Estrogen and Estrogenic Chemicals and Autoimmune Disease

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Chapter 2 Preliminary studies to determine the effects of short-term oral dosing of octylphenol and genistein on the thymus, spleen, and lymph nodes of C57BL/6 or B/W mice

Introduction

There is an extensive body of literature that exhibits estrogenic modulation of the immune system. It includes: the identification of the estrogen receptor on immune cells, gender differences in immune responses, and modulation of the progression of autoimmune disease by estrogen (Grossman, 1984; Ansar Ahmed et al., 1985a; Olsen and Kovacs, 1996). Furthermore, a new class of compounds, EDC, has emerged and is continually growing. Many EDCs, which are able to mimic estrogen in the body, have been found in the environment. octylphenol and genistein are two EDCs that have been shown to mimic estrogen both in vitro and in vivo (Arts et al., 1989; Whitten et al., 1993; White et al., 1994; Levy et al., 1995; Blake and Boockfor, 1997; Coldham et al., 1997). Furthermore, both chemicals possess non-estrogenic properties that may also enable them to modulate the immune system. Many studies have reported the ability of genistein and octylphenol to modulate the reproductive system; however few studies have investigated their ability to modulate the immune system. Preliminary in vitro studies in our laboratory examining the effects of
octylphenol, genistein, methoxychlor, and zearalenone on primary isolations of lymphocytes from the thymus, spleen, and lymph nodes did not modulate the immune system. Therefore, this preliminary study investigates the in vivo effects of genistein and octylphenol on the immune systems of mice.
Male C57BL/6 and the B/W orchiectomized mice were utilized in preliminary studies. The C57BL/6 strain is a ‘normal’ inbred strain and the B/W strain develops a lupus-like autoimmune disease. Both strains have been shown to be sensitive to estrogenic modulation of the immune system (Verthelyi and Ansar Ahmed, 1994, 1997, 1998; Walker, 1999). In vivo estrogen treatment has been shown to effect both B cell and T cell responses in orchiectomized male C57BL/6 mice (Verthelyi and Ansar Ahmed, 1994, 1997, 1998; Hissong et al., unpublished data). Furthermore, estrogen has been shown to accelerate the onset of the lupus-like autoimmune disease in B/W males (Walker, 1999). Since both strains of mice have been shown to be sensitive to estrogen treatment, octylphenol was tested in the B/W strain and genistein was tested in the C57BL/6 strain. Furthermore, endogenous estrogens or androgens may interfere in the effects of the estrogenically weaker EDCs. Therefore, B/W males were orchiectomized in order to decrease the levels of endogenous androgens and estrogens and, hopefully, allow maximal effects of octylphenol.

Specific Aim

The aim of the preliminary study was to determine which, if any, of the lymphoid organs or cell populations of the immune system in the orchiectomized or non-orchiectomized male mouse are sensitive to the effects of octylphenol or genistein. The following morphological and functional properties were examined:
a.) Thymus v Morphology: organ weight, cellularity, proportion of immature (CD4+CD8+, CD4-CD8-) and mature (CD4+CD8-, CD4-CD8+) thymocyte subpopulations v Function: apoptosis and proliferation of unstimulated and stimulated thymocytes
b.) Spleen v Morphology: organ weight, leukocyte cellularity, proportion of T cell and B cell subpopulations v Function: proliferation, very early activation antigen expression (CD69), cytokine production, apoptosis, and entrance into the cell cycle upon stimulation c.) Lymph Nodes v Morphology: T cell and B cell subpopulations v Function: proliferation, very early activation antigen expression (CD69), cytokine production, apoptosis, and entrance into the cell cycle upon stimulation

Materials and Methods

Mice: Eight to ten week old C57BL/6 males were purchased from Charles River (Wilmington, MA) and four to six-week old B/W males were purchased from the Jackson Laboratories (Bar Harbor, ME). All mice were housed two per cage, kept on a 14 hour light, 10 hour dark cycle, and fed a soy-depleted commercial pellet diet (Teklaad 7001 (NIH-31), Harlan Teklaad, Madison, WI) with water ad libitum. One week after arrival the B/W males were anesthetized with 0.625 – 1.25 mg of sodium pentobarbitol and orchiectomized (Ansar Ahmed et al., 1989).
Octylphenol treatment: Five-month old orchiectomized B/W males were fed 10 mg of octylphenol three times a week for three weeks. Previous studies have found this dose to be estrogenic in relation to the reproductive changes in male rats (Blake and Boockfor, 1997). Octylphenol (Sigma, St. Louis, MO) dissolved in ethanol at a stock concentration of 10 mg/20 ml was utilized for treating B/W males. Twenty microliters of the stock was mixed with 10 ml of autoclaved corn oil (ICN, Aurora, OH) and fed to the mice with a disposable tip on a micropipetter three times a week for three weeks. Control mice were fed 20 ml of ethanol mixed with 10 ml of corn oil with a micropipetter three times a week for three weeks. The corn oil was autoclaved to eliminate residual
phytoestrogens and eliminate any contamination. Three days after the last treatment the mice were killed by cervical dislocation as approved by the Virginia Polytechnic Institute and State University Animal Care Committee.
Genistein treatment: Five-month old C57BL/6 males were fed 0.6 mg of genistein everyday for three weeks. The dose of genistein is based on studies in mice that have examined its effect on luteinizng hormone release and DNA adduct formation in bone marrow (Hughes et al., 1991; Giri and Lu, 1995). A 1M (270.2 g/L) stock of genistein (Calbiochem, La Jolla, CA) dissolved in dimethylsulfoxide (DMSO) (Fisher Scientific, Pittsburg, PA) was aliquoted and kept –20°C and utilized for these studies. A 1:7 dilution of the genistein stock solution was made in autoclaved corn oil everyday and the 20 ml was fed to each mouse in the genistein treatment group with a disposable tip on a micropipetter.
The control mice were fed 20 ml of a 1:7 dilution of DMSO in corn oil with a micropipetter. The day after the last treatment the mice were killed by cervical dislocation.
Tissue collection and processing: The mice were weighed upon sacrifice. The spleen, thymus, and lymph nodes were removed under sterile conditions, and the spleen and thymus were weighed sterilely. The body weights taken prior to sacrifice were utilized in the % body weights ratios reported. The spleen, thymus, and lymph nodes were gently teased on a sterile 60-mesh steel screen as described in earlier studies (Ansar Ahmed et al., 1989). All cells were washed
with RPMI media (CellGro, Mediatech, Herndon, VA). The erythrocytes were removed from the splenic cell suspension by lysis in ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4) as previously described (Ansar Ahmed et al., 1994). All cells were then washed in complete RPMI media (RPMI media supplemented with 10% charcoal-stripped fetal bovine serum (Hyclone, Logan, UT), 2mM L-glutamine (ICN, Costa Mesa, CA), 50 IU/ml penicillin (Mediatech), and non-essential amino acids (Fisher)). The lymphocytes in the thymus and leukocytes in the spleen and lymph nodes were then counted and the viability was assessed by trypan blue exclusion. The number of lymphocytes and leukocytes isolated from each organ was noted, and all cells were adjusted to a density of 5 X 106 cells/ml in complete RPMI and kept at 4°C
until utilized in assays.
Flow cytometric analysis of cell markers: Cells from the thymus, spleen, and lymph nodes were stained with appropriate monoclonal antibodies and analyzed by flow cytometry as previously reported (Ansar Ahmed et al., 1994). Briefly, 100 ml of cells at 5 X 106/ml were plated into 96-well round bottom tissue culture plates (Corning, NY). One hundred microliters of diluted fluorochromeconjugated monoclonal antibody in incomplete media, or the appropriate species
and isotype matched control antibody in incomplete RPMI media were added to each well. The cells were incubated with the antibodies in the dark at 4°C for 30 minutes. The cells were then washed and analyzed for fluorescence on an EPICS XL-MXL flow cytometer (Coulter, Hialeah, FL). The following antibodies were utilized in these studies: fluorescein isothyocynate (FITC)-conjugated anti-CD8a (53-6.7), anti-CD90 (Thy1.2, G7), anti-CD11b (Mac-1, M1/70), and a rat IgG2a
isotype control (R35-95) or phycoerythrin (PE)-conjugated anti-CD4 (RM4-5), anti-CD19 (1D3), and rat IgG2a isotype control (R35-95). All monoclonal antibodies were purchased from Pharmigen (San Diego, CA).
Stimulation and expression of Very Early Activation Antigen (CD69):
One hundred microliters of cells from the thymus, spleen, and lymph nodes at a density of 5 X 106 cells/ml were added to 96-well round bottom plates. One hundred microliters of complete RPMI media containing 10 ng/ml of phorbol myristate acetate (Sigma) and 0.5 mg/ml of ionomycin (Calbiochem) (PMA+I) were added to each well. The cells were incubated at 37°C, 5% CO2. After 6
hours, the plate was centrifuged at 300 X g for 6 minutes to pellet the cells and the supernatants were removed. The cells were resuspended in 100 ml of incomplete RPMI media and stained as in the previous section for cell surface markers. The cells were stained with either PE-conjugated anti-CD69 (H1.2F3) (Pharmigen) or PE-conjugated hamster IgG isotype control (A19-3) (Pharmigen)
and analyzed by the flow cytometer for fluorescence.
Stimulation and cell cycle analysis: One hundred microliters of cells from the thymus, spleen, and lymph nodes at a density of 5 X 106 cells/ml were added to 96-well round bottom plates. Thymocytes were stimulated with 100 ml of complete RPMI media containing 15 mg/ml of anti-CD3 antibodies (YCD3, purified in our lab), or complete RPMI media containing 10 ng/ml of phorbol myristate acetate and 0.5 mg/ml of ionomycin (PMA+I). Thymocyte cultures that received 100 ml of complete media only served as base-line controls. Leukocytes from the spleen and lymph nodes were exposed to 100 ml of complete media, complete media containing 10 mg/ml conconavalin A (Con A, ICN), complete RPMI media containing 10 mg/ml lipopolysaccharide (LPS, Sigma), or complete media containing 10 ng/ml of phorbol myristate acetate and 0.5 mg/ml of ionomycin. Plates were incubated at 37°C, 5% CO2 for 8, 12, 24, or 48 hours.
After incubation, the cells were pelleted at 200 X g for 5 minutes and resuspended in Vindelov’s propidium iodide solution followed by the addition of sodium azide buffer as described previously (Robinson et al., 1993; Zhi-June et al., 1998). Samples were incubated overnight at 4°C followed by flow cytometric analysis using DNA cell cycle analysis software. The cells are divided into the
sub G0 (or apoptotic), G1/G0, or S and G2/M phases of the cell cycle. The data are presented as the change from the media baseline of the stimulated cells in the proportion of cells in each phase.
Alamar Blue proliferation assay: Lymphocytes from the thymus and leukocytes from the spleen and lymph nodes were stimulated as described above for cell cycle analysis. Proliferation was determined utilizing the non-radioactive Alamar Blue assay. The Alamar Blue dye (Accumed International, Westlake, OH) contains a redox indicator that changes from a blue to a red color and from non-fluorescent to fluorescent state in the microenvironment of cellular proliferation. The extent of color change or the intensity of fluorescence is a reflection of the cellular proliferation. Studies in our laboratory have shown that the Alamar Blue assay is an indirect method of detecting cellular proliferation that is comparable to the 3H-thymidine incorporation method (Ansar Ahmed et al., 1994, Zhin-Jun et al., 1998). Briefly, 20 ml of Alamar Blue dye were added to each well at the same time the cells and stimulants were added. The cells were incubated with stimulants and Alamar Blue dye for 48 hours at 37°C, 5% CO2.
At 8, 12, 24, and 48 hours of incubation, the fluorescence of each well was determined utilizing the CytoFluor II, fluorescence multiwell plate reader (PerSpectives Biosystems, Framington, MA) at excitation 530 nm, emission 590 nm, and a gain of 35. The data are presented as the change from the media baseline of the stimulated cells in the fluorescence readings. IL-2 and IFN-gg ELISA: Leukocytes isolated from the spleen and lymph nodes were stimulated as described above for cell cycle analysis. Cells were incubated for 24 and 48 hours at 37°C, 5% CO2, and subsequently, cells were pelleted by centrifuging at 300 X g for 6 minutes. The supernatants were removed and frozen for use in an enzyme-linked immunosorbent assay (ELISA) to determine cytokine levels. A sandwich ELISA was utilized to determine the levels of IL-2, and IFN-g. High-binding, flat bottom 96-well Nunc-immunoplates with MaxiSorb surface (Nunc, Denmark) were coated with 50 ml of rat antimouse IL-2 (JES6-1A12, Pharmigen) or rat anti-mouse IFN-g antibodies (R4-6A2, purified in our lab) at 2 mg/ml in PBS. The plates were incubated overnight at 25-27°C and then washed three times with wash buffer (50 mM Tris with 0.2% Tween 20, pH 7.0-7.4). The plates were then blocked with 200 ml of 2% bovine serum albumin (BSA) in PBS per well and incubated for one hour. Supernatants were diluted in complete media and 100 ml/well were added to each plate after three washes. Serial dilutions of known standards were also added to each plate at 100 ml/well. The samples and standards were incubated for 3 hours. After incubation, the plates were washed three times and anti-IL-2 (TES6-5H4, Pharmigen) or anti-IFN-g (XMG1.2, Pharmigen) biotinylated detecting antibody were diluted 1:2000 and added at 100 ml/well to each plate. Plates were incubated for 1 hour and washed again three times. Horse-radish peroxidaseconjugated streptavidin (Vector Labs, Burlingame, CA) was diluted 1:4000 and 100 ml was added/well to each plate. The plates were incubated covered for 30 minutes and then washed three times. The TMB substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was then added at 100 ml/well and incubated in the dark for 30 minutes. One hundred microliters of a 0.18 M H2SO4 solution were added to each well to stop the reaction and the absorbance of the plates was read at 450 nm on the Molecular Devices kinetic plate reader. The concentrations of cytokines per well of the ELISA plate were determined utilizing known standards on each plate and the concentration of cytokines per sample of supernatants was determined by the concentration per well and the dilution utilized for the ELISA.
Statistics: The MIXED procedure of SAS (SAS ver. 6.12, SAS Institute, Cary, NC) was used to perform a mixed effects, repeated measures analysis of variance (ANOVA) on the cell cycle and proliferation data. A first order autoregressive model was used to model covariance among repeated measurements from a mouse. Cell cycle and proliferation data were analyzed as the change from the media baseline. All other data were analyzed utilizing the GLM procedure of SAS to perform a one-way ANOVA. For all data, p<0.05 was considered significant.

Results

Effect of octylphenol treatment on the morphology of the thymus in orchiectomized B/W males: Octylphenol treatment altered the morphology of the thymus in orchiectomized B/W males. The number of thymocytes recovered from the thymus of octylphenol treated mice was significantly less than the number recovered from control mice (Fig 2.1a). However, a comparable decrease in the percent body weight of the thymus was not seen (Fig 2.1b). Furthermore, no change in the proportion of immature (CD4+CD8+) thymocytes or presumably mature (CD4+CD8-, CD4-CD8+) thymocytes was noted (Fig 2.2).
Effect of octylphenol treatment on the function of thymocytes in orchiectomized B/W males: The effect of octylphenol treatment on the proliferation or level of apoptosis of stimulated thymocytes was not statistically significant (Fig 2.3, 2.4). The proliferation of thymocytes induced by anti-CD3 antibodies or PMA+I at 8 and 24 hours of culture was not altered by octylphenol treatment (Fig 2.3). Furthermore, the percent of apoptotic cells at 8 and 24 hours of culture that were induced by stimulation with anti-CD3 antibodies and PMA+I was not statistically significant (Fig 2.4). However, a trend of increased induction of apoptosis compared to control was seen at 8 hours of culture by anti-CD3 stimulation and, to a lesser extent, PMA+I stimulation, but was gone by 24 hours (Fig 2.4).
Effect of octylphenol treatment on the morphology of the spleen and lymph nodes in orchiectomized B/W males: No change in the number of splenic leukocytes or in the percent body weight of the spleen was noted in octylphenol treated mice (Fig 2.1a,b). Furthermore, octylphenol treatment neither altered the proportion of Thy1.2+ T cells or CD19+ B cells (Fig 2.5), nor altered the proportion of the T helper cells (CD4+) and T cytotoxic cells (CD8+) in the spleen or lymph nodes (Fig 2.5). Finally, the CD11b+ developing granulocyte, monocyte and macrophage population in the spleen also was not affected by octylphenol treatment (Fig 2.5).

Chapter 1: Introduction, Rationale, and Literature Review 
a.) Introduction and Rationale
b.) Literature Review
Estrogen
Structure and Function
Estrogen Receptor
Endocrine Disrupting Chemicals
Studies in Wildlife `
Studies in Laboratory Animals
Studies in Humans
Octylphenol
Genistein
Estrogen and Estrogenic Chemicals and the Immune System
Gender Differences
Estrogenic Treatment
Estrogen and Estrogenic Chemicals and the Thymus
Thymus
Estrogenic Effects
Apoptosis
Estrogen and Estrogenic Chemicals and Autoimmune Disease
Gender Differences
Influences of Estrogen
Animal Models
(NZBxNZW)F1 Model for SLE, (B/W)
Chapter 2: Preliminary studies to determine the effects of short-term oral dosing of octylphenol and genistein on the thymus, spleen, and lymph nodes of C57BL/6 or B/W mice 19
a.) Introduction
b.) Specific Aim
c.) Materials and Methods
d.) Results
e.) Discussion
Chapter 3: Dose range studies to determine the effects of short-term oral administration of octylphenol on the thymus of reproductively mature C57BL/6 and B/W males 
a.) Introduction
b.) Specific Aims
c.) Materials and Methods
d.) Results
e.) Discussion
Chapter 4: Route of administration studies to compare the effects of oral and subcutaneous administration of octylphenol and 17b-estradiol in C57BL/6 and B/W Mice 
a.) Introduction
b.) Specific Aims
c.) Materials and Methods
d.) Results
e.) Discussion
Chapter 5: General Discussion 
Literature Cited
Appendix 
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Effects of Short-Term Exposure to Octylphenol and Genistein on the Immune System of C57BL/6 and (NZBxNZW)F1 Mice