Evaluating the bioactivity of the ubiquitous tire preservative 6PPD and degradant, 6PPD-quinone

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-quinone; 6PPD-Q), a transformation product of an antiozonant chemical widely used in automobile tires (6PPD; N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine) was identified as the causative agent for pre-spawn mortality in coho salmon resulting from exposure to stormwater run-off. This study employed a 24 h, 96-well plate based, exposure to larval fathead minnows (5 days post-fertilization) to examine the effects of 6PPD and 6PPD-Q exposure on global gene expression. Concentration-response modeling of the resulting gene expression changes identified a transcriptomic point of departure of 1.3 uM for 6PPD and 2.5 uM for 6PPD-Q, although they are effectively equivalent considering the range of uncertainty. Examination of the functional roles of differentially expressed genes provided little insight into the mechanism of idiosyncratic toxicity observed in coho salmon and certain other salmonid species. Overall, larval fathead minnows do not appear sensitive to the same mechanisms. Overall design: This study was conducted to investigate whether exposure to either the tire rubber antioxidant chemical 6ppd or its quinone-transformation product, 6ppd-q, impacts the transcriptome of fathead minnow and can provide any insight into the mode of toxic action of 6ppd-q or underlying cause of fish species sensitivity differences to 6ppd-q. Comparative gene expression profiling was performed using data obtained from RNA-seq of larval (five day post hatch) fathead minnow exposed for 24 hr in 1-mL, 96-well plate format to either the tire rubber antioxidant chemical 6ppd (n=30) or its quinone transformation product, 6ppd-quinone (6ppd-q; n=40). Exposures were conducted without feeding (reliant on yolk and not independently feeding at this age) in a total volume of 700 µl with 0.3% DMSO per well. Fish were exposed to either six (6ppd) or eight (6ppd-q) concentrations, with 5 replicate fish exposed per plate and triplicate plates (A, B, C). Fish were pulled from either plate A or C for RNA-seq, including a total of 10 non-exposed control samples. Plates were sealed and placed in a 25°C incubator under a 16:8h light routine for 24 h.