XX sex chromosome complement modulates immune responses to heat-killed Streptococcus pneumoniae immunization in a microbiome-dependent manner.

BACKGROUND: Differences in male vs. female immune responses are well-documented and have significant clinical implications. While the immunomodulatory effects of sex hormones are well established, the contributions of sex chromosome complement (XX vs. XY) and gut microbiome diversity on immune sexual dimorphisms have only recently become appreciated. Here we investigate the individual and collaborative influences of sex chromosome complements and gut microbiota on humoral immune activation. METHODS: Male and female Four Core Genotype (FCG) mice were immunized with heat-killed Streptococcus pneumoniae (HKSP). Humoral immune responses were assessed, and X-linked immune-related gene expression was evaluated to explain the identified XX-dependent phenotype. The functional role of Kdm6a, an X-linked epigenetic regulatory gene of interest, was evaluated ex vivo using mitogen stimulation of B cells. Additional influences of the gut microbiome on sex chromosome-dependent B cell activation was also evaluated by antibiotically depleting gut microbiota prior to HKSP immunization. Reconstitution of the depleted microbiome with short-chain fatty acid (SCFA)-producing bacteria tested the impact of SCFAs on XX-dependent immune activation. RESULTS: XX mice exhibited higher HKSP-specific IgM-secreting B cells and plasma cell frequencies than XY mice, regardless of gonadal sex. Although Kdm6a was identified as an X-linked gene overexpressed in XX B cells, inhibition of its enzymatic activity did not affect mitogen-induced plasma cell differentiation or antibody production in a sex chromosome-dependent manner ex vivo. Enhanced humoral responses in XX vs. XY immunized FCG mice were eliminated after microbiome depletion, indicating that the microbiome contributes to the identified XX-dependent immune enhancement. Reconstituting microbiota-depleted mice with select SCFA-producing bacteria enhanced fecal SCFA concentrations and increased humoral responses in XX, but not XY, FCG mice. However, exposure to the SCFA propionate alone did not enhance mitogenic B cell stimulation in ex vivo studies. CONCLUSIONS: FCG mice have been used to assess sex hormone and sex chromosome complement influences on various sexually dimorphic traits. The current study indicates that the gut microbiome impacts humoral responses in an XX-dependent manner, suggesting that the collaborative influence of gut bacteria and other sex-specific factors should be considered when interpreting data aimed at delineating the mechanisms that promote sexual dimorphism.

Male and female immune systems differ in their ability to respond to infectious challenge. While males tend to be more susceptible to infection and produce lower amounts of antibodies in response to vaccination, females are more prone to develop autoimmune and inflammatory diseases. Key contributors to these differences include sex hormones, sex chromosome complement (XX in females vs. XY in males), and distinct gut microbial communities capable of regulating immune activation. While each factor has been studied individually, this research underscores the potential for these factors to collaboratively impact immune activation. Here, possession of an XX vs. XY sex chromosome complement was demonstrated to enhance antibody responses to heat-killed Streptococcus pneumoniae vaccination. While attempting to determine the underlying cause of this immune enhancement, the gut microbiome was identified to play a critical role. In the absence of an intact gut microbiome, XX immune activation was reduced to levels similar to those seen in XY sex chromosome complement-possessing mice. Replacement of the depleted gut microbiomes with select SCFA-producing bacterial species enhanced SCFA levels in antibiotic-treated mice and rescued the XX-dependent immune enhancement, suggesting a SCFA-mediated contribution. Further studies are needed to determine exactly how these select bacteria impact immune activation in a sex chromosome complement-dependent manner. Our findings highlight the need to consider the collaborative effects of individual sex-specific factors when attempting to understand immune sex biases, as a better understanding of these interactions will likely pave the way for improving therapeutics and vaccines tailored to both sexes.

XX sex chromosome complement modulates immune responses to heat-killed Streptococcus pneumoniae immunization in a microbiome-dependent manner.

Background: Differences in male vs. female immune responses are well-documented and have significant clinical implications. While the immunomodulatory effects of sex hormones are well established, the contributions of sex chromosome complement (XX vs. XY) and gut microbiome diversity on immune sexual dimorphisms have only recently become appreciated. Here we investigate the individual and collaborative influences of sex chromosome complements and gut microbiome bacteria on humoral immune activation. Methods: Sham-operated and gonadectomized male and female Four Core Genotype (FCG) mice were immunized with heat-killed Streptococcus pneumoniae (HKSP). Humoral immune responses were assessed, and X-linked immune-related gene expression was evaluated to explain the identified XX-dependent phenotypes. Ex vivo studies investigated the functional role of Kdm6a, an X-linked epigenetic regulatory gene of interest, in mitogenic B cell activation. Additionally, we examined whether gut microbiome communities, or their metabolites, differentially influence immune cell activation in a sex chromosome-dependent manner. Endogenous gut microbiomes were antibiotically depleted and reconstituted with select short-chain fatty acid (SCFA)-producing bacteria prior to HKSP immunization and immune responses assessed. Results: XX mice exhibited higher HKSP-specific IgM-secreting B cells and plasma cell frequencies than XY mice, regardless of gonadal sex. Although Kdm6a was identified as an X-linked gene overexpressed in XX B cells, inhibition of its enzymatic activity did not affect mitogen-induced plasma cell differentiation or antibody production in a sex chromosome-dependent manner ex vivo. Enhanced humoral responses in XX vs. XY immunized FCG mice were eliminated after microbiome depletion, indicating that the microbiome contributes to the identified XX-dependent immune enhancement. Reconstituting microbiota-depleted mice with select SCFA-producing bacteria increased humoral responses in XX, but not XY, FCG mice. This XX-dependent enhancement appears to be independent of SCFA production in males, while female XX-dependent responses relied on SCFAs. Conclusions: FCG mice have been used to assess the influence of sex hormones and sex chromosome complements on various sexually dimorphic traits. The current study indicates that the gut microbiome impacts humoral responses in an XX-dependent manner, suggesting that the collaborative influence of gut bacteria and other sex-specific factors should be considered when interpreting data aimed at delineating the mechanisms that promote sexual dimorphism.

Long-term Immunotoxic Effects of Oral Prenatal and Neonatal Atrazine Exposure.

Atrazine and its metabolites are present at high concentrations in many water supplies in agro-intensive areas. Because residents in these areas drink water from sources fed from these contaminated supplies, we investigated the long-term immunotoxicity of combined prenatal and neonatal (perinatal) exposure to atrazine via drinking water, on the immune system in mice. At 6 months of age, upon immunization with heat-killed Streptococcus pneumoniae, the serum IgG antibody response against the T independent antigen phosphorylcholine was significantly higher in male, but not female, atrazine-exposed mice as compared with that in untreated controls. No alterations were present in all offspring in the serum antibody response against the T-dependent antigen pneumococcal surface protein A (PspA). ELISpot analysis showed only a small, insignificant reduction in PspA-specific IgG producing splenocytes in atrazine-treated male offspring. Interestingly, upon ex vivo stimulation with anti-CD3 and anti-CD28 antibodies, significant decreases in interleukin (IL)-2, tumor necrosis factor-α, interferon-γ, and IL-17A and a decreasing trend in IL-10 were observed in splenocytes from atrazine-exposed male, but not female mice. Analysis of thymic and splenic cell populations showed no effects of atrazine exposure in either sex. This is the first time that long-term changes in the immune response were observed after a perinatal exposure to atrazine and it demonstrates that these early life exposures can result in permanent changes to the immune system as well as a male bias in these effects.

Comparative Pharmacokinetics of High and Low Doses of the Herbicide Propanil in Mice.

We have documented that the herbicide propanil is immunotoxic in mice, and our in vitro tissue culture experiments largely recapitulate the in vivo studies. Laboratory studies on environmental contaminants are the most meaningful when these studies are conducted using concentrations that approximate levels in the environment. Many techniques to measure the distribution and pharmacokinetics (PK) on compounds rely on techniques, such as liquid scintillation counting (LSC) of radio-labeled starting compound, that require concentrations higher than environmental levels. The aim of this study was to compare tissue PK after exposure to propanil concentrations more relevant to levels of exposure to agricultural workers and the general population to concentrations previously reported for laboratory studies. To this end, we conducted a study to measure propanil distribution in three immune organs, using ultrasensitive accelerator mass spectrometry (AMS). We used two doses: the lower dose modeled levels expected in the environment or long-term occupational exposure to low doses, while the higher dose was to model the effects of an accidental exposure. Our results showed that the distribution and PK profiles from these two different concentrations was markedly different. The profile of the high dose (concentration) exposure was indicative of saturation of the detoxifying capability of the animal. In contrast, at the lower environmentally relevant concentration, in vivo concentrations of propanil in spleen, liver, and blood dropped to a very low level by 720 min. In conclusion, these studies highlight the differences in PK of propanil at these two doses, which suggests that the toxicity of this chemical should be re-investigated to obtain better data on toxic effects at doses relevant for humans.

Suppression of arthritis-induced bone erosion by a CRAC channel antagonist.

OBJECTIVE: We have shown in vitro and in vivo that osteoclast maturation requires calcium-release activated calcium (CRAC) channels. In inflammatory arthritis, osteoclasts mediate severe and debilitating bone erosion. In the current study, we assess the value of CRAC channels as a therapeutic target to suppress bone erosion in acute inflammatory arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in mice. The CRAC channel inhibitor 3,4-dichloropropionaniline (DCPA) and a placebo was administered 1 day prior to collagen II booster to induce arthritis. Effects on swelling, inflammatory cell invasion in joints, serum cytokines and bone erosion were measured. RESULTS: Assays, by blinded observers, of arthritis severity showed that DCPA, 21 mg/kg/day, suppressed arthritis development over 3 weeks. Bone and cartilage damage in sections of animal feet was reduced approximately 50%; overall swelling of joints was reduced by a similar amount. Effects on bone density by µCT showed clear separation in DCPA-treated CIA animals from CIA without treatment, while differences between controls without CIA and CIA treated with DCPA differed by small amounts and in most cases were not statistically different. Response was not related to anticollagen titres. There were no adverse effects in the treated group on animal weight or activity, consistent with low toxicity. The effect was maximal 12-17 days after collagen booster, during the rapid appearance of arthritis in untreated CIA. At 20 days after treatment (day 40), differences in arthritis score were reduced and tumour necrosis factor α, interleukin (IL)-1, or IL-6 in the serum of the animals were similar in treated and untreated animals. CONCLUSIONS: DCPA, a novel inhibitor of CRAC channels, suppresses bone erosion associated with acute arthritis in mice and might represent a new treatment modality for acute arthrits.

The XX Sex Chromosome Complement is Required in Male and Female Mice for Enhancement of Immunity Induced by Exposure to 3,4-Dichloropropionanilide.

PROBLEM: The chemical propanil enhances antibody responses to a heat-killed Streptococcus pneumoniae (HKSP) vaccine. The enhanced response is dependent on gonads in females, but independent of gonads in males. The sex differences in the immune response may be due to sexual differentiation of the immune system or sex chromosome complement. METHOD OF STUDY: To test the hypothesis that the immune system is sexually differentiated, newborn C57BL/6 pups were treated with testosterone propionate (TP) or placebo. The role of sex chromosome complement was investigated using the 4-core genotypes (FCG) model of XXF and XYF gonadal females (ovaries), and XXM and XYM gonadal males (testes). For some experiments, mice were gonadectomized or sham gonadectomized. All mice were vaccinated with HKSP, treated with propanil, and the antibody response determined at day seven. RESULTS: Neonatal TP did not alter the response to HKSP. In FCG mice, propanil significantly enhanced the immune response in XXF females and XXM males, but not in XYF females or XYM males. CONCLUSION: The immune system of females was not masculinized by neonatal TP treatment. Sex chromosome complement significantly contributes to the sexually dimorphic immune response after propanil exposure.

Prenatal cadmium exposure produces persistent changes to thymus and spleen cell phenotypic repertoire as well as the acquired immune response.

Cadmium (Cd) is a common environmental contaminant. Adult exposure to Cd alters the immune system, however, there are limited studies on the effects of prenatal exposure to Cd. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl(2) (10 ppm) and the effects on the immune system of the offspring were assessed at 20 weeks of age. Prenatal Cd exposure caused an increase in the percent of CD4(-)CD8(-)CD44(+)CD25(-) (DN1) thymocytes in both sexes and a decrease in the percent of CD4(-)CD8(-)CD44(-)CD25(+) (DN3) thymocytes in females. Females had an increase in the percent of splenic CD4(+) T cells, CD8(+) T cells, and CD45R/B220(+) B cells and a decrease in the percent of NK cells and granulocytes (Gr-1(+)). Males had an increase in the percent of splenic CD4(+) T cells and CD45R/B220(+) B cells and a decrease in the percent of CD8(+) T cells, NK cells, and granulocytes. The percentage of neutrophils and myeloid-derived suppressor cells were reduced in both sexes. The percent of splenic nTreg cells was decreased in all Cd-exposed offspring. Cd-exposed offspring were immunized with a streptococcal vaccine and the antibody response was determined. PC-specific serum antibody titers were decreased in Cd exposed female offspring but increased in the males. PspA-specific serum IgG titers were increased in both females and males compared to control animals. Females had a decrease in PspA-specific serum IgM antibody titers. Females and males had a decrease in the number of splenic anti-PspA antibody-secreting cells when standardized to the number of B cells. These findings demonstrate that very low levels of Cd exposure during gestation can result in long term sex-specific alterations on the immune system of the offspring.

Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells.

Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A 'pro-cancer' gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment.

Prenatal cadmium exposure alters postnatal immune cell development and function.

Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl(2) (10ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2weeks of age. At 7weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4(+)FoxP3(+)CD25(+) (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8(+)CD223(+) T cells were markedly decreased in the spleens in all offspring at 7weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can result in long term detrimental effects on the immune system of the offspring and these effects are to some extent sex-specific.

The role of calcium release activated calcium channels in osteoclast differentiation.

Osteoclasts are specialized macrophage derivatives that secrete acid and proteinases to mobilize bone for mineral homeostasis, growth, and replacement or repair. Osteoclast differentiation generally requires the monocyte growth factor m-CSF and the TNF-family cytokine RANKL, although differentiation is regulated by many other cytokines and by intracellular signals, including Ca(2+). Studies of osteoclast differentiation in vitro were performed using human monocytic precursors stimulated with m-CSF and RANKL, revealing significant loss in both the expression and function of the required components of store-operated Ca(2+) entry over the course of osteoclast differentiation. However, inhibition of CRAC using either the pharmacological agent 3,4-dichloropropioanilide (DCPA) or by knockdown of Orai1 severely inhibited formation of multinucleated osteoclasts. In contrast, no effect of CRAC channel inhibition was observed on expression of the osteoclast protein tartrate resistant acid phosphatase (TRAP). Our findings suggest that despite the fact that they are down-regulated during osteoclast differentiation, CRAC channels are required for cell fusion, a late event in osteoclast differentiation. Since osteoclasts cannot function properly without multinucleation, selective CRAC inhibitors may have utility in management of hyperresorptive states.

Subcellular localization of the amide class herbicide 3,4-dichloropropionanilide (DCPA) in T cells and hepatocytes.

3,4-Dichloropropionanilide (DCPA), or propanil, a post-emergent herbicide used on rice and wheat crops in the United States, is immunotoxic in vivo and in vitro. Although it has been documented that DCPA exerts differential effects on specific immune cell types and is toxic to the liver, the way in which DCPA modulates intracellular functions leading to these effects is less understood. In this study, Jurkat T cells and hepatocytes from C57Bl/6 mice were exposed to 100 microM DCPA for 1.5 h. Following incubation, subcellular fractions of each cell type were isolated. DCPA, when present, was removed from each cell fraction by liquid-liquid extraction. The extraction product was then analyzed for the presence of DCPA using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cellular uptake of DCPA was monitored by detection of the molecular ion and product ion of DCPA. The analyses demonstrate that DCPA, a lipophilic compound, localizes primarily in the cytosol of T cells and hepatocytes. These results indicate that DCPA is able to cross the plasma membrane and is accessible to intracellular immunomodulatory effectors.

Prenatal cadmium exposure dysregulates sonic hedgehog and Wnt/beta-catenin signaling in the thymus resulting in altered thymocyte development.

Cadmium (Cd) is both an environmental pollutant and a component of cigarette smoke. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports in the literature of immunomodulatory effects of prenatal exposure to Cd. The sonic hedgehog (Shh) and Wnt/beta-catenin pathways are required for thymocyte maturation. Several studies have demonstrated that Cd exposure affects these pathways in different organ systems. This study was designed to investigate the effect of prenatal Cd exposure on thymocyte development, and to determine if these effects were linked to dysregulation of Shh and Wnt/beta-catenin pathways. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose (10 ppm) of Cd throughout pregnancy and effects on the thymus were assessed on the day of birth. Thymocyte phenotype was determined by flow cytometry. A Gli:luciferase reporter cell line was used to measure Shh signaling. Transcription of target genes and translation of key components of both signaling pathways were assessed using real-time RT-PCR and western blot, respectively. Prenatal Cd exposure increased the number of CD4(+) cells and a subpopulation of double-negative cells (DN; CD4(-)CD8(-)), DN4 (CD44(-)CD25(-)). Shh and Wnt/beta-catenin signaling were both decreased in the thymus. Target genes of Shh (Patched1 and Gli1) and Wnt/beta-catenin (c-fos, and c-myc) were affected differentially among thymocyte subpopulations. These findings suggest that prenatal exposure to Cd dysregulates two signaling pathways in the thymus, resulting in altered thymocyte development.

A review of the immunotoxicity of the pesticide 3,4-dichloropropionanalide.

The pesticide 3,4-dichloropropionanilide (propanil or, alternatively, DCPA) is a member of the acetanilide chemical family and is predominantly used for the control of weeds on commercial rice crops worldwide. This article was written to provide a brief review of the general toxicity of propanil followed by a detailed summary of the immunotoxicity studies that were performed to date in mammalian in vivo and in vitro models. Propanil affects the immune system at organ, cellular, and molecular levels. Studies demonstrated that it produces thymic atrophy and splenomegaly and decreases developing T- and B-cell populations in the thymus and bone marrow. Natural killer (NK) cells and macrophages are critical components of the innate immune system. NK cell cytotoxicity and the ability of macrophages to phagocytose, kill pathogenic bacteria, and produce inflammatory cytokines are suppressed by propanil. Propanil also affects the respiratory burst of macrophages, inhibiting reactive oxygen and nitrogen species production. Molecular mechanisms responsible for propanil's effects have begun to be elucidated and include alterations in nuclear factor (NF)-kappaB transcription factor activity and intracellular Ca(2+) signaling. Propanil exposure alters a number of functions of mature T lymphocytes and B lymphocytes that impacts the adaptive immune response. T-cell cytotoxic activity and cytokine production are major T-cell functions inhibited by propanil. The humoral antibody response to model antigens and intact bacteria is differentially affected after propanil exposure. How these changes in innate and adaptive immune responses impact the host response to bacterial challenge or vaccination has begun to be examined.

3,4-dichloropropionaniline suppresses normal macrophage function.

Macrophages are a critical part of the innate immune response and natural surveillance mechanisms. As such, proper macrophage function is crucial for engulfing bacterial pathogens through phagocytosis and destroying them by generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The production of a number of cytokines by macrophages, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, plays an important role in the initiation of the acquired immune response creating an inflammatory environment favorable for fighting a bacterial infection. 3,4-Dichloropropionaniline (DCPA) suppresses several inflammatory parameters, including TNF-alpha production through a mechanism where nuclear factor-kappaB (NF-kappaB)-DNA binding is inhibited but not entirely abrogated. The goal of the present study was to evaluate the effects of DCPA on the inflammatory mediators of macrophages, including ROS and RNS in both murine peritoneal exudate cells and the human monocytic cell line, THP-1. The ability to perform phagocytosis and directly kill Listeria monocytogenes was also assessed. The results indicate that DCPA decreases the ability of both types of macrophages to phagocytize beads and generate both types of reactive species, which was correlated with a decrement in listericidal activity. These results demonstrate that DCPA has profound effects on macrophage function and provide insight into the potential mechanisms of immunosuppression by DCPA.

A novel role for 3, 4-dichloropropionanilide (DCPA) in the inhibition of prostate cancer cell migration, proliferation, and hypoxia-inducible factor 1alpha expression.

BACKGROUND: The amide class compound, 3, 4-dichloropropionanilide (DCPA) is known to affect multiple signaling pathways in lymphocyte and macrophage including the inhibition of NF-kappaB ability. However, little is known about the effect of DCPA in cancer cells. Hypoxia-inducible factor 1 (HIF-1) regulates the expression of many genes including vascular endothelial growth factor (VEGF), heme oxygenase 1, inducible nitric oxide synthase, aldolase, enolase, and lactate dehydrogenase A. HIF-1 expression is associated with tumorigenesis and angiogenesis. METHODS: We used Transwell assay to study cell migration, and used immunoblotting to study specific protein expression in the cells. RESULTS: In this report, we demonstrate that DCPA inhibited the migration and proliferation of DU145 and PC-3 prostate cancer cells induced by serum, insulin, and insulin-like growth factor I (IGF-I). We found that DCPA inhibited HIF-1 expression in a subunit-specific manner in these cancer cell lines induced by serum and growth factors, and decreased HIF-1alpha expression by affecting its protein stability. CONCLUSION: DCPA can inhibit prostate cancer cell migration, proliferation, and HIF-1alpha expression, suggesting that DCPA could be potentially used for therapeutic purpose for prostate cancer in the future.

Evidence for a novel endocrine disruptor: the pesticide propanil requires the ovaries and steroid synthesis to enhance humoral immunity.

Steroid hormones are known to affect the humoral immune response to a variety of antigens. However, the mechanisms regulating these effects are poorly understood. The immunotoxic chemical propanil and estrogen have similar effects on the immune system including augmentation of humoral immune responses. Propanil enhances the number of phosphorylcholine (PC)-specific IgG2b, IgG3, and IgM antibody-secreting cells (ASCs) in the spleen four- to sixfold 7 days after vaccination of female C57BL/6 mice with heat-killed Streptococcus pneumoniae. Several experiments were performed to test the hypothesis that propanil increases the response via an estrogenic pathway. Ovariectomy abrogated the effect of propanil on the PC-specific ASC response. Both in vitro and in vivo assays indicate that propanil does not bind either estrogen receptor (ER) alpha or beta. Exogenous estradiol administration in ovariectomized mice failed to restore the effect of propanil on the PC response. Treatment of female mice with a pure ER antagonist, ICI 182,780, or the progesterone antagonist RU486 did not inhibit the increase in ASCs. These data suggest that estrogen and progesterone do not regulate the effect of propanil. However, complete inhibition of steroid synthesis with the gonadotropin-releasing hormone (GnRH) antagonist antide abrogated the increased response in propanil-treated mice, indicating a necessary role for steroid synthesis. Experiments in male mice demonstrated that propanil increased the number of ASCs comparable to female mice. However, orchiectomy did not inhibit this effect, suggesting that androgens do not regulate the amplification of the humoral response. These data suggest a novel role for the ovarian hormones in the regulation of the PC-specific antibody response.

Immunomodulatory effects of maternal atrazine exposure on male Balb/c mice.

Atrazine is a widely used herbicide applied to corn, sugar and other crops as a broad leaf weed inhibitor. Using the Balb/c mouse model, we have determined that prenatal/lactational exposure to atrazine alters adult immune function. Pregnant Balb/c dams were exposed subcutaneously for 21 days via time release pellets to 700 microg per day of atrazine beginning between days 10 and 12 of pregnancy. Prenatal/Lactational exposure caused no overt physical malformations in the offspring and had no effect on the number of litters carried to term or the litter size. Upon reaching early adulthood (approximately 3 months of age), the state of their immune system was evaluated. There were no changes in body weight or in the organ to body weight ratio of the spleen. Additionally, no changes were observed in the number of CD8+ T cell, CD4+ T cell, or B220+ B cell subpopulations in the spleen. T cell function was assessed by measuring proliferation and cytolytic activity after in vitro allogeneic stimulation. Male mice which had been prenatally/lactationally exposed to atrazine had an increase in both T cell proliferation and cytolytic activity. The humoral immune response was assessed after immunization with heat killed Streptococcus pneumoniae (HKSP). There was a significant increase in the number of HKSP-specific IgM secreting B cells in the spleen of prenatal/lactational exposed male mice. Inasmuch as atrazine is a widespread environmental contaminant, this immunopotentiation raises concerns that it may potentiate clinical diseases, such as autoimmune disease and hypersensitivity, and needs to be carefully monitored and studied.

The polysaccharide antibody response after Streptococcus pneumoniae vaccination is differentially enhanced or suppressed by 3,4-dichloropropionanilide and 2,4-dichlorophenoxyacetic acid.

Propanil (3,4-dichloropropionanilide) and 2,4-D (2,4-dichlorophenoxyacetic acid) are commonly used herbicides that have toxic effects on the immune system. The present study determined the effect of exposure to these chemicals on the immune response to a bacterial vaccine. The antibody responses to the T-independent type 2 antigen, phosphorylcholine (PC) and the T-dependent antigen, pneumococcal surface protein A (PspA) were characterized in C57BL/6 mice after heat-killed Streptococcus pneumoniae (HKSP) immunization and single or mixture herbicide exposure. Propanil exposure significantly increased the number of PC-specific IgM, IgG2b, and IgG3 antibody-secreting B cells (ASC) in the spleen 4-6-fold over control animals in a dose-dependent manner. However, the number of ASC in the bone marrow and serum titers were comparable in control and propanil-treated mice. In contrast, 2,4-D exposure decreased the number of PC-specific IgM and IgG bone marrow ASC 2-3-fold from control animals. The decrease in bone marrow ASC in 2,4-D-treated mice corresponded to a 3-4-fold decrease in PC-specific IgM, IgG2b, and IgG3 serum titers compared to control mice. The number of ASC in the spleens of 2,4-D-treated mice was, however, comparable to control mice. The antibody response to PspA was not affected by any of the treatments. There were no mixture interactions between the two herbicides in any of the responses measured. These results characterize the primary PC-specific antibody response in the bone marrow, spleen, and serum after HKSP vaccination and herbicide exposure. The differential effects of propanil and 2,4-D on the antibody response to a bacterial vaccine demonstrate the potential of chemical exposure to augment or suppress immune responses to vaccines and infectious diseases.

Characterization of thymic atrophy and the mechanism of thymocyte depletion after in vivo exposure to a mixture of herbicides.

3,4-Dichloropropionanilide (propanil) and 2,4-dichlorophenoxyacetic acid (2,4-D) are two commonly used herbicides that are marketed as a chemical mixture. It was hypothesized that the interaction between these two herbicides, when administered as a mixture, would result in a greater effect on the immune system than the individual components of the mixture. The present study demonstrates in a murine model that a mixture of propanil and 2,4-D, when compared to single herbicide exposures, exacerbates decreases in thymocyte populations 2 d postexposure and inhibits the repopulation of T-cells in the thymus 7 d postexposure. Exposure to 150 mg herbicide/kg body weight of propanil or 2,4-D alone had no effect on thymus weight. In contrast, decreases in the ratio of thymus weight to body weight (TW:BW) occurred 2 d after treatment with the mixture of 150 mg propanil/kg body weight + 150 mg 2,4-D/kg body weight (150/150). Thymic atrophy was associated with a decrease in the double-positive thymocyte population (CD4+CD8+) and correlated with sera corticosterone levels from 600 to 1000 pg/ml. Therefore, the hypothesis was tested that glucocorticoids, induced after exposure to herbicides, were responsible for the thymic atrophy and depletion of thymocytes. However, similar levels of corticosterone were induced after exposure to 50, 100, or 150 mg propanil/kg body weight, and 50/50 or 100/100 mixture treatments, doses that did not produce thymic atrophy or cell loss. In addition, RU 486, a glucocorticoid receptor blocker, only partially abrogated the thymic atrophy in mice exposed to the 150/150 mixture of herbicides. These results suggest that glucocorticoids are only partially responsible for herbicide-induced thymic atrophy. This study demonstrates that the effects of exposure to a mixture of chemicals cannot always be predicted based on single exposure data and emphasizes the importance of mixture-based studies.

Suppression of cell-mediated immune responses to listeria infection by repeated exposure to diesel exhaust particles in brown Norway rats.

Diesel exhaust particles (DEP) have been shown to alter pulmonary immune responses to bacterial infection. Exposure of rats to 100 mg/m(3) DEP for 4 h was found to aggravate Listeria monocytogenes(Listeria) infection at 3 days postinfection, but the bacteria were largely cleared at 7 days postinfection due to the development of a strong T cell-mediated immunity. In the present study, we examined the effects of repeated DEP exposure at lower doses on pulmonary responses to bacterial infection. Brown Norway rats were exposed to DEP by inhalation at 20.62 +/- 1.31 mg/m 3 for 4 h/day for 5 days, followed by intratracheal inoculation with 100,000 Listeria at 2 h after the last DEP exposure. DEP-exposed rats showed a significant increase in lung bacterial load at both 3 and 7 days postinfection. The repeated DEP exposure was shown to suppress both the innate, orchestrated by alveolar macrophages (AM), and T cell-mediated responses to Listeria. DEP inhibited AM production of interleukin- (IL-) 1beta, tumor necrosis factor- (TNF-) alpha, and IL-12 but enhanced Listeria-induced AM production of IL-10, which has been shown to prolong the survival of intracellular pathogens such as Listeria. DEP exposure also suppressed the development of bacteria-specific lymphocytes from lung-draining lymph nodes, as indicated by the decreased numbers of T lymphocytes and their CD4(+) and CD8(+) subsets. Furthermore, the DEP exposure markedly inhibited the Listeria-induced lymphocyte secretion of IL-2 at day 7, IL-10 at days 3 and 7, and interferon- (IFN-) gamma at days 3 to 10 postinfection when compared to air-exposed controls. These results show a sustained pattern of downregulation of T cell-mediated immune responses by repeated low-dose DEP exposure, which is different from the results of a single high-dose exposure where the acute effect of DEP aggravated bacteria infection but triggered a strong T cell-mediated immunity.