Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene.
BACKGROUND
Ganciclovir exhibits broad-spectrum activity against DNA viruses such as cytomegaloviruses, herpes simplex viruses, varicella-zoster virus, Epstein-Barr virus and human herpes virus-6. Ganciclovir is widely applied for anti-herpetic treatment, cytomegalovirus prophylaxis after organ transplantation, and, more recently, in experimental gene therapy to eradicate cycling cells that express the herpes simplex virus thymidine kinase gene. Although ganciclovir supposedly acts as a chain terminator, there is compelling evidence demonstrating the presence of ganciclovir, but not of acyclovir, incorporated internally into DNA, leaving the precise mechanism by which ganciclovir inhibits DNA synthesis enigmatic.


METHODS
To study the potential involvement of mitochondria in the ganciclovir nucleotide cytotoxicity, we used adenovirus-mediated gene transfer to express herpes simplex virus thymidine kinase in rat liver and administered ganciclovir 2 days post-infection. The integrity and function of mitochondria in the rat liver cells were evaluated by several techniques. In addition, we analyzed the nucleotide pools in cellular extracts and in isolated mitochondria.


RESULTS
We show that ganciclovir nucleotides are abundantly present in the mitochondria of rat livers that express the HSVtk gene. Already 48 h after administration, 10-30% of the total mitochondrial nucleotide pool consists of ganciclovir nucleotides. Their presence is correlated with a lower amount of mitochondrial DNA, a reduced mitochondrial-membrane potential, morphological abnormalities, and liver dysfunction.


CONCLUSIONS
These data provide evidence for the involvement of mitochondria in the hepatotoxicity of the HStk/ganciclovir combination. This may explain the toxicity of the HSVtk/gancilovir combination in some metabolically active but non-proliferating cells, such as liver cells. This toxicity limits the applicability of this enzyme/prodrug combination.
van der Eb
Marjolijn M
MM
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
Geutskens
Sacha B
SB
van Kuilenburg
André B P
AB
van Lenthe
Henk
H
van Dierendonck
Jan-Hein
JH
Kuppen
Peter J K
PJ
van Ormondt
Hans
H
van de Velde
Cornelis J H
CJ
Wanders
Ronald J A
RJ
van Gennip
Albert H
AH
Hoeben
Rob C
RC
D000256:Adenoviridae; D000818:Animals; D000998:Antiviral Agents / Q000009:adverse effects* / Q000378:metabolism / Q000494:pharmacology*; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D015774:Ganciclovir / Q000009:adverse effects* / Q000378:metabolism / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods; D005822:Genetic Vectors; D008099:Liver / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure; D008297:Male; D008928:Mitochondria / Q000737:chemistry* / Q000502:physiology; D010766:Phosphorylation; D011355:Prodrugs; D051381:Rats; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*
D000998:Antiviral Agents; D004272:DNA, Mitochondrial; D011355:Prodrugs; D013937:Thymidine Kinase; D015774:Ganciclovir