Abstract:

Analysis of the oxidative stress markers in red drum after exposure to CEWAF of south Louisiana crude oil. Analysis of DNA damage in red drum after exposure to CEWAF of south Louisiana crude oil.

Suggested Citation:

DL Wetzel, TA Sherwood, RL Medvecky. 2019. Red drum CEWAF uptake/depuration study: Oxidative Stress Assays & DNA Damage Assays. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7J67FDV

Purpose:

Controlled oil exposure study to evaluate the effects of south Louisiana crude oil, via CEWAF, on oxidative stress and DNA damage.

Data Parameters and Units:

Tank Descriptions worksheet: Tank ID, Start and End Date (mm/dd/yyyy), Treatment, Number of Fish. CEWAF Prep worksheet: Date (mm/dd/yyyy), Carboy, Oil (g), Corexit (g), Volume Water (L). Test 113 Sample Key worksheet: Day, Sample Date (mm/dd/yyyy), Sample Location (MAP - Mote Aquaculture Program), Tank ID, Matrix, MML #, Fish ID, Tag #. Fish samples on days 1-10 were composite samples of two fish from the same tank. Therefore, the Tank ID and Day were used to identify these composite samples. Oxidative Stress worksheet: Fish ID, Day, Tank, Treatment, T.A.P. (Total Antioxidant Power) Rep 1, Rep 2, Trolox Conc. (uM). DNA Damage worksheet: Fish ID or MML, Day, Tank, Treatment, BPDE DNA Adducts (ng/mL), AP Sites Rep 1 (AP Sites per 100,000 bp) Avg (AP Sites per 100,000 bp), 8-OHdG Rep 1 (ng/ml), Rep 2 (ng/ml), Avg (ng/ml), Pooled Sample name, n.

Methods:

Sample Collection: plasma Collected 4 mL whole blood into a sodium citrate blood collection tube and gently inverted tube 5 times. Tube was centrifuged at 2000 x g for 10 minutes between 30- 60 minutes from collection time. Supernatant was removed and placed in a clean cryovial then stored in -80 deg C freezer. When samples were removed and thawed for the first time they were aliquotted out to prevent numerous freeze/thaw cycles. Sample Processing: Samples are run according to manufacturer's protocol; Oxford Biomedical Research Total Antioxidant Power Assay (TA02), OxiSelect™ Oxidative DNA Damage Quantitation Kit-AP Sites (STA-324) by Cell Biolabs, and OxiSelect™ Oxidative DNA Damage ELISA- 8- OHdG Quantitation (STA-320) by Cell Biolabs.

Instruments:

DS2 Automated ELISA instrument by Dynex Technologies

Error Analysis:

%CV for the standard curve less than 25% is acceptable, less than 15% for sample replicates.

Individual Files: