DOI: 10.5061/dryad.9p8cz8wn9
title: Contaminated sediment in the Detroit River provokes acclimated responses in wild brown bullhead (Ameiurus nebulosus) populations

description: 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.
description: Brown bullhead (<em>Ameiurus nebulosus</em>) 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.
description: **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.
 (42<sup>o</sup>6’14”N, 82<sup>o</sup>55’47”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.