2 research outputs found
Endocrine Disruption Screening by Protein and Gene Expression of Vitellogenin in Freshly Isolated and Cryopreserved Rainbow Trout Hepatocytes
Xenobiotics
may activate the estrogen receptor, resulting in alteration
of normal endocrine functions in animals and humans. Consequently,
this necessitates development of assay end points capable of identifying
estrogenic xenobiotics. In the present study, we screened the potential
estrogenicity of chemicals via their ability to induce vitellogenin
(VTG) expression in cultured primary hepatocytes from male trout.
A routine method for VTG detection measures the secretion of the protein
by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout
hepatocytes. However, this lengthy (6 days) culturing procedure requires
that hepatocyte isolation is performed each time the assay is run.
We optimized this methodology by investigating the utility of cryopreserved
hepatocytes, shortening the incubation time, performing a quantitative
real-time PCR (qPCR) method for VTG quantification, and verifying
the model system with reference chemicals 17β-estradiol, estrone,
diethylstilbestrol, hexestrol, genistein, and a negative control,
corticosterone. To test the performance of both freshly isolated and
cryopreserved hepatocytes, mRNA was collected from hepatocytes following
24 h treatment for VTG gene expression analysis, whereas cell culture
media was collected for a VTG ELISA 96 h post-treatment. EC<sub>50</sub> values were obtained for each reference chemical except for corticosterone,
which exhibited no induction of VTG gene or protein level. Our results
show linear concordance between ELISA and qPCR detection methods.
Although there was approximately 50% reduction in VTG inducibility
following cryopreservation, linear concordance of EC<sub>50</sub> values
was found between freshly isolated and cryopreserved hepatocytes,
indicating that cryopreservation does not alter the functional assessment
of estrogen receptor activation and therefore VTG expression. These
studies demonstrate that qPCR is a sensitive and specific method for
detecting VTG gene expression that can be used together with cryopreserved
trout hepatocytes for screening estrogenic chemicals, resulting in
a reduction of the time required to perform the assay and enabling
greater access to the model system through the approach of cryopreservation
Intra- and Interlaboratory Reliability of a Cryopreserved Trout Hepatocyte Assay for the Prediction of Chemical Bioaccumulation Potential
Measured
rates of intrinsic clearance determined using cryopreserved
trout hepatocytes can be extrapolated to the whole animal as a means
of improving modeled bioaccumulation predictions for fish. To date,
however, the intra- and interlaboratory reliability of this procedure
has not been determined. In the present study, three laboratories
determined in vitro intrinsic clearance of six reference compounds
(benzoÂ[<i>a</i>]Âpyrene, 4-nonylphenol, di-<i>tert</i>-butyl phenol, fenthion, methoxychlor and <i>o</i>-terphenyl)
by conducting substrate depletion experiments with cryopreserved trout
hepatocytes from a single source. <i>O</i>-terphenyl was
excluded from the final analysis due to nonfirst-order depletion kinetics
and significant loss from denatured controls. For the other five compounds,
intralaboratory variability (% CV) in measured in vitro intrinsic
clearance values ranged from 4.1 to 30%, while interlaboratory variability
ranged from 27 to 61%. Predicted bioconcentration factors based on
in vitro clearance values exhibited a reduced level of interlaboratory
variability (5.3–38% CV). The results of this study demonstrate
that cryopreserved trout hepatocytes can be used to reliably obtain
in vitro intrinsic clearance of xenobiotics, which provides support
for the application of this in vitro method in a weight-of-evidence
approach to chemical bioaccumulation assessment