Diterpenoids from germander, an herbal medicine, induce apoptosis in isolated rat hepatocytes.
OBJECTIVE
Germander was withdrawn from the market after its use for weight control caused an epidemic of hepatitis. Its toxicity was shown to be caused by diterpenoids and their cytochrome P4503A-mediated metabolic activation into electrophilic metabolites that deplete cellular thiols. The aim of the present study was to determine the mechanisms of cell death.


METHODS
Isolated rat hepatocytes were incubated for 2 hours with germander diterpenoids (100 micrograms/mL).


RESULTS
Diterpenoids decreased cell glutathione, increased cytosolic [Ca2+], activated Ca(2+)-dependent tissue transglutaminase forming a cross-linked protein scaffold, and caused internucleosomal DNA fragmentation and the ultrastructural features of apoptosis. Cell death was prevented by decreasing metabolic activation (with troleandomycin), preventing depletion of glutathione (with cystine), blocking activation of Ca(2+)-modulated enzymes (with calmidazolium), or inhibiting internucleosomal DNA fragmentation (with aurintricarboxylic acid). Apoptosis was increased and diterpenoids caused overexpression of p53 and interleukin 1 beta-converting enzyme in rats treated with dexamethasone (cytochrome P4503A inducer). Apoptosis was also increased by a diet deficient in sulfur amino acids.


CONCLUSIONS
The germander furano diterpenoids cause apoptosis within 2 hours in isolated rat hepatocytes. Electrophilic metabolites may stimulate apoptosis by decreasing thiols, increasing [Ca2+], and activating Ca(2+)-dependent transglutaminase and endonucleases.
Fau
D
D
INSERM Unité 24, Hôpital Beaujon, Clichy, France.
Lekehal
M
M
Farrell
G
G
Moreau
A
A
Moulis
C
C
Feldmann
G
G
Haouzi
D
D
Pessayre
D
D
D000603:Amino Acids, Sulfur / Q000172:deficiency; D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003553:Cystine / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004224:Diterpenes / Q000302:isolation & purification / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D007093:Imidazoles / Q000494:pharmacology; D008099:Liver / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D008297:Male; D008954:Models, Biological; D008517:Phytotherapy; D010946:Plants, Medicinal; D010947:Plants, Toxic; D051381:Rats; D017207:Rats, Sprague-Dawley; D014217:Troleandomycin / Q000494:pharmacology
D000603:Amino Acids, Sulfur; D004224:Diterpenes; D004791:Enzyme Inhibitors; D007093:Imidazoles; D001312:Aurintricarboxylic Acid; D003553:Cystine; C031938:calmidazolium; D003907:Dexamethasone; D003513:Cycloheximide; D014217:Troleandomycin; D005978:Glutathione; D002118:Calcium; D019803:Glutathione Disulfide