Anderson's disease (chylomicron retention disease): a new mutation in the SARA2 gene associated with muscular and cardiac abnormalities.

Anderson's disease (AD) or chylomicron retention disease (CMRD) is a rare hereditary lipid malabsorption syndrome linked to SARA2 gene mutations. We report in this study a novel mutation in two sisters for which the Sar1b protein is predicted to be truncated by 32 amino acids at its carboxyl-terminus. Because the SARA2 gene is also expressed in the muscle, heart, liver and placenta, extraintestinal clinical manifestations may exist. For the first time, we describe in this study in the two sisters muscular as well as cardiac abnormalities that could be related to the reported expression of SARA2 in these tissues. We also evaluated six other patients for potential manifestations of the SARA2 mutation. The creatine phosphokinase levels were increased in all patients [1.5-9.4 x normal (N)] and transaminases were moderately elevated in five of the eight patients (1.2-2.6 x N), probably related to muscle disease rather than to liver dysfunction. A decreased ejection fraction occurred in one patient (40%, N: 60%). The muscle, liver and placental tissues that were examined had no specific abnormalities and, in particular, no lipid accumulation. These results suggest that myolysis and other extraintestinal abnormalities can occur in AD/CMRD and that the clinical evaluation of patients should reflect this.

Phosphocreatine synthesis by isolated rat skeletal muscle mitochondria is not dependent upon external ADP: a 31P NMR study.

Phosphocreatine synthesis by mitochondria isolated from rat skeletal muscle was determined in presence of inorganic phosphate, creatine, and either ATP or ADP, using 31P NMR spectroscopy in a new protocol maintaining mitochondria for several hours in a well-coupled state. Maximal velocity of phosphocreatine synthesis was identical with 0.4 mM of ADP or 0.5 mM ATP at a rate of 0.063 mM/min. External ATP and ADP were always present in the spectra, demonstrating that in skeletal muscle cells as in heart muscle cells, mitochondrial creatine kinase coupled to translocase has a very strong amplifying effect on oxidative phosphorylation and converts external inorganic phosphate and creatine into phosphocreatine without net adenine nucleotide consumption. Therefore, adenine nucleotides can be considered as cofactors rather than regulators of mitochondria metabolism. This is in agreement with the "phosphocreatine-circuit" theory.

Maintenance of the biotransformation capacity by cultured human hepatocytes after several daily exposures to drugs.

Adult human hepatocytes were obtained from four different donors and exposed to three new drugs (DPI 201-106, CK 204-933 and SDZ 203-150) for 4 or 5 days with daily renewal of both medium and drug. Both the rates and the routes of metabolism were analysed every day by HPLC and thin-layer chromatography. Metabolism of the three drugs resulted in the formation of several metabolites, which involved various phase I and phase II reactions. For the three drugs, both qualitative and quantitative metabolic capacity was roughly maintained during the period studied. However, some minor metabolites were identified only after two or three incubations, probably because of their low rate of formation and/or multistep synthesis. Individual variations, which were maintained during the whole incubation period, were observed. The maintenance of the same metabolic pathways even after several daily treatments indicates that adult human hepatocytes in primary culture represent a suitable in vitro model system for studies on drug inhibition or induction, drug interactions and long-term drug cytotoxicity.

Study of doxorubicin photodegradation in plasma, urine and cell culture medium by HPLC.

An HPLC method was used to monitor the chemical stability of doxorubicin-HCl (DXR) to light in plasma, urine and cell culture medium at room temperature. The results indicated that DXR was very unstable in cell culture medium and urine when exposed to light. It was more stable in plasma under the same conditions. In all the cases, the decrease in the amount of DXR is greatly dependent on light intensity (no noticeable degradation was observed after 8 h in the dark). These observations may be important for the correct interpretation of the effects and the toxicity of doxorubicin on cells incubated in cell medium, and for determination of urinary or plasma pharmacokinetic parameters.

Different cytotoxicity and metabolism of doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin in cultured human and rat hepatocytes.

Both cytotoxicity and metabolism of five anthracyclines, namely doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin, were investigated in primary cultures of both rat and human adult hepatocytes and, for comparison, in a rat liver epithelial cell line. Toxicity was assessed by morphological examination and measurement of lactate dehydrogenase leakage after 24 hr of treatment. The rank order of toxicity for both rat and human hepatocytes was esorubicin greater than doxorubicin = epirubicin greater than or equal to idarubicin greater than daunorubicin, and for rat epithelial cells: esorubicin greater than or equal to epirubicin greater than idarubicin = daunorubicin = doxorubicin. Human cells were around 2-fold less sensitive than rat hepatocytes to all anthracyclines. Anthracyclines and their metabolites were analyzed by HPLC. Differences in both the percentages and routes of metabolism were demonstrated between rat and human hepatocytes. The main metabolite was the 13-dihydro-derivative (-ol derivative) in both species from daunorubicin, idarubicin and esorubicin. Glucuronides of epirubicin and epirubicinol were found only in human hepatocytes. In addition, several unidentified metabolites were detected of esorubicin, idarubicin and daunorubicin in rat hepatocytes. In human hepatocytes, only one unknown metabolite from daunorubicin and doxorubicin was found to be formed by cells from a different donor. In spite of variations between individuals, human hepatocytes generally metabolized anthracyclines more actively than did rat hepatocytes. Rat liver epithelial cells were only able to convert daunorubicin and idarubicin, the two molecules which have the best affinity for the non-specific NADPH-dependent aldoketoreductase system. Three compounds (doxorubicin, epirubicin and esorubicin) were present in large amounts in the cells as the parent drug, another (idarubicin) as the 13-dihydro-derivative. This comparative study on cytotoxicity and metabolism of five anthracyclines in rat and human hepatocyte cultures emphasises species differences and the importance of this in vitro model system for further analysis of the metabolism and effect of anthracyclines.

Use of hepatocyte cultures for preliminary metabolism and cytotoxicity studies of a new anti-hypertensive agent, oxaminozoline.

1. Adult rat hepatocytes co-cultured with rat liver epithelial cells were used to evaluate chronic cytotoxicity of a new alpha 2 agonist, oxaminozoline (S-3341-3) compared to that of clonidine. The same maximum non-toxic concentration (25 micrograms per ml of medium) was found for both drugs after a daily treatment for 12 days. 2. Oxaminozoline metabolism was analysed in short-term hepatocyte cultures. Four metabolites resulting from oxidation or hydrolysis of the parent drug were identified. Three of the metabolites were identical to those reported in vivo. The presence of an additional minor metabolite in culture may be due to the higher metabolic rate of the drug in this model system.

Identification of metabolic pathways of pindolol and fluperlapine in adult human hepatocyte cultures.

1. Adult human hepatocytes were obtained from a normal donor and a barbiturate-overdose fatality and maintained either in pure culture or in co-culture with rat liver epithelial cells. At varying times of culture, metabolic pathways of pindolol and fluperlapine, two drugs which are transformed at different rates and routes in man and rat, were determined after a 24 h incubation. 2. In both culture conditions, human hepatocytes remained able to form the major urinary metabolites of pindolol and fluperlapine, including oxidized products, glucuronide and sulphate conjugates over the whole period studied (i.e. 5 and 21 days in pure and co-culture respectively). 3. Fluperlapine which has the higher first pass in man and animals was found to be metabolized in vitro more quickly than pindolol. 4. Some qualitative and quantitative differences were found between normal and drug-altered human hepatocytes, indicating that in vivo environmental factors may greatly influence metabolic properties of these cells even after several days in culture.

Differential response of primary cultures of human and rat hepatocytes to aflatoxin B1-induced cytotoxicity and protection by the hepatoprotective agent (+)-cyanidanol-3.

The acute hepatotoxicity induced by aflatoxin B1 (AFB1) and the potential protective effect of (+)-cyanidanol-3 (Catergen) were evaluated in both human and rat hepatocytes in primary cultures. AFB1-induced acute toxicity was visualized by light microscope observation and quantified by measurement of lactic dehydrogenase activity in the medium. Human hepatocytes were susceptible to AFB1-induced cytotoxicity but no evident relationship between the concentration of mycotoxin and the extent of cellular damage was established. (+)-Cyanidanol-3 was not toxic at concentrations up to 2 x 10(-3)M, but no obvious protective effect from AFB1-induced injury was evidenced in human cells. By contrast, rat hepatocytes responded in a dose-related manner to AFB1. (+)-Cyanidanol was toxic at 10(-3)M, but even at this concentration exerted a strong protective effect against AFB1-induced cytotoxicity. Such species differences suggest the existence of metabolic differences in both AFB1 and (+)-cyanidanol-3 activating and deactivating mechanisms.

Species differences in metabolism of ketotifen in rat, rabbit and man: demonstration of similar pathways in vivo and in cultured hepatocytes.

In vitro drug metabolism by cultured rat, rabbit and human adult hepatocytes has been studied, using ketotifen (ZADITEN) as a model substrate because it is biotransformed in vivo by various metabolic pathways in man and animals. The major in vivo pathways were demonstrated in vitro, namely oxidation in rat hepatocytes, oxidation, glucuronidation and sulfation in rabbit hepatocytes, reduction and glucuronidation in human hepatocytes. Human hepatocytes were the most stable in culture, displaying ketotifen biotransformation for at least one week. These results clearly demonstrated that cultured hepatocytes retain their in vivo specific drug metabolizing activities, including inter-species polymorphism, for a few days. Therefore, pure hepatocyte cultures represent a useful system suitable for drug metabolism studies.

Fluorocarbons and cardiac arrhythmia: does difluorodichloromethane (FC 12) inhibit cardiac metabolism?

Certain fluorocarbons, such as difluorodichloromethane (FC 12), depress the cardiovascular system by diminution of all the transmembrane ionic conductances in cardiac tissues. Does FC 12 also inhibit active transport and thus enzymatic activity and cellular energy? We measured phosphocreatine (PC), adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMPc) in rat hearts. Rats were randomly divided into 4 groups; 2 control groups: one breathing a mixture of oxygen (21%) and nitrogen (79%) (group C) and the other breathing the same mixture but simultaneously perfused with 1 microgram/kg/min. epinephrine (groupe E-C); 2 trial groups T and E-T where nitrogen was replaced by FC 12. The maximal FC 12 concentration of 720 micrograms/ml in arterial blood produced no significant difference in the concentrations of these three metabolites compared with controls.

Human hepatocyte cultures: a model of pharmaco-toxicological studies.

Viable adult human hepatocytes were obtained in large yields by perfusion of the liver of kidney donors. The hepatocytes were cultured either alone or in association with rat-liver epithelial cells. In pure culture the survival of hepatocytes did not exceed two to three weeks, while in co-culture they survived for several weeks and better retained the specific liver functions of albumin secretion, cytochrome P-450 content and glucuronidation of drugs. Human hepatocytes, particularly when mixed with rat-liver epithelial cells, may provide a valuable tool for predicting the metabolic pathways and hepatotoxicity of new drugs in man.

Erythromycin toxicity in primary cultures of rat hepatocytes.

Cultured rat hepatocytes were used to study the toxicity of erythromycin base (EB), erythromycin estolate (EE) and a new fluorinated derivative, (8S)-8-fluoroerythromycin A (EF). EF was not cytotoxic after 18 h incubation at concentrations up to 8 X 10(-4) M and EE was much more toxic than EB at all concentrations studied. EE toxicity was greater in a serum-free medium and was not increased by induction of cytochrome P-450 with phenobarbitone. In hepatocytes co-cultured with rat-liver epithelial cells EE, but not EF, raised the cytochrome P-450 content and formed stable cytochrome P-450 complexes with about 40% of the haemoprotein. The lack of correlation between cytochrome P-450 content and cytotoxicity suggests that some of the parent erythromycin drugs and not their metabolites are the toxic entities.

Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type.

The cytochrome P-450 content promptly fell in adult rat hepatocytes cultured in conventional conditions whereas no obvious change was detected over a 10-day period when these cells were co-cultured with another rat liver epithelial cell type. A concomitant maintenance of the amino-pyrine N-demethylase activity and a high aflatoxin B1-induced cytotoxicity was observed, and addition of phenobarbital to the culture medium produced about a 2-fold increase in cytochrome P-450 level. These results indicate that when placed in co-culture, adult hepatocytes remain differentiated for several days and suggest that such a co-culture system is an appropriate model for pharmacotoxicological studies.

[Early cellular alterations induced by myocardial hypoxia: possible role of cyclic AMP (author's transl)]. / Lésions cellulaires précoces induites par l'hypoxie myocardique: rôle éventuel de l'AMP cyclique.

The ability of endogenous myocardial catecholamines to participate in the development of myocardial cellular alterations during a short period of severe hypoxia (30 min) was studied in isolated, Langendorff-perfused rat heart preparation, arrested by a high potassium concentration (16 mM) and perfused in the absence of exogenous substrate (Table I). Tyramine, which accelerated catecholamine depletion, also increased myocardial cell damage as assessed by a higher lactate dehydrogenase (LDH) release and a more marked reduction in cellular levels of high energy phosphates and glycogen (Table II). On the other hand, under conditions of beta-blockade (atenolol), hypoxia-induced tissular damage was reduced (Table II). These changes could be related to modifications in the cellular content of cyclic AMP (cAMP) since cAMP was consistently higher during the first 30 min of hypoxic perfusion than in control normoxic hearts (Table III) whereas cyclic GMP content remained unchanged. Moreover, interventions increasing cellular content of cAMP (theophylline, dibutyryl-cAMP) also increased hypoxic damage (Table IV), whereas N-methyl imidazole which reduced cellular content of cAMP lessened hypoxia-induced cellular alterations (Table IV). It is concluded that cellular lesions developing during the first 30 min of hypoxia in isolated arrested rat heart preparation perfused without exogenous substrate could be related to intracellular accumulation of cAMP occurring under the effect of endogenous catecholamine release.