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• 1. [Article] Antioxidant enzyme response in rainbow trout (Oncorhynchus mykiss) after subchronic exposure to an environmentally-relevant polycyclic aromatic hydrocarbon mixture

  Polycyclic aromatic hydrocarbons (PAHs) are significant pollutants in aquatic environments. Many are carcinogenic and lead to DNA fragmentation and adduct formation in marine and freshwater organisms. ...

  Citation × Citation Citation Abstract Copy Title: Antioxidant enzyme response in rainbow trout (Oncorhynchus mykiss) after subchronic exposure to an environmentally-relevant polycyclic aromatic hydrocarbon mixture Author: Norby, Tyler S. Polycyclic aromatic hydrocarbons (PAHs) are significant pollutants in aquatic environments. Many are carcinogenic and lead to DNA fragmentation and adduct formation in marine and freshwater organisms. Previous research demonstrates that rainbow trout (Oncorhynchus mykiss) compensate to long-term PAH dietary exposures and reduce the DNA damage. The aim of this study was to assess whether upregulation of detoxifying/antioxidant enzymes in rainbow trout at least partially explains compensation to PAH exposure that allows for this reduction in DNA damage. Rainbow trout were fed a diet that contained a mixture of nine high molecular weight PAHs in environmentally relevant ratios and concentrations. Two doses were used: a lower dose of 20 parts per million (ppm) and a higher dose of 200 ppm. Two other treatment formulations were also used containing 20 ppm and 200 ppm concentrations of benzo(α)pyrene. A control group of rainbow trout was also used. 80 rainbow trout per PAH treatment and control were used in this experiment. The fish were fed PAH treatment diets or a control diet daily beginning when their average weight was 20 g. At days 7, 14, 28 and 42 enough fish were sacrificed from each of the four treatment groups plus control to obtain four 1 g pooled liver samples. Body and liver weights were recorded for each sacrificed fish. Various biomarkers and detoxifying/antioxidant enzyme activities were measured. Lipid peroxidation was estimated for all sample groups using a thiobarbituric acidreactive substances (TBARS) assay. NAD(P)H:quinone oxidoreductase 1 [DTdiaphorase], also known as quinone reductase (QR), activities were determined for all sample groups using a modified photometric assay that defines QR levels as the dicoumarol-inhibitable reduction of 2,6-dichlorophenolindophenol (DCPIP). Glutathione peroxidase (GPOX) activities were determined for all sample groups using a modified photometric assay that defines GPOX levels as the reduction of NADPH in cytosolic liver fractions. Catalase (CAT) activities were determined for all sample groups using an assay kit that utilized the reaction CAT has with methanol in the presence of hydrogen peroxide. CAT showed the strongest significant (P < 0.05) response, rising and falling over the time-course of the study in a dose-dependent sinusoidal decay pattern as the system moved toward optimal steady-state activity. GPOX activities increased significantly (P < 0.05) over the time-course of treatments, also showing a sinusoidal decay pattern similar to that seen with CAT, although the GPOX activity followed a slower decay pattern than CAT and was not dose-dependent. QR activities were low overall. This indicated that this detoxifying enzyme was not a major component of the trout’s adaptive response. The TBARS assay was determined to be an insensitive assay for fish tissues due to the harshness and unspecific nature of the test. The mass-spectroscopy-based F2-isoprostanes assay was determined to be a much more appropriate assay for the determination of lipid peroxide levels in fish tissues, based on previous research. Title: Antioxidant enzyme response in rainbow trout (Oncorhynchus mykiss) after subchronic exposure to an environmentally-relevant polycyclic aromatic hydrocarbon mixture Author: Norby, Tyler S. Polycyclic aromatic hydrocarbons (PAHs) are significant pollutants in aquatic environments. Many are carcinogenic and lead to DNA fragmentation and adduct formation in marine and freshwater organisms. Previous research demonstrates that rainbow trout (Oncorhynchus mykiss) compensate to long-term PAH dietary exposures and reduce the DNA damage. The aim of this study was to assess whether upregulation of detoxifying/antioxidant enzymes in rainbow trout at least partially explains compensation to PAH exposure that allows for this reduction in DNA damage. Rainbow trout were fed a diet that contained a mixture of nine high molecular weight PAHs in environmentally relevant ratios and concentrations. Two doses were used: a lower dose of 20 parts per million (ppm) and a higher dose of 200 ppm. Two other treatment formulations were also used containing 20 ppm and 200 ppm concentrations of benzo(α)pyrene. A control group of rainbow trout was also used. 80 rainbow trout per PAH treatment and control were used in this experiment. The fish were fed PAH treatment diets or a control diet daily beginning when their average weight was 20 g. At days 7, 14, 28 and 42 enough fish were sacrificed from each of the four treatment groups plus control to obtain four 1 g pooled liver samples. Body and liver weights were recorded for each sacrificed fish. Various biomarkers and detoxifying/antioxidant enzyme activities were measured. Lipid peroxidation was estimated for all sample groups using a thiobarbituric acidreactive substances (TBARS) assay. NAD(P)H:quinone oxidoreductase 1 [DTdiaphorase], also known as quinone reductase (QR), activities were determined for all sample groups using a modified photometric assay that defines QR levels as the dicoumarol-inhibitable reduction of 2,6-dichlorophenolindophenol (DCPIP). Glutathione peroxidase (GPOX) activities were determined for all sample groups using a modified photometric assay that defines GPOX levels as the reduction of NADPH in cytosolic liver fractions. Catalase (CAT) activities were determined for all sample groups using an assay kit that utilized the reaction CAT has with methanol in the presence of hydrogen peroxide. CAT showed the strongest significant (P < 0.05) response, rising and falling over the time-course of the study in a dose-dependent sinusoidal decay pattern as the system moved toward optimal steady-state activity. GPOX activities increased significantly (P < 0.05) over the time-course of treatments, also showing a sinusoidal decay pattern similar to that seen with CAT, although the GPOX activity followed a slower decay pattern than CAT and was not dose-dependent. QR activities were low overall. This indicated that this detoxifying enzyme was not a major component of the trout’s adaptive response. The TBARS assay was determined to be an insensitive assay for fish tissues due to the harshness and unspecific nature of the test. The mass-spectroscopy-based F2-isoprostanes assay was determined to be a much more appropriate assay for the determination of lipid peroxide levels in fish tissues, based on previous research. Close