Filtration of native and glycated beta2-microglobulin by charged and neutral dialysis membranes.
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BACKGROUND: It has been postulated that protein glycation and formation of advanced glycation end products (AGE) are among toxic factors in chronic uremia, whether the renal disease is of diabetic or nondiabetic origin. In this setting, AGE-modified beta2-microglobulin (beta2m) may favor dialysis beta2m-related dialysis amyloidosis. Consequently, efficient removal of modified beta2m by highly permeable dialysis membranes is as important as removal of native beta2m to postpone the development of dialysis amyloidosis. METHODS: To define the role of dialysis membrane surface electronegativity on plasma protein transfer, an in vitro model was used to test the interactions of native and glycated beta2m with various highly permeable dialysis membranes. An experimental circuit with minidialyzers was used. The neutral high-flux polysulfone membrane (PS), the electronegative polymethylmetacrylate membrane (PMMA), the electronegative AN69 membrane and a modified AN69 membrane, the surface of which was neutralized with polyethyleneimine (AN69-PEI), were tested using both native beta2m and the more acidic glycated beta2m. Protein mass transfer and binding to the membrane were measured. RESULTS: Mass transfer of glycated beta2m was significantly decreased through all membranes tested when compared with native beta2m. This result was due to the increased molecular weight of beta2m, which became less permeable to porous membranes, whereas adsorption of both native and glycated beta2m to membranes, due to ionic interactions, decreased similarly with AN69 and AN69-PEI, but remained unchanged with PS and PMMA. Moreover, surface neutralization of AN69 membrane did not alter its core binding capacity, since beta2m absorption accounted for 98 and 97% and glycated beta2m for 83.7 and 81.4% of the protein removed with AN69 and AN69-PEI, respectively. CONCLUSION: Clearance of glycated beta2m through highly permeable neutral and negatively charged membranes was lower than that of native beta2m, reflecting a decreased sieving coefficient for the neoformed higher molecular weight and conformationally altered molecule. The binding capacity of the neutral PS was roughly half that of the charged membranes. Neutralizing surface electronegativity of the AN69 membrane with PEI did not alter its binding capacity. These results suggest that it would be useful for dialysis protocols to include comparative studies of both serum native and modified beta2m in order to prevent beta2m-amyloidosis. (+info)
Toxicology and carcinogenesis studies of acrylonitrile (CAS No. 107-13-1) in B6C3F1 mice (gavage studies).
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Acrylonitrile is used in the production of acrylic and modacrylic fiber, elastomers, acrylonitrile-butadiene-styrene and styrene-acrylonitrile resins, nitrile rubbers, gas barrier resins, and chemical intermediates such as adiponitrile and acrylamide. Acrylonitrile was nominated for study by the National Institute of Environmental Health Sciences because of its potential for human exposure, its classification as a probable human carcinogen, evidence of its carcinogenicity in rats, and the lack of carcinogenicity studies in a second animal species. Male and female B6C3F1 mice received acrylonitrile (greater than 99% pure) in deionized water by gavage for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, L5178Y mouse lymphoma cells, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse peripheral blood erythrocytes. 14-WEEK STUDY: Groups of 10 male and 10 female mice were administered 0, 5, 10, 20, 40, or 60 mg acrylonitrile/kg body weight in deionized water by gavage, 5 days per week, for 14 weeks. All male and nine female mice in the 60 mg/kg groups and eight male and three female mice in the 40 mg/kg groups died on the first day of the study. The mean body weight gain of 20 mg/kg males was less than that of the vehicle control group. Clinical findings included lethargy and abnormal breathing in the 40 mg/kg groups. Leukocyte and lymphocyte counts were decreased in 20 mg/kg males and 40 mg/kg females, and a minimal hemolytic anemia was observed in 40 mg/kg females. Heart weights of 20 mg/kg males were significantly greater than those of the vehicle controls, and left cauda epididymis weights of 10 and 20 mg/kg males were significantly increased. The incidences of chronic active inflammation and hyperplasia in the forestomach of 40 mg/kg females were significantly increased. 2-YEAR STUDY: Groups of 50 male and 50 female mice were administered acrylonitrile in deionized water by gavage at doses of 0, 2.5, 10, or 20 mg/kg, 5 days per week, for 104 to 105 weeks. Urine from five male and five female mice from each group was collected at 2 weeks and at 3, 12, and 18 months and analyzed for thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations as markers of exposure to acrylonitrile. Survival, Body Weights, and Urinary Metabolite Analyses Survival of 20 mg/kg mice was significantly less than that of the vehicle control groups. Mean body weights of 20 mg/kg males and females were generally less than those of the vehicle controls throughout most of the study. Dose-related increases in urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations occurred in all dosed groups at 2 weeks and at 3, 12, and 18 months. Pathology Findings The incidences of squamous cell papilloma, squamous cell carcinoma, and squamous cell papilloma or carcinoma (combined) of the forestomach occurred with positive trends in males and females, and were present in 50% or greater of mice administered 10 or 20 mg/kg. The incidences of mild focal or multifocal epithelial hyperplasia (combined) of the forestomach in 20 mg/kg males and females and of mild diffuse or focal hyperkeratosis (combined) in 20 mg/kg males were increased. The incidences of harderian gland adenoma and adenoma or carcinoma (combined) were significantly increased in all dosed groups of males and in 10 and 20 mg/kg females, and the incidence of harderian gland hyperplasia was significantly increased in 10 mg/kg males. The incidence of benign or malignant granulosa cell tumor (combined) in the ovary of 10 mg/kg females was greater than that in the vehicle controls. The incidences of atrophy and cyst in the ovary of 10 and 20 mg/kg females were significantly increased. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in 10 mg/kg females was significantly increased. GENETIC TOXICOLOGY: Acrylonitrile was mutagenic in S. typhimurium strains TA100 and TA1535 in the presence of S9 liver enzymes; it was not mutagenic without S9 activation in these two strains. No mutagenic activity was observed in strain TA97 or TA98 with or without S9. Acrylonitrile was mutagenic in mouse lymphoma L5178Y cells in the absence of S9; it was not tested with S9. In cultured Chinese hamster ovary cells, acrylonitrile induced sister chromatid exchanges with and without S9; chromosomal aberrations were significantly increased in the presence of S9 only. Tests for induction of sex-linked recessive lethal mutations in germ cells of male D. melanogaster were negative when acrylonitrile was administered in feed or by injection. A test for induction of reciprocal translocations in male D. melanogaster was negative. In contrast to the induction of chromosomal damage by acrylonitrile in mammalian cells in vitro, no increase in the frequency of micronucleated normochromatic erythrocytes was observed in peripheral blood samples from male or female mice administered acrylonitrile by gavage for 14 weeks. In summary, acrylonitrile induced genetic damage in vitro in bacterial and mammalian cells, but in vivo test results in D. melanogaster and in mice were negative. CONCLUSIONS: Under the conditions of this 2-year gavage study, there was clear evidence of carcinogenic activity of acrylonitrile in male and female B6C3F1 mice based on increased incidences of forestomach and harderian gland neoplasms. Neoplasms of the ovary and lung in female mice may have been related to administration of acrylonitrile. Nonneoplastic lesions of the forestomach and harderian gland in males and of the forestomach and ovary in females were associated with administration of acrylonitrile by gavage for 2 years. (+info)
Mechanisms for the induction of oxidative stress in Syrian hamster embryo cells by acrylonitrile.
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Chronic administration of acrylonitrile to rats resulted in an increase in the incidence of glial neoplasms of the brain. Recent studies have shown that acrylonitrile induces oxidative stress in rat brain and cultured rat glial cells. Acrylonitrile also induces morphological transformation concomitant with an increase in the formation of oxidized DNA in Syrian Hamster Embryo (SHE) cells in a dose-dependent manner. The mechanism for the induction of oxidative stress in SHE cells remains unresolved. The present study examined the effects of acrylonitrile on enzymatic and nonenzymatic antioxidants in SHE cells. SHE cells were treated with subcytolethal doses of acrylonitrile (0, 25, 50, and 75 microg/ml) for 4, 24, and 48 h. Acrylonitrile (50 microg/ml and 75 microg/ml) increased the amount of reactive oxygen species in SHE cells at all time points. Glutathione (GSH) was depleted and catalase and superoxide dismutase activities were significantly decreased in SHE cells after 4 h of treatment. The inhibition of these antioxidants was temporal, returning to control values or higher after 24 and 48 h. Xanthine oxidase activity was increased following 24 and 48 h treatment with acrylonitrile. 1-aminobenzotriazole, a suicidal P450 enzyme inhibitor, attenuated the effects of acrylonitrile on catalase and xanthine oxidase in SHE cells, suggesting that P450 metabolism is required for acrylonitrile to produce its effects on these enzymes. Additional studies showed that in the absence of metabolic sources acrylonitrile had no effect on either catalase or superoxide dismutase activity. These results suggest that the induction of oxidative stress by acrylonitrile involves a temporal decrease in antioxidants and increase in xanthine oxidase activity that is mediated by oxidative metabolism of acrylonitrile. (+info)
Acrylonitrile is a multisite carcinogen in male and female B6C3F1 mice.
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Acrylonitrile is a heavily produced unsaturated nitrile, which is used in the production of synthetic fibers, plastics, resins, and rubber. Acrylonitrile is a multisite carcinogen in rats after exposure via gavage, drinking water, or inhalation. No carcinogenicity studies of acrylonitrile in a second animal species were available. The current studies were designed to assess the carcinogenicity of acrylonitrile in B6C3F1 mice of both sexes. Acrylonitrile was administered by gavage at 0, 2.5, 10, or 20 mg/kg/day, 5 days per week, for 2 years. Urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine were measured as markers of exposure to acrylonitrile. In general, there were dose-related increases in urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations in all dosed groups of mice and at all time points. Survival was significantly (p < 0.001) reduced in the top dose (20 mg/kg) group of male and female mice relative to controls. The incidence of forestomach papillomas and carcinomas was increased in mice of both sexes in association with an increase in forestomach epithelial hyperplasia. The incidence of Harderian gland adenomas and carcinomas was also markedly increased in the acrylonitrile-dosed groups. In female mice, the incidence of benign or malignant granulosa cell tumors (combined) in the ovary in the 10 mg/kg dose group was greater than that in the vehicle control group, but because of a lack of dose response, this was considered an equivocal finding. In addition, the incidences of atrophy and cysts in the ovary of the 10 and 20 mg/kg dose groups were significantly increased. The incidences of alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in female mice treated with acrylonitrile at 10 mg/kg/day for 2 years. This was also considered an equivocal result. In conclusion, these studies demonstrated that acrylonitrile causes multiple carcinogenic effects after gavage administration to male and female B6C3F1 mice for 2 years. (+info)
Cytochrome P450 2E1 (CYP2E1) is essential for acrylonitrile metabolism to cyanide: comparative studies using CYP2E1-null and wild-type mice.
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Acrylonitrile (AN) is a rodent carcinogen and suspected human carcinogen. Metabolism of AN proceeds via conjugation with glutathione or epoxidation via cytochrome P4502E1 (CYP2E1) to cyanoethylene oxide (CEO). It was hypothesized that CEO metabolism via epoxide hydrolase (EH) is the primary pathway for cyanide formation. The objective of this work is to assess the enzymatic basis of metabolism to cyanide. Male wild-type and CYP2E1-null mice received 0, 2.5, 10, 20, or 40 mg of AN/kg by gavage, and cyanide was measured in blood and tissues. CYP2E1 and EH expression were assessed using Western blot analyses. Present results demonstrated that cyanide concentrations in blood and tissues of AN-treated wild-type mice were higher at 1 versus 3 h, increased in a dose-dependent manner, and were significantly higher in AN-treated versus vehicle-treated mice. In contrast, cyanide concentrations in the blood and tissues of AN-treated CYP2E1-null mice were not statistically different from those of vehicle-treated mice. Furthermore, this work showed that EH is expressed in CYP2E1-null and wild-type mice. In conclusion, under the current experimental conditions using CYP2E1-null mice, current work demonstrated for the first time that CYP2E1-mediated oxidation is a prerequisite for AN metabolism to cyanide. Since earlier studies showed that CYP2E1 is the only enzyme responsible for AN epoxidation, it is concluded that AN metabolism to CEO is a prerequisite for cyanide formation, and this pathway is exclusively catalyzed by CYP2E1. Finally, this work confirmed that cyanide plays an essential role in the causation of the acute toxicity/mortality of AN. (+info)
Structure and stereochemistry of the active metabolite of clopidogrel.
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Clopidogrel (SR25990C, PLAVIX) is a potent antiplatelet drug, which has been recently launched and is indicated for the prevention of vascular thrombotic events in patients at risk. Clopidogrel is inactive in vitro, and a hepatic biotransformation is necessary to express the full antiaggregating activity of the drug. Moreover, 2-oxo-clopidogrel has been previously suggested to be the essential key intermediate metabolite from which the active metabolite is formed. In the present paper, we give the evidence of the occurrence of an in vitro active metabolite after incubation of 2-oxo-clopidogrel with human liver microsomes. This metabolite was purified by liquid chromatography, and its structure was studied by a combination of mass spectometry (MS) and NMR experiments. MS results suggested that the active metabolite belongs to a family of eight stereoisomers with the following primary chemical structure: 2-[1-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene]acet ic acid. Chiral supercritical fluid chromatography resolved these isomers. However, only one of the eight metabolites retained the biological activity, thus underlining the critical importance of associated absolute configuration. Because of its highly labile character, probably due to a very reactive thiol function, structural elucidation of the active metabolite was performed on the stabilized acrylonitrile derivative. Conjunction of all our results suggested that the active metabolite is of S configuration at C 7 and Z configuration at C 3-C 16 double bound. (+info)
Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry.
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Mass spectrometry plays a central role in the characterisation of modified nucleotides, but pseudouridine is a mass-silent post-transcriptional modification and hence not detectable by direct mass spectrometric analysis. We show by the use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry that pseudouridines in tRNA can be specifically cyanoethylated by acrylonitrile without affecting the uridines. The tRNA was cyanoethylated and then subjected to digestion with either RNase A or RNase T1. Cyanoethylated digestion fragments were identified by mass spectrometric comparison of untreated and acrylonitrile-treated samples, where the addition of one acrylonitrile resulted in a mass increment of 53.0 Da. The exact modified nucleotide could be identified by tandem mass spectrometry on the cyanoethylated digestion fragment. The methodology was used to identify additional one 4-thiouridine and one pseudouridine in tRNA(TyrII) from Escherichia coli. Furthermore, we observed that RNase A is highly tolerant towards nucleotide modifications, only being inhibited by 2'-O-methylation, whereas RNase T1 cleavage is affected by most nucleotide modifications. (+info)
Physiologically based pharmacokinetic model parameter estimation and sensitivity and variability analyses for acrylonitrile disposition in humans.
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A physiologically based pharmacokinetic (PBPK) model of acrylonitrile (ACN) and cyanoethylene oxide (CEO) disposition in humans was developed and is based on human in vitro data and scaling from a rat model (G. L. Kedderis et al., 1996, TOXICOL: Appl. Pharmacol.140, 422-435) for application to risk assessment. All of the major biotransformation and reactivity pathways, including metabolism of ACN to glutathione conjugates and CEO, reaction rates of ACN and CEO with glutathione and tissues, and the metabolism of CEO by hydrolysis and glutathione conjugation, were described in the human PBPK model. Model simulations indicated that predicted blood and brain ACN and CEO concentrations were similar in rats and humans exposed to ACN by inhalation. In contrast, rats consuming ACN in drinking water had higher predicted blood concentrations of ACN than humans exposed to the same concentration in water. Sensitivity and variability analyses were conducted on the model. While many parameters contributed to the estimated variability of the model predictions, the reaction rate of CEO with glutathione, hydrolysis rate for CEO, and blood:brain partition coefficient of CEO were the parameters predicted to make the greatest contributions to variability of blood and brain CEO concentrations in humans. The main contributor to predicted variance in human blood ACN concentrations in people exposed through drinking water was the Vmax for conversion of ACN to CEO. In contrast, the main contributors for variance in people exposed by inhalation were expected to be the rate of blood flow to the liver and alveolar ventilation rate, with the brain:blood partition coefficient also contributing to variability in predicted concentrations of ACN in the brain. Expected variability in blood CEO concentrations (peak or average) in humans exposed by inhalation or drinking water was modest, with a 95th-percentile individual expected to have blood concentrations 1.8-times higher than an average individual. (+info)