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Biomedical subjects

D Pessayre

Publications and source records attributed to D Pessayre.

At least 19 recordsLinked to original sources
PubMed2000

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates.

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99 degrees C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or TAQ: plus PWO: DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.COLI: exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.

Animals↗
PubMed2000

Opening of the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Fas-mediated hepatic apoptosis in mice.

Although Fas stimulation has been reported to cause outer mitochondrial membrane rupture in Jurkat cells, the mechanism of this effect is debated, and it is not known if outer membrane rupture also occurs in hepatocyte mitochondria. We studied the in vivo effects of Fas stimulation on ultrastructural lesions and mitochondrial function in mice. Four hours after administration of an agonistic anti-Fas antibody (8 microg/animal), caspase activity increased 5.4-fold. Nuclear DNA showed internucleosomal fragmentation, whereas supercoiled mitochondrial DNA was replaced by circular and linear forms. Mitochondrial cytochrome c was partly released into the cytosol. Ultrastructurally, mitochondrial lesions were observed in both apoptotic hepatocytes (with nuclear chromatin condensation/fragmentation) and nonapoptotic hepatocytes (without nuclear changes). In nonapoptotic cells, outer mitochondrial membrane rupture allowed herniation of the inner membrane and matrix through the outer membrane gap. In apoptotic hepatocytes, the matrix became electron-lucent and no longer protruded through the outer membrane gap. Mitochondria clustered around the nucleus, whereas rough endoplasmic reticulum cisternae became peripheral. In liver mitochondria isolated after Fas stimulation, the membrane potential decreased, whereas basal respiration increased. Pretreatment with either z-VAD-fmk (an inhibitor of caspases) or cyclosporin A (a permeability transition inhibitor) totally or mostly prevented mitochondrial outer membrane rupture, membrane potential decrease, cytochrome c release, and apoptosis. In conclusion, in vivo Fas stimulation causes caspase activation, mitochondrial permeability transition (decreasing the membrane potential and increasing basal respiration), mitochondrial matrix expansion (as shown by matrix herniation), outer mitochondrial membrane rupture, and cytochrome c release.

Animals↗
PubMed2000

Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes.

Although cytochrome P-450 (CYP)-generated reactive metabolites can cause hepatocyte apoptosis, the mechanism of this effect is incompletely understood. In the present study, we assessed the hepatotoxicity of skullcap, a diterpenoid-containing herbal remedy. Male rat hepatocytes were incubated for 2 hours with skullcap diterpenoids (100 microg/mL). This treatment decreased cell glutathione and protein thiols and increased cell [Ca(2+)]. This activated Ca(2+)-dependent tissue transglutaminase, forming a cross-linked protein scaffold, and also opened the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, increased cytosolic cytochrome c, activation of procaspase 3, internucleosomal DNA fragmentation, and ultrastructural features of apoptosis. Cell death was increased by a CYP3A inducer (dexamethasone) or a sulfur amino acid-deficient diet increasing glutathione depletion. In contrast, cell death was prevented by decreasing CYP3A activity (with troleandomycin), preventing glutathione depletion (with cysteine or cystine), blocking Ca(2+)-modulated events (with calmidazolium), preventing mitochondrial permeability transition (with cyclosporin A), or inhibiting caspase 3 (with acetyl-Asp-G u-Va-Asp-a dehyde). Both calmidazolium and cyclosporin A also prevented the increase in cytosolic cytochrome c and procaspase 3 activation. In conclusion, CYP3A activates skullcap diterpenoids into reactive metabolites that deplete cellular thiols and increase cell [Ca(2+)]. This activates Ca(2+)-dependent transglutaminase and also opens the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, cytochrome c release, and caspase activation. Preventing mitochondrial permeability transition pore opening and/or caspase activity blocks apoptosis, showing the fundamental role of these final events in metabolite-mediated hepatotoxicity.

Animals↗
PubMed2000

Human microsomal epoxide hydrolase is the target of germander-induced autoantibodies on the surface of human hepatocytes.

Germander, a plant used in folk medicine, caused an epidemic of cytolytic hepatitis in France. In about half of these patients, a rechallenge caused early recurrence, suggesting an immunoallergic type of hepatitis. Teucrin A (TA) was found responsible for the hepatotoxicity via metabolic activation by CYP3A. In this study, we describe the presence of anti-microsomal epoxide hydrolase (EH) autoantibodies in the sera of patients who drank germander teas for a long period of time. By Western blotting and immunocytochemistry, human microsomal EH was shown to be present in purified plasma membranes of both human hepatocytes and transformed spheroplasts and to be exposed on the cell surface where affinity-purified germander autoantibodies recognized it as their autoantigen. Immunoprecipitation of EH activity by germander-induced autoantibodies confirmed this finding. These autoantibodies were not immunoinhibitory. The plasma membrane-located EH was catalytically competent and may act as target for reactive metabolites from TA. To test this hypothesis CYP3A4 and EH were expressed with human cytochrome P450 reductase and cytochrome b(5) in a "humanized" yeast strain. In the absence of EH only one metabolite was formed. In the presence of EH, two additional metabolites were formed, and a time-dependent inactivation of EH was detected, suggesting that a reactive oxide derived from TA could alkylate the enzyme and trigger an immune response. Antibodies were found to recognize TA-alkylated EH. Recognition of EH present at the surface of human hepatocytes could suggest an (auto)antibody participation in an immune cell destruction.

Alkylation↗
PubMed2000

Vesicular transport of newly synthesized cytochromes P4501A to the outside of rat hepatocyte plasma membranes.

Anti-cytochrome P450 (CYP)1A2 autoantibodies are found in dihydralazine-induced hepatitis, and CYPs2B and 2C have been shown to follow vesicular flow to the plasma membrane (PM). However, it is unknown whether other CYPs follow this route, whether NADPH-CYP reductase is present on the hepatocyte surface, and whether autoimmune hepatitis-inducing drugs increase PM CYPs. In this study, we determined the transmembrane topology and transport of CYPs1A in rat hepatocytes. In cultured hepatocytes, colchicine and other vesicular transport inhibitors decreased PM CYPs1A assessed by flow cytometry. Colchicine administration also decreased PM CYPs1A in vivo. Pulse chase experiments with [(35)S]methionine showed that only the newly synthesized CYP molecules are transferred to the PM, whereas microsomal CYP1A2 was stably radiolabeled for several hours. In contrast, radiolabeled CYP1A2 reached the PM and disappeared from the PM with half-lives of less than 30 min. Confocal microscopy, biotinylation, and coimmunoprecipitation experiments showed that PM CYPs1A and CYP reductase are present on the cell surface, and that the reductase is closely associated with PM CYPs. Exposure of whole cells to an anti-CYP1A1/2 antibody at 4 degrees C, before five washes and PM preparation, abolished PM CYPs1A-supported monooxygenase activity, indicating that PM CYPs are mostly located on the external surface. Dihydralazine and other CYPs1A inducers increased PM CYPs1A. In conclusion, newly synthesized CYPs1A follow vesicular flow to the outside of the PM, and NADPH-CYP reductase also is located on the hepatocyte surface. Dihydralazine administration increases PM CYP1A2, its autoimmune target.

Animals↗
PubMed1999

Decrease in hepatic cytochrome P450 after interleukin-2 immunotherapy.

Interleukin-2 (IL-2) has been shown to decrease cytochrome P450 (CYP) mRNAs and proteins in cultured rat hepatocytes, and IL-2 administration decreases CYPs in rats. Although high doses of IL-2 are administered to cancer patients, the effect on human CYPs has not yet been determined. Patients with hepatic metastases from colon or rectum carcinomas were randomly allocated to various daily doses of human recombinant IL-2 (from 0 to 12.10(6) units/m(2)). IL-2 was infused from day 7 to day 3 before hepatectomy and the conservation of a non-tumorous liver fragment in liquid nitrogen. Hepatic CYPs and monooxygenase activities were not significantly decreased in 5 patients receiving daily doses of 3 or 6 10(6) IL-2 units/m2, compared to 7 patients who did not receive IL-2. In contrast, in 6 patients receiving daily doses of 9 or 12 x 10(6) IL-2 units/m2, the mean values for immunoreactive CYP1A2, CYP2C, CYP2E1, and CYP3A4 were 37, 45, 60 and 39%, respectively, of those in controls; total CYP was significantly decreased by 34%, methoxyresorufin O-demethylation by 62%, and erythromycin N-demethylation by 50%. These observations suggest that high doses of IL-2 may decrease total CYP and monooxygenase activities in man.

Adenocarcinoma↗
PubMed1999

An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice.

BACKGROUND & AIMS: Ethanol causes oxidative stress in the hepatic mitochondria of experimental animals and mitochondrial DNA deletions in alcoholics. We postulated that ethanol intoxication may cause mitochondrial DNA strand breaks. METHODS: Effects of an intragastric dose of ethanol (5 g/kg) on hepatic mitochondrial DNA levels, structure, and synthesis were determined by slot blot hybridization, Southern blot hybridization, and in vivo [3H]thymidine incorporation, respectively. RESULTS: Two hours after ethanol administration, ethane exhalation (an index of lipid peroxidation) increased by 133%, although hepatic lipids were unchanged. Mitochondrial DNA was depleted by 51%. Its supercoiled form disappeared, whereas linearized forms increased. Long polymerase chain reaction evidenced lesions blocking polymerase progress on the mitochondrial genome. Mitochondrial transcripts decreased. Subsequently, [3H]thymidine incorporation into mitochondrial DNA increased, and mitochondrial DNA levels were restored. In contrast, nuclear DNA was not fragmented and its [3H]thymidine incorporation was unchanged. Liver ultrastructure only showed inconstant mitochondrial lesions. Ethanol-induced mitochondrial DNA depletion was prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by melatonin, an antioxidant. CONCLUSIONS: After an alcoholic binge, ethanol metabolism causes oxidative stress and hepatic mitochondrial DNA degradation in mice. DNA strand breaks may be involved in the development of mitochondrial DNA deletions in alcoholics.

Alcohol Drinking↗
PubMed1999

Hepatotoxicity due to mitochondrial dysfunction.

Mitochondria are involved in fatty acid beta-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation, which provide most of the cell energy. Mitochondria are also the main source of reactive oxygen species in the cell and are involved in cell demise through opening of the mitochondrial permeability transition pore. It was therefore to be expected that mitochondrial dysfunction could be a major mechanism of drug-induced liver disease. Microvesicular steatosis (which may cause liver failure, coma, and death) is the consequence of severe impairment of mitochondrial beta-oxidation. Endogenous compounds (such as cytokines or female sex hormones) or xenobiotics (including toxins such as ethanol and drugs such as aspirin, valproic acid, ibuprofen, or zidovudine) can inhibit beta-oxidation directly or through a primary effect on the mitochondrial genome or the respiratory chain itself. In some patients, infections and cytokines, or inborn errors of beta-oxidation enzymes or the mitochondrial genome, may favor the appearance of drug-induced microvesicular steatosis. Nonalcoholic steatohepatitis may develop under conditions causing prolonged, microvesicular, and/or macrovacuolar steatosis. In this condition, chronic impairment of mitochondrial beta-oxidation (causing steatosis) and the respiratory chain (increasing the production of ROS) lead to lipid peroxidation, which, in turn, may cause the diverse lesions of steatohepatitis, namely, necrosis, inflammation, Mallory's bodies, and fibrosis. Finally, mitochondria are involved in several forms of drug-induced cytolytic hepatitis, through inhibition or uncoupling of respiration or through a drug-induced or reactive metabolite-induced mitochondrial permeability transition. The latter effect commits hepatocytes to either apoptosis or necrosis, depending on the number of organelles that have undergone the permeability transition.

Animals↗
PubMed1999

Mesalazine (5-aminosalicylic acid) induced chronic hepatitis.

BACKGROUND: Treatment of ulcerative colitis or Crohn's disease with sulphasalazine causes several adverse effects, including hepatitis. Sulphasalazine is cleaved by colonic bacteria into 5-aminosalicylic acid and sulphapyridine. Received wisdom was that 5-aminosalicylic acid was topically active, whereas sulphapyridine was absorbed and caused immunoallergic side effects. Mesalazine, a slow release formulation of 5-aminosalicylic acid, was expected to be a safe alternative. However, several cases of acute hepatitis have been reported. CASE REPORT: A 65 year old man had increased liver enzymes, anti-nuclear and anti-smooth muscle autoantibodies and IgG levels, and lesions of chronic hepatitis after 21 months of mesalazine treatment. Although liver dysfunction had been identified eight months earlier, simvastatin rather than mesalazine had been withdrawn, without any improvement. In contrast, liver enzyme and IgG levels became normal and autoantibodies disappeared after discontinuation of mesalazine administration. CONCLUSION: Contrary to initial expectations, mesalazine can cause most of the sulphasalazine induced adverse effects, and hepatic side effects may be almost as frequent. When liver dysfunction occurs, mesalazine administration should be discontinued to avoid the development of chronic hepatitis and liver fibrosis.

Aged↗
PubMed1999

Interleukin-2 overexpresses c-myc and down-regulates cytochrome P-450 in rat hepatocytes.

The interaction of interleukin-2 (IL-2) with its receptor (IL-2R) decreases cytochrome P-450 (CYP) expression in rat hepatocytes. Because IL-2 increases c-Myc in lymphocytes and because c-myc overexpression represses several genes, we postulated that the IL-2/IL-2R interaction may increase c-Myc and thereby down-regulate CYP in hepatocytes. Cultured rat hepatocytes were exposed for 24 h to IL-2 (350 U/ml) and other agents. IL-2 increased c-myc mRNA and protein but decreased total CYP and the mRNAs and proteins of CYP2C11 and CYP3A. The IL-2-mediated c-myc overexpression and CYP down-regulation were prevented by 1) genistein (a tyrosine kinase inhibitor that blocks the initial transduction of the IL-2R signal), 2) retinoic acid, butyric acid, or dimethyl sulfoxide (three agents that block c-myc transcription), or 3) an antisense c-myc oligonucleotide (which may cause rapid degradation of the c-myc transcript). It is concluded that IL-2 causes the overexpression of c-myc and the down-regulation of CYPs in rat hepatocytes. Block of c-myc overexpression, at three different levels with five different agents, prevents CYP down-regulation, suggesting that c-myc overexpression may directly or indirectly repress CYP in hepatocytes.

Animals↗
PubMed1998

Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes.

BACKGROUND & AIMS: 4,4'-Diethylaminoethoxyhexestrol (DEAEH), amiodarone, and perhexiline cause steatohepatitis in humans. The mechanisms of these effects are unknown for DEAEH and have not been completely elucidated for amiodarone and perhexiline. The aim of this study was to determine these mechanisms. METHODS: Rat liver mitochondria, cultured rat hepatocytes, or rats were treated with these drugs, and the effects on mitochondrial respiration, beta-oxidation, reactive oxygen species formation, and lipid peroxidation were determined. RESULTS: DEAEH accumulated in mitochondria and inhibited carnitine palmitoyl transferase I and acyl-coenzyme A dehydrogenases; it decreased beta-oxidation and caused lipid deposits in hepatocytes. DEAEH also inhibited mitochondrial respiration and decreased adenosine triphosphate (ATP) levels in hepatocytes. DEAEH, amiodarone, and perhexiline augmented the mitochondrial formation of reactive oxygen species and caused lipid peroxidation in rats. CONCLUSIONS: Like amiodarone and perhexiline, DEAEH accumulates in mitochondria, where it inhibits both beta-oxidation (causing steatosis) and respiration. Inhibition of respiration decreases ATP and also increases the mitochondrial formation of reactive oxygen species. The latter oxidize fat deposits, causing lipid peroxidation. We suggest that ATP depletion and lipid peroxidation may cause cell death and that lipid peroxidation products may account, in part, for other steatohepatitis lesions.

Animals↗
PubMed1997

Acute hepatitis caused by alverine associated with anti-lamin A and C autoantibodies.

We report the case of a 67-year-old woman in whom onset and regression of acute hepatitis were closely related to the time of administration and withdrawal of the smooth muscle relaxant alverine. Antinuclear antibodies were positive, and their titer followed the course of hepatitis. They presented a smooth rim-like nuclear immunofluorescence staining pattern. Immunoblot assay showed that they were directed against lamin A and lamin C. This suggests that alverine should be added to the list of drugs known to produce acute hepatitis, and that drug-induced liver injury is a possible cause of antinuclear antibodies specific for lamin A and lamin C.

Acute Disease↗
PubMed1997

Multiple hepatic mitochondrial DNA deletions suggest premature oxidative aging in alcoholic patients.

BACKGROUND/AIMS: A 4977-base pair deletion has been detected in the hepatic mitochondrial DNA of alcoholic patients with microvesicular steatosis, a lesion ascribed to impaired mitochondrial beta-oxidation. However, only a single deletion had been looked for in this previous study, and it could not be determined whether the deletion was preexisting or acquired. Alcohol abuse increases the formation of reactive oxygen species in hepatic mitochondria. If this effect accelerates the oxidative aging of mitochondrial DNA, several other mutations would be expected. METHODS: The mtDNA region extending from nucleotide 8167 to nucleotide 14246 was screened for the presence of large mitochondrial DNA deletions in 58 alcoholic patients and 67 age-matched non-alcoholic controls. Hepatic DNA was subjected to polymerase chain reactions that amplified non-deleted and deleted mitochondrial DNA, respectively, and the boundaries of the mitochondrial DNA deletions were sequenced. RESULTS: Only 3% of the non-alcoholic controls carried a mitochondrial DNA deletion, whereas 24% of all alcoholic patients and 85% of the 13 alcoholic patients with microvesicular steatosis exhibited either single or multiple 4977, 5385, 5039 and 5556-base pair mitochondrial DNA deletions. No deletion(s) were observed, however, in 13 patients with microvesicular steatosis due to other causes. CONCLUSIONS: Diverse mitochondrial DNA rearrangements are observed in alcoholic patients with microvesicular steatosis. We suggest that alcohol abuse leads to premature oxidative aging of mitochondrial DNA. Hypothetically, oxidative damage to mitochondrial constituents (DNA, proteins and lipids) may favor microvesicular fat deposition.

Adult↗
PubMed1997

Impaired mitochondrial function in microvesicular steatosis. Effects of drugs, ethanol, hormones and cytokines.

Microvesicular steatosis occurs in conditions characterized by severe impairment of the mitochondrial beta-oxidation process, due to genetic and/or acquired causes. Drugs and some endogenous compounds can sequester coenzyme A (aspirin, valproic acid), inhibit mitochondrial beta-oxidation enzymes (tetracyclines, several 2-arylpropionate anti-inflammatory drugs, amineptine and tianeptine), or inhibit both mitochondrial beta-oxidation and oxidative phosphorylation (endogenous bile acids, amiodarone, perhexiline and diethylaminoethoxyhexestrol), while female sex hormones have complex, but moderate, effects on mitochondrial structure and function. Other substances impair mitochondrial DNA transcription (interferon-alpha) or mitochondrial DNA replication (dideoxynucleosides), while alcohol abuse might accelerate the normal oxidative aging of mitochondrial DNA. When beta-oxidation is severely impaired, fatty acids, which are poorly oxidized by mitochondria, are mainly esterified into triglycerides, but there is a residual increase in non-esterified fatty acids. Triglycerides (possibly emulsified by a rim of non-esterified fatty acids) accumulate as small vesicles. Impairment of energy production, and the mitochondrial and general toxicity of both non-esterified fatty acids and dicarboxylic acids, may contribute to liver failure, coma and death in severe forms. Although milder forms of microvesicular steatosis have a good short-term prognosis, they can lead to chronic lipid peroxidation and the development of steatohepatitis lesions. Investigational molecules with a carboxylic group or a protonatable amine, or those which might interfere with mitochondrial DNA, should be screened for possible mitochondrial effects.

Animals↗