ToxSci Advance Access originally published online on June 30, 2004
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Toxicological Sciences 80, 216-217 (2004)
Toxicological Sciences vol. 80 no. 2 © Society of Toxicology 2004; all rights reserved.
TOXICOLOGICAL HIGHLIGHT |
Nonlethal Development, Validation, and Application of Cytochrome P4501A1 (CYP1A1) as a Biomarker for Contaminant Exposure
USGS Patuxent Wildlife Research Center, Beltsville Lab, c/o BARC-East, Building 308, 10300 Baltimore Avenue, Beltsville, Maryland 20705
Received May 6, 2004; accepted May 26, 2004
The article highlighted in this issue is "Induction of Cetacean Cytochrome P4501A1 by ß-Naphthoflavone Exposure of Skin Biopsy Slices," by Celine Goddard, Roxanna Smolowitz, Joanna Wilson, Roger Payne, and John Stegeman (pp. 268–275).
The cytochromes P450 (CYPs), particularly the CYP1A family and CYP1A1, have been used as biomarkers of exposure to certain classes of contaminants in both aquatic and terrestrial species.
The types of chemical contaminants that cause induction of the CYP1A family are those that bind to the aryl hydrocarbon receptor (AHR). The classes of compounds that typically cause environmental induction of CYP1A are therefore coplanar polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), tetrachlorodibenzodioxins (TCDDs), and tetrachlorodibenzofurans (TCDFs). Noncoplanar PCBs induce the CYP2B family. The CYPs are thus biomarkers for environmental exposure to these classes of compounds.
Because all animals appear to be capable of the CYP1A induction response, the possible applications of this technique are widespread. It has been used in both the aquatic and the terrestrial areas, and it can be used in regard to questions about contamination in a species or in a habitat.
There are many approaches to evaluating the CYP status of organisms. They vary both in the techniques used to assess CYPs and in the sampling required to perform the assessment. At the most basic level the sampling methods can be divided into lethal and nonlethal. This includes both the development and validation procedures and the utilization of the proven methodology in the field. In some cases the species to be studied are abundant, such as carp (Cyprinus carpio), sucker (Catostomus commersoni), European starling (Sternus vulgaris), tree swallow (Tachycineta bicolor), mallard duck (Anas platyrhynchos), and mouse (Peromyscus sp.) so the sacrifice of individual organisms for the development and validation procedures and the utilization of the proven methodology does not have a serious effect on the resource. (Even for such abundant species, however, it is desirable to be able to utilize nonlethal sampling after methods development and validation.) Some other species, however, due to their rarity (and possible threatened or endangered status), or to their great size, are not candidates for this approach.
The sperm whale (Physeter macrocephalus), the animal studied in the highlighted paper, is an example of an endangered species not being appropriate for sacrifice or in vivo testing, which makes treating 50 sperm whales with an inducer of CYP to develop a dose response curve not possible. In this paper, which is based on field sampling and incubations performed in spring and summer of 1999, a number of techniques have been utilized to develop and validate a CYP monitoring technique in this large cetacean.
Previously Fossi and coworkers have done nonlethal sampling of skin from cetaceans by using a crossbow with a hypodermic needle attached to the bolt (Fossi et al., 1992
, 2003). This provided small samples of skin in which total benzo(a)pyrene monooxygenase (BPMO) activity was measured in skin homogenates, and blubber samples in which organic contaminants such as organochlorines (OCs) were measured. When the BPMO and contaminant levels were compared it was found that, in general, Mediterranean cetaceans that have higher concentrations of OCs in blubber have higher skin BPMO activity (Stenella coeruleoalba > Tursiops truncatus > Delphinus delphis > Balaenoptera physalus) and that in fin whale (B. Physalus) there is a correlation between some blubber OC concentrations and skin BPMO activity. This very interesting, nonlethal approach suggests that contaminants in cetaceans are causing induction of CYP in skin, but did not provide the information needed for method development and validation.
The highlighted work has combined this nonlethal sampling method with the techniques of precision tissue slice incubation and assessment of CYP1A induction via immunohistochemistry for CYP1A. The technique of culture of precision liver slices was initially published in 1985 (Smith et al.). Subsequently, in vitro induction of CYP has been demonstrated in such slices (Drahushuk et al., 1996, 1998; Müller et al., 1996), along with evidence that the tissue retains reasonable integrity for approximately 24 h, depending upon species. The highlighted authors have utilized paired precision skin slices from 50 sperm whales and their expertise in immunological CYP measurement (Smolowitz et al., 1991) to develop a dose response curve for induction of CYP1A by beta-naphthoflavone. The induction of CYP1A1 in these slices was so sensitive that at the lowest exposure concentration, 0.6 µM, the responses were 73, 59, and 69% of maximum for endothelial cells, smooth muscle cells, and fibroblasts, respectively. These results serve as a validation of the use of skin CYP measures as a monitoring tool. Because of the variability in inducibility of CYPs between species it is important to establish the responsiveness of each species. This approach is of value not only for sperm whales and other marine mammals, but suggests that the skin precision slice technique could also be of value for any species where a small piece of skin, but not the individual, could be available for dose response or other toxicological studies or for monitoring.
REFERENCES
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Müller, D., Glöckner, R., and Rost, M. (1996). Monooxygenation, cytochrome P4501A1 and P4501A1-mRNA in rat liver slices exposed to beta-naphthoflavone and dexamethasone in vitro. Exp. Toxicol. Pathol. 48, 433–438.[ISI][Medline]
Smith, P. F., Gandolfi, A. J., Krumdieck, C. L., Putnam, C. W., Zukowski, C. F., III, Davis, W. M., and Brendel, K. (1985). Dynamic organ culture of precision liver slices for in vitro toxicology. Life Sci. 36, 1367–1375.[CrossRef][ISI][Medline]
Smolowitz, R., Hahn, M., and Stegeman, J. (1991). Immunohistochemical localization of cytochrome P-4501A1 induced by 3,3',4,4'-tetrachlorobiphenyl and by 2,3,7,8-tetrachlorodibenzoa-furan in liver and extrahepatic tissues of the teleost Stenotomus chrysops (scup). Drug Metab. Dispos. 19, 113–123.[Abstract]
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