Effects in vitro and in vivo of thioureas on acetylcholine esterase activity of rat tissues

In view of data available that accumulation of acetylcholine in the lung leads to pulmonary edema formation, the effects of thiourea (TU), phenylthiourea (PTU) and α-naphthylthiourea (ANTU) on lung acetylcholine esterase (AchE) activity were examined in vitro and in vivo. ANTU and PTU produced a concentration-dependent inhibition of lung AchE activity in vitro. The Sped₅₀ values of ANTU and PTU were 2.8 × 10^?4 and 5 × 10^?4 M respectively. No significant inhibition in AchE activity of the lung occurred in response to any concentration of TU. The mechanisms of AchE inhibition due to ANTU appeared to be competitive. The apparent K_m for lung AchE averaged 7.23 × 10^?4 M and the K_i for ANTU averaged 1.11 × 10^?4 M. ANTU at 5 × 10^?4 M inhibited the AchE activity of all examined tissues in vitro. An edema-producing dose of ANTU had no effect on AchE activity of any tissue except the brain. A failure to demonstrate the inhibitory effect in vivo of ANTU on AchE activity has been discussed in this paper. Pretreatment with atropine offered no protection against the pulmonary edema-producing ability of ANTU. The data collected in the present report indicate that inhibition of lung AchE is probably not the mechanism of ANTU-induced pulmonary edema. The significance of brain AchE inhibition in response to an edema-producing dose of ANTU is not known.     

氟喹诺酮类药物对大鼠肝外组织GST、GR及GSH-Px活性的影响

目的探讨氟喹诺酮类药物对大鼠肺、脑、肾、小肠中谷胱甘肽S-转移酶、谷胱甘肽还原酶及谷胱甘肽过氧化物酶活性的影响。方法实验分为体外和体内两部份。从大鼠的肺、脑、肾、小肠制备S9,分别测定给予环丙沙星、妥氟沙星、司帕沙星前后三种谷胱甘肽相关酶的酶活性。结果体内实验和体外实验结果显示,三种药物对各组织的G ST活性较对照组均有不同程度的抑制(P<0.05),但体外实验与体内实验中各药物组与对照组的差异有所不同,且不同组织中G ST活性降低的程度也不同;药物组各组织的GR和G SH-Px活性较对照组有降低,但大多无显著性差异。结论氟喹诺酮类药物对肝外组织的G ST活性有一定的抑制作用,且对不同组织的G ST活性的抑制作用存在差别,对GR和G SH-Px活性有抑制的趋势。     

Effect of melatonin on enzyme activities of glutathione reductase from human erythrocytes in vitro and from rat erythrocytes in vivo

The in vivo and in vitro effects of melatonin on enzyme activity of glutathione reductase (Glutathione: NADP(+) oxidoreductase, EC 1.8.1.7; GR) were investigated in this study. Glutathione reductase was purified from human erythrocytes 5.823-fold with a yield of 24% by ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose 4B and gel filtration chromatography on Sephadex G-200. Enzyme activity was determined by the Calberg and Mannervik method using a spectrophotometer at 340 nm. For in vitro experiments, the enzyme activity increased at 0.02 mM and decreased at 0.08 mM melatonin concentration and reached a plateau above 0.08 mM. Melatonin was administered 10 mg/kg intraperitoneally (ip) and had a stimulatory effect on the enzyme. In vivo studies were performed for melatonin in Sprague-Dawley rats and time-dependent effects were demonstrated. Glutathione reductase activity in erythrocytes was increased more by melatonin at 1 and 3 h. These results indicate that pharmacological levels of melatonin increased enzyme activity in erythrocytes. They also indicate that melatonin may be pharmacologically useful in patients with a deficiency of the enzyme in red blood cells causing hemolytic anemia.     

Further investigation on aldehyde reductase activity in ageing rat tissues

Aldehyde reductase (AR) activity was measured in whole brain, different brain areas, kidney and duodenum of 23-26 months old rats and then compared to matched animals of 3 months. No significant changes in AR activity were found in whole brain of old rats with D-glucose as substrate. A significant increase in AR activity in old rats was found in striatum (12%), cerebellum (26%), midbrain (6%), brainstem (19%) and hypothalamus (13%) with p-nitrobenzaldehyde, in striatum (14%), midbrain (10%), brainstem (52%), hypothalamus (9%) and hippocampus (20%) with D-glucuronate, in cerebellum (21%) with D-xylose, in striatum (43%), cerebellum (19%) and brainstem (22%) with DL-glyceraldehyde, and in brainstem (15%) with pyridine 3-aldehyde. A significant decrease in AR activity in old rats was found in midbrain (16%), brainstem (19%) and hypothalamus (19%) with D-xylose, and in midbrain (13%) with DL-glyceraldehyde. A significant decrease in AR activity in old rats was found in kidney with p-nitrobenzaldehyde (30%), D-xylose (31%), DL-glyceraldehyde (33%), pyridine 3-aldehyde (27%) and in duodenum with p-nitrobenzaldehyde (21%), but a significant increase was found in duodenum with pyridine 3-aldehyde (29%). Preliminary experiments show an increase (42%) of sorbitol dehydrogenase activity in whole brain of old rats. The present results clearly indicate that the AR activity increase in the brain of old rats is the high-Km form of aldehyde reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of quinone reductase and glutathione transferase in rat tissues by juglone and plumbagin

The ability of the naturally-occurring naphthoquinone derivatives, juglone and plumbagin, to increase tissue activities of the Phase II detoxification enzymes quinone reductase (QR) and glutathione transferase (GT) has been investigated in rats. Groups of female Sprague-Dawley rats were dosed by oral intubation on 5 consecutive days with either juglone or plumbagin at 12.5, 25, 50, 75, 100 or 125 mumoles/kg/day. The animals were then killed and the activities of QR and GT determined in tissue homogenates. The naphthoquinone derivatives had no significant effect on enzyme activities in the liver, spleen, heart, lung or urinary bladder. Increases in the activities of one or both enzymes were recorded, however, in the caecum, kidney, forestomach, duodenum, colon, glandular stomach and jejunum. The possibility that induction of Phase II enzymes could contribute to the previously-reported ability of juglone and plumbagin to protect animals against chemically-induced intestinal neoplasia is discussed.     

The effects of glutathione and vitamin E on iron toxicity in isolated rat hepatocytes

This study examined the acute toxicity of ferrous sulfate on rat hepatocyte suspensions, the correlation between lipid peroxidation and cell death, and the roles of glutathione and vitamin E in protecting against iron toxicity. Incubation with ferrous sulfate for 2 h produced lipid peroxidation, but did not decrease cell viability in the hepatocytes. When diethyl maleate (DEM) was added to deplete cellular glutathione concentrations, ferrous sulfate treatment (2.0-5.0 mM) did cause cell death and lipid peroxidation developed more extensively, suggesting that iron-mediated hepatotoxicity is influenced by glutathione content. Reduced glutathione (GSH), N-acetylcysteine (NAC) and alpha-tocopherol (vitamin E), alone and in combination, were added to hepatocyte suspensions in an attempt to protect cells against iron-induced damage. In iron-DEM-treated cells, GSH and NAC treatment increased viability by 43 and 36%, respectively, but only the combination of the two agents reduced lipid peroxidation (53% decrease). Vitamin E treatment reduced lipid peroxidation by 39% and also increased cell viability by 12%. The greatest protection against iron-induced lipid peroxidation occurred with the combination of GSH, NAC and vitamin E, which reduced lipid peroxidation by 94% in iron-treated cells, and by 98% in iron-DEM-treated cells. However, this combination did not prevent iron-induced cell death, although it did increase viability by 18%. These results suggest that iron-induced cell death may not be dependent upon lipid peroxidation, at least in short-term exposures. The results also suggest an interaction between GSH and vitamin E in protecting against lipid peroxidation.     

Effects of methiocarb on lipid peroxidation and glutathione level in rat tissues

Methiocarb is an N-methylcarbamate insecticide used worldwide in agriculture and health programs. The aim of this study was to investigate the possible effects of methiocarb to induce lipid peroxidation (LPO) in tissues of male Wistar rats following single and repeated oral exposures. Animals were divided into six different groups, and methiocarb was administered by orally at doses 25, 10, and 2?mg/kg body weight for 1, 5, and 28 days, respectively. Liver, kidney, brain, and testis tissues were taken from the rats for the biochemical examinations. LPO and reduced glutathione (GSH) levels were determined in the tissues. LPO was significantly increased in liver, kidney, brain, and testis after 1-, 5-, and 28-day treatments of methiocarb. GSH levels were significantly increased in the 1-day period and significantly decreased in the 5- and 28-day periods in all tissues after methiocarb administration. It is concluded that methiocarb may induce LPO and produce disturbances on the GSH levels in liver, kidney, testis, and brain of rats. This suggests that methiocarb-induced toxicity may be associated with oxidative stress to cellular membranes. Further studies are required to better understand the role of oxidative stress on methiocarb-induced toxicity.     

In vitro and in vivo studies of the effect of vitamin E on microsomal cytochrome P450 in rat liver

Administration of a diet supplemented with 0.06% vitamin E acetate to male rats over a 6-week period doubled hepatic microsomal stores of alpha-tocopherol over those in control (vitamin E adequate) rat liver. Total cytochrome P450 content and NADPH-cytochrome P450 reductase activity were significantly elevated in hepatic microsomes from vitamin E-supplemented rats to 111% and 123% of respective control values. Androstenedione 16 alpha-hydroxylase activity was increased in these fractions (2.57 +/- 0.31 nmol product/min/mg protein vs 1.81 +/- 0.38 in controls) whereas activities of the 6 beta-, 7 alpha- and 16 beta-hydroxylase pathways were unchanged. Immunoquantitation of the microsomal 16 alpha-hydroxylase, P450 IIC11, indicated a corresponding increase in the hepatic content of the enzyme. In view of the established antioxidant role of tocopherols, the effects of dietary vitamin E manipulation on the concentration of protein sulphydryl groups and the susceptibility of microsomes to ferric sulphate-ADP-NADPH-mediated lipid peroxidation were also assessed. Dietary supplementation did not influence microsomal protein sulphydryl content (68 +/- 10 nmol glutathione equivalents/mg protein) but decreased the extent of lipid peroxidation produced by the ferric sulphate-ADP-NADPH system in vitro. Further in vitro experiments demonstrated that vitamin E acetate (2 microM) protected protein sulphydryl groups and lipids against peroxidation in control microsomes and partially reduced the associated losses of P450-mediated steroid hydroxylase activities. Western immunoquantitation of P450 IIC11 revealed that exogenous vitamin E acetate protected completely against peroxidation-induced apoprotein loss. These studies establish that the in vitro protective effects of vitamin E acetate against sulphydryl and lipid peroxidation extend to protection of the P450 apoprotein but that enzyme activity is only partially protected. This finding suggests that peroxidation-dependent loss of P450 in vitro is mediated by haem degradation from the P450 holoenzyme and is not directly related to lipid/sulphydryl oxidation. In contrast, the in vivo effects of dietary vitamin E on drug metabolizing enzymes are regulatory in nature and are unrelated to effects on lipid peroxidation.     

Effects of ph on the activity of nicotine and nicotine monomethiodide on the rat diaphragm preparation

Results have been obtained which substantiate the view that it is the univalent nicotinium ion rather than the un-ionized base which acts at the neuromuscular junction.     

Effects of some monoterpenes on bovine lens aldose reductase activity

Thirteen monoterpenes were examined for their effects on the bovine lens aldose reductase activity. The monoterpenes tested in this study showed mild inhibitory effects except 3-carene which did not show any significant effect. At the concentration of 10^?3M (+) Pulegon showed 42% inhibition on lens aldose reductase activity, which is the most potent effect among the tested substance.     

In vitro and in vivo anti-picornavirus activity of some p-benzoylphenoxypyridines

Fifteen p-benzoylphenoxypyridines were initially evaluated for their in vitro activity against rhinoviruses (RV) 1A, 2 and 64 and coxsackie virus (Cox) A21 and for their oral prophylactic and therapeutic activity in Swiss albino mice lethally challenged with Cox A21. One compound, (4-[(5-methylsulfonyl-2-pyridinyl)oxy]phenyl) phenyl methanone, was selected for additional evaluation. These studies showed the compound to possess MIC50 values of less than or equal to 5 micrograms/ml against only 6 of 20 (30.0%) RV serotypes tested. In contrast, the compound was active at concentrations of less than or equal to 5.0 micrograms/ml against 10 of 12 (83.3%) enteroviruses evaluated. In vivo studies showed the compound to significantly protect mice lethally infected with Cox A21 after a single oral dose of 37.5 mg/kg (P less than 0.02) and during a regimen of continuous oral doses of at least 4.7 mg/kg per day (P less than 0.001). Mechanism of action studies indicated that the compound inhibits picornavirus uncoating or some earlier virus-host cell-associated event. Isotopic studies show that (4-[(5-methylsulfonyl-2-pyridinyl)oxy]phenyl) phenyl methanone perturbs HeLa cell macromolecular synthesis at concentrations of as low as 3.12 micrograms/ml. This concentration is only 4-fold higher than the concentration of compound necessary to inhibit Cox A21 RNA synthesis by 90%. This narrow therapeutic ratio limits the potential clinical utility of this compound to all but the most serious picornavirus infections.     

Effects of vitamin E deficiency on vasomotor activity and ultrastructural organisation of rat thoracic aorta.

1. The effects of vitamin E deficiency were evaluated in aortic rings isolated from rats maintained on a diet deficient in vitamin E. 2. Endothelium-dependent vasodilator responses to acetylcholine (ACh) and calcium ionophore, A23187, were reduced in preparations from treated animals, compared to the age-matched controls. The maximal vasodilation to ACh was 66.4 +/- 9 (n = 4) and 38.8 +/- 7 (n = 4) % in control and 10 month-treated preparations, respectively. 3. The endothelium-independent vasodilator responses to sodium nitroprusside as well as the concentration-dependent contractile responses to noradrenaline, did not differ between treated and control preparations. 4. Electron microscopic examination of vascular segments and revealed that, following vitamin E deficiency, normal tissue organisation was disrupted, the endothelial monolayer either not being in contact with the underlying tissue or being absent in most of the areas analysed. 5. It is concluded that during vitamin E deficiency both morphological disruption and functional impairment of endothelium occur without observable modification of muscle cell function and morphology.     

Prevention of cadmium-induced effects on regional glutathione status of rat brain by vitamin E

The effect of vitamin E on the cadmium-induced changes of glutathione metabolism was investigated in different brain regions. Daily intraperitoneal injection of cadmium (0.4 mg/kg) for 30 days significantly decreased the concentration of reduced glutathione (GSH), and the activities of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (GPDH) in the cerebellum, cerebral hemispheres and brain stem of rats. Cadmium elevated the levels of oxidized glutathione (GSSG) in cerebellum and cerebral hemisphere regions only, while the GSH/GSSG ratio decreased in all three brain regions. The only effect of intramuscular injections of vitamin E (5 mg/kg) given on alternate days for 30 days was a slight increase in GSH and GR in the cerebral hemispheres. The simultaneous administration of vitamin E and cadmium prevented cadmium-induced changes in GSH and GSSG levels and in the GSH/GSSG ratio, but the cerebellar GSH remained lowered. Furthermore, vitamin E, with the exception of GR in the cerebral hemispheres, did not prevent cadmium-induced changes in enzyme activities. As the simultaneous injections of vitamin E reduced cadmium-induced alterations in glutathione concentration without having any appreciable effect on the activity of related enzymes, it is suggested that the preventive effect of vitamin E is mediated through its antioxidative effect, saving GSH from oxidative destruction in the brain of cadmium-exposed rats.     

Effects of nicotine on hydroxyl free radical formation in vitro and on MPTP-induced neurotoxicity in vivo

Parkinson’s disease (PD) is one of the most frequent disorders of the basal ganglia. From epidemiological studies there is a controversial discussion on the question whether tobacco smoking is correlated with a decreased incidence of PD. The present study aimed to elucidate the role of nicotine and its potential neuroprotective effects in a rodent model of PD. These effects may be related to an altered hydroxyl radical formation; this possibility was studied in vitro. Nicotine and α-phenyl-N-tert-butyl nitrone (PBN) were examined in a cell-free in vitro Fenton system (Fe³⁺/EDTA + H₂O₂) for their radical scavenging properties using the salicylate trapping method. Salicylic acid (0.5 mM) was incubated in the presence and absence of nicotine or PBN and the main products of the reaction of hydroxyl radicals with salicylic acid, namely 2,3- and 2,5-dihydroxybenzoic acid, were immediately determined using HPLC in combination with electrochemical detection. Nicotine and PBN were both able to significantly reduce hydroxyl radical levels at concentrations of 1, 2.5 and 5 mM. Interestingly, at 5 mM nicotine was able to reduce hydroxyl radical levels significantly more than the radical scavenger PBN (5 mM). To investigate the in vivo effects of nicotine, male C57BL/6 mice were used in the MPTP mouse model of PD. Nicotine (0.1 or 0.4 mg/kg s.c.) was administered twice daily for a period of 14 days. On day 8 a single injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg s.c.) was given as well as an enhanced protocol of nicotine treatment (0.1 or 0.4 mg/kg s.c., 30 min before MPTP and 30, 90, 210, 330, 450, 570 min after MPTP) for a total of seven injections of nicotine. High dosage nicotine treatment significantly increased the MPTP-induced loss of body weight and resulted in a significantly decreased striatal dopamine content and an increased dopamine turnover in comparison with the MPTP-treated controls at day 15. However, the lower dosage of nicotine did not significantly alleviate the MPTP-induced effects, although some parameters showed a slight tendency in this direction. These results demonstrate that in vitro nicotine has radical scavenging properties which might suggest neuroprotective effects. In vivo experiments with nicotine, however, showed that a low dosage of nicotine did not alleviate the MPTP-induced dopamine depletion, but a large dosage even enhanced it. Received: 20 March 1998 / Accepted: 23 June 1998     

Effects of smoking and nicotine on human prostacyclin and thromboxane production in vivo and in vitro

We studied the effects of smoking and nicotine on the production of proaggregatory thromboxane A2 (TxA2), antiaggregatory prostacyclin (epoprostenol, PGI2), and on lipid peroxidation in vivo and in vitro. In the in vivo study, serum concentrations of thromboxane B2 (TxB2), a stable metabolite of TxA2, increased immediately after smoking three cigarettes but not after smoking the equivalent amount of tobacco in a pipe, whereas serum lipid peroxide values did not change in either group. In vitro, nicotine (2 X 10(-3) mol/liter) inhibited pulmonary TxB2 production by 70% and simultaneously stimulated the production of 6-keto-prostaglandin F1 alpha, a stable metabolite of PGI2, by 40%, which suggest that nicotine does not exert its effect at the cyclooxygenase level. During aggregation in platelet-rich plasma, TxB2 production was inhibited by 53% with 2 X 10(-3) mol/liter of nicotine, and during whole blood clotting the inhibition was 34% with 2 X 10(-4) mol/liter of nicotine. Thus the rise in cigarette smokers' serum TxB2 was probably caused by some constituent of cigarette smoke other than nicotine. The increased production of TxA2 following cigarette smoking may provide one explanation for the increased incidence of atherosclerosis and its complications in cigarette smokers.     

Effects of some drugs on rat erythrocyte 6-phosphogluconate dehydrogenase: an in vitro and in vivo study

The in vitro and in vivo effects of some drugs on rat erythrocytes 6-phosphogluconate dehydrogenase were investigated in this study. Rat erythrocyte 6-phosphogluconate dehydrogenase was partially purified with ammonium sulfate precipitation. The enzyme activity was determined by Beutler's method. Some drugs such as ampicillin, amikacin sulfate, and netilmicin sulfate inhibited the enzyme activity in in vitro conditions, while metamizole activated it. The I50 values of the inhibiting drugs were 66.2, 5.836, and 0.963 mM, respectively. For the drugs having low I50 values (drug concentrations which produce 50% inhibition) (amikacin sulfate and netilmicin sulfate), in vivo studies were performed in rats (Sprague-Dawley). Amikacin sulfate at 64 mg/kg inhibited the enzyme activity significantly (p < 0.05) 2 h after dosing. Netilmicin sulfate at 6.4 mg/kg also inhibited the enzyme significantly (p < 0.05) 4 h after dosing. Amikacin sulfate and netilmicin sulfate inhibited rat erythrocyte 6-phospogluconate dehydrogenase both in vivo and in vitro. The enzyme was inhibited in vitro by ampicillin and activated in vitro by metamizole.     

Benefits of vitamin E supplementation to Norplant users--in vitro and in vivo studies

Norplant subcutaneous implantation is a contraceptive method used in Indonesia. Endometrial bleeding is one major reason to discontinue the use of Norplant. Angiogenic response in the endometrium of Norplant users was found to be lower than in women with normal menstrual cycle. This disturbance in the angiogenic process may be caused by an imbalance of pro- and antioxidant processes in the endometrium of Norplant users. The aim of this study is to investigate the effect of vitamin E on the endometrial angiogenic activity and to assess the efficacy of vitamin E supplementation in treating endometrial bleeding in Norplant users. Subjects for this study were selected from Norplant users with an exposure of at least 3 months, with endometrial bleeding and recruited on the basis of fully informed consent. TBA reaction was used to measure degradation products of lipid peroxidation. The endometrial angiogenic response was assayed according to Folkman et al. (Folkman et al., 1989. Nature 239, 58-61). Samples from endometrial biopsies were incubated in vitro with vitamin E or placebo before angiogenic measurement. For in vivo supplementation, vitamin E 200 mg/day, or placebo for 10 days/month were given to the subjects with double blind randomisation. The results showed that the blood levels of TBA-reactive substances were significantly higher in Norplant users than in controls. In the endometrium from Norplant users with bleeding problems, in vitro supplementation of vitamin E resulted in a significantly higher angiogenic score than placebo. Although a highly significant reduction of bleeding days in both groups, vitamin E and placebo, was seen during the 2 months of the study, the number of bleeding days was significantly lower in women treated with vitamin E than with placebo.     

Effects of morphine, in vitro and in vivo, on thyrosine hydroxylase activity in rat brain

The effect of morphine on the synthesis of catecholamines was determined in rat brain. In agreement with other studies, morphine produced a dose-dependent increase in the biosynthesis of the catecholamines. To assess whether morphine might enhance the synthesis of norepinephrine and dopamine by a direct chemical interaction with tyrosine hydroxylase, the rate-limiting step in their biosynthesis, the effects of the narcotic on the activity of the enzyme were determined in several regions of rat brain. Morphine, in vitro, from 10^?8 to 10^?2 M had no effect on the activity of tyrosine hydroxylase in any region examined. Moreover, morphine (10^?5 and 10^?3 M) had no effect on the apparent K_m of tyrosine hydroxylase for either substrate or cofactor (6-7-dimethyl-5, 6,7,8-tetrahydropterine). In addition, morphine (10^?5 and 10^?3 M) failed to block the inhibition of tyrosine hydroxylase, in vitro, by norepinephrine and dopamine in the hypothalamus and caudate respectively. The effects of morphine treatment, in vivo, on enzyme activity were also examined. The results of these studies indicated that acute injections of morphine had no effect on tyrosine hydroxylase activity, in vitro, indicating that the drug did not alter the level of an endogenous activator or inhibitor of tyrosine hydroxylase. Further studies indicated that development of tolerance to and physical dependence on morphine was not associated with an increase in the activity of brain tyrosine hydroxylase activity. The results of these studies suggest that morphine does not enhance the biosynthesis of catecholamines by a direct effect of tyrosine hydroxylase and that tolerance to the narcotic is not characterized by an induction of this enzyme.