Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes.
In this study, we investigated the combined effects of EGF and collagen I gel on the phenotype of cultured rat hepatocytes and we focussed our investigations on the regulation of xenobiotic-mediated induction of CYP, cell cycle progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provoked a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitory effect on 3-methylcholantrene (5 microM) and beta-naphtoflavone (25 microM) induction of CYP1A1 activities. In collagen gel sandwich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cycle, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In collagen gel sandwich cultures, we found that neither collagen I nor EGF prevented activation of caspase 8 while collagen I gel inhibited activation of caspase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In contrast, EGF transiently increased caspase 3 activity at day 1 after plating. Altogether, our data demonstrate that collagen I gel triggers intracellular signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibition of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures.
De Smet
K
K
Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090, Brussels, Belgium. kdsmet@redlabs.com
Loyer
P
P
Gilot
D
D
Vercruysse
A
A
Rogiers
V
V
Guguen-Guillouzo
C
C
D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology; D019363:Cytochrome P-450 CYP1A1 / Q000187:drug effects / Q000378:metabolism*; D019362:Cytochrome P-450 CYP2B1 / Q000187:drug effects / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004347:Drug Interactions; D004789:Enzyme Activation; D004790:Enzyme Induction / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D066298:In Vitro Techniques; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012709:Serum Albumin / Q000378:metabolism; D015262:Xenobiotics / Q000494:pharmacology*
D012709:Serum Albumin; D015262:Xenobiotics; D004815:Epidermal Growth Factor; D003094:Collagen; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases