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Enhanced suppression of humoral immunity in DBA2 mice following subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
112, 128-l 32 ( 1992)
Enhanced Suppression of Humoral Immunity in DBA/2 Mice following Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)’ D. L. MORRIS,* N. K. SNYDER,*
V. GOKANI,~
R. E. BLAIR,*
AND M. P. HOLSAPPLE*,’
*Medical College of Virginia/Virginia Commonwealth University, Richmond, Virginia 23298; and tNational Institute for Occupational Health, Gjarat, India Received April 25, 199 1; accepted October 1, 199 1
EnhancedSuppressionof Humoral Immunity in DBA/Z Mice following Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzop-dioxin (TCDD). MORRIS, D. L., SNYDER, N. K., GOKANI, V., BLAIR, R. E., AND HOLSAPPLE, M. P. (1992). Toxicof. Appl. Pharmacol. 112,128-132. Previousstudieshave indicated that mice which differ in their acute susceptibility to responsesmediated by the Ah receptor have a pattern of suppressionof the antibody responsewhich is consistentwith a role by the putative dioxin receptor. The objective of the present investigation wasto comparethe TCDDinduced suppressionof the antibody responsefollowing acute and subchronic exposures in B6C3Fl mice, an Ah-high-responderstrain, and DBA/2 mice, an Ah-low-responder strain. Resultsof our initial studies demonstrate that suppressionof humoral immunity can be enhancedin DBA/2 mice approximately lo-fold following subchronic versusacute exposuresto the samecumulativedosesof TCDD. This changein suppression of the antibody responsein DBA/2 mice was not accompanied by significant changesin liver weight (hepatomegaly), as was observedin the B6C3Fl strain when exposedunder comparable conditions. In contrast, effects on thymus weight (involution) wereenhancedin the DBA/2 mice following subchronicexposure and demonstrateda higher degreeof atrophy than was seenin the B6C3Fl strain (68 versus56% decreasein thymic weight at the 42 pg/kg cumulative dose).Thesefindings suggestthat multiple mechanismsmay be operating to suppresshumoral immunity in viva and that the conditions of exposurecan alter the toxic effects of TCDD in the DBA/Z, Ah-low responsive,mouse strain. 0 1992 Academic Press, Inc.
Acute exposure of mice to the compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)3 leads to changes in organ ’ This work was supported by NIH Grant 1RO 1-ES-O3520, by Training Grant NIH-ES-07087, and by the Thomas F. JefTressand Kate Miller Jeffi Memorial Trust. 2 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, Box 6 13 MCV Station, Medical College of Virginia/VCU, Richmond, VA 23298. ’ Abbreviations used: AFC, antibody forming cell; ip, intraperitoneal; TCDD, 2,3,7,8-tetrachlorodibenzo-pdioxin; 2,7-DCDD, 2,7dichlorodibenzo-pdioxin; AHH, aryl hydrocarbon hydroxylase; SRBC, sheep red blood cell. 0041-008X/92 $3.00 Copyright 0 1992 by Academic Press,Inc. All rights of reproduction in any form reserved.
weights (i.e., specifically those of the liver and thymus, reflecting hepatomegaly and involution, respectively) and a suppression of humoral and cell-mediated immunity that is consistent with a role by the Ah locus (Kerkvliet et al., 1990; Sharma and Gehring, 1979; Vecchi et al., 1983). However, while these findings are fairly descriptive of acute dosing regimens, several reports have indicated that differences may exist in the toxicities produced by TCDD and closely related congeners, depending on the dose (Clark et al., 198 1, 1983), route (Pohjanvirta et al., 1989), and length of exposure (Holsapple et al., 1986) used by various investigators. Of particular interest is this latter phenomenon, in which our laboratory has previously reported that subchronic exposure of B6C3Fl mice to the low affinity Ah-receptor ligand 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) caused suppression of the in
vivo antibody forming cell (AFC) responseto sheepred blood cells (SRBC), which was comparable to that seen with TCDD, even in the absence of significant induction of liver P450 activity (i.e., aryl hydrocarbon hydroxylase activity; AHH; Holsapple et al., 1986). This finding was one of the first to demonstrate that low affinity agonists for the Ah receptor, while having little or no effect on immunity when given acutely, could have dramatic effects on these immune parameters when administered sequentially over a longer period of time. The goal of the current study was to determine if a similar phenomenon would occur following subchronic administration of TCDD to DBA/2 (Ah-low responder) mice. These mice are less responsive to liver P450 enzyme induction and toxicity induced by TCDD following acute exposures than are B6C3Fl mice. However, if sequential dosing can enhance the suppression of humoral immunity in B6C3Fl mice by what appears to be non-Ah-receptor-mediated mechanisms,
as previously observed with subchronic exposure to 2,7DCDD, then subchrohic exposures of DBA/2 mice to TCDD may likewise be able to increase suppression of the AFC response without significant increases in liver weight, an indirector indicator of liver P450 enzyme induction by TCDD. Our findings demonstrate that DBA/Z mice are more susceptible to suppression of humoral immunity following sub128
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TABLE 1 Body and Organ Weights of B6C3Fl and DBA/2 Mice following Acute Exposure to 2,3,7,8-Tetrachlorodihenzo-p-dioxin (2,3,7,8-TCDD) 2,3,7,8-TCDD (&kg) Vehicle (corn oil)
Parameter
4.2
14
42
B6C3Fl Mice Body weight (g) Liver weight (g) % Body weight Thymus weight (mg) % Body weight Spleen weight (mg) % Body weight
20.88 1.09 5.24 47.60 0.23 89.40 0.43
+ + k + k + k
0.57” 0.03 0.05 3.08 0.02 6.82 0.03
20.52 k 0.56 I .09 f 0.04 5.62 f 0.08 ** 43.80 + 1.39 0.21 + 0.01 72.00 _+ 3.24 0.35 + 0.01*
22.44 k 1.38 k 6.17 k 42.80 + 0.19 f 88.40 + 0.36 k
0.95 0.06** 0.08** 3.05 0.01* 9.27 0.01
21.76 1.36 6.25 30.80 0.14 75.20 0.34
Y!Z0.40 k 0.02** rt 0.04** f 1.91** + 0.01** _+4.70 * 0.02**
19.88 ? 0.69 1.03 + 0.05 * 5.19 rfr 0.07; 33.50 + 0.03 0.17 + 0.01 102.20 + 6.28 0.51 + 0.03
19.34 0.95 4.95 26.80 0.14 77.00 0.40
+ 2 It + f k 2
DBA/2 Mice Body weight (g) Liver weight (g) % Body weight Thymus weight (mg) % Body weight Spleen weight (mg) % Body weight
19.40 0.88 4.51 38.40 0.20 96.20 0.50
+ f * + + rt *
0.67 0.03 0.02 0.04 0.03 5.76 0.03
18.88 0.85 4.51 38.40 0.20 89.60 0.47
+ 0.49 5~0.03 & 0.13 * 0.02 + 0.0 1 f 5.52 + 0.02
0.64 0.03 0.20 0.01’ 0.01* 5.02* 0.02*
Note. Significance, as determined by the Dunnett’s t test, is indicated. *p < 0.05; **p < 0.0 1. a Values represent the mean Itr SE of organ weights derived from four or ftve animals/treatment group.
chronic versus acute exposures to TCDD, without concommitant effects on liver weight (i.e., hepatomegaly). This is in direct contradiction to effects found following acute exposures in either strain, where hepatomegaly and immune suppression appear to be closely paralleled. The importance of these studies is discussed from the standpoint that suppression of in vivo antibody responses in Ah-low responsive mice is directly dependent upon the length and repetitive nature of the exposure to TCDD. MATERIALS
AND METHODS
Animals. (C57BL/6 X C3H)Fl (B6C3FI) mice were purchased through the National Cancer Institute (Frederick, MD). DBA/2 mice were purchased from Charles River Laboratories (Raleigh, NC). Mice arrived at between 5 and 7 weeks of age and were housed four/cage in a humidity and temperatureregulated atmosphere with alternating 12-hr light/dark cycles. Food and water were available ad libitum. Particular care was exerted to guarantee that both strains of animal were age- and weight-matched at between 19 and 22 g at the time of experimentation. Chemicals. 2,3,7,8-Tetrachlorodibenzo-p-dioxin was obtained through the National Toxicology Program and Bio-Rad Laboratories (Rockville Centre, NY) and kept as a 100 &ml solution in corn oil (vehicle; Sigma). Chemical treatmenfs. For acute exposures, animals were administered either corn oil or 4.2, 14, or 42 &kg TCDD via oral gavage on Day 0 of the study. For subchronic (14 day) exposures, animals were administered either corn oil or 0.3, 1.0, or 3.0 &kg TCDD/day via oral gavage for 14 consecutive days (cumulative doses of 4.2, 14, and 42 pg/kg). Organ necropsies and antibody response determinations were performed on Day 15, 24 hr after the last treatment of the subchronically exposed animals.
Necropsies and antiMy forming cell responses. On Day 11, both acutely and subchronically exposed animals were sensitized to the T-dependent antigen SRBC by administration of 5 X lo* SRBC in 1 ml of saline via ip injection. On Day 15, animals were terminated, body and organ weights were recorded, and antiSRBC AFC responses were determined as previously described (Holsapple et al., 1986). We have historically observed that this antibody response peaks 4 days after sensitization. Statistics. Dunnett’s t test was performed on both body and organ weight data and the AFC responses obtained in these studies in order to determine significant treatment effectsas compared to corn oil-treated control animals.
RESULTS
Body and organ weights and Day 4 anti-SRBC AFC responsesof BK3Fl and DBA/2 mice following acute exposuresto TCDD. Consistent with previous reports, Table 1 illustrates the changes that occur in body and organ weight parameters following acute exposure of B6C3Fl (Ah-high responder) and DBA/2 (Ah-low responder) mice to TCDD. Of particular importance are the abrupt changes in liver (hepatomegaly), thymus, and spleen weights in the B6C3Fl mice which occurred in a dose-related manner (i.e., when normalized to percentage of body weight). In contrast, DBAf 2 mice demonstrated little change in liver, thymus, or spleen weight except at the highest dose tested (42 pg/kg). Table 2 shows that the antibody response to SRBC in B6C3Fl mice is likewise reduced in a fashion which dosedependently follows the changes in both liver and thymus weights. In contrast, a significant suppression of the antibody
130
MORRIS ET AL.
TABLE 2 Day 4 IgM Antibody Forming Cell Response in B6C3Fl and DBA/Z Mice following Acute Exposure to 2,3,7,8-Tetrachlorodibenzo-pdioxin (2,3,7,8-TCDD) 2,3,7,8-TCDD bglk) Parameter
Corn oil
4.2
14
42
B6C3Fl Mice Cells/spleen (X 10’) IgM AFC/spleen (X103) IgM AFC/ I O6 recovered cells
12.21 f 0.76” 151 + 26 1163 f 158
10.22 f 0.49 54 + 5** 539 f 71**
11.13 + 0.45 49 f 5** 435 2 33**
10.55 _t 0.5 1 26 f 5** 249 5~ 61**
15.56 -c 1.06 82+ 19 5 18 + 90.67
1 I .50 + 0.7 I** 33 f 2** 287 f 20**
DBA/Z Mice Cells/spleen (X 10’) IgM AFC/spleen (X 103) IgM AFC/ 10 6 recovered cells
17.20 + 1.38 145 f 29 827 + 137
14.48 + 1.15 105 f 17 723 f 107
Significance, as determined by the Dunnett’s t test, is indicated. **p < 0.0 1. a Values represent the mean + SE of the Day 4 anti-SRBC AFC response derived from four or five animals/treatment group.
Note.
response occurred only at the highest dose in the DBA/2 mice, although a dose-related trend was apparent across all doses tested. Body and organ weights and Day 4 anti-SRBC AFC responses of B6C3Fl and DBA/2 mice following subchronic (14 day) exposures to TCDD. In close parallel to acute exposures, body and organ weights in the BK3Fl mice were similarly affected following subchronic (14 day) exposures to TCDD. These effects were characterized by significant and dose-related increases in liver weight parameters and coordinate decreases in both thymus and spleen weights (Table 3). In a similar manner, the Day 4 anti-SRBC AFC response was significantly and dose-dependently suppressed at all doses tested and correlated with the degree of hepatomegaly as assessed by the dose-related increases in liver weight as a function of body weight (Table 4). Subchronic exposures in DBA/2 mice produced changes in both lymphoid organ weight and humoral immune parameters which were inconsistent either with results obtained following acute exposures or with changes in the liver weights of these mice; the latter were only slightly elevated following the 14&y regimen (as compared to acute) and remained at a nonsignificant level at all of the doses tested. In addition, effects on thymus weights (involution) were greatly enhanced (significance was observed in all dose groups) and demonstrated a higher degree of atrophy than was seen even in the B6C3Fl strain (68 versus 56% suppression at the 42 &kg dose) following either dose regimen. This effect on thymus weight was consistent in a second experiment, where exposure to the three cumulative doses of 4.2, 14, and 42 &kg gave thymus weights as percentages of body weight which were 0.25, 0.22, and 0. 1 l%, respectively, as compared to a control value of 0.4 1%.
More consistent with effects on the thymus, as opposed to those on the liver, suppression of the Day 4 anti-SRBC AFC response in the DBA/2 mice was greatly enhanced following the 1Cday exposure. More descriptively, the enhanced suppression resulted in an approximately 1O-fold increase in the magnitude of suppression as related to the same dose administered acutely. As indicated above, a second experiment has confirmed these findings on the suppression of the Day 4 anti-SRBC AFC response in the DBA/2 mice, where the levels of responses in the second study were found to be 793 + 108, 135 k 17 (17%), 65 + 14 (8%), and 61 _+ 34 (7.6%) AFC/ lo6 splenocytes for the corn oil, 4.2, 14, and 42 pg/kg cumulative dose groups, respectively. DISCUSSION Results of the current investigation demonstrate that the conditions of exposure are important in determining the mechanisms that are involved in suppression of humoral immunity by TCDD. This conclusion is based on the finding that subchronic exposure in DBA/2 mice leads to an enhanced suppression of the Day 4 anti-SRBC AFC response in the absence of significant changes in liver weight (i.e., hepatomegaly). These findings are also consistent with previous reports which have demonstrated that subchronic exposure of mice (i.e., B6C3Fl) to either 2,7-DCDD (as previously discussed; Holsapple et al., 1986) or any of several polycyclic aromatic hydrocarbons (PAHs; White et al., 1985) can produce suppression of the anti-SRBC AFC response that is comparable to that produced following acute exposures to TCDD. This latter study is especially noteworthy with respect to the current investigation because of the differential effects that were seen in DBA/2 mice, as opposed to those in the B6C3Fl strain. In particular, the results by
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IMMUNITY
BY TCDD
131
TABLE 3 Body and Organ Weights of B6C3Fl and DBA/2 Mice following Subchronic (14 Day) Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) 2,3,7,8-TCDD (&kg) Parameter
Vehicle (corn oil)
4.2
14
42
B6C3Fl Mice Body weight (g) Liver weight (g) % Body weight Thymus weight (mg) % Body weight Spleen weight (mg) % Body weight
22.38 i 0.18” 1.13 * 0.02 5.06 + 0.13 57.00 i- 2.63 0.25 k 0.01 99.00 +- 3.69 0.44 2 0.02
2 1.94 1.28 5.84 65.60 0.30 82.40 0.37
f f f f f f +
0.40 0.04** 0.07 ** 4.50 0.02 3.56 0.01
23.02 k 1.48 f 6.40 k 46.40 f 0.20 + 85.60 + 0.37 k
0.71 0.06** 0.08** 2.20 0.01 5.91 0.03
22.84 f 1.50 f 6.53 k 25.80 + 0.11 + 76.80 + 0.33 f
0.75 0.06** 0.08** 3.58** 0.01** 5.43* 0.01**
DBA/Z Mice Body weight (g) Liver weight (g) % Body weight Thymus weight (mg) % Body weight Spleen weight (mg) %Body weight
2 I .30 2 0.5 1 1.05 k 0.03 4.94 k 0.10 54.00 k 7.07 0.25 +- 0.04 85.00 + 5.06 0.40 + 0.02
22.28 + 0.43 1.15 -t 0.05 5.16 +- 0.21 32.00 f 1.47* 0.14 f 0.01* 84.00 rf: 2.5 1 0.38 rt 0.01
20.87 + 0.15 1.17 l?r0.05 5.62 + 0.21 26.00 k 0.58 * 0.12 f 0.01** 74.67 + 2.02 0.36 + 0.01
21.57 1.20 5.57 18.30 0.08 61.67 0.29
f 0.59 zk 0.30* + 0.09 f 2.73** + O.Ol** -+-2.96: f 0.02*
Note. Significance, as determined by the Dunnett’s t test, is indicated. *p G 0.05; **p < 0.0 1. a Values represent the mean t SE of organ weights derived from four or five animals/treatment group.
White et al. (1985) indicated that subchronic exposure to either 3-methylcholanthrene or 7,12-dimethylbenz[alanthracene resulted in a higher degree of suppression of the Day 4 antibody response in DBA/2 mice than was seen in the B6C3Fl strain. Similar results were observed in one of the two trials with DBA/2 mice in the present study. This finding is important from the standpoint that the PAHs are
also thought to induce their immunosuppressive effects, at least in part, through Ah-receptor-mediated mechanisms. However, as the results of the current study have demonstrated, these findings are also suggestive of additional mechanisms in suppression of humoral immunity by both the polycyclic and the polyhalogenated aromatic hydrocarbon compounds.
TABLE 4 Day 4 IgM Antibody Forming Cell Responsein B6C3Fl and DBA/2 Mice following Subchronic (14 Day) Exposure to 2,3.7.8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) 2,3,7.8-TCDD (&kg) Parameter
Corn oil
4.2
14
42
15.19 ?z 0.89 80+7** 528 f 50**
13.66 + 0.60* 38 +_ 5** 283 k 42**
12.27 + 0.74 14 + 6** 106 f 49**
10.73 + 0.50 10 f 5** 86 f 37**
B6C3Fl Mice Cells/spleen (X 10’) IgM AFC/spleen (X10-‘) IgM AFC/ 1O6 recovered cells
16.48 f 0.52” 315 * 55 1891 f 302
Cells/spleen (X 10 7, IgM AFC/spleen (X10’) IgM AFC/106 recovered cells
13.24 + 0.55 82+ IO 626 ? 71
14.09 +- 0.49 70 -+ 6** 496 f 49** DBA/Z Mice 12.30 t 1.20 36? II* 271 i 83*
Significance, as determined by the Dunnett’s t test, is indicated. *p < 0.05; **p d 0.0 1. n Values represent the mean + SE of the Day 4 anti-SRBC AFC response derived from four or five animals/treatment group. Note.
132
MORRIS ET AL.
Moreover, this study is important in raising a concern about the relevancy of subchronic exposures in immunotoxicity evaluations of TCDD. Previous investigators have demonstrated that a subchronic exposure regimen based on dosing once per week for several weeks produces a greater suppression in C57BL/6 mice than is observed in the DBA/ 2 strain (Vecchi et al., 1983). A comparison of these results with those obtained in the present study indicates that a subchronic regimen based on weekly exposures produces a profile of immunotoxicity that is more like the one associated with an acute (single) exposure (Clark et al., 198 1; Vecchi et al., 1983). Therefore, when exposures to human populations are considered, a supposed Ah-low responder population, subchronic studies in low-responsive animal models (i.e., DBA/2 mice) may better depict the dose regimen and body burden attained following repeated low level environmental exposures as would have occurred, for example, in Times Beach, Missouri (Knutson, 1984). This is in contrast to exposures that have taken place following large scale industrial accidents as in Seveso, Italy (Pocchiari et al., 1979; Tognoni and Bonaccorsi, 1982), in which acute exposures to large doses of TCDD may be more appropriately applied. In this regard, human populations exposed to low doses over an extended period of time (i.e., months to years) may be at increased risk to immunotoxic effects by these chemicals through additional and presently unidentified mechanisms. REFERENCES Clark, D. A., Gauldie, .I., Szewczuk, M. R., and Sweeney, G. (1981). Enhanced suppressor cell activity as a mechanism of immunosuppression
by 2,3,7,8-tetrachlorodibenzo-pdioxin. Proc. Sot. Exp. Biol. Med. 168, 290. Clark, D. A., Sweeney, G., Safe, S., Hancock, E., Kilbum, D. G., and Gauldie, J. (1983). Cellular and genetic basis for suppression of cytotoxic T cell generation by haloaromatic hydrocarbons. Immunopharmacology6,143. Holsapple, M. P., McCay, J. A., and Barnes, D. W. ( 1986). Immunosuppression without liver induction by subchronic exposure to 2,7-dichlorodibenzo-pdioxin in adult female B6C3Fl mice. Toxicol. Appl. Pharmacol. 83, 445. Kerkvliet, N. I., Steppan, L. B., Brauner, J. A., Deyo, J. A., Henderson, M. C., Tomar, R. S., and Buhler, D. R. (1990). Influence of the Ah ~CXXIS on the humoral immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: Evidence for Ah-receptor-dependent and Ah-receptor-independent mechanisms of immunosuppresion. Toxicol. Appl. Pharmacol. 105,26. Knutson, A. P. (1984). Immunologic effectsofTCDD exposure in humans. Bull.
Environ.
Contam.
Toxicol.
33,673.
Pocchiari, F., Silano, V., and Zampieri, A. (1979). Human health effects from accidental release of tetrachlorodibenzo-pdioxin. Ann. N. Y. Acad. Sci. 320, 31 I. Pohjanvirta, R., Tuomisto, L., and Tuomisto, J. (1989). The central nervous system may be involved in TCDD toxicity. Toxico[ogy 58, 167. Sharma, R. P., and Gehring, P. J. (1979). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on splenic lymphocyte transformation in mice after single and repeated exposures. Ann. N. Y. Acad. Sci. 320,487. Tognoni, G.. and Bonaccorsi, A. (1982). Epidemiological problems with TCDD (A critical review). Drug Metab. Rev. 13(3), 447. Vecchi, A., Sironi. M., Canegrati, M. A., Recchia, M., and Garattini, S. ( 1983). Immunosuppressive effects of 2,3,7,8-tetrachlorcdibenzo-p-dioxin in strains of mice with different susceptibility to induction of aryl hydrocarbon hydroxylase. Toxicol. Appl. Pharmacol. 68, 434. White, K. L., Lysy, H. H., and Holsapple, M. P. (1985). Immunosuppression by polycyclic aromatic hydrocarbons: A structure-activity relationship in B6C3Fl and DBA/2 mice. Immunopharmacology 9, 155.
AND
APPLIED
PHARMACOLOGY
112, 128-l 32 ( 1992)
Enhanced Suppression of Humoral Immunity in DBA/2 Mice following Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)’ D. L. MORRIS,* N. K. SNYDER,*
V. GOKANI,~
R. E. BLAIR,*
AND M. P. HOLSAPPLE*,’
*Medical College of Virginia/Virginia Commonwealth University, Richmond, Virginia 23298; and tNational Institute for Occupational Health, Gjarat, India Received April 25, 199 1; accepted October 1, 199 1
EnhancedSuppressionof Humoral Immunity in DBA/Z Mice following Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzop-dioxin (TCDD). MORRIS, D. L., SNYDER, N. K., GOKANI, V., BLAIR, R. E., AND HOLSAPPLE, M. P. (1992). Toxicof. Appl. Pharmacol. 112,128-132. Previousstudieshave indicated that mice which differ in their acute susceptibility to responsesmediated by the Ah receptor have a pattern of suppressionof the antibody responsewhich is consistentwith a role by the putative dioxin receptor. The objective of the present investigation wasto comparethe TCDDinduced suppressionof the antibody responsefollowing acute and subchronic exposures in B6C3Fl mice, an Ah-high-responderstrain, and DBA/2 mice, an Ah-low-responder strain. Resultsof our initial studies demonstrate that suppressionof humoral immunity can be enhancedin DBA/2 mice approximately lo-fold following subchronic versusacute exposuresto the samecumulativedosesof TCDD. This changein suppression of the antibody responsein DBA/2 mice was not accompanied by significant changesin liver weight (hepatomegaly), as was observedin the B6C3Fl strain when exposedunder comparable conditions. In contrast, effects on thymus weight (involution) wereenhancedin the DBA/2 mice following subchronicexposure and demonstrateda higher degreeof atrophy than was seenin the B6C3Fl strain (68 versus56% decreasein thymic weight at the 42 pg/kg cumulative dose).Thesefindings suggestthat multiple mechanismsmay be operating to suppresshumoral immunity in viva and that the conditions of exposurecan alter the toxic effects of TCDD in the DBA/Z, Ah-low responsive,mouse strain. 0 1992 Academic Press, Inc.
Acute exposure of mice to the compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)3 leads to changes in organ ’ This work was supported by NIH Grant 1RO 1-ES-O3520, by Training Grant NIH-ES-07087, and by the Thomas F. JefTressand Kate Miller Jeffi Memorial Trust. 2 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, Box 6 13 MCV Station, Medical College of Virginia/VCU, Richmond, VA 23298. ’ Abbreviations used: AFC, antibody forming cell; ip, intraperitoneal; TCDD, 2,3,7,8-tetrachlorodibenzo-pdioxin; 2,7-DCDD, 2,7dichlorodibenzo-pdioxin; AHH, aryl hydrocarbon hydroxylase; SRBC, sheep red blood cell. 0041-008X/92 $3.00 Copyright 0 1992 by Academic Press,Inc. All rights of reproduction in any form reserved.
weights (i.e., specifically those of the liver and thymus, reflecting hepatomegaly and involution, respectively) and a suppression of humoral and cell-mediated immunity that is consistent with a role by the Ah locus (Kerkvliet et al., 1990; Sharma and Gehring, 1979; Vecchi et al., 1983). However, while these findings are fairly descriptive of acute dosing regimens, several reports have indicated that differences may exist in the toxicities produced by TCDD and closely related congeners, depending on the dose (Clark et al., 198 1, 1983), route (Pohjanvirta et al., 1989), and length of exposure (Holsapple et al., 1986) used by various investigators. Of particular interest is this latter phenomenon, in which our laboratory has previously reported that subchronic exposure of B6C3Fl mice to the low affinity Ah-receptor ligand 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) caused suppression of the in
vivo antibody forming cell (AFC) responseto sheepred blood cells (SRBC), which was comparable to that seen with TCDD, even in the absence of significant induction of liver P450 activity (i.e., aryl hydrocarbon hydroxylase activity; AHH; Holsapple et al., 1986). This finding was one of the first to demonstrate that low affinity agonists for the Ah receptor, while having little or no effect on immunity when given acutely, could have dramatic effects on these immune parameters when administered sequentially over a longer period of time. The goal of the current study was to determine if a similar phenomenon would occur following subchronic administration of TCDD to DBA/2 (Ah-low responder) mice. These mice are less responsive to liver P450 enzyme induction and toxicity induced by TCDD following acute exposures than are B6C3Fl mice. However, if sequential dosing can enhance the suppression of humoral immunity in B6C3Fl mice by what appears to be non-Ah-receptor-mediated mechanisms,
as previously observed with subchronic exposure to 2,7DCDD, then subchrohic exposures of DBA/2 mice to TCDD may likewise be able to increase suppression of the AFC response without significant increases in liver weight, an indirector indicator of liver P450 enzyme induction by TCDD. Our findings demonstrate that DBA/Z mice are more susceptible to suppression of humoral immunity following sub128
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IMMUNITY
BY TCDD
129
TABLE 1 Body and Organ Weights of B6C3Fl and DBA/2 Mice following Acute Exposure to 2,3,7,8-Tetrachlorodihenzo-p-dioxin (2,3,7,8-TCDD) 2,3,7,8-TCDD (&kg) Vehicle (corn oil)
Parameter
4.2
14
42
B6C3Fl Mice Body weight (g) Liver weight (g) % Body weight Thymus weight (mg) % Body weight Spleen weight (mg) % Body weight
20.88 1.09 5.24 47.60 0.23 89.40 0.43
+ + k + k + k
0.57” 0.03 0.05 3.08 0.02 6.82 0.03
20.52 k 0.56 I .09 f 0.04 5.62 f 0.08 ** 43.80 + 1.39 0.21 + 0.01 72.00 _+ 3.24 0.35 + 0.01*
22.44 k 1.38 k 6.17 k 42.80 + 0.19 f 88.40 + 0.36 k
0.95 0.06** 0.08** 3.05 0.01* 9.27 0.01
21.76 1.36 6.25 30.80 0.14 75.20 0.34
Y!Z0.40 k 0.02** rt 0.04** f 1.91** + 0.01** _+4.70 * 0.02**
19.88 ? 0.69 1.03 + 0.05 * 5.19 rfr 0.07; 33.50 + 0.03 0.17 + 0.01 102.20 + 6.28 0.51 + 0.03
19.34 0.95 4.95 26.80 0.14 77.00 0.40
+ 2 It + f k 2
DBA/2 Mice Body weight (g) Liver weight (g) % Body weight Thymus weight (mg) % Body weight Spleen weight (mg) % Body weight
19.40 0.88 4.51 38.40 0.20 96.20 0.50
+ f * + + rt *
0.67 0.03 0.02 0.04 0.03 5.76 0.03
18.88 0.85 4.51 38.40 0.20 89.60 0.47
+ 0.49 5~0.03 & 0.13 * 0.02 + 0.0 1 f 5.52 + 0.02
0.64 0.03 0.20 0.01’ 0.01* 5.02* 0.02*
Note. Significance, as determined by the Dunnett’s t test, is indicated. *p < 0.05; **p < 0.0 1. a Values represent the mean Itr SE of organ weights derived from four or ftve animals/treatment group.
chronic versus acute exposures to TCDD, without concommitant effects on liver weight (i.e., hepatomegaly). This is in direct contradiction to effects found following acute exposures in either strain, where hepatomegaly and immune suppression appear to be closely paralleled. The importance of these studies is discussed from the standpoint that suppression of in vivo antibody responses in Ah-low responsive mice is directly dependent upon the length and repetitive nature of the exposure to TCDD. MATERIALS
AND METHODS
Animals. (C57BL/6 X C3H)Fl (B6C3FI) mice were purchased through the National Cancer Institute (Frederick, MD). DBA/2 mice were purchased from Charles River Laboratories (Raleigh, NC). Mice arrived at between 5 and 7 weeks of age and were housed four/cage in a humidity and temperatureregulated atmosphere with alternating 12-hr light/dark cycles. Food and water were available ad libitum. Particular care was exerted to guarantee that both strains of animal were age- and weight-matched at between 19 and 22 g at the time of experimentation. Chemicals. 2,3,7,8-Tetrachlorodibenzo-p-dioxin was obtained through the National Toxicology Program and Bio-Rad Laboratories (Rockville Centre, NY) and kept as a 100 &ml solution in corn oil (vehicle; Sigma). Chemical treatmenfs. For acute exposures, animals were administered either corn oil or 4.2, 14, or 42 &kg TCDD via oral gavage on Day 0 of the study. For subchronic (14 day) exposures, animals were administered either corn oil or 0.3, 1.0, or 3.0 &kg TCDD/day via oral gavage for 14 consecutive days (cumulative doses of 4.2, 14, and 42 pg/kg). Organ necropsies and antibody response determinations were performed on Day 15, 24 hr after the last treatment of the subchronically exposed animals.
Necropsies and antiMy forming cell responses. On Day 11, both acutely and subchronically exposed animals were sensitized to the T-dependent antigen SRBC by administration of 5 X lo* SRBC in 1 ml of saline via ip injection. On Day 15, animals were terminated, body and organ weights were recorded, and antiSRBC AFC responses were determined as previously described (Holsapple et al., 1986). We have historically observed that this antibody response peaks 4 days after sensitization. Statistics. Dunnett’s t test was performed on both body and organ weight data and the AFC responses obtained in these studies in order to determine significant treatment effectsas compared to corn oil-treated control animals.
RESULTS
Body and organ weights and Day 4 anti-SRBC AFC responsesof BK3Fl and DBA/2 mice following acute exposuresto TCDD. Consistent with previous reports, Table 1 illustrates the changes that occur in body and organ weight parameters following acute exposure of B6C3Fl (Ah-high responder) and DBA/2 (Ah-low responder) mice to TCDD. Of particular importance are the abrupt changes in liver (hepatomegaly), thymus, and spleen weights in the B6C3Fl mice which occurred in a dose-related manner (i.e., when normalized to percentage of body weight). In contrast, DBAf 2 mice demonstrated little change in liver, thymus, or spleen weight except at the highest dose tested (42 pg/kg). Table 2 shows that the antibody response to SRBC in B6C3Fl mice is likewise reduced in a fashion which dosedependently follows the changes in both liver and thymus weights. In contrast, a significant suppression of the antibody
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MORRIS ET AL.
TABLE 2 Day 4 IgM Antibody Forming Cell Response in B6C3Fl and DBA/Z Mice following Acute Exposure to 2,3,7,8-Tetrachlorodibenzo-pdioxin (2,3,7,8-TCDD) 2,3,7,8-TCDD bglk) Parameter
Corn oil
4.2
14
42
B6C3Fl Mice Cells/spleen (X 10’) IgM AFC/spleen (X103) IgM AFC/ I O6 recovered cells
12.21 f 0.76” 151 + 26 1163 f 158
10.22 f 0.49 54 + 5** 539 f 71**
11.13 + 0.45 49 f 5** 435 2 33**
10.55 _t 0.5 1 26 f 5** 249 5~ 61**
15.56 -c 1.06 82+ 19 5 18 + 90.67
1 I .50 + 0.7 I** 33 f 2** 287 f 20**
DBA/Z Mice Cells/spleen (X 10’) IgM AFC/spleen (X 103) IgM AFC/ 10 6 recovered cells
17.20 + 1.38 145 f 29 827 + 137
14.48 + 1.15 105 f 17 723 f 107
Significance, as determined by the Dunnett’s t test, is indicated. **p < 0.0 1. a Values represent the mean + SE of the Day 4 anti-SRBC AFC response derived from four or five animals/treatment group.
Note.
response occurred only at the highest dose in the DBA/2 mice, although a dose-related trend was apparent across all doses tested. Body and organ weights and Day 4 anti-SRBC AFC responses of B6C3Fl and DBA/2 mice following subchronic (14 day) exposures to TCDD. In close parallel to acute exposures, body and organ weights in the BK3Fl mice were similarly affected following subchronic (14 day) exposures to TCDD. These effects were characterized by significant and dose-related increases in liver weight parameters and coordinate decreases in both thymus and spleen weights (Table 3). In a similar manner, the Day 4 anti-SRBC AFC response was significantly and dose-dependently suppressed at all doses tested and correlated with the degree of hepatomegaly as assessed by the dose-related increases in liver weight as a function of body weight (Table 4). Subchronic exposures in DBA/2 mice produced changes in both lymphoid organ weight and humoral immune parameters which were inconsistent either with results obtained following acute exposures or with changes in the liver weights of these mice; the latter were only slightly elevated following the 14&y regimen (as compared to acute) and remained at a nonsignificant level at all of the doses tested. In addition, effects on thymus weights (involution) were greatly enhanced (significance was observed in all dose groups) and demonstrated a higher degree of atrophy than was seen even in the B6C3Fl strain (68 versus 56% suppression at the 42 &kg dose) following either dose regimen. This effect on thymus weight was consistent in a second experiment, where exposure to the three cumulative doses of 4.2, 14, and 42 &kg gave thymus weights as percentages of body weight which were 0.25, 0.22, and 0. 1 l%, respectively, as compared to a control value of 0.4 1%.
More consistent with effects on the thymus, as opposed to those on the liver, suppression of the Day 4 anti-SRBC AFC response in the DBA/2 mice was greatly enhanced following the 1Cday exposure. More descriptively, the enhanced suppression resulted in an approximately 1O-fold increase in the magnitude of suppression as related to the same dose administered acutely. As indicated above, a second experiment has confirmed these findings on the suppression of the Day 4 anti-SRBC AFC response in the DBA/2 mice, where the levels of responses in the second study were found to be 793 + 108, 135 k 17 (17%), 65 + 14 (8%), and 61 _+ 34 (7.6%) AFC/ lo6 splenocytes for the corn oil, 4.2, 14, and 42 pg/kg cumulative dose groups, respectively. DISCUSSION Results of the current investigation demonstrate that the conditions of exposure are important in determining the mechanisms that are involved in suppression of humoral immunity by TCDD. This conclusion is based on the finding that subchronic exposure in DBA/2 mice leads to an enhanced suppression of the Day 4 anti-SRBC AFC response in the absence of significant changes in liver weight (i.e., hepatomegaly). These findings are also consistent with previous reports which have demonstrated that subchronic exposure of mice (i.e., B6C3Fl) to either 2,7-DCDD (as previously discussed; Holsapple et al., 1986) or any of several polycyclic aromatic hydrocarbons (PAHs; White et al., 1985) can produce suppression of the anti-SRBC AFC response that is comparable to that produced following acute exposures to TCDD. This latter study is especially noteworthy with respect to the current investigation because of the differential effects that were seen in DBA/2 mice, as opposed to those in the B6C3Fl strain. In particular, the results by
SUPPRESSION OF HUMORAL
IMMUNITY
BY TCDD
131
TABLE 3 Body and Organ Weights of B6C3Fl and DBA/2 Mice following Subchronic (14 Day) Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) 2,3,7,8-TCDD (&kg) Parameter
Vehicle (corn oil)
4.2
14
42
B6C3Fl Mice Body weight (g) Liver weight (g) % Body weight Thymus weight (mg) % Body weight Spleen weight (mg) % Body weight
22.38 i 0.18” 1.13 * 0.02 5.06 + 0.13 57.00 i- 2.63 0.25 k 0.01 99.00 +- 3.69 0.44 2 0.02
2 1.94 1.28 5.84 65.60 0.30 82.40 0.37
f f f f f f +
0.40 0.04** 0.07 ** 4.50 0.02 3.56 0.01
23.02 k 1.48 f 6.40 k 46.40 f 0.20 + 85.60 + 0.37 k
0.71 0.06** 0.08** 2.20 0.01 5.91 0.03
22.84 f 1.50 f 6.53 k 25.80 + 0.11 + 76.80 + 0.33 f
0.75 0.06** 0.08** 3.58** 0.01** 5.43* 0.01**
DBA/Z Mice Body weight (g) Liver weight (g) % Body weight Thymus weight (mg) % Body weight Spleen weight (mg) %Body weight
2 I .30 2 0.5 1 1.05 k 0.03 4.94 k 0.10 54.00 k 7.07 0.25 +- 0.04 85.00 + 5.06 0.40 + 0.02
22.28 + 0.43 1.15 -t 0.05 5.16 +- 0.21 32.00 f 1.47* 0.14 f 0.01* 84.00 rf: 2.5 1 0.38 rt 0.01
20.87 + 0.15 1.17 l?r0.05 5.62 + 0.21 26.00 k 0.58 * 0.12 f 0.01** 74.67 + 2.02 0.36 + 0.01
21.57 1.20 5.57 18.30 0.08 61.67 0.29
f 0.59 zk 0.30* + 0.09 f 2.73** + O.Ol** -+-2.96: f 0.02*
Note. Significance, as determined by the Dunnett’s t test, is indicated. *p G 0.05; **p < 0.0 1. a Values represent the mean t SE of organ weights derived from four or five animals/treatment group.
White et al. (1985) indicated that subchronic exposure to either 3-methylcholanthrene or 7,12-dimethylbenz[alanthracene resulted in a higher degree of suppression of the Day 4 antibody response in DBA/2 mice than was seen in the B6C3Fl strain. Similar results were observed in one of the two trials with DBA/2 mice in the present study. This finding is important from the standpoint that the PAHs are
also thought to induce their immunosuppressive effects, at least in part, through Ah-receptor-mediated mechanisms. However, as the results of the current study have demonstrated, these findings are also suggestive of additional mechanisms in suppression of humoral immunity by both the polycyclic and the polyhalogenated aromatic hydrocarbon compounds.
TABLE 4 Day 4 IgM Antibody Forming Cell Responsein B6C3Fl and DBA/2 Mice following Subchronic (14 Day) Exposure to 2,3.7.8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) 2,3,7.8-TCDD (&kg) Parameter
Corn oil
4.2
14
42
15.19 ?z 0.89 80+7** 528 f 50**
13.66 + 0.60* 38 +_ 5** 283 k 42**
12.27 + 0.74 14 + 6** 106 f 49**
10.73 + 0.50 10 f 5** 86 f 37**
B6C3Fl Mice Cells/spleen (X 10’) IgM AFC/spleen (X10-‘) IgM AFC/ 1O6 recovered cells
16.48 f 0.52” 315 * 55 1891 f 302
Cells/spleen (X 10 7, IgM AFC/spleen (X10’) IgM AFC/106 recovered cells
13.24 + 0.55 82+ IO 626 ? 71
14.09 +- 0.49 70 -+ 6** 496 f 49** DBA/Z Mice 12.30 t 1.20 36? II* 271 i 83*
Significance, as determined by the Dunnett’s t test, is indicated. *p < 0.05; **p d 0.0 1. n Values represent the mean + SE of the Day 4 anti-SRBC AFC response derived from four or five animals/treatment group. Note.
132
MORRIS ET AL.
Moreover, this study is important in raising a concern about the relevancy of subchronic exposures in immunotoxicity evaluations of TCDD. Previous investigators have demonstrated that a subchronic exposure regimen based on dosing once per week for several weeks produces a greater suppression in C57BL/6 mice than is observed in the DBA/ 2 strain (Vecchi et al., 1983). A comparison of these results with those obtained in the present study indicates that a subchronic regimen based on weekly exposures produces a profile of immunotoxicity that is more like the one associated with an acute (single) exposure (Clark et al., 198 1; Vecchi et al., 1983). Therefore, when exposures to human populations are considered, a supposed Ah-low responder population, subchronic studies in low-responsive animal models (i.e., DBA/2 mice) may better depict the dose regimen and body burden attained following repeated low level environmental exposures as would have occurred, for example, in Times Beach, Missouri (Knutson, 1984). This is in contrast to exposures that have taken place following large scale industrial accidents as in Seveso, Italy (Pocchiari et al., 1979; Tognoni and Bonaccorsi, 1982), in which acute exposures to large doses of TCDD may be more appropriately applied. In this regard, human populations exposed to low doses over an extended period of time (i.e., months to years) may be at increased risk to immunotoxic effects by these chemicals through additional and presently unidentified mechanisms. REFERENCES Clark, D. A., Gauldie, .I., Szewczuk, M. R., and Sweeney, G. (1981). Enhanced suppressor cell activity as a mechanism of immunosuppression
by 2,3,7,8-tetrachlorodibenzo-pdioxin. Proc. Sot. Exp. Biol. Med. 168, 290. Clark, D. A., Sweeney, G., Safe, S., Hancock, E., Kilbum, D. G., and Gauldie, J. (1983). Cellular and genetic basis for suppression of cytotoxic T cell generation by haloaromatic hydrocarbons. Immunopharmacology6,143. Holsapple, M. P., McCay, J. A., and Barnes, D. W. ( 1986). Immunosuppression without liver induction by subchronic exposure to 2,7-dichlorodibenzo-pdioxin in adult female B6C3Fl mice. Toxicol. Appl. Pharmacol. 83, 445. Kerkvliet, N. I., Steppan, L. B., Brauner, J. A., Deyo, J. A., Henderson, M. C., Tomar, R. S., and Buhler, D. R. (1990). Influence of the Ah ~CXXIS on the humoral immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: Evidence for Ah-receptor-dependent and Ah-receptor-independent mechanisms of immunosuppresion. Toxicol. Appl. Pharmacol. 105,26. Knutson, A. P. (1984). Immunologic effectsofTCDD exposure in humans. Bull.
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Pocchiari, F., Silano, V., and Zampieri, A. (1979). Human health effects from accidental release of tetrachlorodibenzo-pdioxin. Ann. N. Y. Acad. Sci. 320, 31 I. Pohjanvirta, R., Tuomisto, L., and Tuomisto, J. (1989). The central nervous system may be involved in TCDD toxicity. Toxico[ogy 58, 167. Sharma, R. P., and Gehring, P. J. (1979). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on splenic lymphocyte transformation in mice after single and repeated exposures. Ann. N. Y. Acad. Sci. 320,487. Tognoni, G.. and Bonaccorsi, A. (1982). Epidemiological problems with TCDD (A critical review). Drug Metab. Rev. 13(3), 447. Vecchi, A., Sironi. M., Canegrati, M. A., Recchia, M., and Garattini, S. ( 1983). Immunosuppressive effects of 2,3,7,8-tetrachlorcdibenzo-p-dioxin in strains of mice with different susceptibility to induction of aryl hydrocarbon hydroxylase. Toxicol. Appl. Pharmacol. 68, 434. White, K. L., Lysy, H. H., and Holsapple, M. P. (1985). Immunosuppression by polycyclic aromatic hydrocarbons: A structure-activity relationship in B6C3Fl and DBA/2 mice. Immunopharmacology 9, 155.