Formation of 4,4-dialkoxycyclohexa-2,5-dienone N-(thiol-S-yl)imine during reaction of 4-alkoxynitrosobenzenes with thiols in alcoholic solvents

During the interaction of nitrosoarenes with glutathione in aqueous media, intermediate generation of a highly resonance-stabilized sulfenamide cation has been repeatedly suggested. Most intermediates and end products could be explained by reactions of this sulfenamide cation with different nucleophiles such as excess thiol, solvent water, and metabolically produced arylamine. The present paper presents evidence for adduct formation of the sulfenamide cation with solvent alcohol at neutral pH. Sulfenamide cations generated from 4-nitrosophenetole and 4-nitrosoanisole, respectively, are strongly suggested to form the metastable ketals 4-ethoxy-4-methoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine and 4,4-dimethoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine, respectively, during reaction with solvent methanol. Reaction of the two sulfenamide cations in ethanol yielded 4,4-diethoxycyclohexa-2, 5-dienone N-(glutathion-S-yl)imine and 4-ethoxy-4-methoxycyclohexa-2, 5-dienone N-(glutathion-S-yl)imine, respectively. Although the metastability of the ketals did not allow isolation of pure solid material, chromatographic and chemical behavior as well as tandem MS fragmentation substantiate a ketal structure of these intermediates. To confirm the proposed structure, new compounds, 2, 6-dimethyl-4-nitrosophenetole, 2,6-dimethyl-4-nitrophenetole, 2, 6-dimethyl-4-phenetidine, and N-(glutathion-S-yl)-N-hydroxy-4-aminoacetophenone, were synthesized and included in supportive experiments. In summary, the detection of ketals corroborates once more the occurrence of a sulfenamide cation which obviously not only reacts with soft nucleophiles such as GSH but, to a limited extent, also reacts with hard nucleophiles. The toxicological significance of this result is discussed.     

Nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol in Fischer 344 rats: comparisons with 2- and 4-chloroaniline and 2- and 4-aminophenol

Nephrotoxicity occurs following intraperitoneal (i.p.) administration of 2-chloroaniline or 4-chloroaniline hydrochloride to Fischer 344 rats, but the nephrotoxicant chemical species and mechanism of nephrotoxicity are unknown. The purpose of this study was to evaluate the in vivo and in vitro nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol, metabolites of 4-chloroaniline and 2-chloroaniline. A comparison was also made between the nephrotoxic potential of the aminochlorophenols and the corresponding aminophenols to examine the effect of adding a chloride group on the nephrotoxic potential of the animophenols. Male Fischer 344 rats (4/group) were given an i.p. injection of a chloroaniline or aminochlorophenol hydrochloride (1.5 mmol/kg), and aminophenol (1.0 or 1.5 mmol/kg), or vehicle, and renal function monitored at 24 and 48 h. Both aminochlorophenols induced smaller and fewer renal effects that the parent chloroanilenes in vivo. Also, 4-aminophenol was markedly more potent as a nephrotoxicant that 4-amino-3-chlorophenol, while 2-aminophenol and 2-amino-5-chlorophenol induced only mild change in renal function. In vitro, the phenolic compounds reduce p-aminohippurate accumulation by renal cortical slices at bath concentrations of 0.01 mM, while a bath concentration of 0.50 mM or greater was required for the chloroanilines. However, all compounds reduced tetraethylammonium accumulation at bath concentrations of 0.1-0.5 mM or greater. These results indicate that extrarenally-produced aminochlorophenol metabolites do not contribute to the mechanism of chloroaniline nephrotoxicity. Also, the reduced nephrotoxic potential of 4-amino-3-chlorophenol compared to 4-aminophenol could result from an altered ability of the aminochlorophenol to redox cycle or form conjugates.     

Formation of catechol estrogen glutathione conjugates and gamma-glutamyl transpeptidase-dependent nephrotoxicity of 17beta-estradiol in the golden Syrian hamster

In an animal model of hormone-mediated carcinogenesis, male golden Syrian hamsters develop renal carcinoma following prolonged exposure to 17beta-estradiol. The basis for the species and tissue specificity is unclear. Detailed information on the disposition of 17beta-estradiol in this model is lacking. Because catechol estrogens have been implicated in this model of carcinogenesis, we investigated the metabolism and nephrotoxicity of 17beta-estradiol in golden Syrian hamsters, with emphasis on the formation of catechol estrogen thioethers. 17beta-Estradiol (50 micromol/kg, i.p.) is a mild nephrotoxicant, causing significant elevations in the urinary excretion of gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase, glutathione S-transferase (GST) and glucose. Increases in renal protein carbonyls and lipid hydroperoxides, which are markers of oxidative damage, also occur after administration of 17beta-estradiol (50 micromol/kg, i.p.). 17beta-Estradiol-mediated nephrotoxicity is reduced by treating animals with acivicin, an inhibitor of gamma-GT, implying that toxicity is mediated by metabolites requiring metabolism by this enzyme. Following administration of 17beta-[14C]estradiol (100 micromol/kg) to hamsters, 9.7% of the dose is recovered in bile after 5 h, the majority (7.9%) representing aqueous metabolites. Seven catechol estrogen GSH conjugates were identified, 2-hydroxy-1,4-bis-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-estrone, 2-hydroxy-1-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-17beta-estradiol, and 2-hydroxy-1-(glutathion-S-yl)-17beta-estradiol. At 5.4 micromol/kg of 17beta-estradiol, a dose-reflective of daily exposure levels in the hamster model of nephrocarcinogenicity, 12% of the dose is recovered within 5 h as a combination of GSH conjugates of 2- and 4-hydroxy-17beta-estradiol and 2- and 4-hydroxyestrone. In summary, oxidation of catechol estrogens, followed by GSH conjugation, occurs in vivo and 17beta-estradiol is a mild nephrotoxicant in a manner dependent on the activity of gamma-GT.     

Identification of four new metabolic products of metoclopramide using mass spectrometry

1. N-(Diethylaminoethyl)-4-amino-5-chloro-2-methoxybenzamide (metoclopramide, I) N-(ethylaminoethyl)-4-amino-5-chloro-2-methoxybenzamide (II), N-(aminoethyl)-4-amino-5-chloro-2-methoxybenzamide (III), N-(2'-hydroxyethyl)-4-amino-5-chloro-2-methoxybenzamide (IV), N-(diethylaminoethyl)-4-amino-5-chloro-2-hydroxybenzamide (V) and N-(ethylaminoethyl)-4-amino-5-chloro-2-hydroxybenzamide (VI) were obtained from chloroform extracts of incubation mixtures of I or II (HCl salts) with 9000 g liver microsomal preparations from male rabbits.     

Identification of hepatic metabolites of two highly carcinogenic polycyclic aza-aromatic compounds, 7,9-dimethylbenz[c]acridine and 7,10-dimethylbenz[c]acridine

The hepatic microsomal metabolites of the highly carcinogenic dimethylbenzacridines, 7,9-dimethylbenz[c]acridine (7,9-DMBAC), and 7,10-dimethylbenz[c]acridine (7,10-DMBAC) were obtained with preparations from 3-methylcholanthrene-pretreated rats. Metabolites were separated by reversed-phase HPLC and characterized using UV spectral data and chemical ionization-mass spectrometry after trimethylsilylation and GC. Comparisons with products formed in the presence of the epoxide hydrolase inhibitor, 1,1,1-trichloropropane 2,3-oxide and with those formed from the three synthetic alcohol derivatives of each parent compound, aided the assignment of firm or tentative structures to 16 products from 7,9-DMBAC found in 22 reversed-phase chromatographic peaks, and for 17 products of 7,10-DMBAC found in 19 chromatographic peaks. The more abundant metabolites were derived from oxidation of the methyl groups. Other metabolites were dihydrodiols, epoxides, phenols and secondary metabolites. The 9-methyl group prevented dihydrodiol formation at the 8,9-position from 7,9-DMBAC, and for each carcinogen, the 3,4-dihydrodiol was formed. As well, 3,4-dihydrodiols of methyl oxidized compounds were found.     

Urinary metabolites of 3,3-dimethyl-1-phenyltriazene

Urinary metabolites excreted after a subcutaneous injection of 3,3-dimethyl-1[14C] phenyltriazene (DM[1-14C]PT) to rats accounted for 82% of the applied radioactivity. We have isolated aniline (1-2%), 2-hydroxyaniline (5-7%), 3-hydroxyaniline (about 1%) and 4-hydroxyaniline (31-37%) from ethyl acetate extracts of acid-hydrolysed urine, UV spectrometric determination of 4-hydroxyaniline, using the indophenol reaction, showed that the most abundant metabolite accounted for 56 to 61% of the applied dose. We have also demonstrated the excretion of metabolites containing the intact triazene structure (0.9-1.1%) by cold acid cleavage of these compounds, followed by coupling of the released arenediazonium cations with N-ethyl-1-naphthylamine (EN). The coloured derivatives of these metabolites, 4-benzeneazo-N-ethyl-1-naphthylamine (BAEN) (0.6-0.7%), 4-(2-hydroxybenzeneazo)-N-ethyl-1-napthylamine (2-HO-BAEN) (0.02%) and 4-(4-hydroxybenzeneazo)-N-ethyl-1-naphthylamine (4-HO-BAEN) (0.3-0.4%) were isolated. The identification of BAEN as the principal azo derivative of the excreted triazene metabolites is in full agreement with the proposed in vivo activation of 3,3-dimethyl-1-phenyltriazene (DMPT) to a carcinogenic methylating agent. The hydroxylation of the methyl group at N-3 yields the corresponding aminol, some of which is covalently bonded to a water-soluble compound.     

Reactivity of haloketenes and halothioketenes with nucleobases: chemical characterization of reaction products

Halothioketenes and haloketenes are postulated as intermediates in haloolefin bioactivation. Little is known about the interactions of these reactive intermediates with macromolecules such as DNA. DNA binding, however, may be relevant in the toxicity of the parent olefins since they or their proximate metabolites are genotoxic. This prompted us to elucidate the structures and properties of potential DNA adducts formed. Adenine, cytosine, guanine, and thymine were reacted with chloro- and dichlorothioketene, chloro- and dichloroketene, and chloro- and dichloroacyl chloride. While thymine did not react, adenine and cytosine formed stable DNA base adducts with all reaction partners as demonstrated by HPLC analysis. Guanine yielded only products with chloroketene and chloroacetyl chloride. The pH-dependent UV spectra, 1H and 13C NMR, FT-IR, and elemental analysis showed (i) nucleophilic attack of the exocyclic amino groups of the DNA bases yielded haloacyl (thio)amides with all reactants as clearly demonstrated by the FT-IR spectra; (ii) the sulfur in the initial thioamides seems to be rapidly exchanged with oxygen; (iii) the acyl chlorides form identical products but in lower yields as compared to the haloketenes. Reactions of the nucleosides with haloketenes showed the formation of similar nucleoside adducts upon HPLC and MS analysis. Beside the modification of the base moieties, additional peaks in the reaction mixtures analyzed suggested acylation of the deoxyribose hydroxyl groups. In aqueous solutions at pH 7 N6-(chloroacetyl)adenine, N4-(chloroacetyl)cytosine, and N2-(chloroacetyl)guanine are not stable and cleaved to the original base or form 1,N6-acetyladenine, 3,N4-acetylcytosine, 1,N2-acetylguanine, and N2,3-acetylguanine. Under the same conditions, N6-(dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine were completely hydrolyzed to adenine and cytosine, respectively. All haloacyl DNA base adducts proved to be stable at pH 5 but were rapidly degraded at neutral or alkaline pH. The compounds with an additional five-membered ring remained unchanged after 1 week at room temperature. All synthesized DNA base adducts except N2-(chloroacetyl)guanine and 1,N2-acetylguanine were fluorescent. The characterized compounds, especially the etheno (epsilon) base adduct-related derivatives, may represent potential DNA adducts formed as a consequence of haloolefin bioactivation.     

Determination of the major urinary metabolite of flurazepam in man by high-performance liquid chromatography

A high performance liquid chromatographic method for the determination of N-1-hydroxyethylflurazepam, the major urinary metabolite of flurazepam, in human urine is described. Urine specimens were incubated enzymatically to deconjugate N-1-hydroxyethylflurazepam glucuronide (metabolite) and were then extracted at pH 9.0 to extract the metabolite. The extracts were chromatographed on a microparticulate silica gel column using automatic sample injection, isocratic elution at ambient temperature and UV monitoring at 254 nm. The internal standard was 7 chloro-5(2'-chlorophenyl) 1,3-dihydro-1-2-dimethylaminoethyl-2H-1,4-benzodiazepine-2-one. The recovery from urine, in the 0.5-25.0 microgram/ml range, was 96.5 +/- 11.5% (S.D.), and the sensitivity limit was 0.5 microgram/ml. The method was found to be specific for N-1-hydroxyethylflurazepam in the presence of intact flurazepam and other possible urinary metabolites of flurazepam. The method was successfully applied to urine specimens collected from human subjects following the administration of 30-mg single oral doses of flurazepam dihydrochloride.     

Oxidation of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine, a 1-amino analog of MPTP, by type A and B monoamine oxidase

A 1-amino analog of MPTP, 1(N)-amino-4-phenyl-1,2,3,6-tetrahydropyridine, was synthesized and the oxidation was examined using human synaptosomal mitochondria as sources of type A and B monoamine oxidase. An oxidation product, 1-amino-4-phenylpyridinium ion, was quantified by high-performance liquid chromatography-fluorometric detection. The amino analog was a substrate of both type A and B monoamine oxidase and the oxidation depended linearly on the enzyme amount and the reaction time with an optimal pH around 7.5. After the systemic injection of the amino analog in C57/black mice for one week, 1-amino-4-phenylpyridinium ion was detected in the brain. 1(N)-Amino-4-phenyl-1,2,3,6-tetrahydropyridine was proved to be cytotoxic to pheochromocytoma PC12 cells, and it may be a new neurotoxin bioactivated through the oxidation by type A and B monoamine oxidase.     

Clinical experience with the holmium:YAG laser for transmyocardial laser revascularization and myocardial denervation as a mechanism

Several novel N-(4,5-diphenylthiazol-2-yl)-N'-aryl or alkyl (thio)ureas and N-(4,5-diphenylthiazol-2-yl)alkanamides were prepared as potential acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitors. Synthesis was accomplished by reaction of 2-amino-4,5-diphenylthiazole with the suitable isocyanate, isothiocyanate or acyl chloride. Some analogues without the 5-phenyl substituent or both the phenyl groups in 4 and 5 position of the thiazole ring were also prepared. Moreover, some bioisosters of the title compounds in which the thiazole ring was replaced by an imidazole were synthesized starting from the 2-amino-4,5-diphenyl-1H-imidazole. The ability of synthesized compounds to inhibit ACAT was evaluated in vitro by measuring the formation of cholesteryl[14C]oleate from cholesterol and [1-14C]oleoyl-CoA in rat liver microsomes. Among the tested compounds, only some thiazole ureas were able to inhibit ACAT in a reasonable degree. N-(4,5-diphenylthiazol-2-yl)- N'-[2,6-bis(2-methylethyl)phenyl] urea (11) and N-(4,5-diphenylthiazol-2-yl)-N'-n-butyl urea (16) were the most active compounds in the series showing IC50 values in the low micromolar range.     

N-[4-(R',R"-phenylazo)aryl]aminolupinanes with antitubercular activity

A set of twelve N-[4-(R-phenylazo)aryl]aminolupinanes (10a-c; 11a-i) was prepared by coupling the suitably substituted phenyldiazonium salts with N-(2,3-xylyl)-aminolupinane and with N-(5,6,7,8-tetrahydronaphth-1-yl)- aminolupinane. These compounds, as well as the N-(4-amino-2,3-xylyl)aminolupinane and the N-(4-amino-5,6,7,8-tetrahydronaphth-1-yl)-aminolupinane, that could be obtained from the former through an in vivo reductive cleavage of the azo group, exhibited a high activity against Mycobacterium tuberculosis H37RV with M.I.C. in the range 0.1-1 microgram/ml.     

Detection of PCB adducts by the 32P-postlabeling technique

The purpose of this study was to determine whether lower chlorinated biphenyls would be bioactivated to electrophilic metabolites by microsomes alone or in combination with peroxidase. Monochloro- and dichlorobiphenyls were incubated with liver microsomes of rats treated with phenobarbital and beta-naphthoflavone, an NADPH-regenerating system, and deoxyguanosine 3'-monophosphate (dGp). The resultant adducts were analyzed by 32P-postlabeling either following microsomal incubation alone ("preoxidized") or coupled with subsequent oxidation with horseradish peroxidase/H2O ("oxidized"). The incubation of 4-monochlorobiphenyl (4-MCB) resulted in the formation of two minor adducts by microsomal activation alone. However, the oxidized sample showed two additional major adducts. Formation of the latter adducts was almost completely (> 80%) inhibited when the oxidation reaction was performed in the presence of ascorbic acid. The other test mono- and dichlorobiphenyls also formed 1-3 major adducts. Compared with microsomal activation alone, these adducts were enhanced after the oxidation reaction or detected only in the oxidized samples. These data suggest that (1) some adducts of the lower chlorinated biphenyls are derived from arene oxides and (2) many adducts may be formed by metabolism of the parent compounds to catechol and p-hydroquinone species, which are oxidized to semiquinones and/or quinones. The involvement of quinones and/or semiquinones was supported by UV/vis spectroscopic measurements, which showed that metabolites of 4-MCB can be oxidized to products with spectra characteristic of quinones. These data raise the possibility that lower chlorinated biphenyls may be genotoxic and may explain the fact that commercial polychlorinated biphenyl mixtures are complete rodent carcinogens.     

In vitro microsomal metabolic studies on secondary aromatic amides

Previous studies showed that amides are metabolites arising from certain secondary aromatic amines. However, some analogue amines did not lead to the formation of the corresponding amides when metabolised under identical conditions. We, therefore, wished to establish the factors preventing detection of amides. In the present study, we thought that amide detection as metabolites from secondary anilines may depend on the hydrolytic rate of the corresponding amide. We studied the in vitro hepatic microsomal metabolism of four aromatic amides i.e. N-(4-nitrobenzoyl)aniline (N4NBZA), N-benzoyl-4-nitroaniline (NBZ4NA), N-benzoylaniline (NBZA) and N-benzoyl-2,4,6-trimethylaniline (NBZTMA) which were (or not) detected following microsomal metabolism of secondary anilines in previous studies. Following the preparation, characterisation and separation of substrates and potential metabolites, incubations were carried out using rabbit microsomal preparations fortified with NADPH. The substrates and potential metabolites were extracted into dichloromethane and analysed by TLC, HPLC and UV. The results indicated that both steric and electronic factors may influence hydrolysis of amides. Three amides i.e. N4NBZA, NBZ4NA and NBZA yielded hydrolytic metabolites, whereas, NBZTMA did not. Para hydroxylated metabolites were also detected from N4NBZA and NBZA. These observations support the concept that one reason for not detecting amides as metabolites from secondary anilines in previous studies could be due to their rapid hydrolysis to the corresponding primary amines.     

DPC4 splice variants in neuroblastoma

Nitrogen mustard (HN2) is a bifunctional alkylating agent which is thought to cause cytotoxicity by covalently binding to DNA. Most studies to date have looked at qualitatively determining the presence of DNA-HN2 adducts from reactions with native DNA. The adduct which is predominately formed in these reactions is N-[2-(hydroxyethyl)-N-(2-(7-guaninyl)ethyl]methylamine (N7G). A simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of N7G from DNA using ultraviolet detection is described. DNA samples having been exposed to HN2 treatment were hydrolyzed and preseparated from high-molecular-mass material by filtration using a molecular mass cut-off of 3000. The mobile phase consisted of methanol-26 mM ammonium formate, pH 6.5 (24:76, v/v). N7G, as well as the internal standard, methoxyphenol, were separated within 30 min. The recovery of N7G after hydrolysis of the DNA reaction product was quantitative and limits of detection and quantification of 10 and 20 ng/ml, respectively, were calculated. The method was validated in DNA-HN2 dose response experiments. The N7G reaction product appears to be the first reaction product formed at lower ratios of HN2/DNA but its production plateaus at higher ratios of HN2/DNA probably due to increased formation of hitherto unknown adducts. The method is simple and sensitive and for this reason, may be suited for the determination of DNA/HN2 reaction products.     

A comparison of guanosine-quartet inhibitory effects versus cytidine homopolymer inhibitory effects on rat neointimal formation

Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-l-3-(2-naphthyl)-alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH- N -glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make some comparisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N -Glycan-BNAH and-BAP derivatives have, overall, comparable reactivities with lectins which recognize N -glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non-reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl- N- glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide beta-glycoside) can substitute for the natural beta-glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl-hydrazide-beta-glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N -glycans.     

Comparative effect of tiaprofenic acid and piroxicam alone and as adjuvants to praziquantel in Schistosoma mansoni infected mice

A simple, rapid and sensitive two column-switching high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (AY4166, I) and its seven metabolites in human plasma and urine. Measurements of I and its metabolites were carried out by two column-switching HPLC, because metabolites were classified into two groups according to their retention times. After purification of plasma samples using solid-phase extraction and direct dilution of urinary samples, I and each metabolite were injected into HPLC. The calibration graphs for plasma and urinary samples were linear in the ranges 0.1 to 10 microg ml(-1) and 0.5 to 50 microg ml(-1), respectively. Recoveries of I and its seven metabolites were over 88% by the standard addition method and the relative standard deviations of I and its metabolites were 1-6%.     

Zn~（2＋）催化氟虫腈水解的产物及其结构表征

在溶剂热条件下,利用路易斯酸Zn2＋对氟虫腈（5-氨基-1-（2,6-二氯-4-三氟甲基苯基）-4-三氟甲基亚磺酰基吡唑-3-腈）进行原位水解反应,得到其结晶产物5-氨基-1-（2,6-二氯-4-三氟甲基苯基）-4-三氟甲基亚磺酰基吡唑-3-羧酸.对该化合物进行了元素分析、红外、紫外-可见光谱测定及X-射线单晶衍射等表征.晶体结构解析表明该化合物属单斜晶系,空间群为P2（1）/n,晶胞参数为：a=12.7457（8）,b=8.8244（6）,c=15.7322（11）,α=90°,β=100.2310（10）°,γ=90°,Z=4,V=1741.3（2）3,F（000）=856.化合物中存在的弱相互作用力分子间氢键将其形成一个稳定的三维层状结构.紫外-可见光谱分析表明该化合物在220 nm和281 nm处有最大吸收峰.     

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes

The potent rat colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), unlike other food-borne heterocyclic amines, does not induce tumors in rat liver. This correlates with an extremely low level of PhIP-DNA adducts formed in this tissue, and together these observations suggest that PhIP is efficiently detoxified in the liver. In order to identify possible detoxification mechanisms, we assessed the effect of inhibition of glucuronidation, glutathione (GSH) conjugation and sulfation on PhIP metabolism and PhIP-induced DNA damage in rat hepatocytes. Hepatocytes isolated from rats pretreated with Aroclor 1254 metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIP N3-glucuronide and N-hydroxy-PhIP N2-glucuronide were major and minor metabolites respectively. 32P-Postlabeling analysis of DNA from the PhIP-treated hepatocytes indicated the presence of two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP, and one minor adduct. There was no unscheduled DNA synthesis (UDS) in these cells. However, pretreatment of the hepatocytes with 1-bromoheptane and buthionine sulfoximine, which depletes GSH and prevents its resynthesis, resulted in a 15-fold increase in the formation of PhIP-DNA adducts, as well as in a high level of UDS. GSH depletion had no effect on the formation of detectable PhIP metabolites. Hepatocyte pretreatment with D-galactosamine, which inhibits glucuronidation, increased the formation of DNA adducts two-fold and UDS was increased similarly. D-Galactosamine decreased the formation of the two N-glucuronides of N-hydroxy-PhIP by 50-60%, but had no effect on other metabolites. Pentachlorophenol, which strongly inhibits sulfotransferases, decreased adduct formation slightly, but had essentially no effect on UDS or on the formation of PhIP metabolites. These results indicate that metabolic conjugation pathways involving GSH and glucuronidation may play an important role in protecting rat liver against PhIP carcinogenesis.     

Transient monocular blindness and antiphospholipid antibodies in systemic lupus erythematosus

Cation binding to the monovalent cation selective channel, gramicidin A, is shown to induce changes in the dipolar and chemical shift observables from uniformly aligned samples. While these changes could be the result of structural or dynamic changes, they are shown to be primarily induced by through-bond polarizability effects when cations are solvated by the carbonyl oxygens of the peptide backbone. Upon cation binding partial charges are changed throughout the peptide plane, inducing large changes in the 13C1 chemical shifts, smaller changes in the 15N chemical shifts, and even smaller effects for the 15N-13C1 and 15N-2H dipolar interactions. These conclusions are substantiated by characterizing the 15N chemical shift tensors in the presence and absence of cations in fast-frozen lipid bilayer preparations of gramicidin A.     

Blood levels in methaqualone in man following chronic therapeutic doses

Human serum was analyzed for methaqualone (MTQ) and hydroxylated metabolites by gas liquid chromatographic (GLC), ultraviolet spectrophotometric (UV) and spectrofluorimetric (SF) procedures. Intact methaqualone was found to be the major circulating drug component after administration of multiple 300 mg daily doses over a 28-day period. Hydroxylated methaqualone metabolites, if present, were estimated to be in extremely low concentrations. After acute ingestion of large quantities of methaqualone (2.4-3.0 g), at least one methaqualone metabolite, [2-methyl-3-(2' hydroxymethylphenyl)-4(3H)-quinazolinone] was present in serum obtained from subjects with a history of chronic drug abuse.