Jacques Tulliez

Flavonoids and the inhibition of PKC and PI 3-kinase

Flavonoids provide a large number of interesting natural compounds that are consumed daily and exhibit more or less potent and selective effects on some signaling enzymes as well as on the growth and proliferation of certain malignant cells in vitro. Among the identified signal transducers, phosphoinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC) are now considered key players in many cellular responses including cell multiplication, apoptosis, and transformation. Despite their lack of strict specificity, some flavonoids provide valuable bases for the design of analogues that could be used to specifically block particular isoforms of PI 3-kinase or PKC and their downstream-dependent cellular responses.

Mechanism of Sulforaphane-Induced Cell Cycle Arrest and Apoptosis in Human Colon Cancer Cells

Sulforaphane (SFN) is a natural micronutrient found in cruciferous vegetables that has been shown to possess antitumoral properties in carcinogen-treated rats. In vitro, SFN regulates phase II enzymes, cell cycle, and apoptosis. In the present study, we investigated the relationship between SFN induction of apoptosis and cell cycle arrest in HT29 human colon carcinoma cells. In previously published data, a significant increase in the G2/M phase of the cell cycle has been observed in SFN-treated cells that was associated with increased cyclin B1 protein levels. In the present study, our results show that SFN induced p21 expression. Moreover, preincubation of HT29 cells with roscovitine, a specific cdc2 kinase inhibitor, blocked the G2/M phase accumulation of HT29 cells treated with SFN and abolished its apoptotic effect (22.2 ± 4 of floating cells in SFN-treated cells vs. 6.55 ± 2 in cells treated with both SFN and roscovitine). These results suggest that the cdc2 kinase could be a key target for SFN in the regulation of G2/M block and apoptosis. Moreover, in SFN-treated cells the retinoblastoma tumor suppressor protein (Rb) is highly phosphorylated. Inhibition of the cdc2 kinase by roscovitine did not change the phosphorylation status of Rb in SFN-treated cells, suggesting that this cyclin-dependent kinase may not be involved. In our study, we did not observe any significant change in the proteasomal activity between control and SFN-treated cells. Moreover, inhibition of proteasomal activity through the use of MG132 diminished SFN-induced HT29 cell death, suggesting that the apoptotic effect of SFN requires a functional proteasome-dependent degradation system. In summary, we have elucidated part of the mechanism of action of SFN in the concomitant regulation of intestinal cell growth and death.

SELECTIVE CYTOSTATIC AND CYTOTOXIC EFFECTS OF GLUCOSINOLATES HYDROLYSIS PRODUCTS ON HUMAN COLON CANCER CELLS IN VITRO

Glucosinolates hydrolysis products are attracting increasing attention since many studies have suggested that they may be involved in the anticarcinogenic property of cruciferous vegetables. In this study, we show that diindolylmethane (DIM) and sulforaphane, produced during the hydrolysis of glucobrassicin and glucoraphanin, respectively, exert a dose-dependent cytotoxicity on human colon adenocarcinoma HT29 cells. Moreover, these products are able to inhibit quiescent cells to re-enter the cell cycle. Interestingly, our results clearly show that low doses of DIM and sulforaphane, although very effective on undifferentiated intestinal HT29 cells, do not affect the viability of the differentiated CaCo2 cells. The reversibility of their effects has also been tested and is discussed.

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.

Metabolites of the naphthenic hydrocarbon dodecylcyclohexane in rainbow trout liver and their incorporation into lipids

Rainbow trout (Salmo gairdneri R.) were force-fed 0.1 mCi (0.5 mg)3H-dodecylcyclohexane and sacrificed at 12, 24, 48 hr and 1 week after feeding in order to study the metabolic utilization of a naphthenic hydrocarbon. One week after dosing, approximately 25% of the ingested radioactivity was stored in the carcass and 3/4 of this radioactivity was due to unchanged hydrocarbon. In the liver, only 25% of the3H present after one week was associated with hydrocarbon. The incorporation of radioactivity in hepatic lipids 24 hr after ingestion of3H-dodecylcyclohexane revealed that radioactivity was equally incorporated into the phospholipids and neutral lipids. In neutral lipids, the free fatty acids were the most labeled fraction, whereas in phospholipids the greater deposition of radioactivity occurred in phosphatidyl choline. The major biotransformation products were characterized in the liver by thin layer chromatography, radio-gas chromatography and mass spectrometric analysis. Four metabolites, resulting from the oxidation of the alkyl chain or of the cyclohexane ring were identified; namely, 3-dodecylcyclohexanol, 4-dodecylcyclohexanol, cyclohexyldodecane-2-ol, and cyclohexyldodecanoic acid. This latter metabolite accounted for 30% of the liver radioactivity 24 hr after dosing. The toxicological relevance of this pathway is discussed.

Growth reduction in trout induced by naphthenic and isoprenoid hydrocarbons (dodecylcyclohexane and pristane)

Rainbow trout were fed a diet containing 1% dodecylcyclohexane or pristane for 9 weeks. Feed intake was recorded daily and weight gain every 3 weeks. These animals were compared with fish receiving a hydrocarbon-free diet (groups fed ad libitum and pair-fed groups for which the ration provided was the amount of food consumed by the hydrocarbon-contaminated fish, the day before). The total food ingested by the pristane and dodecylcyclohexane groups amounted to 66 and 70%, respectively, of that eaten by the controls. The final mean weight of the controls was twofold their initial weight. The average weight gain of the dodecylcyclohexane and pristane groups was 37 and 25%. During the same period the fish of the pair-fed groups gained approximately 70% of their initial weight. Significant effects of hydrocarbon consumption on food conversion factors, viscerosomatic index, hepatosomatic index, and liver lipid concentration were observed. The results are discussed in relation to the possible causes of such changes.

Chronic ingestion of saturated hydrocarbons by rainbow trout: Influence of dodecylcyclohexane and pristane on lipid metabolism

The influence of alkanes on total lipid content and fatty acid composition of trout was investigated inSalmo gairdneri Richardson fed for 9 months or 10 months a diet containing 1% of dodecylcyclohexane or pristane. Results were compared with a control group fed a standard diet. Total lipids in the whole carcass and muscle were lower in the two test groups than in the control group.Cyclohexyldodecanoic acid was detected among the fatty acids of carcass, fat, and liver of trout receiving dodecylcyclohexane; the highest proportion of this unusual cyclic fatty acid was found in the liver.During the 2 months depuration period, one-half of the trout in each group were fed a hydrocarbon-free diet, and the others were starved. After starvation, both controls and dodecylcyclohexane fish had lost about 20% of the initial stored lipids whereas the loss in the pristane group was 54%. When the trout were fed the standard diet, an increase of lipid content was observed in dodecylcyclohexane and control groups (×l.3) whereas no variation occurred in the pristane-fed trout.

Optimization of the separation of β-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.

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.

Hydroxylation of pristane by isolated hepatocytes of rainbow trout: a comparison with in vivo metabolism and biotransformation by liver microsomes

Abstract In vitro models may represent a useful approach for studying the biotransformation of xenobiotics in animals. However, the value of such models can only be established by relating in vitro results with in vivo data. In fish, several investigators have compared xenobiotic metabolism in vivo with xenobiotic metabolism in subcellular liver fractions. In contrast, the biotransformation of chemicals in isolated fish hepatocytes has been scarcely studied. The objective of this work carried out in rainbow trout was to compare the metabolism of a widespread branched alkane (pristane) in isolated hepatocytes with metabolism in vivo and in liver microsomal fractions. The incubation of hepatocytes with [3H] pristane led to pristanol, pristane-diol and pristanic acid, while pristanol appeared to be the only product in microsomal preparations. In the in vivo experiment, liver contained significant amounts of pristanol and pristanic acid whereas conjugates of pristanol and pristane-diol were found in bile. Thus, the metabolism of pristane in hepatocytes suspension correlates with in vivo biotransformation better than does metabolism in microsomes.