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Journal Publication: “Gene expression in two hepatic cell lines, cultured primary
hepatocytes and liver slices compared to the in vivo liver gene expression in rats:
Possible implications for toxicogenomics use of in vitro systems.” Franziska
Boess, Markus Kamber, Simona Romer, Rodolfo Gasser, Dieter Muller, Silvio
Albertini, Laura Suter. Toxicological Sciences Manuscript 02-0413
Data analysis for the above mentioned manuscript can be downloaded as an Excel or
tab-delimited file from http://www.roche.com/science-download.htm. The name of the
file is web_TOXSCI_020413_data.xls.
The genes are identified by Affymetrix IDs, as provided by the microarray supplier
(GeneChip RG-U34A, Affymetrix, Santa Clara, USA, http://www.affymetrix.com)
and Genebank accession numbers. Gene expression analysis was performed using an
in-house developed software tool (RACE-A, F. Hoffmann-La Roche, Basel,
Switzerland) which allows expression values of biological replicates (n per group) to
be normalized before the group mean and the p-value of an unpaired, two-tailed
Student’s t-test is calculated. Thus, gene expression results are expressed as
ChangeFactors (chgf) between the means of the in vitro systems and the in vivo liver
tissue at the 6h time point. Chgf reports the change from condition 1 (in vivo) to
condition 2 (in vitro). In case of an increase, it is the ratio (cond2/cond1) minus 1. In
case of a decrease, it is -(cond1/cond2) plus 1 (the addition/subtraction of 1 avoids a
gap in the scale between +1 and -1). Positive values indicate that expression in the
respective in vitro system was higher than in vivo at the 6h time point, negative
values indicate that expression in vitro was lower than in vivo (baseline).
Example (for a non-paired T-test): if mean in vitro is 100% of mean in vivo, the chgf
is 0 (200%=1; 300%=2; 500%=4; 50%=-1; 33%=-2; 25%=-3).
In addition, the expression levels are given in arbitrary units. In our hands,
expression levels under 200 are considered below the detection limit of the system.
However, values between 200 and 20 were used for chgf calculation. Values below
20 were set to 20 for chgf calculation.
Corresponding author:
Dr. Franziska Boess
F. Hoffmann-La Roche AG, PRNS
Buliding 68/110
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CH- 4070 Basle
Switzerland
Phone: +41-616871880
Fax: +41-616882908
E-mail:
franziska.boess@roche.com
Abstract
Microarray technology allows the simultaneous analysis of mRNA expression levels
of thousands of genes. In the field of Toxicogenomics this technology could help to
identify potentially unsafe compounds based on the changes in mRNA expression
patterns they induce. Rodent in vivo and in vitro systems are currently the
experimental models of choice for predictive toxicology, especially in early phases
of development.
This study characterizes several hepatic in vitro systems based on mRNA expression
profiles, comparing them to gene expression in liver tissue. The in vitro systems
investigated comprise two rat liver cell lines (BRL3A and NRL clone 9), primary
hepatocytes in conventional monolayer or in sandwich culture, and liver slices. The
results demonstrate that liver slices exhibit the strongest similarity to liver tissue
regarding mRNA expression, whereas the two cell lines are quite different from the
whole liver. We were able to identify genes with strong changes in expression levels
in all or at least one of the in vitro systems relative to whole liver. In particular, for
some cytochrome P450s the differences observed on the mRNA expression level
were paralleled by protein expression and enzymatic activity. In addition, the effect
of time in culture was assessed. We were able to show a profound effect of the
duration of culture. Expression patterns change most rapidly soon after cell isolation
and culture initiation and stabilize with time in culture. The findings are discussed
with respect to the usefulness of the various hepatic in vitro systems for microarraybased toxicological testing of compounds.
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