Metabolic activation of pesticides with proestrogenic activity

The role of metabolism in the estrogenic activity of chlorinated hydrocarbon pesticides was examined. Whether the estrogenic activity in technical grade preparations of the pesticide methoxychlor is due to methoxychlor or to contaminants was also investigated. Identified compounds in technical methoxychlor were examined by an in vitro method to determine whether they are estrogens or proestrogens. This method showed that purified methoxychlor and MDDE, an olefinic derivative of methoxychlor, are proestrogens and that monohydroxymethoxychlor and monohydroxy-MDDE are estrogens. Thus, the estrogenic activity in technical methoxychlor is due to both methoxychlor and contaminants. MDDE is an in vivo metabolite of methoxychlor, and the mono- and bishydroxy derivatives of methoxychlor and MDDE are metabolites of methoxychlor and MDDE, respectively. These metabolites exhibited in vitro estrogenic activity in the following order of potency: bis-OH-MDDE greater than bis-OH-methoxychlor greater than mono-OH-MDDE greater than mono-OH-methoxychlor. A similar order of potency was observed in vivo, demonstrating that metabolites of methoxychlor are potent estrogens. In addition to phenolic products, hepatic monooxygenases metabolize methoxychlor and MDDE to reactive intermediates that bind covalently to microsomal proteins. Further studies are needed to determine the factors controlling the two pathways of methoxychlor metabolism and determine whether covalent binding is associated with cellular and organ toxicity.     

Inhibition of the mutagenic activity of some heterocyclic dietary carcinogens and other mutagenic/carcinogenic compounds by rat organ preparations

The mutagenic activity of some dietary mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), was inhibited in the Salmonella-plate test preincubated with heat-inactivated rat intestinal preparations. A similar inhibition was observed by preincubating intestinal preparations with 2-acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P). The effect was not specific for small intestine and was also obtained with spleen, liver, lung, colon and stomach preparations. Mutagenic activity was not inhibited by beef muscle proteins. Lipids extracted from intestinal mucosa preparations were equally effective as inhibitors of the mutagenic activity. Lipid fractions from intestinal mucosa were capable of inhibiting the formation of activated IQ by mammalian S9, and other components of the intestinal preparations were able to bind the promutagens and their active metabolites. The mutagenic activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was also inhibited by intestinal preparations, but not by their lipid fractions. A binding of IQ to intestinal preparations was also demonstrated with HPLC techniques. The data indicate that tissue components may reduce the mutagenic activity of chemicals by interfering with the activation process and by reducing the concentration of the promutagens and their active metabolites at target sites.     

In vitro and in vivo induction of chromosome aberrations by alpha- and beta-zearalenols: comparison with zearalenone

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by Fusarium fungi. It contaminates different components of the food chain and can cause serious economic and public health problems. The major metabolites of ZEN in various animal species are alpha- and beta-zearalenol (α-, β-ZOL). Some in vivo studies have shown that these two metabolites are as toxic as the mother molecule (ZEN), but other investigations have demonstrated that α- and β-ZOL are less toxic than ZEN. Thus, the aim of the present study was to evaluate cytotoxicity and genotoxicity of α- and β-ZOL in vivo, in mouse bone-marrow cells and in vitro, in cultured HeLa cells, and to compare it with ZEN. ZEN showed the same cytotoxicity as α-ZOL and both are more cytotoxic than β-ZOL. Genotoxicity of ZEN and its derivatives was assessed by the chromosome aberration assay. Our results show that ZEN as well as α- and β-ZOL increased the percentage of chromosome aberrations in mouse bone-marrow cells and in HeLa cells. In the two systems, ZEN and α-ZOL exhibited the same range of genotoxicity and both were more genotoxic than β-ZOL. Furthermore, our results show that either ZEN or its two metabolites inhibited cell viability in a dose-dependent manner. We conclude that biotransformation of ZEN may be considered as only a partial detoxification pathway since the resulting metabolites remain relatively toxic.     

Benzodiazepines and their metabolites: relationship between binding affinity to the benzodiazepine receptor and pharmacological activity

Experiments were carried out to study the relationship between binding affinity to the benzodiazepine receptor and pharmacological activity, especially anti-anxiety activity, of clinically useful benzodiazepines. In the in vitro experiments, fludiazepam showed the highest affinity to the benzodiazepine receptor with 4 times more potency than that of diazepam, which paralleled the in vivo activity. Diazepam and nimetazepam also bound with high affinities as expected from their in vivo activities. On the contrary, medazepam and cloxazolam showed extremely low affinities and oxazolam showed no affinity, although they showed moderate in vivo activity. However, their metabolites were found to have both high affinity and in vivo activities. These results strongly suggest that in the case of medazepam, cloxazolam and oxazolam, their metabolites may bind to receptor sites in the brain and then elicit pharmacological action. This conclusion was supported by the fact that a good correlation between the binding affinity and the anti-anxiety activity of the tested compounds was observed.     

Applications of LC-MS in the study of the uptake, distribution, metabolism and excretion of bioactive polyphenols from dietary supplements

Specific and quantitative analyses of the bioactive components and their metabolites in body fluids are essential to assess the interaction between groups of compounds in dietary supplements and preparations of psychoactives. Reverse-phase LC separations combined with tandem mass spectrometry provide the necessary specificity and sensitivity. In this paper, applications of these methods are described for the analysis of isoflavones, salvinorin A, synephrine isomers and their metabolites in serum, urine and aqueous humor.     

Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin. III. The serological activity and immunochemical properties of irradiated unpurified toxin

The immunochemical properties and serological activity of irradiated preparations of crude cholera exotoxin have been studied. This study has revealed that with the increase of the dose of ionizing radiation changes occur in the physico-chemical properties of the preparations of the toxin, which leads to an increase in the electrophoretic motility of the protein components of the toxin, to the aggregation and polymerization of individual fragments. The preparations of antigen exotoxins have been shown to retain their serological activity within the range of radiation doses under study (10-350 kGy).     

Echinacea stimulates macrophage function in the lung and spleen of normal rats

Echinacea plant extract has been used for immunostimulation for many years but the evidence supporting its therapeutic potential is still controversial. Using male Sprague-Dawley rats (425-475 g), an in vivo study was conducted to examine the immunomodulatory effects of preparations of Echinacea containing its components cichoric acid, polysaccharides and alkylamides in different concentrations. The rats were gavaged orally with these preparations, two times/day for 4 days. Phagocytic activity of alveolar macrophage was increased with increasing concentrations of the Echinacea components. A trend of increase in TNF-alpha and nitric oxide release by the alveolar macrophages following an in vitro stimulation with LPS was also evident. An enhanced release of cytokines (such as TNF-alpha and IFN-gamma) in response to Echinacea components, was also apparent in rat's spleen macrophage, but at higher concentrations. These results suggest that the Echinacea preparations containing optimal concentrations of cichoric acid, polysaccharides and alkylamides are potentially effective in stimulating an in vivo, non-specific immune response in normal rats.     

Gas chromatographic–mass spectrometric procedures for determination of the catechol-O-methyltransferase (COMT) activity and for detection of unstable catecholic metabolites in human and rat liver preparations after COMT catalyzed in statu nascendi derivatization using S-adenosylmethionine

A procedure is presented for determination of the catechol-O-methyltransferase (COMT) activity in liver cytosolic preparations using 3,4-dihydroxyphenethylamine as substrate and by quantifying the product 3-methoxy-4-hydroxyphenethylamine (3-MHP). For quantification of 3-MHP in liver cytosolic preparations a gas chromatographic–mass spectrometric procedure after liquid–liquid extraction and acetylation was established and validated. The intra- and inter-day accuracy and precision were better than 15% and 20%, respectively. Extraction efficiency and selectivity were also sufficient. For in statu nascendi derivatization of unstable catecholic metabolites in liver microsome preparations, cytosolic preparations with COMT activities of at least 1 nmol product/min/mg protein were used after addition of S-adenosylmethionine. Such catecholic metabolites, which are claimed to be responsible for toxic effects in vivo, e.g., neurotoxicity or carcinogenesis, must not be overlooked in in vitro metabolism studies. Using this trick, gas chromatography–mass spectrometry (GC–MS) was suitable for the determination of catecholic metabolites in human and rat liver preparations after the same sample preparation as for 3-MHP quantification. The applicability was exemplified for the antidepressant paroxetine.     

Evaluation of the cytogenetic effect of the herbicide treflan and of a number of its metabolites on mammalian somatic cells

Investigation of the cytogenetic effect of two samples of trifluralin, a herbicide, (chemically pure active ingredient and 25% emulsion concentrate) and its eight metabolites in the culture of human peripheral lymphocytes in vitro and in the bone marrow cells of mice in vivo has revealed that trifluralin itself does not induce chromosome aberrations both in vitro and in vivo, but some of its derivatives possess moderate (metabolites III and VII) or weak (metabolites II and XI) clastogenic activity. The results obtained should be taken into account for the hygienic regulation of trifluralin use allowing for the reality of entrance of its mutagenic derivatives into the human organism and for the degree of its genetic danger.     

虫草及相关真菌的次生代谢产物及其活性

全面系统地总结了目前在虫草及相关真菌中次生代谢产物研究方面的最新成果,发现目前在虫草及其密切相关的虫生真菌中共报道272个有具体结构的化合物,其活性涉及杀虫、抗癌、抗菌、免疫调节和抗疟原虫等多方面。这些化合物主要来源于20余种虫草、虫草无性型或与虫草密切相关的虫生真菌。虽然近年来有关虫草中生物活性代谢物的研究受到世界各国广泛关注,但仍有90%以上虫草及相关虫生真菌种类中尚无任何具体成分的研究报道,因此有关研究尚需要进一步深入。     

Recent advances in the in vivo and in vitro metabolism of retinoic acid.

Retinoic acid, a natural metabolite of retinol, has previously been shown to be capable of supporting growth and maintaining proper differentiation in epithelial tissues. Recently, investigation into the in vivo and in vitro metabolism of retinoic acid in hamsters, using both tracheal organ culture and subcellular preparations derived from intestinal mucosa, liver, and testis, has revealed the production of several metabolites more polar than the parent compound. Two of the early products of this metabolic pathway have been identified as 4-hydroxy- and 4-keto-retinoic acid. The formation of these metabolites is maximal in vitamin A-deficient hamsters that have been pretreated with retinoic acid and in vitamin A-normal animals. This fact, together with the decreased biological activity of the two compounds relative to retinoic acid in a tracheal organ culture assay, suggested that oxidative attack at carbon-four of the cyclohexenyl ring may be the first step in the elimination of retinoic acid from tissues. In addition, observations both in vivo and in vitro indicate that all-trans- and 13-cis-retinoic acid at low concentrations may be sharing a common metabolic pathway that includes an isomer of 4-keto-retinoic acid.     

Identification of placental transforming growth factor-beta and bikunin metabolites as contaminants of pharmaceutical human chorionic gonadotrophin preparations by proteomic techniques

A contaminant protein complex found in pharmaceutical urinary human chorionic gonadotrophin preparations is reported to have anti-human immunodeficiency virus-associated Kaposi's sarcoma activity. The aim of this study was to isolate and characterize this protein complex by proteomic approaches. Size exclusion chromatography was used in the isolation of these human chorionic gonadotrophin-associated fragments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of a protein complex that dissociated into two protein bands under reducing conditions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of this complex showed three polypeptides at approximately 6.2, 11.4, and 15.8 kDa. Peptide mass mapping and N-terminal amino acid sequencing identified two polypeptides as metabolites of placental transforming growth factor-beta (11.4 kDa) and bikunin (15.8 kDa). Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of the anti-human immunodeficiency virus-associated Kaposi's sarcoma active preparations CG-10 (Sigma), Pregnyl (Organon), and Profasi (Serono) revealed the presence of metabolites of placental transforming growth factor-beta in all three; no other non-human chorionic gonadotrophin-related protein species were observed in these preparations. Our findings present evidence that urinary human chorionic gonadotrophin preparations are contaminated with metabolites of placental transforming growth factor-beta, which may have transforming growth factor-beta agonist actions, and metabolites of bikunin, which is a protease inhibitor. In combination these molecules may be responsible for the anti-human immunodeficiency virus-associated Kaposi's sarcoma activity demonstrated for these urinary human chorionic gonadotrophin preparations.     

The oxygen-centered radicals scavenging activity of sulfasalazine and its metabolites. A direct protection of the bowel.

Oxygen-centered radicals, such as superoxide (O2-) and hydroxyl radicals (.OH) generated by phagocytes have been suggested to be involved in the pathogenesis of chronic inflammations of the bowel, such as Crohn's disease and colitis ulcerosa. Recently, sulfasalazine (SASP) and its metabolites have been reported to exert their effects as a direct scavenger of oxygen-centered radicals in the bowel. To scavenge oxygen-centered radicals in vivo, however, SASP and its metabolites have to react with O2- and/or .OH in vitro very rapidly, furthermore they have to reach an appropriate (possible millimolar) concentration range at the site of inflammation. To test this possibility, we investigated the direct O2- and .OH scavenging activity of SASP and its metabolites using the specific electron paramagnetic resonance/spin trapping method, and we compared the 50% inhibition rates of SASP and its metabolites with their known concentrations in the bowel and in the human plasma. It was found that SASP and its metabolites, such as 5-amino-salicylic acid (5-ASA), and acetyl-5-amino-salicylic acid (AC-5-ASA), but not sulfapyridine (SP) and acetyl-sulfapyridine (Ac-SP) have a direct O2- and .OH scavenging activity in vitro systems. Among the compounds, SASP and 5-ASA can reach a concentration which is appropriate to scavenge oxygen-centered radicals in the bowel but not in the human plasma. It was concluded that the in vivo antiinflammatory effects of SASP and its metabolites are, at least partly, due to the direct oxygen-centered scavenging activity of these drugs.     

Bioalkylation and biooxidation of anthracene, in vitro and in vivo

Anthracene undergoes biomethylation in rat liver cytosol preparations in vitro and in rat subcutaneous tissue, in vivo. The in vitro reaction is dependent on cytosol preparations fortified by the addition of S-adenosyl-L-methionine. The products of the reaction are the meso-anthracenic or L-region derivatives 9-methylanthracene and 9,10-dimethylanthracene. The latter compound may be the simplest polynuclear aromatic hydrocarbon carcinogen known. These reactive methylated metabolites are readily oxidized in cytosol preparations and in subcutaneous tissue, in vivo, to hydroxymethyl and formyl derivatives. Oxidation takes place mainly on the methyl groups since ring oxidized products were not detected.     

Cytogenetic effects of cyclophosphamide on human lymphocytes in vivo and in vitro

A comparative study of cytogenetic effects in human lymphocytes caused in vivo by cyclophosphamide (CP) after intravenous injection and in vitro by exposure of plasma of the same patients was carried out. It was found that the frequency of induced chromosome aberrations (CA) and sister-chromatid exchanges (SCE) increased linearly for SCE and exponentially for CA within the 'dose' of alkylating activity of CP metabolites. Parameters of 'cytogenetic effect-dose' in vivo and in vitro coincided. The intensity of cytogenetic effects varied between individuals.     

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction

After force-feeding a protein-free diet to male rats for 5-7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.