EFFECTS OF LATENT IN UTERO AND PERINATAL TCDD EXPOSURE IN POST-NATAL C57BL/6 AND SNF1 MURINE 2 ND LITTER OFFSPRING

1. EFFECTS OF LATENT IN UTERO AND PERINATAL TCDD EXPOSURE IN POST-NATAL C57BL/6 AND SNF1 MURINE 2ND LITTER OFFSPRING M.B.Goff1, A. Mustafa1, S.D. Holladay1, R.P. Kerr1, R.M. Gogal Jr.1. 1Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Disease, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia. Discussion On PND1, the neonatal liver still serves as a hematopoietic organ. Evaluation of the neonatal liver showed a dose-dependent decrease in cellular density across both low affinity and high affinity AhR murine strains. Interestingly, the stem cell population appeared to decline in the C57BL/6 strain yet increased in the SNF1. This would suggest that AhR affinity can influence the degree and direction of immune dysregulation. On PND 14, thymus evaluation revealed a notable shift in the double positive (DP) expression profile of immature thymocytes such that a dosed dependent decrease was seen in the C57BL/6 mice whereas an increase was seen in the SNF1 mice. These opposite response patterns would continue to enforce the assumptions noted above concerning AhR affinity. The change in double positive expression is also important to note because dioxin’s effect on the thymus is presumed to be most pronounced during cortical thymocyte maturation. Thymocytes mature from being double negative (DN, CD4-CD8-) to being double positive within the cortex. Therefore, DP expression would be the first measureable endpoint for cell surface protein expression after the stage at which dioxin is presumed to attack the thymus. Analysis of splenic lymphocytes on PND 21 revealed that immune functionality, as measured by response to mitogens, showed a decreasing trend in both the high and low affinity strains. In previously reported data sets, expression patterns were altered such that opposing trends occurred in the high and low dose strains respectively. In this instance, the data suggest that both T and B lymphocyte functionality as a whole is impaired following a latent dioxin exposure much lower than the primary exposure. Environmental Implications TCDD is a well-known mutagen, teratogen, and carcinogen in which other environmental chemicals are compared against for measuring toxicity. In this study, we have shown that a single environmentally relevant dose of TCDD administered during gestation of a first pregnancy can adversely affect the immune system of offspring from a second pregnancy in the same mother. The consequence being an impaired immune system, which could lead to a variety of disorders ranging from a reduced ability to respond to protectively against pathogens to enhanced susceptibility to developing auto immune disease. Abstract TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and its congeners have a long and well-documented history of causing damage from both acute and chronic exposure. However, the potential risks associated with latent in utero and perinatal exposure to dioxin have not been investigated. In this preliminary study, we evaluated the latent effect of TCDD exposure in utero on the select immune parameters of murine offspring on postnatal day 1 (PND1), postnatal day 14 (PND14), and postnatal day 21 (PND 21). C57BL/6 mice, a known high-affinity aromatic hydrocarbon receptor (AhR) strain, and SNF1 (NZB x SWR) mice, an autoimmune disease predisposed strain with a low-affinity AhR receptor, were used. Murine dams (C57BL/6 and SWR) were acutely exposed to either corn oil vehicle or TCDD (C57BL/6, 2.5, 5 or 10µg/kg; SNF1, 40 or 80µg/kg) on gestation day 12. Post-parturition, the dams nursed their first litter for 21-25 days upon which the pups were weaned. At approximately 32 days post-exposure, TCDD-exposed C57BL/6 and SWR mothers were rebred. The pups from these litters were randomly evaluated on PND 1, PND 14, and PND 21 for alterations in immune phenotype. C57BL/6 mice showed dose dependent decrease in splenic weight, liver cellularity, CD44+ expression in the liver, CD8+ expression in the thymus, and CD90+ expression in the spleen. In contrast, SNF1 mice revealed increases in thymic cellularity, CD4+CD8+ and CD4+ expression in the thymocytes, and splenic cellularity and mature B-cell population. Data from this preliminary study indicate that latent TCDD exposure results in immune dysregulation in both C57BL/6 and SNF1 mice. Figure 4: Thymic CD4+CD8+ Expression (PND14) Figure 1: Experimental Timeline A B METHODS: General timeline of prenatal and postnatal exposure showing initiation of breeding, TCDD exposure time, and estimated TCDD half life in the body. Dioxin’s estimated half life is 12-28 days in mice. Based on this timeline, we project that two half lives had occurred prior to exposure to the second litter resulting in an exposure of approximately one-quarter the original dose. If each mouse is approximately 25 g and dosing is based on µg/kg, then we divide the dose by 40 to get an approximate mass/mouse. Literature further suggests that 0.5% is able to cross the placenta and expose an average of 8 pups. Based on these calculations involving dose and half-life, we predict that each pup received between 0.01 pg and 0.32 pg transplacentally before data collection at PND1. Introduction TCDD is the most biologically active compound of a family of immunotoxic congeners to which humans and animals are unavoidably exposed due to their ubiquitous nature. According to EPA estimates as of 2000, the lifetime cancer risk associated with dioxin exposure is between 1 in 1000 and 1 in 100. Additionally, “safe levels” of dioxin exposure are 300-600 times less than the present average daily human exposure. In general, mammalian species show an prenatal sensitivity far greater than that of an adult. Furthermore, fetal damage in the womb can have long term consequences. In this study, we examined the effects of latent TCDD exposure on immune development postnatally in 2nd litter murine offspring. Depending on the mouse strain, the reported half-life of dioxin ranges from 7-31 days, with averages at 14 days. This latent exposure model works under a hypothesis that prior to organogenesis the dioxin levels present in the dam has been subject to multiple half-lives, thus significantly reducing its concentration. However, even with this reduction in dioxin load, there is belief that the exposure both transplacentally and via lactation will result in abnormal immune development most notably seen as immunosuppressive effects and as inappropriate enhancement of immune function. Figure 2: Liver Density (PND 1) B A RESULTS: Double positive (CD4+CD8+) expression of cell surface antigens decreases slightly in the C57BL/6 mice while it increase slightly in the SNF1. METHODS: After collection of organs and enrichment of lymphocytic cells, PE conjugated CD4+ and FITC conjugated CD8+ were mixed with cells subsequently examined using flow cytometry. Using a simultaneous two color analysis, cytometer is able to elucidate percent expressionbased upon fluorescent expression. Figure 5: Splenocyte mitogenic proliferation (PND 21) A • Materials and Methods • Mice: Pregnant C57BL/6 (high affinity AhR) and SNF1 (low affinity AhR) mice were orally dosed with TCDD at 0.0, 5.0 and 10 g/kg and 0.0, 40.0 and 80.0 g/kg, respectively. After parturition of the first litter, juvenile mice were weaned at 21-25 days for a separate study and dams rebred to generate the second litters. At postnatal day (PND) 1, four mice were collected from each dam with tissues of three mice pooled for immune analysis while the fourth was used for histopathology. At PND 14 and 21, two juvenile mice were randomly selected (one for histopathology and one for immune phenotyping). • Organ and Body Weights: Organ and body weights were collected at PND 1, 14, and 21. At PND 1 the organ weight for thymus, spleen, and liver were pooled while at PND 14 and 21 • Organ Dissociation and Cellularity: Organ dissociation was accomplished by manual technique using a 60 nm stainless steel screen.Cell enumeration was performed on a Beckman-Coulter Multisizer 3 with cell suspensions adjusted to 5.0 x 106 cells/mL. • Flow Cytometric Analysis: An aliquot of cells (5x105/100 µl/well) was added to each well of a microtiter plate containing fluorescent antibodies for thymic lymphocytes (PE-anti-CD4 and FITC-anti-CD8a), splenic lymphocytes (PE-anti-CD45 and FITC-anti-CD90 (Thy1.2)) and hepatocytes (PND1 only,FITC-anti-CD44). Cells were incubated at 4ºC, 45 min on an orbital mixer at a slow speed in the dark. Cells were washed at • 250 x g 10 min, resuspended in 200 µL of PBS and analyzed on an Epics XL/MXL flow cytometer. • Proliferation Analysis: Cell aliquots (5x105/100/well) in complete media (RPMI + glutamine + HEPES + penicillin/streptomycin + MEM + heat inactivated FBS) were added to wells of a microtiter tissue culture plate. Media, concanavalin A (ConA), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate + ionomycin (PMI) were added to cell-containing wells at 100 µl aliquots in triplicate and cultured for 72 hr.. Cell proliferation was measured with the AlamarBlue™ assay. RESULTS: In both the C57BL/6 (A) mice and the SNF1 (B) mice, a decrease at PND 1 in liver density is seen as the TCDD dose increases. METHODS: Liver density is calculated by taking the cellularity as obtained on the Coulter Multisizer and dividing it by the weight of the pooled organs from three pups at PND 1 • References • Holladay SD, Luster MI. alterations in fetal thymic and liver hematopoietic cells as an indicator of exposure to developmental immunotoxicants. Environmental Health Perspectives 104:809-813, 1996. • Kakkanaiah VN, Pyle RH, Nagarkatti M, Nagarkatti PS. Evidence for major alterations in the thymocyte subpopulations in murine models of autoimmune diseases. J Autoimmunity 3:27-28, 1990 • Rocha B, Bassalli P, Guy-Grand D. The extrathymic T-cel development pathway. Immunol Today 13:449-454, 1992. • Silverstone AE, Frazier DE Jr, Gasiewicz TA. Alternate immune system targets for TCDD: Lymphocyte stem cells and extrathymic T-cell development. Exp Clin Immunogenet 11:94-101, 1994. Figure 3: Liver (PND 1) Absolute CD44+ Expression (PND1) B B A RESULTS: Both the C57BL/6 (A) and the SNF1(B) mice show a decreasing response to mitogens Con A (T-cell subset), LPS (B-cell subset) and PMI (pan-lymphocyte). METHODS: After cells are enriched and treated with complete media to ensure viability, they are put into 96-well plates and mixed with mitogens at standard concentrations. After 24 hours of culture at 37ºC and 4.5% CO2, Alamar BlueTM is added to each well. After another 48 hours, the plates are read for absorbance at 570 nm. Change in absorbance (∆Abs) is based on media without added mitogens. RESULTS: The C57BL/6 (A) mice showed a decrease in CD44+ bright expression in the total number of cells examined whereas the SNF1 (B) mice showed an increase. METHODS: Absolute expression is calculated by taking the number of cells generated enumerated and multiplying this by the percent expression of selected fluorescence as determined through flow cytometry. • Acknowledgement • Funded by NIH R21-PAR-03-121