Progress 01/24/20 to 09/30/21
Outputs
Target Audience:The studies are complete. The target audience for the publications is the scientific community in the field of environmental health and toxicology. Changes/Problems:In some experiments,we decided to use DiNP instead of DEHP as our model phthalate because DiNP is replacing DEHP in products and it is environmentally relevant. Last year, some of our progress was delayed due to the COVID-19 pandemic. However, we were able to complete all of the proposed experiments on DEHP plus additional experiments on DiNP. What opportunities for training and professional development has the project provided?The project served as part of the disseration work for two graduate students. The graduate students learned to generate hypotheses, design experiments to test their hypotheses, conduct experiments, and write manuscripts on the findings. In addition, the students learned to present the findings at local and national scientific conferences. How have the results been disseminated to communities of interest?We published fourpeer-reviewed manuscripts in scientific journals. These journals are read by researchers in the fields of toxicology, environmental health, animal health, and nutrition. We also presented the work at several local and national scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the funding period, we completed the proposed studies and published threepeer-reviewed manuscripts and onepeer-reviewed review article. Below is a summary of the findings. Scientific Reports (Chiu et al., 2000) Di-isononyl phthalate (DiNP), a common plasticizer used in polyvinyl chloride products, exhibits endocrine-disrupting capabilities. It is also toxic to the brain, reproductive system, liver, and kidney. However, little is known about how DiNP impacts the gastrointestinal tract (GIT). It is crucial to understand how DiNP exposure affects the GIT because humans are primarily exposed to DiNP through the GIT. Thus, this study tested the hypothesis that subacute exposure to DiNP dysregulates cellular, endocrine, and immunological aspects in the colon of adult female mice. To test this hypothesis, adult female mice were dosed with vehicle control or DiNP doses ranging from 0.02 - 200 mg/kg for 10-14 days. After the treatment period, mice were euthanized during diestrus, and colon tissue samples were subjected to morphological, biochemical, and hormone assays. DiNP exposure significantly increased histological damage in the colon compared to control. Exposure to DiNP also significantly decreased sICAM-1 levels, increased Tnf expression, decreased a cell cycle regulator (Ccnb1), and increased apoptotic factors (Aifm1andBcl2l10) in the colon compared to control. Colon-extracted lipids revealed that DiNP exposure significantly decreased estradiol levels compared to control. Collectively, these data indicate that subacute exposure to DiNP alters colon morphology and physiology in adult female mice. Toxicological Sciences (Chiu et al., 2021) Di-isononyl phthalate (DiNP) is a high-molecular-weight phthalate commonly used as a plasticizer for polyvinyl chloride and other end products, such as medical devices and construction materials. Most of our initial exposure to DiNP occurs by ingestion of DiNP-contaminated foods. However, little is known about the effects of DiNP on the colon. Therefore, the goal of this study was to test the hypothesis that DiNP exposure alters immune responses and impacts specialized epithelial cells in the colon. To test this hypothesis, adult female mice were orally dosed with corn-oil vehicle control or doses of DiNP ranging from 20 µg/kg/d to 200 mg/kg/d for 10-14 days. After the dosing period, mice were euthanized in diestrus, and colon tissues and sera were collected for histological, genomic, and proteomic analysis of various immune factors and specialized epithelial cells. Subacute exposure to DiNP significantly increased protein levels of Ki67 and MUC2, expression of a Paneth cell marker (Lyz1), and estradiol levels in sera compared to control. Gene expression of mucins (Muc1, Muc2, Muc3a, and Muc4), Toll-like receptors (Tlr4 and Tlr5), and specialized epithelial cells (ChgA, Lgr5, Cd24a, and Vil1) were not significantly different between treatment groups and control. Cytokine levels of IL-1RA and CXCL12 were also not significantly different between DiNP treatment groups and control. These data reveal that DiNP exposure increases circulating estradiol levels and gene expression in specialized epithelial cells with immune response capabilities (e.g., goblet and Paneth cells) in the mouse colon, which may initiate immune responses to prevent further damage in the colon. Toxics (Chiu et al., 2021) Di-2-ethylhexyl phthalate (DEHP) is a plasticizer commonly found in polyvinyl chloride, medical equipment, and food packaging. DEHP has been shown to target the reproductive system and alter the gut microbiome in humans and experimental animals. However, very little is known about the impact of DEHP-induced microbiome changes and its effects during pregnancy. Thus, the objective of this study was to investigate the effects of DEHP exposure during pregnancy on the cecal microbiome and pregnancy outcomes. Specifically, this study tested the hypothesis that subacute exposure to DEHP during pregnancy alters the cecal microbiome in pregnant mice, leading to changes in birth outcomes. To test this hypothesis, pregnant dams were orally exposed to corn oil vehicle or 20 µg/kg/day DEHP for 10 days and euthanized 21 days after their last dose. Cecal contents were collected for 16S Illumina and shotgun metagenomic sequencing. Fertility studies were also conducted to examine whether DEHP exposure impacted birth outcomes. Subacute exposure to environmentally relevant doses of DEHP in pregnant dams significantly increased alpha diversity and significantly altered beta diversity. Furthermore, DEHP exposure during pregnancy significantly increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Deferribacteres compared with controls. The affected taxonomic families included Deferribacteraceae, Lachnospiraceae, and Mucisprillum. In addition to changes in the gut microbiota, DEHP exposure significantly altered 14 functional pathways compared with the control. Finally, DEHP exposure did not significantly impact the fertility and birth outcomes compared with the control. Collectively, these data indicate that DEHP exposure during pregnancy shifts the cecal microbiome, but the shifts do not impact fertility and birth outcomes. Review in Toxicological Sciences (Chiu et al., 2020) Since the surge of microbiome research in the last decade, many studies have provided insight into the causes and consequences of changes in the gut microbiota. Among the multiple factors involved in regulating the microbiome, exogenous factors such as diet and environmental chemicals have been shown to alter the gut microbiome significantly. Although diet substantially contributes to changes in the gut microbiome, environmental chemicals are major contaminants in our food and are often overlooked. Herein, we summarize the current knowledge on major classes of environmental chemicals (bisphenols, phthalates, persistent organic pollutants, heavy metals, and pesticides) and their impact on the gut microbiome, which includes alterations in microbial composition, gene expression, function, and health effects in the host. We then discuss health-related implications of gut microbial changes, which include changes in metabolism, immunity, and neurological function.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chiu, K., Warner, G., Nowak, R.A., Flaws, J.A. and Mei, W. 2020. The impact of environmental chemicals on the gut microbiome. Toxicological Sciences 176:253-284.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chiu, K., Bashir, S.T., Nowak, R.A., Mei, W. and Flaws, J.A. 2020. Subacute exposure to di-isononyl phthalate alters the morphology, endocrine function, and immune system in the colon of adult female mice. Scientific Reports 10:18788.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Chiu, K., Bashir, S.T., Chiu, J., Nowak, R.A. and Flaws, J.A. 2021. The impact of di-isononyl phthalate exposure on specialized epithelial cells in the colon. Toxicological Sciences (In Press).
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Chiu, K., Bashir, S.T., Gao, L., Gutierrez, J., de Godoy, M.C., Drnevich, J., Fields, C., Cann, I., Flaws, J.A. and Nowak, R. 2021. Subacute exposure to an environmentally relevant dose of di-(2-ethylhexyl) phthalate during gestation alters cecal microbiome in pregnant mice. Toxics (In Press).
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Progress 01/24/20 to 09/30/20
Outputs
Target Audience:The target audience is the scientific community. We disseminated information to the scientific community in the form of onemanuscript and oneabstract so far. Below is the information on the dissemination of information. Manuscript: Chiu, K., Warner, G., Nowak, R.A., Flaws, J.A. andMei, W. 2020. The impact of environmental chemicals on the gutmicrobiome.Toxicological Sciences (In Press). Abstract: Chiu, K., Meling, D.D., Chiang, C. andFlaws, J.A. 2020. Diisononyl phthalate exposure affects colonic health in adult female mice. Society of Toxicology 2020. Changes/Problems:We elected to use DiNP instead of DEHP as our model phthalate. This is because DiNP is replacing DEHP in products and it is environmentally relevant. Further, both DEHP and DiNP are known endocrine disrupting chemicals with similar structures and functions. Due to the pandemic, my laboratory was completely shutdown for about 2.5 months and then allowed only to work in limited capacity for another 1.5 months. This delayed some of our progress. That said, my lab is fully operational now and I am hopeful that we will complete the remaining work during the next funding period. What opportunities for training and professional development has the project provided?This project is forming the basis of the dissertation for a PhD candidate in the Division of Nutritional Sciences Graduate Program at the University of Illinois at Urbana-Champaign. It is a useful tool to train the student on how to conduct research in the field. How have the results been disseminated to communities of interest?We have published the findings for presentation at a scientfic conference. We also published a review manuscript on the topic. What do you plan to do during the next reporting period to accomplish the goals?To complete Specific Aim 1, adult CD-1 female mice (39-40 days) will be orally dosed with vehicle (tocopherol-stripped corn oil), DiNP (20 and 200 µg/kg/day; 20 and 200 mg/kg/day) in acute and chronic dosing paradigms (n=8/group/time point). We will use 20 and 200 mg/kg/day because these doses are environmentally relevant, and 20 and 200 mg/kg/day, which represent occupational exposure.We will begin dosing at 39-40 days of age when CD-1 mice are sexually mature. We will use oral dosing because it is the common route of human exposure.In the acute dosing arm, mice will be exposed for 10 or 30 days to vehicle, or doses of DINP. This acute dosing period will help us understand whether the effects of phthalates are permanent or reversible with time and it will help identify the early effects of exposure.In the chronic dosing arm, mice will be continually exposed to vehicle or phthalates for 3-12 months. This will allow us to determine the effects of chronic exposure to phthalates on reproductive outcomes and mimic chronic exposure that occurs in women. Mouse gastrointestine tract (GIT), i.e., stomach, ileal, cecum, and colonic digesta will be subjected to microbial 16S rRNA gene and metagenomic DNA sequencing. Multivariate supervised and unsupervised methods will be used to uncover simultaneous changes of transcriptome and metabolome and to identify associations among these variables and reproductive health outcomes. To integrate these datasets, we will utilize methods robust to common issues such as multicollinearity and potential Type I (false discovery) errors. Initially, we will utilize Random Forest (RF) analysis, as this is a useful and simple machine learning approach that does not require extensive parameter modification and handles mixed variables readily. We also anticipate more advanced approaches will be required to integrate the multiomic data that we will generate in this study. We will assess multiple approaches for integrated analyses using packages developed for microbiome and metagenome analysis such as those in MetaMLand the Bioconductor package mixOmicsohar. To complete Specific Aim 2, adultCD-1 female mice (39-40 days) will be orally dosed with vehicle (tocopherol-stripped corn oil), DiNP (20 and 200 µg/kg/day; 20 and 200 mg/kg/day) in acute and chronic dosing paradigms as described in Aim 1 (n=16/group/time point).In both the acute and chronic dosing arms, eightof the exposed females for each treatment group and time-point (half of the n=16) will be euthanized in diestrus for collection of serum, ovaries, uteri, stomach, intestinal ileum, and cecal digesta. The eightremaining females for each treatment group and time-point will be mated with non-exposed adult proven male breeders. These breeding trials will occur immediately after dosing as well as at selected times after dosing ends. This schedule will help determine whether phthalate exposed mice experience subfertility/infertility and reproductive senescence at times earlier than controls. We will compare time to pregnancy, gestation length, numbers of live and dead pups, and the sexes of the pups in the treatment groups over time using ANOVA as published by the PI. At each time-point, we will collect sera for follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), inhibin B, and estradiol (E2) measurements as published by the PI.FSH levels are indicators of impending ovarian failure because they increase during onset of reproductive senescence in women and mice.AMH and inhibin B are markers of ovarian aging.We will compare differences in FSH, AMH, inhibin B, and E2levels over time in the treatment groups using ANOVA. A subset of samples will also be assayed for C-reactive protein and interleukin-1 (IL-1) as general markers of inflammation. Serum samples collected from mice not undergoing breeding trials that will be euthanized at the specific timepoints (10 and 30 days; 3, 6, 9, 12 months) will be analyzed for the same hormones to monitor changes as the mice age. Ovarian, uterine and intestinal ileum tissues collected at these timepoints will be processed for histological evaluation. All mice will be euthanized in diestrus determined by monitoring vaginal cytology. Ovaries will be evaluated for numbers of healthy and atretic primordial, primary, preantral, and antral follicles as published by the PI.Apoptosis will be assessed by qPCR for the apoptosis genesBcl-2andBaxand calculation of theBax/Bcl-2ratio. To help determine if DINP blocks ovulation, we will count corpora lutea using methods.We will also analyze the ovaries for numbers of inflammatory macrophages using an F4/80 antibody and for the presence of ovarian cysts. Effects on oxidative stress will be assessed by qPCR for expression of the antioxidant enzymes SOD1, glutathione peroxidase (GPX), and catalase, which we have shown are decreased in response to treatment with a phthalate mixture in vitro. The uteri will be analyzed using methods described in our publications. Specific endpoints that will be assessed include changes in uterine cell proliferation using Ki67 as a biomarker; expression of estrogen receptor-α and progesterone receptor; quantitation of numbers of uterine glands and presence of dilated or cystic glands; presence of epithelial hyperplasia; integrity of endometrial blood vessels; presence of endometrial glandular nests; influx of inflammatory macrophages using F4/80 antibody, and evidence of fibrotic areas in the endometrium or myometrium using a collagen type I antibody. The intestinal ileum will be analyzed since the cells residing here are in more direct contact with orally administered DINP and the gut microbiota reside there. Ileum samples will be assessed for changes in the height of the microvilli; cell proliferation in the microvilli using Ki67; numbers of goblet cells; numbers of intestinal stem cells using the antibodies LGR5 and BMi1; and numbers of macrophages as a marker for inflammation using F4/80 antibody. To explore the link between the microbiome and host reproductive pathologies, the digesta samples from the different sections of the GIT will be processed to DNA for both 16S rRNA genes and community genomic DNA sequencing. Community RNA will also be extracted for metatranscriptomic analysis. All methods will be performed as described in detail in our preliminary data section. In addition, we will perform Multivariate Association with Linear Models (MaAsLin2) analysis to determine associations between physiological metadata and microbial meta'omic features (e.g., abundance and function).Permutation based multivariate analysis of variance (PERMANOVA) will also be used to assess the influence of DINP on associations between physiological and reproductive outcomes and the GIT microbiota of mice.
Impacts
What was accomplished under these goals?
Progress Summary/Accomplishments: During the funding period, we focused on experiments outlined in Specific Aim 1. Specifically, we tested the hypothesis that that subacute exposure to diisononyl phthalate (DiNP) alters the morphology, immune system, and endocrine function of the colon. Please note that we decided to use DiNP instead of DEHP as our model phthalate because DiNP is replacing DEHP in products and it is environmentally relevant. To test the hypothesis, adult female mice were dosed with vehicle control or DiNP doses ranging from 0.02 - 200 mg/kg for 10-14 days. After the treatment period, mice were euthanized during diestrus, and colon tissue samples were subjected to morphological, biochemical, and hormone assays. The results of these experiments are described below. Gross Measurements: In all mice, the colon length ranged from 5.8 to 10.2 cm. DiNP exposure at all doses did not significantly affect colon length compared to control. Similarly, DiNP exposure did not markedly alter colon weight compared to control. Exposure to DiNP also did not significantly alter the colon weight-to-length ratio compared to control. Histopathology: Histological analysis revealed that DiNP exposure increased colonic damage compared to control. Specifically, DiNP doses at 0.02, 0.2, 2, and 200 mg/kg significantly increased colonic damage compared to control. Interestingly, the changes at the low doses of DiNP (0.02 and 0.2 mg/kg/day) were mainly due to cellular infiltration and aberrant colon walls, whereas the changes at high doses of DiNP (2, 20, and 200 mg/kg/day) were mainly attributed to edema. Further, some enterocyte sloughing occurred in the 0.02 - 2 mg/kg DiNP treatment groups. Hormone levels: DiNP exposure at most doses did not significantly alter testosterone levels in the colon compared to control, but 0.2 mg/kg/day DiNP marginally decreased testosterone levels compared to control. DiNP exposure significantly decreased estradiol concentrations at 0.2, 20, and 200 mg/kg/day compared to control in the colons. However, DiNP exposure at 0.02 and 2 mg/kg/day did not alter estradiol levels in the colon significantly compared to control. Cell Cycle Regulation and Cell Health: Expression of cell cycle factors that promote the cell cycle such as cyclin A2 (Ccna2), cyclin B1 (Ccnb1), cyclin D2 (Ccnd2), cyclin E2 (Ccne1), and cyclin dependent kinase 4 (Cdk4) were examined in the colon. The highest dose of DiNP decreased Ccnb1 expression compared to control. However, DiNP treatment did not alter expression of Ccna2, Ccnd2, Ccne1, and Cdk4 compared to control. Further, DiNP treatment did not alter expression of cyclin dependent kinase inhibitor 1a or Cdkn1a, a cell cycle inhibitor, compared to control. Expression of apoptotic factors including Aifm1 and Bcl2l10 was also examined to determine the health of cells. DiNP exposure at an environmentally relevant dose (0.2 mg/kg) increased expression of Aifm1 compared to control. However, DiNP at other doses did not affect expression of Aifm1 compared to control. In contrast, DiNP exposure at 20 mg/kg significantly increased Bcl2l10 expression compared to control. In addition to examining apoptotic factors, cell survival factors including Bcl2 were examined in each treatment group. DiNP exposure did not alter expression of Bcl2 compared to control. Further, DiNP exposure did not significantly alter Ki67 expression compared to control at any dose. Because DiNP increased expression of two pro-apoptotic factors, Aifm1 and Bcl2l10, compared to control, we conducted TUNEL staining to examine whether DiNP treatment caused the DNA fragmentation that occurs during apoptosis. Interestingly, DiNP exposure did not affect TUNEL staining compared to control. Inflammation: Expression of the following cytokines was measured from the distal colon: Il4, Il5, Il6, Il13, Il17a, Tnf, and Ifng. DiNP exposure did not significantly alter expression of Il4, Il5, Il6, Il13, and Il17a compared to control. However, environmentally relevant doses of DiNP (0.02 and 0.2 mg/kg/day) borderline increased the expression of interferon gamma (Ifng). Interestingly, an environmentally relevant dose (0.2 mg/kg/day) of DiNP exposure significantly increased Tnf expression compared to control. Although DiNP exposure significantly increased the expression of Tnf compared to control, it did not alter TNF-α protein levels. Interestingly, the gene expression and protein level for this cytokine showed a similar trend and dose-response curve. The lowest dose of DiNP exposure borderline increased sICAM-1 levels compared to control. On the other hand, DiNP at the highest dose significantly decreased sICAM-1 compared to control. Tight junctions play a role in mediating immune responses. Thus, we examined expression of tight junction proteins such as Zo-1, Zo-2, Zo-3, Cldn1, Cldn4, and Ocln. DiNP exposure did not affect the expression of Zo-1, Zo-2, Cldn1, Cldn4, and Ocln. However, DiNP exposure at 200 mg significantly decreased expression of Zo-3 compared to control. Collectively, these data indicate that subacute exposure to DiNP alters colon morphology and physiology in female mice. Thus, during the next funding period, we will determine whether any of the observed DiNP-induced effects on the colon impact the gut microbiome and/or reproductive outcomes. Problems: Due to the pandemic, my laboratory was completely shutdown for about 2.5 months and then allowed only to work in limited capacity for another 1.5 months. This delayed some of our progress. That said, my lab is fully operational now and I am hopeful that we will complete the remaining work during the next funding period.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2020
Citation:
Chiu, K., Warner, G., Nowak, R.A., Flaws, J.A. and Mei, W. 2020. The impact of environmental chemicals on the gut microbiome. Toxicological Sciences (In Press).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Chiu, K., Meling, D.D., Chiang, C. and Flaws, J.A. 2020. Diisononyl phthalate exposure affects colonic health in adult female mice. Society of Toxicology 2020.
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