Interspecies difference in liver-specific functions and biotransformation of testosterone of primary rat,porcine and human hepatocyte in an organotypical sandwich culture

Interspecies difference is an important issue in toxicology research. We compared the potential in vitro metabolism of human, porcine and rat hepatocytes over 2 weeks in culture in an organotypical culture model which reflects the in vivo situation. All three species show similar LDH-rates. Albumin measurements showed that rat cells are about twice as active as human and porcine hepatocytes. The ethoxyresorufin-O-deethylase (EROD) activity of the rat hepatocytes is with about 14 μU/10⁶ cells distinctly higher than those of porcine and human cells (1.8 and 0.5 μU/10⁶ cells respectively), furthermore, the activity of the rat EROD increases slightly during the prolonged time in culture, whereas those of porcine and human enzymes slightly decrease. Concerning ethoxycoumarin-O-deethylase (ECOD), the enzyme activities are found to be in three different ranges where rat cells show the highest activity with 66 μU/10⁶ cells, porcine hepatocytes exhibit an activity of about 23 μU/10⁶ cells, and human activity is lowest with 0.7 μU/10⁶ cells. All three species show a similar decreasing trend of ECOD during the period of study. Regarding the biotransformation of testosterone, human and porcine liver cells form three major metabolites whereas rat cells form a mixture of all measured metabolites. Hence, in vitro metabolism using porcine hepatocytes would be much more scientific sense than one using rat hepatocytes since the metabolic pathways are much closer to human metabolism.     

Comparison of the activity of 2'-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)adenine in vivo.

The inhibition of P388 cell deamination of arabinosyladenine (ara-A) in vivo by the adenosine deaminase inhibitors 2′-deoxycoformycin (dCF) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and their subsequent effects on ara-A metabolism were determined and compared. A single i.p. injection of EHNA (3 mg/kg, 10.9 μmoles/kg) initially inhibited ara-A deamination in vivo by 96 per cent with recovery to 50 per cent of control values within 30 min. In comparison, dCF (0.2 mg/kg, 0.75 μmole/kg) inhibition of ara-A deamination was initially low (4 per cent), but maximized (96 per cent) after 15 min. This inhibition was sustained for 2 hr and did not recover to 50 per cent of control values until after 10 hr. Injected alone, the $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of ara-A in the peritoneal ascitic fluid was less than 1 min, but was increased to 7 min when injected with EHNA and to 12 min when injected 15 min after dCF. The rate of efflux of ara-A and its metabolites from the peritoneal cavity ($\text{T}{}_{\mathrm{<mtext>case1</mtext><mtext>2</mtext>}}\text{= 15?18}\text{min}$) was not affected significantly by either deaminase inhibitor. Cellulat ara-ATP concentrations were elevated and the extent and duration of inhibition of DNA synthetic capacity were increased identically in cells of mice treated with ara-A and either deaminase inhibitor as compared with those treated with ara-A alone. Sustained deaminase inhibition after intraperitoneal concentrations of ara-A had been diminished by otherwise normal disposition did not augment the biochemically demonstrable activity of ara-A. Therefore, it appears that maintenance of the initial high concentrations of ara-A is the primary function of a deaminase inhibitor in increasing the therapeutic efficacy of this analog.     

Inhibition of human recombinant T-type calcium channels by the endocannabinoid N-arachidonoyl dopamine

Background and purpose:

N-arachidonoyl dopamine (NADA) has complex effects on nociception mediated via cannabinoid CB₁ receptors and the transient receptor potential vanilloid receptor 1 (TRPV1). Anandamide, the prototypic CB₁/TRPV1 agonist, also inhibits T-type voltage-gated calcium channel currents (I_Ca). These channels are expressed by many excitable cells, including neurons involved in pain detection and processing. We sought to determine whether NADA and the prototypic arachidonoyl amino acid, N-arachidonoyl glycine (NAGly) modulate T-type I_Ca

Experimental approach:

Human recombinant T-type I_Ca (Ca_V3 channels) expressed in HEK 293 cells and native mouse T-type I_Ca were examined using standard whole-cell voltage clamp electrophysiology techniques.

Key results:

N-arachidonoyl dopamine completely inhibited Ca_V3 channels with a rank order of potency (pEC₅₀) of Ca_V3.3 (6.45) ≥ Ca_V3.1 (6.29) > Ca_V3.2 (5.95). NAGly (10 µmol·L⁻¹) inhibited Ca_V3 I_Ca by approximately 50% or less. The effects of NADA and NAGly were voltage- but not use-dependent, and both compounds produced significant hyperpolarizing shifts in Ca_V3 channel steady-state inactivation relationships. By contrast with anandamide, NADA and NAGly had modest effects on Ca_V3 channel kinetics. Both NAGly and NADA inhibited native T-type I_Ca in mouse sensory neurons.

Conclusions and implications:

N-arachidonoyl dopamine and NAGly increase the steady-state inactivation of Ca_V3 channels, reducing the number of channels available to open during depolarization. These effects occur at NADA concentrations at or below to those affecting CB₁ and TRPV1 receptors. Together with anandamide, the arachidonoyl neurotransmitter amides, NADA and NAGly, represent a new family of endogenous T-type I_Ca modulators.     

Effects of the interferon inducing agents tilorone and polyriboinosinic acid · polyribocytidylic acid (poly IC) on the hepatic monooxygenase systems of the developing neonatal rat

This paper describes the effects of the interferon inducing agents tilorone and polyriboinosinic acid · polyribocytidylic acid (poly IC) on the postnatal development of hepatic cytochrome P-450-linked monooxygenase systems of male rats from birth through early adolescence. The administration of tilorone to rats on days 1and 2 postpartum modified the changes in the activities of hepatic monooxygenase systems that occur normally during the first four days postpartum. Thus, aniline hydroxylase activity, which develops very rapidly during the first 2 days postpartum, was depressed markedly by tilorone, ethylmorphine N-demethylase activity was depressed moderately, and benzo[a]pyrene hydroxylase, normally the slowest of the three monooxygenase activities to develop, was induced. These changes in monooxygenase activities occurred without a significant change in the cytochrome P-450 content. These observations suggest that not all species of neonatal cytochrome P-450 are affected equally by tilorone administration. By day 7 postpartum, the cytochrome P-450 content and all three monooxygenase activities were depressed in rats that had received tilorone on days 1 and 2 postpartum. All three monooxygenase systems were depressed by the administration of a single dose of poly IC (10 mg/kg) in 1-, 2-, 21-, 28- and 56-day-old rats. The length of the period between maximal depression and complete recovery of cytochrome P-450 systems was shown to be a function of the age of the rat; it increased from about 6 hr in 1-day-old rats to 48 hr in 56-day-old rats. Protein is synthesized more rapidly and degraded more slowly in neonate than in adult animals; this may account for the more rapid recovery of poly IC-induced depression of monooxygenase systems in neonates.     

Potential of long-term dietary administration of rosemary in improving the antioxidant status of rat tissues following carbon tetrachloride intoxication

In this study, 24 Wistar rats were allocated to 4 groups of 6 animals each. Groups 1 and 2 were fed a basal diet, while groups 3 and 4 were fed the basal diet supplemented further with ground rosemary at 1% level. Following 6-weeks feeding, groups 2 and 4 were injected 1 ml CCl₄/kg bw and after six hours all animals were sacrificed. Results showed that feeding rosemary before CCl₄ treatment resulted in decline (P < 0.05) of the increased aspartate transaminase, alanine transaminase and alkaline phosphatase activities and increase (P < 0.05) of the reduced cholesterol and triacylglycerols in serum. It also decreased (P < 0.05) lipid peroxidation and increased (P < 0.05) the reduced hydroxyl anion radical and hydrogen peroxide scavenging activities in serum, liver, kidney and heart tissues. In addition, it increased (P < 0.05) the reduced ABTS radical cation and the superoxide anion scavenging activities in all tissues except in heart and in kidney and heart tissues, respectively. These results suggest that dietary rosemary has the potential to become a promising functional food component.     

Acute doxorubicin cardiotoxicity alters cardiac cytochrome P450 expression and arachidonic acid metabolism in rats

Doxorubicin (DOX) is a potent anti-neoplastic antibiotic used to treat a variety of malignancies; however, its use is limited by dose-dependent cardiotoxicity. Moreover, there is a strong correlation between cytochrome P450 (CYP)-mediated arachidonic acid metabolites and the pathogenesis of many cardiovascular diseases. Therefore, in the current study, we have investigated the effect of acute DOX toxicity on the expression of several CYP enzymes and their associated arachidonic acid metabolites in the heart of male Sprague-Dawley rats. Acute DOX toxicity was induced by a single intraperitoneal injection of 15 mg/kg of the drug. Our results showed that DOX treatment for 24 h caused a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A1, CYP4A3, CYP4F1, CYP4F4, and EPHX2 gene expression in the heart of DOX-treated rats as compared to the control. Similarly, there was a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A, and sEH proteins after 24 h of DOX administration. In the heart microsomes, acute DOX toxicity significantly increased the formation of 20-HETE which is consistent with the induction of the major CYP ω-hydroxylases: CYP4A1, CYP4A3, CYP4F1, and CYP4F4. On the other hand, the formation of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) was significantly reduced, whereas the formation of their corresponding dihydroxyeicosatrienoic acids was significantly increased. The decrease in the cardioprotective EETs can be attributed to the increase of sEH activity parallel to the induction of the EPHX2 gene expression in the heart of DOX-treated rats. In conclusion, acute DOX toxicity alters the expression of several CYP and sEH enzymes with a consequent alteration in arachidonic acid metabolism. These results may represent a novel mechanism by which this drug causes progressive cardiotoxicity.     

Increases in cytochrome P-450 in cultured hepatocytes mediated by 3- and 4-carbon alcohols

The amount of cytochrome P-450 was increased to different extents after treatment of cultured chick embryo hepatocytes with n-propanol, isopropanol, n-butanol, or isobutanol, These increases were associated with increases in benzphetamine demethylase activity, a cytochrome P-450-catalyzed oxidation, and glucuronidation of phenol red, catalyzed by UDP-glucuronyl transferase. The responses were similar to those obtained with ethanol or propylisopropylacetamide, which are phenobarbital-like inducers. Pretreatment of cells with cycloheximide prevented the increases in both cytochrome P-450 and glucuronidation of phenol red, indicating that protein synthesis was required for these responses.     

Serotonin synthesis with rat brain synaptosomes. Effects of L-dopa, L-3-methoxytyrosine and catecholamines

Experiments were done with a fraction of rat brain containing mitochondria and synaptosomes. L-DOPA∥ (K_t 0·3 mM) and L-3-methoxytyrosine (K_t 0·5 mM) were found to be competitive inhibitors of tryptophan accumulation, while dopamine and l-noradrenaline had no effect on the accumulation of tryptophan. Furthermore, L-DOPA (K_t 5·6 μM) was about 100 times more potent than L-3-methoxytyrosine in inhibiting the synthesis of 5HT from tryptophan. The inhibition of 5HT synthesis by L-DOPA appeared to be competitive to tryptophan and was not linear at concentrations of L-DOPA higher than 1 μM. L-DOPA also interfered with the rate of deamination of 5HT synthesized in vitro. Dopamine and l-noradrenaline also inhibited the synthesis of 5HT.     

Antimicrobial Activity of Snake β-Defensins and Derived Peptides

β-defensins are antimicrobial peptides presenting in vertebrate animals. They participate in innate immunity, but little is known about them in reptiles, including snakes. Although several β-defensin genes were described in Brazilian snakes, their function is still unknown. The peptide sequence from these genes was deduced, and synthetic peptides (with approximately 40 amino acids and derived peptides) were tested against pathogenic bacteria and fungi using microbroth dilution assays. The linear peptides, derived from β-defensins, were designed applying the bioisosterism strategy. The linear β-defensins were more active against Escherichia coli, Micrococcus luteus, Citrobacter freundii, and Staphylococcus aureus. The derived peptides (7–14 mer) showed antibacterial activity against those bacteria and on Klebsiella pneumoniae. Nonetheless, they did not present activity against Candida albicans, Cryptococcus neoformans, Trychophyton rubrum, and Aspergillus fumigatus showing that the cysteine substitution to serine is deleterious to antifungal properties. Tryptophan residue showed to be necessary to improve antibacterial activity. Even though the studied snake β-defensins do not have high antimicrobial activity, they proved to be attractive as template molecules for the development of antibiotics.     

Genotoxic effects of monocrotophos,an organophosphorous pesticide,on an estuarine bivalve, Meretrix ovum

Genotoxicity studies evaluate the effects of pollutants on organisms and consequently, their implications on human health. Meretrix ovum was exposed to different concentrations of monocrotophos, viz. 5.5, 11.0 and 16.5 mg/L continuously for four different time periods viz. 2, 3, 7 and 14 days. Gills of these animals were collected immediately after exposure at the above time intervals and analyzed for genotoxic effect employing micronucleus test and effect on somatic growth by estimating the total RNA/DNA ratio. Data were analyzed employing Student’s ‘t’ test and ANOVA. Significant increase of micronuclei observed in the present study in a dose dependant manner indicates the possible chromosomal damage induced by monocrotophos in this species and thereby reveals its genotoxic potency. Significant reduction of the total RNA/DNA ratio observed in a time dependant manner indicates a considerable retardation of somatic growth in monocrotophos treated mussels. Results of the present study indicate that monocrotophos is genotoxic on M. ovum and also induces a pollution stress related retardation of somatic growth of this mussel. Further, this study indicated the possibility of using MN test in bivalves as a marker for screening/monitoring the genotoxic potential of pollutants in aquatic ecosystems.     

Effect of metal chelating agents on the storage of norepinephrine in vitro by cerebral synaptic vesicles

The inhibitory effects of fourteen different metal chelating agents on the storage in vitro of [³H]dl-norepinephrine (NE) by the isolated synaptic vesicles from rat brain were investigated with a view to explore the possible importance of metal chelation phenomena. The chelators under examination belong to the classes of polyamines, aminocarboxylic and hydroxy carboxylic acids exhibiting a wide range of metal-binding strengths. Values of ic₅₀, viz., the amount of the chelator which will inhibit 50 per cent of the storage of [³H]dl-NE, were determined for the different compounds. The strengths of chelate formation of the “synaptosomal and vesicular metal ions”, viz. Cu²⁺, Fe²⁺, Fe³⁺, Zn²⁺, Mg²⁺ and Ca²⁺, with the above compounds were compared with their inhibition in vitro of the vesicular storage of [³H]dl-NE in order to determine if a correlation existed between the two. On the basis of regression analyses of the data on the stabilities of the individual metal chelates (log K_ML) and the inhibitory effects of the different chelators (log ic₅₀/NE), significant correlation coefficients were obtained with the Cu²⁺ and Zn²⁺ amine systems, i.e. 0.98 with P < 0.02, and 0.88 with P < 0.02 respectively. In regard to the metal-aminoacids systems, Mg²⁺ gave the most satisfactory correlation coefficient, i.e. 0.91, P < 0.01; with Fe(II) = 0.84, P < 0.02 and the mixed-metal system Cu + Zn + Fe + Mg + Ca = 0.87, P < 0.05. The existence of such correlation is considered significant from the point of view of the metal coordination hypothesis for the vesicular binding and storage of NE.     

Identification of 24-O-β-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms,Dinophysis and Prorocentrum Cultures,and Shellfish in Europe,North America and Australasia

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-β-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1–5, the 24-O-β-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4–6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.     

N-acetyl, 4-O-methyldopa,a major metabolite of L-4-O-methyldopa in man and rat

N-Acetyl, 4-O-methyldopa was identified as a major urinary metabolite of $\text{L}\text{-4-O-}\text{methyldopa}$, both in man and in the rat. The urinary metabolites were examined in man after oral and in rat after intraperitoneal administration of $\text{L}\text{-4-O-}\text{methyldopa}$, $\text{L}\text{-3-O-}\text{methyldopa}$, L-dopa and N-acetyl, 4-O-methyldopa. 4-Mdopa was found to be converted mainly to N-acetyl, 4-Mdopa and iso-HVA and very little to the corresponding pyruvic and lactic acids, whereas 3-Mdopa was metabolized to its pyruvic, lactic and acetic (HVA) derivatives and practically not acetylated. It is suggested therefore that the 3-hydroxy,4-methoxy group (the iso-vanyl structure) prevents transamination and that N-acetylation represents an alternative metabolic pathway. The lack of N-acetyl,4-O-methyldopa after the L-dopa loads shows that L-dopa is not 4-O-methylated and therefore that 4-O-methyldopa is not, or only in a minute amount if any, an in vivo metabolite of L-dopa. N-Acetyl,4-Mdopa administration to rats resulted in excretion of the compound almost unchanged. These results agree with a previous suggestion by the authors of partly distinct metabolic routes for the O-methyl catecholamines according to whether the methyl group is bound on the meta or on the para position and raises the question as to what extent iso-HVA levels in body fluids are representative of a para-O-methylation of dopamine, implicated in neuropsychiatric disorders.     

An in vitro study of hemicholinium-3 on phospholipid metabolism of Krebs II ascites cells

With [Me-¹⁴C]choline as marker and after separation of choline and phosphocholine by ion-exchange column chromatography or thin layer chromatography on alumina, it is shown that 40 μM hemicholinium-3 (HC-3) inhibits the cytosolic choline-kinase of rat liver and Krebs cells. This inhibition is competitive (Km different, Vm similar) in the first case and mixed in the second (Km and Vm different). Despite this general inhibition of the phosphocholine formation, the synthesis of phosphatidylcholine (PC) by post-nuclear supernatants of rat liver and Krebs cells is different when tested with HC-3. It is unaffected in rat liver; however, HC-3 induces a PC deficiency in Krebs cells which is time-course dependent between 15 and 120min and proportional to the drug concentrations in the interval 5–40 μM. Incorporation of AT-[γ³²P] or [2-¹⁴C]ethanolamine into phospholipids shows that the sequential methylation pathway is not detectable in Krebs cells. These results are discussed in relation to those established concerning HC-3 action on phospholipid metabolism in other tissues.     

Avicholic Acid: A Lead Compound from Birds on the Route to Potent TGR5 Modulators

相似文献   

The effect of arsenic exposure and the efficacy of DMSA on the proteins and lipids of the gill tissues of Labeo rohita

Arsenic is a naturally occurring, highly toxic environmental pollutant. Fourier transform infrared (FT-IR) spectroscopy is a non-disturbing technique which provides quantitative information about the molecular composition of biological samples. The aim of this work is to study the compositional and structural changes at the molecular level occurring in gill tissues of Labeo rohita fingerlings due to arsenic exposure for various exposure periods by using FT-IR spectroscopic technique. The results of the present study suggest that arsenic exposure causes significant changes on the major biochemical constituents such as proteins, lipids and nucleic acids in the gill tissues of L. rohita. The changes are more pronounced as the period of exposure is increased. The significant decrease in the intensity and area of the amide I peak and CH₃ asymmetric stretching band suggests an alteration in the protein profile and lipid levels respectively, due to arsenic exposure. The amide A peak shifts suggests a change in the level of protein amide hydrogen bonding due to arsenic exposure. Further, the treatment with meso-2,3-dimercaptosuccinic acid (DMSA) improves the levels of biochemical constituents significantly, which suggest that DMSA treatment reduces the toxic effects and helps the recovery of gill tissues and its return to the level of the control.     

Loss of msnA, a Putative Stress Regulatory Gene, in Aspergillus parasiticus and Aspergillus flavus Increased Production of Conidia, Aflatoxins and Kojic Acid

Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells.     

An in vitro model for the pro-fibrotic effects of retinoids: mechanisms of action

BACKGROUND AND PURPOSE

Retinoids, including all-trans retinoic acid (tRA), have dose-dependent pro-fibrotic effects in experimental kidney diseases. To understand and eventually prevent such adverse effects, it is important to establish relevant in vitro models and unravel their mechanisms.

EXPERIMENTAL APPROACH

Fibrogenic effects of retinoids were assessed in NRK-49F renal fibroblasts using picro-Sirius red staining for collagens and quantified by spectrophotometric analysis of the eluted stain. Other methods included RT-qPCR, immunoassays and matrix metalloproteinase (MMP) activity assays.

KEY RESULTS

With or without TGF-β1, tRA was dose-dependently pro-fibrotic, notably increasing collagen accumulation. tRA and TGF-β1 additively suppressed expression of mRNA for MMP2, 3 and 13 and suppressed MMP activity. tRA, in the presence of TGF-β1, induced plasminogen activator inhibitor-1 (PAI-1) mRNA and they additively induced PAI-1 protein expression. A PAI-1 inhibitor, a pan-retinoic acid receptor (RAR) antagonist and a pan-retinoid X receptor (RXR) antagonist each partially prevented the pro-fibrotic effect of tRA. The dose-dependent pro-fibrotic effects of a pan-RXR agonist were similar to those of tRA. A pan-RAR agonist showed weaker, less dose-dependent pro-fibrotic effects and the pro-fibrotic effects of RARα and RARβ-selective agonists were even smaller. An RARγ-selective agonist did not affect fibrogenesis.

CONCLUSIONS AND IMPLICATIONS

An in vitro model for the pro-fibrotic effects of retinoids was established in NRK-49F cells. It was associated with reduced MMP activity and increased PAI-1 expression, and was probably mediated by RXR and RAR. To avoid or antagonize the pro-fibrotic activity of tRA, further studies on RAR isotype-selective agonists and PAI-1 inhibitors might be of value.     

Prophylactic effect of α-linolenic acid and α-eleostearic acid against MeHg induced oxidative stress,DNA damage and structural changes in RBC membrane

The present study was undertaken to evaluate under in vivo condition the effects of α-linolenic acid and α-eleostearic acid against methyl mercury (MeHg) induced oxidative stress. Male albino rats were divided into six groups. Group 1 was under normal control and Group 2 was treated with methyl mercury chloride (MeHgCl; 5 mg/kg BW/day). Groups 3, 4, 5 and 6 were orally treated with different doses of the two fatty acids (0.5% and 1.0% of total lipid given for each kind of linolenic acid isomer) along with MeHgCl (5 mg/kg BW). Comet assay of blood lymphocytes showed that administration of α-linolenic acid reduced DNA damage significantly (P < 0.05). Results also showed that activity of antioxidant enzymes of plasma and brain tissue and total antioxidant capacity in plasma decreased significantly due to oxidative stress generated by MeHgCl. Administration in higher dose of both kind of linolenic acid restored all the activities of the antioxidant enzymes and also reduced lipid peroxidation and increased total antioxidant capacity in plasma. Both kinds of linolenic acid successfully maintained the RBC membrane integrity which was totally disrupted and became flat due to MeHgCl stress. α-Linolenic acid was more efficient antioxidant than α-eleostearic acid against oxidative DNA damage.     

Inhibitory effects of ω-3 polyunsaturated fatty acids on receptor-mediated non-selective cation currents in rat A7r5 vascular smooth muscle cells

1. The effects of ω-3 polyunsaturated fatty acids on receptor-mediated non-selective cation current (I_cat) and K⁺ current were investigated in aortic smooth muscle cells from foetal rat aorta (A7r5 cells). The whole-cell voltage clamp technique was employed.
2. With a K⁺-containing solution, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, 30 μM) produced an outward current at a holding potential of −40 mV. This response was inhibited by tetraethylammonium (20 mM) or Cs⁺ in the patch pipette solution, and the reversal potential of the EPA-induced current followed the K⁺ equilibrium potential in a near Nernstian manner.
3. Under conditions with a Cs⁺-containing pipette solution, both vasopressin and endothelin-1 (100 nM) induced a long-lasting inward current at a holding potential of −60 mV. The reversal potential of these agonist-induced currents was about +0 mV, and was not significantly altered by the replacement of the extracellular or intracellular Cl⁻ concentration, suggesting that the induced current was a cation-selective current (I_cat).
4. La³⁺ and Cd²⁺ (1 mM) completely abolished these agonist-induced I_cat, but nifedipine (10 μM) failed to inhibit it significantly.
5. ω-3 polyunsaturated fatty acids (3100 μM), EPA, DHA and docosapentaenoic acids (DPA), inhibited the agonist-induced I_cat in a concentration-dependent manner. The potency of the inhibitory effect was EPA>DHA>DPA, and the half maximal inhibitory concentration (IC₅₀) of EPA was about 7 μM.
6. Arachidonic and linoleic acids (10, 30 μM) showed a smaller inhibitory effect compared to ω-3 fatty acids. Also, oleic and stearic acids (30 μM) did not show a significant inhibitory effect on I_cat.
7. A similar inhibitory action of EPA was observed when I_cat was activated by intracellularly applied GTPγS in the absence of agonists, suggesting that the site of action of ω-3 fatty acids is not located on the receptor.
8. These results demonstrate that ω-3 polyunsaturated fatty acids can activate a K⁺ current and also effectively inhibit receptor-mediated non-selective cation currents in rat A7r5 vascular smooth muscle cells. Thus, the data suggest that ω-3 fatty acids may play an important role in the regulation of vascular tone.