Master of Science (MS), Wright State University, 2021, Anatomy

Small changes in antibody expression have broad impacts on human health and disease. An environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces profound immune suppression in animal models in an aryl hydrocarbon receptor (AhR)-mediated fashion. In a human Burkitt's lymphoma cell line (CL-01), TCDD inhibited IgG secretion and differentially impacted Ig heavy chain expression. In addition to TCDD, the AhR has several endogenous, dietary, and bacterial ligands such as 6-formylindolo[3,2-b]carbazole (FICZ) and indole, which are present within the human body at varied concentrations (0.30 mM- 6.64 mM). Both FICZ (100 nM) and indole (100 µM) significantly reduced IgG secretion. Overall antibody secretion is less sensitive to endogenous ligands of the AhR than TCDD, which inhibits IgG secretion in the nM concentration range. However, antibody secretion is inhibited by FICZ, suggesting a biological role of the AhR in antibody expression.

Committee: Courtney Sulentic Ph.D. (Advisor); Nancy Bigley Ph.D. (Committee Member); Thomas Brown Ph.D. (Committee Member); Eric Bennett Ph.D. (Other) Subjects: Biology

MS, University of Cincinnati, 2003, Medicine : Environmental Health Sciences

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmentally persistent compound that causes cancer and toxicity in multiple organ systems. Because of its potency, other structurally similar hazardous toxicants are compared to TCDD if they act through the same toxicological mechanism. Coplanar polyhalogenated biphenyls (PHBs) have TCDD toxic equivalency (TEQ), whereas noncoplanar PHBs do not. The TEQ-associated toxicological mechanism involves an oxidative stress response, but the intracellular source of reactive oxygen species (ROS) is unconfirmed. The requirement for activation of the aromatic hydrocarbon receptor (AHR) is well established, however. The AHR is a cytosolic factor that is activated upon binding TCDD-like compounds. Activation results in translocation into the nucleus and transcription of the "AHR" gene battery. Phase I monooxygenase enzymes, cytochromes P4501A1 and P4501A2 (CYP1A1 and CYP1A2), are among "AHR" gene battery products. Enzymatic uncoupling of substrate hydroxylation from oxygen consumption generates ROS and might contribute to TEQ-associated toxicity. Various substrates, including TCDD and PHBs, are examined in this study for their ability to induce monooxygenase uncoupling in isolated microsomes containing or lacking AHR-controlled cytochromes P450. Microsomes were isolated from "wild-type" mice, "Cyp1a1 (-/-)" knockout mice and "Cyp1a2 (-/-)" knockout mice to examine relative CYP isoform-specific contributions to ROS generation. Results show increased uncoupling of CYP1A1 in the presence of highly halogenated, coplanar substrates only. Thus, CYP uncoupling likely contributes to TEQ-associated toxicity since previously identified TCDD-like PHBs are shown to cause CYP uncoupling rates comparable to TCDD-induced rates. Non-AHR-controlled CYP2E1 is identified as the predominant CYP isoform responsible for basal levels of ROS generation in the absence of substrates. Surprisingly, CYP1A2 is found to act in an anti-oxidant manner by atte (open full item for complete abstract)

Committee: Dr. Howard Shertzer (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

Acute myeloid leukemia (AML) is an aggressive and relatively common form of leukemia with poor survival outcomes despite modern advances in targeted and cellular therapies. For patients able to tolerate induction therapy and go on to receive a bone marrow transplant, at least half will eventually relapse, underscoring the critical need to better understand the immune-related mechanisms likely underpinning this process. Natural killer (NK) cells are a type of innate lymphoid cell (ILC) with potent anti-leukemic functions, however AML patients often have hypomature and hypofunctional circulating NK cells which correlate with poor outcomes. Other ILCs lack the cytotoxic function of NK cells but possess more immunomodulatory functions. All these ILC populations share a common developmental precursor, and thus the overall goal and hypothesis of this research was that the hypomature NK cell phenotype observed in AML patients may be indicative of developmental rerouting away from the NK cell lineage and towards other ILCs. The results of these studies characterize the developmental reprogramming of ILC precursors (ILCPs) by AML-mediated activation of the aryl hydrocarbon receptor (AHR) pathway to skew development away from NK cells and towards both the ILC1 and ILC3 lineage. We utilize both patient samples and in vivo models to identify expansion of ILC populations and then use a combination of ex vivo, in vivo, and in vitro modeling approaches to demonstrate expansion of these populations is due at least in part to skewed development of ILCPs via an AHR-dependent process. First, by phenotyping the ILC compartment of a mouse model of AML that recapitulates human disease, we identified expansion of ILC1s in the bone marrow and liver. Functional testing of this population revealed that they produced levels of IFNγ lower than what would be produced by an NK cell. When we cultured human ILCPs in differentiating conditions with AML cell lines or AHR agonists, we observed in (open full item for complete abstract)

Committee: Bethany Mundy-Bosse (Advisor); Aharon Freud (Committee Co-Chair); Michael Caligiuri (Committee Member); Susheela Tridandapani (Committee Member); Eugene Oltz (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; Immunology; Molecular Biology

Master of Science (MS), Wright State University, 2022, Pharmacology and Toxicology

The 3'IGHRR is thought to be responsible for the transcription of the immunoglobulin heavy chain (IGH) gene locus, which is essential for the production of antibodies. The human 3'IGHRR, which is duplicated in humans, contains the hs3, hs4, and hs1.2 enhancers. Additionally, the hs1.2 enhancer is polymorphic in humans and consists of a 53 bp invariant sequence that can be repeated one to four times. Previous experiments in a mouse and human B-cell line model have shown that the high affinity AhR ligand and environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce hs1.2 enhancer activity in an AhR-dependent manner. The AhR is a transcription factor that can modulate gene transcription either directly through its transactivation domain or indirectly by interacting with other transcription factors. Further studies with a human B-cell line model (CL-01) that lacks AhR transactivation function demonstrated TCDD-induced activation of the hs1.2 enhancer that was not influenced by the number of the invariant sequence repeat. These studies suggest that a functional transactivation domain may not be necessary for the AhR to mediate TCDD-induced activation of the hs1.2 enhancer. The objective of this study is to assess whether a functional AhR transactivation domain enhances TCDD-induced hs1.2 enhancer activity and alters the transcriptional impact of an increased number of invariant sequences. For this experiment, wildtype CL-01 cells lacking AhR transactivation function and CRISPR/Cas9-edited CL-01 cells expressing an AhR with a functional transactivation domain were transfected via electroporation with luciferase reporter plasmids that contain one of the four human polymorphic hs1.2 enhancer alleles (α1A, α1B, α1C, or α1D corresponding to one, two, three, or four invariant sequence repeats, respectively) and treated with different concentrations of TCDD. Results demonstrated no difference in TCDD-induced activation of the hs1.2 reporter in cells with (open full item for complete abstract)

Committee: Courtney E.W. Sulentic Ph.D. (Advisor); Ravi P. Sahu Ph.D. (Committee Member); Mike Kemp Ph.D. (Committee Member); Khalid Elased Pharm.D., Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

Bachelor of Science, Wittenberg University, 2021, Biology

Insecticide use is steadily increasing in the United States. Of these, the insecticide Sevin™ has the active ingredient carbaryl, which is a xenobiotic. This is a reverse inhibitor of acetylcholinesterase, which prevents nerve excitation causing paralysis and death in insects. Though insects are the target species, carbaryl has many routes of exposure such as run-off, inhalation, and physical contact making it easy to encounter for non-target species like aquatic life and humans. Carbaryl has been studied within in vitro models to test its effects on zebrafish. The insecticide has already been shown to act on the acetylcholinesterase (AChE) pathway, but it also has potential to act on a second pathway: the aryl hydrocarbon receptor (AhR) pathway. Zebrafish genes and genetic pathways are homologous to humans, allowing for insight on how carbaryl could be affecting humans as well. This study set out to use an in vivo model to show how carbaryl is inducing cytochrome P450 or other AhR pathway gene expression. Gene expression of carbaryl treated embryos were compared to untreated embryos to examine this possible relationship. Cyp1a and cyp1b gene expression was increased with an increase in carbaryl concentration suggesting that carbaryl may be causing induction of the AhR pathway.

Committee: Michelle McWhorter (Advisor); Matthew Collier (Committee Member); Daniel Marous (Committee Member) Subjects: Agricultural Chemicals; Biochemistry; Biology; Developmental Biology; Environmental Science; Genetics; Molecular Biology; Toxicology

Master of Science (MS), Wright State University, 2020, Microbiology and Immunology

Aryl hydrocarbon receptor (AhR) mediates the immunosuppressive effects of 2,3,7,8 -tetrachlorodibenzodioxin (TCDD) in murine B cells. The effects of AhR activation on the regulation of expression of human immunoglobulin isotypes (μ, γ1-4, α1-2 and ε) and Ig secretion is unclear. Our previous results using CL-01 cell-line originating from a Burkitt's lymphoma patient, demonstrated an inhibitory effect of TCDD on IgG expression but a surprising and marked loss of IgG secretion when the AhR was knocked out by siRNA or CRISPR/Cas9 gene editing. To determine if the AhR is a critical mediator of IgG expression, current study is focused on characterizing IgG expression in another human B-cell line (SKW 6.4 or SKW WT) originating from a different, non-related Burkitt's lymphoma. We confirmed that SKW WT cells do not have endogenous expression of AhR using PCR analysis and Western blotting. We also demonstrated that SKW 6.4 cells can be stimulated in-vitro using CD40L and IL-4 to produce more IgM antibodies as detected by ELISA assays. Further, we demonstrate that total IgG secretion induced by CD40L and IL-4 stimulation is severely impaired in SKW WT cells. Conversely, the Qt-PCR studies show that the expression of ε, γ2-4 transcripts that code for IgE and IgG2-4 respectively is significantly increased with stimulation as compared to un-stimulated SKW cell lines. The expression of γ1 was low in naive as well as stimulated SKW WT cells. The α1-2 transcripts coding for IgA1-2 respectively are not expressed at all in SKW cells regardless of stimulation. To further investigate, we used CL-01 AhRTA cells that express AhR with functional TAD, to compare the expression of different isotypes. It was found that the expression of γ1-4 and ε transcripts was significantly higher in AhR expressing CL-01 AhRTA cells as compared to SKW WT cells. Our observations imply that AhR plays a critical role in expression of the IgH gene.

Committee: Courtney Sulentic Ph.D. (Advisor); Thomas Brown Ph.D. (Committee Member); Nancy Bigley Ph.D. (Committee Member) Subjects: Immunology; Toxicology

Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences

Acetaminophen (APAP) toxicity is a major cause of acute liver failure (ALF). Although circulating miR-122 is a sensitive biomarker of ALF, the role of this most abundant liver-specific miRNA in ALF has not been elucidated. Here, we show that miR-122 is downregulated in liver biopsies of ALF patients compared to non-ALF biopsies and in the livers of mice treated with APAP. A dramatic decrease in the primary-miR-122 expression occurs upon APAP overdose in mice due to suppression of its two key trans-activators, HNF4a and HNF6. More importantly, the mortality rates of both male and female liver-specific miR-122 knockout (LKO) (Mir122loxP/loxP; Alb-Cre) mice were significantly higher than that of the age- and sex-matched control (Mir122loxP/loxP) mice when injected intraperitoneally with a dose of APAP lethal to LKO mice. Higher basal levels of CYP2E1 and CYP1A2 that convert APAP to highly reactive N-acetyl-p-benzoquinone imine (NAPQI) are contributing factors to the sensitivity of LKO mice to APAP overdose. Upregulation of Cyp1a2 primary transcript and mRNA in LKO livers correlated with elevation of Ahr and Med1, two trans-activators of Cyp1a2 gene. Analysis of ChIP-seq data in the ENCODE database identified the association of CTCF with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1 or Ctcf in Mir122-/- hepatocytes reduced Cyp1a2 expression. Glycerol density gradient centrifugation and pulse-chase studies showed that Cyp2e1 is upregulated in LKO hepatocytes at the protein level. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to APAP toxicity that correlated with upregulation of AHR, MED1 and CYP1A2 expression. Collectively, these results suggest a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in ALF. Cyp1a2 expression. Glycerol density gradient centrifugation and pulse-chase studies showed that Cyp2e1 is upregulated (open full item for complete abstract)

Committee: Kalpana Ghoshal (Advisor); Samson Jacob (Committee Member); Tsonwin Hai (Committee Chair); Ramesh Ganju (Committee Member) Subjects: Biomedical Research; Molecular Biology

Master of Science (MS), Wright State University, 2016, Microbiology and Immunology

Macrophages are crucial for ridding the body of debris and foreign cells. The aryl hydrocarbon receptor (AhR) also plays a critical role in immunity. This study examined the effect of the AhR on the expression of major histocompatiability complex class I (MHCI) and MHC class II (MHCII) in two murine macrophage cell lines. This study used Raw264.7 and J774A.1 murine macrophage cell lines. The Raw264.7 cells are from male BALB/c mice while the J774A.1 cells are from female BALB/cN mice. The addition of the AhR anatagonist CH-223191 (AhRa) showed that the AhR does not significantly impact MHCI expression. However, MHCII expression was decreased by the addition of AhRa in Raw264.7 cells, while MHCII expression was significantly increased in J774A.1 cells after AhRa addition. During the course of the study, trypan blue results showed increased cell survival in classically activated macrophages. Therefore, early apopotosis, late apoptosis, and necrosis were examined by annexin V and propidium iodide analysis. Cell death analysis showed increases in late apoptosis for both cell lines after the addition of AhRa, suggesting the AhR plays a role in cell survival during macrophage activation. This study shows that even basal levels of AhR expression can impact MHCII expression and apoptosis of murine macrophages.

Committee: Nancy Bigley Ph.D. (Advisor); Barbara Hull Ph.D. (Committee Member); Courtney Sulentic Ph.D. (Committee Member) Subjects: Biology; Immunology

Master of Science (MS), Wright State University, 2015, Microbiology and Immunology

The aryl hydrocarbon receptor (AhR) is a ligand-activated cytosolic transcription factor that regulates xenobiotic-metabolizing enzymes. It mediates the toxicity of various environmental chemicals such as 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD). TCDD inhibits the differentiation of B cells into antibody-secreting cells and inhibits immunoglobulin (Ig) expression in various animal models. We have previously determined that TCDD-induced inhibition of the mouse Ig heavy chain gene (mo-Igh) is AhR-dependent. This inhibition may be mediated by binding of the AhR to dioxin response elements (DREs) within the 3'Igh regulatory region (3'IghRR) and inhibition of 3'IghRR activity, a significant transcriptional regulator of Ig expression. However, there are structural differences between the mouse and human 3'IghRR. The mouse contains four enhancers (hs3A; hs1,2; hs3B; and hs4), whereas the human contains three (hs3; hs1,2; and hs4). In addition, the human hs1,2 is known to be highly polymorphic and has been associated with several autoimmune diseases. The current study focuses on elucidating the role of the AhR in human Ig expression and class switch recombination (CSR). We disrupted the AhR signaling pathway in a human B-cell line (CL-01) using two different shRNA constructs or with the chemical AhR antagonist (CH-223191). Although the CL-01 AhR has three heterozygous single nucleotide polymorphisms (SNPs) that results in loss of CYP1A1 gene induction, TCDD significantly inhibits IgG expression, whereas IgM expression has very low sensitivity to TCDD. Interestingly, decreased AhR protein levels results in low IgG expression, while there was no change in IgM expression. In contrast, the AhR antagonist induced greater IgG secretion in stimulated B cells, which was not replicated by the AhR knockdown suggesting a mechanistic difference between the chemical antagonist and AhR knockdown. Reduced AhR levels caused an isotype-specific inhibition of the CSR to IgG1, but not to I (open full item for complete abstract)

Committee: Courtney Sulentic Ph.D. (Advisor); Barbara Hull Ph.D. (Committee Member); Nancy Bigley Ph.D. (Committee Member); Katherine Excoffon Ph.D. (Committee Member) Subjects: Immunology; Microbiology; Toxicology

Doctor of Philosophy (PhD), Wright State University, 2014, Environmental Sciences PhD

Transcriptional regulation of the murine immunoglobulin heavy chain gene involves several regulatory elements including the 3'Igh regulatory region (3'IghRR) composed of at least four enhancers (hs3A; hs1,2; hs3B; hs4). Enhancers hs1,2 and hs4 contain binding sites for several transcription factors including NF-κB/Rel proteins and the AhR. Interestingly, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) profoundly inhibits 3'IghRR and hs1,2 activation induced by the B-cell activator lipopolysaccharide (LPS), but enhances the activation of the hs4. Within the hs4, the AhR binding site overlaps an NF-κB/Rel binding site suggesting that both the AhR and the NF-κB together may modulate of the 3'IghRR. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR following LPS stimulation and TCDD treatment on 3'IghRR, hs1,2, and hs4. In our studies we utilized the CH12.LX B cell line; the CH12.IκBαAA cell line, which expresses an inducible IκBα super repressor (IκBαAA); the CH12.γ2b-3'IghRR cell line that stably expresses a γ2b-3'IghRR-regulated γ2b transgene reporter; and splenocytes derived from B6C3F1 mice. The stimulation of the CH12.γ2b-3'IghRR cell line with Toll-like receptor (TLR) agonists LPS, Resiquimod (R848), or Cytosine-phosphate-Guanine (CpG)-oligodeoxynucleotides combined with a co-treatment of TCDD significantly inhibited the TLR-induced activation of the 3'IghRR. Utilizing transiently expressed luciferase reporters, we found induction of IκBαAA expression partially attenuated LPS-induced activation of the 3'IghRR and hs4, partially reversed the effects of a TCDD and LPS co-treatment on the activity of the 3'IghRR and hs4, and the addition of an AhR antagonist, CH223191, markedly reversed the LPS and TCDD induced inhibition of the 3'IghRR and inhibited the synergistic activation of the hs4. Chromatin immunoprecipitation analysis of CH12.LX and murine splenocytes demonstrated a LPS and a LPS co-treatment with TCDD-dependent increase in Rel (open full item for complete abstract)

Committee: Courtney Sulentic Ph.D. (Advisor); Michael Leffak Ph.D. (Committee Member); Mill Miller Ph.D. (Committee Member); David Cool Ph.D. (Committee Member); Rodney DeKoter Ph.D. (Committee Member); Don Cipollini Ph.D. (Other) Subjects: Environmental Science; Immunology; Molecular Biology; Toxicology

Master of Science (MS), Wright State University, 2013, Pharmacology and Toxicology

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant that inhibits immunoglobulin (Ig) expression and Ig heavy (IgH) chain gene transcription. Transcription of the IgH gene involves several regulatory elements including the 3'lgh regulatory region (3'lghRR) which is composed of four enhancers (hs3A, hs1,2, hs4, and hs3B). Dioxin responsive elements (DRE) in the hs4 and hs1,2 enhancers of the 3lghRR that bind the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates dioxin sensitive genes suggest that the 3'lghRR may be a transcriptional target of TCDD. The current study utilized an IgA secreting mouse B-cell line that stably expresses a γ 2b transgene regulated by the 3'lghRR (CH12.γ 2b-3'lghRR cells). Both shRNA knock down of AhR and an AhR antagonist (CH-223191) reduced TCDD-induced inhibition of endogenous IgA and the γ 2b transgene expression. With the growing number of immune-related disorders correlated with polymorphisms of the human hs1,2 enhancer, ubiquitously found AhR ligands, and sensitivity of human Ig expression to TCDD, our findings may provide indispensible information for human health risk assessment and insight into the development of therapeutic interventions for immune-related disease.

Committee: Courtney Sulentic Ph.D. (Committee Chair); Michael Leffak Ph.D. (Committee Member); Katherine Excoffon Ph.D. (Committee Member) Subjects: Immunology; Pharmacology; Toxicology

Master of Science (MS), Wright State University, 2010, Microbiology and Immunology

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a disrupter, of B-cell differentiation, induces binding of the aryl hydrocarbon receptor (AhR) nuclear complex to dioxin responsive elements (DRE) within the mouse immunoglobulin heavy chain regulatory region (3'IgHRR), and produces a marked inhibition of 3'IgHRR activation, IgH expression, and antibody secretion in a well-characterized mouse B-cell line (CH12.LX). The mouse 3'IgHRR consists of at least four enhancers (hs3a; hs1,2; hs3b; hs4), and is highly homologous with the three enhancers (hs3; hs1,2; hs4) of the human 3'IgHRR. A polymorphism of the human hs1,2 enhancer (resulting in varying numbers of tandem repeats containing a DRE and κB site) has been correlated with several autoimmune diseases. Although the human and mouse hs1,2 enhancers are share a ~90% identity, luciferase reporter studies in mouse CH12.LX B-cells showed that TCDD inhibited LPS stimulation of the mouse hs1,2 enhancer but co-treatment with TCDD and LPS synergistically activated human hs1,2 enhancer activity. To evaluate transcriptional differences between the human and mouse hs1,2 enhancers, our objectives were to characterize the IM-9 cells as a potential human B-cell model, and to evaluate TCDD-induced transcriptional regulation of the polymorphic human hs1,2 enhancer in a human cell line. We confirmed AhR expression and TCDD-induced CYP1A1 induction in IM-9 cells. Then we transiently transfected IM-9 cells with the human hs1,2 reporters and determined that TCDD activates the human hs1,2 enhancer in IM-9 B-cells, as seen in CH12.LX B-cells. However, the TCDD-induced fold-activation in human IM-9 cells appeared less compared to results in mouse CH12.LX B-cells perhaps due to differences between the mouse and human AhR. Our data suggests that the TCDD-induced inhibition of the mouse hs1,2 enhancer versus the activation of the human hs1,2 enhancer may be related to an inhibitory BSAP site located on the mouse hs1,2 enhancer that is absent from th (open full item for complete abstract)

Committee: Courtney E Sulentic PhD (Advisor); Michael Leffak PhD (Committee Member); Nancy Bigley Phd (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Environmental Science; Immunology; Microbiology; Molecular Biology; Pharmacology; Toxicology

Master of Science (MS), Wright State University, 2007, Biological Sciences

The immune system is critical to human survival. However, assessing alterations of immune function by potential immunotoxicants is complicated by the diffuse nature of the immune system, which is composed of various effector cells each with differing effector functions. Current immunotoxicity testing is limited to animal studies. We have developed a model, which may provide an in vitro alternative to animal studies in identifying immunotoxicants that specifically target B cell function (i.e., alteration of immunoglobulin (Ig) or expression and antibody secretion). This model consists of a well-characterized B cell line, CH12.LX, which appears to appropriately model primary B cell function. We have stably transfected the CH12.LX cell line with a transgene regulated by an enhancer, the 3'Ig heavy chain regulatory region (3'IgH RR), purported to control Ig heavy chain gene expression. Our previous work has identified the 3'IgH RR as a sensitive target of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) possibly mediated by activation of the aryl hydrocarbon receptor (AhR) signaling pathway. We have tested our model with several structurally diverse chemicals shown to activate the AhR signaling pathway and found an association between AhR activation and inhibition of both 3'IgH RR activation and Ig protein expression. We have also tested two chemicals previously shown to activate humoral immunity through non-AhR receptors. Results indicate that our model appropriately identifies immunomodulators of two receptor-signaling pathways, each leading to altered immunoglobulin expression. (Supported by the Colgate-Palmolive Grants for Alternative Research and the Boonshoft School of Medicine, WSU)

Committee: Courtney Sulentic (Advisor) Subjects:

PhD, University of Cincinnati, 2010, Medicine: Cell and Molecular Biology

Dioxin and dioxin like compounds (DLCs) are environmental contaminants released during many industrial processes. Halogenated and polycyclic aromatic hydrocarbons, such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), DLCs, and benzo[a]pyrene (BaP) are known or suspected human carcinogens and developmental teratogens and induce expression of the cytochrome P450 encoded by the substrate responsive CYP1A1 gene. CYP1A1 gene induction requires trans-activation by the heterodimeric transcriptional complex formed by ligand bound Aromatic Hydrocarbon Receptor (AHR) and the AHR Nuclear Translocator proteins (ARNT). We have shown that with BaP induced activation of Cyp1a1 gene expression in mouse hepatoma Hepa-1 cells, there is a concomitant change in the pattern of histone modifications associated with the Cyp1a1 promoter and distal enhancer. At the promoter of the Cyp1a1 gene this pattern of histone modification that accompanies BaP induced Cyp1a1 transcription includes trimethylation of Lys4 of histone H3 (3MeK4H3) and hyperacetylation of Lysine 14 of histone H3 (AcK14H3). At the distal enhancer of the Cyp1a1 gene BaP leads to an increase in phosphorylation of Ser10 on histone H3(pS10H3) and a hyperacetylation of Lysine 16 of histone H4 (AcK16H4). Here we find that in mouse embryonic fibroblast, treatment with BaP shows a similar increase in Cyp1a1 transcription as in Hepa-1 cells accompanied by at the same pattern of histone modifications at both the distal enhancer and proximal promoter as in Hepa-1 cells. In MEFs these changes are dependent on the AHR binding to BaP and MEFs with an AHR allele which encodes an AHR protein with low binding affinity for ligand do not elicit the same modifications of histone marks in the Cyp1a1 gene promoter or distal enhancer as those with an allele encoding an AHR protein with higher affinity for ligand. We determine that treatment with various different AHR ligands all lead to similar patterns of histone modifications upstream of the Cyp (open full item for complete abstract)

Committee: Alvaro Puga PhD (Committee Chair); Sohaib Khan PhD (Committee Member); Ying Xia PhD (Committee Member); Daniel Nebert MD (Committee Member); Susan Waltz PhD (Committee Member) Subjects: Cellular Biology

PhD, University of Cincinnati, 2007, Medicine : Toxicology (Environmental Health)

Polychlorinated biphenyls (PCBs) are persistent organic pollutants linked to numerous human health problems, including learning and memory deficits in children of exposed mothers. PCBs exist in the environment as complex mixtures. Both coplanar and non-coplanar PCBs are reported to have neurotoxic effects in animal studies, but individual congeners do not always produce the same effects as PCB mixtures. We used a mixture of eight PCBs to model human exposures based on their reported concentrations in human tissue, breast milk, and the human food supply. Individual risk to PCBs varies by genetic makeup. We developed a mouse model to explore the role of two genes that may be responsible for some of the reported inter-individual differences: the aryl hydrocarbon receptor (AHR) and CYP1A2. There are >12-fold differences between humans with regard to AHR affinity and >60-fold differences in hepatic basal CYP1A2 levels. Our mouse model used Cyp1a2(+/+)wild-type and Cyp1a2(-/-)knockout mice with either a high-affinity or poor-affinity Ahrgene. We hypothesized that high-affinity Ahr bCyp1a2(-/-)would be most susceptible and poor-affinity dCyp1a2(+/+)most resistant to PCB-induced developmental neurotoxicity. Using GC-ECD, we determined that offspring of Cyp1a2(-/-)mothers were exposed to higher levels of coplanar PCBs during development and that Ahr bmice metabolized all PCB congeners more quickly. Differences in PCB exposure were closely correlated with changes in CYP1A1 and CYP1A2 mRNA and protein levels and with well-known endpoints of AHR-mediated toxicity: immunosuppression and hepatotoxicity. In addition, we conducted behavioral phenotyping of exposed offspring. We found significant deficits in learning and memory in Ahr bCyp1a2(-/-)mice, including impairments in novel object recognition and an increased failure rate in the Morris water maze. However, all PCB-treated groups and all genotypes showed significant differences in at least one measure of learning (open full item for complete abstract)

Committee: Dr. Daniel Nebert (Advisor) Subjects: