Bio-availability and metabolism of n-3 fatty acid rich garden cress (Lepidium sativum) seed oil in albino rats.

The ratio of fatty acids namely linoleic acid (LA, 18:2, n-6) and alpha linolenic acid (ALA, 18:3, n-3) in the diet plays an important role in enrichment of ALA in tissues and further conversion to long-chain polyunsaturated fatty acids (LC-PUFA) like eicosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3). Garden cress seed oil (GCO) is one of the richest sources of omega-3 fatty acid and contains 29-34.5% of ALA. In this study, dietary supplementation of GCO on bio-availability and metabolism of alpha-linolenic acid was investigated in growing rats. Male wistar rats were fed with semi-purified diets supplemented with 10.0% sunflower oil (SFO 10%); 2.5% GCO and 7.5% SFO (GCO 2.5%); 5% GCO and 5% SFO (GCO 5.0%); 10% GCO (GCO 10%) for a period of 8 weeks. There was no significant difference with regard to the food intake, body weight gain and organ weights of rats in different dietary groups. Rats fed with GCO showed significant increase in ALA levels in serum and tissues compared to SFO fed rats. Feeding rats with 10% GCO lowered hepatic cholesterol by 12.3% and serum triglycerides by 40.4% compared to SFO fed group. Very low density lipoprotein cholesterol (VLDL-C) and low density lipoprotein cholesterol (LDL-C) levels decreased by 9.45% in serum of 10% GCO fed rats, while HDL remained unchanged among GCO fed rats. Adipose tissue showed incorporation of 3.3-17.4% of ALA and correlated with incremental intake of ALA. Except in adipose tissue, the EPA, DHA levels increased significantly in serum, liver, heart and brain tissues in GCO fed rats. A maximum level of DHA was registered in brain (11.6%) and to lesser extent in serum and liver tissues. A significant decrease in LA and its metabolite arachidonic acid (AA) was observed in serum and liver tissue of rats fed on GCO. Significant improvement in n-6/n-3 fatty acid ratio was observed in GCO based diets compared to diet containing SFO. This is the first study to demonstrate that supplementation of GCO increases serum and liver ALA, EPA, DHA and decreases LA and AA in rats. Therefore, the GCO can be considered as a potential, alternate dietary source of ALA.

Eugenol--the active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells.

Polymorphonuclear leukocytes (PMNL) play an important role in the modulation of inflammatory conditions in humans. PMNL cells recruited at the site of inflammation, release inflammatory mediators such as leukotrienes, proteolytic enzymes and reactive oxygen species. Among these, leukotrienes are implicated in pathophysiology of allergic and inflammatory disorders like asthma, allergic rhinitis, arthritis, inflammatory bowel disease and psoriasis. 5-lipoxygenase (5-LO) is the key enzyme in biosynthetic pathway of leukotrienes. Our earlier studies showed that spice phenolic active principles significantly inhibit 5-LO enzyme in human PMNLs. In this study we have further characterized the inhibitory mechanism of eugenol, the active principle of spice-clove on 5-LO enzyme and also its effect on leukotriene C((4)) (LTC(4)). Substrate dependent enzyme kinetics showed that the inhibitory effect of eugenol on 5-LO was of a non-competitive nature. Further, eugenol was found to significantly inhibit the formation of LTC(4) in calcium ionophore A23187 and arachidonic acid (AA) stimulated PMNL cells. These data clearly suggest that eugenol inhibits 5-LO by non-competitive mechanism and also inhibits formation of LTC(4) in human PMNL cells and thus may have beneficial role in modulating 5-LO pathway in human PMNL cells.

Antiproliferative and apoptotic effect of ascorbyl stearate in human glioblastoma multiforme cells: modulation of insulin-like growth factor-I receptor (IGF-IR) expression.

Human glioblastomas (gliomas) are characterized as highly invasive and rapidly growing brain tumors. In this study, we present data on in vitro effect of ascorbyl stearate (Asc-S), a liphophilic derivative of ascorbic acid on cell proliferation, transformation, apoptosis and modulation of expression of insulin-like growth factor-I receptor (IGF-IR) in human glioblastoma multiforme (T98G) cells. Asc-S showed significant inhibition of fetal bovine serum and human recombinant insulin-like growth factor-I (IGF-I) dependent cell proliferation in a dose dependent manner. Treatment of T98G cells with 0, 50, 100 and 150 microM Asc-S for 24h slowed down the cell multiplication cycle with significant accumulation of cells at late S/G2-M phase of cycle. Asc-S treatment (100 microM) reversed the transformed phenotype as determined by clonogenecity in soft agar and also induced apoptosis of T98G. These changes were found to be associated with significant decrease in IGF-IR expression in dose and time dependent manner compared to untreated controls. The data clearly demonstrate that Asc-S has antiproliferative and apoptotic effect on T98G cells probably through modulation of IGF-IR expression and consequent facilitation of programmed cell death.

Purification and partial characterization of peroxidase from human term placenta of non-smokers: metabolism of benzo(a)pyrene-7, 8-dihydrodiol.

Peroxidase (Donor: H(2)O(2)oxidoreductase EC 1.11.1.7) from human term placentae of non-smokers was purified to homogeneity by a combination of NH(4)Cl extraction, affinity chromatography, (NH(4))(2)SO(4)precipitation, ion-exchange and gel filtration chromatography. The homogeneity of purified human placental peroxidase (HTPP) was confirmed by gel filtration, reverse phase high performance liquid chromatography (HPLC) and SDS-PAGE. Peroxidase was found to be a membrane bound enzyme. A high concentration of NH(4)Cl (1.2 m) was needed to extract and solublize the enzyme. Removal of the salt resulted in irreversible precipitation of the enzyme. The protein exhibited a molecular mass of 126 000 kDa according to gel filtration and approximately 60 000 kDa by SDS-PAGE, indicating that the peroxidase is a homodimer. The purified peroxidase showed an optimum pH range of 7 to 8.5 and the K(m)for H(2)O(2)and guaiacol were found to be 0.08 m m and 10.0 m m, respectively. The purified peroxidase oxidized several substrates, namely potassium iodide, tetramethyl benzidine, guaiacol, ortho dianisidne and tyrosine. The enzyme was resistant to thermal denaturation up to 70 degrees C and also to chaotropic agents, guanidinium chloride and urea. Spectral properties indicated the presence of Soret band at 433 which shifted to 451 nm on complexation with cyanide. The circular dichroism studies showed that HTPP has a predominantly helical secondary structure. The enzyme showed similarities to the myeloperoxidase with regard to spectral and catalytical properties but differed significantly in amino acid composition, the R(z)value and molecular mass. Purified HTPP differed from eosinophil peroxidase in all physico-chemical properties indicating that it is not of eosinophil origin, but may represent a distinct, constitutive peroxidase in human placenta. Further, purified peroxidase catalyzed oxidation of benzo(a)pyrene-7, 8-dihydrodiol in presence of tyrosine and hydrogen peroxide to BP-tetrols, the hydrolytic products of BP-diol-epoxides, demonstrating the ability of peroxidase in bioactivation of benzo(a)pyrene in human placenta.

Antioxidant activity of selected Indian spices.

Spices and vegetables possess antioxidant activity that can be applied for preservation of lipids and reduce lipid peroxidation in biological systems. The potential antioxidant activities of selected spices extracts (water and alcohol 1:1) were investigated on enzymatic lipid peroxidation. Water and alcoholic extract (1:1) of commonly used spices (garlic, ginger, onion, mint, cloves, cinnamon and pepper) dose-dependently inhibited oxidation of fatty acid, linoleic acid in presence of soybean lipoxygenase. Among the spices tested, cloves exhibited highest while onion showed least antioxidant activity. The relative antioxidant activities decreased in the order of cloves, cinnamon, pepper, ginger, garlic, mint and onion. Spice mix namely ginger, onion and garlic; onion and ginger; ginger and garlic showed cumulative inhibition of lipid peroxidation thus exhibiting their synergistic antioxidant activity. The antioxidant activity of spice extracts were retained even after boiling for 30 min at 100 degrees C, indicating that the spice constituents were resistant to thermal denaturation. The antioxidant activity of these dietary spices suggest that in addition to imparting flavor to the food, they possess potential health benefits by inhibiting the lipid peroxidation.

Brain CT and MRI findings after carbon monoxide toxicity.

The neuropathologic sequelae of carbon monoxide (CO) toxicity have been well described in postmortem examinations. Globus pallidus damage as well as diffuse white matter lesions and encephalopathic changes occur. Brain CT has provided imaging correlates to the premortem changes. MRI is more sensitive and provides more specificity. Cerebral edema changes may occur early with subsequent demonstration of globus pallidus lesions and white matter changes. Globus pallidus lesions in many cases do not correlate directly to clinical status and outcome; however, the presence of diffuse white matter disease is a more reliable index of both. These changes are seen in patients in both accidental exposures to CO and in suicide attempts.

Antioxidant effect of anthocyanin on enzymatic and non-enzymatic lipid peroxidation.

In vitro enzymatic and non-enzymatic polyunsaturated fatty acid peroxidation was significantly inhibited in a dose dependent manner by purified anthocyanin, a deep-red colour pigment from carrot cell culture. The kinetics showed that anthocyanin is a non-competitive inhibitor of lipid peroxidation. Anthocyanin has been found to be a potent antioxidant compared to classical antioxidants such as butylated hydroxy anisole (BHA), butylated hydroxy toulene (BHT) and alpha tocopherol. This natural agent, in addition to imparting colour to the food, might prevent autooxidation of lipids as well as lipid peroxidation in biological systems.

Entry rate kinetics of D-mannitol and carboxyl-inulin for transfer across the blood-cerebrospinal fluid barrier.

This study evaluates the entry rate kinetics of hydrophilic compounds [3H]-D-mannitol and [14C]-carboxyl-inulin across the blood-cerebrospinal fluid (CSF) barrier in a rabbit experimental model. To maintain steady state levels of these tracers in circulation, 100 microCi of [3H]-D-mannitol and 150 microCi of [14C]-carboxyl-inulin were administered as a bolus and by slow infusion for four hours via a femoral venous catheter. Entry rate kinetics of [3H]-D-mannitol and [14C]-carboxyl-inulin from plasma into cisterna magna CSF were computed using a mathematical equation described by Davson. [3H]-D-mannitol and [14C]-carboxyl-inulin maintained steady state levels throughout the experiment. Entry rates for mannitol and carboxyl-inulin were represented by a straight line, from the slope of which K(out) (or K(in)) were computed: K(in) values for mannitol and carboxyl-inulin were 0.06820 hr(-1) and 0.00023 hr(-1), respectively. Differences in the entry rate of mannitol and carboxyl-inulin may be explained by the molecular size and effective radius of these tracers.

Epinephrine increases spinal cord concentrations of [3H]-clonidine hydrochloride in rabbits after epidural infusion.

Epinephrine is often given with epidurally administered drugs to prolong and enhance analgesia, which is partly attributed to alpha-adrenergic processes. This investigation evaluates the effect of epinephrine on the distribution of epidurally administered [3H]-clonidine hydrochloride (clonidine HCl) in serum and in the central nervous system. After placing a lumbar epidural catheter via a laminectomy, rabbits were randomly assigned to receive 20 microCi of clonidine HCl with epinephrine (1:200,000) (n = 5) or without (control; n = 5) for 90 min. During the administration, which included bolus and slow infusion, blood samples were collected at 15-min intervals. At the end of the administration, rabbits were perfused with normal saline, leading to exsanguination. Brain and spinal cord tissues were excised for radiometric analysis. In both groups, the concentration of clonidine HCl was greatest in the lumbar cord. Epinephrine further enhanced accumulation of clonidine HCl into the lumbar cord but did not alter the concentration of clonidine HCl in serum, brain, cervical cord, and thoracic cord. We conclude that lumbar administration of epidural clonidine HCl leads to increased concentrations in the lumbar cord, which is further enhanced by epinephrine. The increased spinal cord accumulation of clonidine may be another mechanism by which epinephrine improves epidural analgesia.

Modulation of glioma cell growth and 5-lipoxygenase expression by interferon.

The effect of mouse interferon-alpha/beta (MuIFN-alpha/beta on growth/viability, cell cycle regulation, 5-lipoxygenase (5-LO) protein expression, leukotriene B4 (LTB4) biosynthesis and glial fibrillary acidic protein (GFAP) expression of mouse glioma (G-26) cells in vitro was studied. The G-26 cells were treated with 800 IU/ml of MuIFN-alpha/beta for 1, 2, 3 and 4 days. The growth and viability of glioma cells was evaluated by [3H]-thymidine incorporation and MTT (3(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazoliumbromi de) assay, resulted in a time dependent decrease in [3H]-thymidine incorporation into DNA and MTT formazan formation, respectively. The cell cycle regulation measured by flow cytometry with propidium iodide staining revealed that the cell multiplication cycle was slowed down due to accumulation of cell in S-phase of the cell cycle, leading to inhibition in G0/G1 phase of the cell cycle. The 5-LO protein expression (measured by Western immunoblot analysis) and LTB4 biosynthesis (measured by enzyme immunoassay) were found to be increased by 2 to 2.4 fold and several fold respectively on days 3 and 4 of MuIFN-alpha/beta treatment. The GFAP protein expression was also found to be increased at least by 3 fold on day 4 of the MuIFN-alpha/beta treatment. These results suggest that inhibition in growth and thereby slowing of the cell multiplication cycle of glioma cells has resulted in upregulation of GFAP expression and 5-LO pathway.

Epinephrine increases the selective permeability of epidurally administered [3H]-D-mannitol and [14C]-carboxyl-inulin across the blood-spinal cord barrier.

A rabbit experimental model was used to ascertain how epinephrine influences uptake of epidurally-administered [3H]-D-mannitol and [14C]-carboxyl-inulin into regions of brain and spinal cord. [3H]-D-mannitol and [14C]-carboxyl-inulin, 20 microCi each, were distributed in normal saline (control) and normal saline with 0.03 microM (1:200,000 diluted) epinephrine. These tracers were administered as bolus and slow epidural infusion for 90 min. Epinephrine decreased the uptake of [3H]-D-mannitol and [14C]-carboxyl-inulin into the serum collected at 15, 30, 45, 60, 75 and 90 min intervals during the administration of tracers. Epinephrine did not alter the uptake of [3H]-D-mannitol and [14C]-carboxyl-inulin into the regions of the brain (cerebrum, cerebellum, brain stem including midbrain and pons) and upper regions of the spinal cord (mid-cervical and mid-thoracic) compared to controls. The uptake of [3H]-D-mannitol and [14C]-carboxyl-inulin into the mid-lumbar region is significantly increased compared to the control. The differential uptake of [3H]-D-mannitol and [14C]-carboxyl-inulin into 1 cm thick sections of lumbo-sacral cord was significantly increased by epinephrine at the site of the epidural catheter placement, while sections of the cord distal on either end (thoracic and sacral ends) did not show significant differences compared to the control. The efficacy of epinephrine in increasing spinal cord uptake of hydrophilic [3H]-D-mannitol and [14C]-carboxyl-inulin may be attributed to its vasoconstrictive properties and its ability to increase vascular endothelial cell permeability across the blood-spinal cord barrier.

Eugenol--an inhibitor of lipoxygenase-dependent lipid peroxidation.

The effect of eugenol on enzymatic lipid peroxidation catalyzed by soybean lipoxygenase was studied in an in vitro system. Lipid peroxidation was inhibited by eugenol in a concentration-dependent manner. The half-maximal inhibition (IC50) was found to be 380 microM eugenol. Enzyme kinetic studies showed that eugenol non-competitively inhibited lipid peroxidation by altering the maximum velocity (Vmax) and without any change in Michaelis-Menten constant (Km) values. The inhibitory mechanism implies that eugenol does not inactivate the enzyme directly but may interfere with fatty acid radical intermediates due to its hydroxy radical scavenging ability and thus play a role in inhibiting the propagation of lipid peroxidation.

Selective permeability of [3H]-D-mannitol and [14C]-carboxyl-inulin across the blood-brain barrier and blood-spinal cord barrier in the rabbit.

Selective permeability across the blood-brain and blood-spinal cord barriers was studied in a rabbit experimental model. To maintain steady state levels of the tracers in the circulation, 50muCi each of [3H]-D-mannitol and [14C]-carboxyl-inulin were administered as a bolus and by slow intravenous infusion for 60, 90 and 120 minutes; 90 min proved to be the optimal equilibration time for uptake of radioactive tracers into central nervous system (CNS) regions. Kinetic transfer of [3H]-D-mannitol and [14C]-carboxyl-inulin from blood into CNS was computed as apparent transfer constant (KD). The KD for [3H]-D-mannitol in regions of brain (cerebrum, cerebellum, mid-brain, pons and brain stem), and spinal cord (cervical, thoracic and lumbar) were 0.033 to 0.054 microliter/min/g-1 and 0.053 to 0.065 microliter/min/g-1, respectively. The KD for [14C]-carboxyl-inulin into brain and spinal cord regions were 0.016 to 0.033 and 0.037 to 0.054 microliter/min/g-1, respectively. Of the regions of spinal cord and brain examined, lumbar cord appears to be the most permeable. The KD values pooled for samples of the spinal cord show a significant increase in uptake of [3H]-D-mannitol (p = 0.0043) and [14C]-carboxyl-inulin (p = 0.0001) compared to samples of brain. The blood-spinal cord barrier was more permeable to these substances than the blood-brain barrier.

Polycyclic aromatic hydrocarbons in placenta, maternal blood, umbilical cord blood and milk of Indian women.

Human placenta, umbilical cord blood, maternal blood and breast milk samples from mothers were analysed for the presence of selected polycyclic aromatic hydrocarbons (PAHs). Benzo(a) pyrene (B(a)P), dibenzo(a,c)anthracene (DBA) and chrysene (Chy) were detected in all the four types of sample. Levels of dibenzo(a,c)anthracene were higher in the above samples compared with the other two PAHs. Umbilical cord blood and breast milk samples showed relatively high concentrations of all the three PAHs and thus demonstrated that the developing foetus/new born were exposed to these carcinogenic environmental contaminants. The possible implications of PAHs in relation to human health are discussed.

Effect of metalaxyl on heart rate in rats: role of alpha 1-adrenoreceptors.

Intraperitoneal injection of metalaxyl (250 mg/kg) produced a decrease in heart rate lasting for more than 60 min in anesthetized rats. Pretreatment of rats with phentolamine (a nonselective alpha-adrenoreceptor antagonist, ip at 20 mg/kg) and prazosin (an alpha 1-adrenoreceptor antagonist, ip at 5 mg/kg) significantly reduced the bradycardia induced by metalaxyl. Yohimbine (an alpha 2-adrenoreceptor antagonist, ip at 10 mg/kg) did not change the effect of metalaxyl on heart rate. The results suggest that alpha 2-adrenoreceptors mediate the bradycardic effect of metalaxyl.

Inhibition of monoamine oxidase by the fungicide metalaxyl.

The in vitro effect of metalaxyl on monoamine oxidase (MAO) activity in rat heart was studied. Metalaxyl decreased MAO activity in a dose-dependent manner. The inhibitory concentration (IC50) for metalaxyl was found to be 19 mumol. Substrate-dependent kinetic studies demonstrated noncompetitive inhibition as evidenced by decreased velocity of enzyme activity (Vmax) without significant change in enzyme-substrate affinity (Km). The inhibition of MAO activity by metalaxyl suggests interference with amine metabolism.

Metalaxyl-induced bradycardia in rats: mediated by alpha-adrenoreceptors.

The effect of the fungicide metalaxyl [methyl N-(2-methoxyacetyl)-N-(2,6-xylyl)-DL-alaniate] on cardiac activity was investigated in albino rats anesthetized with phentobarbitone. Metalaxyl caused dose-dependent bradycardia, and at higher doses (250 and 300 mg/kg body weight) the sustained bradycardia led to cardiac arrest. The acetylcholinesterase activities in brain and heart were not affected in metalaxyl-treated rats. However, the bradycardia induced by the fungicide was blocked by pretreatment of rats with the alpha-adrenoreceptor antagonist phentolamine (20 mg/kg, ip). The data suggest that the bradycardia-inducing effect of metalaxyl was not mediated by the cholinergic system but through alpha-adrenoreceptors.

Cardiotoxic effects of dichlorvos (DDVP) in albino rats.

Dichlorvos (O, O-dimethyl O-(2,2-dichlorovinyl phosphate: DDVP 76.6 X EC) an organophosphate pesticide had a profound effect on cardiac activity of albino rats. Adult male rats anesthetized with pentobarbitone were administered 30, 50, 70 and 90 mg/kg body weight of dichlorvos. The heart rate and electrocardiogram were monitored and acetylcholinesterase activity was measured in heart and brain. Dichlorvos produced abnormalities in ECG, decrease in heart rate, cardiac arrest and inhibition of cholinesterase activity. It is suggested that cardiotoxic effect of DDVP may be mediated by the accumulated acetylcholine as a result of cholinesterase inhibition.