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Photosensitization by psoralens: Mitochondria structural modifications in fanconi anemia fibroblasts as compared to normal human fibroblasts
Colloque SBCF-ATIPWCNRS
Biologie du Developpement, mars 1995 MITOCHONDRIA CHONDRIA-BOUND
PEROXISOME PROLIFERATION IN RODENTS AND HUMAN: A MODEL OF CELL ORGANELLE BIOGENESIS PACOT Corinne, PASSILLY Patricia, CLEMENCET Marie-Claude, BARDOT Olivier, CAIRA Francoise, CHERKAOU! MALK! Mustapha, JANNIN Brigitte et LATRUFFE Norben, Universitt
de Bourgogne,
LBMC,
BP 138,21004
D/JON
CEDEX.
Mammalian peroxisomes are important in lipid and oxidative metabolism, especially in very long chain fatty acid R-oxidation (LATRUFFE
N. (1992). MCdecineiSciences , 8,239-24X).
The interest of peroxisomes is outlined by the phenomenon of rodent hepatic peroxisome proliferation under the effects of xenobiotics such as hypolipemic drugs, plasticizers and herbicides (CHERKAOUI MALKl M., ASSAKA L.,PACOTC.,BARDOTO.elLATRUFFE 123-133).
N.(1991).
Cell.Mo[.
Biol., 37,
This peroxisomal proliferation is characterized by enhanced peroxisomes biogenesis and specific genes activation. In one hand, our current knowledges on biogenesis indicate that peroxisomal number increase derives from the fission of pre-existing peroxisomes provoked by the import in the organelle of newly svnthesized oroteins and lioids. The oolvueptides neo-svnthesized on free cytosolic poiysomes are addressed io pyroxisomes, either by C-terminal label: the SKL amino acids motif, or by N-terminal signal. Two membrane receptors for polypeptides translocation have been already identified (MANNAERTS G., Belgium and SUBRAMANlS.,USA). On the other hand. neroxisome oroliferation is associated with eenes activation under n’u’clear recep’tors control: PPAR (Peroxisome Proliferator Activated Recentor) and RXR (9-cis Retinoid Acid Receptor) (BARD~T~.,ALDRIDOET..
BiochcmEidphy~.
Moreover, by using enzymatic methods, peroxisome isolation, and blotting methods, our studies on the effect of ciprofibrate, a highly potent hypolipemic drug, show only very weak peroxisome proliferation in guinea pigs liver and human hepatoblastoma cell line (HepGZ), in contest to treated rats ttver i/t viva and to rat hepatoma cell line (Fao). Supported Botlr~o&we.
by GIS To,wolo,~~ie
Cvllultrire
de Dijon
nntl
Conseil
Rkgional
de
PHOTOSENSITIZATION BY PSORALENS: MITOCHONDRIA STRUCTURAL MODIFICATIONS IN FANCONI ANEMIA FIBROBLASTS AS COMPARED TO NORMAL HUMAN FIBROBLASTS. ROUSSET Sotanze, NOCENTINI Silvano and MOUSTACCHI Ethel. CNRS URA 1292, France.
Institut
Curie,
26 rue d’Ulm,
7S231 Paris
Ceder
OS,
Fanconi anemia (FA), a rare human disorder with a recessive pattern of inheritance, is characterized by progressive pancytopenia, physical malformations and cancer proneness. FA cells demonstrate chromosomal aberrations, hypersensitivity to DNA cross-linking agents, anomalies in cytokine expression, and, under defined conditions modifications of their oxidative metabolism. For this last reason, structural modifications of mitochondria were investigated in FA libroblasts, from complementation group A, cultured under normal or reduced oxygen tension (20% or 5%, respectively), and photoreacted with 8-methoxypsoralen (g-MOP) plus UVA (365nm), compared to normal human fibroblasts subjected to the same treatment. Changes in the mitochondrial structures were found in FA fibroblasts by comparison with normal libroblasts. At time 0 the electron ooacitv of the mitochondrial matrix in FA cells, measured bv optical density, appeared significantly increased afte; UVA alone (36Snm), at both oxygen tensions. This condensation of the matrix was enhanced after S-MOP photoreaction as a function of the g-MOP concentration. No statistically significant differences were observed in mitochondria of normal cells when untreated cultures were compared to UVA or g-MOP olus UVA treated cultures. The averaged distance between cristae wis increased in untreated FA control cel& relative to normal control cells. This value was slightly reduced in FA cells after UVA alone or g-MOP plus UVA under the low oxygen tension, and in parallel the surface of the cristea was smaller. Some dense granules were detected in the mitochondrial matrix esoeciallv in FA cells. Their number varied with the oxygen tension as -wet! ai with the different treatments. The data underline the importance of UVA and psoralen induced extragenomic lesions for the physiology of FA cells.
de Vaqirard,
Paris
95
STRUCTURAL MODIFICATIONS, HEXOKINASE, GLYCOLYSIS
Ceder
MITOAND
15, France.
Glioblastoma multiforme, a malignant primary brain tumor, is characterized by an increase in aerobic glycolysis and in nucleic acid metabolism. In highly glycolytic tumors! 60% of ATP supply is provided by glycolysis. The phosphorylatton by ATP of glucose to glucose-6-phosphate is catalyzed by a clue enzyme: hexokinase 1 (HK), whose activity is clearly reduced in gliomas. The increase of HKdependent ATP supply could be related to defective mitochondria (mt) functions expressed by oxidative respiration alterations and/or reflected by mt ultrastructure re-modelling. We have examined the correlation between morphological mt characteristics. glvcolvsis metabolism and -< oxidative phosphorylation, in four human xenografted gliomas expressing different levels of mt-bound HK (mHK) fraction. Glioma samples were highly glycolytic, as shown by the lactate/pyruvate ratio, by comparison with rat brain tissue taken as control. The four gliomas. according to their mHK fraction, could be separated into two-distinct low and high mHK fraction groups. Biochemical results showed that human glioma xenografts did-not present major impairements in mt electron transfer system and respiratory capacities. Glioma mt were examined for their ultrastructural morphometry, relative number, organite relationships, matrix density and cristea distribution. The mt averaged surface of gliomas was not statistically different from that of rat brain mt. Less mt were in close contacts with the nuclear membrane in gliomas from the low mHK fraction group. In addition, the length of the contact zones was reduced in gliomas compared with rat brain. The mt fraction in contact with the rough endoplasmic reticulum was independent of the corresponding glioma group. The optical density of matrix electron opaque material was higher in the low mHK fraction group than in the high one, where values were very similar to those obtained with rat brain mt. Mean cristae distance was constantly higher in gliomas. The results suggest that gliomas -all being glycolytic- from the low mHK fraction group have a predominant oxidative phosphorylation, and that those from the high mHK fraction group have a glycolytic deviation.
Ultrastructural mitochondrial alterations in kidney and white stem cells of bone marrow of rats after intravenous administration of 239 Pu-citrate *THANNOO Danee Raj, *BERRY Jean Pierre, *GALLE **PAQUET Francois. *SC 27 de I’INSERM, facultC de Mddecine FRANCE. ** CEA, B.P. 12, 91680 Bruyeres Le Chatel
de Cre’teil,
Pierre and
94010
Crtfteil
FRANCE.
Most of the knowledge on the toxicity of plutonuim comes from animal experimentation, namely studies made on alpha track microdosimetry in lungs ( SANDERS C.L., LAUHALA K.E.. MC DONALD K.E. & SANDERS G.A. 1993 He&h Physics 64 509 - 521 ) and on the localisation of plutonium in interstitial tkSUe Of testis in rats (MILLER S.C. & BOWMAN B.M. 1983 Rndiat. Res. 94 416 - 426 ) and in the lysosomes of hepatic cells of the Chinese hamster ( HOT2 G & SEIDEL A. 1987 Int. L Radiat. Biol52 723 - 734 1. However‘little studies have dealt with damages induced by plutonium at the subceltular level. In our studv we are concerned with the 239 isotope of plutonium (half life of 244od years ), mainly an a particle emitter. it is a by-product of uranium in nuclear reactors, and it is also disseminated in the atmosphere during explosions of nuclear weapons. Here we investigate the subcellular alterations uroduced after administration of intravenous $tonium citrate in adult male Sprague-Dawley rats. Two groups of fifteen animals were used. The first group received an activitv of 2.3 KBa oer animal and uer iniection while the second received an act& of 9.25 i(Bq per animal and p& injection. Each animal received 5 injections, each at a week of interval. Two animals of each activity group were sacrified ( first sacrifice: one week after the first injection, last sacrifice: one week after the last injection ) . The tissues studied were the kidneys and the bone marrow, they were prepared according to the conventional methods of electron microscopy. We observed swelling of mitochondria both in the proximal tube of kidney and in white stem cell samples.These mitochondria bore lesions in their cristae. No significant alterations were observed in the nuclei. In the proximal tubes of our kidney samples, we have come across perfectly healthy cells adjacent to cells bearing mitochondrial alterations. The respective roles of the chemical or the radiological toxicity in the induction of the observed mitochondrial alterations remain to be determined.
Biologie du Developpement, mars 1995 MITOCHONDRIA CHONDRIA-BOUND
PEROXISOME PROLIFERATION IN RODENTS AND HUMAN: A MODEL OF CELL ORGANELLE BIOGENESIS PACOT Corinne, PASSILLY Patricia, CLEMENCET Marie-Claude, BARDOT Olivier, CAIRA Francoise, CHERKAOU! MALK! Mustapha, JANNIN Brigitte et LATRUFFE Norben, Universitt
de Bourgogne,
LBMC,
BP 138,21004
D/JON
CEDEX.
Mammalian peroxisomes are important in lipid and oxidative metabolism, especially in very long chain fatty acid R-oxidation (LATRUFFE
N. (1992). MCdecineiSciences , 8,239-24X).
The interest of peroxisomes is outlined by the phenomenon of rodent hepatic peroxisome proliferation under the effects of xenobiotics such as hypolipemic drugs, plasticizers and herbicides (CHERKAOUI MALKl M., ASSAKA L.,PACOTC.,BARDOTO.elLATRUFFE 123-133).
N.(1991).
Cell.Mo[.
Biol., 37,
This peroxisomal proliferation is characterized by enhanced peroxisomes biogenesis and specific genes activation. In one hand, our current knowledges on biogenesis indicate that peroxisomal number increase derives from the fission of pre-existing peroxisomes provoked by the import in the organelle of newly svnthesized oroteins and lioids. The oolvueptides neo-svnthesized on free cytosolic poiysomes are addressed io pyroxisomes, either by C-terminal label: the SKL amino acids motif, or by N-terminal signal. Two membrane receptors for polypeptides translocation have been already identified (MANNAERTS G., Belgium and SUBRAMANlS.,USA). On the other hand. neroxisome oroliferation is associated with eenes activation under n’u’clear recep’tors control: PPAR (Peroxisome Proliferator Activated Recentor) and RXR (9-cis Retinoid Acid Receptor) (BARD~T~.,ALDRIDOET..
BiochcmEidphy~.
Moreover, by using enzymatic methods, peroxisome isolation, and blotting methods, our studies on the effect of ciprofibrate, a highly potent hypolipemic drug, show only very weak peroxisome proliferation in guinea pigs liver and human hepatoblastoma cell line (HepGZ), in contest to treated rats ttver i/t viva and to rat hepatoma cell line (Fao). Supported Botlr~o&we.
by GIS To,wolo,~~ie
Cvllultrire
de Dijon
nntl
Conseil
Rkgional
de
PHOTOSENSITIZATION BY PSORALENS: MITOCHONDRIA STRUCTURAL MODIFICATIONS IN FANCONI ANEMIA FIBROBLASTS AS COMPARED TO NORMAL HUMAN FIBROBLASTS. ROUSSET Sotanze, NOCENTINI Silvano and MOUSTACCHI Ethel. CNRS URA 1292, France.
Institut
Curie,
26 rue d’Ulm,
7S231 Paris
Ceder
OS,
Fanconi anemia (FA), a rare human disorder with a recessive pattern of inheritance, is characterized by progressive pancytopenia, physical malformations and cancer proneness. FA cells demonstrate chromosomal aberrations, hypersensitivity to DNA cross-linking agents, anomalies in cytokine expression, and, under defined conditions modifications of their oxidative metabolism. For this last reason, structural modifications of mitochondria were investigated in FA libroblasts, from complementation group A, cultured under normal or reduced oxygen tension (20% or 5%, respectively), and photoreacted with 8-methoxypsoralen (g-MOP) plus UVA (365nm), compared to normal human fibroblasts subjected to the same treatment. Changes in the mitochondrial structures were found in FA fibroblasts by comparison with normal libroblasts. At time 0 the electron ooacitv of the mitochondrial matrix in FA cells, measured bv optical density, appeared significantly increased afte; UVA alone (36Snm), at both oxygen tensions. This condensation of the matrix was enhanced after S-MOP photoreaction as a function of the g-MOP concentration. No statistically significant differences were observed in mitochondria of normal cells when untreated cultures were compared to UVA or g-MOP olus UVA treated cultures. The averaged distance between cristae wis increased in untreated FA control cel& relative to normal control cells. This value was slightly reduced in FA cells after UVA alone or g-MOP plus UVA under the low oxygen tension, and in parallel the surface of the cristea was smaller. Some dense granules were detected in the mitochondrial matrix esoeciallv in FA cells. Their number varied with the oxygen tension as -wet! ai with the different treatments. The data underline the importance of UVA and psoralen induced extragenomic lesions for the physiology of FA cells.
de Vaqirard,
Paris
95
STRUCTURAL MODIFICATIONS, HEXOKINASE, GLYCOLYSIS
Ceder
MITOAND
15, France.
Glioblastoma multiforme, a malignant primary brain tumor, is characterized by an increase in aerobic glycolysis and in nucleic acid metabolism. In highly glycolytic tumors! 60% of ATP supply is provided by glycolysis. The phosphorylatton by ATP of glucose to glucose-6-phosphate is catalyzed by a clue enzyme: hexokinase 1 (HK), whose activity is clearly reduced in gliomas. The increase of HKdependent ATP supply could be related to defective mitochondria (mt) functions expressed by oxidative respiration alterations and/or reflected by mt ultrastructure re-modelling. We have examined the correlation between morphological mt characteristics. glvcolvsis metabolism and -< oxidative phosphorylation, in four human xenografted gliomas expressing different levels of mt-bound HK (mHK) fraction. Glioma samples were highly glycolytic, as shown by the lactate/pyruvate ratio, by comparison with rat brain tissue taken as control. The four gliomas. according to their mHK fraction, could be separated into two-distinct low and high mHK fraction groups. Biochemical results showed that human glioma xenografts did-not present major impairements in mt electron transfer system and respiratory capacities. Glioma mt were examined for their ultrastructural morphometry, relative number, organite relationships, matrix density and cristea distribution. The mt averaged surface of gliomas was not statistically different from that of rat brain mt. Less mt were in close contacts with the nuclear membrane in gliomas from the low mHK fraction group. In addition, the length of the contact zones was reduced in gliomas compared with rat brain. The mt fraction in contact with the rough endoplasmic reticulum was independent of the corresponding glioma group. The optical density of matrix electron opaque material was higher in the low mHK fraction group than in the high one, where values were very similar to those obtained with rat brain mt. Mean cristae distance was constantly higher in gliomas. The results suggest that gliomas -all being glycolytic- from the low mHK fraction group have a predominant oxidative phosphorylation, and that those from the high mHK fraction group have a glycolytic deviation.
Ultrastructural mitochondrial alterations in kidney and white stem cells of bone marrow of rats after intravenous administration of 239 Pu-citrate *THANNOO Danee Raj, *BERRY Jean Pierre, *GALLE **PAQUET Francois. *SC 27 de I’INSERM, facultC de Mddecine FRANCE. ** CEA, B.P. 12, 91680 Bruyeres Le Chatel
de Cre’teil,
Pierre and
94010
Crtfteil
FRANCE.
Most of the knowledge on the toxicity of plutonuim comes from animal experimentation, namely studies made on alpha track microdosimetry in lungs ( SANDERS C.L., LAUHALA K.E.. MC DONALD K.E. & SANDERS G.A. 1993 He&h Physics 64 509 - 521 ) and on the localisation of plutonium in interstitial tkSUe Of testis in rats (MILLER S.C. & BOWMAN B.M. 1983 Rndiat. Res. 94 416 - 426 ) and in the lysosomes of hepatic cells of the Chinese hamster ( HOT2 G & SEIDEL A. 1987 Int. L Radiat. Biol52 723 - 734 1. However‘little studies have dealt with damages induced by plutonium at the subceltular level. In our studv we are concerned with the 239 isotope of plutonium (half life of 244od years ), mainly an a particle emitter. it is a by-product of uranium in nuclear reactors, and it is also disseminated in the atmosphere during explosions of nuclear weapons. Here we investigate the subcellular alterations uroduced after administration of intravenous $tonium citrate in adult male Sprague-Dawley rats. Two groups of fifteen animals were used. The first group received an activitv of 2.3 KBa oer animal and uer iniection while the second received an act& of 9.25 i(Bq per animal and p& injection. Each animal received 5 injections, each at a week of interval. Two animals of each activity group were sacrified ( first sacrifice: one week after the first injection, last sacrifice: one week after the last injection ) . The tissues studied were the kidneys and the bone marrow, they were prepared according to the conventional methods of electron microscopy. We observed swelling of mitochondria both in the proximal tube of kidney and in white stem cell samples.These mitochondria bore lesions in their cristae. No significant alterations were observed in the nuclei. In the proximal tubes of our kidney samples, we have come across perfectly healthy cells adjacent to cells bearing mitochondrial alterations. The respective roles of the chemical or the radiological toxicity in the induction of the observed mitochondrial alterations remain to be determined.