An investigation into the binding of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one to DNA in vitro.
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After metabolic activation the carcinogen 15,16-dihydro-11-[3H]methylcyclopenta[a]phenanthren-17-one binds to DNA in vitro, and this binding is prevented by 7,8-benzoflavone. Radioactivity cannot be removed from the DNA with organic solvents or by chromatography on Sephadex G-50, even after heat denaturation of the DNA. Enzymatic hydrolysis yields radioactive fractions, which elute from a column of Sephadex LH-20 immediately after the natural nucleosides. At least two species of reactive metabolites are involved in this bending, those with a half-life of a few hr and others with greater stability. After extraction from the aqueous incubation mixture, they could be detected in discrete polar fractions from separations of the complex metabolite mixture by high-pressure liquid chromatography. Their ability to bind to DNA decreased with time at ambient temperature, and they were rapidly deactivated by acid. 7,8-Benzolflavone acted by suppressing the formation of polar metabolites derived from enzymatic oxidation of the aromatic double bonds. The inhibitor had no effect on the enzymes hydroxylating saturated carbon; hence it is unlikely that metabolism of the methyl group is important in conversion of this carcinogen to its proximate form, although the presence of the 11-methyl group is essential for carcinogenic activity in this series. (+info)
Contrasting effects of a nonionic surfactant on the biotransformation of polycyclic aromatic hydrocarbons to cis-dihydrodiols by soil bacteria.
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The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria, Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae B8/36, under conditions which facilitate mass-transfer limited substrate oxidation. Both of these strains are mutants that accumulate cis-dihydrodiol metabolites under the reaction conditions used. The effects of the nonpolar solvent 2,2,4, 4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined. HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates. The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene. Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate. However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions. The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results. The surfactant inhibited growth of the wild-type strain S. yanoikuyae B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro. (+info)
Antimalarial activities of various 9-phenanthrenemethanols with special attention to WR-122,455 and WR-171,669.
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Pilot appraisals of the activities of 16 specially selected 9-phenanthrenemethanols against acute infections with Plasmodium falciparum in owl monkeys showed that all were more active than the reference compound, WR-33,063. WR-122,455, the most active derivative, and WR-171,669, ranked sixth, were selected for study in human volunteers. To assist this undertaking, appraisals of both compounds in owl monkeys infected with various strains of P. falciparum were expanded. These assessments showed: (i) that WR-122,455 was four times as active as chloroquine against infections with chloroquine-sensitive strains and that WR-171,669 equalled chloroquine in activity; (ii) that these compounds were fully active against infections with strains resistant to chloroquine, pyrimethamine, or quinine, or to all three standard drugs; (iii) that the activity of WR-122,455 was a function of total dose, single doses being as effective as the same amounts delivered in three or seven daily fractions; and (iv) that a single dose of WR-122,455 conferred extended, although only partial, protection against challenges with trophozoites. Complementary experiments in rhesus monkeys inoculated with sporozoites of P. cynomolgi showed that the activity of WR-122,455 was limited to blood schizonts and did not extend to early or late tissue schizonts. These evaluations were compatible with the results of preliminary studies of the activities of WR-122,455 and WR-171,669 in human volunteers. (+info)
Inhibition of GABA-gated chloride channels by 12,14-dichlorodehydroabietic acid in mammalian brain.
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1. 12,14-dichlorodehydroabietic acid (12,14-Cl2DHA) reduced GABA-stimulated uptake of 36Cl- into mouse brain synaptoneurosomes suggesting inhibition of mammalian GABA(A) receptor function. 2. 12,14-Cl2DHA did not affect the binding of [3H]-muscimol to brain membranes but displaced specifically bound [3H]-EBOB. The inhibitory effect on [3H]-EBOB binding was not reversible. 12,14-Cl2DHA reduced the availability of [3H]-EBOB binding sites (Bmax) without changing the KD of the radioligand for remaining sites. 12,14-Cl2DHA did not affect the rate of association of [3H]-EBOB with its chloride channel receptor, but increased the initial rate of [3H]-EBOB dissociation. 3. 12,14-Cl2DHA enhanced the incidence of EPSCs when rapidly applied to cultured rat cortical neurones. Longer exposures produced block of IPSCs with marked increases in the frequency of EPSCs and min EPSCs. 12,14-Cl2DHA also irreversibly suppressed chloride currents evoked by pulses of exogenous GABA in these cells. 4. Ultimately, 12,14-Cl2DHA inhibited all synaptic traffic and action currents in current clamped cells indicating that, in contrast to picrotoxinin (which causes paroxysmal bursting), it is not fully selective for the GABA(A) receptor-chloride channel complex. 5. The depolarizing block seen with 12,14-Cl2DHA in amphotericin-perforated preparations implicates loss of Ca2+ buffering in the polarity change and this may account for inhibition of spontaneous action potentials. 6. Our investigation demonstrates that 12,14-Cl2DHA blocks GABA-dependent chloride entry in mammalian brain and operates as a non-competitive insurmountable GABA(A) antagonist. The mechanism likely involves either irreversible binding of 12,14-Cl2DHA to the trioxabicyclooctane recognition site or a site that is allosterically coupled to it. We cannot exclude, however, the possibility that 12,14-Cl2DHA causes localized proteolysis or more extensive conformational change within a critical subunit of the chloride channel. (+info)
A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila BKME-9.
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Pseudomonas abietaniphila BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The ditA1, ditA2, and ditA3 genes, which encode the alpha and beta subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9. 2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditC. A Tn5 insertion in the alpha subunit gene, ditA1, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene, ditA3, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in Escherichia coli of ditA1A2A3, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11, 12-dihydroxy-8,13-abietadien acid, which was identified by 1H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the alpha subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases. (+info)
Effect of selected antimalarial drugs and inhibitors of cytochrome P-450 3A4 on halofantrine metabolism by human liver microsomes.
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Halofantrine (HF) is used in the treatment of uncomplicated multidrug-resistant Plasmodium falciparum malaria. Severe cardiotoxicity has been reported to be correlated with high plasma concentrations of HF but not with that of its metabolite N-debutylhalofantrine. The aim of this study was to investigate the effects of other antimalarial drugs and of ketoconazole, a typical cytochrome P-450 3A4 inhibitor, on HF metabolism by human liver microsomes. Antimalarial drug inhibitory effects were ranked as follows: primaquine > proguanil > mefloquine > quinine > quinidine > artemether > amodiaquine. Artemisine, doxycycline, sulfadoxine, and pyrimethamine showed little or no inhibition of HF metabolism. Mefloquine, quinine, quinidine, and ketoconazole used at maximal plasma concentrations inhibited N-debutylhalofantrine formation noncompetitively with Ki values of 70 microM, 49 microM, 62 microM, and 0.05 microM resulting in 7%, 49%, 26%, and 99% inhibition, respectively, in HF metabolism. In conclusion, we showed that quinine and quinidine coadministered with HF might inhibit its metabolism resulting in the potentiation of HF-induced cardiotoxicity in patients. This requires a close monitoring of ECG. For the same reasons, the concomitant administration of HF and ketoconazole must be avoided. By contrast, none of the other antimalarials studied inhibited HF metabolism and, by extrapolation, cytochrome P-450 3A4 activity. (+info)
Immunosuppressant PG490 (triptolide) inhibits T-cell interleukin-2 expression at the level of purine-box/nuclear factor of activated T-cells and NF-kappaB transcriptional activation.
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PG490 (triptolide) is a diterpene triepoxide with potent immunosuppressive and antiinflammatory properties. PG490 inhibits interleukin(IL)-2 expression by normal human peripheral blood lymphocytes stimulated with phorbol 12-myristate 13-acetate (PMA) and antibody to CD3 (IC50 of 10 ng/ml), and with PMA and ionomycin (Iono, IC50 of 40 ng/ml). In Jurkat T-cells, PG490 inhibits PMA/Iono-stimulated IL-2 transcription. PG490 inhibits the induction of DNA binding activity at the purine-box/antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target sequence but not at the NF-kappaB site. PG490 can completely inhibit transcriptional activation at the purine-box/ARRE/NF-AT and NF-kappaB target DNA sequences triggered by all stimuli examined (PMA, PMA/Iono, tumor necrosis factor-alpha). PG490 also inhibits PMA-stimulated activation of a chimeric transcription factor in which the C-terminal TA1 transactivation domain of NF-kappaB p65 is fused to the DNA binding domain of GAL4. In 16HBE human bronchial epithelial cells, IL-8 expression is regulated predominantly by NF-kappaB, and PG490 but not cyclosporin A can completely inhibit expression of IL-8. The mechanism of PG490 inhibition of cytokine gene expression differs from cyclosporin A and involves nuclear inhibition of transcriptional activation of NF-kappaB and the purine-box regulator operating at the ARRE/NF-AT site at a step after specific DNA binding. (+info)
PG490 (triptolide) cooperates with tumor necrosis factor-alpha to induce apoptosis in tumor cells.
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Progress in the treatment of solid tumors has been slow and sporadic. The efficacy of conventional chemotherapy in solid tumors is limited because tumors frequently have mutations in the p53 gene. Also, chemotherapy only kills rapidly dividing cells. Members of the tumor necrosis factor (TNF) family, however, induce apoptosis regardless of the p53 phenotype. Unfortunately, the cytotoxicity of TNF-alpha is limited by its activation of NF-kappaB and activation of NF-kappaB is proinflammatory. We have identified a compound called PG490, that is composed of purified triptolide, which induces apoptosis in tumor cells and sensitizes tumor cells to TNF-alpha-induced apoptosis. PG490 potently inhibited TNF-alpha-induced activation of NF-kappaB. PG490 also blocked TNF-alpha-mediated induction of c-IAP2 (hiap-1) and c-IAP1 (hiap-2), members of the inhibitor of apoptosis (IAP) family. Interestingly, PG490 did not block DNA binding of NF-kappaB, but it blocked transactivation of NF-kappaB. Our identification of a compound that blocks TNF-alpha-induced activation of NF-kappaB may enhance the cytotoxicity of TNF-alpha on tumors in vivo and limit its proinflammatory effects. (+info)