Vitellogenin as a biomarker of environmental contamination by xenoestrogens: Production and characterization of polyclonal anti-vitellogenin of Caiman latirostris

REY F, RAMOS JG, STOKER C, RODRÍGUEZ H, BUSSMANN L, LUQUE EH, MUÑOZ DE TORO MM

Buenos Aires

Congreso; VI Reunión Anual de SETAC Latinoamérica (Society of Environmental Toxicology and Chemistry); 2003

SETAC LA (Sociedad de Toxicología y Química Ambiental Lationamérica

The impact of endocrine disruptors (ED) on ecosystem health is an important issue in contemporary environmental toxicology. Endocrine disruption is defined as a perturbation of the endogenous hormonal milieu by exogenous chemicals. Among the best understood of the environmental ED are those that mimic estrogen action affecting reproductive processes and fetal development of humans and wildlife. Vitellogenin (Vtg) is a large phospholipoglycoprotein synthesized in the liver of female oviparous vertebrates in response to estrogens. In immature and male oviparous animals Vtg is undetectable since the absence of circulating estrogens prevents the transcription of the Vtg gene. However, high levels of Vtg could be induced in males exposed to exogenous estrogens. Thus, the presence of Vtg in serum of male animals of oviparous species could serve as a useful biomarker for assessing xenoestrogens exposure. Caiman latirostris, is widely distributed in northeastern Argentina. The ecological features of C. latirostris, such as like lifespan, predator habits and distribution, make this species as a potential sentinel of estrogen contamination. The aims of the present study were: a) to produce and characterize a polyclonal antibody that recognizes C. latirostris Vtg, b) to develop a quantitative western blot reaction and an immunohistochemistry (IHC) protocol to assess the estrogenic induction of Vtg in caimans. Vtg arising from E2 treated C. latirostris females was identified in polyacrylamide gels stained with Coomassie blue and Stains all®, isolated from plasma by double precipitation with MgCl2-EDTA and distilled water, and purified using ion exchange chromatography. The purified Vtg fraction was loaded in polyacrylamide gels and transferred to nitrocellulose membranes. In order to obtain immune serum, pulverized nitrocellulose diluted in PBS buffer was injected sc to rabbits. The western blot protocol was optimized to evaluate the specificity and sensibility of the anti-Vtg immune serum. The lowest concentration of Vtg detected was 0.3 mg/ml and a high range of linearity (0.3 mg/ml to 70 mg/ml, r2: 0.95) was scanned when dilutions of the purified standard were used. Using different Vtg-induction protocols either in males or females the antibody showed high specificity, resulting appropriate to assess low levels of circulating Vtg. Moreover, the antiserum was useful to detect Vtg expression in paraffin-embedded sections of liver tissue. Vtg was detected as dispersed and peripherical granules located in the inner border of the cytoplasmic membrane of the hepatocytes. The antiserum was able to detect very low levels of C. latirostris Vtg in quantitative western blot. The assay developed could be used to assess Caiman latirostris exposure to environmental estrogens. These findings support the utility of C. latirostris as a sentinel to monitor estrogenic contamination in aquatic environment