There are numerous known chemicals, present in surface waters, which may pose a significant risk to the health of fish living in aquatic environments. These chemicals can impair a variety of physiological process and to date, screening assays for evaluating acute, chronic, reproductive toxicity, and endocrine disruption have been well developed. However, screening assays capable of identify chemicals that can adversely impact immune function have yet to be developed. Developing such assays is necessary given that immune system disruption can increase the incidence of disease and death. Thus, the purpose of this study was to develop and validate a fish-based neutrophil migration assay that can be utilized to rapidly screen chemical for immunotoxicity. The specific objective of this study was to develop a neutrophil migration assay featuring adult fathead minnows (a commonly-used toxicity testing model organism). Specifically, this study sought to optimize methods for two key steps of the neutrophil migration assay – tail injury and depigmentation of fish tails for neutrophil visualization. Three tail injury methods were evaluated including partial amputation, a tail nick, and a biopsy punch. The methods of depigmentation evaluated were H2O2/KOH treatment alone, H2O2/KOH and acetone, and H2O2/KOH treatment in combination with acetone and FlyClear solution 1.1 (Triton X, THEED, and urea). Results showing which of these methods is best suited for neutrophil migration assays featuring adult fathead minnows will be presented.

Oxidative stress is an imbalance of reactive oxygen species (ROS) and antioxidant defenses resulting in cell damage and chronic inflammation. It contributes to many pathologies including neurodegenerative disorders, cardiovascular disease, diabetes, and cancer. Macrophages and microglia are phagocytic immune cells that destroy pathogens while releasing inflammatory mediators, such as pro-inflammatory cytokines and ROS. While inflammation is initially a protective mechanism, chronic inflammation is damaging to tissues. To counter oxidative stress, cells express nuclear factor-erythroid 2-related factor (Nrf2) to mitigate excess ROS production. Nrf2 is a transcription factor that promotes the expression of numerous antioxidant enzymes. Our study targets the expression and activation of Nrf2 in cells treated with L2, a compound created by Dr. Kayla Green (TCU Chemistry). Our lab is attempting to determine the molecular mechanism in which L2 may protect phagocytic cells from oxidative stress, and if this mechanism involves the Nrf2 pathway. This research could provide preliminary evidence for the efficacy of this compound as a treatment option for diseases involving oxidative stress.

With increasing global industrialization and subsequent pollution, there are mounting concerns regarding the presence and impacts of reproductive endocrine disrupting chemicals (REDCs), including environmental estrogens. These concerns have led to new international regulations (i.e. REACH) which require that chemicals be screened for endocrine disrupting activity. A variety of in vivo and in vitro screening currently exist; however, the in vivo methods are time-intensive and expensive and the in vitro methods may fail to detect estrogenic compounds with unique modes of action. Thus, there is a need for in vivo estrogen screen assays that are quick and inexpensive. The objective of this study is to validate the newly-developed Rapid Estrogen ACTivity In Vivo (REACTIV) Assay as a reliable approach for the detection of chemicals with estrogenic activity. This assay employs Japanese Medaka (Oryzias latipes) that have been genetically modified to co-express green fluorescent protein and choriogenin (an egg precursor protein). In the assay, the transgenic medaka embryos are exposed to a chemical of interest for 24 hours after hatch and then imaged under a fluorescent microscope. To validate the performance of the assay, tests were performed using two chemicals with known estrogenic activity (i.e., bisphenol A, estradiol) and two inert chemicals (i.e., saccharin and cefuroxime). Results showed that larvae exposed to the estrogenic compound experienced dose-dependent increases in liver fluorescence, while those exposed to the inert chemicals did not. Overall, these results indicate that the REACTIV assay produces predicable results and thus, may be appropriate for use as a standardized estrogen screen method.

BRCA1 is a gene found in humans that, when mutated, has been linked to breast and ovarian cancer. A homolog version of this gene, known as brc-1, exists in an organism called the Caenorhabditis elegans. This is a species of nematode worm that has the potential to be used as a model organism to study this homolog gene that is associated with human breast cancer. Previous studies with C. elegans have shown links between the brc-1 gene and DNA damage responses, cytochrome p450, or cyp, transcription levels, and ratios of male phenotype worms. This project focused on studying whether these brc-1 functions are dictated by the enzymatic activity of the protein made by this gene. To measure these phenotypes, we used a strain of C. elegans with a brc-1 mutation engineered to lack enzymatic activity of the BRCA1 protein toward nucleosomes. In order to determine how this lack of enzymatic activity affects brc-1 functions, we measured levels of reactive oxygen species (serving as a proxy for DNA damage), numbers of male offspring, and cyp levels in the mutant and wild-type C. elegans. Our initial results indicate the effects of enzymatic activity towards nucleosomes on the aforementioned phenotypes.

Staphylococcus aureus is the causative agent of many skin infections and the leading cause of death due to infectious disease in the United States. Additionally, S. aureus is known to rapidly gain antibiotic resistance, as seen with methicillin resistant Staphylococcus aureus (MRSA). Zinc oxide (ZnO), a nontraditional antibiotic, demonstrates antimicrobial action against S. aureus. While the exact mechanism of ZnO antibacterial action is currently unknown, production of reactive oxygen species (ROS) is a commonly proposed mechanism. We find that S. aureus ΔkatA, a mutant susceptible to hydrogen peroxide (H2O2) due to a deletion in the catalase gene, exhibits comparable growth to wild type S. aureus in ZnO. This suggests that production of H2O2 is not vital to the antimicrobial action of ZnO. To further test this, we generated a ZnO resistant mutant (ZnOR) that demonstrates less susceptibility to ZnO. We find that the ZnOR mutant demonstrates comparable growth to wild type S. aureus in H2O2, making H2O2 production an unlikely toxicity mechanism of ZnO. To evaluate the role of ROS besides H2O2, susceptibility of ZnOR and wild type S. aureus to two other ROS, bleach and paraquat was evaluated. We are currently investigating whether N-Acetyl-Cysteine (NAC), a compound that stimulates production of antioxidants and is protective against a wide range of ROS, protects S. aureus from ZnO mediated toxicity. Our data suggests that ROS formation is not the dominant mechanism of antimicrobial action by ZnO and future studies should focus on other potential mechanisms of action.