Contaminated sediment in the Detroit River provokes acclimated responses in wild brown bullhead (Ameiurus nebulosus) populations
Data files
Sep 11, 2023 version files 213.91 KB
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GSTdataDRYAD.xlsx
26.27 KB
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README.md
5.90 KB
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sedimentanalysisDRYAD.xls
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WeatherDRYAD.xlsx
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WesternblotdensitometryvaluesDRYAD.xlsx
45.43 KB
Nov 27, 2023 version files 213.72 KB
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GSTdataDRYAD.xlsx
26.27 KB
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README.md
5.71 KB
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sedimentanalysisDRYAD.xls
119.30 KB
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WeatherDRYAD.xlsx
17.01 KB
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WesternblotdensitometryvaluesDRYAD.xlsx
45.43 KB
Abstract
In a previous study, adaptive responses to a single polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), were identified in brown bullhead (Ameiurus nebulosus) captured from contaminated sites across the Great Lakes. The tumor suppressor p53 and phase I toxin metabolizing CYP1A genes showed a protective and refractory response, respectively, up to the F1 generation (Williams and Hubberstey, 2014). As an extension to the first study, bullhead were exposed to sediment collected from sites along the Detroit River to see if these adaptive responses are attainable when fish from a contaminated site are exposed to a mixture of contaminants, instead of a single compound. p53 and CYP1A proteins were measured in both studies with the addition of phase II glutathione-s-transferase (GST) activity in the second. Three treatment groups were measured: acute (treated immediately), cleared (depurated for three months and subsequent treatment), and farm raised F1 offspring. All three treatment groups were exposed to clean and contaminated sediment for 24 and 96 hours. Acute fish from contaminated sites exposed to contaminated sediment revealed an initial elevated p53 response that was not reached in cleared fish exposed to contaminated sediment. Instead, cleared and F1 bullhead from clean and contaminated sites had overlapping p53 expression patterns in response to contaminated sediment by 96 hours. Acute fish from contaminated sites exposed to contaminated sediment revealed refractory CYP1A expression, which disappeared in cleared fish and whose F1 refractory response overlapped with clean site F1 offspring. Decreasing GST activity was evident in both clean and contaminated fish over time, with clean site fish responding to contaminated sediment more deliberately. By 96 hours, the response patterns of F1 offspring from clean and contaminated sites to clean and contaminated sediment exposures were similar.
Because p53, CYP1A and GST activity responses to contaminated sediment dosing overlapped in clean and contaminated farm-raised F1 offspring, these results suggest that contaminated fish have acclimated to the contaminants present in their environments by reaching a tolerance threshold and no evidence of adaptation was detected in these biomarkers.
README
Contaminated Sediment in the Detroit River selects for evolved CYP1A and p53 responses in wild brown bullhead (Ameiurus nebulosus) populations
Access this dataset on Dryad: doi:10.5061/dryad.9p8cz8wn9
The dataset contains three workbooks:
1. Western blot densitometry values for p53\, CYP1A and loading control actin protein expression in brown bullhead (Ameiurus nebulous) fish retrieved from four sites in the Great Lakes (Pêche Isle (PI; 42o20’38”N, 82o55’43”W), Belle River (BR; 42o17’39”N, 82o42’43”W), Trenton Channel (TC; 42o10’33”N, 83o9’16”W), Belle Isle (BI; 42o20’57”N, 82o58’31”W). Fish were exposed to two classes of sediment: contaminated sediment and clean sediment. Contaminated sediment was collected from Trenton Channel (Detroit River, USA) and clean sediment was collected from Pêche Island (Detroit River, USA). Three categories of fish were exposed to both classes of sediment for two time points before they were sacrificed: 24 and 96 hours. Acute fish were taken directly from all four sites and exposed to sediment immediately; Cleared fish were collected from sites and housed in man made ponds for three months before sediment exposure, and F1 offspring were reared from parents collected the previous year and exposed to sediment at time of experimentation, with Acute and Cleared fish. Optical density values of p53, CYP1A and loading control actin gene expression was analyzed in categories of fish from all four sites and exposed to Trenton Channel (contaminated) and Pêche Island (clean) sediment for 24 and 96 hours. The order of tabs in the workbook represent the category of fish (Acute, Cleared, F1), the gene of interest (p53, CYP1A, actin), and the length of exposure (24 or 96 hour time point): p53_acute, CYP1A_acute24, CYP1A_acute96, p53_cleared, CYP1A_cleared24, CYP1A_cleared96, p53_F1, CYP1A_F124, and CYP1A_F196. In each sheet, the order of columns from left to right represent: a description including the site origin of the fish, category and sediment treatment (contaminated or clean), the numerical ID of the fish named by researchers (FishID), the weight of the fish (Weight (g)), the length of the fish (Length (cm)), optical density values of the gene of interest (p53 or CYP1A), the optical density of the loading control (actin optical density), the ratio of the gene of interest and the loading control (p53/CYP1A:actin), the average optical densities of the n of fish that were part of that treatment group, and the standard error of the average optical density values (SE).
2. Glutathione-s-transferase (GST) enzyme activity data for Trenton Channel and Pêche Island acute, cleared and F1 fish exposed to clean and contaminated sediment for 24 and 96 hours. Tabs correspond to the same categories of fish (acute, cleared and F1) described in Workbook1; Acute 96 hours, Acute 24 hours, Cleared 96 hours, Cleared 24 hours, F1 96 hours, F1 24 hours. Each sheet contains duplicates of samples and FishIDs are grouped together and labeled according to site of origin, category and treatment overtop. Alternatively, FishIDs (including weight and length) can be cross checked in Workbook1. The first column of the sheet gives the 6 timepoints at which fluorescent readings were recorded every minute for 6 minutes for each sample. Each FishID (defined in Workbook1) has 6 corresponding fluorescent readings, 1 per minute beginning at 0:00 and ending at 5:00. Mastermix includes the reaction mixture without an enzyme sample and the Control was provided by Sigma, Canada. All other samples include the master mix and enzyme sample from the FishID. Fluorescent values represent glutathione-s-transferase activity at 340 nm and were used as instructed by Sigma, Canada (CS0410) to compute GST specific activity.
3. Sediment analysis of contaminated and clean treatment sediment collected from Trenton Channel (contaminated) and Peche Island (clean) sites used for treatment of Acute, Cleared and F1 Trenton Channel categories of fish from Pêche Island, Belle River, and Belle Island fish for 24 and 96 hours.
Data is organized into 4 tabs: SITE PCBs, OCs; SITE PAHs; POND, POOL, PCBs, OCs; and POND, POOL PAHs. ‘SITE’ tabs represent analysis of sediment from actual sites in the Great Lakes where fish were retrieved (Belle River, Trenton Channel, Pêche Isle, Belle Isle) and ‘POND, POOL’ tabs represent sediment analysis of ponds used to rear F1 offspring and hold cleared fish for 3 months (pond) and kiddie pools where experimentation took place (pool). %TOC - based on Loss on Ignition (1-muffled weight/dry weight)100. **SRM 1944* - NIST certified dry sediment standard; 0.5g was run in the same way sediment unknown samples were analyzed and compared to published values. PCB 34 - internal standard; 200ng is spiked into the sample before extraction takes place. The value is reported as %recovery based on the amount that the sample was spiked with (200ng). Lab codes are unique identifiers specific to the Sample ID that was determined by the Great Lakes Institute of Environmental Research (GLIER) where samples were analyzed. PBDE-71 and d10-AN were used to check the efficiency of extraction procedures and %recoveries are reported from each sample. ND - no data.
4. Weather data during experimentation in kiddie pools at Leadley Environmental Ltd. (42o6’14”N, 82o55’47”W); average temperature (F), humidity, and pressure are included; can be cross-listed with FishID in Workbook 1, 2, & 3.
Any cells with **N/A* values are void of data or values and this annotation was included to improve readability in our workbooks.
Methods
Brown bullhead (Ameiurus nebulosus) were collected from two reference 'clean' (Peche Island, Belle River) and contaminated sites (Trenton Channel, Belle Isle) in the Great Lakes and exposed to clean (Peche Island) and contaminated (Trenton Channel) sediment for 24 or 96 hours to confirm previously identified adaptive properties between different bullhead populations in the Detroit River. Instead of exposing fish to a single polycyclic aromatic hydrocarbon (Benzo[a]pyrene) as in the previous study, we exposed fish to either clean or contaminated sediment from sites where fish were collected using a petite ponar.
Liver tissue samples were preserved in RNA later or flash frozen in liquid nitrogen at time of collection.
Differences in CYP1A and p53 protein expression (phase I metabolism) and glutathion-s-transferase (phase II) enzyme activity was measured and quantified in F1 farm-raised, Acute and Cleared (for 3 months) fish. Protein expression in liver tissue was measured using western blotting technique and ANOVA was used to identify significant differences in test groups. Microsomal preparation and cytosolic fractionation of liver tissue was used to measure glutathion-s-transferase (GST) activity differences between fish populations and significance was identified by unpaired t-tests.