Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells.

Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.

[Oxidation of a naphthenic hydrocarbon, dodecylcyclohexane, in the rat]. / Mise en évidence de l'oxydation par le rat d'un hydrocarbure naphténique, le dodécylcyclohexane

Naphthenic hydrocarbons, mainly of fossil origin, are widespread in our environment, and contaminate the food chains; they are also used as food additives. Their fate in mammals is unknown, except for the fact that they are absorbed and accumulate in tissues. Only a few microorganisms have been shown capable or oxidising n-alkyl substituted cycloparaffins. In this study, dodecylcylohexane has been chosen as a typical monocycloparaffin, and has been administered orally to rats. The GLC and GLC-MS analysis of the methylesters of body and hepatic fatty acids led to the identification of cyclohexyldodecanoic and its decanoïc and octanoïc homologs. The alkyl chain undergoes a terminal oxidation followed by the classical beta-oxidation process. After administration of one 200 mg dose, or incorporation of 0.1% of the cycloparaffin in the diet for 2 months, these acids were found at low levels in neutral lipids and phospholipids. Their subsequent metabolic pathway and their possible interaction with the biochemical mechanisms involving phospholipids are under investigation.

Optimization of the separation of beta-agonists by capillary electrophoresis on untreated and C18 bonded silica capillaries.

The conditions of the separation of ten beta-agonists by capillary zone electrophoresis were studied. Several buffers were tested at different ionic strengths and different pH values. The experiments were carried out on two different supports, i.e. an untreated fused-silica capillary and a C18 covalently bonded silica capillary. The results showed that the optimum pH value was the same for the two capillaries. Separation efficiencies were slightly better for the fused-silica capillary whereas better selectivity and repeatability were obtained with the C18 bonded capillary, under optimal conditions.

Hydrocarbon hydroxylation system in liver microsomes from four animal species.

The in vitro metabolism of n-heptadecane was investigated using hepatic microsomes from Hubbard chickens, New Zealand rabbits, Wistar rats and rainbow trout. Incubations with the 14C-labelled alkane for 1 hr showed that the rate of oxidation varied between species; the rate (per mg protein) in chickens was roughly 20-fold greater than the rate in trout, and roughly 10-fold greater than the rates in rats and rabbits. On the basis of cytochrome P-450 content, the rate of heptadecane metabolism was roughly 20-fold greater in the chicken than in the other species. Moreover, the lambda max Soret band of the reduced cytochrome P-450 CO-complex was observed at 452 nm in the chicken. Correlation between the rate of heptadecane metabolism and in vivo storage levels is discussed.

Metabolism of naphthenic hydrocarbons. Utilization of a monocyclic paraffin, dodecylcyclohexane, by rat.

3H-Dodecylcyclohexane was incorporated in rat diet in order to study the metabolic utilization by mammals of a monocycloparaffin chosen as a typical naphthenic constituent of mineral oils. Dodecylcyclohexane was largely absorbed. No elimination of the hydrocarbon was observed in urine while an extended excretion of 3H occurred via this route. About 7% of absorbed dodecylcyclohexane was stored in the carcass, while the rest was omega-oxidized to cyclohexyldodecanoic acid, which incorporated into neutral lipids and phospholipids. The aliphatic chain of this unusual fatty acid underwent the normal fatty acid degradation pathways, leading to even fatty acids, squalene, cholesterol and nonlipid resynthesis, while the cyclohexyl ring was eliminated as urinary metabolites. Incorporation of omega-cyclohexyl fatty acids in phospholipids may raise a point of toxicological significance which is to be investigated.

Metabolism of a n-paraffin, heptadecane, in rats.

14C-heptadecane incorporated in rat diet was largely absorbed, and a balance study showed extensive 14CO2 excretion (65%). There was no elimination of the hydrocarbon in the urine, and only minute quantities of labeled metabolites. Radioactivity in the feces was entirely in heptadecane. About 7% of the heptadecane absorbed was stored in the carcass, whereas the rest was omega-oxidized to heptadecanoic acid. This fatty acid was incorporated into neutral lipids and phospholipids, underwent the normal fatty acid degradation pathway, and contributed to the synthesis of lipids, including fatty acids, squalene and cholesterol, and nonlipids (7-10%). Heptadecanoic acid was desaturated to heptadecenoic acid. The even distribution of radioactivity in the fatty acids of the various phospholipid classes indicated that heptadecane did not interfere with the biochemical mechanisms of these functional lipids.

Mitochondrial hydroxylation of the cyclohexane ring as a result of beta-oxidation blockade of a cyclohexyl substituted fatty acid.

Among the urinary metabolites of dodecylcyclohexane or cyclohexylacetic acid, the glycine conjugate of 1-hydroxy-cyclohexylacetic acid was identified and its origin studied, using cyclohexylacetic acid as the starting molecule, as it results from beta-oxidation of cyclohexyldodecanoic acid produced by terminal oxidation of the alkyl chain of the cycloparaffin. Three hypotheses were tested: (a) hydroxylation by the liver microsomal mixed-function oxidases involved in detoxication mechanisms; (b) hydroxylation by a cyt. P450-containing mitochondrial hydroxylase; and (c) beta-oxidation blockade after the reaction catalyzed by enoyl-CoA-hydratase. Liver microsomal or mitochondrial fractions were prepared and incubated in the presence of [14C] cyclohexylacetic acid, glucose-6-phosphate dehydrogenase and a NADPH-producing system. On the other hand, mitochondria were incubated in a suitable respiratory medium with or without cofactors required for ATP production. The reaction products were extracted and analyzed by thin layer radiochromatography and radio gas chromatography. Evidence is given that hydroxylation of cyclohexylacetic acid in position 1 is a mitochondrial step requiring activation in the acyl-CoA form and results from beta-oxidation blockade, the cyclohexane ring hindering hydroxyacyl-CoA-dehydrogenase action.

Disposition and metabolism of pristane in rat.

The fate of pristane (2,6,10,14-tetramethylpentadecane), a widespread isoprenoid hydrocarbon, has been studied in rats after a single per os administration of 3H-labeled pristane. The balance study showed an extensive fecal excretion (66%) mainly as unchanged hydrocarbon, whereas about 14% of ingested pristane was excreted in urine as pristane metabolites and tritiated water. After one wk, 8.3% of the ingested 3H still was stored in the carcass, and radioactive distribution in tissues and organs showed a preferential incorporation into adipose tissue and liver. Over 75% of the radioactivity stored in the carcass was associated with pristane metabolites and tritiated water. Tissue metabolites were characterized by thin layer chromatography, gas chromatography and mass spectrometric analyses. Four metabolites were identified: pristan-1-ol, pristane-2-ol, pristanic acid and 4,8,12-trimethyltridecanoic acid. These demonstrate that this isoprenoid hydrocarbon undergoes subterminal hydroxylation or terminal oxidation followed by the classical beta-oxidation process. Incorporation of metabolites in phospholipids and more particularly in the phosphatidylserine fraction has been observed and is discussed.

[Carbon monoxide poisoning. Neurologic aspects]. / Les intoxications a l'oxyde de carbone. Le point de vue du neurologue.

It is most often during winter months that carbon monoxide intoxications occur. Poorly functioning heating systems are the leading cause. We hereby summarize the mechanisms of CO toxicity which leads to treatment basis, and immediate neurological signs of this intoxication. We emphasize the delayed neuropsychiatric syndrome typical of this intoxication, often under or misdiagnosed. Two observations are presented.

[Diagnosis of internal carotid artery dissection. Two case reports]. / Approche diagnostique de la dissection carotidienne. A propos de deux cas.

Two cases of acute internal carotid dissection are presented. Typical symptoms, pathogeny and imaging features are reviewed. Magnetic Resonance is actually the best technique for the diagnosis of internal carotid artery dissection, which should be searched in young patients presenting neurologic and cervico-facial symptoms.

Assessment of metastable atom bombardment (MAB) ionization mass spectrometry for the fast determination of heterocyclic aromatic amines in cooked meat.

An investigation of metastable atom bombardment (MAB) ionization mass spectrometry for the fast characterization of mutagenic/carcinogenic heterocyclic aromatic amines (HAAs) formed during heating processes of meats is presented. The aim of our study was to use the selective ionization of MAB to develop a detection method for HAAs in non-purified meat extracts, thus avoiding purification and concentration steps and reducing analysis time. Sample introduction into the MAB ion source was achieved by pyrolysis, allowing the direct and fast insertion of complex food extracts into the mass spectrometer. Analysis conditions were optimized on standard HAAs by using different ionization gases for the MAB process. Metastable nitrogen was selected as the best MAB gas for the analysis of HAAs. Ionization selectivity is shown by the detection of heterocyclic amines in non-purified chicken meat extracts spiked with HAAs. A quantitative approach is also presented by using pyrograms as chromatograms for quantification purposes. HAAs determination using Py-MAB-ToF was finally performed on cooked chicken breast extracts and compared to an LC-APCI-MS/MS method. Although Py-MAB-ToF sensitivity remains to be improved in the present state of development of our prototype device, only 2 h from the cooking were required to obtain quantitative results in good agreement with HAAs concentrations measured by LC-MS/MS in 36 h. Figure Experimental set-up for pyrolysis-MAB-ToF mass spectrometry experiments.

[Determination of 3, 4-benzopyrene in spiruline algae produced and treated by various procedures]. / Determination du 3, 4-benzopyrene dans les algues spirulines produites et traitees suivant differents procedes

Spirulina algae grow in highly salted natural lakes. Artificial cultivation in tanks with addition of carbon dioxide (natural gas, burned gases) has been studied in order to improve the biosynthesis. A possible 3, 4-benzopyrene (BaP) contamination must be then considered. Several BaP determinations have been performed in batches of algae from bath origins, prepared following different processes (filtration, spray, cylinder drying). BaP contents are very low (2 to 3 ppb) and comparable between batches.

[Metabolism of paraffinic and naphthalenic hydrocarbons in higher animals. I. Retention of paraffins (normal, cyclo and branched) in rats]. / Métabolisme des hydrocarbures paraffiniques et naphténiques chez les animaux supérieurs. I. Rétention des paraffines (normal, cyclo et ramifiées) chez le rat

Apparent retention of aliphatic hydrocarbons in the Rat is studied. Animals weighing 150 g are put into metabolism cages and given a 15 mg dose of one of the paraffins studied in solution in the peanut oil of the ration. There is no urinary excretion, and fecal elimination is low (5-10%), as long as the carbon chain stays below 24 atoms. Real retention is measured on animals recieving a single dose of 15 mg in the same conditions. Branched paraffins give only very low residual amounts 15 days after ingestion, while n-paraffins and cyclo-paraffins remain unchanged in the carcass, as long as the number of carbon atoms is not greater than 20; the proportion of the latter paraffins is about 8% of the dose ingested (5 days as 21 days after intake). Beyond C 20, real retention decreases rapidly to zero.

A fast and simple high-performance liquid chromatographic assay for aryl hydrocarbon hydroxylase.

A simple high-performance liquid chromatographic method using fluorescence detection of the remaining substrate is described for the determination of benzo[alpha]pyrene hydroxylase activity. This assay is far simpler than the previous ones, as it does not require extraction or centrifugation and the measurement occurs directly after dilution of the total incubation medium. The aryl hydrocarbon hydroxylase (AHH) activities in rat liver microsomes are in agreement with those obtained by radioactive assays. Moreover, this assay allows the routine determination of the AHH activity in animal tissues.

Electrospray ionization-mass spectrometry as a tool for characterization of glutathione S-transferase isozymes.

Reversed-phase high-performance liquid chromatography coupled to electrospray ionization-mass spectrometry (ESI-MS) was used for the first time to determine the molecular masses of nine rat liver cytosolic glutathione S-transferase (GST) subunits. The precision of the measurements was +/- 3-4 mass units which, in practice, allowed discrimination between monomers differing by more than 8 Da. Mass accuracy was improved by replicates in the measurements. Comparison of experimental values to cDNA or protein-deduced data available reveals slight differences. N-terminal sequence analyses and interstrain mass comparisons tend to show that primary structures of liver cytosolic GSTs are well conserved from Sprague-Dawley to Wistar rats. Moreover, ESI-MS analysis enabled identification of two minor additional subunits present in both strains, one of which belongs to the mu-class. In addition to rapid and accurate mass determination of GST monomers, and direct determinations achieved on heterodimeric forms, this technique provides precise information on minor structural differences or modifications of these proteins. As such, it constitutes a useful tool for rapid characterization of purified GSTs in comparative studies.

Metabolism of n-alkanes and their incorporation into lipids in the rainbow trout.

The metabolic utilization by fish of n-alkanes was investigated in Salmo gairdneri R. receiving per os a single dose of [14C]heptadecane. This alkane was largely absorbed and radioactivity in the intestinal content was entirely in heptadecane. No elimination of the hydrocarbon was observed in urine. One week after dosing, ca. 50% of the ingested radioactivity was stored in the carcass and two-thirds of this radioactivity was due to heptadecane. The highest concentration of 14C was found in adipose tissue and liver, in which 77 and 22%, respectively, of the radioactivity originated from hydrocarbon. The rest was omega-oxidized to heptadecanoic acid. This fatty acid was incorporated into neutral lipids (mainly as free fatty acid) and phospholipids.

Urinary metabolites of dodecylcyclohexane in Salmo gairdneri: evidence of aromatization and taurine conjugation in trout.

1. The urinary metabolites of 3H-dodecylcyclohexane were investigated in rainbow trout, Salmo gairdneri R. after a single intragastric dose. In 72 h, 14% of the ingested radioactivity was excreted in urine. 2. Cyclohexylacetic acid, 1-hydroxy-, 3-hydroxy- and 4-hydroxy-cyclohexylacetic acids were present in the unconjugated fraction. 3. In the glucuronide fraction (1.2% dose) labelled aglycones were cyclohexylacetic acid and phenylacetic acid. 4. More than 30% of the urinary 3H was present as phenylacetic and cyclohexylacetic acids conjugated with taurine.