Transcript Slide 1

EFFECTS OF LATENT IN UTERO AND PERINATAL TCDD
ND
EXPOSURE IN POST-NATAL C57BL/6 AND SNF1 MURINE 2
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.
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Check Dams for
Plugs
Expose to
TCDD via
Oral Gavage
First litter of
pups born
Dams
remated
Second Litter Postnatal
of Pups Born Day 1
Experiment
Postnatal
Day 14
Experiment
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 cellcontaining wells at 100 µl aliquots in triplicate and cultured for 72 hr..
Cell proliferation was measured with the AlamarBlue™ assay.
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Percentage±SEM
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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, CD4CD8-) 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.
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Dose (ug/kg)
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RESULTS: Double positive (CD4+CD8+) expression of cell surface antigens decreases slightly
in the C57BL/6 mice while it increase slightly in the SNF1.
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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 expression
based upon fluorescent expression.
Figure 5: Splenocyte mitogenic proliferation (PND 21)
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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
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References
48 LPS
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48 PMI
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.
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5
10
Dose (ug/kg)
Figure 3: Liver (PND 1) Absolute CD44+ Expression (PND1)
B
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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.
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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.
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Environmental Implications
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B
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∆Abs
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.
Postnatal
Day 21
Experiment
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Dose (ug/kg)
Figure 2: Liver Density (PND 1)
Millions of Cells/gram±SEM
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
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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 1228 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.
Millions of Cells±SEM
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).
5th TCDD Halflife
Day 78
4th TCDD Halflife
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Day 64
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Materials and Methods
3rd TCDD Halflife
Day 43
2nd TCDD Halflife
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Discussion
Percentage±SEM
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1st TCDD Halflife
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Day 21
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Day 12
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Figure 4: Thymic CD4+CD8+ Expression (PND14)
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∆Abs
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.
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Millions of Cells/gram±SEM
Introduction
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Day 0
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.
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Millions of Cells/gram±SEM
Abstract
Figure 1: Experimental Timeline
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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
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Rocha B, Bassalli P, Guy-Grand D.
The extrathymic T-cel
development pathway. Immunol Today 13:449-454, 1992.
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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.
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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.
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.
Acknowledgement
Funded by NIH R21-PAR-03-121