ORCID

https://orcid.org/0000-0001-5262-5457

Date of Award

2023

Document Type

Dissertation

Degree Name

Philosophy (Ph.D)

Department

Pharmaceutical Sciences

First Advisor

Louis D Trombetta

Second Advisor

Joseph M Cerreta

Third Advisor

Diane Hardej

Abstract

Copper, as an essential trace element, is crucial for various biological processes but has also been utilized as a constituent of various pesticides for decades. Copper (II) octanoate is a pesticide utilized as a fungicide and is approved for use in the production of certified organic foods. Previous studies have implicated copper as an inducer of oxidative stress and activator of the Nrf2-ARE pathway. Nrf2 is essential in regulating the transcription of various genes to alleviate toxicity such as those involved in glutathione synthesis and detoxification. Due to the limited toxicological data, this study into the potential induction of oxidative injury and activation of Nrf2-mediated mechanisms was performed on copper (II) octanoate in vitro. Astrocytes were the chosen model as these cells are vital for ion homeostasis and support overall neuronal survival. Astrocytes were treated with 100-1000 μM of copper (II) octanoate for 24 hours. Cell viability was assessed and the LC25 and LC50 determined as 300 and 350 μM, respectively. Fluorescence microscopy of astrocytes treated with 300 or 350 μM copper (II) octanoate indicated an increase of Nrf2 translocation into the nucleus from the cytosol as compared to untreated cells. Cell treated with 300 or 350 μM copper (II) octanoate also resulted in a significant increase in total glutathione levels, a decrease in the GSH/GSSG ratio, and a statistically significant increase in heme oxygenase 1 (HO-1) protein levels in both treatment groups as compared to control. A statistically significant upregulation in glutamate cysteine ligase catalytic (Gclc) and modifier (Gclm) subunits of glutamate cysteine ligase, heme oxygenase 1 (Hmox1), and NAD(P)H quinone dehydrogenase 1 (Nqo1) mRNA levels were observed in cells treated with 350 μM copper (II) octanoate as compared to control. Intracellular metal analysis by inductively coupled plasma-optical emission spectroscopy (ICP-OES) of astrocytes treated with 300 or 350 μM copper (II) octanoate revealed a concentration-dependent increase in copper and zinc content as compared to control. Thus, activation of the Nrf2-ARE pathway by copper (II) octanoate suggests toxicity may be associated with accumulation of intracellular copper, cellular redox dysregulation, and the induction of Nrf2-mediated antioxidant/detoxification pathways to mitigate cellular injury.

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