense mechanisms, we studied the effect of this chemical (30 ,ug/kg i.p.) on NK cell activity, a cell-mediated reaction possibly representing an in vitro correlate of in vivo natural resistance to tumors and infections (5,13). Spleen NK activity per unit number of lymphoid cells was similar in TCDD-treated and control mice (Table V). However, since in agreement with previous data (12, 15-17), total spleen cell numbers were significantly reduced 7 and 14 days after TCDD, total / spleen cytotoxic capacity expressed as LU 33/organ was less in mice exposed to this chemical. Table V shows cytotoxicity data obtained with YAC-1 tumor target cells 1" and 14 days after TCDD. Similar results were obtained on day 23 in one experiment using the RLCfl lymphoma (5) as target. �7- DISCUSSION The results presented here indicate that TCDD does not affect the functional status of murine macrophages and NK cells as judged from their cytotoxic activity per unit number of effector cells. However, although macrophage-mediated and NK cell-mediated cytotoxicity on tumor cells was not modified on a per cell basis, fewer macrophages and spleen cells were recovered from TCDD-treated mice. Therefore the total cytotoxic capacity expressed as LU/organ was significantly reduced after exposure to TCDD but recovery was complete by day 47. This effect of TCDD on NK activity is similar ot that of irradiation (5,6) and of cytotoxic agents such as Adriamycin (11). In contrast, hydrocortisone, cyclophosphamide and azathioprine markedly suppress NK activity on a per cell basis too (5,6,11). TCDD has been shown to result in thymic atrophy and in cell depletion of thymus dependent areas of lymphoid organs in rodents (2,15-17). In fact, thymus weight was consistently lower in the TCDD-treated mice (results not presented). The nature of NK cells has not been completely elucidated but recent evidence suggests that they may belong to the T cell lineage (5). Therefore the observed lack of inhibition of NK activity per unit number of lymphoid cells is somewhat suprising and suggests a relative resistance to this agent of the T cell subset (prethymic T cells) which has been proposed as effectors of NK activity (5). Macrophages and NK cells derive from bone marrow precursors (3,13). Microscopic examination of the bone marrow of TCDD-treated animals revealed marked hypocellularity (12). Thus the bone marrow toxicity of TCDD might at least partially account for the smaller number of LU recovered from the spleen and peritoneal cavity of mice exposed to this agent. There is evidence that macrophages and NK cells represent important mechanisms of in vivo resistance against infection and neoplasia (5,13). �8- Thus impairment of these effector mechanisms by TCDD might play a role in the decreased resistance to infection of mice exposed to this chemical (14) and in its carcinogenic and cocarcinogenic activity (1,7). ACKNOWLEDGEMENT This work was supported by Regione Lombardia, Milan, Italy- Special Research Program on TCDD. We than Mr. R. Motta and Mr. L. Vaghi for skillful technical assistance in the animal house. am/ �9 - Table I - Effect of TCDD on mouse peritoneal exudate cells (PEC). Day after TCDD TCDD (H9/kg) Vehicle 7 1-2 6 30 Vehicle 1.2 14 14 6 30 Vehicle ,7 47 45 54 1 4.9 + 0.3 4.9 + 0.2 4.8 + 0.5 5.8 + 0.9 37 35 34 39 29 32 25 26 34 33 41 35 4.1 + 0.2 2.0 + 0.6 2.7 — 0.7 + 1.3 + 0.2* 58 NT NT 54 40 NT NT 45 2 NT NT 1 49 • NT NT 52 50 NT NT 45 1 NT NT 3 52 55 48 44 0 1 + + + + 6 30 3.1 1.3 1.3 1.0 Vehicle 30 2.7 + 0.2 2.0 + 0.1 1.2 . Macrophages % Polymorphs 2.1 + 0.3 - 2fi °/ 10 % Lymphocytes PEC (xlO6) 0.4 0.2* ± 0.01 0.07* p<0.05 versus mice given vehicle NT = not tested �Table II - Effect of TCDD on macrophage-mediated cytolytic activity. ay after TCDD Macrophages (xlO6) 1.2 6 30 1.9 1.7 1.6 2.2 32.4 + 31.5 + 30.2 + 34;0 _+ 30 7 TCDD (jug/kg) 24 ^ 0.7 1 5 14 26 47 30 30 % specific lysis 10:1 20:1 L U20/mouse 5:1 1.5 1.8 0.3 1.2 15.4 + 0.3 12.9 + 1.9 14.7 + 0.5 16.8 + 1.4 24, 19 21 36 20.5 + 1.4 22.4 + 1.9 18.4 + 2.8 19.1 + 0.3 14.8 + 1.1 16.7 + 0.9 14 6 ' 0.5 19.8 + 0.3 17.4 + 2.4 17.4+1.2 16.9 + 0.2 12.5+0.8 14.1 + 1.6 8 2 1.4 1.1 22.7 + 4.5 37.9 + 4.3 18.0 + 3.5 27.8+2.9 NT NT 12 14 2.3 0.5 2.4 1.5 24.5 20.3 27.6 28.4 + + + + 0 1 �11 - Table III - Effect of TCDD on in vitro stimulation of macrophage cytolytic activity by endotoxin. Day after TCDD Endotoxin TCDD (jug/kg) _ (10 jLig/ml) 1.2 30 6 30. 2 + 2.4 34. 0 ± 1 .5 44.4+2.7* 46. 2 + 1 . 2 * 45. 1 + 4 .5 * 20. 5 + 1 .4 NT NT 22. 4 + 1 .9 31. 4 + 2 .5* NT NT 34. 3 + 2 .9* 19. 8 + 0 .3 NT NT 17. 4 + 2 .4 33. 5 + 1 .4* NT NT 31. 6 + 1 .6* 32. 4 + 2 .3 31.5 45. 0 + 1 .6* + 0.5 7 + 14 + 26 + Results are % specific H-TdR release from prelabelled mKSA TU5 target cells (mean +_ S.D.). The A:T ratio was 20:1 """Salmonella typhosa 0901 (Difco) *p <0.05 versus samples without endotoxin �12 - Table IV - Effect of TCDD on macrophage-mediated cytostatic activity Day after TCDD 1.2 30.5 + 3 .4 14 + 21 + Results are presented (mean _+ S.E.) 25 .3 + 4.3 34. 3 + 2.1 58.0 _+ 0.4* 64.0 + 3.2* 63 .0 ± 3.1* 59.4 +_ 3.4* 19.2 + 2.3 16 .4 __ 0.6 + 13. 9 + 0.4 66.3 + 3 .2* 73.4 + 4.5* 75 .3 + 4.2* 64.6 24. 3 ~~ 0.9 + + 31.4 + 0.9 15. 0 ~~ 1 .2 + 7 + + TCDD (jug/kg) 6 30 Endotoxin (10 yg/ml) 24 + 1.5 18 .3 __ 3.6 + 22.7 + 3.2 56.8 + 2 .3* 52.3 + 1.3* — 1.4* —. — 59 .4 + 1.9* 56. 1 + 1.7* as % inhibition of H-TdR uptake by SL2 lymphoma cells *p < 0.05 versus samples without endotoxin 4- �Table V - Effect of TCDD on spleen NK activity against YAC-1 lymphoma cells. Day after TCDD TCDD (30 jig/kg) % specific Spleen cells (xlO 6 ) 50:1 51 Cr release LU33/spleen 20:1 10:1 79.2+11 + 42.9 + 6.9 38.7 + 3.5 24.2 + 5.9 105 52.6 + 9* 43.6 + 4.7 40.0 + 5.6 31.3 + 3.9 87 75.3+5.5 7 39.2 + 1.8 26.9 +_ 0.6 20.4 + 1.1 50 48.1 + 3* 36.2 + 2.8 30.3+4.3 20.0 + 1.2 32 14 + p<0.05 versus controls. GO �14 - SUMMARY C57B1/6 J mice (6-8 weeks old) were given single i.p. doses (1.2, 6 and 30 jug/kg) of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and macrophagemediated and natural killer (NK) cell-mediated cytotoxicity was evaluated at different times after treatment. Peritoneal macrophage cytolytic 3 activity was measured as H-thymidine release from prelabelled mKSATU5 target cells in a 48 h assay; macrophage-mediated cytostasis was assessed o in terms of inhibition of H-thymidine uptake by SL2 lymphoma cells. Spleen NK activity was measured using Cr-labelled YAC-1 lymphoma cells as targets. TCDD did not modify spontaneous macrophage-mediated and NK cell-mediated cytotoxicity per unit number of effector cells nor did 'it affect the macrophages' capacity to express increased cytolytic and cytostatic activity in the presence of endotoxin. Lower numbers of peritoneal macrophages and splenocytes were recovered from TCDD treated mice. Thus the total numbers of lytic units recovered from animals exposed to TCDD were lower than controls. Impairment of these cellular effector mechanisms, due to cell loss rather than inhibition of functions might play a role in the lowered resistance to bacterial infection of mice given TCDD and in the carcinogenic and cocarcinogenic activity of this chemical. �15 - RESUME A des souris C57B1/6 (agees de 6-8 semaines) on a administre des doses uniques en i.p. (1,2,6 et 30 ug/kg) de 2,3,7,8 tetrachlorodibenzo-p-dioxine (TCDD), T'activite cytolytique des macrophages et des cellules natural killer a ete est evaluee a differents moments apres le traitement. o La liberation de H thymidine par des cellules cibles marquees m kSATUS, permet, apres 48 h de meaurer 1'activite cytolytique des macrophages. L 1 inhibition de la capture de H thymidine par les cellules du lymphome SL2 permet de measurer la cytostase due aux macrophages. L'activite destructice de la rate est meauree en utilisant des cellules du lymphome YAC-1 marquees par 51 Cr. L'activite cytotoxique spontane, exprimee par unite de cellule effectrice, n'est pas modifiee par le TCDD, il n'y a pas inhibition de 1'augmentation de 1'activite cytolytique et de la cytostase en presence d'endotoxine. Un nombre plus fiable de macrophages peritoneaux et de splenocytes fut denombre sur des souris traitees au TCDD. Ainsi 1'activite cytotoxique total etait plus faible chez animaux traite que chez les temoins. L'affaiblissement de ces mecanismes cellulaires serait du a une diminution de cellules plutot qu'a une inhibition de fonction, et pourrait jouer un role as la diminution de resistance aux affections bacteriennes des souris ayant regu du TCDD ainsi que dans 1'activite carcinogene et cocarcinogene de la substance. �16 - REFERENCES 1. Di Giovanni J., Viaje A., Berry D.L., Slaga T.J. & Juchau M.R. Tumor-initiating a b i l i t y of 2,3,7,8-tetrachIorodibenzo-pd i o x i n (TCDD) and arochlor 1254 in the two-stage system of mouse skin careinogenesis. B u l l , environ. Contam. Toxicol,, 1977, H, 552. 2. Faith R.E., Luster M.I. & Moore J.A. Chemical separation of helper c e l l function and delayed hypersensitivity responses. C e l l . I" mmuno I., 1978, 40, 275. 3. H a l l e r 0. & W i g z e l l H. Suppression of natural k i l l e r c e l l activity with radioactive strontium: Effector c e l l s are marrow dependent. J. ImmunoI., 1977, 118, 1503. 4. Hay A, Toxic cloud over Seyeso. Nature, 1976, 5. Herberman R.B. & Holden H.T. Natural eel I-mediated immunity. Advanc. Cancer Res., 1978, 27, 305. 6. K i e s s l i n g R«, Hochman P.S., H a l l e r 0., Shearer G.M., W i g z e l l H. & Cudkowicz G. Evidence for a s i m i l a r or common mechanism for natural k i l l e r c e l l activity and resistance to hemopoietic grafts. Europ. J. ImmunoI., 1977,_7, 655« 7. Kouri R.E., Rude T.H., Joglekar R., Dansette P.M., Jering D.M., Atlas S.A., Owens I.S. & Nebert D.W. 2,3,7,8-TetrachIorodibenzop-dioxin as cocarcinogen causing 3-methyIchoIanthreneinitiated subcutaneous tumors in mice g e n e t i c a l l y "nonresponsive" at aji locus. Cancer Res,, 1978, 38, 2777. 8. Mantovani A. Effects on in vitro tumor growth of murine macrophages isolated from sarcoma l i n e s differing in immunogenicity and metastasizing capacity. I n t « J. Cancer, 1978, 22, 741. 262, 636. 9. Mantovani A. In vitro and in vivo cytotoxicity of adriamycin and -'daunomycin for murine macrophages. Cancer Res., 1977, 37_f 815. 10. Mantovani A., J e r r e l l s T.R., Dean J.H. & Herberman R.B. Cytolytic and cytostatic activity on tumor c e l l s of circulating human monocytes. Int. J „ Cancer, in press. �17 - 11. Mantovani A,, L u i n i W«, Peri G., Vecchi A & Spreafico F. Effect of chemotherapeutic agents on natural eel I-mediated cytotoxicity in mice. J « nat. Cancer Inst., 1978, 6l_r 1255« 12. McConnelI E.E., Moore J.A., Haseman J.K. & Harris M.W. The comparative toxicity of chlorinated dibenzo-p-dioxins isomers in mice and guinea pigs. Toxicol. appI. Pharmacol. 1978, 44, 335. 13. Nelson D.S. Ed. ImmunobioIogy of the Macrophage, Academic Press, New York, 1976. 14. Thigpen J.E., Faith R.E., McConnelI E.E, & Moore J.A. Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-tetrachIorodibenzo-p-dioxin. Infect. Immun., 1975, 12,, 1319. 15» Vos J.G. & Moore J.A. Suppression of c e l l u l a r immunity in rats and mice by maternal treatment with 2,3,7,8-tetrachIorodibenzop-dioxin. Int. Arch, A l I e r g y , 1974, 47, 777. 16. Vos J.G., Moore J.A. & Z i n k l J.G. Effect of 2,3,7,8-tetrachIorodi benzo-p-di oxi n on the immune system of laboratory animals. Environ. Health Perspect., 1973, 5 , 149. . 17. Vos J.G., Moore J.A. & Zinkl J.G. Toxicity of 2,3,7,8tetrachIorodibenzo-p-dioxin (TCDD) in C57BJ./6 mice. Tox i co I . app I . Pharmaco I . , 1974/ 2£, 2 9 . 2' 1.8« Walsh J. Seveso: The questions persist where d i o x i n created a wasteland. Sci ence, 1977, 197, 1064. � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 2415 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 Mantovani, A. A. Vecchi W. Luini M. Sironi G. P. Candiani F. Spreafico S. Garattini Date A point or period of time associated with an event in the lifecycle of the resource 1979 Title A name given to the resource Typescript: Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Macrophage and Natural Killer Cell-Mediated Cytotoxicity 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 104 Folder The folder containing the original item. 2823 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 Rizzardini, M. M. Romano F. Tursi M. Salmona A. Vecchi M. Sironi F. Gizzi E. Benfenati S. Garattini R. Fanelli Source A related resource from which the described resource is derived Chemospere Date A point or period of time associated with an event in the lifecycle of the resource 1983 Title A name given to the resource Toxicological Evaluation of Urban Waste Incinerator Emissions 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 166 Folder The folder containing the original item. 4958 Series The series number of the original item. Series VII 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 Fanelli, R. M. G. Castelli G. P. Martelli A. Noseda S. Garattini Source A related resource from which the described resource is derived Bulletin of Environmental Contamination and Toxicology 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 Presence of 2,3,7,8-Tetrachlorodibenxo-p-dioxin in Wildlife Living Near Seveso, Italy: A Preliminary Study 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 120 Folder The folder containing the original item. 3265 Series The series number of the original item. Series VI Subseries II 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 Fanelli, R. M. G. Castelli G. P. Martelli A. Noseda S. Garattini Source A related resource from which the described resource is derived Bulletin of Environmental Contamination and Toxicology 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 Presence of 2,3,7,8 -Tetraclorodibenzo -p- dioxin in Wildlife Living near Seveso 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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