Document Type

Dissertation

Date of Award

8-2021

School/College

College of Science, Engineering, and Technology (COSET)

Degree Name

Ph.D. in Environmental Toxicology

Committee Chairperson

Zivar ZY Yousefipour

Committee Co-Chairperson

Kasturi KR Ranganna

Committee Member 1

Shodimu-Emmanuel SO Olufemi

Committee Member 2

Erica EC Cassimere

Keywords

Acrolein DNA Methylation, DNA Methyltransferases(DNMTs), Epigenetics, N-acetylcysteine, Ten Eleven Translocation Enzyme (TET)

Abstract

Acrolein, an alpha-beta unsaturated aldehyde, and a very reactive and toxic compound is released into the environment from different sources. As a pervasive environmental pollutant, acrolein poses a serious environmental health threat acknowledged by investigators, health, and environmental government agencies. Acrolein is released into the environment through the burning of organic compounds. The main sources of acrolein pertinent to human health and toxicity include cooked foods, combustion of fossil fuels, cigarette smoke, overheating of frying oil, endogenously produced via lipid peroxidation, metabolism of polyamine, and anticancer drug cyclophosphamide. Exposure of humans to acrolein is mainly through inhalation of contaminated air, ingestion of contaminated food, and water and dermal contact. Exposure and buildup of acrolein in the biological system have been linked to different disease conditions such as cancer, cardiovascular diseases, multiple sclerosis, spinal cord injury, and Alzheimer’s diseases. Several mechanisms have been proposed for the toxicity of acrolein including inflammation, protein abduction, oxidative stress, DNA abduction, and membrane disruption. Some studies have linked acrolein-induced toxicity to epigenetic modifications. Epigenetics is the modifications in gene expression that do not alter the DNA sequence of a gene. This alteration could occur naturally or by factors such as age, environmental exposure, individual lifestyle, and disease condition. DNA methylation is an epigenetic process that ensues via the addition of a methyl (CH3) group to DNA and frequently alter the role of the gene and transcription factor and as such influence gene manifestation. DNA methyltransferases ( DNMTs) are a group of enzymes that control the addition of methyl groups at different levels in cells. There are three major types of DNMTs; DNMT1 is the methylation enzyme responsible for the maintenance of established patterns of DNA methylation while DNMT3A and DNMT3B mediate the establishment of new or de novo DNA methylation patterns. On the other hand, Ten Eleven Translocation (TET) enzymes counter the activities of DNA methyltransferases via stepwise biochemical conversion of 5- methylcytosine to 5-carboxyl cytosine, which is then modified by thymine DNA glycosylase (TDG) making it a basic cytosine which allows the regeneration of unmethylated cytosine through base excision repair thereby abolishing DNA methylation. N- Acetylcysteine (NAC) is an antioxidant that is vital for the reduction of oxidative stress and the downstream negative effects that are associated with it. NAC has shown potency in the inhibition of acrolein-induced toxicity based on the recent studies done in our laboratory and other investigators. NAC beneficial effects are through improvement in the production of cellular glutathione, a natural antioxidant enzyme known to defend the body against the invasion of xenobiotics such as acrolein. Our earlier work using Vascular Smooth Muscle Cells (VSMCs) has indicated that acrolein induces cytotoxicity and modifies histones specifically histone H3, through epigenetic modification via methylation and acetylation and NAC blocks the toxicity and inhibits histone modification. Based on current information, we are hypothesizing that acrolein toxicity is through changes in DNA methylation and NAC prevents this toxicity by inhibiting DNA methylation. Specific aims of the study are: Demonstrate that acrolein induces toxicity in rats is through DNA methylation, Evaluate the activities of DNA methylation/demethylation enzymes, Evaluate the effect of N-acetylcysteine (NAC) on the prevention of DNA methylation and subsequent changes. To investigate our hypothesis, male Sprague-Dawley rats were treated with 2mg/kg/day of acrolein for seven days (i.p) in the presence or absence of 600mg/kg/day of NAC (i.p). The dose and duration of exposure were selected based on our previous studies. At the end of the study period, kidney tissue was collected, nuclear protein and DNA samples were extracted for biochemical analysis. Lipid peroxidation was measured using MDA (Malondialdehyde)- based colorimetric assay. 5-mC hydroxylase TET activity, DNMT activity, Thymine DNA glycosylase activity, DNA Demethylase activity, and DNA Damage Quantification (8-OHdG) were measured by ELISA-based colorimetric and fluorometric assay from Epigentek. Global DNA methylation and global DNA hydroxymethylation were measured by ELISA-based colorimetric assay from Abcam and protein expression by western blotting. Our results revealed that acrolein-treated rats showed about a 169% increase in lipid peroxide level when compared to control. The addition of NAC decreases lipid peroxidation level to about 52% compared to the acrolein group. There was about a 67% decrease in 5-mC TET Hydroxylase activity in the acrolein treated group compared to control, the addition of NAC increased 5-mC TET Hydroxylase activity by 28% compared to the acrolein group. Consistently, there was about a 14% decrease in demethylase activity in the acrolein group compared to the control group and the addition of NAC increased demethylase activity to about 20% compared to the acrolein group. Similarly, there was about a 22% decrease in TDG activity in the acrolein treated group compared to the control and the addition of NAC showed an approximately 15% increase in TDG activity compared to the acrolein group. On the contrary, there was about a 30% increase in DNMT activity in the acrolein group compared to the control. The addition of NAC indicated about 32% increase compared to the acrolein group. The reason behind the increase in DNMT activity after the addition of NAC is yet to be understood since NAC is known to repeal acrolein toxicity. There was up to 16% increase in the global 5-mC level in the acrolein treated group compared to the control and the addition of NAC increased it by 16% when compared to the control. The same trend was observed in global 5-HmC where there was a 22% decrease in 5-HmC activity in the acrolein group compared to the control. The addition of NAC increased the level of 5-HmC approximately to 130% compared to the acrolein group. The decrease in global 5-mC and 5-HmC agrees with many investigators’ reports (hypomethylation) as the prognosis of different cancer development. Finally, there was about an 18% decrease in the 8-OHdG level in the acrolein group compared to the control and NAC addition increased the 8-OHdG level by 167% compared to the acrolein group. Based on the present data, we are concluding that epigenetic changes observed in acrolein-treated rats are the results of increased generation of ROS, which contribute to increasing oxidation level, alteration of TET enzymes and their products, and increased DNA methylation and subsequent DNA damage.

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