Statistical analysis of cytochrome P4501A biomarker measurements in fish

Abstract

Abstract-Induction of the cytochrome P4501A (CYP1A) enzyme system in fish is a common biomarker of exposure to aromatic hydrocarbons. Induction of CYP1A can be measured at a number of steps in the transcription-translation-functional protein pathway using a variety of techniques. The present study examined the range of these measurements from 94 published papers in an attempt to examine the statistical characteristics of each method. Cytochrome P4501A induction, as measured by catalytic ethoxyresorufin-O-deethylase (EROD) activity, protein levels (enzyme-linked immunosorbent assay, Western blot analysis, and immunohistochemistry), and mRNA levels (Northern blot analysis and reverse transcription-polymerase chain reaction), was analyzed. When possible, the variance structure, effect size determination, and dose-response modeling of each method of measurement in the laboratory and field were examined. Conclusions from this analysis include: 1) Because of interlaboratory and interspecies variability, general end-point determinations will need to be defined in terms of the statistically detectable fold-change of measurements relative to control or reference values, and 2) fold-change in EROD activity provides the most robust measure of the dose responsiveness of aromatic hydrocarbons within specific chemical classes (e.g., polycyclic aromatic hydrocarbons). The relationship between the ability to measure statistical differences in induction level and the biological significance of those measurements has yet to be defined. To utilize these biomarkers in a risk assessment context, this relationship must be addressed at the scientific and management levels.

Cytochrome P4501A mRNA and protein induction in striped bass (Morone saxatilis)

“…It has also repeatedly been reported that EROD can substantially be inhibited by elevated levels of PCBs, fluoranthene (a prevalent PAH), dioxins and heavy metals (e.g., Viarengo et al 1997;Gooch et al 1989;Willett et al 2001;Ortiz-Delgado et al 2008). EROD activity is technically easy to measure, and it is still considered as a robust biomarker (Oris and Roberts 2007); however, this enzymatic activity biomarker shows some limitations that can make interpretation sometimes difficult in field studies (Spearow et al 2011). Although several studies have focused separately on Cyp1a induction at different organizational levels such as gene expression (e.g.…”

“…Cytochrome P4501A can also be activated by other pathways and plays a strong role in steroid hormone biosynthesis, tryptophan and retinol metabolism (review in Monostory et al 2009). Induction of Cyp1a varies between fish species (Wirgin et al 1996;Willett et al 1997;Oris and Roberts 2007), so that a specific evaluation needs to be performed before using this biomarker on a sentinel species in environmental monitoring programs. In this study, we thus focus on both Cyp1a gene expression and protein content in order to provide a combined biomarker specific for the striped bass Morone saxatilis.…”

Chronic hydrocarbon exposure of harlequin ducks in areas affected by the Selendang Ayu oil spill at Unalaska Island, Alaska

“…In 2008 the ratio was 220.0. Thus, all analyses were based on comparisons of EROD values between oiled and reference sampling locations within years [8,14]. This approach accounted for any systematic bias in laboratory procedures between years.…”

Exposure of sea otters and harlequin ducks in Prince William Sound, Alaska, USA, to shoreline oil residues 20 years after the Exxon Valdez oil spill

“…4A, B) [10,28], a much greater and statistically significant difference in EROD activity in harlequin ducks from oiled and unoiled sites is needed to demonstrate exposure to ERODinducing chemicals [57]. By this metric, we conclude that the EROD data for harlequin ducks from oiled and unoiled areas are all within the range of natural variability of background levels and, therefore, do not prove continuing exposure of harlequin ducks in the NKI area to EVOS PAH residues, particularly in the absence of a proven exposure pathway.…”