Kinetics of inhibition of angiotensin converting enzyme by captopril and by enalapril diacid

Using a continuous spectrophotometric assay, inhibition of angiotensin converting enzyme by captopril and by the active diacid derivative of enalapril was reinvestigated. The onset of inhibition was comparatively slow, but the inhibition achieved was stronger than previous estimates: approximate Ki-values were 0.3 nM for captopril and 0.06 nM for enalapril diacid. The rate-constants for association and dissociation of these enzyme-inhibitor complexes were estimated, and half-times of approximately 12 min for the captopril complex and 60 min for the enalapril diacid complex were calculated. The rate of dissociation of the captopril-enzyme complex was measured directly by reacting the thiol group in free captopril with 5,5'-dithiobis(2-nitrobenzoic acid) and observing the reactivation of the enzyme; a half-time of approximately 30 min was obtained. Therefore the release of these inhibitors from the enzyme may be slow enough to affect the duration of their hypotensive action.     

Effect of old age on paracetamol-induced lipid peroxidation in rat liver

Post-mitochondrial supernatants isolated from the livers of mature rats (3 to 6 months old) 2 h or more after the administration of a single large oral dose of paracetamol (800 mg/kg) showed rapid rates of lipid peroxidation when incubated in vitro. In similar experiments with old rats (27-30 months old) the time between administration of paracetamol and the onset of lipid peroxidation was much longer (6 h or more). In both age groups, lipid peroxidation was dependent on the depletion of glutathione from the liver.     

Tolerance to the carbamate insecticide propoxur

Male mice were given the carbamate insecticide propoxur (2-isopropoxyphenyl methylcarbamate; Baygon®) in drinking water at weekly increasing concentrations (from 50 to 2000 ppm), for a period of 6 weeks. At the end of the treatment the LD₅₀ for propoxur was significantly higher in the treated animals as compared with controls. Propoxur-treated animals were also resistant to the hypothermic effect of an acute administration of the same compound. Groups of mice were challenged with the cholinergic agonist carbachol at intervals during the drinking water dosing and at its end. No differences in sensitivity to carbachol acute toxicity were found between control and treated animals. Propoxur-tolerant animals were also not resistant to the hypothermic effect of oxotremorine, another cholinergic agonist. [³H]Quinuclidinyl benzilate ([³H]QNB) binding (a measure of muscarinic receptor density and affinity) in forebrain, hindbrain and ileum never differed in control and treated mice. The possibility that repeated administrations of propoxur induced increased metabolic inactivation was tested by measuring hexobarbital sleeping time and carboxylesterase activity in treated and control mice. No changes in tissue carboxylesterase activities occurre but hexobarbital sleeping time was significantly reduced in propoxur treated animals suggesting an induction of hepatic microsomal enzymes. These results suggest that tolerance to propoxur is not mediated by a decrease of cholinergic receptors, as reported for other acetylcholinesterase inhibitors, but possibly by an enhancement of its metabolism.     

Effect of bongkrekic acid, a product of Pseudomonas cocovenenans, on thiol proteases

—The in vitro effect of bongkrekic acid on stem bromelain, papain and ficin was studied. The hydrolysis of casein by these enzymes was inhibited by bongkrekic acid, but the inhibition was always incomplete even with a large excess of the effector. Using a fully activated specimen of stem bromelain, purified on an organomercurial agarose affinity column, the inhibition by bongkrekic acid was not stoichiometric. The SH group of cysteine remained intact after incubation with an excess of bongkrekic acid at 24°C for 20 min. However, partial inhibition of stem bromelain by bongkrekic acid was reversed by incubation at 37°C for 5 min with 5 mM cysteine or 2-mercaptoethanol. Ethylene glycol and glycerol had no such restorative effect. These results indicate that molecules of bongkrekic acid are non-covalently bound to a thiol protease, only partially and reversibly shielding its essential SH group.     

Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide

The amount of glutathione present in hepatic protein mixed disulfides was determined to be 20–30 nmole/g liver. This was established using two specific enzymatic methods: (a) the coupled assay with DTNB and glutathione (GSSG) reductase and (b) a newly developed test using GSH transferase and 1-chloro-2,4-dinitrobenzene for the estimation of GSH released from proteins after borohydride treatment; further, these results were confirmed by HPLC analysis. Thus, authentic glutathione makes up only 2–6% of the value for total protein mixed disulfides. The latter were determined with the generally employed o-phthalaldehyde assay, which is not necessarily specific for GSH. The amount of glutathione mixed disulfides depends linearly on the content of glutathione disulfide in the liver cell in the range studied. By increasing the GSSG levels from 20 to about 60 nmole/g liver with paraquat, nitrofurantoin or t-butyl hydroperoxide, glutathione protein mixed disulfides are increased by a similar amount.     

Role of carnitine in promoting the effect of antidiabetic biguanides on hepatic ketogenesis.

Effects of biguanides on carnitine content of rat and guinea pig liver and on capacity of rat liver slices for ketogenesis were studied. In acute experiments, fed. 24-hour and 48-hour fasted male rats were given a single dose ofbuformin and the carnitine and acetylearnitine level in the tissues were determined 1 or 3 hr afterwards. The same was performed on fed guinea pigs. In all the 1-hr groups we found an increase ranging from 30 to 50 per cent in hepatic carnitine level. In chronic experiments rats were treated with buformin or metformin for 6 days. The carnitine content, carnitine acetyltransferase and carnitine palmitoyltransferase activities were determined. The respective carnitine levels in the buformin- and metformin-treated groups were 4 times and 2.5 times the control value expressed on a per gram basis. In addition, carnitine acetyltransferase activity, given as mU/mg mitochondrial protein, increased 2-fold in the buformin-treated group. The increase in carnitine content strongly suggests that liver has enhanced capacity for oxidation of fatty acids and consequently for production of ketone bodies. The latter has been verified in the chronic experiments by the following observations: (1) The buformin administration increased the total ketone body content of the freeze-clamped liver specimens to 210 per cent of the control value. The calculated mitochondrial NAD⁺/NADH ratio was reduced from 10.6 to 5.96 in the same specimens. (2) the liver slices from treated animals formed 30–40 per cent more ketone bodies than those from control ones during the 30-min and 60-min incubations. (3) The ketone body associated radioactivity deriving from Na-[1¹⁴-C] palmitate accounted for 90.5 per cent of water soluble radioactivity in slices from treated animals, whereas it accounted for 66.8 per cent in slices from control ones.     

Vinyl chloride-induced depression of hepatic non-protein sulfhydryl conten and effects on bromosulphalen (BSP) clearance in rats

Rats were exposed to atmospheres of 2000, 1000, 250, 150, 50 and 10 ppm vinyl chloride (VC) for 1–7 h to determine the effect of VC on the hepatic non-protein sulfhydryl content. Exposure to 2000, 1000, 250 and 150 ppm VC caused a progressive depression of the hepatic non-protein sulfhydryl content. Following exposure to 50 ppm VC for 7 h to depression was inconsistent, and no depression was observed after 10 ppm VC for 7 h. Also, exposure to 1000 ppm VC did not alter the serum clearance of bromosulphalein (BSP).     

Reactions of isodimethoate with human red cell acetylcholinesterase

Isodimethoate is a thermal decomposition product that is present in usual pesticide formulations of dimethoate. Owing to its PO structure the compound is a direct anticholinesterase agent whose properties, to the best of our knowledge, are presented here for the first time. Isodimethoate shows an inhibition rate constant towards human red blood cell acetylcholinesterase (AChE) of 2.3x10(3) M(-1) min(-1) (pH 7.4, 37 degrees C), indicating a somewhat higher potency than found with omethoate, the CYP450-mediated active metabolite of pure dimethoate. Isodimethoate-inhibited AChE shows fast spontaneous reactivation and aging kinetics (half-life 2.3 and 25 min, respectively). The inhibited, non-aged enzyme is readily reactivated by obidoxime (k(r)=9 min(-1); K(D)=0.1 mM) but hardly by pralidoxime at therapeutic concentrations. Interestingly, isodimethoate hydrolyzes readily in buffered solutions at pH 7.4 and 37 degrees C with liberation of methylmercaptan (half-life 16 min). Liberation of N-(methyl)mercaptoacetamide, the expected leaving group, was not observed. These properties make isodimethoate a hit-and-run agent that renders part of AChE non-reactivatable within a short period of time. The clinical consequences of exposure to or intentional ingestion of isodimethoate-containing dimethoate formulations are a partly untractable AChE shortly after incorporation. In fact, aging of AChE in dimethoate-poisoned patients on admission was much more advanced than expected from the reaction with omethoate. Manufacturers, researching scientists and clinical toxicologists should be aware of this problem.     

The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin on glutathione s-transferase activity of rat liver cytosol

Specific activities and apparent Michaelis-Menten kinetic parameters were determined for glutathione (GSH) S-transferase activity (E.C. 2.5.1.18) in rat liver cytosol, towards styrene oxide (STOX), 1,2-butylene oxide (BOX) and 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrates, before and after pretreatment with the drug-metabolizing enzyme inducers phenobarbital (PB), 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The measured GSH S-transferase activities appear to obey Michaelis-Menten kinetics. In non-induced animals the apparent K_m values of the transferase activities were equal for STOX vs GSH, but they differed by a factor of 2 for CDNB vs GSH and by a factor of 14 for BOX vs GSH. The apparent V_max values in each combination of GSH and electrophilic substrate were equal, but differed by one order of magnitude for the mutual substrate combinations. Pretreatment of the rats with MC resulted in enhancement of all measured activities expressed in terms of cytosol protein, while TCDD only enhanced the activities expressed as per gram body wt. PB enhanced both activities when STOX was employed as substrate, but when CDNB was used as the substrate, only the activity per gram body wt increased. All pretreatments increased the V_max values using CDNB as the substrate, while PB and MC had an enhancing effect using STOX; the V_max using BOX was enhanced after TCDD administration only. The K_m values using BOX as the substrate was lowered after MC pretreatment; TCDD pretreatment decreased the K_m using STOX, while it increased the K_m using CDNB. It is concluded that the GSH S-transferase system is inducible, but in contrast to the induction of the mixed function oxidase system, qualitative differences between the inducing effects of PB and MC were not observed. Use of TCDD as inducing agent, however. resulted in a different induction pattern, which may indicate that during induction with this agent different types of GSH S-transferases are involved.     

Ameliorative effect of methanol extract of Rumex vesicarius on CCl4-induced liver damage in Wistar albino rats

Abstract

Context: Rumex vesicarius L. (Polygonaceae), an edible plant, is reported to have many bioactive phytochemicals, especially flavonoids and anthraquinones with antioxidant and detoxifying properties.

Objective: This study evaluated the methanolic extract of R. vasicarius (MERV) for hepatoprotective activity in rats against CCl₄-induced liver damage.

Materials and methods: The whole plant extract was prepared and investigated for its hepatoprotective activity. Rats were pretreated with MERV (100 and 200?mg/kg, p.o.) for 7?d prior to the induction of liver damage by CCl₄. Animals were then sacrificed 24?h after CCl₄ administration for the biochemical (AST, ALT, and ALP activity in serum; lipid peroxidation (LPO) and glutathione (GSH) levels in liver tissue) and histological analyses.

Results: CCl₄-induced hepatotoxicity was confirmed by an increase (p?<?0.05) in serum AST (4.55-fold), ALT (3.51-fold), and ALP (1.82-fold) activities. CCl₄-induced hepatotoxicity was also manifested by an increase (p?<?0.05) in LPO (3.88-fold) and depletion of reduced glutathione (3.14-fold) activity in liver tissue. The multiple dose MERV administration at 200?mg/kg showed promising hepatoprotective activity as evident from significant decrease levels of serum AST (230.01?±?13.21), serum ALT (82.15?±?5.01), serum ALP (504.75?±?19.72), hepatic LPO (3.38?±?0.33), and increased levels of hepatic glutathione (0.34?±?0.04) towards near normal. Further, biochemical results were confirmed by histopathological changes as compared with CCl₄-intoxicated rats.

Discussion and conclusion: The results obtained from this study indicate hepatoprotective activity of Rumex plant against CCl₄-induced liver toxicity; hence, it can be used as a hepatoprotective agent.     

Development of a dynamic model for real-time determination of membrane-bound acetylcholinesterase activity upon perfusion with inhibitors and reactivators

Quantitative predictions of the course of acetylcholinesterase (AChE) activity, following interference of inhibitors and reactivators, are usually obscured by the time-dependent changes of all reaction partners. To mimic these dynamics we developed an in vitro model. Immobilized human erythrocyte ghosts in a bioreactor were continuously perfused while AChE activity was monitored by a modified Ellman method. The perfusion system consisted of two HPLC pumps with integrated quaternary low-pressure gradient formers that were programmed by a computer using commercial HPLC software. The combined eluates passed a particle filter (Millex-GS, 0.22 microm) containing a thin layer of erythrocytes that was immersed in a temperature-controlled water bath. The effluent passed a flow cell in a UV-vis detector, the signal of which was digitized, written to disc and calculated with curve fitting programs. AChE activity decreased by 3.4% within 2.5 h. The day-to-day variation of the freshly prepared bioreactor using the same enzyme source was +/-3.3%. Residual activity of 0.2% marked the limit of quantification. Following perfusion with paraoxon, pseudo first-order rate constants of inhibition were established that did not differ from results obtained in conventional assays. The same holds true for reactivation with obidoxime. The set-up presented allows freely programmable time-dependent changes of up to eight solvents to mimic pharmacokinetic profiles without accumulation of products. Due to some hysteresis in the system, reaction half-lives should be >3 min and concentration changes in critical compounds should exceed half-lives of 5 min. Otherwise, the system offers much flexibility and operates with high precision.     

Effect of ranitidine on ileal myenteric plexus preparation and on acetyl- and butyrylcholinesterase

Ranitidine at concentrations from 1 microM to 0.1 mM brought about a dose-dependent potentiation of the twitch responses elicited by electrical stimulation of the ileal myenteric preparation. At higher concentrations (0.3-3 mM) ranitidine also caused irregular slow contractions of the unstimulated ileal preparation which were potentiated by eserine and blocked by atropine and tetrodotoxin. In order to identify the mechanism of these apparently cholinomimetic actions, the effects of ranitidine on AChE and BuChE were studied. Ranitidine showed an instantaneous and promptly reversible inhibitory action at concentrations between 0.5 and 30 microM. Double reciprocal plots were prepared and equilibrium dissociation constants calculated. It appears that ranitidine exerts an inhibition of the "mixed" type on both AChE and BuChE, but the dissociation constants for BuChE were markedly higher than those for AChE. Since AChE inhibition occurs in the same concentration range potentiating the twitch responses on the ileal myenteric preparation, it may explain the cholinomimetic effect of ranitidine.     

Mechanisms of the metabolic disturbances caused by hypoglycin and by pent-4-enoic acid in vitro studies

1. In the presence of the hypoglycin metabolites methylenecyclopropylpyruvate (MCPP) and methylenecyclopropylacetate (MCPA), rat liver mitochondria oxidized palmitoyl-carnitine only as far as butyrate and at a decreased rate. Although pent-4-enoic acid (pent-4-enoate) inhibited the rate of β-oxidation of palmitoyl-carnitine by mitochondria, the oxygen uptake was consistent with the complete oxidation of the substrate. 2. The inhibition of β-oxidation by pent-4-enoate was partially reversed by very high concentrations of l-carnitine. Conditions were also defined for the sustained oxidation of pent-4-enoate by mitochondria. 3. Pent-4-enoate inhibited pyruvate oxidation, but only at concentrations much higher than those needed to inhibit β-oxidation. MCPA had no effect on either pyruvate or 2-oxoglutarate oxidation in mitochondria. 4. Soluble extracts of rat or ox liver mitochondria completely oxidized pent-4-enoyl-CoA and the oxidation of butyryl-CoA added subsequently was unaffected. Incubation of soluble extracts with pent-4-enoyl-CoA in the absence of cofactors caused inhibition of acetoacetyl-CoA thiolase activity. However, this enzyme was not inhibited in intact mitochondria by pent-4-enoate. 5. MCPA specifically inhibited butyryl-CoA dehydrogenase in both intact mitochondria and in soluble extracts supplemented with ATP and CoASH. 6. Both MCPP and MCPA (1 mM) caused a rapid decrease in CoASH concentrations in mitochondria; acetyl-CoA concentrations were unaffected. Concentrations of pent-4-enoate (20 μM) sufficient to inhibit β-oxidation caused only a slight decrease in CoASH whereas higher concentrations (0.1–1.0 mM) caused a more extensive depletion of CoASH. However, evidence is presented to suggest that CoASH depletion is not the mechanism by which these compounds inhibit β-oxidation. 7. Pent-4-enoate and MCPA were substrates for butyryl-CoA synthetase. K_m and V_max values for several unusual, straight and branched chain fatty acids were determined. 8. Some short-chain acyl-CoA esters were substrates for an acyl-CoA hydrolase located in the mitochondrial matrix. 9. Some short-chain acyl-CoA esters competitively inhibited the activation of pyruvate carboxylase by acetyl-CoA. 10. The possible mechanisms by which hypoglycin and pent-4-enoate cause inhibition of β-oxidation and hypoglycaemia in vivo are discussed.     

胆宁片对实验性急慢性肝损伤的保护作用

目的：研究胆宁片对实验性小鼠急性肝损伤预防作用，及对大鼠慢性肝损伤的预防和治疗作用。方法：分别用D-氨基半乳糖和四氯化碳复制小鼠急性肝损伤和大鼠慢性肝损伤模型，检测小鼠和大鼠血清谷草转氨酶（AST）和谷丙转氨酶（ALT）的改变，观察胆宁片对大鼠血清总蛋白、白蛋白、A／G的影响、同时观察肝脏病理学改变。结果：胆宁片可明显抑制D-氨基半乳糖和四氯化碳引起的ALT、AST升高（P〈0．05或P〈0．01）；可显著升高四氯化碳引起的血清总蛋白和白蛋白含量降低（P〈0．05或P〈0．01）；肝脏病理组织学检查显示胆宁片可减轻肝细胞脂肪变性程度和纤维化程度。结论：胆宁片对小鼠D-氨基半乳糖急性肝损伤有较好的保护作用，对四氯化碳所致大鼠慢性肝损伤有一定的预防及治疗作用。     

Influence of liver S-9 preparations from rats and rainbow trout on the activity of four mutagens

The mutagenicity of four chemical compounds to strain TA100 of S. typhimurium was affected differently by liver S-9 preparations from untreated Sprague-Dawley rats and from rainbow trout (Salmo gairdneri). These two species were equally effective in decreasing the direct mutagenicity of sodium dichromate. Rat preparations were totally inactive and trout preparations were slightly active in producing mutagenic metabolites from benzo(a)pyrene (BP). Conversely, rat homogenates were significantly more efficient in activating aflatoxin B1 (AFB1) and, in particular, 2-aminofluorene (2-AF).     

The identity of enzymes reducing a thiamine disulfide derivative and cystine derivatives via thiol-disulfide exchange

In earlier work¹, we have studied a labile enzyme activity catalyzing an exchange between thiol and acceptor. Glutathione (GSH) was used as the thiol and a number of low molecular weight substances such as cystine and GSH-disulfide derivatives, S-sulfocysteine (CySSO₃H), S-sulfoglutathione and 5,5′-dithiobis(2-nitrobenzoa (DTNB) were used as acceptor substrates in the thiol transfer reaction. This broad substrate specificity led us to the tentative suggestion that thiamine disulfide derivatives also were acceptor substrates to the thioltransferase² activity, which is confirmed in this study. The methods used for the resolution of enzymes and substrate specificity were: (1) isoelectric focusing, (2) CM-cellulose chromatography, (3) labelling of the thioltransferase with [³⁵S]GSH, (4) gel filtration on Bio-Gel P-150, and (5) investigation of ratios of the specific activities of GSH-linked enzymes in different tissues. Generally it was found that bovine tissue had higher specific thioltransferase activity than rat tissue. GSH S-aryltransferase (EC 2.5.1.13) had quite different activity ratios from those obtained with the enzyme involved in cystine and thiamine disulfide reduction. This result, and dissimilar Chromatographic behavior, indicate that GSH S-aryltransferase is not involved in disulfide reduction.     

Effect of nitroheterocyclic drugs on lipid peroxidation and glutathione content in rat liver extracts

Incubation of rat liver cell-free extracts with an NADPH-generating system and with nifurtimox or benznidazole (two nitroheterocyclic drugs used in the treatment of Chagas' disease) produced oxidation of reduced glutathione (GSH) and increased lipid peroxidation, as shown by the generation of thiobarbituric-acid-reacting intermediates. Nifurtimox and benznidazole inhibited GSSG-reductase, but not GSH-peroxidase, the former inhibition contributing to GSH depletion. In every case, nifurtimox was more effective than benznidazole. Addition of GSH or free-radical scavengers (catalase, superoxide dismutase, mannitol, sodium benzoate or L-histidine) prevented the effect of nifurtimox on lipid peroxidation reactions. These results support the assumption [M. Dubin, S. N. J. Moreno, E. E. Martino, R. Docampo and A. O. M. Dubin, Biochem. Pharmac.32, 483 (1983)] that, in the rat liver, GSH exerts a protective action against oxygen radicals generated by the nitroheterocyclic drugs.     

Effect of parathion,malathion, endosulfan and chlordane on porphyrin accumulation and alasynthetase in chick embryo liver

In the present study we investigated the porphyrinogenic ability of 4 pesticides: chlordane, endosulfan, parathion and malathion, all of which are widely used in agriculture.In order to determine whether they affect the heme biosynthetic pathway we studied, in 17-day old chick embryo liver “in ovo”, their effects on the amount of porphyrins and on the activity of δ-ALA synthetase (ALA-S), the first and rate limiting enzyme of this pathway.All of them induced hepatic porphyrin accumulation to a different extent compared with dimethylsulfoxide (DMSO) controls. Parathion as well as endosulfan promoted remarkable increases, chlordane raised porphyrin level in a lower degree and malathion slightly modified it. However, the accumulation observed with malathion was markedly enhanced if the period of incubation was extended to 48 h and, even more, if a second dose was injected during these 48 h.When ALA-S activity was analyzed in the chick embryos treated with parathion or endosulfan no alteration could be found in spite of producing noticeable accumulation of porphyrins. In contrast, chlordane promoted a statistically significant elevation of ALA-S as well as malathion which produced the highest induction observed.These results show that not only organochlorinated but also organophosphorous pesticides affect heme metabolism and that induction of ALA-S and porphyrin accumulation are not parallel.     

Effects of PR toxin on liver cells in culture.

We studied the effects on liver cells in culture of PR toxin, a substance produced from Penicillium roqueforti. PR toxin displayed cytotoxicity which increased as a function of its concentration but the form of such toxicity differed, depending on the toxin's concentration. Thus, cells only underwent quick retraction and intensive vacuolization when treated with low drug concentrations, and they came away from the substrate easily under these conditions. By contrast, the major events observed in the case of high concentrations were loss of structure of the nuclei and strong adhesiveness of dead cells to the support. PR toxin already inhibited cell multiplication at low concentrations and became toxic when the concentration was raised; growth inhibition decreased but the toxic effect increased when cells passed from the exponential growth phase to a phase of slower growth. PR toxin inhibited tritiated precursor incorporation into DNA, RNA and proteins in a similar time and concentration-dependent manner. Inhibition of DNA synthesis persisted even after removal of the drug from the medium.