The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA.

Direct sequencing of human mitochondrial tRNALysshows the absence of editing and the occurrence of six modified nucleotides (m1A9, m2G10, Psi27, Psi28 and hypermodified nucleotides at positions U34 and A37). This tRNA folds into the expected cloverleaf, as confirmed by structural probing with nucleases. The solution structure of the corresponding in vitro transcript unexpectedly does not fold into a cloverleaf but into an extended bulged hairpin. This non-canonical fold, established according to the reactivity to a large set of chemical and enzymatic probes, includes a 10 bp aminoacyl acceptor stem (the canonical 7 bp and 3 new pairs between residues 8-10 and 65-63), a 13 nt large loop and an anticodon-like domain. It is concluded that modified nucleotides have a predominant role in canonical folding of human mitochondrial tRNALys. Phylogenetic comparisons as well as structural probing of selected in vitro transcribed variants argue in favor of a major contribution of m1A9 in this process.

Molecular analysis of carnitine palmitoyltransferase II deficiency with hepatocardiomuscular expression.

Carnitine palmitoyltransferase (CPT) II deficiency, an inherited disorder of mitochondrial long-chain fatty-acid (LCFA) oxidation, results in two distinct clinical phenotypes, namely, an adult (muscular) form and an infantile (hepatocardiomuscular) form. The rationale of this phenotypic heterogeneity is poorly understood. The adult form of the disease is commonly ascribed to the Ser-113-Leu substitution in CPT II. Only few data are available regarding the molecular basis of the infantile form of the disease. We report herein a homozygous A-2399-C transversion predicting a Tyr-628-Ser substitution in a CPT II-deficient infant. In vitro expression of mutant cDNA in COS-1 cells demonstrated the responsibility of this mutation for the disease. Metabolic consequences of the SER-113-Leu and Tyr-628-Ser substitutions were studied in fibroblasts. The Tyr-628-Ser substitution (infantile form) resulted in a 10% CPT II residual activity, markedly impairing LCFA oxidation, whereas the Ser-113-Leu substitution (adult form) resulted in a 20% CPT II residual activity, with out consequence on LCFA oxidation. These data show that CPT II activity has to be reduced below a critical threshold in order for LCFA oxidation in fibroblasts to be impaired. The hypothesis that this critical threshold differs among tissues could provide a basis to explain phenotypic heterogeneity of CPT II deficiency.

Impairment of the mitochondrial respiratory chain activity in diethylnitrosamine-induced rat hepatomas: possible involvement of oxygen free radicals.

Alterations in the energy metabolism of cancer cells have been reported for many years. However, the deleterious mechanisms involved in these deficiencies have not yet been clearly proved. The main goal of this study was to decipher the harmful mechanisms responsible for the respiratory chain deficiencies in the course of diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis, where mitochondrial DNA abnormalities had been previously reported. The respiratory activity of freshly isolated hepatoma mitochondria, assessed by oxygen consumption experiments and enzymatic assays, presented a severe complex I deficiency 19 months after DENA treatment, and later on, in addition, a defective complex III activity. Since respiratory complex subunits are encoded by both nuclear and mitochondrial genes, we checked whether the respiratory chain defects were due to impaired synthesis processes. The specific immunodetection of complex I failed to show any alterations in the steady-state levels of both nuclear and mitochondrial encoded subunits in the hepatomas. Moreover, in vitro protein synthesis experiments carried out on freshly isolated hepatoma mitochondria did not bring to light any modifications in the synthesis of the mitochondrial subunits of the respiratory complexes, whatever the degree of tumor progression. Finally, Southern blot analysis of mitochondrial DNA did not show any major mitochondrial DNA rearrangements in DENA-induced hepatomas. Because the synthetic processes of respiratory complexes did not seem to be implicated in the respiratory chain impairment, these deficiencies could be partly ascribed to a direct toxic impact of highly reactive molecules on these complexes, thus impairing their function. The mitochondrial respiratory chain is an important generator of noxious, reactive oxygen free radicals such as superoxide and H2O2, which are normally catabolized by powerful antioxidant scavengers. Nineteen months after DENA treatment, a general collapse of the antioxidant enzymatic system was demonstrated in the hepatomas, as recurrently observed in cancer cells. This oxidant versus antioxidant imbalance was characterized by the establishment of oxidative stress in the course of hepatocarcinogenesis, as partly shown by the important decrease of glutamine synthetase activity, an enzyme whose function is highly sensitive to oxidant reactions. This disequilibrium would result in a net increase of the steady-state concentration of superoxide generated between respiratory complexes I and III in the mitochondria. Once generated, superoxide would likely inactivate complexes I and III via oxidant reactions on their superoxide-sensitive [4Fe, 4S] clusters. The role of mitochondrial respiratory chain impairment in chemical carcinogenesis and/or the persistence of the cancerous state is further discussed.

Defect in the lipoyl-bearing protein X subunit of the pyruvate dehydrogenase complex in two patients with encephalomyelopathy.

Among the many metabolic encephalomyelopathies caused by deficiencies in the pyruvate dehydrogenase complex (PDHC), nearly all involve its E1 subunit. We describe two new familial cases of PDHC deficiency with encephalomyelopathy, chronic lactic acidemia, and a normal E1 subunit of PDHC but deficiency in another component. Activity of PDHC was measured in cultured skin fibroblasts and skeletal muscle, and immunoblot studies were performed on mitochondrial extracts from skin fibroblasts. Spectra of muscle tissue, obtained in vivo with phosphorus 31 nuclear magnetic resonance, were recorded both at rest and with exercise. The PDHC activity was markedly reduced to 10% to 20% of normal values in both cultured skin fibroblasts and skeletal muscle. Immunoblotting of skin fibroblast mitochondrial extracts showed a specific deficiency in the protein X component of PDHC but normal E1, E2, and E3 components. Spectra obtained with 31P nuclear magnetic resonance showed alterations compatible with those found in mitochondrial myopathies. This is the second description of an encephalomyelopathy associated with a specific absence of the lipoyl-containing protein X component, which has a structural role in the formation of a functional PDHC.

Presence of proteins recognized by mammalian cytochrome P-450 antibodies in Euglena gracilis.

We have attempted to probe three microsomal cytochrome P-450 isozymes in Euglena gracilis using immunochemical methods. They cross-react with anti-rat cytochrome P4502C11, cytochrome P4502E, and cytochrome P4502B. Activities of alkoxyphenoxazone dealkylation have been tested in living cells. In untreated cultures, the amount of proteins recognized by anti-cytochrome P4502C11 or anti-cytochrome P4502E is high. Phenobarbital treatment increased the levels of microsomal proteins recognized by antibody to cytochrome P4502B, as well as dealkylases of pentoxyresorufin, but decreased the level of proteins recognized by anti-cytochrome P450C11 or cytochrome P4502E. These results suggest that these unicellular algae may contain different isozymes of microsomal cytochromes P-450, comparable to those in mammalian liver. They are cytochrome P-450 equivalents of mammalian isoenzymes 2C, 2E and 2B. However, we could not demonstrate ethanol induction of cytochrome P-450 equivalent to isoenzyme 2E. Its role in xeno- or endobiotic metabolism remains to be elucidated.

Major pathway of imipramine metabolism is catalyzed by cytochromes P-450 1A2 and P-450 3A4 in human liver.

The metabolism of imipramine by human liver microsomes was examined using a combination of five strategies. Human hepatic microsomes produced N-desmethylimipramine (84%), 2-hydroxyimipramine (10%), and 10-hydroxyimipramine (6%). Preincubation of human hepatocytes in culture with beta-naphthoflavone and macrolides exclusively induced the formation of desmethylimpramine (552%, p < 0.05, and 234%, p < 0.003, respectively). Correlations were obtained between rates of imipramine demethylation and cytochrome P-450 (P-450) 1A2 (r = 0.88, p < 0.001) and P-450 3A (r = 0.80, p < 0.02) concentrations in human liver microsomal preparations from 13 different subjects. Anti-P-450 1A2 and anti-P-450 3A antibodies selectively inhibited N-demethylation (80% and 60%, respectively). N-Demethylation was completely inhibited when anti-1A2 and anti-3A were added simultaneously. Kinetic studies with human microsomes confirm the contribution of two different enzymes in the N-demethylation. The Km of 1A2 was similar to the high affinity Km in human liver microsomes, whereas the Km of 3A was similar to the low affinity Km in human liver microsomes. P-450 1A2 was apparently more efficient than 3A4 (lower Km and higher Vmax) but was expressed in much lower concentration. Human P-450s 1A2 and 3A4 expressed in yeast efficiently produced desmethylimipramine. These results suggest that P-450 1A2 and P-450 3A4 are the major enzymes involved in imipramine N-demethylation in human hepatic microsomes. Similar experiments were conducted using P-450 2D6, and they confirmed that P-450 2D6 catalyzes imipramine 2-hydroxylation. Interindividual variations in 3A4 hepatic content may explain the large variations in imipramine blood levels observed after uniform dosages and thus may explain the variations in clinical efficacy. Caution might be advised in the clinical use of tricyclic antidepressants when drugs are also administered that induce or inhibit P-450s 3A4 and 1A2.

E1 pyruvate dehydrogenase deficiency in a child with motor neuropathy.

We report the case of a boy who developed a motor neuropathy during infectious episodes at 18 mo and 3 y of age. When he was 7 y old, he suffered persistent weakness and areflexia; his resting lactate and pyruvate values were 3.65 mM and 398 microM, respectively (controls: 1.1 +/- 0.3 mM and 90 +/- 22 microM), and an exercise test demonstrated a lactic acidosis (13.6 mM; controls: 6.4 +/- 1.3 mM) with a high pyruvate level (537 microM; controls: 176 +/- 15 microM) and a low lactate/pyruvate ratio (24.2; controls: 35 +/- 2). The results of polarographic studies on muscle mitochondria suggested a defect in pyruvate oxidation (pyruvate 17 ng atom O/min/mg protein; controls: 115 +/- 42), whereas glutamate, palmitoylcarnitine, and succinate were good respiratory substrates. The activity of total pyruvate dehydrogenase complex (PDHC) in muscle mitochondria and in fresh mononuclear cells was markedly decreased (9.7 and 0.054 nmol 14CO2/min/mg protein, respectively; controls: 123 +/- 4.5 and 0.733 +/- 0.03, respectively). Immunochemical analysis in muscle mitochondria demonstrated an absence of the alpha and beta E1 PDHC subunits. After 2 y of treatment with 500 mg/d thiamine, the patient was clinically improved. A genetic study of the main regions of mutations (exon 10 and 11) in the X chromosome encoding for the E1 alpha subunit of PDHC did not show any mutation. These data indicate that, although genetically different, this case enters in a very rare category of patients with PDHC deficiency without cerebral dysfunction and improved by thiamine + L-carnitine therapy.

Cytochrome P450 induction and mutagenicity of 2-aminoanthracene (2AA) in rat liver and gut.

The aim of our study was to establish a relationship between the ability of rat liver and gut to activate 2-aminoanthracene (2AA) into mutagens and their P450 enzyme composition. Rats were orally pretreated with beta-naphthoflavone (beta NF), phenobarbital (PB), dexamethasone (DEX) or acetone (AT). Mutagenic activation of 2AA was detected in the Ames test. P450IA1, IA2, IIB1/B2 and IIE1 were immunochemically quantified by Western blots. All the results were compared to those obtained in untreated rats. In all tissues, beta NF treatment considerably increased the mutagenicity of 2AA. PB treatment significantly reduced the mutagenicity of 2AA in the liver but not in the intestine. By contrast, AT treatment significantly decreased the number of revertants in the duodenum but not in the liver whereas DEX treatment significantly decreased the number of revertants in both tissues. 2AA appears to be metabolized by various P450s in both organs. In the liver, reactive metabolites may be produced after metabolism by the P450IA subfamily. The other P450 enzyme seems to play a part in the metabolism of 2AA leading to formation of either mutagenic or non-mutagenic metabolites.

Hormonal regulation of microsomal flavin-containing monooxygenase: tissue-dependent expression and substrate specificity.

The substrate- and tissue-dependent hormonal regulation of flavin-containing monooxygenase (EC 1.14.13.8) was studied in male and female rats. Hypophysectomy of males reduced liver microsomal N,N-dimethylaniline N-oxidation, thiobenzamide S-oxidation, and imipramine N-oxidation, although the reduction was not as marked with the latter substrate. Castration also reduced flavin-containing monooxygenase-dependent activities, but not to the same extent as hypophysectomy. Administration of growth hormone or testosterone to hypophysectomized males only partially restored basal activities. In female rats, hypophysectomy had no effect on N,N-dimethylaniline N-oxidation or thiobenzamide S-oxidation and actually stimulated imipramine N-oxidation (98%). These effects were demonstrated to be tissue- and sex-dependent. For example, hypophysectomy markedly (300%) enhanced imipramine N-oxidation in male kidney and significantly decreased the same activity in male and female lung. Correlations between levels of the enzyme determined by immunoquantitation (with antibody to the rat liver enzyme) and activities toward these three substrates, in male and female liver, lung, and kidney, also provide evidence for the existence of multiple forms of flavin-containing monooxygenase, which appear to be under different hormonal regulation.

Drug-metabolizing enzyme expression in human normal, peritumoral and tumoral colorectal tissue samples.

In the present study, we investigated Phase I (cytochrome P450; DT-diaphorase, DTD) and Phase II (epoxide hydrolase, EH; glutathione-S-transferases, GSTs) enzymes in normal colon from patients without colorectal adenocarcinoma and in peritumoral and tumoral tissues from patients with colorectal adenocarcinoma. No significant changes in levels of cytochrome P450IIIA4 (the only P450 detectable in this tissue), EH, GSTs and DTD activity were found between normal and peritumoral tissues. In tumoral tissue, compared with peritumoral tissues, we observed significant decreases in cytochrome P450IIIA4 (-50%, P less than 0.002) and EH (-60%, P less than 0.03), no change in DTD activity and significant increases in GST pi (+40%, P less than 0.03) and total GST activity (+30%, P less than 0.01). The numerous changes observed in tumoral tissues suggest that variations in drug-metabolizing enzyme expression in colorectal adenomatous polyps could represent pretumoral markers. Moreover, a better understanding of the expression of these enzymes in tumoral tissues would help us to choose the most appropriate colon tumor cell lines for the testing of new anti-cancer drugs.

Infantile form of carnitine palmitoyltransferase II deficiency with hepatomuscular symptoms and sudden death. Physiopathological approach to carnitine palmitoyltransferase II deficiencies.

Reported cases of carnitine palmitoyltransferase II (CPT II) deficiency are characterized only by a muscular symptomatology in young adults although the defect is expressed in extra-muscular tissues as well as in skeletal muscle. We describe here a CPT II deficiency associating hypoketotic hypoglycemia, high plasma creatine kinase level, heart beat disorders, and sudden death in a 3-mo-old boy. CPT II defect (-90%) diagnosed in fibroblasts is qualitatively similar to that (-75%) of two "classical" CPT II-deficient patients previously studied: It resulted from a decreased amount of CPT II probably arising from its reduced biosynthesis. Consequences of CPT II deficiency studied in fibroblasts differed in both sets of patients. An impaired oxidation of long-chain fatty acids was found in the proband but not in patients with the "classical" form of the deficiency. The metabolic and clinical consequences of CPT II deficiency might depend, in part, on the magnitude of residual CPT II activity. With 25% residual activity CPT II would become rate limiting in skeletal muscle but not in liver, heart, and fibroblasts. As observed in the patient described herein, CPT II activity ought to be more reduced to induce an impaired oxidation of long-chain fatty acids in these tissues.

Immunoquantitative analysis of human carnitine palmitoyltransferase I and II defects.

Carnitine palmitoyltransferase deficiency realizes two distinct clinical forms. We previously showed and confirmed in the present work that CPTII (identified as the carnitine palmitoyltransferase activity assayable in detergent conditions) is decreased in the muscular form whereas it is unaffected and CPTI is decreased in the hepatic form. The antibody previously prepared against human liver mitochondrial CPTII recognizes the same enzyme in muscle, liver, and fibroblasts. Immunoprecipitation experiments were performed in fibroblasts from patients with the muscular and hepatic forms of the defect. As compared with controls, cell lines from two patients with the hepatic form of the defect did not exhibit any qualitative nor quantitative abnormality of cross-reacting material, whereas cell lines from two patients with the muscular form of the defect exhibited a decreased amount of cross-reacting material. These data suggest that CPTII deficiency could result from a decreased production of protein. The amount of cross-reacting material in the two sets of patients only correlates with CPTII activity, which is decreased in the muscular presentation and unaffected in the hepatic form. These results strengthen the hypothesis of distinct proteins supporting CPTI and CPTII activities.

Cytochrome P 450 isoenzymes, epoxide hydrolase and glutathione transferases in rat and human hepatic and extrahepatic tissues.

The organ distribution of microsomal cytochrome P 450 isoenzymes (P 450), microsomal epoxide hydrolase (EH) and cytosolic glutathione-S-transferases was investigated by immunoblotting and enzyme measurements in rats and humans. In rats, P 450 IA1 was detected only in the duodenum, and P 450 IA2 and IIC11 were detected only in the liver. The highest concentrations of P 450 IIB1/B2 were found in the lung and in the duodenum; pentoxyresorufin-O-dealkylase activity was closely correlated with the amounts of P 450 IIB1/B2 in the different organs. P 450 IIE1 was present in liver, kidney and lung, whereas EH was found in liver, intestine and kidney. In humans, P 450 IIIA4 was detected in all tissues investigated; the highest concentrations were found in liver and intestine. The P 450 IIIA4 level was closely correlated with that of erythromycin demethylase and pentoxyresorufin-O-dealkylase activities. P 450 IIC8-10, IIE1 and IID6 were expressed in liver and intestine, P 450 9 in liver and kidney and P 450 IA2 in liver. EH was identified only in liver, intestine and kidney. In both species, concentrations and total amounts of P 450 isoenzymes and EH were much lower in all extrahepatic tissues than in the liver. Conversely, glutathione-S-transferase-pi was abundant in human intestine and colon compared to liver. Glutathione-S-transferase-mu polymorphism was confirmed in all tissues investigated. This extensive study showed that the pattern of (iso) enzymes was different in all tissues studied; consequently, xenobiotic metabolism would appear to be very different in each type of tissue.

A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

Ontogeny and regulation of drug metabolism in humans. Phase I: Monooxygenases.

Most data concerning the development and regulation of cytochrome P-450-dependent monooxygenases were obtained in experimental animals, especially rats, by two successive approaches: first, the assay of total liver cytochrome P-450 and of related monooxygenase activities; then, more recently, the immunoquantification of molecular forms of cytochrome P-450, and the estimation of corresponding mRNAs with nucleic acid probes. Results were compared, before and after birth, in untreated animals, and in animals treated by inducers, mainly phenobarbital and polycyclic aromatic hydrocarbons. For evident ethical reasons, fewer data could be obtained in human liver during development. However, the availability of specific antibodies and nucleic acid probes recently allowed some progress in the comparison of the isoenzyme pattern of cytochromes P-450 in fetal and adult human liver.

Purification and characterization of a cytochrome P-450 isozyme isolated from the liver of rats pretreated with clofibrate.

1- Using a methodology of purification consisting of only one chromatographic step (phenyl-Sepharose) we have purified a cytochrome P-450 isozyme from liver of clofibrate - treated rats. It was called cytochrome P-450 clo. 2- A single polypeptide of mol.wt. 50,000 was visible after sodium dodecyl sulphate polyacrylamide gel electrophoresis. 3- Antiserum raised against the pure enzyme specifically recognized P-450 clo and inhibited more than 90% of the 11- and 12- laurate hydroxylase activities present in clofibrate - treated rats. 4- Clofibrate treatment of the rats resulted in a six fold increase in microsomal cytochrome P-450 clo as judged by immunochemical quantification. This result is in agreement with the increase of laurate hydroxylase activity after treatment by clofibrate.

Purification and comparison of liver microsomal flavin-containing monooxygenase from normal and streptozotocin-diabetic rats.

Liver microsomal flavin-containing monooxygenase (FMO) activity towards thiobenzamide is two-fold increased in streptozotocin diabetic (insulin deficient) rats and mice and, to a lesser degree in congenital insulin resistant Ob/Ob mice. No difference in thermal stability appears between microsomal FMOs from both normal and diabetic rats. FMO has been purified to homogeneity from these two sources, with a 50-fold increase of specific activity. Their apparent molecular weight is respectively 50,000 and 49,000 and a discrete modification appears in the HPLC profiles of tryptic peptides from purified FMOs. They appear immunochemically very similar and present in equal quantity in microsomal membranes from both normal and diabetic rats, so that the increased activity cannot be ascribed to an increased concentration of the enzyme protein.