Intraamniotic Ethyl Docosahexaenoate Administration Protects Fetal Rat Brain from Ischemic Stress

Abstract: Studies were conducted on the prenatal rat given a single intraamniotic injection of ethyl docosahexaenoate (Et-DHA; 9.6–12 mmol per fetus) or subjected to an n-3 fatty acid-deficient diet to assess the role of docosahexaenoate on oxidative stress during episodes of ischemia. A time-dependent decrease in the ability of brain slices from animals treated with Et-DHA to produce thiobarbituric acid-reactive substance (TBARS), most pronounced after 1 day (from 58.1 ± 4.22 to 15.9 ± 1.6 nmol/mg of DNA), was noticed on stimulation with Fe²⁺. Brain slices from fetuses treated for 1 day with Et-DHA and those from untreated fetuses produced TBARS levels of 46.7 ± 6.5 and 114.8 ± 10.8 nmol/mg of DNA, respectively, after a 20-min occlusion of the fetal-maternal circulation at embryonic day 20, suggesting a protective effect of Et-DHA. The protective effect of a single dose of Et-DHA in utero remained high up to 3 days after injection ( p < 0.001) and was long-lasting, yet not significant, up to 3 days following birth. In agreement with a reduction in TBARS production by slices, the endogenous levels of TBARS in brains of Et-DHA-treated animals were lower than in the controls. Et-DHA-injected fetuses exhibited significantly higher levels of esterified DHA than the non-injected controls. n-3-deficient diet given to dams for 2 weeks before birth did not affect the levels of TBARS production in control fetal brain slices but abolished the increase caused by ischemia. Et-DHA administration for 24 h to n-3-deficient fetuses reduced the amount of TBARS produced by the fetal brain slices from 49.1 ± 8.5 to 31.7 ± 4.1 nmol/mg of DNA. A protective effect from oxidative damage after postischemic oxidative stress in fetal brain following DHA supplements is suggested, whereas the effect of n-3 fatty acid deficiency in this regard is more ambiguous.     

Enhanced free radical scavenging and decreased lipid peroxidation in the rat fetal brain after treatment with ethyl docosahexaenoate

In order to explore possible mechanisms to explain previously observed decreases in fetal brain lipid peroxidation (LPO) following intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to near term fetuses, the hydroxyl radical trapping capacity of Et-DHA treated fetal brain preparations was compared to control ethyl oleate injected fetuses by electron spin resonance using 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) probe. Lipid extracts from control brains showed little hydroxyl radical scavenging activity, whereas those from the Et-DHA injected animals exhibited an almost 70% decrease in the amount of DMPO-OH adducts. A marked decrease (58%) in LPO formation was noticed in the Et-DHA treated animals compared to controls. The Et-DHA treatment related trapping capacity resided in the phospholipid fraction of the lipid extract, which was enriched in both docosahexaenoic acid and aminophospholipid contents. The decreased LPO production, as well as increased production of prostaglandin E(2) and nitric oxide by the fetal brain following Et-DHA administration, could be mimicked by a synthetic quinone possessing both hydroxyl radical producing and LPO propagation inhibiting properties. The data are consistent with the possibility that the neuroprotective effect of Et-DHA might be due to possible free radical scavenging ability of the brain tissue and interference with LPO propagation.     

Oxidant-increased proteolysis in rat liver slices: effect of bromotrichloromethane, antioxidants and effectors of proteolysis

Proteolysis and lipid peroxidation were evaluated in rat liver slices incubated in the presence of the oxidant bromotrichloromethane and effectors of proteolysis. Proteolysis was evaluated by S-amino acids and lipid peroxidation by thiobarbituric acid-reactive substances (TBARS) released into the incubation medium. The increased release of S-amino acids by BrCl3C depended on incubation time and oxidant concentration. S-Amino acid release increased 30% over control value and TBARS increased from 22 to 124 nmol/g liver by incubation for 120 min with 1 mM BrCl3C. Release of S-amino acids and TBARS was decreased when liver slices were treated with nor-dihydroguaiaretic acid (NDG), butylated hydroxyanisole (BHA), Trolox C, or N,N'-diphenyl-1,4-phenylenediamine (DPPD) immediately prior to addition of oxidant, suggesting participation of lipid-soluble free radicals. Oxidant-induced release of S-amino acids but not of TBARS was decreased by mannitol, suggesting participation of hydroxyl radical or a species with similar reactivity; and by superoxide dismutase and catalase, suggesting participation of superoxide and hydrogen peroxide, respectively. The decrease of S-amino acid release by sodium fluoride, sodium arsenate, 2,4-dinitrophenol, chloroquine, leupeptin, phenylmethylsulfonyl fluoride, EDTA and o-phenanthroline was variable, suggesting the presence in liver of several proteases to remove oxidatively-modified proteins.     

Damage to protein synthesis concurrent with lipid peroxidation in rat liver slices: effect of halogenated compounds, peroxides, and vitamin E1

Protein synthesis and lipid peroxidation were evaluated in rat liver slices incubated in the presence of oxidants and protein synthesis inhibitors. Protein synthesis by rat liver slices was evaluated by [3H]leucine incorporation into the trichloroacetic acid (TCA)-insoluble material, and lipid peroxidation was evaluated by thiobarbituric acid-reactive substances (TBARS) released into the incubation medium. Protein synthesis inhibition by bromotrichloromethane (BrCCl3) or t-butyl hydroperoxide (t-BOOH) depended on the incubation time and oxidant concentration. [3H]Leucine incorporation was decreased to 20 and 47% of control values and TBARS were enhanced from the control value of 16.9 to 45.3 and 62.5 nmol/g of liver by incubation for 1 h with 1 mM BrCCl3 and t-BOOH, respectively. Following incubation, both protein synthesis damage and lipid peroxidation were decreased in control and oxidant-treated slices prepared from rats injected with 200 mg of DL-alpha-tocopherol/kg of body wt. Release of lactate dehydrogenase was not enhanced by oxidant treatment. Protein synthesis inhibitors reversibly decreased [3H]leucine incorporation, but the effect of oxidants on protein synthesis was irreversible. Cumene hydroperoxide and methyl ethyl ketone peroxide, but not hydrogen peroxide, damaged protein synthesis and induced lipid peroxidation. The ability of carbon tetrabromide, benzyl chloride, bromoform, bromobenzene, carbon tetrachloride, chloroform, dichloromethane, and bromochloromethane to inhibit protein synthesis was correlated with their ability to induce lipid peroxidation, and with their LD50. The results suggest that oxidant-induced lipid peroxidation and protein synthesis damage occurred concurrently, and that protein synthesis inhibition may be involved in cell injury or death mediated by free radicals.     

Halogenated compounds as inducers of lipid peroxidation in tissue slices

Twenty-seven halogenated compounds were screened as potential inducers of lipid peroxidation in rat liver, kidney, spleen, and testes slices. In addition to the known lipid peroxidation inducers--carbon tetrachloride and bromotrichloromethane--the novel compounds carbon tetrabromide, p-bromobenzyl bromide, and benzyl bromide increased lipid peroxidation in each of the tissues studied. Lipid peroxidation was measured by release of thiobarbituric acid-reactive substances (TBARS) from the tissue slices. The amount of TBARS released from liver slices incubated with bromotrichloromethane, carbon tetrabromide, dichloromethane, bromobenzene, chloroform, bromoform, benzyl chloride, bromochloromethane, and carbon tetrabromide correlated with the lethality of these compounds as evaluated by their oral LD50 in rats. The lethality of a number of the compounds tested did not correlate with their capacity to induce lipid peroxidation.     

The roles played by crucial free radicals like lipid free radicals, nitric oxide, and enzymes NOS and NADPH in CCl(4)-induced acute liver injury of mice

Mice were administered a single dose of carbon tetrachloride (CCl(4)) to induce acute liver injury. We found that lactate dehydrogenase (LDH) and glutamic pyruvic transaminase (GPT) levels in serum, as well as the level of thiobarbituric acid reaction substances (TBARS) in liver homogenate increased significantly in a manner both dose dependent and time dependent after CCl(4) administration. Such results suggest that the liver is susceptible to CCl(4) treatment and that lipid peroxidation is associated with CCl(4)-induced liver injury. The spin-trapping electron paramagnetic resonance (EPR) method was used to detect nitric oxide (NO) level in liver. The chemiluminescence method was also employed to measure the NO(2)(-)/NO(3)(-) concentration in serum. The NO levels in liver tissues and NO(2)(-)/NO(3)(-) concentration in serum were found to decrease significantly both in a dose-dependent manner and in time course after CCl(4) treatment. The nitric oxide synthase (NOS) II activity in the liver, in contrast, was found to increase significantly. Our study suggests that not only should the expression of NOS be analyzed but NO organ and blood concentration must be measured in the study of diseases involving nitric oxide. L-arginine treatment had no significant effect on the liver function of CCl(4)-treated mice. It was found that NO donor sodium nitroprusside (SNP; 50 or 100 microg/kg) treatment resulted in decreases of LDH, GPT, and TBARS levels, leading to a protective effect on CCl(4)-treated mice. On the other hand, N(G)-nitro-L-arginine methyl ester (L-NAME, 100 or 300 mg/kg) treatment caused more severe liver damage. Moreover, we have found in an in vitro EPR study that SNP could scavenge lipid peroxyl radical LOO&z.rad;. The above results together suggest that NO may protect CCl(4)-induced liver injury through scavenging lipid radical, inhibiting the lipid peroxidation chain reaction. On the basis of our analysis, we put forth two explanations for the stated discrepancy between NOS II and NO production: (i) NO was used up gradually in terminating lipid peroxidation and (ii) NADPH was depleted (on the basis of correlation evidence only).     

Docosahexaenoic acid-deficient phosphatidyl serine and high alpha-tocopherol in a fetal mouse brain over-expressing Cu/Zn-superoxide dismutase

The over-expressed Cu/Zn-superoxide dismutase (Cu/Zn-SOD) gene has been found in some circumstances phenotypically deleterious and associated with oxidative injury-mediated aberrations while in other studies it was considered neuroprotective. In this work we examine a number of biochemical markers in fetal and adult brain from transgenic (tg) mice expressing the human Cu/Zn-SOD gene, which may determine this dual characteristic. These markers include the polyunsaturated fatty acid (PUFA) profile in discrete phospholipid species, the alpha-tocopherol levels, a marker for lipid anti-oxidant status, and thiobarbituric acid reactive substance (TBARS), a marker for the tissue oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg and nontransgenic (ntg) animals of either fetal or adult brain. Serine-phosphoglycerides, however, showed a marked decrease from 20. 07+/-0.53 to 14.92+/-0.87 wt% and 14.52+/-1.15 wt% in docosahexaenoic acid (DHA; 22:6 n3), in the tg 51 and tg 69 fetal brains, respectively, but not in the comparable adult tissues. The alpha-tocopherol levels were significantly higher in the fetal compared to the adult brain. There were no differences in the anti-oxidant levels between the ntg and tg fetal brains, but there were differences in the adult animals; the tg mice were higher by at least two-fold than the control animals. The basal TBARS in the tg 51 fetal brain was 35% lower than that of ntg mouse and in the presence of Fe(2+), brain slices from the former released less TBARS (57% reduction) into the medium than the latter. These results suggest that higher dosages of Cu/Zn-SOD gene are compatible with increased alpha-tocopherol levels, reduced basal TBARS levels and a DHA deficiency in the fetal, but not the adult, tg brain.     

Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes

Liver slices were used to measure lipid peroxidation induced by bromotrichloromethane, tert-butyl hydroperoxide (t-BOOH), or ferrous iron. The responses of liver homogenates and microsomes to oxidative conditions were compared with the response of tissue slices. Lipid peroxidation was evaluated by the production of thiobarbituric acid-reactive substances (TBARS). As was observed in homogenates and microsomes, TBARS production by liver slices depended upon the amount of tissue, the incubation time, inducer, the amount of inducer, and the presence of antioxidant. Control liver slices incubated at 37 degrees C for 2 h produced 19 nmol of TBARS per g of liver. When slices were incubated in the presence of 1 mM BrCCl3, 1 mM t-BOOH, or 50 microM ferrous iron, TBARS production increased 4.6-, 8.2-, or 6.7-fold over the control value, respectively. Comparable induction of TBARS by liver homogenates and microsomes was observed when these preparations were incubated with the same inducers. Addition of 5 microM butylated hydroxytoluene (BHT) prevented the induction of TBARS by 50 microM ferrous iron by liver slices. The results indicate the usefulness of tissue slices to measure lipid peroxidation. The usefulness of tissue slices is emphasized when a number of compounds or tissues are studied and tissue integrity is desired as in toxicological, pharmacological, and nutritional studies where reduced numbers of experimental animals is a relevant issue.     

延龄草对LPS诱导大鼠肝损伤的保护作用及机制

目的：研究延龄草（TTM）对脂多糖（LPS）诱导大鼠氧化应激与肝损伤的保护作用。方法：SD大鼠60只,按体重随机分成TTM高、中、低剂量组、模型组、地塞米松磷酸钠（DEX）对照组及空白对照组（n=10）。TTM高、中及低剂量组按（8、4、2） g/（kg·d） TTM灌胃,模型组、DEX对照组及空白对照组灌胃等量蒸馏水,每隔5 d,TTM高、中、低剂量组、模型组、DEX对照组按1 mg/kg腹腔注射LPS,DEX对照组同时腹腔注射DEX（5 mg/kg）,空白对照组注射等量生理盐水。30 d后,测定大鼠胸腺指数、脾脏指数,对血清一氧化氮合酶（NOS）、超氧化物歧化酶（SOD）活性与一氧化氮（NO）、谷胱甘肽（GSH）、硫代巴比妥酸反应产物（TBARS）、白细胞介素6（IL-6）、IL-10及肿瘤坏死因子α（TNF-α）含量,肝组织SOD、谷胱甘肽过氧化氢酶（GSH-Px）活性与GSH、TBARS含量进行检测。结果：与模型组相比,TTM高剂量组在（19~30） d体重显著降低（P<0.05）,TTM高、中、低剂量组胸腺指数,TTM高剂量组脾脏指数显著降低（P<0.05）,TTM高、中、低剂量组血清NOS活性与TBARS、NO含量显著降低（P<0.05）,TTM高剂量组血清SOD活性及中、高剂量组GSH含量显著上升（P<0.05）,TTM高、中剂量组血清IL-6、TNF-α含量显著降低,IL-10含量显著升高（P<0.05）,TTM中、高剂量组肝脏TBARS含量显著降低,TTM各剂量组肝脏SOD活性与中、高剂量组GSH-Px活性,高剂量组GSH含量显著升高（P<0.05）。结论：TTM对LPS所致大鼠的胸腺、脾脏萎缩有一定的延缓作用,能有效降低血清中NOS活性,减少NO生成,提升SOD、GSH-Px活性与GSH含量,减轻脂质过氧化,降低IL-6、TNF-α过量分泌、提升IL-10含量,有抗炎护肝的功能。     

Lipopolysaccharide-induced increase of prostaglandin E(2) is mediated by inducible nitric oxide synthase activation of the constitutive cyclooxygenase and induction of membrane-associated prostaglandin E synthase

NO produced by the inducible NO synthase (NOS2) and prostanoids generated by the cyclooxygenase (COX) isoforms and terminal prostanoid synthases are major components of the host innate immune and inflammatory response. Evidence exists that pharmacological manipulation of one pathway could result in cross-modulation of the other, but the sense, amplitude, and relevance of these interactions are controversial, especially in vivo. Administration of 6 mg/kg LPS to rats i.p. resulted 6 h later in induction of NOS2 and the membrane-associated PGE synthase (mPGES) expression, and decreased constitutive COX (COX-1) expression. Low level inducible COX (COX-2) mRNA with absent COX-2 protein expression was observed. The NOS2 inhibitor aminoguanidine (50 and 100 mg/kg i.p.) dose dependently decreased both NO and prostanoid production. The LPS-induced increase in PGE(2) concentration was mediated by NOS2-derived NO-dependent activation of COX-1 pathway and by induction of mPGES. Despite absent COX-2 protein, SC-236, a putative COX-2-specific inhibitor, decreased mPGES RNA expression and PGE(2) concentration. Ketoprofen, a nonspecific COX inhibitor, and SC-236 had no effect on the NOS2 pathway. Our results suggest that in a model of systemic inflammation characterized by the absence of COX-2 protein expression, NOS2-derived NO activates COX-1 pathway, and inhibitors of COX isoforms have no effect on NOS2 or NOS3 (endothelial NOS) pathways. These results could explain, at least in part, the deleterious effects of NOS2 inhibitors in some experimental and clinical settings, and could imply that there is a major conceptual limitation to the use of NOS2 inhibitors during systemic inflammation.     

The cyclooxygenase hydroperoxide product PGG(2) activates synaptic nitric oxide synthase: a possible antioxidant response to membrane lipid peroxidation

Nitric oxide is a potent inhibitor of membrane lipid peroxidation. It is unknown, however, whether nitric oxide synthase (NOS) activity increases under conditions of membrane lipid peroxidation. Importantly, cyclooxygenase (COX)-catalyzed peroxidation of arachidonic acid is well-established to be increased by lipid hydroperoxides. The results of the present study demonstrate that the COX hydroperoxide product prostaglandin G(2) (PGG(2)) greatly stimulated NOS activity in synaptosomal membrane fractions from rat brain in a dose-dependent (EC(50) = 0.2 microM) manner in the presence of ATP and the antioxidant urate. NOS activation was also produced, albeit to a lesser extent, by 15-hydroperoxyeicosatetraenoic acid (15-HPETE) but not by the corresponding hydroxy compounds PGH(2) and 15-HETE or by hydrogen peroxide. These findings demonstrate that PGG(2)-activated synaptic NOS by a hydroperoxide-mediated pathway and support the view that NOS activation may be an important physiological response to lipid peroxidation.     

Lipid peroxidation and growth inhibition of human microvascular endothelial cells

Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n - 3), arachidonic acid (C20:4. n - 6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.     

Signal transduction pathways involved in non-genomic action of estrone on vascular tissue

Previously we demonstrated that estrone non-genomically regulates rat aortic NOS and COX activity and that this effect depends on ovarian activity. The purpose of the present study was to characterize this effect and investigate the participation of phospholipase C and phophatidylinositol-3-kinase system in the intracellular transduction pathway involved in the response. Using aortic strips isolated from female fertile rats we showed that estrone stimulate nitric oxide synthase and cyclooxygenase in a short time interval (5-20 min), and that NO production was dependent in part on PGI2 production since 1 microM indomethacin significantly reduced this free radical production. Injection of 17-beta-estradiol to ovariectomized rats restored tissue capacity to rapidly increase NO production in response to "in vitro" treatment with 1 nM estrone. We also demonstrated that in aortic strips isolated from intact animals estrone elicited a rapid phospholipase C activation, inducing a biphasic increase in diacylglycerol generation (peaking at 45 s and 5 min). The presence of protein kinase C inhibitor chelerythrine did not prevent the increase of NO released in response to hormone treatment. We proved that PI3K-Akt system does not mediate NOS and COX activation. However, PLC activation was dependent on PI3K since presence of LY 294002 in the incubation medium abolished estrone-induced DAG increment. We concluded that, estrone rapid action on vascular tissue involves a cross talk between NOS and COX system, and the activation of PLC/DAG/PKC transduction pathways.     

The effect of iron overload on urinary excretion of immunoreactive prostaglandin E2

The effect of in vivo lipid peroxidation on the excretion of immunoreactive prostaglandin E2 (PGE2) in the urine of rats was studied. Weanling, male Sprague-Dawley rats were fed a vitamin E-deficient diet containing 10% tocopherol-stripped corn oil (CO) or 5% cod liver oil (CLO) with or without 40 mg dl-alpha-tocopheryl acetate/kg. To induce a high, sustained level of lipid peroxidation, some rats were injected intraperitoneally with 100 mg of iron as iron dextran after 10 days of feeding. Iron overload stimulated in vivo lipid peroxidation in rats, as measured by the increase in expired ethane and pentane. Dietary vitamin E reversed this effect. Rats fed the CLO diet excreted 9.5-fold more urinary thiobarbituric acid-reactive substances (TBARS) than did rats fed the CO diet. Iron overload increased the excretion of TBARS in the urine of rats fed the CO diet, but not in urine of rats fed the CLO diet. Dietary vitamin E decreased TBARS in the urine of rats fed either the CO or the CLO diet. Iron overload decreased by 40% the urinary excretion of PGE2 by rats fed the CO diet, and dietary vitamin E did not reverse this effect. Iron overload had no statistically significant effect on urinary excretion of PGE2 by rats fed the CLO diet. A high level of lipid peroxidation occurred in iron-treated rats, as evidenced by an increase in alkane production and in TBARS in urine in this study, and by an increase in alkane production by slices of kidney from iron-treated rats in a previous study [V. C. Gavino, C. J. Dillard, and A. L. Tappel (1984) Arch. Biochem. Biophys. 233, 741-747]. Since PGE2 excretion in urine was not correlated with these effects, lipid peroxidation appears not to be a major factor in renal PGE2 flux.     

The effects of nitric oxide on prostanoid production and release by human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) express and synthesize both constitutive and inducible nitric oxide synthase (NOS) and cyclo-oxygenase (COX) enzymes, and have been extensively used as an in vitro model to investigate the role of these enzymes in the patho-physiology of placenta-fetal circulation. In this study we investigated the role of NO in regulating prostanoid production and release from HUVEC. Both untreated and IL-1beta-treated HUVEC were exposed to various NOS inhibitors and NO donors in short-term (1 or 3 hours) experiments, and the effects on prostanoid production were evaluated through the measurement of prostaglandins (PG) I2, E2 and F2alpha released in the incubation medium. We found that the inhibition of inducible NOS but not endothelial NOS antagonizes the IL-1beta-induced increase in PGI2 release. However, NOS inhibitors do not modify baseline PGI2 production. Pharmacological levels of NO, obtained with various NO donors, inhibit basal and IL-1beta-stimulated PG release.     

Halogenated hydrocarbon and hydroperoxide-induced peroxidation in rat tissue slices

Tissue slices were used to compare relative peroxidation capacity of bromotrichloromethane (BrCCl₃) and t-butyl hydroperoxide (BHP) by measurement of both peroxidation products and biochemical indices of damage. In liver and testes slices, BHP increased thiobarbituric acid reactive-substances (TBARS) and total aldehydes, measured as cyclohexanedione-reactive substances (CHDRS), to a greater extent than did an equimolar amount of BrCCl₃. GSH was decreased more by BHP than by BrCCl₃. Neither compound released lactate dehydrogenase or glutamic-pyruvicf transaminase from liver slices. Treatment of rats with cynamide, an aldehyde dehydrogenase inhibitor, increased the total CHDRS in liver slices and medium after incubation with BHP or BrCCl₃. HPLC of the CHDRS showed hexanal and propanal increased to the greatest extent. The hydroperoxide, BHP, which does not require metabolism to an active species, was a better initiator of peroxidation than the halogenated hydrocarbon, BrCCl₃, which must be metabolized to a radical species before it can initiate peroxidation.     

NO and prostaglandin interactions during hemodynamic stress in the fetal ovine pulmonary circulation

Nitric oxide (NO) and prostacyclin (PGI(2)) are potent fetal pulmonary vasodilators, but their relative roles and interactions in the regulation of the perinatal pulmonary circulation are poorly understood. We compared the separate and combined effects of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibition during acute hemodynamic stress caused by brief mechanical compression of the ductus arteriosus (DA) in chronically prepared fetal lambs. Nitro-L-arginine (L-NNA; NOS antagonist), meclofenamate (Mec; COX inhibitor), combined drugs (L-NNA-Mec), or saline (control) was infused into the left pulmonary artery (LPA) before DA compression. In controls, DA compression decreased pulmonary vascular resistance (PVR) by 43% (P < 0.01). L-NNA, but not Mec, treatment completely blocked vasodilation and caused a paradoxical increase in PVR (+31%; P < 0.05). The effects of L-NNA-Mec and L-NNA on PVR were similar. To determine if the vasodilator effect of PGI(2) is partly mediated by NO release, we studied PGI(2)-induced vasodilation before and after NOS inhibition. L-NNA treatment blocked the PGI(2)-induced rise in LPA blood flow by 73% (P < 0.001). We conclude that NO has a greater role than PGs in fetal pulmonary vasoregulation during acute hemodynamic stress and that PGI(2)-induced pulmonary vasodilation is largely mediated by NO release in the fetal lung.     

Lipid Peroxides are Generated by the Fetal Rat Brain After Episodes of Global Ischemia in Utero

Complete arrest of maternal-fetal blood supply for up to 30 min caused a time-dependent increase in the endogenous levels of lipid peroxides (LPO) in fetal brain and liver extracts and fetal blood and amniotic fluids as indicated by the appearance of thiobarbituric acid reactive substances (TBARS), A steady increase of TBARS from 48.0 ± 2.2 pmol/g wet weight to 75.0 ± 5.6 pmol/g wet weight up to 30 min restriction was noticed in the fetal brain. The fetal liver TBARS values increased by approximately 69% after 5 min restriction and remained high, above the control level, for 30 min. After two days reperfusion following 30 min restriction, the TBARS levels in the fetal brain were 1.8 fold higher above the control, while those of the liver returned to control values. The levels of the lipid-soluble antioxidant -tocopherol were reduced by about 40% and 50% in the placenta and brain tissues after 5 min restriction, respectively. Slices of fetal brain incubated at 37°C in DMEM under oxygen in the presence of 50 M Fe²⁺ were able to generate LPO in a time- and tissue concentration-dependent manner. After 15 min incubation, about 6.3 fold increase in total TBARS levels could be measured in the presence of 50 M Fe²⁺, most of which was released in the medium. The iron chelator desferrioxamine (25 M) and the antioxidant -tocopherol (10 M) added to the incubation medium, each inhibited by about 88% TBARS production. After 20 min episode of ischemia, fetal brain slices released into the medium 138.5 ± 9.8 nmol/15 min/mg DNA compared to 75.9 ± 4.5 nmol/15 min/mg DNA released by the sham preparations. After 3 h reperfusion, brain slices from fetuses exposed to 20 min ischemia continued to produce TBARS above control levels, whereas those of brief ischemia (5 min) returned to control levels. The data indicate that the limited resistance of the fetal brain to brief, rather than prolonged, periods of ischemia, is likely due to a lack of free FA for LPO generation, rather than the levels of tissue lipid antioxidants.     

Irradiation of the rabbit cornea with UVB rays stimulates the expression of nitric oxide synthases-generated nitric oxide and the formation of cytotoxic nitrogen-related oxidants

Until now, the role of nitric oxide (NO) in cornea irradiated with UVB rays remains unknown. Therefore, we investigated nitric oxide synthase isomers (NOS), enzymes that generate NO, nitrotyrosine (NT), a cytotoxic byproduct of NO, and malondialdehyde (MDA), a byproduct of lipid peroxidation, in rabbit corneas repeatedly irradiated with UVB rays (312 nm, 1x daily for 6 days, the dose per day 1.01 J/cm2) using immunohistochemical methods. The biochemical measurement of nitrite and nitrate has been used for the indirect investigation of NO concentration in the aqueous humor. Results show that in contrast to normal corneas, where of the NOS isomers only endothelial nitric oxide synthase (NOS3) was expressed in a significant amount (in the epithelium and endothelium), in irradiated corneas all NOS isomers (also brain nitric oxide synthase, NOS1, and inducible nitric oxide synthase, NOS2) as well as an indirect measure of ONOO-formation and MDA were gradually expressed, first in the epithelium, the endothelium and the keratocytes beneath the epithelium and finally in the cells of all corneal layers and the inflammatory cells that invaded the corneal stroma. This was accompanied by an elevated concentration of NO in the aqueous humor. In conclusion, repeated irradiation with UVB rays evoked the stimulation of NO production, peroxynitrite formation (demonstrated by NT residues) and lipid peroxidation (evaluated by MDA staining).     

铅胁迫下三叶鬼针草内源一氧化氮的生成及其对氧化损伤的缓解效应

对不同浓度铅(Pb)胁迫下三叶鬼针草(Bidens pilosa L.)叶、茎和根中内源一氧化氮(NO)和活性氧(ROS)的生成机制及根系活力的变化,内源NO对Pb胁迫下三叶鬼针草幼苗氧化损伤的缓解效应进行了研究。结果显示,在0~1000 mg/L范围内,随着Pb浓度的增加,叶片中NO含量呈升高趋势,根中NO含量呈先升高后降低的趋势,但仍高于对照,Pb浓度在0~400 mg/L范围内,茎中NO含量与对照持平,Pb浓度大于600 mg/L时,茎中NO含量低于对照;600 mg/L Pb处理能显著增强叶、茎和根中一氧化氮合成酶(NOS)和硝酸还原酶(NR)活性,显著增加叶和茎中亚硝酸根离子(NO_2~-)和类胡萝卜素(Car)含量,NOS、NR、NO_2~-和Car均能促进叶片中内源NO的生成,NOS是根中内源NO生成的主要途径。Pb胁迫使超氧阴离子(O_2~(·-))产生速率、过氧化氢(H_2O_2)含量、丙二醛(MDA)含量和相对电导率(REC)显著升高,从而造成幼苗严重的膜脂过氧化损伤,而胁迫诱发产生的NO能降低根中ROS的产生,促进幼苗根系活力,进而缓解胁迫造成的膜脂过氧化损伤。