Placental transfer of SR49059 in the human dually perfused cotyledon in vitro.

SR49059 is an antagonist of vasopressin V(1a)receptors currently developed as a tocolytic drug. We investigated the transplacental transfer of SR49059 in vitro using the single pass dually perfused human cotyledon model. Thirteen placentae were collected from normal term pregnancies immediately after delivery. The placental transfer of SR49059 was tested at steady state at three different concentrations (100 ng/ml, 200 ng/ml and 500 ng/ml) along with that of antipyrine 20 mg/l as a reference substance. Concentrations were assayed by liquid chromatography with UV (antipyrine) or mass spectrometry (SR49059) detection. At steady state, the mean+/-s.d. fetal transfer rate of SR49059 was 10.80+/-4.33 per cent, 9.34+/-4.41 per cent, and 11.78+/-3.26 per cent at 100 ng/ml, 200 ng/ml and 500 ng/ml, respectively. The corresponding clearance indices were 0.29+/-0.14, 0.25+/-0.08, and 0.31+/-0.06, respectively. The absence of saturation kinetics is consistent with a passive mechanism of transfer. Moderate amounts of SR49059 are transferred from the maternal to the fetal circulation. The clearance index of SR49059 appeared to be very similar to that of ritodrine, which is currently used as a tocolytic.

[Carcinoma arising within mammary fibroadenomas. A study of six patients]. / Carcinomes développés sur adénofibromes mammaires. A propos de six observations.

We report six cases of carcinomas arising within fibroadenomas. Fibroadenoma is a benign neoplasm occurring in young women. Its association with carcinomas is unfrequent and particularly reported in older women. Few data are available on the histologic features of fibroadenomas harboring malignant lesions. In this study, most cases of fibroadenomas showed cysts, sclerosing adenosis, epithelial calcifications or papillary apocrine changes. These fibroadenomas are classified as complex and are a long-term risk factor for breast cancer. The complex fibroadenoma may be specific of fibroadenoma associated with carcinoma.

Cytochrome P4502E1 inducibility and hydroxyethyl radical formation among alcoholics.

BACKGROUND/AIMS: Animal studies have shown that the induction of cytochrome P4502E1 (CYP2E1) modulates oxidative damage induced by ethanol. Since CYP2E1 activity varies substantially in humans, we have investigated whether differences in CYP2E1 activity might influence the formation of hydroxyethyl free radicals and the stimulation of lipid peroxidation among alcohol abusers. METHODS: Chlorzoxazone oxidation, an index of CYP2E1 activity, and the levels of antibodies reacting with hydroxyethyl radical and malonyldialdehyde protein adducts were investigated in 51 alcoholic patients. RESULTS: We observed that in 40 out of 51 (78%) alcoholics, chlorzoxazone oxidation was increased over the control levels, consistently with CYP2E1 induction by ethanol. However, in the remaining 22% of the patients, in spite of a similar alcohol intake, chlorzoxazone oxidation was within the control range, indicating a lack of CYP2E1 inducibility. IgG reacting with hydroxyethyl free radical-protein adducts were absent in subjects without CYP2E1 induction, while they were significantly increased in alcoholics with induced CYP2E1 activity. IgG against malonyldialdehyde protein-adducts were increased in all patients, irrespective of CYP2E1 inducibility. Moreover, chlorzoxazone oxidation was significantly lower in alcoholics without clinical and biochemical signs of liver disease as compared to patients with alcoholic liver disease. CONCLUSIONS: These results indicate that CYP2E1 activity greatly influences the formation of hydroxyethyl radicals in humans, and suggest a possible role of CYP2E1 in the development of alcoholic liver disease.

Plasma membrane hydroxyethyl radical adducts cause antibody-dependent cytotoxicity in rat hepatocytes exposed to alcohol.

BACKGROUND & AIMS: We reported previously that patients with alcoholic liver disease (ALD) have circulating immunoglobulins reacting with cytochrome P4502E1 (CYP2E1) complexed with hydroxyethyl free radicals. The aim of this study was to investigate whether hydroxyethyl radical adducts are present on the plasma membranes of ethanol-treated hepatocytes and their role in antibody-dependent cytotoxicity. METHODS: Immunofluorescence confocal laser microscopy, Western blotting, and antibody-dependent cell-mediated cytotoxicity assay were used. RESULTS: Isolated rat hepatocytes incubated in vitro with ethanol or obtained from ethanol-treated animals showed strong surface fluorescence when exposed to rabbit anti-hydroxyethyl radical serum or sera from patients with ALD. No surface fluorescence was evident on control hepatocytes or after scavenging hydroxyethyl radicals with 4-pyridyl-1-oxide-t-butyl nitrone. The presence of CYP2E1-hydroxyethyl radical adducts on hepatocyte plasma membranes was shown by Western blot and by immunofluorescence using double staining for human and rabbit anti-CYP2E1 immunoglobulin G. Cytotoxicity was observed in ethanol-treated hepatocytes incubated with immunoglobulin G from patients with ALD and normal human blood mononuclear cells. This effect was blocked by preabsorbing the sera with human albumin complexed with hydroxyethyl radicals, which also eliminated the antibody reaction with the plasma membranes. CONCLUSIONS: Hydroxyethyl radicals bound to CYP2E1 on hepatocyte plasma membranes can target immune reactions triggered by alcohol abuse.

Lipid peroxidation, CYP2E1 and arachidonic acid metabolism in alcoholic liver disease in rats.

The role of cytochrome P450 metabolism of fatty acids and lipid peroxidation in the alterations of the fatty acid composition of the liver and liver pathology was investigated. The CYP2E1 inhibitors partially prevented CYP2E1 induction by ethanol and completely blocked lipid peroxidation. However, the liver pathology induced by ethanol was only partially prevented as was the decrease in arachidonic acid in total liver lipid, triglycerides and cholesterol esters. This means that liver peroxidation induced by ethanol can not completely account for the liver pathology or the decrease in arachidonic acid caused by ethanol. Lauric acid omega-1 hydroxidation by the liver microsomes in vitro was increased by ethanol and partially blocked by CYP2E1 inhibitors. However, although ethanol feeding increased the total hydroxidation and epoxidation of arachidonic acid, these were not inhibited by CYP2E1 inhibitors. Thus the ethanol-induced arachidonic acid depletion is not likely due to CYP2E1 metabolism of arachidonic acid, since the severity of liver pathology correlated negatively with the decrease in arachidonic acid in the ethanol-fed rats. The increase in its metabolism by microsomes and decrease in synthesis may be an important mechanism of ethanol-induced liver injury.

Free radicals and not acetaldehyde influence the circulating levels of glutathione after acute or chronic alcohol abuse: in vivo and in vitro studies.

BACKGROUND: The oxidation of ethanol and acetaldehyde enhances the production of various free radicals involved in membrane lipoperoxidation, and decreases glutathione levels. AIMS: We evaluated the effects of acute and chronic ethanol use in vivo, with or without the administration of S-adenosyl-methionine (SAME, 2 g I.v.), and the effects of ethanol and acetaldehyde in vitro, on the erythrocyte levels of malonyldialdehyde and glutathione, and of its principal synthesizing enzymes, gamma-glutamyl-cysteine-synthetase and glutathione-synthetase. METHODS: Twelve healthy volunteers (age range 26-44 years, median 32 years) and 20 chronic alcohol abusers without liver disease (age range 26-57 years, median 44 years) were studied. Malonyldialdehyde was evaluated by thiobarbituric acid; glutathione and its enzymes by high performance liquid chromatography using a fluorescent detector. RESULTS: In the healthy subjects, an acute load of ethanol induced a significant decrease in plasma levels of glutathione, which was inhibited by the infusion of S-adenosyl-methionine. In the erythrocytes of alcoholic patients, glutathione and glutathione-synthetase were decreased while malonyldialdehyde was increased. In vitro, acetaldehyde did not affect either the glutathione or the glutathione-related enzyme levels. CONCLUSIONS: Our data suggest that the alterations in glutathione metabolism in the erythrocytes of alcoholics may be due principally to the production of free radicals, as supported by the high levels of malonyldialdehyde observed.

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics.

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.

Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol.

We have previously shown that the treatment with diallyl sulfide (DAS) and phenylethyl isothiocyanate (PIC) of rats receiving ethanol in the alcohol tube-feeding model effectively suppressed the induction of cytochrome P4502E1 (CYP2E1) by ethanol. Here we report that rat treatment with DAS and PIC significantly decreased the trapping of hydroxyethyl free radicals in liver microsomes incubated in vitro with ethanol. Furthermore, these inhibitors also greatly reduced the production of hydroxyethyl radical-derived epitopes detectable in vivo in the liver of ethanol-fed rats. The action of DAS and PIC on the formation of hydroxyethyl radicals paralleled their inhibitory effect on lipid peroxidation as monitored using, respectively, liver malonildialdehyde (MDA) and plasma lipid hydroperoxide levels as well as by the titers of antibodies versus MDA adducts to proteins. Thus, these results indicated a link between the induction of CYP2E1 by ethanol, the formation of hydroxyethyl radicals and the stimulation of lipid peroxidation. The pathological scores in the livers of rats fed with ethanol plus or minus DAS and PIC also correlated with levels of hydroxyethyl radical-derived epitopes. Rats fed intragastrically with ethanol developed antibodies and the formation of these antibodies was greatly reduced by DAS and PIC. Taken together these results suggest that CYP2E1 plays an important role in the generation of hydroxyethyl radicals during chronic alcohol feeding and that ethanol-derived free radicals might play a role in the onset of liver injury in this model of alcohol administration.

Modulation of experimental alcohol-induced liver disease by cytochrome P450 2E1 inhibitors.

This study was done to determine if a relationship exists between CYP2E1 induction by ethanol, lipid peroxidation, and liver pathology in experimental alcohol-induced liver disease in the rat. Rats were fed ethanol with or without diallyl sulfide (DAS) or phenethyl isothiocyanate (PIC) intragastrically for 1 month. CYP2E1 induction by ethanol was correlated with lipid peroxidation, liver microsomal CYP2E1 hydroxylation of paranitrophenol, and the liver pathology score using the data from the PIC-fed rats. Some of the data from the ethanol and DAS-fed rats were not included here because they have been reported elsewhere. Microsomal CYP2E1 protein levels induction by ethanol was decreased by PIC ingestion. Similarly, PIC reduced the increase microsomal reduced form of nicotinamide-adenine dinucleotide (NADPH)-dependent lipid peroxidation and p-nitrophenol hydroxylase (PNPH) activity, induced by ethanol feeding. The lipid peroxidation was reduced to below control levels; however, the pathology score was partially but not significantly reduced by isothiocyanate feeding. CYP2E1 messenger RNA (mRNA) was decreased by both inhibitors of CYP2E1. Immunohistochemical staining of liver for CYP2E1 protein showed that the lobular distribution of the isozyme changed from the centrilobular to a diffuse pattern, with an increase in the periportal region when the CYP2E1 inhibitors were fed with ethanol, and that this change correlated with the change in the distribution of fat in the lobule. The data support the idea that there is a link between CYP2E1 induction by ethanol and the early phase of ethanol-induced liver injury in this rat model. This link may involve lipid peroxidation, but other factors related to CYP2E1 induction must also be involved.

Activation of alkylhydrazines to free radical intermediates by ethanol-inducible cytochrome P-4502E1 (CYP2E1).

Electron spin resonance (EPR) spectroscopy analysis using the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) was used to measure the formation of free radical intermediates during NADPH-dependent oxidation of 1-methyl-, 1-ethyl-, and 1-isopropylhydrazine in rat liver microsomes and in reconstituted enzyme systems. The experiments in microsomes revealed that the specific activation of the hydrazines, as measured by the EPR signal intensities, was about two-fold higher, when expressed per nmol of P-450, in microsomes from rats treated with ethanol (EtOH) as compared to membranes isolated from either phenobarbital (PB)-, beta-naphthoflavone (beta-NF)-treated or control rats. Furthermore, kinetic experiments revealed that EtOH-microsomes had an apparent affinity for 1-ethylhydrazine about one order of magnitude higher than PB-microsomes. In reconstituted vesicular systems composed of phospholipids, NADPH cytochrome P-450 reductase and P-450, the intensities of EPR signals produced by the formation of the methyl-, ethyl- and isopropyl-free radicals, were 3- to 5-fold more intense in membrane vesicles containing ethanol-inducible CYP2E1 than phenobarbital-inducible CYP2B1. By contrast, CYP1A2, CYP2B4 and CYP2C4 were inefficient catalysts of radical formation. Desferrioxamine, catalase and superoxide dismutase did not influence the extent of ethyl radicals formed in EtOH-microsomes, indicating that hydroxyl radicals are not involved in the CYP2E1-dependent activation of 1-ethylhydrazine. Addition of cytochrome b5, an efficient donor of the second electron to P-450 and hence an inhibitor of the formation of the oxy-cytochrome P-450 complex, increased to be consistent with the results, did not influence the amount of ethyl radicals trapped. In liver microsomes from untreated rats selective substrates of CYP2E1, such as diethyl-dithiocarbamate and p-nitrophenol, as well as anti-CYP2E1-IgG, inhibited the free radical formation from 1-ethylhydrazine by about 60%. The anti-CYP2E1 IgG used significantly inhibited ethyl radical production also in human liver microsomes incubated with 1-ethylhydrazine and 4-POBN. Taken together, these results indicate that CYP2E1, as compared to other rat liver cytochromes P-450, is an efficient catalyst of transformation of alkylhydrazines to free radical intermediates, a finding that might be of importance in the development of the toxicity of these compounds.

Detection of antibodies against proteins modified by hydroxyethyl free radicals in patients with alcoholic cirrhosis.

BACKGROUND/AIMS: We have previously shown that hydroxyethyl free radicals produced during cytochrome P4502E1-mediated oxidation of ethanol covalently bind to microsomal proteins. The present study examined whether alkylation of proteins by hydroxyethyl radicals induces an immunologic response in alcoholic patients. METHODS: A microplate enzyme-linked immunosorbent assay was developed using as antigen human serum albumin or bovine fibrinogen reacted with chemically produced hydroxyethyl radicals. RESULTS: This assay showed that the sera of alcoholic cirrhotics contained both immunoglobulin (Ig) Gs and IgAs that recognized proteins modified by hydroxyethyl radicals, whereas practically no reaction was observed in the sera of healthy controls or cirrhotics without evidence of alcohol abuse. The reactivity of the sera from alcoholic patients was not influenced by the protein to which hydroxyethyl radicals were bound. The sera of alcoholic cirrhotics also contained antibodies directed against acetaldehyde-modified albumin. However, the reaction of alcoholic sera with hydroxyethyl radical epitopes was not inhibited by increasing concentrations of acetaldehyde-modified albumin produced under either reducing or nonreducing conditions. CONCLUSIONS: The results indicate that a new group of antigens that do not cross-react with antibodies against acetaldehyde-derived epitopes is formed by the alkylation of protein by hydroxyethyl radicals and is involved in the development of immunologic reactions in alcoholic patients.

Monitoring oxidative damage in patients with liver cirrhosis and different daily alcohol intake.

This study looked at the possible association between alcohol abuse and free radical mediated oxidative injury by examining the presence of oxidative damage, as monitored by erythrocyte malonildialdehyde and plasma lipid hydroperoxides, in patients with liver cirrhosis and different lifetime daily alcohol intake. All patients with an alcohol intake above 100 g/day (ALC) showed concentrations of malonildialdehyde and lipid hydroperoxide on average four to fivefold higher than cirrhotic patients with alcohol intake below 100 g/day (NAC) or healthy controls. Further subgrouping of ALC patients showed that those with alcohol intake ranging between 100 and 200 g/day (ALC1) had malonildialdehyde and lipid hydroperoxide concentrations significantly lower than those with an intake higher than 200 g/day (ALC2). These differences were not related to the extent of liver injury or to the liver derangement as assessed by Child's classification. The increase in lipid peroxidation markers in ALC cirrhotic patients was associated with a decrease in, respectively, plasma alpha-tocopherol and erythrocyte glutathione concentrations. Significant differences were also seen between ALC1 and ALC2 groups in plasma alpha-tocopherol, but not in erythrocyte glutathione concentrations. The concentrations of alpha-tocopherol and glutathione in the blood of NAC patients were in contrast not substantially different from those of healthy controls. The close association between oxidative damage and alcohol abuse suggested that free radical intermediates produced during ethanol metabolism might be responsible for causing oxidative damage.

Risks and benefits of splenectomy in myelofibrosis: an analysis of 39 cases.

From 1980 to 1993, 39 splenectomies were performed in the Department of Visceral Surgery of Saint-Louis Hospital, in patients referred for myelofibrosis associated with myeloid splenomegaly. The short term morbidity was considerable: 33 serious haemorrhagic, infectious or thrombotic complications including 5 fatal accidents were observed in 18 patients. Severe thrombotic or infectious complications leading to 6 further deaths occurred in 8 patients over the two years following splenectomy, while six cases of acute leukaemia appeared between 6 months and 3 years after splenectomy. In 40% of cases with regular follow-up, the operation did not provide any haematological improvement and all these patients died. Only patients with minimally progressive or stable myelofibrosis and residual marrow activity in isotope studies showed an amelioration of general status with relief of pain and reduction of transfusional requirements. The indication for splenectomy should therefore probably be limited to such cases.

Free radical activation of acetaldehyde and its role in protein alkylation.

The formation of carbon centered free radicals, identified as methylcarbonyl species, was observed using ESR spectroscopy and the spin trapping agent 4-pyridyl-1-oxide-N-t-butyl nitrone (4-POBN) during the oxidation of acetaldehyde by xanthine oxidase. The reaction was dependent upon the presence of OH. radicals and was inhibited by the addition of superoxide dismutase, catalase or OH. radical scavengers. The generation of methylcarbonyl radicals was associated with a doubling of stable acetaldehyde adducts with serum albumin, and 4-POBN or superoxide dismutase and catalase, completely blocked this effect. Thus, methylcarbonyl radicals contributed to acetaldehyde-mediated protein alkylation which is involved in causing toxic as well as immunological reactions ascribed to acetaldehyde.

Specificity of autoantibodies against oxidized LDL as an additional marker for atherosclerotic risk.

BACKGROUND: LDL oxidation is a crucial step in the development and progression of atherosclerotic lesions. The detection of an increase in the anti-oxidized LDL antibody titre may thus represent a biological marker of enhanced LDL oxidation in vivo. METHODS: The occurrence of anti-oxidized LDL autoantibodies was investigated in control patients, in patients with atherosclerotic coronary artery disease, in those without clinically relevant signs of atherosclerosis, but considered at risk, and in patients with chronic alcohol-related liver disease. RESULTS: Anti-oxidized LDL autoantibodies were present in the plasma of the majority of patients with overt coronary atherosclerosis. An increased antibody titre can also be detected well before the onset of clinically relevant signs of the atherosclerotic disease in patients classically considered at risk, indicating the occurrence of in-vivo LDL oxidation during atherosclerosis development. The specificity of molecular targets (LDL) for oxidative modifications is supported by the demonstration that anti-oxidized LDL autoantibodies are absent in the plasma of alcoholic patients who exhibit a marked increase in biological markers of oxidative stress but do not classically develop atherosclerosis. CONCLUSION: These data demonstrate that the occurrence of anti-oxidized LDL autoantibodies could be specifically related to the promotion and progression of atherosclerosis and is not a simple epiphenomenon of any oxidative process occurring in vivo.

Ethanol-inducible cytochrome P4502E1: genetic polymorphism, regulation, and possible role in the etiology of alcohol-induced liver disease.

In the Tsukamoto-French model, ethanol causes an important 10-20-fold induction of ethanol-inducible cytochrome P4502E1 (CYP2E1), mediated through enzyme stabilization and increased rate of gene transcription. The CYP2E1 induction results in a pronounced increase in the rate of NADPH-dependent microsomal lipid peroxidation, an elevation which is not seen after simultaneous administration of the CYP2E1 inhibitor diallylsulfide. Increased amounts of lipid peroxides are seen in plasma and red blood cells of both rats and humans during high ethanol intake. A mechanism for ethanol-dependent liver damage is proposed which involves the CYP2E1-dependent lipid peroxide formation, either directly by its capability to induce NADPH-dependent peroxidation in the microsomal membranes or indirectly by a hypoxia-mediated transformation of xanthine dehydrogenase to xanthine oxidase, in activation of Ito cells and Kupffer cells to yield cytokine and collagen production. The CYP2E1 gene is polymorphic among Caucasians. Four different unrelated or partially linked polymorphisms have been observed. One polymorphism in the 5'-flanking region has been described to be associated with altered enzyme expression in vitro, and the rare allele was found to be less frequent among Swedish patients having lung cancer when compared to two different control groups. Another polymorphism, detectable with Dra I restriction endonuclease fragment length polymorphism (RFLP), was localized to intron 6, and the rare allele was less common among Italian alcoholics with clinical signs of liver cirrhosis, as compared to controls. Several other mutations in the CYP2E1 gene were found to be associated with this allele. However, further research is needed to relate the CYP2E1 gene polymorphism with incidence of liver cirrhosis.

Possible role of free radical intermediates in hepatotoxicity of hydrazine derivatives.

Hydrazine derivatives constitute a wide group of compounds and have found application in industry, agriculture, and (as therapeutical agents) medicine. In spite of their widely spread use, several hydrazine derivatives are known to exert hepatotoxic effects and are carcinogenic. Free radical species are produced during the hepatic biotransformation of alkylhydrazines by both rat and humans liver microsomes. Cytochrome P-450 dependent monoxygenase system is responsible for the production of these reactive species and specific cytochrome P-450 isoenzymes appear to catalyze the formation of free radical intermediates. Free radicals generated during the metabolism of alkylhydrazines are capable of inducing oxidative stress in isolated hepatocytes and might contribute to the development of cell injury.

Splenectomy in human immunodeficiency virus-related thrombocytopenia.

To evaluate the efficacy and safety of splenectomy in patients with human immunodeficiency virus (HIV)-related thrombocytopenia, 30 HIV-infected patients with thrombocytopenia (platelet count < 50 x 10(9)/l) who underwent splenectomy were followed prospectively for a mean period of 42 months. There were no perioperative deaths and morbidity was minimal. Twenty-one patients had a persistent complete response, six had a partial response and were asymptomatic after splenectomy, and only three showed no response. Three patients developed acquired immune deficiency syndrome during follow-up, an incidence that was no different from that expected. Splenectomy is a safe and effective treatment in HIV-infected patients with severe symptomatic thrombocytopenic purpura resistant to medical therapy.