Reduced cytochrome P4501A activity and recovery from oxidative stress during subchronic benzo[a]pyrene and benzo[e]pyrene treatment of rainbow trout

Abstract

This study assessed the role of aryl hydrocarbon receptor (AHR) affinity, and cytochrome P4501A (CYP1A) protein and activity in polyaromatic hydrocarbon (PAH)-induced oxidative stress. In the 1-100nM concentration range benzo[a]pyrene (BaP) but not benzo[e]pyrene (BeP) competitively displaced 2nM [(3)H]2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin from rainbow trout AHR2α. Based on appearance of fluorescent aromatic compounds in bile over 3, 7, 14, 28 or 50days of feeding 3μg of BaP or BeP/g fish/day, rainbow trout liver readily excreted these polyaromatic hydrocarbons (PAHs) and their metabolites at near steady state rates. CYP1A proteins catalyzed more than 98% of ethoxyresorufin-O-deethylase (EROD) activity in rainbow trout hepatic microsomes. EROD activity of hepatic microsomes initially increased and then decreased to control activities after 50days of feeding both PAHs. Immunohistochemistry of liver confirmed CYP1A protein increased in fish fed both PAHs after 3days and remained elevated for up to 28days. Neither BaP nor BeP increased hepatic DNA adduct concentrations at any time up to 50days of feeding these PAHs. Comet assays of blood cells demonstrated marked DNA damage after 14days of feeding both PAHs that was not significant after 50days. There was a strong positive correlation between hepatic EROD activity and DNA damage in blood cells over time for both PAHs. Neither CYP1A protein nor 3-nitrotyrosine (a biomarker for oxidative stress) immunostaining in trunk kidney were significantly altered by BaP or BeP after 3, 7, 14, or 28days. There was no clear association between AHR2α affinity and BaP and BeP-induced oxidative stress.

Figure 1

Competitive binding of BaP and BeP to rainbow trout AHR2α. Trout AhR2α was synthesized by in vitro transcription and translation and incubated (18 h at 4°C) with [³H]TCDD (2 nM) and increasing concentrations of competitor, followed by analysis by velocity sedimentation as described in Materials and Methods. (a) Representative binding curves showing [³H]TCDD binding to AhR2α in the absence or presence of increasing concentrations of BaP (left panel) or BeP (right panel) or to unprogrammed lysate (UPL; a measure of non-specific binding). (b) Specific binding of [³H]TCDD in the presence of increasing concentrations of competitor, expressed as percent control specific binding (binding in the absence of competitor). Error bars indicate the range of values, from two independent binding assays.

Figure 2

Mean concentrations of fluorescent aromatic compounds (FACs) (±SE) measured at benzo(a)pyrene wavelengths 380/430(BaP-FACs) (a) and phenanthrene wavelengths (PHE-FACs) (b) in bile of juvenile rainbow trout fed either 3 μg BaP/g fish/day, 3 μg BeP/g fish/day or control diet for 50 days. Measurements (n=4 for each treatment) indicated with asterisk (*) are significantly different than control (ANOVA, p<0.05). The double asterisk (**) indicates significant difference from other times in this treatment group.

Figure 3

(a) Changes in ethoxyresorufin-O-deethylase (EROD) activity at 3, 7, 14, 28 and 50 days in liver microsomes of juvenile rainbow trout fed either 3 μg BaP/g fish/day, 3 μg BeP/g fish/day or control diet for 50 days. Measurements (n=3) indicated with asterisk (*) are significantly different than control (ANOVA, p<0.05). (b) Staining intensity (mean ±SE of CYP1A in liver of juvenile rainbow trout at 3, 7, 14 and 28 days fed either 3 μg BaP/g/fish, 3 μg BeP/g/fish or control diet. Measurements (n=3–5) indicated with asterisk (*) are significantly different than control (ANOVA, p<0.05).

Figure 4

Results of the single cell electrophoresis (comet assay) expressed in terms of the percentage of DNA in the comet tail measured in blood cells of juvenile rainbow trout fed either 3 μg BaP/g fish/day, 3 μg BeP/g fish/day or control diet for 14, 28 or 50 days. Results are expressed as mean ±SE for n=4 samples. Measurements indicated with asterisk (*) are significantly different than control (ANOVA, p<0.05).

Figure 5

Simple linear regression of tail DNA from comet assays on whole blood (dependent variable) versus hepatic microsomal EROD activity (independent variable) from rainbow trout fed BaP or BeP for 14, 28, or 50 days. The coefficient of determination (r²) was 0.93 with p=0.00003.