INVESTIGADORES
RODRIGUEZ Horacio Adolfo
congresos y reuniones científicas
Título:
Vitellogenin as a biomarker of environmental contamination by xenoestrogens: production and characterization of a polyclonal anti-vitellogenin of Caiman latirostris (yacaré overo)
Autor/es:
REY F; RAMOS JG; STOKER C; RODRIGUEZ HA; BUSSMANN, L; LUQUE EH; MUÑOZ DE TORO M
Lugar:
Buenos Aires
Reunión:
Congreso; VI Congreso SETAC (Society of Environmental Toxicology and Chemistry) Latinoamérica; 2003
Institución organizadora:
SETAC: Sociedad de Toxicología y Química Ambiental
Resumen:
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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 g/ml and a high range of linearity (0.3 g/ml to 70 g/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.