毛细管GC法测定还原型谷胱甘肽中有机溶剂的残留量

目的建立毛细管气相色谱法测定还原型谷胱甘肽中4种有机溶剂的残留量。方法采用气相色谱法,DB-624毛细管柱(60m×0.25mm×0.25μm),FID检测器,柱温为程序升温,以DMF-水(3∶7)为溶剂,测定了还原型谷胱甘肽原料药中甲醇、乙醇、乙酸乙酯、四氢呋喃4种有机溶剂的残留量。结果 4种有机溶剂均能完全分离,在所考察的范围内线性关系良好,加样回收率均满意。结论该方法灵敏、准确,适用于还原型谷胱甘肽原料中有机溶剂残留量的检测。     

The formation of glutathione conjugate derived from propranolol

Mixed function oxidases in liver microsomes from 3-methylcholanthrene-pretreated guinea pigs converted S-propranolol to 4-hydroxypropranolol, 5-hydroxypropranolol, and desisopropyl-propranolol. The addition of glutathione and cytosol from rat liver to the system resulted in the formation of a water-soluble metabolite. Evidence that the metabolite was a glutathione adduct was obtained by showing that treatment with gamma-glutamyltranspeptidase changed its HPLC retention time. The formation of the glutathione adduct required glutathione S-transferases in cytosol and was accompanied by a decrease in the formation of 5-hydroxypropranolol, but not of 4-hydroxypropranolol or desisopropylpropranolol. The formation of the propranolol-glutathione adduct was confirmed by two independent approaches. When [14C]glutathione and [3H]S-propranolol were used, the relationship between 14C and 3H in the metabolite indicated that it contained equal amounts of glutathione and propranolol. When a pseudoracemic mixture of S-propranolol and [2,4-2H2]R-propranolol was used, thermospray LC/MS analysis of the glutathione adduct revealed two peaks having different retention times. The first peak, representing a [2,4-2H2]R-propranolol-GSH adduct, gave fragment ions at m/z 294, 278, 262, while the second, which represented the S-propranolol-GSH adduct, gave fragment ions having 2 mass units less than those obtained with [2,4-2H2]R-propranolol. There was no evidence of any loss of deuterium during the formation of these fragment ions. The material in both peaks gave ions of m/z 308, 147, and 130, which is consistent with the presence of a glutathione group.     

由一株放线菌产生的对谷胱苷肽转移酶具有抑制作用的活性化合物的研究

谷胱苷肽-S-转移酶-π （GST-π的活性在某些类型的癌症患者中有显著的升高并且在癌症细胞对抗癌药物耐药性的形成过程中起着重要的作用。抑制过量表达的GST—π的酶活性将有助于克服肿瘤细胞的耐药性，因此，GST-π被认为是一个潜在的药物作用靶点。利用我所建立的GST-π抑制剂高通量筛选模型，在筛选针对GST—π的新的抗癌药物增敏剂的过程中，发现由一株放线菌550产生的发酵代谢产物对来源于人的GST-π产生明显的抑制活性。经有机溶媒萃取、葡聚糖凝胶LH-20柱层析、HPLC制备等方法，从该菌的发酵液中分离到了一个对GST-π有中等程度抑制活性的化合物550-a，其IC50为21．4μg／ml，经各种理化性质及NMR分析确定该化合物与木黄酮（genistein）同质，该化合物对GST-π的酶抑制活性以前尚未见报道。     

Structure-activity relationships of alloxan-like compounds derived from uric acid

The diabetogenic activity of a range of alloxan-like compounds derived from uric acid has been investigated. The classes of derivatives were: 5-substituted-isouric acids; 4,5-disubstituted-4, 5-dihydrouric acids; 5-substituted-pseudouric acids; salts of dehydro-uramil hydrate; salts of dehydro-isouramil hydrate; alloxan derivatives. Compounds were tested by intravenous injection into rats and diabetogenic activity assessed by production of persistent hyperglycaemia and glycosuria. The only essential structural feature common to all active compounds was the presence of a quinonoid pyrimidine system or its hydrated equivalent. The presence of the five-membered ring of uric acid (or an opened form thereof) did not abolish and in some compounds enhanced diabetogenic activity.     

Reactive intermediates and the dynamics of glutathione transferases.

Reactive intermediates are a continuous burden in biology and several defense mechanisms have evolved. Here we focus on the functions of glutathione transferases (GSTs) with the aim to discuss the quantitative aspects of defense against reactive intermediates. Humans excrete approximately 0.1 mmol of thioether conjugates per day. As the amount of GST active sites in liver is approximately 0.5 mmol, it appears that glutathione transferase catalysts are present in tremendous excess. In fact, the known catalytic properties of GSTs reveal that the enzymes can empty the liver glutathione (GSH) pool in a matter of seconds when provided with a suitable substrate. However, based on the urinary output of conjugates (or derivatives thereof), individual GSTs turn over (i.e., catalyze a single reaction) only once every few days. Glutathione transferase overcapacity reflects the fact that there is a linear relation between GST enzyme amount and protection level (provided that GSH is not depleted). Put in a different perspective, a few reactive molecules will always escape conjugation and reach cellular targets. It is therefore not surprising that signaling systems sensing reactive intermediates have evolved resulting in the increase of GSH and GST levels. Precisely for this reason, more moderately reactive electrophiles (Michael acceptors) are receiving growing interest due to their anticarcinogenic properties. Another putative regulatory mechanism involves direct activation of microsomal GST1 by thiol-reactive electrophiles through cysteine 49. The toxicological significance of low levels of reactive intermediates are of interest also in drug development, and here we discuss the use of microsomal GST1 activation as a surrogate detection marker.     

Tolerance during inhalation of organic solvents

Inhalation of several different halogenated solvents stimulated motor activity in mice. During prolonged exposure acute tolerance developed. The development of tolerance depended both on the schedule of exposure, and on the solvent. Exposure to trichloroethylene induced both stimulation and tolerance while the same degree of stimulation induced by 1,1,1-trichloroethane caused no tolerance. Thus the mechanisms which induce stimulation do not always initiate tolerance. Slow steady increases in the concentration of trichloroethylene could be maintained for several hours without any stimulation of motor activity. At the end of such exposures concentrations were reached which, if applied directly, would have induced considerable stimulation. Thus tolerance may develop without motor stimulation. Inhalation of ethanol also stimulated motor activity initially. During constant exposure the stimulation was followed by a considerable reduction in motor activity. This resulted in a hypoactive period, which in turn was followed by a second increase in motor activity, indicating the existence of not only two but several counteracting mechanisms. Development of metabolites with sedative effects counteracting the stimulating effect of the pure solvents seems to be one explanation for the results.     

Effect of some organic solvents on oxidative phosphorylation in rat liver mitochondria: Choice of organic solvents

The effect of acetone, acetonitrile, dimethyl sulfoxide (DMSO), ethanol and methanol on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria has been studied. All the organic solvents inhibited the oxidative phosphorylation in a concentration dependent manner, but with differences in potencies. Among the tested organic solvents, acetonitrile and acetone were more potent than ethanol, methanol, and DMSO. There was no significant difference in oxidative phosphorylation, compared to controls, when the concentrations of acetone was below 1% (v/v), of acetonitrile below 2% (v/v), of DMSO below 10% (v/v), of ethanol below 5% or of methanol below 2%, respectively. There was complete inhibition of oxidative phosphorylation at 50% (v/v) of acetone, acetonitrile and ethanol. But in the case of DMSO and methanol there were some residual activities observed at the 50% concentration level. DMSO showed least effect on oxidative phosphorylation with an IC₅₀ value of 13.3 ± 1.1% (v/v), followed by methanol (IC₅₀ value 8.3 ± 1.0), ethanol (IC₅₀ value 4.6 ± 1.1), acetone (IC₅₀ value 4.3 ± 1.0) and finally acetonitrile (IC₅₀ value 2.1 ± 1.0).All the organic solvents showed modulatory effects on 2,4-dinitrophenol (DNP) mediated inhibition of oxidative phosphorylation with potentiation of the action of DNP. Acetonitrile showed the highest potentiation effect followed by acetone, ethanol, methanol, and DMSO in presence of DNP. The use of organic solvents for investigation of the effects of compounds on oxidative phosphorylation in mitochondria should therefore include the use of relevant concentrations of the organic solvent in order to validate the contribution.     

Characterization of glutathione conjugates derived from reactive metabolites of seven silymarin isomers

1. Silymarin refers to a class of flavonoid lignans occurring in the fruits and seeds of the Silybum manalttlm (L). Gaertn, and is widely used in dietary supplements.

2. The main active ingredients of silymarin are silychristins A and B, silydianin, silybins A and B, and isosilybins A and B. However, the metabolism of silymarin has never been investigated. The major objectives of the present study were to investigate the metabolic pathways of silymarin isomers and to identify reactive metabolites.

3. Fourteen glutathione (GSH) conjugates were detected in rat/human liver microsomes incubations containing NADPH, GSH and seven individual isomers. Seven GSH conjugates (M1-M7) resulted from demethylated silymarin. M8-M14 originated from hydroxylated silymarin. Moreover, we found that GSH was probably conjugated on either ring A or ring E of silymarin based on the mass spectrometric fragments. In addition, recombinant enzyme incubation experiments demonstrated that CYP3A4 was the predominant P450 responsible for the metabolism of silymarin.

4. Several P450 enzymes were reportedly inactivated by some of bioactive constituents of silymarin to some extent. Our findings facilitate the understanding of mechanisms of the reported inactivation of P450 enzymes induced by silymarin.     

卡培他滨中有机残留溶剂的检测

目的建立气相色谱法测定卡培他滨原料药中二氯甲烷、乙醇、甲醇和乙酸乙酯4种有机溶剂。方法采用DB-624毛细管柱,FID检测器,以DMSO为溶剂。结果各有机溶剂的平均加样回收率为93.7%～97.6%,RSD为2.5%～3.7%。结论该方法稳定准确,可用于卡培他滨中有机溶剂残留的检测。     

气相色谱法检测有机溶剂残留量

目的建立气相色谱测定化工产品中N,N二甲基乙酰胺、乙醇、乙酸乙酯、1.4-二氧六环的检测方法。方法进样温度220℃。柱温为250℃,载气为氮气,以氢火焰离子化检测器进行检测。结果N,N二甲基乙酰胺、乙醇、乙酸乙酯、14二氧六环的检测浓度分别为20.0～100μg·mL^-1（r=0.9997）,25～125μg·mL^-1（r=0.99991）,25～125μg·mL^-1（r=0.99996）,10～50μg·mL^-1（r=0.9994）范围内线性关系良好：平均回收率分别为98.9%（RSD=1.4%）、93.6%（RSD=2.1%）、99.2%（RSD=2.0%）、92.8%（RSD=1.6%）三批样品中有机溶媒残留药均符合规定。结论本方法简单、灵敏可靠分离度好、检出限低。     

Enzymatic transformation of mercapturic acids derived from halogenated alkenes to reactive and mutagenic intermediates

The metabolism of the mercapturic acids S-pentachlorobutadienyl-N-acetylcysteine (N-Ac-PCBC), S-trichlorovinyl-N-acetylcysteine (N-Ac-TCVC) and S-dichlorovinyl-N-acetylcysteine (N-Ac-DCVC) by subcellular fractions from male rat liver and kidney homogenates was studied. As a model compound, N-Ac-PCBC, 14C labelled, was synthesised. It was intensively metabolised by cytosolic but not by microsomal enzymes from rat liver and kidney. The major metabolite identified by GC/MS was pentachlorobutadienylcysteine, the amount produced being highest in kidney cytosol. Metabolic conversion of 14C-N-Ac-PCBC by kidney and liver cytosol resulted in covalent binding of radioactivity to protein, binding was strongly inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA). N-Ac-TCVC and N-Ac-DCVC were also transformed by cytosolic enzymes to the corresponding cysteine conjugates (trichlorovinylcysteine and dichlorovinylcysteine). The three mercapturic acids tested were strong mutagens in the Ames-test after addition of rat kidney cytosol. In the absence of cytosol, N-Ac-TCVC and N-Ac-DCVC were weakly but definitely mutagenic, whereas N-Ac-PCBC was not. In contrast to N-Ac-PCBC, the "direct" mutagens N-Ac-TCVC and N-Ac-DCVC were both transformed to pyruvate by bacterial (S. typhimurium TA100) homogenate 100,000 g supernatants. It is concluded that mercapturic acids are deacetylated to the corresponding cysteine conjugates by cytosolic (N-Ac-PCBC, N-Ac-TCVC and N-Ac-DCVC) and bacterial enzymes (N-Ac-TCVC and N-Ac-DCVC) and further cleaved to reactive and mutagenic intermediates by mammalian and/or bacterial beta-lyase. The observed activation mechanisms for the mercapturic acids, whose formation from hexachlorobutadiene, tetrachloroethylene and trichloroethylene has been proven, might contribute to the nephrotoxicity and nephrocarcinogenicity of the parent alkenes.     

Reversible interaction of a reactive intermediate derived from furazolidone with glutathione and protein

Swine liver microsomes convert the nitrofuran furazolidone into N-(4-cyano-2-oxo-3-butenylidene)-3-amino-2-oxazolidone, a reactive open-chain acrylonitrile derivative. This derivative may be trapped with such thiol-group-containing agents as glutathione and mercaptoethanol. However, this reaction is reversible; e.g., adding an excess of mercaptoethanol to an aqueous solution (pH 7.4) of the glutathione conjugate results in conversion of 43% of this compound into the mercaptoethanol conjugate. In addition, when microsomal protein is added to the glutathione conjugate or the mercaptoethanol conjugate, 36 and 44%, respectively, become covalently bound to the protein. The amount of this covalently bound radioactivity decreases again on prolonged incubation at 37 degrees C (42% disappearance within 24 hr), suggesting that the acrylonitrile derivative also reacts reversibly with thiol groups of microsomal protein. Indeed an excess of mercaptoethanol could remove covalently bound radioactivity from microsomal protein resulting in the formation of the mercaptoethanol conjugate. The reversibility of the reaction is dependent on pH, as is demonstrated for the mercaptoethanol conjugate. Below pH 2 this conjugate is stable; optimal exchange to microsomal protein is found between pH 7 and 10. At very high pH (greater than 11) no binding to protein is found, although the conjugate disappears rapidly. The mercaptoethanol conjugate exhibits mutagenic activity in the Salmonella/microsome test indicating that the acrylonitrile derivative of furazolidone also interacts with DNA.     

The carcinogenic significance of reactive intermediates derived from 3-acetoxy- and 5-acetoxy-2-hydroxy-N-nitrosomorpholine

N-Nitroso-2-hydroxymorpholine (NHMOR), a relatively reactive metabolite of two potent carcinogens, N-nitrosodiethanolamine (NDELA) and N-nitrosomorpholine (NMOR), has been reported to not be carcinogenic. Two isomeric acetate esters of the alpha-hydroxynitrosamines expected to be produced from the cytochrome P450-mediated metabolism of NHMOR have been synthesized, and their hydrolytic decomposition products, hydrolysis rates, and deoxyguanosine (dG) reaction adducts have been determined. N-Nitroso-3-acetoxy-2-hydroxymorpholine was prepared in high yield from the reaction of N-nitroso-2,3-dehydromorpholine with dry peracetic acid in glacial acetic acid or by the reaction of its dimethyldioxirane-produced epoxide with glacial acetic acid. The hydrolysis of this alpha-acetoxynitrosamine gave acetaldehyde (10%), ethylene glycol (55%), glyoxal (95%), and acetic acid. The pH rate profile for the hydrolysis of this nitrosamine was abnormal in that it exhibited pronounced base-catalyzed hydrolysis beginning at pH 5. The mechanism of hydrolytic decomposition is proposed to involve neighboring group participation with the formation of a reactive epoxide intermediate. N-Nitroso-3-acetoxy-2-hydroxymorpholine reacted with dG to give these guanine adducts after acidic deglycosylation: 1,N2-glyoxal (65%), 7-(2-hydroxyethyl)guanine (9%), and O6-hydroxyethylguanine (3%). N-Nitroso-5-acetoxy-2-hydroxymorpholine was synthesized from 2-hydroxyethylvinylnitrosamine by its oxidative conversion to the corresponding aldehyde followed by reaction with dry peracetic acid in glacial acetic. The hydrolytic decomposition products of this nitrosamine were 2-acetoxyacetaldehyde (65%), a rearrangement product, glycol aldehyde (15%), a trace of glyoxal, and acetic acid. The pH rate profile for the hydrolysis of this acetate is similar to other alpha-acetoxynitrosamines in that it exhibits a pH-independent region which gives way to base-catalyzed ester hydrolysis beginning at pH 7. The lower pH ( approximately 7 < 9) onset of base catalysis is proposed to involve base-catalyzed opening of the hemiacetal and intramolecular acyl transfer to give an unstable alpha-hydroxynitrosamine. N-Nitroso-5-acetoxy-2-hydroxymorpholine was less reactive toward dG and gave the 1,N2-etheno-dG adduct (44%). The products from both of the isomeric alpha-acetoxy nitrosamines were judged to arise from diazonium ions produced from unstable alpha-hydroxynitrosamine intermediates. The high yield of the rearrangement product 2-acetoxyacetaldehyde could explain the low carcinogenic potential of NHMOR if it is mainly alpha-hydroxylated at the 5 carbon. Hydroxylation of NHMOR at carbon 3 is expected to yield a carcinogenic outcome.     

Occupational neurotoxicology of organic solvents and solvent mixtures

The results of two field studies in painters and spray painters, the outcomes of examinations of workers with suspected work-related disease due to solvents, as well as data from an evaluation of an epidemiologic study in painters with confirmed occupational disease, are presented and discussed. The results of these studies and the experiences in occupational medicine in the Federal Republic of Germany do not support the assumption of high neurotoxic risks in solvent-exposed workers, which can be postulated from various epidemiologic studies from Scandinavian countries. Several factors may explain the different conclusions: 1) lower solvent exposures of German painters in the past decades; 2) false positive diagnosis of a toxic encephalopathy; 3) aetiological misclassification; 4) differences in legislation relevant for the acknowledgement of occupational diseases. In conclusion, there is a need for further well-designed epidemiologic studies in occupationally solvent-exposed workers. Suggestions regarding assessment of exposure and neurobehavioral tests are given.