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PubMed1995

Dethiobiotin synthetase: the carbonylation of 7,8-diaminonanoic acid proceeds regiospecifically via the N7-carbamate.

Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of dethiobiotin from (7R,8S)-7,8-diaminononanoic acid (7,8-diaminopelargonic acid, DAPA), CO2, and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of CO2. However, four lines of evidence together indicated that only the N7-carbamate of DAPA was an intermediate in the reaction catalyzed by DTBS. (1) Addition of diazomethane to mixtures of DAPA and [14C]CO2 yielded a mixture of the N7- and N8-methyl carbamate esters, consistent with carbamate formation in free solution. In the presence of excess DTBS (over DAPA), the ratio of N7:N8-methyl carbamate esters recovered was roughly doubled, suggesting that the enzyme preferentially bound the N7-DAPA-carbamate. (2) Both N7- and N8-DAPA-carbamates were observed directly by 1H and 13C NMR in solutions containing DAPA and [13C]CO2. In the presence of excess DTBS (over DAPA) only one carbamate was observed, showing that carbamate binding to the enzyme was regiospecific. 13C NMR of mixtures containing enzyme, [7-15N]DAPA, and [13C]CO2 showed that the enzyme-bound carbamate was at N7 of DAPA. In addition, pulse-chase experiments showed that the binary complex of DTBS and N7-DAPA-carbamate became kinetically committed upon addition of MgATP. (3) The N7-DAPA-carbamate mimic, 3-(1-aminoethyl)nonanedioic acid, in which the carbamate nitrogen was replaced with a methylene group, cyclized to the corresponding lactam in the presence of DTBS and ATP; ADP and P(i) were also formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids, Diamino↗
PubMed1981

The carbamate equilibrium of alpha- and epsilon-amino groups of human hemoglobin at 37 degrees C.

We have investigated the carbamate equilibrium of human adult hemoglobin, human cord blood hemoglobin, methemoglobin, and carbamylated hemoglobin using a stopped flow, rapid reaction pH apparatus described previously. The carbamate formation of human adult hemoglobin at 37 degrees C and ionic strength 0.15 was measured at pH values ranging from 6.2 to 8.8 and at CO2 partial pressures between 15 and 140 Torr. From experiments with unmodified hemoglobin as well as with hemoglobin specifically carbamylated at the four NH2 termini, it was found that already at pH 8, the epsilon-amino groups contribute significantly to carbamate formation in addition to the alpha-amino groups. At pH 8.5, about 70% of the total carbamate is due to epsilon-amino groups. The carbamate formation of alpha- and epsilon-amino groups can be suppressed by complete carbamylation of the hemoglobin. The results obtained from human adult deoxy- and oxyhemoglobin were used to calculate the equilibrium constants governing carbamate formation of these hemoglobins: Kc, the carbamate equilibrium constant, Kz, the R-NH2 ionization constant, and n, the number of binding sites per hemoglobin tetramer. Accordingly, two types of alpha-amino groups, each comprising two groups per tetramer, participate in carbamate formation of deoxyhemoglobin, one of low CO2 affinity (pKc = 5.2; pKz = 7.1; n = 2) and one of high CO2 affinity (pKc = 4.4; pKz = 6.1; n = 2). The pKz values derived from carbamate measurements agree within experimental error with figures obtained by difference titration of unmodified and specifically carbamylated hemoglobin. In addition to the alpha-amino groups, 15 epsilon-amino groups with pKc = 5.0 and pKz = 9.8 form carbamate in deoxyhemoglobin. In oxyhemoglobin, the carbamate data could be fitted with only two similar alpha-amino groups per tetramer in addition to 15 epsilon-amino groups, the latter with pKc = 4.7 and pKz = 10.2. The difference titration of the alpha-amino groups of oxyhemoglobin showed abnormal titration behavior of the beta-chain alpha-NH2. The pKc and pKz values obtained for the epsilon-amino groups of unmodified hemoglobin also provide a good description of the carbamate formed by hemoglobin specifically carbamylated at the four alpha-amino groups. The oxylabile carbamate, according to these results and in agreement with earlier reports, is only formed by alpha-amino groups; it amounts to 0.18 mol/mol of hemoglobin monomer at physiological conditions of pH 7.2 and pCO2 = 40 Torr. The amount of CO2 bound by methemoglobin equals that of oxyhemoglobin. Experiments carried out in the presence of 2,3-diphosphoglycerate provided evidence for competitive binding of CO2 and diphosphoglycerate to adult and cord blood oxy- as well as deoxyhemoglobin, the sites of competition probably being alpha-amino groups.

Carbamates↗
PubMed1986

Modification of DNA and metabolism of ethyl carbamate in vivo: formation of 7-(2-oxoethyl)guanine and its sensitive determination by reductive tritiation using 3H-sodium borohydride.

The modification of liver DNA of mice and rats by ethyl carbamate and its putative proximate metabolite, vinyl carbamate, has been investigated. Following treatment with [ethyl-1-14C]-ethyl carbamate, the main radioactive DNA adduct was identified as 7-(2-oxoethyl)guanine by cochromatography with the authentic marker in several separation systems. After reduction by sodium borohydride (NaBH4) to 7-(2-hydroxyethyl)guanine, the radioactive material again cochromatographed with the respective marker. Reduction of modified liver DNA by 3H-NaBH4, following administration of unlabelled ethyl carbamate or vinyl carbamate, allowed the quantitation of 7-(2-oxoethyl)guanine (as 7-(2-hydroxy-2-[3H]-ethyl)guanine). Vinyl carbamate led to about 100 times as much 7-(2-oxoethyl)guanine (on a molar basis) as did ethyl carbamate. Both the formation of 7-(2-oxoethyl)guanine by ethyl carbamate and vinyl carbamate, and the much higher activity of the latter compound, strongly support the existence of the metabolic activation pathway, ethyl carbamate----vinyl carbamate----epoxyethyl carbamate, as proposed by Dahl et al. (1978, 1980). The possible role of 7-(2-oxoethyl)guanine in the initiation of the carcinogenic process is discussed in view of the structural equilibrium with its hemiacetal conformation, O6,7-(1'-hydroxyethano)guanine. In the latter conformation, it is assumed to represent a promutagenic lesion. In addition, intrastrand cross-links between modified guanine and adjacent cytosine or adenine seem possible and may have promutagenic consequences. Replication of DNA containing such lesions may lead to the induction of mutations. This may be a critical event in the initiation, and eventually progression, of the carcinogenic process as determined by ethyl carbamate and other carcinogens, such as vinyl chloride, which lead to the same DNA modification.

Animals↗
PubMed1991

N-Butyl-N-methyl-4-nitrophenyl carbamate as a specific active site titrator of bile-salt-dependent lipases.

The effect of a series of synthetic carbamates on the human (milk or pancreatic) bile-salt-dependent lipase (cholesterol esterase) was examined. N-isopropyl-O-phenyl, N-methyl-O-phenyl, N-butyl-(4-nitrophenyl), N-phenyl-(4-nitrophenyl), N-butyl-N-methyl and N-pentyl-O-phenyl carbamates were inhibitors of the enzyme activity, while O-isopropyl-N-phenyl, O-methyl-N-phenyl, O-benzyl-N-isopropyl and O-cyclohexyl-N-phenyl carbamates were not even recognized by the enzyme. The N-alkyl chain length is essential for the enzyme inhibition and N-butyl-(4-nitrophenyl) or N-pentyl-O-phenyl carbamates are more potent inhibitors than N-methyl-O-phenyl or N-isopropyl carbamates. The inhibition by reactive carbamates fits the criteria for mechanism-based inhibition: the inhibition is first-order with time, shows saturation kinetics with increasing carbamate concentration and leads to an inactive stoichiometric enzyme-inhibitor complex; the enzyme activity can be protected by a competitive inhibitor. Evidence is shown that the enzymatic nucleophilic attack of carbamates is directed at the carbonyl carbon atom and not the nitrogen atom. The inhibition of bile-salt-dependent lipase does not occur consecutive to the formation of a reactive isocyanate derivative of carbamate but via a tetrahedral intermediate involving essential residues implicated in the enzyme catalytic site. This intermediate evolves by liberation of alcohol (or phenol) and formation of an inactive carbamyl enzyme. Among the carbamates tested, N-butyl-N-methyl-(4-nitrophenyl) carbamate specifically inhibits the bile-salt-dependent lipase; the release of 4-nitrophenol from this carbamate is directly proportional to the enzyme inhibition and it may be defined as a specific active-site titrator for bile-salt-dependent lipases.

Animals↗
PubMed1996

Effects of ethyl carbamate and its metabolites on the antibody response in splenocyte cultures from female Balb/C mice.

To investigate a possible role by cytochrome P450 (P450) in ethyl carbamate-induced immunosuppression, an attempt to assess the ability of ethyl carbamate, its metabolites produced by P450 (i.e., ethyl N-hydroxycarbamate and vinyl carbamate), and methyl carbamate to suppress the polyclonal antibody response induced by bacterial lipopolysaccharide was made in splenocyte cultures isolated from female Balb/C mice. The results showed that vinyl carbamate and ethyl N-hydroxycarbamate were more immunosuppressive compared to ethyl carbamate. A structurally related analogue, methyl carbamate, did not suppress the antibody response. These results indicate that metabolism of ethyl carbamate by P450 may produce more immunosuppressive metabolites as in ethyl carbamate-induced carcinogenicity. A pre-incubation study with phenobarbital-induced liver microsomes in the presence of NADPH-generating system showed that the antibody response was suppressed by ethyl carbamate when splenocytes were pre-incubated with ethyl carbamate and microsomes simultaneously. Moreover, the suppression was completely recovered by the addition of a P450 inhibitor, aminoacetonitrile, in the pre-incubation. Taken together, the present results indicate that metabolism of ethyl carbamate by P450 enzyme(s) may be an important pathway to cause immunosuppression.

Animals↗
PubMed1982

The relationship between binding to cytochrome P-450 and metabolism of n-alkyl carbamates in isolated rat hepatocytes.

The binding constants of an homologous series of n-alkyl (C2-C10) carbamates (see formula in text) to the cytochrome P-450 of suspensions of isolated, viable rat hepatocytes have been measured. All the carbamates except ethyl and propyl carbamate produced type I difference spectra and their binding affinities (1/Ks) were found to be directly dependent upon their lipophilicity. These binding affinities were similar to those determined in rat liver microsomes. Maximum development of the binding spectrum in hepatocytes was always within one second of the addition of each carbamate, indicating that for these carbamates membrane permeability was not rate limiting for access to, and metabolism by, cytochrome P-450 and that much of the cells' cytochrome P-450 was unoccupied by endogenous substrates. Th major metabolites of C4-C8 carbamates were unconjugated omega-1 oxidation products. Below hexyl carbamate only the omega-1 hydroxylated metabolite was observed but for the more lipophilic carbamates the keto metabolite was also a major product. The same products were found in blood after i.p. dosing of rats with hexyl carbamate. A direct relationship was observed between the affinity constant of the carbamate for cytochrome P-450 and the total rate of oxidative metabolism in the omega-1 position. Hydrolysis of the carbamate group was a minor metabolic pathway in contrast to the in vivo situation.

Animals↗
PubMed1999

Structure-reactivity probes for active site shapes of cholesterol esterase by carbamate inhibitors.

4,4'-Biphenyl-di-N-butylcarbamate (1), (S)-1,1'-bi-2-naphthyl-2, 2'-di-N-butylcarbamate (S-2), (S)-1, 1'-bi-2-naphthyl-2-N-butylcarbamate-2'-butyrate (S-3), 2, 2'-biphenyl-di-N-butylcarbamate (4), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-octylcarbamate (5), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-phenylcarbamate (6), 2, 2'-biphenyl-2-N-butylcarbamate-2'-butyrate (7), 2, 2'-biphenyl-2-N-butylcarbamate-2'-ol (8), 2, 2'-biphenyl-2-N-octylcarbamate-2'-ol (9), (R)-1, 1'-bi-2-N-naphthyl-2-butylcarbamate-2'-ol (R-10), and glyceryl-1,2, 3-tri-N-butylcarbamate (11) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase. All inhibitors are irreversible inhibitors of the enzyme. Carbamates 1-3 and 7-10 are the first alkyl chain and esteratic binding site-directed irreversible inhibitors due to the fact that the reactivity of the enzyme is protected by the irreversible inhibitor, trifluoroacetophenone in the presence of these carbamates. Carbamate 1 is the least potent inhibitor for the enzyme probably due to the fact that the inhibitor molecule adopts a linear conformation and one of the carbamyl groups of the inhibitor molecule covalently interacts with the first alkyl chain binding site of the enzyme while the other carbamyl group of the inhibitor molecule exposes outside the active site. With near orthogonal conformations at the pivot bond of biaryl groups, one carbamyl group of carbamates S-2, S-3, and R-10 covalently binds to the first alkyl chain binding site of the enzyme while the other carbamyl, butyryl, or hydroxy group can not bind covalently to the second alkyl chain binding site probably due to the orthogonal conformations. Carbamates 4-9 and 11 are very potent inhibitors for the enzyme probably due to the fact that all these molecules freely rotate at the pivot bond of the biphenyl or glyceryl group and therefore can fit well into the bent-shaped first and second alkyl chains binding sites of the enzyme. Although, carbamates 4-6 and 11 are irreversible inhibitors of cholesterol esterase, the enzyme is not protected but further inhibited by trifluoroacetophenone in the presence of these carbamates. Therefore, carbamates 4-6 and 11 covalently bind to the first alkyl chain binding site of the enzyme by one of the carbamyl groups and may also bind to the second alkyl chain binding site of the enzyme by the second carbamyl group. Besides the bent-shaped conformation, the inhibition by carbamate 6 is probably assisted by a favorable pi-pi interaction between Phe 324 at the second alkyl chain binding site of the enzyme and the phenyl group of the inhibitor molecule. For cholesterol esterase, carbamates 8-10 are more potent than carbamates S-2, 4, and 5 probably due to the fact that the inhibitor molecules interact with the second alkyl chain binding site of the enzyme through a hydrogen bond between the phenol hydroxy group of the inhibitor molecules and the His 435 residue in that site.

Binding Sites↗
PubMed2000

Immunotoxicity of ethyl carbamate in female BALB/c mice: role of esterase and cytochrome P450.

Ethyl carbamate, a potent carcinogen, has been characterized to be metabolized by cytochrome P450 (P450) and esterase. It has recently been demonstrated that P450 may activate ethyl carbamate to immunotoxic metabolites. To investigate the role of esterase in ethyl carbamate-induced immunosuppression, mice were pretreated intraperitoneally with an esterase inhibitor, diazinon, at 20 mg/kg 30 min prior to the administration of ethyl carbamate intraperitoneally at 100 and 400 mg/kg for 7 consecutive days. Pretreatment with diazinon completely blocked the serum esterase activity. Histopathologically splenic and thymic atrophy was observed when mice were treated with ethyl carbamate, which was potentiated by the pretreatment with diazinon. In spleen, lymphocytes in the periarteriolar lymphoid sheath and the marginal zone appeared to be depleted in the white pulps. In thymus, ethyl carbamate caused a marked depletion of cells in cortex. The antibody response to sheep red blood cells (SRBCs) was more suppressed by ethyl carbamate in diazinon-pretreated groups than in corn oil-pretreated groups. These results suggest that the metabolism of ethyl carbamate by esterase may be an inactivation pathway in ethyl carbamate-induced immunosuppression. In addition, ethyl N-hydroxycarbamate, a P450 metabolite, suppressed the lymphoproliferative response induced by lipopolysaccharide and concanavalin A in splenocyte cultures. These results indicate that the metabolism of ethyl carbamate by P450 may be an activation pathway in immunosuppression by ethyl carbamate.

Animals↗
PubMed2000

N-carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea. Thermodynamics and kinetics of the spontaneous reaction.

N-carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v1) and cleavage (v2) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k1* of carbamate formation from 'MFR'(MFR + MFRH+) and 'CO2' (CO2 + H2CO3 + HCO3-+ CO32-) was found to be 7 M-1.s-1 (v1 = k1* ['MFR'] ['CO2']) while the pseudo first order rate constant k2* of carbamate cleavage was 12 s-1 (v2 = k2* [carbamate]). The equilibrium constant K* = k1*/k2* = [carbamate]/['MFR']['CO2'] was 0.6 M-1 at pH 7.0 corresponding to a free energy change DeltaG degrees ' of + 1.3 kJ.mol-1. The pH and temperature dependence of k1*, of k2* and of K* were determined. From the second order rate constant k1* it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO2 formation from HCO3- in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3- which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N-carboxymethanofuran to N-formylmethanofuran. From the concentrations of 'CO2' and of 'MFR' determined by 1D-NMR spectroscopy and the pKa of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k1 (v1 = k1 [MFR] [CO2]). The second order rate constant k1 was found to be approximately 1000 M-1 x s-1 at 300 K and pH values between 7.0 and 8. 0 which is in the order of k1 values determined for other carbamate forming reactions by stopped flow.

Aldehyde Oxidoreductases↗
PubMed1993

Molecular dissection of cholinesterase domains responsible for carbamate toxicity.

Carbamate compounds marked for their cholinesterase (ChE) inhibition are widely used as therapeutics and as insecticides. Groups of closely related carbamate molecules provide an important tool in the understanding of the domains responsible for binding these ligands to ChEs. Comparative inhibition profiles were derived for five N-methyl carbamates, mostly carbofuran derivatives, differing in length and branching of their hydrocarbonic chain towards human erythrocyte acetylcholinesterase (H.AChE), human serum butyrylcholinesterase (H.BChE) in its normal form or in a mutant form containing the point mutation Asp70-->Gly, and Drosophila nervous system ChE. Carbofuran was more toxic to all three ChEs than any of the other derivatives, with IC50 values which differed by more than 1000-fold. Drosophila ChE appeared to be most sensitive to all of the examined carbamates, and H.AChE was consistently more sensitive than H.BChE. Moreover, inhibition efficiency for H.BChE decreased more effectively than it did for H.AChE with increased length and complexity of the side chain, indicating less flexible carbamate binding site in BChE as compared with AChE. The Asp70-->Gly mutation had no apparent effect on H.BChE inhibition by N-methyl carbamates, suggesting that the Asp70 domain localized near the rim of the active site groove is not important in carbamate binding. Comparison of the carbamate IC50 values with published LD50 values demonstrated correlation between the in vivo toxicity and inhibition of BChE by carbamates, suggesting a biological in addition to scavenging importance for BChE in mammals. Pinpointing different domains characteristic of carbamate binding in each member of the ChE family can thus shed light on the variable toxicity of these inhibitors to insects and mammals, predict the toxicity of yet untested inhibitor molecules and help in designing novel and improved ChE inhibitors.

Acetylcholinesterase↗
PubMed1995

Effects of cigarette smoke and disulfiram on tumorigenicity and clastogenicity of ethyl carbamate in mice.

Exposure of male Balb/C mice to mainstream cigarette smoke for 4 months, starting 10 or 30 days before the administration of ethyl carbamate (0.3% in drinking water for 3 weeks), resulted in an up to 57.6% (P < 0.05) decrease of lung adenoma multiplicity. However, the number of ethyl carbamate-induced lung tumors was not significantly affected by exposure to cigarette smoke when ethyl carbamate was injected i.p. in single doses of 0.5 or 1.0 g/kg, irrespective of the different treatment schedules used, i.e. (a) 10 days before and 4 days after the ethyl carbamate injection; (b) throughout the experiment starting 10 days before the ethyl carbamate injection, and (c) until the end of the experiment, starting 30 days after the ethyl carbamate injection. Disulfiram (500 mg/kg), given by gavage 24 h and 1 h before the ethyl carbamate injection, decreased by 88.5% (P < 0.001) the multiplicity of lung adenomas but had no effect on tumorigenesis when administered after the carcinogen injection. Proadifen (SKF-525 A, 50 mg/kg) injected i.p. 24 h and 1 h before and 24 h and 48 h after the injection with ethyl carbamate tended to decrease the multiplicity of lung adenomas, but not to a significant extent. Furthermore, disulfiram given 24 h and 1 h before the i.p. administration of ethyl carbamate completely prevented its clastogenicity in mouse bone marrow. On the other hand, cigarette smoke, which was per se a weak clastogen in bone marrow erythroblasts, synergistically potentiated the clastogenic response to ethyl carbamate in a more than additive fashion.

Animals↗
PubMed1996

Molecular recognition by cholesterol esterase of active site ligands: structure-reactivity effects for inhibition by aryl carbamates and subsequent carbamylenzyme turnover.

Interactions of mammalian pancreatic cholesterol esterases from pig and rat with a family of aryl carbamates CnH2n+1NHCOOAr [n = 4-9; Ar = phenyl, p-X-phenyl (X = acetamido, bromo, fluoro, nitro, trifluoromethyl), 2-naphthyl, 2-tetrahydronaphthyl, estronyl] have been investigated, with an aim of delineating the ligand structural features which lead to effective molecular recognition by the active site of the enzyme. These carbamates inhibit the catalytic activity of CEase by rapid carbamylation of the active site, a process that shows saturation kinetics. Subsequent slow decarbamylation usually leads to full restoration of activity, and therefore aryl carbamates are transient inhibitors, or pseudo-substrates, of CEase. Structural variation of carbamate inhibitors allowed molecular recognition in the fatty acid binding and steroid binding loci of the extended active site to be probed, and the electronic nature of the carbamylation transition state to be characterized. Optimal inhibitory activity is observed when the length of the carbamyl function is n = 6 and n = 7 for porcine and rat cholesterol esterases, respectively, equivalent to eight- and nine-carbon fatty acyl chains. In contrast, inhibitory activity increases progressively as the partial molecular volume of the aromatic fragment increases. Hammett plots for p-substituted phenyl-N-hexyl carbamates indicate that the rate-determining step for carbamate inhibition is phenolate anion expulsion. Effects of the bile salt activator taurocholate on the kinetically resolved phases of the pseudo-substrate turnover of aryl carbamates were also studied. Taurocholate increases the affinity of the carbamate for the active site of cholesterol esterase in the reversible, noncovalent complex that precedes carbamylation and increases the rate constants of the serial carbamylation and decarbamylation steps. Structural variation of the N-alkyl chain and of the aryl fused-ring system provides an accounting of bile salt modulation of the fatty acid and steroid binding sites, respectively. In that pseudo-substrate turnover of aryl carbamates proceeds by a three-step mechanism that is analogous to that for rapid turnover of lipid ester substrates, these investigations illuminate details of ligand recognition by the extended active site of cholesterol esterase that are prominent determinants of the substrate specificity and catalytic power of the enzyme.

Animals↗
PubMed2005

Role of hydrogen bonds in controlling the morphology of self-assembling carbamate systems.

With a view to understand the role of hydrogen bonding in controlling the morphology of self-assembling carbamate systems, N-octadecylcarbamate dodecyl ester was blended individually with a low molecular weight polyethylene and two commercial clarifiers, namely Kemamide S and Kemamide E 180. The effect of blending on the morphology of this long chain carbamate was investigated using optical microscopy, differential scanning calorimetry, and X-ray diffraction. The crystal structure of the carbamate was not affected by the addition of polyethylene or Kemamide S. The heterogeneous nucleation of the carbamate by the polyethylene or Kemamide S resulted in the reduction of the spherulite size of the carbamate, but it did not improve the transparency of the sample due to phase separation. On the other hand, significant improvement of transparency was achieved when the carbamate was blended with Kemamide E 180. Blending reduced the crystallite and spherulite size, heat of fusion, and crystallinity. An exchange of hydrogen bonds between the carbamate and Kemamide E is indicated in the IR spectra, and this affects the packing of the alkyl chains of the carbamates. This heterogeneous blending shows similar effects on the morphology as was achieved by blending two homologous carbamates in our previous study (J. Phys. Chem. B 2003, 107, 8416).

Amides↗
PubMed2005

Retrospective trends and current status of ethyl carbamate in German stone-fruit spirits.

Ethyl carbamate (urethane, C(2)H(5)OCONH(2)) is a known genotoxic carcinogen of widespread occurrence in fermented food and beverages with highest concentrations found in stone-fruit spirits. Between 1986 and 2004, 631 cherry, plum or mirabelle (yellow plum) spirits were analysed for ethyl carbamate using gas chromatography in combination with mass spectrometry after extrelut extraction. The ethyl carbamate concentration of the samples ranged between 0.01 mg l(-1) and 18 mg l(-1) (mean 1.4 mg l(-1)). After exposure of the samples to UV light, significantly (p=0.001) higher concentrations between 0.01 mg l(-1) and 26 mg l(-1) (mean 2.3 mg l(-1)) were found. The ethyl carbamate concentration increased on average by 1.3 mg l(-1). A linear correlation between the year of sampling and ethyl carbamate concentration showed a statistically significant but very slight decrease (R=-0.10, p=0.024). However, if only samples which officially were non-compliant were considered exceeding the upper limit of 0.4 mg l(-1) more than twice, a significant reduction (R =-0.56, p=0.018) of the quota was evident. This shows that measures to reduce ethyl carbamate were successfully introduced in many distilleries. However, nearly 20 years after the first warnings about ethyl carbamate in spirit drinks, the problem persists especially in products derived from small distilleries. During experimental production of stone-fruit spirits using state-of-the-art technologies, it was shown that the occurrence of ethyl carbamate in stone fruit spirits is preventable. Even for small distilleries, simple possibilities like destoning exist to minimize the ethyl carbamate content.

Alcoholic Beverages↗
PubMedDate not supplied

Ethyl carbamate metabolism: in vivo inhibitors and in vitro enzymatic systems.

The metabolism of ethyl carbamate and the localization of its metabolites have been shown to be almost completely inhibited by ethanol in the mouse [Waddell, Marlowe, Pierce: Food Chem. Toxicol.25, 527 (1987); Yamamoto, Pierce, Hurst, Chen, Waddell: Drug Metab. Dispos. 16, 355 (1988)]. The enzyme system catalyzing this metabolism which is inhibited by ethanol now has been further investigated in both in vivo and in vitro studies. There is a direct, highly significant relationship between the extent of metabolism of ethyl carbamate and covalent binding of metabolites to liver protein. Paraoxon, carbaryl, CCl4 ethanol, methimazole, 4-methylpyrazole, diethyl maleate, ethyl N-hydroxycarbamate, and t-butyl carbamate inhibit, to different extents, the metabolism of ethyl carbamate in vivo; SKF-525A, CoCl2, Cacyanamide, chloral hydrate, 2-oxo-4-thiazolidine carboxylic acid, allopurinol, and methyl carbamate do not. Porcine liver esterase, yeast aldehyde dehydrogenase and mouse liver catalase catalyzed the metabolism in vitro; dog or bovine catalase, acid phosphatase, alcohol dehydrogenase, or carbonic anhydrase did not under the conditions tested. Paraoxon, 4-methylpyrazole, carbaryl, and NaF significantly inhibited the hydrolytic activity of mouse liver homogenates toward p-nitrophenyl acetate; ethanol or ethyl carbamate did not. However, each of these, except 4-methylpyrazole, inhibited the metabolism of ethyl carbamate by mouse liver homogenate or porcine liver esterase to about the same extent. Ion exchange chromatography of mouse liver cytosol revealed that the fraction with ability to metabolize ethyl carbamate co-chromatographed almost exactly with the ability to hydrolyze p-nitrophenyl acetate. It is proposed that ethyl carbamate is metabolized in the mouse, at least partially, by esterases; however, metabolism by other enzyme systems cannot be excluded.

Animals↗
PubMed2006

Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.

Animals↗
PubMed1991

Screen of five alkyl carbamates for initiating and promoting activity in rat liver.

Five alkyl carbamates, methyl, ethyl, propyl, hydroxypropyl and ethylhexyl, were tested for initiating and promoting activity in rat liver. The test for initiating activity consisted of administering the carbamate by gavage to male Sprague--Dawley rats at either 6 or 18 h after a 2/3 partial hepatectomy. One week later, the rats received 500 ppm sodium phenobarbital in their drinking water until killed 10 weeks later. None of the carbamates at 1/5 the LD50 induced gamma-glutamyltranspeptidase (GGT)-positive foci, indicating the lack of initiating activity. The tumor promoting activity test consisted of initiation with 80 mmol/kg diethylnitrosamine administered 18 h after a partial hepatectomy. One week later, the rats received one of the the carbamates at either 1/10 or 1/20 the LD50 5 days per week until sacrificed 10 weeks later. The alkyl carbamates increased the volume of the foci, the percent of the liver occupied by the foci, the number of foci/cm3 (except ethylhexyl carbamate), and the number of foci/liver (except ethylhexyl carbamate). These results suggest that the alkyl carbamates are tumor promoters and not tumor initiators in rat liver.

Animals↗
PubMed2003

Selective effects of carbamate pesticides on rat neuronal nicotinic acetylcholine receptors and rat brain acetylcholinesterase.

Effects of commonly used carbamate pesticides on rat neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. The potencies of these effects have been compared to the potencies of the carbamates to inhibit rat brain acetylcholinesterase. The potency order of six carbamates to inhibit alpha4beta4 nicotinic receptors is fenoxycarb > EPTC > carbaryl, bendiocarb > propoxur > aldicarb with IC50 values ranging from 3 microM for fenoxycarb to 165 microM for propoxur and >1 mM for aldicarb. Conversely, the potency order of these carbamates to inhibit rat brain acetylcholinesterase is bendiocarb > propoxur, aldicarb > carbaryl > EPTC, fenoxycarb with IC50 values ranging from 1 microM for bendiocarb to 17 microM for carbaryl and > mM for EPTC and fenoxycarb. The alpha4beta2, alpha3beta4, and alpha3beta2 nicotinic acetylcholine receptors are inhibited by fenoxycarb, EPTC, and carbaryl with potency orders similar to that for alpha4beta4 receptors. Comparing the potencies of inhibition of the distinct subtypes of nicotinic acetylcholine receptors shows that the alpha3beta2 receptor is less sensitive to inhibition by fenoxycarb and EPTC. The potency of inhibition depends on the carbamate as well as on a combination of alpha and beta subunit properties. It is concluded that carbamate pesticides affect different subtypes of neuronal nicotinic receptors independently of acetylcholinesterase inhibition. This implicates that neuronal nicotinic receptors are additional targets for some carbamate pesticides and that these receptors may contribute to carbamate pesticide toxicology, especially after long-term exposure.

Acetylcholinesterase↗