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Anal. Real Acad. Farm. 200, 66:

In Vitro Induction of Apoptosis in Rat Hepatocytes
by Cyclosporine A

ARMIN WOLF, SIBYLLE GRUB, ELKE PERSOHN,
Novartis Pharma AG, Experimental Toxicology, CH-4002 Basel,

Switzerland,
WOLFGANG E. TROMMER
Department of Chemistry, University of Kaiserslautern, Germany

ABSTRACT

In rat hepatocytes and isolated liver mitochondrial fractions, CsA is often used
as a specific inhibitor of mitochondrial Ca2+ release and as a specific blocker of
mitochondrial membrane potential and permeability transition (MPT), which are all
processes involved in the inhibition of apoptosis. However, neither inhibition nor
induction of apoptosis by CsA has yet been described in the rat hepatocyte primary
culture during incubation for 4 and 20 hours. It was the purpose of the present study to
examine by means of morphological and biochemical criteria the effects of CsA on
apoptosis, and to characterize the underlying mechanisms.

Rat hepatocytes were cultured for 4 or 20 hours with CsA at concentrations of
0, 10, 25 and 50 µM. Chromatin condensation and fragmentation, DNA fragmentation
(TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3 and -6
activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release
into the cytosol were investigated.

Four hours after CsA treatment, chromatin condensation and fragmentation, and
the number of TUNEL- and Annexin V-positive cells increased dose dependently