Induction of Cetacean Cytochrome P4501A1 by  -Naphthoflavone Exposure of Skin Biopsy Slices

Abstract

Marine mammals can accumulate environmental contaminants in their blubber at concentrations harmful to laboratory animals. Induction of the cytochrome P450 1A1 (CYP1A1) enzyme is widely used as a biomarker of exposure and molecular effects in animal species, yet the validity of this biomarker has not been established in marine mammals. In vivo studies are generally precluded in these protected species, but skin biopsies (epidermis and dermis) can be collected in a minimally invasive way. We developed an in vitro assay using skin biopsy slices to examine CYP1A1 protein induction in marine mammals in response to chemical exposure. Skin biopsies from sperm whale (Physeter macrocephalus) were exposed for 24 h to beta-naphthoflavone (BNF), a prototypical CYP1A1 inducer, and CYP1A1 induction was detected by immunohistochemical staining in endothelial cells, smooth muscle cells, and fibroblasts. Biopsy slices were exposed to a range of BNF concentrations (0.6-600 microM), and a significant concentration-effect relationship was observed in both endothelial and smooth muscle cells (p = 0.05). This is the first study using skin biopsy slices to examine exposure of cetacean tissue to a CYP1A1 inducer. It demonstrates a causal relationship between chemical exposure and CYP1A1 induction and therefore validates the use of CYP1A1 expression in skin biopsies as a biomarker in cetaceans. Our protocol can be adapted to the investigation of chemicals, mixtures, concentrations, incubation times, or biological endpoints of choice. This should prove particularly relevant for these and other protected species that cannot be studied in the laboratory.

Cytochrome P4501A is induced in endothelial cell lines from the kidney and lung of the bottlenose dolphin, Tursiops truncatus

“…The induction of CYP1A1 in endothelium by βNF was observed also in vitro in skin tissue slices from whales (Godard et al,2004). In cetacean populations, in vivo, an elevated level of AHR receptor agonists can be correlated with the expression of CYP1A1detected immunohistochemically in integument samples (Angell et al,2004).…”

“…In cetaceans, exposure to toxicants also results in differential expression of CYP1A1in endothelium, i.e., primarily in the microvascular or capillary endothelium, in the skin (Angell et al,2004;Godard et al,2004) and in other organs (Wilson et al,2005). The availability of bottlenose dolphin lung and kidney endothelial cells now allows studies of toxicant response in cetacean endothelial cells from two organs.…”

“…Godard et al (2004) demonstrated that in vitro induction of CYP1A1expression by β-naphthoflavone in skin biopsy slices from sperm whale, Physeter macrocephalus, was dose dependent in endothelial tissue.…”

“…However, there are no studies with endothelial cells cultured from cetacean tissues which describe the dose dependent responses to toxicant exposure. This is an important objective given the biological roles of endothelium, the frequent detection of CYP1A1 in endothelium in biopsies and its potential as a marker of exposure in marine mammals (Ben-David et al,2001;Angell et al,2004;Godard et al,2004).…”

First detection of CYP1A1 and CYP2B induction in Mediterranean cetacean skin biopsies and cultured fibroblasts by Western blot analysis

“…CYP2B as semi-quantitative detection tools, in biopsy samples, to explore different exposure to planar (polycyclic aromatic hydrocarbons and planar halogenated aromatic hydrocarbons) (Angell et al, 2004;Godard et al, 2004;Wilson et al, 2007) and globular (globular halogenated aromatic hydrocarbons) lipophilic contaminants, respectively, in three subpopulations of Mediterranean Stenella coeruleoalba. Here the calibration curves were validated (as pilot experiment) for semi-quantitative analysies of CYP1A1 (Fig.…”