Metabolism of (R)-(+)-pulegone and (R)-(+)-menthofuran by human liver cytochrome P-450s: evidence for formation of a furan epoxide. (9/1563)

(R)-(+)-Pulegone, a monoterpene constituent of pennyroyal oil, is a hepatotoxin that has been used in folklore medicine as an abortifacient despite its potential lethal effects. Pulegone is metabolized by human liver cytochrome P-450s to menthofuran, a proximate hepatotoxic metabolite of pulegone. Expressed human liver cytochrome (CYP) P-450s (1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4) were tested for their ability to catalyze the oxidations of pulegone and menthofuran. Expressed CYP2E1, CYP1A2, and CYP2C19 oxidized pulegone to menthofuran, with respective Km and Vmax values of 29 microM and 8.4 nmol/min/nmol P-450 for CYP2E1, 94 microM and 2.4 nmol/min/nmol P-450 for CYP1A2, and 31 microM and 1.5 nmol/min/nmol P-450 for CYP2C19. The human liver P-450s involved in the metabolism of menthofuran are the same as pulegone except for the addition of CYP2A6. These P-450s were found to oxidize menthofuran to a newly identified metabolite, 2-hydroxymenthofuran, which is an intermediate in the formation of the known metabolites mintlactone and isomintlactone. Based on studies with 18O2 and H218O, 2-hydroxymenthofuran arises predominantly from a dihydrodiol formed from a furan epoxide. CYP2E1, CYP1A2, and CYP2C19 oxidized menthofuran with respective Km and Vmax values of 33 microM and 0.43 nmol/min/nmol P-450 for CYP2E1, 57 microM and 0.29 nmol/min/nmol P-450 for CYP1A2, and 62 microM and 0.26 nmol/min/nmol P-450 for CYP2C19.  (+info)

Reactivity of Cl-P(+)-Cl toward cyclic organic ethers. (10/1563)

The dichlorophosphenium ion (Cl-P(+)-Cl) undergoes a variety of reactions with cyclic organic ethers in the gas phase in a Fourier-transform ion cyclotron resonance mass spectrometer. Most of the reactions are initiated by Cl-P(+)-Cl-induced heterolytic C-O bond cleavage. However, the observed final products depend on the exact structure of the ether. For saturated ethers, e.g., tetrahydropyran, tetrahydrofuran, and 2-methyltetrahydrofuran, the most abundant ionic product corresponds to hydroxide abstraction by Cl-P(+)-Cl. This unexpected reaction is rationalized by a multistep mechanism that involves an initial heterolytic C-O bond cleavage accompanied by a 1,2-hydride shift, and that ultimately yields a resonance-stabilized allyl cation and HOPCl2. The process is estimated to be highly exothermic (AM1 calculations yield delta H = -(33-38) kcal mol(-1) for the ethers mentioned above). However, the adducts formed from most of the unsaturated ethers are unable to undergo hydride shifts and hence cannot react via this pathway. In some of these cases, e.g., for 2,5-dihydrofuran and 2,5-dihydro-3,4-benzofuran, the C-O bond heterolysis is followed by oxygen/chlorine exchange to yield the O=PCl radical and a resonance-stabilized carbocation (AM1 calculations yield delta H = -14 kcal mol(-1) for the reaction of 2,5-dihydro-3,4-benzofuran). Hydride abstraction by Cl-P(+)-Cl also yields an abundant product for these two ethers. On the other hand, the ethers with low ionization energies, such as 2,3-dihydrofuran and 2,3-dihydrobenzofuran, react with Cl-P(+)-Cl by electron transfer. Finally, a unique pathway, addition followed by elimination of HCl, dominates the reaction with furan. The observed reactions are rationalized by thermochemical data obtained from semiempirical molecular orbital calculations.  (+info)

Promoting effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone on rat glandular stomach carcinogenesis initiated with N-methyl-N'-nitro-N-nitrosoguanidine. (11/1563)

The modifying effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a mutagenic by-product in chlorinated water, on the development of glandular stomach cancers were investigated in Wistar rats. A total of 120 males, 6 weeks of age, were divided into six groups. After initiation with 100 ppm N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) solution and 5% NaCl diet for 8 weeks, 30 rats each in groups 1-3 were given MX in the drinking water at concentrations of 30, 10, or 0 ppm for the following 57 weeks. Ten animals each in groups 4-6 were administered the MX without prior carcinogen exposure. There were no statistical significant differences in final body weights between the groups. The incidences and multiplicities of adenocarcinomas in the glandular stomachs were significantly higher (P < 0.05) in the initiated 30 ppm MX group than those in the MNNG/NaCl group. The incidences of atypical hyperplasias in the glandular stomachs were also significantly increased (P < 0.05 or 0.01) by the MX treatments. With their multiplicity, the effects were clearly dose dependent. Interestingly, the 30 ppm MX alone itself induced atypical hyperplasias in the pylorus, although the incidences and severity were low. Moreover, MX showed a tendency to enhance the development of intrahepatic cholangiocellular tumors and thyroid follicular cell tumors in the MNNG-treated animals. The results of the present study thus indicate that MX exerts promoting effects when given during the postinitiation phase of two-stage glandular stomach carcinogenesis in rats.  (+info)

The platelet-activating factor receptor antagonist L-659,989 inhibits phospholipase D activity. (12/1563)

The platelet-activating factor (PAF) receptor antagonist L-659,989 [(+/-)-trans-2-(3-methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran)] has been reported to be a specific inhibitor of the PAF receptor and as such, it is widely used for assessment of PAF receptor mediated biological effects. We report here that L-659,989 may not be as specific as previously reported because it is also a potent inhibitor of phospholipase D activity. At concentrations of 30 micrograms/ml, L-659,989 inhibited basal and agonist-stimulated phospholipase D activity by about 55% and 70-100% respectively, through a mechanism that may involve the generation of intracellular ceramides. Another PAF receptor antagonist, WEB-2086, did not affect phospholipase D activity at concentrations up to 50 micrograms/ml. Either of these inhibitors when present at 20 micrograms/ml are reported to fully block the effects of PAF. Furthermore, L-659,989 directly inhibited the activity of bacterial PLD in vitro. These results indicate that caution is required in the interpretation of results derived from the use of L-659,989.  (+info)

Effect of media constituents on the formation by halophilic yeast of the 2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone aroma component specific to miso. (13/1563)

The formation of HEMF [2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone] by yeast was examined in an attempt to investigate its mechanism and involved factors. HEMF formation was promoted by yeast cultivation in a heat-sterilized medium which included glucose, ribose, and a nitrogenous compound such as an extract of shoyu koji, poly-peptone, casamino acid, or an amino acid (glutamic acid, threonine, serine, or alanine).  (+info)

Nonactin biosynthesis: the product of nonS catalyzes the formation of the furan ring of nonactic acid. (14/1563)

Nonactin is the parent compound of a group of ionophore antibiotics, known as the macrotetrolides, produced by Streptomyces griseus subsp. griseus ETH A7796. Nonactin is a significant compound because of its inhibitory effects on the P170 glycoprotein-mediated efflux of chemotherapeutic agents in multiple-drug-resistant cancer cells. Nonactin is also significant in that it is a highly atypical polyketide. Very little is presently known about the genes of the nonactin biosynthesis cluster. In this paper we describe our efforts to establish a connection between the product of a gene from the nonactin biosynthesis cluster and a known biochemical transformation in nonactin biosynthesis. Nonactate synthase is the enzyme which catalyzes the formation of nonactic acid from an acyclic precursor in nonactin biosynthesis. We have synthesized the substrate for this enzyme and have detected the in vitro cyclization activity of the substrate in cell-free preparations of S. griseus subsp. griseus ETH A7796. Previous studies by R. Plater and J. A. Robinson (Gene 112:117-122, 1992) had suggested, based on sequence homology, that the product of a partial open reading frame found close to the tetranactin resistance gene of S. griseus could be the nonactate synthase. We have therefore cloned, sequenced, and heterologously expressed this full gene (nonS), and we have shown that the gene product, NonS, does indeed catalyze the formation of the furan ring of nonactic acid as hypothesized.  (+info)

Development and characterization of an in vitro ovulation model using mouse ovarian follicles. (15/1563)

To investigate ovulation, an in vitro model with cultured mouse follicles was developed and compared with an in vivo ovulation model. In this model, secondary follicles were grown in vitro with immature mouse serum (5%) and recombinant human FSH. Addition of ascorbic acid and selenium to the medium increased follicular survival (from 29% to 86%) and resulted in the development of healthy preovulatory follicles (> 400 microm) producing estradiol. Depending on the starting size of the follicles, the preovulatory stage was reached after 4-6 days. The ovulatory response to hCG was maximal in follicles exceeding a diameter of 400 microm. The in vitro-ovulated oocytes could be fertilized and were able to develop to the blastocyst stage. Ovulation induced by hCG was dose dependent, reaching a maximum of 80% at 1 IU/ml. Concomitantly, progesterone production increased from 3.6 +/- 0.5 to 29 +/- 2 ng/ml. Both in vivo and in vitro, hCG induced expression of the progesterone receptor and the prostaglandin endoperoxide synthase-2 (PGS-2) gene within 3 h. Ovulation could be completely blocked with the anti-progestogen Org-31710 and partially (50%) with the PGS inhibitor indomethacin in vitro and in vivo. Org-31710 and indomethacin did not affect progesterone production. In summary, a physiologically relevant in vitro ovulation model of cultured mouse follicles that can be used to study the process of follicular rupture has been developed.  (+info)

DNA protein cross-links produced by NSC 652287, a novel thiophene derivative active against human renal cancer cells. (16/1563)

2, 5-bis(5-Hydroxymethyl-2-thienyl)furan (NSC 652287), is a representative of a series of thiophene derivatives that exhibit potent and selective antitumor activity against several tumor cell lines in the National Cancer Institute Anticancer Drug Screen. NSC 652287 has noticeable activity for the renal cell lines and produces cures in certain corresponding xenografts. The cellular mechanisms of action of NSC 652287 were therefore investigated in this study in greater detail. The most sensitive renal carcinoma cell line, A498, exhibited cell cycle arrest in G(0)-G(1) and G(2)-M at 10 nM NSC 652287, with increased p53 and p21(WAF1) protein. At higher concentrations, NSC 652287 still induced p53 elevation but with p21(WAF1) reduction and massive apoptosis. These results collectively suggested that NSC 652287 induced DNA damage. Using alkaline elution techniques, we found that NSC 652287 induced both DNA-protein and DNA-DNA cross-links with no detectable DNA single-strand breaks. These DNA-protein cross-links (DPC) persisted for at least 12 h after drug removal and their frequency was correlated with cytotoxicity in the renal cell lines studied. The most sensitive cells (A498) produced the highest DPC followed by the cell line with intermediate sensitivity (TK-10). DPC were minimal in the two resistant cell lines, ACHN and UO-31. Nonetheless, a similar degree of DPC occurred at doses imparting equitoxic effects. These results indicate that DNA is a primary target for the novel and potent anticancer thiophene derivative, NSC 652287. NSC 652287 did not cross-link purified DNA or mammalian topoisomerase I suggesting the importance of active metabolite(s) for the cross-linking activity.  (+info)