S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate

S-(N-methylcarbamoyl)glutathione, a chemically-reactive glutathione conjugate, has been isolated from the bile of rats administered methyl isocyanate and characterized, as its N-benzyloxycarbonyl dimethylester derivative, by tandem mass spectrometry. The ability of this glutathione adduct to donate an N-methylcarbamoyl moiety to the free -SH group of cysteine was evaluated in vitro with the aid of a highly specific thermospray LC/MS assay procedure. The glutathione adduct reacted readily with cysteine in buffered aqueous media (pH 7.4, 37 degrees C) and after 2 hr, 42.5% of the substrate existed in the form of S-(N-methylcarbamoyl)cysteine. The reverse reaction, i.e. between the cysteine adduct and free glutathione, also took place readily under these conditions. It is concluded that conjugation of methyl isocyanate with glutathione in vivo affords a reactive S-linked product which displays the potential to carbamoylate nucleophilic amino acids. The various systemic toxicities associated with exposure of animals or humans to methyl isocyanate could therefore be due to release of the isocyanate from its glutathione conjugate, which thus may serve as a vehicle for the transport of methyl isocyanate in vivo.     

S-(2-chloroacetyl)glutathione, a reactive glutathione thiol ester and a putative metabolite of 1,1-dichloroethylene

Conversion of the toxic vinyl halide 1,1-dichloroethylene (DCE) to S-(2-S-glutathionyl-acetyl)glutathione (GSCH2COSG) involves sequential acylation and alkylation of two glutathione (GSH) molecules by the microsomal DCE metabolite ClCH2COCl. To examine its possible role in DCE biotransformation, we synthesized the putative intermediate S-(2-chloroacetyl)glutathione (ClCH2COSG). In aqueous buffer, ClCH2COSG did not hydrolyze to release GSH, but instead underwent a two-step rearrangement to yield a cyclic product. Product analyses by liquid secondary ion mass spectrometry and 1H-13C heteronuclear correlation nuclear magnetic resonance spectroscopy indicated that rearrangement involved initial transfer of the chloroacetyl moiety from the cysteinyl thiol to the gamma-glutamyl alpha-amine. The cysteinyl thiol then displaced chloride from the 2-chloroacetyl methylene carbon to yield the cyclic product. Incubation of 2 mM ClCH2COSG with 20 mM GSH yielded approximately 4.5-fold more cyclic product than GSCH2COSG. ClCH2COSG alkylated oxytocindithiol and N-acetyl-L-cysteine to yield S-[2-(alkylthio)acetyl]glutathione adducts analogous to GSCH2COSG. S-2-Chloroacetylation products were absent. In reacting with thiols by alkylation and in decomposing by rearrangement, ClCH2COSG displayed properties strikingly different from those of ClCH2COCl. Although much less reactive than its acyl halide precursor, ClCH2COSG may display greater selectivity in covalent modification of cellular targets in DCE intoxication.     

Identification of N-(2-propenal)ethanolamine as a urinary metabolite of malondialdehyde

N-(2-propenal)ethanolamine was isolated from rat and human urine using anion exchange, cation exchange, size exclusion and high performance liquid chromatography. Acid hydrolysis of the isolate yielded malondialdehyde (MDA) and ethanolamine (E) in a 1:1 molar ratio. A 1:1 E-MDA adduct was synthesized and found to be chromatographically inseparable from the urinary metabolite. Its NMR and UV spectra and lack of fluorescence were consistent with those of an enaminal formed by a Schiff's base reaction. The identification in urine of an adduct of MDA with ethanolamine, and the previous identification of an adduct with serine, constitutes direct evidence for the oxidative decomposition in vivo of polyunsaturated fatty acids present in the relevant phospholipids. The absence in urine of MDA adducts with other alpha-amino compounds (at least in comparable amounts) indicates that the ethanolamine and serine derivatives are formed in situ and not as a result of reactions with MDA generated in enzymatic processes.     

Identification of N-(2-propenal) serine as a urinary metabolite of malondialdehyde

N-2-(Propenal) serine (S-MDA) was synthesized by reacting serine with malondialdehyde (MDA) and was shown to be a 1:1 adduct of the starting materials. The synthetic compound was found to be identical to a metabolite of MDA excreted in rat and human urine. The identity of the metabolite was confirmed by isolation and hydrolysis to yield equimolar quantities of serine and MDA. The presence of S-MDA in urine constitutes direct evidence for oxidative decomposition of phospholipids by lipid peroxidation in vivo.     

Identification of N-acetyltaurine as a novel metabolite of ethanol through metabolomics-guided biochemical analysis

The influence of ethanol on the small molecule metabolome and the role of CYP2E1 in ethanol-induced hepatotoxicity were investigated using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics platform and Cyp2e1-null mouse model. Histological and biochemical examinations of ethanol-exposed mice indicated that the Cyp2e1-null mice were more resistant to ethanol-induced hepatic steatosis and transaminase leakage than the wild-type mice, suggesting CYP2E1 contributes to ethanol-induced toxicity. Metabolomic analysis of urinary metabolites revealed time- and dose-dependent changes in the chemical composition of urine. Along with ethyl glucuronide and ethyl sulfate, N-acetyltaurine (NAT) was identified as a urinary metabolite that is highly responsive to ethanol exposure and is correlated with the presence of CYP2E1. Subsequent stable isotope labeling analysis using deuterated ethanol determined that NAT is a novel metabolite of ethanol. Among three possible substrates of NAT biosynthesis (taurine, acetyl-CoA, and acetate), the level of taurine was significantly reduced, whereas the levels of acetyl-CoA and acetate were dramatically increased after ethanol exposure. In vitro incubation assays suggested that acetate is the main precursor of NAT, which was further confirmed by the stable isotope labeling analysis using deuterated acetate. The incubations of tissues and cellular fractions with taurine and acetate indicated that the kidney has the highest NAT synthase activity among the tested organs, whereas the cytosol is the main site of NAT biosynthesis inside the cell. Overall, the combination of biochemical and metabolomic analysis revealed NAT is a novel metabolite of ethanol and a potential biomarker of hyperacetatemia.     

Why walnut flies superparasitize: time savings as a possible explanation

 This study evaluated a possible fitness advantage, specifically time savings, that might account for an unusual propensity in walnut flies (Rhagoletis spp.) to superparasitize their walnut hosts and to place eggs into existing egg-laying cavities. The first part of this study demonstrated that, in laboratory assays, females of two walnut fly species, R. boycei and R. juglandis, save time when cavities are reused and that in R. juglandis, where it was examined in detail with in vivo staining of eggs, time saving was not an artifact of differences in the size of clutches deposited at new versus existing sites. We further demonstrated that time savings reflected a reduction in the time required to generate the cavity itself. In the second part of the study, we evaluated the possibility that, in the field, time saved by reusing existing cavities is nullified by extra time spent mating associated with a previously described tendency for males to guard these cavities. Field observations of R. juglandis indicated that use of existing sites was, as expected, associated with increased mating. Yet, despite the added time spent mating, in observations of similar length females attempting to lay eggs at existing sites deposited clutches more often than females attempting to lay eggs at new sites. We discuss these results in the context of the more common pattern of superparasitism avoidance observed in host-specific insects. Received: 25 January 1996 / Accepted: 25 June 1996     

Identification of N alpha-acetyl-epsilon-(2-propenal)lysine as a urinary metabolite of malondialdehyde

Although orally administered malondialdehyde (MDA), a reactive hepatotoxic and mutagenic product of lipid peroxidation, is extensively metabolized to CO2, a portion is excreted in the urine in acid labile "bound" forms. Since much of the MDA in the diet is apparently bound to protein, the metabolism of protein-bound MDA was investigated. MDA was reacted with serum albumin and fed to rats. A urinary metabolite was detected which was shown to be identical to a metabolite of the lysine-MDA enaminal N epsilon-(2-propenal)lysine. After isolation by ion exchange and high performance liquid chromatography the metabolite was identified using high field nuclear magnetic resonance spectroscopy and fast atom bombardment-mass spectroscopy as N alpha-acetyl-epsilon-(2-propenal)lysine. This compound also was a major urinary metabolite of the Na enol salt of MDA administered by stomach intubation, and was excreted in increased amounts by rats fed a diet containing a highly peroxidizable oil (cod liver oil). It was also detected in the urine of fasted animals after injection with NaMDA, indicating that it is formed as a product of lipid peroxidation in vivo as well as of peroxidation of dietary lipids.     

Identification and quantification of 6 beta-hydroxydexamethasone as a major urinary metabolite of dexamethasone in man

Identification of 6 beta-hydroxydexamethasone as a major urinary metabolite of dexamethasone in man has been accomplished by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Mass fragmentographic measurements revealed that more than 30% of the intravenously or orally administered dexamethasone dose was excreted in the 24-h urine as 6 beta-hydroxydexamethasone, while only a small fraction of the dose was excreted as unchanged dexamethasone and its glucuronic acid conjugate.     

Coherent dynamics in water as a possible explanation of biological membranes formation

The formation of a biological molecular aggregate such as a membrane, is discussed as a collective process emerging from the dispersive dynamics arising from coherent quantum electrodynamics. The essential role of liquid water is stressed.     

Identification of dichloromethyl carbene as a metabolite of carbon tetrachloride

Although indirect evidence has suggested that liver microsomal cytochrome P-450 can reductively dehalogenate several compounds to carbene metabolites, there has been no direct proof for the formation of these reactive species. We report in this paper that carbenes can be chemically trapped and identified as metabolites. For example, 1,1-dichloro-2,2,3,3-tetramethylcyclopropane was identified as a metabolite by gas chromatography mass spectrometry when carbon tetrachloride (CCl₄) was incubated anaerobically with rat liver microsomes, NADPH and 2,3-dimethyl-2-butene. The reaction required NADPH and was inhibited by carbon monoxide. These findings show that cytochrome P-450 in rat liver microsomes can reductively metabolize CCl₄ to dichloromethyl carbene (:CCl₂) which can be trapped with 2,3-dimethyl-2-butene to form 1,1-dichloro-2,2,3,3-tetramethylcyclopropane. A similar approach may be used for the identification of carbene metabolites of other compounds.     

Oestrogen conjugation in mid trimester human fetal brain: a possible role in its sexual differentiation

Mid-trimester human fetal brain cytosol incubated with [3H]-oestradiol gave a radioactive peak on 5% polyacrylamide gels, which migrated with the bromophenol blue marker (anodic peak) and which could be suppressed by the addition of 100 times molar excess of unlabelled ligand to the incubate. This anodic peak could only be destroyed by proteolytic enzymes if they were added at the beginning of the incubation, but not subsequently, indicating that it did not represent a protein-bound oestradiol. Competition studies show that the anodic peak can be suppressed with natural oestrogens and ethinyloestradiol, but not by the synthetic oestrogens, androgens and progestins tested. Butanol extraction of incubates, followed by t.l.c. in a number of systems, indicates that both oestrone and oestradiol are sulphated. The parent steroids could be liberated by hydrolysis of incubates with either 1N sulphuric acid or aryl sulphatase. This conjugation may effectively curtail the action in fetal tissues of high levels of oestrogens and hence play a role in brain sexual differentiation in the human.     

Distribution of Proteus (Amphibia: Urodela: Proteidae) and its possible explanation

Approximately 250 localities of the nominal species Proteus anguinus Laurenti 1768 have been evaluated and listed. The species is limited to the Dinaric Karst; it ranges from the Isonzo-Soĉa River in southeastern Venezia Giulia, Italy, through the southern half of Slovenia, southern Croatia, and parts of Bosnia and Hercegovina, to the Trebišnica River in eastern Hercegovina. In some regions, populations have been extinguished or endangered by pollution or human-induced hydrographical changes. The distribution of Proteus is compared with those of some cave Crustacea: Troglocaris (Crustacea: Decapoda), Monolistra , and Titanethes (Crustacea: Isopoda). The similarity of distribution patterns within this ecologically diverse assemblage supports their paleogeographic rather than ecological foundation. The paleogeographical and paleoclimatological data, in combination with the physiological requirements of Proteus, strongly suggest that these animals invaded the caves, at least in the NW parts of their ranges, only after the last glaciations, within the last 10,000 years. It is suggested that the high heterozygosity of populations can best be explained by fusion of some locally restricted immigration waves. The close morpho- logical similarity of nearly all populations of Proteus is probably due to the convergent evolution of previously differentiated populations (or even species) after their withdrawal underground.     

DNA photolyase of enterococci: possible explanation for its low sunlight inactivation rate

DNA photolyase is perhaps the most ancient and direct arsenal in curing the UV-induced dimers formed in the microbial genome. Out of two cofactors of the enzyme, catalytic and light harvesting, differences in the latter have provided basis for categorizing photolyases of prokaryotes as folate and deazaflavin types. In the present study, the homology modeling of DNA photolyase of Enterococcus faecalis was undertaken. The predicted models were structurally compared with the crystal structure coordinates of photolyases from Escherichia coli (folate type) and Anacystis nidulans (deazaflavin type). Discrepancies present in the multiple sequence alignment and tertiary structures, particularly at the light harvesting cofactor (methenyltetrahydrofolic acid, MTHF; 8-hydroxy-5-deazaflavin, 8-HDF) binding sites indicated the mechanistic nature of enterococcal photolyase. Concisely, despite the greater holistic homology with folate-type photolyase, enterococcal photolyase was characterized as deazaflavin-type. The presence of 8-HDF binding sites and groove architecture of substrate binding sites were also found supportive in this regard. The inter cofactor distance and/or orientation also implied to the efficient energy transfer in photolyase of Enterococcus in comparison with E. coli. In addition, we observed relatively high protein deformability in the enterococcal genome, which may favors the repair action of photolyase. The findings are expected to provide molecular insights into the difference in sunlight inactivation rate of two important fecal contamination indicators, namely Enterococcus and E. coli.     

Mutagenicity of industrial compounds: Styrene and its possible metabolite styrene oxide

Styrene and its presumed metabolite, styrene oxide, were tested for their mutagenic effect on a forward mutation system of yeast and of Chinese hamster cells, and on a gene-conversion system of yeast. Experiments with liver microsomal preparations and host-mediated assay with yeast were also carried out.Styrene oxide was mutagenic in all test systems. Styrene was mutagenic only in the host-mediated assay.     

Identification of geosmin as a volatile metabolite of Penicillium expansum.

Cultures of Penicillium expansum produce a musty, earthy odor. Geosmin [1,10-trans-dimethyl-trans(9)-decalol] was identified by gas chromatography-mass spectrometry from headspace samples of P. expansum cultures. Olfactory comparison of P. expansum cultures with a geosmin standard indicated geosmin is the primary component of the odor associated with P. expansum.     

Identification of geosmin as a volatile metabolite of Penicillium expansum.

Cultures of Penicillium expansum produce a musty, earthy odor. Geosmin [1,10-trans-dimethyl-trans(9)-decalol] was identified by gas chromatography-mass spectrometry from headspace samples of P. expansum cultures. Olfactory comparison of P. expansum cultures with a geosmin standard indicated geosmin is the primary component of the odor associated with P. expansum.     

Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling

Changes in gene expression associated with skeletal muscle atrophy due to aging are distinct from those due to disuse, suggesting that the response of old muscle to inactivity may be altered. The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-mo) and old (32-mo) male Brown Norway/F344 rats by 2 wk of hindlimb suspension (HS), and soleus muscles were analyzed by cDNA microarrays. Overall, similar changes in gene expression with HS were observed in young and old muscles for genes encoding proteins involved in protein folding (heat shock proteins), muscle structure, and contraction, extracellular matrix, and nucleic acid binding. More genes encoding transport and receptor proteins were differentially expressed in the soleus muscle from young rats, while in soleus muscle from old rats more genes that encoded ribosomal proteins were upregulated. The gene encoding the cold-shock protein RNA-binding motif protein-3 (RBM3) was induced most highly with HS in muscle from old rats, verified by real-time RT-PCR, while no difference with age was observed. The cold-inducible RNA-binding protein (Cirp) gene was also overexpressed with HS, whereas cold-shock protein Y-box-binding protein-1 was not. A time course analysis of RBM3 mRNA abundance during HS showed that upregulation occurred after apoptotic nuclei and markers of protein degradation increased. We conclude that a cold-shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss.     

Reduced satellite cell density and myogenesis in Wagyu compared with Angus cattle as a possible explanation of its high marbling

Mechanisms responsible for excellent marbling in Japanese black cattle, Wagyu, remain to be established. Because both muscle cells and intramuscular adipocytes are developed from mesenchymal progenitor cells during early muscle development, we hypothesized that intramuscular progenitor cells in Wagyu cattle have attenuated myogenic capacity in favor of adipogenesis, leading to high marbling but reduced muscle growth. Biceps femoris muscle biopsy samples were obtained from both Angus (n=3) and Wagyu (n=3) cattle at 12 months of age. Compared with Angus, the density of satellite cells was much lower in Wagyu muscle (by 45.8±10%, P<0.05). Consistently, the formation of myotubes from muscle-derived progenitor cells was also lower (by 64.2±12.9%, P<0.05), but adipogenic capacity was greater in Wagyu. The average muscle fiber diameter was larger in Wagyu (by 23.9±6.8%, P=0.089) despite less muscle mass, suggesting less muscle fiber formation in Wagyu compared with Angus cattle. Because satellite cells are derived from fetal myogenic cells, the reduction in satellite cell density together with lower muscle fiber formation suggests that myogenesis was attenuated during early muscle development in Wagyu cattle. Given the shared pool of mesenchymal progenitor cells, the attenuated myogenesis likely shifts progenitor cells to adipogenesis during early development, which may contribute to high intramuscular adipocyte formation in Wagyu cattle.