Patrick Rouimi

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.

Characterization of hepatic and extrahepatic glutathione S-transferases in rainbow trout (Oncorhynchus mykiss) and their induction by 3,3′,4,4′-tetrachlorobiphenyl

Liver, kidney, gill and olfactory epithelium cytosolic fractions of rainbow trout (Oncorhynchus mykiss) were examined for glutathione S-transferase (GST) contents. Proteins retained on a glutathione (GSH)-affinity matrix were separated as monomers by reversed-phase HPLC and characterized by immunoblotting, mass spectrometry and partial amino acid sequence. For each organ concerned, a specific pattern of these proteins was determined and appeared similar for liver and kidney on one hand, and for gill and olfactory epithelium on the other hand. It was confirmed that the prominent hepatic GST is a class π enzyme, also constitutively expressed as a major isoform in the four organs studied. Moreover, a class π variant and two new class μ GST subunits were characterized in minor fractions. An unknown protein, which was found major in gills and olfactory epithelium, exhibited some characteristics of class θ GSTs. Occurrence of possible GSH-adduct formation observed on two distinct monomers in specific experimental conditions is discussed. These results and methods were used to investigate the effect of 3,3′,4,4′-tetrachlorobiphenyl (TCB), a polychlorinated biphenyl (PCB), on GST expression in trout liver. From HPLC-profiling, significant co-induction of the major class π and the two minor class μ GST subunits was observed in trout after waterborne exposure to TCB which was followed by a slight increase in 1-chloro-2,4-dinitrobenzene (CDNB) activity. The present work allows qualitative evaluation of the specific detoxification potential of rainbow trout. The use of HPLC-profiling of GSTs as a possible tool for the biomonitoring of polluted aquatic environment is suggested.

Comparative study of bisphenol A and its analogue bisphenol S on human hepatic cells: a focus on their potential involvement in nonalcoholic fatty liver disease.

For several decades, people have been in contact with bisphenol A (BPA) primarily through their diet. Nowadays it is gradually replaced by an analogue, bisphenol S (BPS). In this study, we compared the effects of these two bisphenols in parallel with the positive control diethylstilbestrol (DES) on different hepatocyte cell lines. Using a cellular impedance system we have shown that BPS is less cytotoxic than BPA in acute and chronic conditions. We have also demonstrated that, contrary to BPA, BPS is not able to induce an increase in intracellular lipid and does not activate the PXR receptor which is known to be involved in part, in this process. In parallel, it failed to modulate the expression of CYP3A4 and CYP2B6, the drug transporter ABCB1 and other lipid metabolism genes (FASN, PLIN). However, it appears to have a weak effect on GSTA4 protein expression and on the Erk1/2 pathway. In conclusion, in contrast to BPA, BPS does not appear to induce the metabolic syndrome that may lead to non-alcoholic fatty liver disease (NAFLD), in vitro. Although we have to pay special attention to BPS, its use could be less dangerous concerning this toxicological endpoint for human health.

Impacts of low doses of pesticide mixtures on liver cell defence systems.

Low amounts of residual pesticides are present in the environment, often as mixtures of chemicals which contaminate drinking water and food, being a source of chronic exposure for humans and a growing matter of concern in public health policy. Despite of the needs and growing investigation, little is known about the impact of low doses and mixtures of these chemicals on human health. The purpose of this study was to enlighten if modifications of liver cell metabolic- and/or defence-related capacities could occur under such exposures. In vitro perturbations of several metabolic, stress and survival pathways in human and mice cultured hepatocytes and liver cells were evaluated under exposure to low doses of single molecules or equimolecular combinations of the three pesticides, atrazine, chlorpyrifos and endosulfan. Mainly phases I and II enzymes of detoxification were found modulated, together with apoptotic process deregulation. Hence, CYP3A4 and CYP3A11 were upregulated in primary cultured human and mouse hepatocytes, respectively. These inductions were correlated to an anti-apoptotic process (increased Bcl-xL/Bax ratio, inhibition of the PARP protein cleavage). Such disturbances in pathways involved in cell protection may possibly account for initiation of pathologies or decrease in drugs efficiency in humans exposed to multiple environmental contaminants.

Analysis and characterization of glutathione S-transferase subunits from wheat (Triticum aestivum L.)

Abstract The total subunit complement of glutathione S -transferases (GSTs) of wheat ( Triticum aestivum L.) was isolated by glutathione-agarose affinity chromatography. The proteins bound to the affinity column were analysed by SDS-PAGE, which separated six to seven protein bands. Reverse phase-HPLC analysis revealed six major and about 15 minor peaks. The amounts of three major components were increased upon treatment with the herbicide safener naphthalic anhydride. The six major components were isolated. Their relative molecular masses, determined by electrospray ionization mass spectrometry, were between 23 140±2 and 24 957±3 Da. The sequences of the 20 N-terminal amino acids were also determined, and revealed strong similarities with GST subunits from various monocotyledonous plants. Wheat GST subunits can be assigned to two groups: constitutive subunits, with identical N-termini and molecular masses around 23.2 kDa, and constitutive and inducible subunits, with some differences in N-terminal sequences, and molecular masses around 24.9 kDa.

Obesogen effects after perinatal exposure of 4,4′-sulfonyldiphenol (Bisphenol S) in C57BL/6 mice

Bisphenol A were removed from consumer products and replaced by chemical substitutes such as Bisphenol S (BPS). Based on their structural similarity, BPS may be obesogen like Bisphenol A in mice. Our objective was to determine the impact of BPS on lipid homeostasis in C57Bl/6 mice after perinatal and chronic exposure. Pregnant mice were exposed to BPS via the drinking water (0.2; 1.5; 50μg/kg bw/d). Treatment began at gestational day 0 and continued in offspring up to 23-weeks old. Then, offspring mice were fed with a standard or high fat diet. The body weight, food consumption, fat mass and energy expenditure were measured. A lipid load test was performed to check the postprandial triglyceridemia. Plasma parameters and mRNA gene expression in adipose tissues were also analysed. BPS induced overweight in male mice offspring fed with a HFD at the two highest doses. There was no change in food intake and energy expenditure. The overweight was correlated to the fat mass, hyperinsulinemia and hyperleptinemia. The plasma triglyceride clearance was significantly increased with BPS and tyloxapol(®) (triglyceride clearance inhibitor) reversed this phenomenon. BPS induced alteration in mRNA expression of marker genes involved in adipose tissue homeostasis: hormone sensitive lipase, PPARγ, insulin receptor, SOCS3 and adiponectin. This is the first time that BPS is described as obesogenic at low doses and after perinatal and chronic exposure in male mice. BPS potentiated the obesity induced by a HFD by inducing the lipid storage linked to faster lipid plasma clearance.

Purification and characterisation of glutathione S-transferases from the freshwater clam Corbicula fluminea (Müller).

Glutathione S-transferases (GSTs) are involved in the phase II detoxification metabolism. To provide a molecular basis for their use as biomarkers of pollution, cytosolic GSTs from the freshwater clam Corbicula fluminea have been purified by glutathione-Sepharose affinity chromatography, anion-exchange chromatography (AEC) and reversed-phase (RP) HPLC. SDS-PAGE of visceral mass (VM) affinity-purified extracts revealed four subunits with apparent molecular masses (MW) of 30.2, 29.2, 28.5 and 27.2 kDa. Analysis by non-denaturing PAGE revealed three acidic dimeric proteins with apparent MW of 64, 55 and 45 kDa, named GSTc1, GSTc2 and GSTc3, respectively, based on their elution order by AEC. Only GSTc2 and GSTc3 exhibited GST activity towards 1-chloro-2,4-dinitrobenzene. A tissue-specific subunit pattern was obtained by RP-HPLC of affinity-purified extracts from VM and gills (GI): three major peaks were resolved, one of which was common to both tissues. MW of each VM subunit was determined by electrospray ionisation-mass spectrometry: 23602+/-1 Da for the major subunit and 23289+/-1 Da for the minor ones. Immunoblot analysis revealed all subunits from both tissues were related to the Pi-class GSTs. In addition, minor VM subunits were slightly related to the Mu-class ones. The interest of such molecular studies in biomonitoring programs is discussed.

Increased expression of prostaglandin reductase 1 in hepatocellular carcinomas from clinical cases and experimental tumors in rats

To identify novel tumor-associated proteins, we analyzed the protein expression patterns from experimental hepatocellular carcinoma (HCC) that were induced using hepatocarcinogenesis models in rats. Rats were subjected to two previously described protocols of hepatocarcinogenesis using diethylnitrosamine as a carcinogen: the alternative Solt-Farber (aS&F) protocol, which induces HCC within 9 months, and Schiffers model, which induces cirrhosis and multifocal HCC within 18 weeks. The patterns of protein expression from tumors and normal liver tissue were examined by SDS-PAGE and the bands identified at 33-34 kDa were analyzed by mass spectrometry. The prostaglandin reductase 1 (PTGR1) showed the highest number of peptides, with a confidence of level >99%. The increased expression of PTGR1 in tumors was confirmed in these two models by Western blotting and by increase in alkenal/one oxidoreductase activity (25-fold higher than normal liver). In addition, the gene expression level of Ptgr1, as measured by qRT-PCR, was increased during cancer development in a time-dependent manner (200-fold higher than normal liver). Furthermore, PTGR1 was detected in the cytoplasm of neoplastic cells in rat tumors and in 12 human HCC cases by immunohistochemistry. These analyses were performed by comparing the expression of PTGR1 to that of two well-known markers of hepatocarcinoma, Glutathione S-transferase pi 1 (GSTP1) in rats and glypican-3 in humans. The increased expression and activity of PTGR1 in liver carcinogenesis encourage further research aimed at understanding the metabolic role of PTGR1 in HCC and its potential application for human cancer diagnosis and treatment.

Glutathione-S-transferase subunits pattern in rainbow trout isolated hepatocytes

Abstract Cytosolic GSTs are homo- or heterodimeric enzymes which have been assigned to four separate families designated Alpha, Mu, Pi and Theta on the basis of primary structure, substrate specificity and immunological properties. In fish, there are several cytosolic forms of the enzyme. The objective of the present work was to compare the GST subunit patterns in cytosolic fractions from liver and freshly isolated hepatocytes. Trout hepatocytes were isolated from male immature rainbow trout ( Oncorhynchus mykiss ) weighing 250–300 g, using a collagenase perfusion procedure. Cytosolic fractions were obtained from sonicated hepatocytes or homogenized liver by differential centrifugation. GSTs were purified by a glutathione affinity chromatography and the activity of retained fractions was detected with chlorodinitrobenzene as substrate. The GST subunits were separated and characterized by a combination of HPLC and electrospray-ionization mass spectrometry analysis. HPLC analysis of affinity fractions from freshly isolated hepatocytes exhibited five GST subunits with highly reproducible retention times of 14, 19, 21, 25 and 26 min respectively. This profile was qualitatively identical to that obtained from the cytosol prepared from the liver. Quantitatively, the major form in isolated cells as well as in the liver corresponded to the peak eluting at 14 min and could correspond to a Pi-class GST subunit. The less polar peaks, eluting at 25 and 26 min are minor in the liver cytosol and often present as traces in hepatocytes.

Tumor promoting and co-carcinogenic effects in medium-term rat hepatocarcinogenesis are not modified by co-administration of 12 pesticides in mixture at acceptable daily intake.

The purpose of this investigation was to evaluate the possible influence of a mixture of pesticides on medium-term carcinogenesis using improved hepatocarcinogenesis protocols. We performed a 12 commercially available pesticides combination with alachlor, atrazine, carbofuran, chlorpyrifos, diazinon, dicofol, endosulfan, iprodione, mancozeb, maneb, procymidone and rotenone. The mixture was given at 1-fold and 10-fold the acceptable daily intake (ADI) level in a set of Solt-Farber-derived protocols involving diethylnitrosamine, 2-acetylaminofluorene treatments and a partial hepatectomy. Co-carcinogenic effect and promoting activity were evaluated using gamma-glutamyl transpeptidase (GGT) positive altered hepatocyte foci, as well, protein and mRNA levels of glutathione S-transferase P (GSTP) in liver extracts as molecular biomarkers of carcinogenic effects. The pesticide treatments when compared to vehicle treatments always produced the same number of hepatocyte lesions and an equal GSTP expression on liver extracts independently of carcinogenic-protocol utilized. On this base, we concluded that the pesticide mixture evaluated in this report does not have tumor promoting activity or co-carcinogenic effect in the rat medium-term liver carcinogenesis. Altogether these data contribute to the confidence that the ADI represents a safe intake level to mixture of pesticides at dietary exposure.