Contribution of human cytochrome P450 to benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol metabolism, as predicted from heterologous expression in yeast.

The metabolism of benzo[a]pyrene (B[a]P) and its proximate mutagen B[a]P-7,8-dihydrodiol (7,8-diol) was investigated in the presence of human microsomal epoxide hydrolase and P450 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4 expressed in the yeast Saccharomyces cerevisiae. P450 1A1 had the highest turnover numbers for the formation of all B[a]P metabolites, including phenols and dihydrodiols. P450 1A2, 2C8, 2C9, 2C18, 2C19 and 3A4, which are well represented in the liver, gave rise to the formation of appreciable amounts of 3-hydroxy-B[a]P and of some dihydrodiols from B[a]P. When 7,8-diol was used as substrate, P450 1A1 also exhibited the highest turnover numbers for the formation of tetrols, the hydrolysis products of the diolepoxides, whereas P450 1A2, 2C8, 2C19 and 3A4 showed moderate activities. In order to test the validity of the yeast system, the contribution of each P450 isoform to B[a]P and 7,8-diol metabolism was evaluated as the product of the turnover numbers of recombinant P450s by specific contents of each P450 in human liver microsomes. Calculated formation rates for each B[a]P and 7,8-diol metabolite globally matched experimental values. There is evidence that P450 3A4 and 2C9 play a major role in the formation of 3-hydroxy-B[a]P from B[a]P. Accumulation of the proximate mutagen 7,8-diol was predicted to be mainly driven by P450 1A2, 2C9 and 2C19, while formation of the genotoxic diolepoxides from 7,8-diol appeared to be dependent on P450 1A2 and 3A4 in the liver.

Human CYP2B6: expression, inducibility and catalytic activities.

Human cytochrome (CYP)2B6 cDNA was cloned and expressed in bacteria and in yeast. Its expression in Saccharomyces cerevisiae enabled us to obtain, at a high level, an active yeast-expressed CYP2B6 protein, so as to assess its role in the metabolism of ethoxyresorufin, pentoxyresorufin, benzyloxyresorufin, ethoxycoumarin, testosterone and cyclophosphamide. Kinetic analysis showed that human CYP2B6 preferentially metabolized benzyloxyresorufin and pentoxyresorufin, although other CYPs also metabolized these substrates in human liver microsomes. CYP2B6 also manifested a strong 4-hydroxycyclophosphamide activity. Its expression in Escherichia coli enabled us to produce a very specific anti-human CYP2B6 antibody. No cross reactivity of this antibody was observed with CYPs1A1, 1A2, 3A4, 3A5, 2C8, 2C9, 2C18, 2C19, 2D6 or 2E1. This antibody enabled us to study the hepatic and extrahepatic expression of CYP2B6 in man, as well as its expression and inducibility in primary cultured human hepatocytes and in different human cell lines. Immunoblot analysis revealed that the CYP2B6 protein was expressed in 43 of the 48 human liver samples tested, with levels ranging from 0.4 to 8 pmol/mg of microsomal protein with a mean of 1.7 pmol/mg protein. CYP2B was also expressed in human brain, intestine and kidney, and at a lower level in the lung. CYP2B mRNA was detected in human liver, kidney, lung, trachea and intestine. We also found that CYP2B6 is induced at protein and mRNA levels by phenobarbital (2 mM) and cyclophosphamide (1 mM), an anticancer drug known to be metabolized by CYP2B6. No expression or inducibility of CYP2B6 was observed in any of the human cell lines tested.

Oxidative damage and stress response from ochratoxin a exposure in rats.

Ochratoxin A (OTA) is a mycotoxin found in some cereal and grain products. It is a potent renal carcinogen in male rats, although its mode of carcinogenic action is not known. Oxidative stress may play a role in OTA-induced toxicity and carcinogenicity. In this study, we measured several chemical and biological markers that are associated with oxidative stress response to determine if this process is involved in OTA-mediated toxicity in rats. Treatment of male rats with OTA (up to 2 mg/ 24 h exposure) did not increase the formation of biomarkers of oxidative damage such as the lipid peroxidation marker malondialdehyde in rat plasma, kidney, and liver, or the DNA damage marker 8-oxo-7,8-dihydro-2' deoxyguanosine in kidney DNA. However, OTA treatment (1 mg/kg) did result in a 22% decrease in alpha-tocopherol plasma levels and a 5-fold increase in the expression of the oxidative stress responsive protein haem oxygenase-1, specifically in the kidney. The selective alteration of these latter two markers indicates that OTA does evoke oxidative stress, which may contribute at least in part to OTA renal toxicity and carcinogenicity in rats during long-term exposure.

Involvement of cytochrome P450 3A enzyme family in the major metabolic pathways of toremifene in human liver microsomes.

The anti-estrogen toremifen-Fc-1157a or 4-chloro-1,2-diphenyl-1-[4-[2(N,N-dimethylamino)ethoxy]-phenyl]-1- butene is now used for the treatment of breast cancer. This drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-demethyl-(DMTOR), 4-hydroxy-(4OH-TOR) and deamino-hydroxy-(TOR III) toremifene metabolites. The specific forms of cytochrome P450 involved in these oxidation reactions were examined in 32 human liver microsomal preparations previously characterized with respect to their contents of several known P450 enzymes. Toremifene was demethylated with an apparent Km of 124 microM while it was hydroxylated with an apparent Km of 139 microM. The metabolic rates were 71 +/- 56, 13 +/- 9 and 15 +/- 4 pmol/min/mg microsomal protein, respectively, for DMTOR, 4-OH-TOR and TOR III. The N-demethylation activity was strongly correlated with estradiol 2-hydroxylation (r = 0.75), nifedipine oxidation (r = 0.86), tamoxifen N-demethylation (r = 0.73), testosterone 6 beta-hydroxylation (r = 0.78) and erythromycin N-demethylation (r = 0.84), all these monooxygenase activities known to be supported by CYP3A4 isoform. Furthermore, the CYP3A content of liver microsomal samples, measured by western blot analysis using a monoclonal anti-human CYP3A4 antibody, was strongly correlated with DMTOR formation (r = 0.80). Compounds such as cyclosporin, triacetyl-oleandomycin and testosterone inhibited the N-demethylation of toremifene metabolism at 80, 89 and 56% vs control, respectively, while the formation of TOR III was inhibited at 78, 82 and 73% vs control and the 4-hydroxylation pathway was inhibited no more than about 50% vs control. Prior incubation of microsomes with 100 microM gestodene, known to be a selective mechanism-based inhibitor of CYP3A4 in the presence of NADPH, led to 76 +/- 6 and 76 +/- 5% (N = 5 samples) reductions in the N-demethylation and formation of TOR III, respectively. Polyclonal antibody directed against human CYP3A enzymes inhibited formation of DMTOR and TOR III by 60 and 46%, respectively. The metabolism of toremifene was not activated by alpha-naphthoflavone. Finally, the use of yeasts genetically engineered for expression of human P4501A1, 1A2, 2C9 and 3A4 allowed us to demonstrate that DMTOR and TOR III formations are mediated by P4501A and 3A4 enzymes and by contrast these enzymes are not involved in the 4-hydroxylation pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of P450 enzymes involved in metabolism of verapamil in humans.

The calcium channel blocker verapamil[2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6- azaoctanitrile] is widely used in the treatment of hypertension, angina pectoris and cardiac arrhythmias. The drug undergoes extensive and variable hepatic metabolism in man with the major metabolic steps comprising formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile] and norverapamil [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile]. The enzymes involved in metabolism of verapamil have not been characterized so far. Identification of these enzymes would enable estimation of both interindividual variability in verapamil metabolism introduced by the respective pathway and potential for metabolic interactions. We therefore characterized the enzymes involved in formation of D-617 and norverapamil. The maximum rate of formation of D-617 and norverapamil was determined in the microsomal fraction of 21 human livers which had been previously characterized for the individual expression of various P450 enzymes (CYP1A2, CYP2C, CYP2D6, CYP2E1 and CYP3A3/4) by means of Western blotting. Specific antibodies directed against CYP3A were used to inhibit formation of D-617 and norverapamil. Finally, formation of both metabolites was investigated in microsomes obtained from yeast cells which were genetically engineered for stable expression of human P450. Formation of D-617 was correlated with the expression of CYP3A (r = 0.85; P < 0.001) and CYP1A2 (r = 0.57; P < 0.01) in the microsomal fraction of 21 human livers after incubation with racemic verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunotoxicology and expression of human cytochrome P450 in microorganisms.

Drug-induced hepatitis can be caused by an abnormal immunological response. In the case of tienilic acid- and dihydralazine-induced hepatitis, we postulated a scheme in which a P450 produced a reactive metabolite (step 1); this reactive metabolite bound to the P450 producing it (step 2) leading to a neoantigen triggering the immune response (step 3); the autoantibodies produced during the immune response recognized the P450 producing the reactive metabolite (step 4). The use of microorganisms (yeast or bacteria) expressing cloned human P450 helped in proving some steps of this postulated scheme, particularly steps 1 and 4.

Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies.

Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics. Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate. Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies. These antibodies were characterized for their specificity and sensitivity by immunoblots; anti-CYP3A4 was immunoinhibitor. These antibodies could be used in association with other means to identify the CYPs responsible for production of a given metabolite. The use of our human recombinant CYP1A2 as antigen and the corresponding specific antibody enabled us to quantify the CYP1A2 content in 43 human livers. The average level was 69 pmol of CYP1A2/mg of microsomal proteins. Finally, these antibodies were also used to evaluate the level of heme incorporation in human microsomal CYP expressed in yeasts.

Genetically engineered yeast cells and their applications.

The first generation of yeast expression systems relies on inducible expression cassettes borne by multicopy plasmids for production of unmodified human P450s and on the endogenous NADPH-P450 reductase to support activities. A second generation of engineered yeast involved targeted genomic modifications allowing overexpression of the yeast reductase and coexpression of human cytochrome b5 and of a phase II enzyme such as epoxide hydrolase. These features allow improved P450 turnover numbers and simulation of some phase I-phase II couplings. In the third generation, the human reductase was substituted for the yeast reductase by genome engineering. Simultaneously, induction procedures were optimized to reach high P450 specific contents. Dramatic improvements (1000-fold) of yeast-expressed P450 activities have thus been obtained. To get more insight into complex metabolic events, such as that of a typical pollutant: benzo[a]pyrene, an approach was designed which involves a complementary use of yeast expression and computer simulations.

End-chain radiolabeling and in vitro stability studies of radiolabeled poly(hydroxy acid) nanoparticles.

In order to study the tissue distribution of biodegradable nanoparticles after oral administration in animals, end-chain-radiolabeled poly(D,L-lactides) were prepared. Two groups of polymers (Mn = 7500, I = 2.4 and Mn = 28000, I = 1.4 as determined by organic size-exclusion chromatography) were chemically modified by reaction of [14C]acetic anhydride with hydroxyl end-chain groups. The activities of both resulting radioactive poly(D,L-lactides) varied from 57 to 1140 microCi/g. Poly(D,L-lactide) or poly(D,L-lactide-co-glycolide) nanoparticles containing various amounts of radioactive polymer were prepared according to the solvent evaporation process with acetone as cosolvent with methylene chloride in the organic phase. Their mean diameter was 133 +/- 25 nm, measured by photon correlation spectroscopy. The radiolabeled-end-group stability of these particles in buffer solutions was found to be greater when the matrix was made from the radiolabeled poly(D,L-lactide) having the highest molecular weight and the lowest polydispersity index. The polymer-chain stability was totally retained for at least 1 week in a phosphate buffer, pH 7.4, i.e. for the selected experiment time.

Effects of true density, compacted mass, compression speed, and punch deformation on the mean yield pressure.

Compressibility properties of pharmaceutical materials are widely characterized by measuring the volume reduction of a powder column under pressure. Experimental data are commonly analyzed using the Heckel model from which powder deformation mechanisms are determined using mean yield pressure (Py). Several studies from the literature have shown the effects of operating conditions on the determination of Py and have pointed out the limitations of this model. The Heckel model requires true density and compacted mass values to determine Py from force-displacement data. It is likely that experimental errors will be introduced when measuring the true density and compacted mass. This study investigates the effects of true density and compacted mass on Py. Materials having different particle deformation mechanisms are studied. Punch displacement and applied pressure are measured for each material at two compression speeds. For each material, three different true density and compacted mass values are utilized to evaluate their effect on Py. The calculated variation of Py reaches 20%. This study demonstrates that the errors in measuring true density and compacted mass have a greater effect on Py than the errors incurred from not correcting the displacement measurements due to punch elasticity.

[VVI mode cardiac pacing: cause or risk factor of cerebral embolism?]. / La stimulation cardiaque VVI: cause ou facteur de risque d'embolies cérébrales?

Epidemiological studies suggest that VVI pacing is associated with a higher risk of embolic complications than atrial or dual chamber pacing. However, no studies have been performed on pacemaker patients admitted to a neurological department with a cerebral embolism. The authors report the cases of 8 patients (6 men and 2 women) with an average age of 74 years and having the following characteristics: 1) a cerebral embolism, 2) a permanent cardiac pacemaker (7 VVI and 1 DDD mode). The average delay between implantation of the pacemaker and the neurological complication was 31 months. Cardiological investigations at the time of admission showed: a clinically evident cause of cardiac embolism in 3 cases (2 with VVI and 1 with DDD pacing); paroxysmal or permanent atrial fibrillation in 4 cases with VVI pacing at the time of the embolic event (in only one case at the time of implantation); various echocardiographic abnormalities in 6 of the 7 patients who underwent this examination, mainly left atrial dilatation (6/7), septal wall motion abnormalities in all related to ventricular pacing and unexplained left ventricular dilatation in 2 patients with VVI pacemakers. These results suggest that although the etiology of cerebral embolism was probably multifactorial in some patients, VVI packing probably a predisposing role, though not the only cause, and could be considered to be an embolic risk factor as suggested by previous epidemiological studies. These preliminary retrospective data should be interpreted cautiously taking into account the small population size. Prospective studies on pacemaker patients with cerebral embolism are required.

[Interatrial septal aneurysm. Echocardiographic diagnosis]. / Anévrysme de la cloison interauriculaire. Diagnostic échocardiographique.

Interatrial septal aneurysm is a rare abnormality and can now be diagnosed by echocardiography. We report the case of a 52 year old woman in whom this condition was diagnosed after an embolic cerebrovascular accident. M mode recordings showed a linear echo in the left atrial cavity in early and mid systole. The aneurysm was directly visualised by 2D echo as a hemispherical bulge in the mid portion of the interatrial septum, which was mobile and had a to-and-fro motion between the two atria in relation to the different phases of the cardiac cycle. The diagnosis was confirmed by angiography, and at surgery. A feature of this case was the close correlation between echocardiographic, angiographic and operative findings. The pathogenesis of this type of aneurysm remains conjectural as does its role in the production of cerebral embolism, the evidence for which was circumstantial in the absence of other demonstrable causes and in the light of previously reported cases.

[Vascular attacks]. / Accidents vasculaires.

The present paper is aimed at presenting an overview of the studies on cerebrovascular diseases made by Charcot and his collaborators and later by the successive Professors in the Chair. The significance of studies from abroad is also briefly reviewed. Charcot's major interest in the field was cerebral haemorrhage and the evolution of the ideas about the aneurysms of Charcot-Bouchard is reviewed. During Charcot's Professorship, Henri Duret gave one of the first modern anatomical studies of the cerebral arteries. The study of lacunes was one among numerous important contributions of Pierre Marie to Neurology. Later the works of Charles Foix and his collaborators on cerebral infarction were prominent. Since the fifties, pathological, physiopathological and therapeutic studies have been one of the main flow of research in the Chair. Brief accounts of studies on hypertensive encephalopathy and vascular accidents of the spinal cord are given.

[Thrombolytic agents in cerebral infarctions]. / Les thrombolytiques dans les infarctus cérébraux.

Thrombolytic agents are aimed at restoring arterial patency thus reducing the risk of cerebral infarction in case of arterial occlusion by blood thrombus or embolus. A review of data from the literature is presented, and the authors have tried to answer the following questions: 1) What are the probabilities of early mortality (first 30 days) and of morbidity due to untreated or medically (thrombolysis excluded) treated cerebral infarction? 2) What are the morbidity and mortality rates after systemic thrombolysis or selective intraarterial thrombolysis? 3) Are the results of thrombolysis affected by the site of arterial occlusion (carotid or vertebrobasilar system)? 4) Does thrombolysis increase the risk of cerebral haemorrhage? 5) Can thrombolysis be regarded as an effective treatment and, if so, in which cases? This study of systemic thrombolysis yielded a mean mortality rate of 22.2%, with satisfactory functional outcome in 58.2% of the survivors. The corresponding figures for selective intraarterial thrombolysis were 29.7% and 84.5% respectively. However when the arterial territories occluded were taken into account, they became 10.4% and 84.4% respectively in the carotid system and 61.5% and 85.00% in the vertebrobasilar system. Comparisons between the natural history of cerebral infarctions, as far as it is known, and the results of thrombolysis suggest that thrombolytic agents are: 1) probably justified by the selective intraarterial route when the occlusion lies in the carotid system; 2) not indicated by the systemic route with urokinase or streptokinase but trials with tPA are being performed. These conclusions, however, must be interpreted with caution as they rest on general data leaving out a number of factors that are often neglected in reports particularly the exact site of arterial occlusion, chiefly in the vertebrobasilar system. From 3 limited cohorts it was possible to evaluate at about 14% the mortality rate in thrombolysis for basilar artery occlusion. This figure, compared with the 70-80% estimate found in the literature for untreated occlusions, suggests that the selective intraarterial thrombolysis may perhaps be indicated also in basilar artery occlusions. It must be noted that selective intraarterial thrombolysis is still an exceptional treatment which requires sophisticated techniques, highly qualified neuroradiologists and prompt application i.e. emergency stroke units.

[Dystonia musculorum deformans. Favorable effect of bromocriptine]. / Dystonia musculorum deformans. Effet favorable de la bromocriptine.

A 25 year-old woman suffered from a severe dystonia musculorum deformans since the age of 14. No similar cases were recorded in the family. Examination showed torsion spasm and a cogwheel phenomenon in both superior limbs. Signs disappeared on bromocriptine 22,5 mg/day then reappeared when dosages where progressively reduced. With 12.5 mg/day after 1 year of treatment, the patient leads a normal life including university studies.

[Dissecting aneurysm of the internal carotid artery and isolated Claude Bernard-Horner syndrome]. / Anévrysmes disséquants de l'artère carotide interne et syndrome de Claude Bernard-Horner isolé.

In 2 cases of dissecting aneurysm of the internal carotid artery the only clinical sign was a Claude Bernard-Horner's syndrome, with pain in the neck and face.

[Gelastic epilepsy. Probable diencephalic hamartoma]. / Epilepsie gélastique. Probable hamartome diencéphalique.

We report a of gelastic epilepsy. MRI showed a probable hamartoma in the diencephalic region.

[Comparison between x-ray computed tomography and magnetic resonance imaging after transient ischemic accidents]. / Comparaison du scanner X et de l'imagerie par résonance magnétique après des accès ischémiques transitoires.

We studied 18 patients with transient ischemic accidents (TIA) and normal CT scan during the first 24 hours. MRI was performed 9 days later on average. MRI revealed abnormalities in 10 patients (56 p. 100), but only 5 of them (27 p. 100) had lesions corresponding to TIAs, and in every case these were in the carotid territory. In the 7 patients with clinical signs of TIA in the vertebro-basilar territory, MRI was always negative. In 5 patients (27 p. 100), MRI showed abnormalities that were unrelated to TIA. This study suggests that MRI is of limited value in detecting brain tissue abnormalities and is perhaps not more sensitive than CT in vertebro-basilar TIA. However, in view of the small number of patients these results must be interpreted with caution.

[Carotid auscultation. Correlation in 200 patients with 332 angiograms]. / Auscultation carotidienne. Corrélations chez 200 patients avec 332 angiographies

1. Results of auscultation of carotid arteries have been reviewed in 200 adults patients with 332 carotid angiograms. Cases with abnormal cardiac bruits of supraclavicular buits were excluded. There was no case with stenosis of the external artery or significant anemia. 2. The present series of patients comprises : 100 with internal carotid artery (ICA) stenosis, 50 with ICA occlusion and normal contralateral ICA, 50 with cerebral space-occupying lesions. 3. A bruit was present in 80/132 ICA stenosis (60 per cent), 60/84 ICA stenoses reducing the lumen by more than 75 per cent (71 per cent), 20/48 ICA stenoses reducing the lumen by less than 75 per cent (41 per cent). It appeared likely that the presence of an ICA stenosis plays a role in the presence of a contralateral bruit where a contralateral stenosis or occlusion are present. 4. An ipsilateral bruit was present in 50/50 ICA occlusions. A contralateral bruit was present in 7 patients the ICA angiogram being normal. A bruit was present on both sides in 4 patients and it may be that some patients are somewhat apt to have bruits. 5. There was no bruit in 49 patients with normal carotid angiograms and a cerebral space-occupying lesion. Data from the present series suggest that where both ICA's are normal auscultation is falsely positive in less than 10 per cent of patients.

[Images of acute intracranial arterial occlusions obtained by CT scan]. / Images d'occlusions arterielles intracrâniennes aiguës obtenues par le scanner X.

In 1 case of occlusion of the basilar artery and 1 case of occlusion of the left middle cerebral artery a CT Scan performed within 24 hours of onset showed a high density area corresponding to the segmental arterial occlusion. In both cases on repeat CT Scans 5 and 9 days after the onset the high density area had disappeared.