Thiol-quinone adduct formation in myofibrillar proteins detected by LC-MS

Protein oxidation in meat is considered to decrease meat tenderness due to protein disulfide cross-link formation of thiol-containing amino acid residues. An LC-MS method for detection of thiol-quinone adducts (RS-QH(2)) in myofibrillar proteins was developed to investigate the interaction between phenols, as protective antioxidants, and proteins from meat under oxidative conditions using aqueous solutions of (i) cysteine (Cys), (ii) glutathione (GSH), (iii) bovine serum albumin (BSA), or (iv) a myofibrillar protein isolate (MPI). The aqueous solutions were incubated at room temperature (30 min) with 4-methyl-1,2-benzoquinone (4MBQ) prepared from oxidation of 4-methylcatechol (4MC) by periodate resin or incubated at room temperature (5 h) with 4MC and Fe(II)/H(2)O(2). GSH, BSA, and MPI were hydrolyzed (6 N HCl, 110 °C, 22 h) after incubation, and the cysteine-quinone adduct, Cys-QH(2) (m/z 244.2) was identified according to UV and mass spectra after separation on an RP-C18 column. The thiol-quinone adduct was present in all thiol systems after incubation with 4MBQ or 4MC oxidized by Fe(II)/H(2)O(2). Direct reaction with 4MBQ resulted in each case in increased Cys-QH(2) formation compared to simultaneous oxidation of thiol source and 4MC with Fe(II)/H(2)O(2). The covalent bonds between quinones and thiol groups may act as a potential antioxidant by inhibiting disulfide protein cross-link formation.     

Protective activities of stilbene glycosides from Acer mono leaves against H2O2-induced oxidative damage in primary cultured rat hepatocytes

In our previous study, we isolated two new hepatoprotective stilbene glycosides, 5-O-methyl-(E)-resveratrol 3-O-beta-D-glucopyranoside (MRA) and 5-O-methyl-(E)-resveratrol 3-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (MRAG), from the methanolic extract of Acer mono leaves. Thereby, we have attempted to elucidate the hepatoprotective mechanism of these compounds, focusing on antioxidative effects, using hydrogen peroxide (H2O2)-injured primary cultures of rat hepatocytes. Both MRA and MRAG showed potent hepatoprotective activities in pretreatment but showed little effects in posttreatment. In addition, they increased the glutathione (GSH) level in the normal control cultures and significantly prevented the depletion of GSH in H2O2-injured primary cultured rat hepatocytes. Moreover, these compounds significantly restored the level of GSH depleted by buthionine sulfoximine or diethylmaleate in the presence or absence of H2O2. Furthermore, these compounds preserved the activities of antioxidant enzymes such as superoxide dismutase, glutathione reductase, and glutathione peroxidase reduced by H2O2 insults. Meanwhile, MRA and MRAG showed moderate scavenging effects with IC50 values of 103.6 and 80.5 microM, respectively, as determined by 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging activity. Taken together, these results suggest that MRG and MRAG exert significant hepatoprotective activities against H2O2-induced hepatotoxicity by maintaining the antioxidative defense system rather than scavenging free radicals.     

Toxicological and analytical investigations of noni (Morinda citrifolia) fruit juice

Morinda citrifolia (noni) is known to contain genotoxic anthraquinones in the roots. Because of the widespread use of noni juice, the possible genotoxic risk was examined through a battery of short-term tests. Noni juice was also chemically analyzed for the possible presence of anthraquinones. Noni juice extract in the Salmonella microsome assay showed a slight mutagenic effect in strain TA1537, due to the presence of flavonoids. No mutagenicity was observed in the mammalian mutagenicity test with V79 Chinese hamster fibroblasts. Rats treated with a noni juice concentrate did not show DNA repair synthesis (UDS) in primary rat hepatocytes, nor could DNA adducts or DNA strand breaks be observed. HPLC analysis of noni juice for anthraquinones was negative, with a sensitivity of <1 ppm. In summary, chemical analysis and genotoxicity tests reveal that noni juice does not have a genotoxic potential and that genotoxic anthraquinones do not exist in noni juice.     

Studies on the metabolism of the plant lignans secoisolariciresinol and matairesinol

The plant lignans secoisolariciresinol and matairesinol occur in numerous foods such as oil seeds, whole grains, vegetables, and fruits. We have studied the hitherto unknown oxidative metabolism of secoisolariciresinol and matairesinol in hepatic microsomes from untreated and Aroclor 1254-induced Wistar rats and from humans. Five oxidative metabolites of secoisolariciresinol and 10 oxidative metabolites of matairesinol were detected in rat liver microsomes, and their chemical structures were elucidated. The pathways in the metabolism of both secoisolariciresinol and matairesinol included aliphatic and aromatic hydroxylation, whereas oxidative demethylation was only observed for matairesinol. Human hepatic microsomes were able to metabolize secoisolariciresinol whereas matairesinol was only poorly metabolized. This study clearly shows that secoisolariciresinol and matairesinol are substrates of cytochrome P450-mediated metabolism. However, from preliminary experiments with rats dosed orally with secoisolariciresinol and matairesinol, it appears that the intestinal absorption and subsequent oxidative metabolism of these plant lignans occur only to a very small extent due to the highly efficient conversion of secoisolariciresinol and matairesinol to the mammalian lignans enterodiol and enterolactone by the gut microflora.     

Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo.

The soy isoflavones daidzein and genistein are found in high concentrations in human plasma and urine after soy consumption. However, in vitro and in vivo data regarding the oxidative metabolism of isoflavones in humans are scarce. Therefore, we have studied the oxidative metabolites of these compounds formed in human liver microsomes and excreted in urine of male and female humans ingesting soy products for 2 days. Human liver microsomes transformed the soy isoflavone daidzein to three monohydroxylated and three dihydroxylated metabolites according to GC/MS analysis. On the basis of a previous study with rat liver microsomes and with the help of reference substances, these metabolites were identified as 6,7,4'-trihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone, 7,8,4'-trihydroxyisoflavone, 7,8,3',4'-tetrahydroxyisoflavone, 6,7,8,4'-tetrahydroxyisoflavone, and 6,7,3',4'-tetrahydroxyisoflavone. Significant amounts of the same metabolites except 6,7,8,4'-tetrahydroxyisoflavone were also found in urine of female and male volunteers after soy intake. Genistein was metabolized by human liver microsomes to six hydroxylation products. The main metabolites were the three aromatic monohydroxylated products 5,6,7,4'-tetrahydroxyisoflavone, 5,7,8,4'-tetrahydroxyisoflavone and 5,7,3',4'-tetrahydroxyisoflavone. The aliphatic monohydroxylated metabolite 2,5,7,4'-tetrahydroxyisoflavone and two aromatic dihydroxylated metabolites, 5,7,8,3',4'-pentahydroxyisoflavone and 5,6,7,3',4'-pentahydroxyisoflavone, were formed in trace amounts. The same hydroxylated genistein metabolites except the aliphatic hydroxylated one could also be detected in human urine samples. Methylated forms of the catechol metabolites, which were generated by incubations with catechol-O-methyltransferase in vitro could be detected only in trace amounts in the urine samples. This implies that this reaction does not play a major role in the biotransformation of the hydroxylated daidzein and genistein metabolites in vivo. Most of these oxidative metabolites are described as human in vivo metabolites for the first time. Their biological significance remains to be established.     

Inhibition of acrylamide toxicity in mice by three dietary constituents

The inhibitory effects of three dietary constituents, tea polyphenols, resveratrol, and diallyl trisulfide, on acrylamide-biomacromolecule (liver DNA, protamine, and hemoglobin) adduct formation at human exposure level were studied by accelerator mass spectrometry. The results demonstrated that the three dietary constituents all significantly inhibited the formation of acrylamide adducts with liver DNA, whereas tea polyphenols and diallyl trisulfide reduced protamine and hemoglobin adducts, respectively. Further biochemical studies showed that acrylamide could significantly inactivate creatine kinase and glutathione S-transferase and deplete glutathione. When the inhibitors were cotreated with acrylamide, all of them could effectively recover the activities of creatine kinase. In addition, tea polyphenols and diallyl trisulfide could increase glutathione S-transferase activity remarkably. On the basis of these results, mechanisms of the effects are discussed. This study might provide a beneficial guide to people's diet for the purpose of reducing the harmful effect of acrylamide.     

Urolithins, ellagic acid-derived metabolites produced by human colonic microflora, exhibit estrogenic and antiestrogenic activities

Urolithins A and B (hydroxy-6H-dibenzo[b,d]pyran-6-one derivatives) are colonic microflora metabolites recently proposed as biomarkers of human exposure to dietary ellagic acid derivatives. Molecular models suggest that urolithins could display estrogenic and/or antiestrogenic activity. To this purpose, both urolithins and other known phytoestrogens (genistein, daidzein, resveratrol, and enterolactone) were assayed to evaluate the capacity to induce cell proliferation on the estrogen-sensitive human breast cancer MCF-7 cells as well as the ability to bind to alpha- and beta-estrogen receptors. Both urolithins A and B showed estrogenic activity in a dose-dependent manner even at high concentrations (40 microM), without antiproliferative or toxic effects, whereas the other phytoestrogens inhibited cell proliferation at high concentrations. Overall, urolithins showed weaker estrogenic activity than the other phytoestrogens. However, both urolithins displayed slightly higher antiestrogenic activity (antagonized the growth promotion effect of 17-beta-estradiol in a dose-dependent manner) than the other phytoestrogens. The IC(50) values for the ERalpha and ERbeta binding assays were 0.4 and 0.75 microM for urolithin A; 20 and 11 microM for urolithin B; 3 and 0.02 for genistein; and 2.3 and 1 for daidzein, respectively; no binding was detected for resveratrol and enterolactone. Urolithins A and B entered into MCF-7 cells and were metabolized to yield mainly urolithin-sulfate derivatives. These results, together with previous studies regarding absorption and metabolism of dietary ellagitannins and ellagic acid in humans, suggest that the gut microflora metabolites urolithins are potential endocrine-disrupting molecules, which could resemble other described "enterophytoestrogens" (microflora-derived metabolites with estrogenic/antiestrogenic activity). Further research is warranted to evaluate the possible role of ellagitannins and ellagic acid as dietary "pro-phytoestrogens".     

Development of a sensitive enzyme-linked immunosorbent assay for the determination of ochratoxin A

Polyclonal antibodies for ochratoxin A (OTA) were generated from rabbits after the animals had been immunized with either OTA-gamma-globulin or OTA- keyhole limpet hemocyanin (KLH). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of OTA in various agricultural commodities. The antibody titers in the serum of rabbits immunized with OTA-gamma-globulin were considerably higher than those in rabbits immunized with OTA-KLH. The antibodies from the rabbits immunized with OTA-gamma-globulin were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of OTA-horseradish peroxidase to the antibodies by OTA, ochratoxin B (OTB), and ochratoxin C (OTC) were found to be 0.90, 110, and 0.54 ng/mL, respectively. When 10 to 250 ng/g of standard OTA was spiked to soybean samples and then extracted with 50% aqueous methanol, the recovery rate of OTA was found to be 85.9% in the cdELISA. Analysis of OTA in various agricultural commodities showed that 12 of the 20 examined samples were contaminated with OTA at levels from 16 to 160 ng/g. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method.     

Effect of ethanol and red wine on ochratoxin a-induced experimental acute nephrotoxicity

Ochratoxin A (OTA), is a nephrotoxic mycotoxin present in wine, which is nephrotoxic in humans. Our working hypothesis is that natural substances in wine may counteract OTA toxicity. Thirty-six rats were randomized to OTA dissolved in saline, red wine, or 13.5% ethanol or to OTA-free wine, ethanol, or saline. OTA (289 microg/kg of body weight/48 h) was administered by gastric gavage for 2 weeks. Serum creatinine, tubular enzymuria, renal lipohydroperoxides (LOOH), reduced (GSH) and oxidized (GSSG) glutathione, and renal superoxide dismutase activity (SOD) were determined in renal tissue. OTA alone produced significant increases in renal lipoperoxides and significant decreases in SOD and GSH/GSSG ratio. In red wine or ethanol, OTA was less nephrotoxic, reducing oxidative damage as revealed by LOOH. In OTA-wine and OTA-ethanol groups, SOD activity was higher than in the OTA-treated one, suggesting that both ethanol and nonalcoholic fractions may preserve antioxidant reserve. GSH/GSSG ratio was significantly preserved only in the OTA-wine group and not in OTA-ethanol. Red wine may exert a protective effect against OTA nephrotoxicity by limiting oxidative damage. The ostensible protection afforded by ethanol deserves further investigation.     

Some sulfur-containing metabolites of tri-n-butyltin chloride in male rats.

In an attempt to elucidate metabolic destination of TBTO, sulfur-containing metabolites were investigated in the urine. Tri-n-butyltin chloride (TBTC), tri-n-butyltin oxide (TBTO), and their in vitro metabolites in rat liver microsomal enzyme systems, di-n-butyl(3-hydroxybutyl)tin chloride (T3OH), di-n-butyl(3-oxobutyl)tin chloride (T3CO), dibutyltin dichloride (DBTC), and monobutyltin trichloride (MBTC), were intraperitoneally administered to rats. In particular, administration of T3OH and T3CO gave higher amounts of mercapturic acid derivatives, such as N-acetyl-S-(3-oxobutyl)-L-cysteine (3CO-MA) and N-acetyl-S-(3-hydroxybutyl)-L-cysteine (3OH-MA), than TBTC or TBTO. On the other hand, DBTC and MBTC did not yield measurable amounts of 3CO-MA and/or 3OH-MA. The appearance of organotin metabolites in urine indicates that T3OH, T3CO, and hypothesized secondary metabolites, such as n-butyl(3-hydroxybutyl)(3-oxobutyl)tin chloride, n-butyl(3-hydroxybutyl)(4-hydroxybutyl)tin chloride, etc., are subject to the action of glutathione S-transferase to give mercapturic acid derivatives. These sulfur-containing metabolites (3CO-MA and 3OH-MA) were also found in control rat urine.     

DNA adducts as biomarkers for oxidative and genotoxic stress from pesticides in crop plants

Plant studies have been carried out to identify the nature and extent of the formation of adducts with DNA bases when treated with pesticide formulations. DNA extracted from crop plants after treatment with pesticide formulations has yielded evidence of adduct formation. The extent of DNA modification has been established by (32)P postlabeling studies. The radiochromatograms from (32)P postlabeling of isolated plant DNA from grapes, bush beans, soybeans, pumpkins, and cucumbers show elevated adduct levels in treated vegetable plants as compared with untreated controls. A number of different adduct spots appear, likely indicating adduct formation with pesticide molecules or their metabolites. The DNA adducts from hexenal and 4-hydroxy-2-nonenal were clearly observed, indicating oxidative stress and lipid peroxidation in the plant.     

Urinary and biliary metabolic patterns of chlorothalonil in germ-free and conventional rats.

The metabolic fate of chlorothalonil, a broad spectrum fungicide that is known to be metabolized via glutathione conjugation, was examined through the analysis of urine and bile metabolites. The role of digestive microflora in the metabolism of chlorothalonil was assessed by comparing the metabolic patterns in germ-free and conventional rats. Low urinary and biliary excretion of radioactivity was observed in both conventional and germ-free rats. However, the urinary excretion of radioactivity was higher in conventional than in germ-free rats. Radio-HPLC analysis of urine and bile showed a complex metabolic profile in both conventional and germ-free rats. Methylthio metabolites of chlorothalonil were determined in ethyl acetate extracts of urine and bile of conventional and germ-free rats. These metabolites were excreted in a higher amount in the urine of conventional rats than in the urine of germ-free rats. This study shows the complexity of chlorothalonil metabolism and the role of the digestive microflora in chlorothalonil metabolism.     

Topoisomerase I and II enzyme inhibitory aqueous extract of Ardisia compressa and ardisin protect against benomyl oxidation of hepatocytes

Tea preparations of Ardisia compressa (AC) have been used in folk medicine against liver disorders. The objective of this study was to evaluate the in vitro topoisomerase I and II enzyme inhibition and the antioxidant effect of an aqueous extract from dry leaves of AC and a pure component (ardisin) purified from AC on benomyl (Be)-induced cytotoxicity in primary culture rat hepatocytes. Lipid peroxidation (malondialdehyde), antioxidant enzyme activities of glutathione reductase, glutathione peroxidase, and superoxide dismutase, and glutathione levels were studied. Topoisomerase I and II enzyme inhibition was used to guide purification of ardisin, which was purified using TLC, MPLC, and preparative and analytical HPLC methods. Benomyl increased malondialdehyde (58% change in comparison to the control) and glutathione peroxidase (10%), producing a significant consumption of endogenous antioxidant glutathione (65%, P < 0.05). A 94% hepatocyte protection was observed when cells were first exposed to ardisin (0.27 microg/mL), followed by Be (35 microg/mL). Cell protection by the tea extract of AC (AE) was greater than that by (-)-epigallocatechin 3-gallate (EGCG). Ardisin showed a clear inhibition of topoisomerases I and II catalytic activity in Saccharomyces cerevisiae mutant cells JN 394, JN394t(-)(1), and JN394t-(2)(-)(5). The potency of ardisin was superior to that of AE and EGCG as an antioxidant, protecting rat hepatocytes when exposed to Be. On the basis of the effective concentrations of equivalents to [+]catechin found in the present study, it can be estimated that, in order to gain antioxidative protection, a person would need to ingest approximately 1 L of AC tea per day, with a total content of 10.8 g of plant material.     

Identification of metabolites of [1,2,3-(13)C]Propargyl alcohol in rat urine by (13)C NMR and mass spectrometry

Little is known about the metabolism of acetylenic (C&tbd1;C) compounds commonly used in the formulation of pesticides. To better understand the in vivo reactivity of this bond, we examined the metabolism of propargyl alcohol (PA), 2-propyn-1-ol, used extensively in the chemical industry. [1,2,3-(13)C, 2,3-(14)C]PA was administered orally to male Sprague-Dawley rats. Approximately 56% of the dose was excreted in urine by 96 h. Major metabolites were characterized, directly, in the whole urine by one- and two-dimensional (13)C NMR. To determine the complete structures of metabolites of PA, rat urine was also subjected to TLC followed by purification of separated TLC bands on HPLC. The purified metabolites were identified by (13)C NMR and mass spectrometry and by comparison with available synthetic standards. The proposed metabolic pathway involves oxidation of propargyl alcohol to 2-propynoic acid and further detoxification via glutathione conjugation to yield as final products: 3, 3-bis[(2-(acetylamino)-2-carboxyethyl)thio]-1-propanol, 3-(carboxymethylthio)-2-propenoic acid, 3-(methylsulfinyl)-2-(methylthio)-2-propenoic acid, 3-[[2-(acetylamino)-2-carboxyethyl]thio]-3-[(2-amino-2-carboxyethyl)t hio]-1-propanol and 3-[[2-(acetylamino)-2-carboxyethyl]sulfinyl]-3-[2-(acetylamino)-2-car boxyethyl]thio]-1-propanol. These unique metabolites have not been reported previously and represent the first example of multiple glutathione additions to the carbon-carbon triple bond.     

Screening of commercial hydrolases for the degradation of ochratoxin A

Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin. The toxin is a common contaminant of various foods and feeds and poses a serious threat to the health of both humans and animals. A number of commercial hydrolases were screened for the ability to degrade OTA to nontoxic compounds. A crude lipase from Aspergillus niger (Amano A) proved to substantially hydrolyze OTA to the nontoxic OTalpha and phenylalanine, as confirmed by HPLC with fluorescence detection. The enzyme was purified by anion exchange chromatography to homogeneity. Activity staining of the purified enzyme with alpha-naphthyl acetate/Fast Red revealed only one band exhibiting hydrolytic activity. The specific activity of the purified enzyme toward OTA was 2.32 units/mg.     

Removal of atrazine from water using covalent sequestration

Monochlorotriazines including atrazine and its major metabolites, deethylatrazine and deisopropylatrazine, are susceptible to nucleophilic aromatic substitution. Competitive reactions to rank the relative reactivity of nucleophiles with atrazine reveal that constrained secondary amines are the most reactive. When the nucleophile is attached to a solid support, atrazine can be sequestered from solution. As proof of concept, polystyrene resins displaying constrained secondary amines are shown to sequester atrazine, deethylatrazine, and deisopropylatrazine from water. Sequestration can be followed spectrophotometrically or using a liquid chromatography mass spectrometry protocol. The kinetics of sequestration are similar to that of granulated charcoal. Evidence for covalent bond formation comes from control experiments with unreactive herbicides and degradation analysis of the solid support. Using both (1)H NMR spectroscopy and mass spectrometry, covalent adducts are identified in ratios close to what is calculated theoretically. This method for sequestration is effective at removing atrazine from pond water.     

Sulfated ferulic acid is the main in vivo metabolite found after short-term ingestion of free ferulic acid in rats

The bioavailability of ferulic acid (FA; 3-methoxy-4-hydroxycinnamic acid) and its metabolites was investigated in rat plasma and urine after an oral short-term ingestion of 5.15 mg/kg of FA. Free FA, glucuronoconjugates, and sulfoconjugates were quickly detected in plasma with a peak of concentration found 30 min after ingestion. Sulfoconjugates were the main derivates ( approximately 50%). In urine, the cumulative excretion of total metabolites reached a plateau 1.5 h after ingestion, and approximately 40% were excreted by this way. Free FA recovered in urine represented only 4.9 +/-1.5% of the native FA consumed by rats. Glucuronoconjugates and sulfoconjugates represented 0.5 +/- 0.3 and 32.7 +/- 7.3%, respectively. These results suggested that a part of FA incorporated in the diet was quickly absorbed and largely metabolized in sulfoconjugates before excretion in urine.     

Secondary metabolites from Glycine soja and their growth inhibitory effect against Spodoptera litura

The wild soybean (Glycine soja Sieb. et Zucc) has been reported to be relatively resistant to insect and pathogenic pests. However, the responsible secondary metabolites in the aerial part of this important plant are largely unknown. From the aerial part of G. soja, 13 compounds were isolated and identified, including seven isoflavonoids (1-7), a cyclitol (8), two sterol derivatives (9 and 10), and three triterpenoids (11-13). Compound 7 is a new isoflavonoid, and compounds 9 and 10 are reported as natural products for the first time. The growth inhibitory activity of 1, 3, 4, and 8 against the larvae of Spodoptera litura was investigated. The most abundant isoflavonoid in the aerial part of G. soja, daidzein (1), which could not be metabolized by S. litura, was found to inhibit the insect larvae growth significantly in 3 days after feeding diets containing the compound. Compounds 3, 4, and 8, which could be partially or completely metabolized, were inactive. Our results suggested that the isoflavonoid daidzein (1) might function as a constitutive defense component in G. soja against insect pests.     

Novel oxidations of (+)-catechin by horseradish peroxidase and laccase

Horseradish peroxidase (HRP; EC 1.11.1.7) catalyzed the H(2)O(2)-dependent oxidative coupling of (+)-catechin 1 to form three different biphenyl C-C dimers 2-4, whereas Rhus vernicifera laccase catalyzed the formation of two new catechin-hydroquinone adducts 5 and 6. Spectroscopic evidence showed that HRP dimers were linked through position 8 of the A-ring of one catechin moiety to C-5' of ring B in 2 and 4 and to C-2 of ring C in 3. The unusual catechin dicarboxylic acid dimer 4 was obtained by ortho cleavage of the E-ring. Hydroquinone served as both a shuttle oxidant and a reactant by coupling at C-2' and C-5' of the catechin B-ring during laccase oxidations. HRP and laccase oxidation products were compared to D,L-alpha-tocopherol and (+)-catechin for their abilities to inhibit iron-induced lipid peroxidation in rat brain homogenates and Fe(3+)-ADP/NADPH in rat liver microsomes, as measured by the intensity of thiobarbituric acid reactive substance. All metabolites exhibited anti-lipid peroxidation with IC(50) values approximately 2-8 times higher than those of standard compounds. Characteristic reaction products may prove to be novel markers for (+)-catechin antioxidant reactions in living systems.