Rainbow trout primary epidermal cell proliferation as an indicator of aquatic toxicity: an in vitro/in vivo exposure comparison.
Little or no work has been carried out on primary cell cultures in terms of cellular proliferation and toxicity studies. Cell proliferation represents one of the most relevant cellular functions. Anti-PCNA antibodies have aroused considerable interest recently as potential immunocytochemical markers of proliferation for use in toxicity studies. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to rainbow trout (Oncorhynchus mykiss (R.)) primary cultured epidermal cells exposed in vivo i.e. whole animal exposures and in vitro for the study of the ecotoxicological potential of the aromatic amine, 2,4-dichloroaniline (2,4-DCA), a member of a little studied and widespread class of aquatic pollutants. There are many approaches to assess the proliferative activity of cells. Immunocytochemical methods offer a high sensitivity and specificity. The immunohistochemical avidin-biotin complex (ABC) method was used for the detection and quantification of PCNA, one of the best-known endogenous proliferation markers, applying the mammalian monoclonal antibody PC-10 to formalin-fixed primary cultures of rainbow trout skin. Here we describe our experience with the immunocytochemical detection and quantification of this proliferation marker. Results indicate that the antibody cross reacts with the corresponding rainbow trout epitope and that the alterations in PCNA labelling in the in vivo and in vitro exposed cultures followed similar patterns. This paper presents data on the validation of rainbow trout primary epidermal culture as an in vitro ecotoxicity model with epidermal proliferation as an endpoint. It can be concluded that cellular proliferation could be used as an indicator of the aquatic toxicity potential of xenobiotics. Correlations between cellular proliferation responses in primary cultures derived from in vivo exposed rainbow trout and primary cultures exposed in vitro were assessed. A dose-response was evidenced in both approaches, however the in vivo exposures appeared to be approximately two orders of magnitude more sensitive than the in vitro exposures. Responses in vitro occurred between 200 and 1000 micro M while in vivo responses were between 2 and 10 micro M. The good qualitative correspondence between the in vitro and in vivo results indicates that studies using trout epidermal cells allow the identification of xenobiotic effects in fish skin. However, further work is required before quantitative predictions i.e. effective concentrations in vivo, can be made from in vitro studies. This study suggests that the in vitro exposed rainbow trout primary cultured cell model with proliferation as an endpoint can be used as an alternative testing procedure to the whole animal assay.
Environmental and Radiation Science Centre, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland. m.kilemade@ucc.ie
Michael
M
Kilemade
Maria
M
Lyons-Alcantara
Tina
T
Rose
Richard
R
Fitzgerald
Carmel
C
Mothersill
D000814:Aniline Compounds / Q000633:toxicity; D000818:Animals; D000917:Antibody Formation; D018929:Cell Culture Techniques; D002455:Cell Division / Q000187:drug effects*; D000078404:Epidermal Cells; D007118:Immunoassay / Q000379:methods; D007150:Immunohistochemistry; D017686:Oncorhynchus mykiss / Q000502:physiology*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity; D018675:Toxicity Tests / Q000379:methods; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D015262:Xenobiotics / Q000633:toxicity*
D000814:Aniline Compounds; D018809:Proliferating Cell Nuclear Antigen; D014874:Water Pollutants, Chemical; D015262:Xenobiotics; C054619:2,4-dichloroaniline