Persistence of Benzo(a)pyrene disruption of cardiac function, metabolism, and gene expression in rainbow trout and zebrafish
Date
2020-05-13
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Doctoral
Abstract
Benzo(a)pyrene (BaP)is a ubiquitous environmental pollutant that can be created through natural processes and anthropogenic activities, often involving uncontrolled combustion or petroleum extraction, shipment, storage or usage. Rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) are both considered excellent vertebrate models for investigating mechanisms of toxicity in environmental toxicology. BaP has been reported to induce persistent cardiac and metabolic effects in fish. Juvenile rainbow trout were injected once daily for 2 consecutive days with corn oil (control vehicle) or B(a)P (0.1 and 1 mg/kg; n=20 per group). On days 4 and 7 (n=10/group/day), fish underwent cardiac ultrasound, and were then euthanized, necropsied, and arterial blood, heart, liver, and red muscle were collected for analysis of BaP residues in liver and muscle (HPLC), analysis of tissue glycogen and triglycerides content; liver and muscle enzyme activities of lipoprotein lipase (hydrolyzation of triglycerides), 3-hydroxyacyl-CoA dehydrogenase (-oxidation of fatty acids), citrate synthase (aerobic metabolism), lactate dehydrogenase (anerobic metabolism), and L-lactate level in blood, liver, and muscle (marker of anaerobic metabolism). Most cardiac effects were transient and correlated with the BaP residue in the body that was detected at Day 4, but not Day 7. Trout exposed to BaP underwent a dose-dependent shift toward more glycolytic metabolism, detected with changes in enzyme activities, but not mRNA expression, that persisted until day 7 despite BaP residues in tissues returning to control levels. The research presented here demonstrated that BaP induces disruption in metabolism that persists after the parent compound has been cleared from tissues in rainbow trout. Several mechanisms can be responsible for these effects including, but not limited to, epigenetics, pre- and post-translational modifications. In a second series of experiments, the compound 5-azacytidine (5aza) was used in combination with BaP because it has been used extensively in mammalian research on epigenetics. However, effects of 5aza alone were compared to BaP effects. Therefore, results of this second study were inconclusive and data could not be used to support an epigenetic mechanism.
In the third study of this thesis, adult zebrafish were aqueously exposed to 162 g/L of BaP (dissolved in DMSO) or vehicle control (0.1%) for 48h and reared in clean, dechlorinated, municipal water for another 24 or 72h. Even though on day 2 there were significantly higher residues of BaP in whole body, the effects of exposure to the contaminant were minimal. In the muscle, citrate synthase was increased on day 2 and lactate dehydrogenase mRNA abundance was increased on day 3, showing an acute increase in aerobic metabolism and posterior upregulation of transcription of the gene encoding the enzyme regarded as a biomarker for anaerobic metabolism. Moreover, in the heart, there was an increase in mRNA abundance of citrate synthase, denoting an increase in aerobic metabolism in cardiac tissue. Overall, the adult zebrafish demonstrated minimal disruption of metabolism and gene expression due to acute exposure to BaP and high capacity for recovery from exposure.
The main findings of this thesis are that BaP has the potential to induce deleterious effects in rainbow trout metabolism that persist beyond the existence of the compound in the fish, which suggests pre- and/or post-translational changes, rendering the fish burdened with modified metabolic demands. Moreover, these studies further demonstrate that the zebrafish is highly resistant to BaP. The persistent metabolic effects observed in both fish species have the potential to negatively affect individual organisms by disrupting reproduction, growth, and development, thus potentially affecting entire populations and ecosystems.
Description
Keywords
rainbow trout, zebrafish, benzo(a)pyrene, persistence, metabolism
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Toxicology Centre
Program
Toxicology