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Actively Acquired Tolerance to Dinitrochlorobenzene121From the Department of Dermatology of the New York University Post-Graduate Medical School and the Skin and Cancer Unit of the University Hospital, New York, New York.2The details of this study will be published in the Journal of Immunology.This study was aided by a grant from the National Institutes of Health.

1961; Elsevier BV; Volume: 37; Issue: 4 Linguagem: Inglês

10.1038/jid.1961.114

1523-1747

Leonard C. Harber, Stanley A. Rosenthal, Rudolf L. Baer,

Effects and risks of endocrine disrupting chemicals

The principle of actively acquired tolerance postulated by Burnet (1) and classically demonstrated by Medawar (2) has been verified and expanded by numerous investigators (3, 4, 5). Such studies have involved various experimental technics which have resulted in the in utero exposure of a fetus to a substance that is usually or often allergenic to a mature organism. As a consequence of such an exposure, the animals failed to respond or responded immunologically to a lesser degree, when reexposed to the homologous allergen in adult life. Our investigation was undertaken as an extension of the work of Baer, Rosenthal and Hagel (6) whose studies indicated a slight but consistent trend toward a reduction in the allergic response to dinitrochlorobenzene [DNCB] in guinea-pig offspring whose mothers had been fed this chemical during gestation. METHOD AND MATERIALS During a one year period, 168 pregnant guinea-pigs of the white Hartley strain received a single or weekly intraperitoneal injections of 0.1 ml of 5% DNCB dissolved in acetone, t Another 168 pregnant animals served as a control group and received injections of 0.1 ml of acetone alone. After reaching a weight of 100 gms, the 264 offspring from both of these groups were kept in individual cages until they were at least 30 days old. An attempt was then made to sensitize them to DNCB by application of 0.05 ml of 2% DNCB in absolute alcohol to the clipped nuchal area once daily for five consecutive days. All offspring were skin tested 24 t Radioactive DNCi4B was injected intraperitoneal by Doctors Cyril March and Theodore Tromovitch into a pregnant guinea pig. The animal was sacrificed 4 hours later and the uterine sac removed in toto. Significant radioactivity was demonstrable in the amniotic fluid, fetus, and maternal blood, indicating that in utero exposure to DNCB or its metabolites had occurred. days later with 0.2% DNCB in olive oil, 0.09% DNCB in olive oil, 0.05% DNCB in olive oil, and olive oil alone. The resulting reactions were read at twenty-four hours, the degree of erythema being graded from 0 to 4. A comparison was made between the mean DNCB skin test erythema scores at the 0.2%, 0.09% and 0.05% DNCB test sites of the 124 guinea-pigs born to mothers who had received DNCB during pregnancy and the mean skin test erythema scores of the 140 guinea pigs whose mothers had received acetone alone. These 264 offspring were skin tested in 13 groups varying in size from 12 to 38 animals during the 10 month period of the study with the results summarized in Table I. In 12 of the 13 groups, the degree of hypersensitivity to DNCB as expressed in the mean erythema scores was lower in the offspring of DNCB injected animals than in the offspring of the control animals. This difference was statistically significant, P < .01, when analyzed with the Wilcoxon Rank Test (7).§ In order to ascertain whether or not the tolerance to DNCB was a specific phenomenon, the same test offspring were exposed to sensitizing doses of citraconic anyhdride [CA], a compound which is immunologically unrelated to DNCB. The sensitizing procedure consisted of 4 intracutaneous injections of 0.1 ml of 0.1% CA. Skin tests were performed 28 days later by applying 20%, 10% and 5% CA solutions to the backs of all animals. The procedures used in testing and erythema scoring were identical with those used in the DNCB skin tests. A comparison of the skin test erythema scores elicited with CA in guinea pigs whose mothers were exposed to DNCB during pregnancy as compared to the offspring of the control animals showed that in none of the 7 studies comprising a total of 148 offspring was there a statistically significant difference in CA sensitivity between the DNCB and control group. In addition, the average CA skin erythema scores in these 7 groups were evenly divided between negative § The statistical analysis of these studies was made by Miss Lee Herrera of the Department of Medical Statistics. 241 242 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY TABLE I Mean DNCB erythema scores of offspring of guinea-pigs exposed during pregnancy to DNCB or acetone Number of Injections DNCB Acetone DNCB-Acetone Group Number of guinea-pigs Mean erythema score Number of guinea-pigs Mean erythema score Differences in Mean Score 1 2 3 4 5-6 7 8 9 10 11 12 13 MJ M M M M M M S S s s M 8 7 8 19 12 5 7 12 15 15 13 3 2.12 4.50 2.06 2.05 1.96 2.10 1.36 1.71 1.73 2.37 3.19 1.00 10 5 6 8 26 7 19 7 14 20 9 9 4.20 5.30 1.92 3.06 2.79 2.21 2.89 3.93 2.11 4.28 3.94 3.28 ~2.08t -0.80 +0.14 -1.01 -0.83 -0.11 -1.53* -2.22f -0.38 -l.Qlt -0.75 -2.28 * Statistically significant [P < .05]. t Statistically significant [P < .01]. IM = multiple injections; S = single injection. and positive differences in reactivity. Thus, there was no evidence of actively acquired tolerance to CA in the offspring of animals exposed to DNCB during pregnancy. Another test for the specificity of the actively acquired tolerance to DNCB was to carry out skin tests with 2,4-dinitrofluorobenzene [DNFB] in 50 offspring of mothers injected during pregnancy with DNCB in acetone and 40 offspring of mothers injected during pregnancy with acetone alone. These animals previously had been subjected to sensitizing exposures of DNCB only [See Table I, Groups 4, 7 9 and 11]. The individual erythema scores of each of the 90 animals' skin tested to 0.2%, 0.09%, and 0.05% DNFB were then totaled. In each of the 4 test groups, erythema elicited by DNFB was less in the animals born to DNCB injected mothers than in the control animals. Statistical analysis of the results in the 90 animals showed a significance in the neighborhood of P < .05. These data were interpreted as “cross-tolerance' ' between DNCB and DNFB. SUMMARY In confirmation of previous studies (6) which utilized dinitrochlorobenzene [DNCB] force feeding of pregnant guinea-pigs, the data pre- sented here indicate that the susceptibility to allergic contact sensitization to DNCB was significantly reduced in the offspring of guinea-pigs which were intraperitoneally injected with DNCB during pregnancy. This actively acquired tolerance appeared to be specific since it did not extend to citraconic anhydride, an immunologically unrelated compound. Actively acquired “cross-tolerance” was demonstrated between DNCB and dinitrofluorobenzene.