Abstract:

Analysis of the oxidative stress and DNA damage in adult Florida pompano after reproduction and exposure to chemically enhanced water accommodated fraction (CEWAF) of south Louisiana crude oil.

Suggested Citation:

DL Wetzel, TA Sherwood, CA Miller, RL Medvecky. 2019. CEWAF-exposed Florida pompano reproduction: Oxidative stress and DNA damage assays. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7PZ5793

Purpose:

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

Data Parameters and Units:

Sample Key worksheet: MML, Matrix, Field ID (Fish Tag), Collected date (MM/DD/YYYY), Treatment, Gender, Spawning Tank. Exposure Tank Descriptions worksheet: Tank ID, Start date (MM/DD/YYYY), End date (MM/DD/YYYY), Treatment (High CEWAF, Low CEWAF, Control, Number of Fish. Spawning Tank Description worksheet: Tank ID, Start date (MM/DD/YYYY), End date (MM/DD/YYYY), Treatment (Control, Low, High), Number of Fish. CEWAF Prep worksheet: Start Date (MM/DD/YYYY), End Date (MM/DD/YYYY), Carboy, Oil (g), Corexit (g), Volume Water (L), Treatment (High, Low), Amt Stock Soln (L), Amt Seawater (L). Oxidative Stress worksheet: MMLs with two Field IDs represent two plasma samples combined to run the test due to low volume (For example: CII-16-2754/4280). GSSG and MDA data were not collected for Fish Tag 124B. Spawn #, Fish Tag (Field ID), Treatment (Control, Low, High), Gender, Spawning Tank, MML, T.A.P. (Total Antioxidant Power), Trolox concentration (uM), LiHep with aprotinin, GSH (glutathione uM), GSSG (oxidized glutathione uM), MDA HPLC (malondialdehyde high performance liquid chromatographic method uM). DNA Damage worksheet: Spawn Event, Fish Tag (Field ID), Treatment (Control, Low, High), Gender, Spawning Tank, MML, Matrix, AP Sites per 100,000 bp, 8-OHdG (ng/ml).

Methods:

Plasma Sample Collection: 4mL whole blood was collected into a sodium citrate blood collection tube and inverted 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 which was stored in -80 deg C freezer or in LN2. When samples were removed and thawed for the first time they were aliquotted out to prevent numerous freeze/thaw cycles. Sample Processing: Samples were run according to the manufacturer's protocol; Total Antioxidant Power Colorimetric Microplate Assay (TA02) by Oxford Biomedical Research, GSH/GSSG Microplate Assay (GT40) by Oxford Biomedical Research, 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: