In vitro evaluation of anticancer effects of different probiotic strains on HCT-116 cell line.

AIM: Since the evolution of man, microbes are associated with humans, playing a vital role in the maintenance of good health. However, an imbalance in the gut microbial ecosystem is associated with several diseases including colorectal cancer (CRC). The supplementation with probiotics has been proven to be beneficial in improving CRC. In this study, we have evaluated the anticancer effects of 11 probiotic strains on human colorectal carcinoma cell line (HCT-116). METHODS AND RESULTS: In this study, HCT-116 cells were treated with various concentrations (0·5, 5, 10, 20 and 200 million CFU per ml) of probiotic strains. The viability was analysed using a MTT assay and IC50 values were determined. Besides this, we evaluated the expression of multiple genes involved in the apoptosis and stress tolerance by real-time PCR. Lactobacillus reuteri (UBLRu-87), Saccharomyces boulardii (Unique-28), Bacillus clausii (UBBC-07), Bacillus coagulans (Unique-IS2), Streptococcus salivarius (UBSS-01), Lactobacillus fermentum (UBLF-31), Lactobacillus salivarius (UBLS-22), Bifidobacterium bifidum (UBBB-55) and Lactobacillus plantarum (UBLP-40) exhibited potent cytotoxicity on HCT 116 cells. Furthermore, UBLF-31 and Unique-28 induced the expression of CJUN, CFOS and CASP-9, and downregulated the expression of BCL6. UBLRu-87 and UBBB-55 induced the expression of CJUN, CFOS and CASP-9 but not BCL-6. UBLP-40, UBBC-07, UBLS-22, and Unique-IS2 induced the expression of CJUN and CASP-9 and downregulated the expression of BCL-6. CONCLUSION: These studies indicate the anticancer effects of selected probiotic strains by inducing apoptosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The probiotic strains with the anticancer effects identified in this study can be proposed as potential candidates in the treatment of CRCs.

Anti-angiogenic effect of chebulagic acid involves inhibition of the VEGFR2- and GSK-3ß-dependent signaling pathways.

Inhibition of angiogenesis is a useful strategy to prevent cancer growth by targeting new vessels that grow to nourish actively proliferating tumor cells. Endothelial cells can use a number of different pathways to cause angiogenesis, and each step in these pathways can be targeted. The use of multi-targeted drugs is gaining much importance in this scenario. Our previous results have shown that chebulagic acid (a benzopyran tannin present in the fruits of Terminalia chebula) has anti-angiogenic properties. Thus, this study was designed to examine the molecular mechanism for the anti-angiogenic effects of chebulagic acid. Results from our investigations using molecular docking studies and human umbilical vein endothelial cells in culture suggested that chebulagic acid inhibits both GSK-3ß-dependent ß-catenin phosphorylation (an important mediator of VE-cadherin-ß-catenin signaling) and VEGFR2 phosphorylation, which is an important step in VEGF signaling. Chebulagic acid inhibits angiogenesis by blocking both the VEGF-VEGFR2 complex and cell-cell contact dependent downstream signaling pathways.

Effect of 15-lipoxygenase metabolites on angiogenesis: 15(S)-HPETE is angiostatic and 15(S)-HETE is angiogenic.

OBJECTIVE: 15(S)-Hydroxyeicosatetraenoic acid [15(S)-HETE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] are the products of arachidonic acid formed in the 15-lipoxygenase pathway. They have opposing effects on the inflammatory process. The present study was designed to examine the role of these metabolites on angiogenesis, which is critically associated with inflammatory conditions. METHODS: Chick chorio-allantoic membrane (CAM), rat aortic rings and human umbilical vein endothelial cells (HUVECs) in culture were used to study the effect of 15(S)-HETE and 15(S)-HPETE on angiogenesis. Biochemical markers of angiogenesis were analysed by ELISA. RESULTS: 15(S)-HETE increased vessel density in chick CAM, induced sprouting in rat aortic rings and increased endothelial cell-cell contact and formation of tubular network-like structures in HUVECs. Furthermore, it up-regulated the expression of CD31, E-selectin and vascular endothelial growth factor (VEGF) in HUVECs, indicating its pro-angiogenic effect. 15(S)-HPETE, on the other hand, decreased vessel density in chick CAM, down-regulated the expression of E-selectin (<35 %), VEGF (<90 %) and CD31 (<50 %) and did not produce sprouting in aortic rings, suggesting an anti-angiogenic property. 15(S)-HETE-mediated up-regulation of CD 31 and VEGF was reversed by treatment with 15(S)-HPETE. CONCLUSION: These results indicate the divergent effects of hydroxy and hydroperoxy products of 15-LOX on angiogenesis, highlighting the role of these products in the co-dependence of inflammation and angiogenesis.

Minimum MD simulation length required to achieve reliable results in free energy perturbation calculations: case study of relative binding free energies of fructose-1,6-bisphosphatase inhibitors.

In an attempt to establish the criteria for the length of simulation to achieve the desired convergence of free energy calculations, two studies were carried out on chosen complexes of FBPase-AMP mimics. Calculations were performed for varied length of simulations and for different starting configurations using both conventional- and QM/MM-FEP methods. The results demonstrate that for small perturbations, 1248 ps simulation time could be regarded a reasonable yardstick to achieve convergence of the results. As the simulation time is extended, the errors associated with free energy calculations also gradually tapers off. Moreover, when starting the simulation from different initial configurations of the systems, the results are not changed significantly, when performed for 1248 ps. This study carried on FBPase-AMP mimics corroborates well with our previous successful demonstration of requirement of simulation time for solvation studies, both by conventional and ab initio FEP. The establishment of aforementioned criteria of simulation length serves a useful benchmark in drug design efforts using FEP methodologies, to draw a meaningful and unequivocal conclusion.

CoMFA and CoMSIA studies on 5-hydroxyindole-3-carboxylate derivatives as 5-lipoxygenase inhibitors: generation of homology model and docking studies.

In this study, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on a series of 2-substituted 5-hydroxyindole-3-carboxylate derivatives as potent 5-LOX inhibitors with IC(50) values ranging from 0.031 to 13.4 µM. Two datasets of same molecules were prepared with two different partial atomic charges; one with Gasteiger-Huckel and another with the ESPFIT charges obtained from the gaussian package. CoMFA and CoMSIA models were generated for both the datasets and the results were analysed. With regard to the non-cross validated r(2) values (r(ncv)(2)) and cross-validated q(2) values (q(cv)(2)) of the resulting QSAR models, the dataset with ESPFIT charges yielded higher values; hence it was further used in the study. The CoMFA and CoMSIA models have been further validated for their stability and robustness using group validation and bootstrapping techniques and for their predictive abilities using an external test set of ten compounds. The predictive power of the CoMSIA model was higher than the CoMFA model, the high predictive r(2) values of the test set reveals that the models prove to be useful tools for activity prediction of newly designed 5-LOX inhibitors. The ESPFIT-derived charges yielded better models than those based on charges calculated from Gasteiger-Huckel charges. We generated a homology model for human 5-LOX and identified the key residues at the binding site. The 3D-QSAR models were compared with the interactions at the active site to further elucidate the accuracy of the models. The data generated from 3D-QSAR study was used to design potential 5-LOX inhibitors.

High-throughput screening assays for cyclooxygenase-2 and 5-lipoxygenase, the targets for inflammatory disorders.

High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify 'hit' molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer antiinflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.

Pharmacophore modeling and virtual screening for designing potential 5-lipoxygenase inhibitors.

Inhibitors of the 5-Lipoxygenase (5-LOX) pathway have a therapeutic potential in a variety of inflammatory disorders such as asthma. In this study, chemical feature based pharmacophore models of inhibitors of 5-LOX have been developed with the aid of HipHop and HypoGen modules within Catalyst program package. The best quantitative pharmacophore model, Hypo1, which has the highest correlation coefficient (0.97), consists of two hydrogen-bond acceptors, one hydrophobic feature and one ring aromatic feature. Hypo1 was further validated by test set and cross validation method. The application of the model shows great success in predicting the activities of 65 known 5-LOX inhibitors in our test set with a correlation coefficient of 0.85 with a cross validation of 95% confidence level, proving that the model is reliable in identifying structurally diverse compounds for inhibitory activity against 5-LOX. Furthermore, Hypo1 was used as a 3D query for screening Maybridge and NCI databases within catalyst and also drug like compounds obtained from Enamine Ltd, which follow Lipinski's rule of five. The hit compounds were subsequently subjected to filtering by docking and visualization, to identify the potential lead molecules. Finally 5 potential lead compounds, identified in the above process, were evaluated for their inhibitory activities. These studies resulted in the identification of two compounds with potent inhibition of 5-LOX activity with IC(50) of 14 microM and 35 microM, respectively. These studies thus validate the pharmacophore model generated and suggest the usefulness of the model in screening of various small molecule libraries and identification of potential lead compounds for 5-LOX inhibition.

Co-administration of C-Phycocyanin ameliorates thioacetamide-induced hepatic encephalopathy in Wistar rats.

Fulminant hepatic failure (FHF) is a condition with a sudden onset of necrosis followed by degeneration of hepatocytes, without any previously established liver disease, generally occurring within hours or days. FHF is associated with a wide spectrum of neuropsychiatric alterations ranging from stupor to coma, culminating in death. In the present study FHF was induced in rats by the administration of thioacetamide (TAA). Oxidative stress is thought to play a prominent role in the pathophysiology of cerebral changes during FHF leading to the assumption that antioxidants might offer protection. Hence, in the present study the protective effect of C-Phycocyanin (C-PC), a natural antioxidant, was evaluated on TAA-induced tissue damage. C-Phycocyanin was administered intraperitoneally twice at 24 h interval (50 mg/kg body weight) along with the hepatotoxin TAA (300 mg/kg body weight). The animals were sacrificed 18 h after the second injection of TAA treatment and various biochemical parameters were analysed in liver, serum and brain tissues. These studies revealed significant prevention of TAA-induced liver damage by C-PC, as evidenced by a) increase in survival rate; b) the prevention of leakage of liver enzymes (AAT and AST) and ammonia into serum; c) increase in prothrombin time and d) liver histopathology. Ultrastructural studies of astrocytes of different regions of brain clearly showed a decrease in edema after C-PC treatment. TAA-induced histopathological lesions in different regions of the brain namely cerebral cortex, cerebellum and pons medulla were significantly reduced by the co-administration of C-PC with TAA. Further C-PC treatment resulted in a) decrease in the levels of tryptophan and markers of lipid peroxidation and b) elevation in the activity levels of catalase, glutathione peroxidase in different regions of brain. These studies reveal the potential of C-PC in ameliorating TAA-induced hepatic encephalopathy by improving antioxidant defenses.

Metabolism of arachidonic acid in sheep uterus: in vitro studies.

Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins - PGF2alpha and prostacyclin derivative 6-keto PGF1alpha. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.

Phospholipid and cholesterol alterations accompany structural disarray in myelin membrane of rats with hepatic encephalopathy induced by thioacetamide.

Fulminant hepatic failure is often associated with a wide range of neurological symptoms which are collectively referred to as hepatic encephalopathy. Fulminant hepatic failure with associated hepatic encephalopathy has a poor prognosis with the currently available sure treatment being only liver transplantation. This is largely owing to the lack of understanding of critical factors involved in the etiology of the condition. Lipid changes have been implicated in cerebral derangements characteristic of hepatic encephalopathy. About 79% of the brain lipid is concentrated in the myelin fraction where they play an important role in ion balance and conduction of nerve impulses. Hence, in the present study we aimed to investigate changes in myelin lipid composition and structure. Myelin was isolated by sucrose density gradient centrifugation from cerebral cortex of male Wistar rats (250-300 g body weight) treated with 300 mg/kg body weight thioacetamide administered twice at 24h interval to induce hepatic encephalopathy. Significant decrease was observed in the cholesterol and phospholipids content of myelin from treated rats. Sphingomyelin, phosphatidylserine and phosphatidylethanolamine content also decreased significantly following 18 h of thioacetamide administration. However, phosphatidylcholine levels remained unaltered. Transmission electron microscopic observation of myelin membrane from cerebral cortex sections showed considerable disorganization in myelin structure. Increase in malondialdehyde levels precede lipid changes leading to the speculation that oxidative damage may be the critical factor leading to decrease in the anionic phospholipids. Changes in myelin were evident only in later stages of hepatic encephalopathy indicating that myelin alteration may not play a role in early stages of hepatic encephalopathy. Nevertheless, myelin alteration may have a crucial role to play in various psycho-motor alterations during later stages of hepatic encephalopathy.

Differential effects of 15-HPETE and 15-HETE on BHK-21 cell proliferation and macromolecular composition.

Arachidonate and/or linoleate metabolites have been implicated in modulating cell growth, replication and cell transformations. In studies with BHK-21 cells, we found lipoxygenase and cyclooxygenase inhibitors (NDGA and indomethacin, respectively) to be antiproliferative. Studies on the metabolism of arachidonic acid in BHK-21 cells have demonstrated that prostaglandin D2 is the major cyclooxygenase product, and 15-hydroxyeicosatetraenoic acid (15-HETE) is the major lipoxygenase product. Addition of D2 showed a significant decrease in the BHK-21 cell number showing antiproliferative action. Addition of lipoxygenase products, on the other hand, showed differential effects in that 15-HPETE decreased the cell number while 15-HETE increased. NDGA and 15-HPETE decreased DNA, RNA and protein contents, while 15-HETE significantly increased them. 5-HPETE and 5-HETE also showed similar results but were less potent than 15-H(P)ETEs. The differential effects of 15-HPETE and 15-HETE could be due to the generation of free radicals by the hydroperoxide and mitogenic response by hydroxide.

Differential formation of octadecadienoic acid and octadecatrienoic acid products in control and injured/infected potato tubers.

Lipoxygenases in plants have been implicated in the activation of defense responses against injury/infection. Pathogen-derived polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid and their metabolites have been shown to elicit defense responses against pathogen infection in plants. However, not much is known about the role of host-derived fatty acids and their metabolites in plant defense responses. In this study, isolation and characterisation of endogenous lipoxygenase metabolites formed in potato tubers in response to injury/infection was undertaken. While 9-hydroperoxyoctadecadienoic acid (9-HPODE), derived from octadecdienoic acid (linoleic acid) is the major lipoxygenase product formed in control potato tubers, 9-hydroperoxyoctadecatrienoic acid (9-HPOTrE), derived from octadecatrienoic acid (alpha-linolenic acid) is the major lipoxygenase product formed in potato tubers in response to injury or infection with Rhizoctonia bataticola. As a result, the relative ratio of 9-HPODE to 9-HPOTrE showed a shift from 4:1 in control to 1:2 and 1:4.5 in injured and infected potato tubers respectively. From this study, it is proposed that lipoxygenase metabolites of octadecadienoic acid may be involved in physiological responses under control conditions, while octadecatrienoic acid metabolites are mediating the defense responses. This forms the first report on the differential formation of endogenous lipoxygenase products in potato tubers under control and stress conditions.

Anti-proliferative and apoptotic effects of celecoxib on human chronic myeloid leukemia in vitro.

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is the only non-steroidal anti-inflammatory drug so far which has been approved by the FDA for adjuvant treatment of patients with familial adenomatous polyposis. The molecular mechanism responsible for the anti-cancer effects of celecoxib is not fully understood. There is little data on the potential role of COX-2 in lymphoma pathogenesis. In view of the reported induction of apoptosis in cancer cells by cyclooxygenase-2 inhibitors, the present study is undertaken to test the effect of celecoxib on human chronic myeloid leukemia cell line, K562 and other hematopoietic cancer cell lines like Jurkat (human T lymphocytes), HL60 (human promyelocytic leukemia) and U937 (human macrophage). Treatment of these cells with celecoxib (10-100 microM) dose-dependently, reduced cell growth with arrest of the cell cycle at G0/G1 phase and induction of apoptosis. Further mechanism of apoptosis induction was elucidated in detail in K562 cell line. Apoptosis was mediated by release of cytochrome c into the cytoplasm and cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). This was followed by DNA fragmentation. The level of anti-apoptotic protein Bcl-2 was decreased without any change in the pro-apoptotic Bax. Celecoxib also inhibited NF-kB activation. Celecoxib thus potentiates apoptosis as shown by MTT assay, cytochrome c leakage, PARP cleavage, DNA fragmentation, Bcl-2 downregulation and possibly by inhibiting NF-kB activation.

Evidence for the association of synaptotagmin with glutathione S-transferases: implications for a novel function in human breast cancer.

OBJECTIVE: To analyze the pattern of changes in GSTs in cancerous and adjacent non-cancerous tissues obtained from breast cancer patients undergoing surgery. DESIGN AND METHODS: Cytosolic GST purification, assay of GST, protein expression levels, and GST-synaptotagmin association were analyzed using standard biochemical techniques like GSH-affinity purification, spectrophotometry, SDS-PAGE, Western blots, and matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF). RESULTS: GST activity in cancerous tissues (0.26 U/mg protein) was significantly higher (P < 0.05) as compared to those from adjacent non-cancerous tissues (0.14 U/mg protein) of breast cancer patients. Further analysis of GST subunits on SDS-PAGE and Western blots using class-specific GST antibodies revealed significant elevation in GST-pi levels in cancer tissues with no appreciable changes in GST-alpha and GST-mu. Along with the elevation of GST-pi levels, high molecular weight proteins (approximately 70 kDa) cross reacting with GST antibodies were detected only in surgically resected tumor biopsies but not in the non-cancerous tissues adjacent to the tumor. Based on MALDI-TOF analysis, the high molecular weight band was identified as synaptotagmin V bound to GST-M1 with 47% sequence coverage after processing on an MS-FIT search engine. CONCLUSIONS: Our results suggest a novel putative functional role for the GST-synaptotagmin complex in human breast cancers. As this association of GST M1-synaptotagmin was not seen in adjacent non-cancerous tissues, this can be used as a marker for breast cancers.

Novel functional association of rat testicular membrane-associated cytosolic glutathione S transferases and cyclooxygenase in vitro.

AIM: To analyze the role of cytosolic glutathione S-transferases cGSTs and membrane-associated cytosolic GSTs macGSTs in prostaglandin biosynthesis and to evaluate the possible interaction between glutathione S-transferases GSTs and cyclooxygenase (COX) in vitro. METHODS: SDS-PAGE analysis was undertaken for characterization of GSTs, thin layer chromatography (TLC) to monitor the effect of GSTs on prostaglandin biosynthesis from arachidonic acid (AA) and spectrophotometric assays were done for measuring activity levels of COX and GSTs. RESULTS: SDS-PAGE analysis indicates that macGSTs have molecular weights in the range of 25-28 kDa. In a coupled assay involving GSTs, arachidonic acid and cyclooxygenase-1, rat testicular macGSTs produced prostaglandin E2 and F2alfa, while the cGSTs caused the generation of prostaglandin D2, E2 and F2alfa. In vitro interaction studies on GSTs and COX at the protein level have shown dose-dependent inhibition of COX activity by macGSTs and vice versa. This effect, however, is not seen with cGSTs. The inhibitory effect of COX on macGST activity was relieved with increasing concentrations of reduced glutathione (GSH) but not with 1-chloro 2,4-dinitrobenzene (CDNB). The inhibition of COX by macGSTs, on the other hand, was potentiated by glutathione. CONCLUSION: We isolated and purified macGSTs and cGSTs from rat testis and analyzed their involvement in prostaglandin biosynthesis. These studies reveal a reversible functional interaction between macGSTs and COX in vitro, with possible interactions between them at the GSH binding site of macGSTs.

Inhibition of 5-lipoxygenase by vitamin E.

Purified 5-lipoxygenase from potato tubers was inhibited strongly by vitamin E and its analogs. The inhibition by d-alpha-tocopherol was found to be irreversible and non-competitive with respect to arachidonic acid. An IC50 of 5 microM was calculated for d-alpha-tocopherol. The inhibition appears to be unrelated to its antioxidant function. Binding studies with 14C-labelled d-alpha-tocopherol revealed that there is a strong interaction between vitamin E and 5-lipoxygenase. Tryptic digestion and peptide mapping of 5-lipoxygenase-vitamin E complex indicate that vitamin E binds strongly to a single peptide. These studies suggest that cellular vitamin E levels may have profound influence on the formation of leukotrienes.

Prostaglandin metabolism during growth and differentiation of the regenerating vertebrate appendage.

House lizards are able to regenerate their tails. This is an ideal model to study the growth and differentiation of an organ. Prostaglandins (PGs) are local hormones having diverse and potent biological activities. In an effort to understand PG metabolism during the growth and differentiation of the regenerating lizard tail, we analysed the fatty acid (FA) composition of phospholipids are free FAs by GC, the activity of two rate-limiting enzymes (phospholipases A and C), the activity of the enzyme responsible for the oxygenation of polyunsaturated fatty acids to PGs (cyclooxygenase) and characterized the endogenous PGs by HPLC. It was observed that on the 20th day, i.e. the tissue differentiation period, there was an increase in phospholipase A activity, together with a sudden fall in the free arachidonic acid (AA) level, an increase in cyclooxygenase activity and the appearance of endogenous PGE2. PGE2 can stimulate cyclic adenosine monophosphate (cAMP) production and it may stimulate a cascade of events associated with tissue differentiation.

Role of environmental estrogens in the deterioration of male factor fertility.

OBJECTIVE: To evaluate the role of the environmental estrogens polychlorinated biphenyls (PCBs) and phthalate esters (PEs) as potential environmental hazards in the deterioration of semen parameters in infertile men without an obvious etiology. DESIGN: Randomized controlled study. SETTING: Tertiary care referral infertility clinic and academic research center. PATIENT(S): Twenty-one infertile men with sperm counts <20 million/mL and/or rapid progressive motility <25% and/or <30% normal forms without evidence of an obvious etiology and 32 control men with normal semen analyses and evidence of conception. Semen and blood samples were obtained as part of the treatment protocol. MAIN OUTCOME MEASURE(S): Evaluation of semen parameters such as ejaculate volume, sperm count, motility, morphology, vitality, osmoregulatory capacity, sperm chromatin stability, and sperm nuclear DNA integrity. RESULT(S): PCBs were detected in the seminal plasma of infertile men but not in controls, and the concentration of PEs was significantly higher in infertile men compared with controls. Ejaculate volume, sperm count, progressive motility, normal morphology, and fertilizing capacity were significantly lower in infertile men compared with controls. The highest average PCB and PE concentrations were found in urban fish eaters, followed by rural fish eaters, urban vegetarians, and rural vegetarians. The total motile sperm counts in infertile men were inversely proportional to their xenoestrogen concentrations and were significantly lower than those in the respective controls. CONCLUSION(S): PCBs and PEs may be instrumental in the deterioration of semen quality in infertile men without an obvious etiology.

Fulminant hepatic failure induced oxidative stress in nonsynaptic mitochondria of cerebral cortex in rats.

Fulminant hepatic failure (FHF) is a condition with sudden onset of necrosis of hepatocytes and degeneration of liver tissue without any established liver disease. FHF is associated with increased ammonia levels in blood and brain, which is supposed to be neurotoxic, ultimately leading to neuronal death. Evidences from previous studies suggest for mitochondrial dysfunctions under hyperammonemic conditions. In the present investigation, on thioacetamide-induced FHF rat models, studies were undertaken on cerebral nonsynaptic mitochondrial oxidative stress. The results of the present study reveal elevated lipid peroxidation along with reduced total thiol levels in the cerebral cortex mitochondria of experimental animals compared to saline treated control rats. In addition, the enzymatic activities of glutathione peroxidase and glutathione reductase were decreased, with an elevation in Mn-SOD activity. Overall, thioacetamide-induced FHF in rats enhanced the levels of lipid peroxidation coupled with impaired antioxidant defenses in the cerebral nonsynaptic mitochondria.

Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption.

Cell cultures of Plumbago rosea were immobilized in calcium alginate and cultured in Murashige and Skoog's basal medium containing 10 mM CaCl(2) for the production of plumbagin, an important medicinal compound. Studies were carried to find out the impact of immobilization on the increased accumulation of this secondary metabolite. Immobilization in calcium alginate enhanced the production of plumbagin by three, two and one folds compared to that of control, un-crosslinked alginate and CaCl(2) treated cells respectively. Cell loading at a level of 20% to the polymer volume (Na-alginate) was optimal and maximum plumbagin was obtained. At higher cell loading (40-50%), lower plumbagin accumulation was noticed. Addition of 200 mg l(-1) chitosan as an elicitor to the immobilized cells resulted in eight and two folds higher accumulation of plumbagin over control and immobilized cells. Also, more than 70% of the plumbagin was released into the medium, which is highly desirable for easy recovery of the product. Sucrose utilization rate of the cells was higher when cells were subjected to in situ product removal using Amberlite XAD-7. This may indicate that the toxicity of plumbagin was reduced on cells when it was removed from the medium. Cells subjected to combined treatments of chitosan, immobilization and in situ extraction showed a synergistic effect and yielded 92.13 mg g(-1) DCW of plumbagin which is 21, 5.7, 2.5 times higher than control, immobilized, immobilized and elicited cells respectively.