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Azospirillum brasilense colonizes plant roots and improves productivity, but the molecular mechanisms behind its phytostimulation properties remain mostly unknown. Here, we uncover an important role of TARGET OF RAPAMYCIN (TOR) signaling on the response of Arabidopsis thaliana to A. brasilense Sp245. The effect of the bacterium on TOR expression was analyzed in the transgenic line TOR/tor-1, which carries a translational fusion with the GUS reporter protein, and the activity of TOR was assayed thought the phosphorylation of its downstream signaling target S6K protein. Besides, the role of TOR on plant growth in inoculated plants was assessed using the ATP-competitive inhibitor AZD-8055. A decrease in growth of the primary root correlates with an improved branching and absorptive capacity via lateral root and root hair proliferation 6 days after transplant to different concentrations of the bacterium (10 3 or 10 5 CFU/mL). Bacterization increased the expression of TOR in shoot and root apexes and promoted phosphorylation of S6K 3 days after transplant. The TOR inhibitor AZD-8055 (1 μM) inhibited plant growth and cell division in root meristems and in lateral root primordia, interfering with the phytostimulation by A. brasilense. In addition, the role of auxin produced by the bacterium to stimulate TOR expression was explored. Noteworthy, the A. brasilense mutant FAJ009, impaired in auxin production, was unable to elicit TOR signaling to the level observed for the wild-type strain, showing the importance of this phyhormone to stimulate TOR signaling. Together, our findings establish an important role of TOR signaling for the probiotic traits elicited by A. brasilense in A. thaliana., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Epidermal growth factor and vascular endothelial growth factor-2 are important targets of tyrosine kinase for the treatment of various cancerous diseases. Combination of inhibition of both targets to produce synergy in the signal pathway is a critical approach to identify novel tyrosine kinase inhibitors. In this study, a series of new compounds derived from the 4-aminoquinoline as dual inhibitors were synthesized. The obtained results of cytotoxicity assay against human carcinoma cell lines indicated 0.8 µM for 4c against A549 showing its high efficiency in comparison to erlotinib. Pharmacophore modeling as a structure-based method was investigated on dual inhibitors and 4c which was compared with co-crystallized in the active site of EGFR and VEGFR-2. They have shown the same binding orientation as vandetanib, erlotinib and sorafenib. Molecular dynamics simulation results approved that Met769, Lys721, Asp1046, and Lys868 are key residues in two binding sites for dual activity. Ala1050 and Pro968 were identified as new amino acid interaction sites for dual inhibition. 4c showed more favorable stability than vandetanib in VEGFR-2 receptor for a 50 ns dynamic simulation. The high correlation between essential pharmacophoric features of designed compounds and lead inhibitors interactions provided a deeper insight into the structural basis of 4-aminoquinoline inhibition., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We demonstrate an innovative approach for optimization of kinase inhibitor potency and selectivity utilising kinase mini-panels and kinome-wide panels. We present a focused case study on development of a selective inhibitor of cyclin G associated kinase (GAK) using the quin(az)oline inhibitor chemotype. These results exemplify a versatile, efficient approach to drive kinome selectivity during inhibitor development programs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: To understand the pathways involved in drug clearance from the body, quantitative evaluations of the hepatobiliary transport of drugs are important. The organic anion transporting polypeptide (OATP) family transporter, particularly OATP1B1 and 1B3, are considered to play an important role in hepatic uptake of organic anion compounds. Pitavastatin is a substrate of OATP, and it includes a fluorine group. Therefore, it represents an acceptable positron-emission tomography (PET) tracer using fluorine-18 to image in vivo hepatic transporter functions., Method: [ 18 F]Pitavastatin was synthesized using the method we previously reported. To evaluate the potential of [ 18 F]pitavastatin in PET imaging of in vivo OATP functions, we investigated the hepatic uptake with/without rifampicin as an OATP inhibitor after administration in normal SD rats. [ 18 F]Pitavastatin metabolite was evaluated using reverse-phase thin-layer chromatography (TLC) autoradiography. We subsequently analyzed the PET image results and demonstrated that [ 18 F]pitavastatin selectively accumulated in the liver post-administration. Result and discussion In metabolite analysis using reverse-phase TLC, we found that the radioactivity detected in the plasma, liver (>90% intact), and bile mostly originated from the parent pitavastatin of the PET study (~40 min). [ 18 F]pitavastatin's hepatic uptake decreased (approximately 76%) with rifampicin co-administration in PET analysis. Because [ 18 F]pitavastatin has lower clearance in rats than other previously reported OATP1B PET s, it holds the potential of an imaging tracer that has a higher sensitivity in monitoring hepatic OATP1B function's changes., Conclusion: Compared with the previously reported OATP imaging tracers, [ 18 F]pitavastatin is more suitable for the sensitive detection of functional changes in OATP transporters. We believe that [ 18 F]pitavastatin enables quantitative analysis of the hepatobiliary transport system for organic anion compounds., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Melanization of carbon stressed Aspergillus nidulans cultures were studied. Melanin production showed strong positive correlation with the activity of the secreted chitinase and ß-1,3-glucanase. Deletion of either chiB encoding an autolytic endochitinase or engA encoding an autolytic ß-1,3-endoglucanase, or both, almost completely prevented melanization of carbon stressed cultures. In contrast, addition of Trichoderma lyticase to cultures induced melanin production. Synthetic melanin could efficiently inhibit the purified ChiB chitinase activity. It could also efficiently decrease the intensity of hyphal fragmentation and pellet disorganization in Trichoderma lyticase treated cultures. Glyphosate, an inhibitor of L-3,4-dihydroxyphenylalanine-type melanin synthesis, could prevent melanization of carbon-starved cultures and enhanced pellet disorganization, while pyroquilon, a 1,8-dihydroxynaphthalene-type melanin synthesis inhibitor, enhanced melanization, and prevented pellet disorganization. We concluded that cell wall stress induced by autolytic cell wall hydrolases was responsible for melanization of carbon-starved cultures. The produced melanin can shield the living cells but may not inhibit the degradation and reutilization of cell wall materials of dead hyphae. Controlling the activity of autolytic hydrolase production can be an efficient approach to prevent unwanted melanization in the fermentation industry, while applying melanin synthesis inhibitors can decrease the resistance of pathogenic fungi against the chitinases produced by the host organism., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Akathisia is a subset of the larger antipsychotic side effect profile known as extrapyramidal syndrome (EPS). It is associated with antipsychotic treatment and is characterized as a feeling of inner restlessness that results in a compulsion to move. There are currently no primate models available to assess drug-induced akathisia; the present research was designed to address this shortcoming. We developed a novel rating scale based on both the Barnes Akathisia Rating Scale (BARS) and the Hillside Akathisia Scale (HAS) to measure the objective, observable incidence of antipsychotic-induced akathisia-like behavior in Cebus apella non-human primates (NHPs). To induce akathisia, we administered the atypical antipsychotic aripiprazole (1 mg/kg) or the selective phosphodiesterase 10A (PDE10A) inhibitor MP-10 (1-3 mg/kg). Treatment with both compounds produced significantly greater akathisia scores on the rating scale than vehicle treatment. Characteristic behaviors observed included vocalizations, stereotypies, teeth grinding, restless limb movements, and hyperlocomotion. Adenosine A2A receptor antagonists have previously been shown to be effective in blocking antipsychotic-induced EPS in primates. The selective A2A receptor antagonist, SCH 412348 (10-30 mg/kg), effectively reduced or reversed akathisia-like behavior induced by both aripiprazole and MP-10. This work represents the first NHP measurement scale of akathisia and demonstrates that NHPs are responsive to akathisia-inducing agents. As such, it provides a useful tool for the preclinical assessment of putative antipsychotics. In addition, these results provide further evidence of the utility of A2A receptor antagonists for the treatment of antipsychotic-induced movement disorders., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Perturbation of hydroxyl radical accumulation by subinhibitory concentrations of 2,2'-bipyridyl plus thiourea protects Escherichia coli from being killed by 3 lethal antimicrobial classes. Here, we show that 2,2'-bipyridyl plus thiourea delays and/or reduces antimicrobial killing of Staphylococcus aureus by daptomycin, moxifloxacin, and oxacillin. While the protective effect of 2,2'-bipyridyl plus thiourea varied among strains and compounds, the data support the hypothesis that hydroxyl radical enhances antimicrobial lethality.

According to published in vitro studies, cytochrome P450 3A4 catalyzes montelukast 21-hydroxylation (M5 formation), whereas CYP2C9 catalyzes 36-hydroxylation (M6), the primary step in the main metabolic pathway of montelukast. However, montelukast is a selective competitive CYP2C8 inhibitor, and our recent in vivo studies suggest that CYP2C8 is involved in its metabolism. We therefore reevaluated the contributions of different cytochrome P450 (P450) enzymes, particularly that of CYP2C8, to the hepatic microsomal metabolism of montelukast using clinically relevant substrate concentrations in vitro. The effects of P450 isoform inhibitors on montelukast metabolism were examined using pooled human liver microsomes, and montelukast oxidations by human recombinant CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were investigated. The results verified the central role of CYP3A4 in M5 formation. The CYP2C8 inhibitors gemfibrozil 1-O-β glucuronide and trimethoprim inhibited the depletion of 0.02 μM montelukast and formation of M6 from 0.05 μM montelukast more potently than did the CYP2C9 inhibitor sulfaphenazole. Likewise, recombinant CYP2C8 catalyzed montelukast depletion and M6 formation at a 6 times higher intrinsic clearance than did CYP2C9, whereas other P450 isoforms produced no M6. On the basis of depletion of 0.02 μM montelukast, CYP2C8 was estimated to account for 72% of the oxidative metabolism of montelukast in vivo, with a 16% contribution for CYP3A4 and 12% for CYP2C9. Moreover, CYP2C8 catalyzed the further metabolism of M6 more actively than did any other P450. In conclusion, CYP2C8 plays a major role in the main metabolic pathway of montelukast at clinically relevant montelukast concentrations in vitro.

Background and Purpose: Intracerebroventricularly injected tachykinin NK(3) receptor (R) antagonists normalize mean arterial blood pressure (MAP) in spontaneously hypertensive rats (SHR). This study was pursued to define the role played by NK(3)R located on dopamine neurones of the ventral tegmental area (VTA) in the regulation of MAP in SHR., Experimental Approach: SHR (16 weeks) were implanted permanently with i.c.v. and/or VTA guide cannulae. Experiments were conducted 24 h after catheterization of the abdominal aorta to measure MAP and heart rate (HR) in freely behaving rats. Cardiovascular responses to i.c.v. or VTA-injected NK(3)R agonist (senktide) and antagonists (SB222200 and R-820) were measured before and after systemic administration of selective antagonists for D(1)R (SCH23390), D(2)R (raclopride) or non-selective D(2)R (haloperidol), and after destruction of the VTA with ibotenic acid., Key Results: I.c.v. or VTA-injected SB222200 and R-820 (500 pmol) evoked anti-hypertension, which was blocked by raclopride. Senktide (10, 25, 65 and 100 pmol) elicited greater increases of MAP and HR when injected in the VTA, and the cardiovascular response was blocked by R-820, SCH23390 and haloperidol. VTA-injected SB222200 prevented the pressor response to i.c.v. senktide, and vice versa, i.c.v. senktide prevented the anti-hypertension to VTA SB222200. Destruction of the VTA prevented the pressor response to i.c.v. senktide and the anti-hypertension to i.c.v. R-820., Conclusions and Implications: The NK(3)R in the VTA is implicated in the maintenance of hypertension by increasing midbrain dopaminergic transmission in SHR. Hence, this receptor may represent a therapeutic target in the treatment of hypertension., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

Several health benefits, including protection from tumors at various anatomic sites, such as the lungs, stomach, and prostate gland, have been attributed to tomatoes and tomato-based products. Among tomato carotenoids, lycopene is the most active antioxidant, although it has many other biological effects, but data on its antimutagenic effects are scarce and often discrepant. The aim of our work was to determine the protective effects of lycopene, with regard to mutagenicity, via two indirect mutagens/carcinogens-2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and aflatoxin B₁ (AFB₁) and the direct mutagen/carcinogen N-nitroso-N-methylurea (MNU)--using the Ames and micronucleus tests. The significant, dose-dependent, antimutagenic effects of two concentrations of lycopene (30 μg and 300 μg per plate) were demonstrated at various concentrations of both AFB₁ and IQ in two strains of Salmonella typhimurium (TA98 and TA100). The protective effects of lycopene relative to MNU were lower in comparison to its protective effects relative to AFB₁ and IQ. Mice treated for 3 days with different doses of lycopene (either 25 or 50 mg/kg of body weight) prior to administration of individual mutagens resulted in a significant reduction of micronuclei numbers in the micronucleus test. Tomato purée (tested using the Ames test and AFB(1)) revealed a much stronger, dose-dependent, antimutagenic effect compared with corresponding doses of pure lycopene. Results indicate that lycopene has antimutagenic effects, although the effects are lower than that of tomato purée, which contains a complex mixture of bioactive phytochemicals. The antimutagenic effect is connected with the chemoprotective role of lycopene, tomatoes, and tomato products in the prevention of carcinogenesis.

ARQ-197 is an oral, selective c-Met inhibitor under development by ArQule Inc, in partnership with Daiichi Sankyo Co Ltd and Asian licensee Kyowa Hakko Kirin Co Ltd, for the potential treatment of solid tumors, including NSCLC, hepatocellular carcinoma and pancreatic cancer, as well as microphthalmia transcription factor-driven tumors. c-Met, a key cell surface receptor tyrosine kinase involved in diverse regulatory functions, is often aberrantly activated in human cancers. While the precise mechanism of action of ARQ-197 remains undefined, data from preclinical studies have demonstrated that ARQ-197 inhibits c-Met activation in numerous human tumor cell lines and specifically targets c-Met in various cancer types; uniquely, ARQ-197 inhibits c-Met in a non-ATP-competitive manner. Phase I/II clinical trials demonstrated promise in terms of both tolerability and tumor response. Intriguingly, dose-limiting adverse effects were hematological in nature. Combinational trials are also ongoing to take advantage of the signaling crosstalk between c-Met and other oncogenic signaling systems. Prioritization of the clinical development of c-Met inhibitors, such as ARQ-197, among different tumor disease types is a key challenge at present; an improved understanding of the prediction of molecular determinants in tumors with respect to c-Met kinase as the driver oncogenic receptor, and of the prediction of tumor response, is still urgently needed.

A previous report has shown that mosquito sterol carrier protein-2 inhibitors (SCPIs) are larvicidal to larvae of the yellowfever mosquito, Aedes aegypti (L.) (J. Lipid Res. 46: 650-657, 2005). In the current study, we tested SCPI-1 in an additional four mosquito species for larvicidal activities: Culex pipiens pipiens, Anopheles gambiae, Culex restuans, and Aedes vexans. Cholesterol accumulation in SCPI-treated Ae. aegypti fourth instars was examined. SCPI-1 is lethal to all tested mosquito species, with the LC50 value ranging from 5.2 to 15 microM when treatments started at the first to third instar. However, LC50 values increase to from 5.2 to 38.7 microM in treatments started at first and fourth instar, respectively. The results indicate that the lethal effect of SCPI-1 decreases with the growth of larvae, which suggests that SCPI-1 is more effective before the larvae reach final growth period (the last instar). SCPI-1 suppressed cholesterol uptake in Ae. aegypti fourth instars, suggesting that one of the modes of action of SCPI-1 is via reduction in cholesterol absorption.

Studies have indicated that protective effects of statins (HMG-CoA reductase inhibitor) are associated with the regulation of endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS) in heart and liver diseases. Statins have been reported to enhance hepatic NO production and decrease the vascular tone in patients with cirrhosis. However, it is unclear which NOS contributes to the increased NO production. We hypothesized that statins are involved in the up-regulation of iNOS in inflammatory liver, resulting in decreased hepatic resistance. Primary cultured rat hepatocytes were treated with pro-inflammatory cytokine interleukin (IL)-1beta in the presence or absence of pitavastatin. Pretreatment of cells with pitavastatin resulted in up-regulation of iNOS induction by IL-1beta, followed by increased NO production. Pitavastatin had no effects on the degradation of IkappaB or activation of NF-kappaB. However, pitavastatin super-induced the up-regulation of type I IL-1 receptor (IL-1RI), which is essential for iNOS induction in addition to the IkappaB/NF-kappaB pathway. Mevalonate and geranylgeranylpyrophosphate blocked the stimulatory effects of pitavastatin on iNOS and IL-1RI induction. Transfection experiments revealed that pitavastatin increased the stability of iNOS mRNA rather than its promoter transactivation. In support of this observation, pitavastatin increased the antisense-transcript corresponding to the 3'-UTR of iNOS mRNA, which stabilizes iNOS mRNA by interacting with the 3'-UTR- and RNA-binding proteins. These findings demonstrate that pitavastatin up-regulates iNOS by the stabilization of its mRNA, presumably through the super-induction of IL-1RI and antisense-transcript. This implies that statins may contribute to a novel potentiated treatment in liver injuries including cirrhosis.

Background: The long duration of the current tuberculosis (TB) treatment is demanding and warrants the development of new drugs. Moxifloxacin shows promising results and may be combined with rifampicin to shorten the duration of TB treatment. Rifampicin induces the phase II metabolic enzymes that are involved in the biotransformation of moxifloxacin. Therefore, the interaction between rifampicin and moxifloxacin should be investigated., Patients and Methods: Nineteen Indonesian patients with pulmonary TB who were in the last month of their TB treatment completed a 1-arm, 2-period, fixed-order pharmacokinetic study. In phase 1 of the study, they received 400 mg of moxifloxacin every day for 5 days in addition to 450 mg of rifampicin and 600 mg of isoniazid 3 times per week. In phase 2 of the study, after a 1-month washout period, patients received moxifloxacin for another 5 days (without rifampicin and isoniazid). A 24-h pharmacokinetic curve for moxifloxacin was recorded on the last day of both study periods, and its pharmacokinetic parameters were evaluated for an interaction with rifampicin, using a bioequivalence approach., Results: Coadministration of moxifloxacin with rifampicin and isoniazid resulted in an almost uniform decrease in moxifloxacin exposure (in 18 of 19 patients). The geometric means for the ratio of phase 1 area under the curve to phase 2 area under the curve and for the ratio of phase 1 peak plasma concentration to phase 2 peak plasma concentration were 0.69 (90% confidence interval, 0.65-0.74) and 0.68 (90% confidence interval, 0.64-0.73), respectively. The median time to reach peak plasma concentration for moxifloxacin was prolonged from 1 h to 2.5 h when combined with rifampicin and isoniazid (P=.003)., Conclusions: Coadministration of moxifloxacin with intermittently administered rifampicin and isoniazid results in reduced moxifloxacin plasma concentrations, which is most likely the result of induced glucuronidation or sulphation by rifampicin. Further studies are warranted to evaluate the impact of the interaction on the outcome of TB treatment.

The monkey is often used to predict metabolism of drugs in humans since it generally shows a metabolic pattern similar to humans. However, metabolic profiles different from humans are occasionally seen in monkeys for some drugs including pitavastatin. Recently, we have successfully identified a monkey-specific cytochrome P450 (CYP) 2C76, which possibly accounts for a species difference between monkeys and humans because of its sequence and functional uniqueness. The present study on the role of CYP2C76 and other monkey CYP2Cs in pitavastatin metabolism, as an example, has revealed that CYP2C76 is important for the metabolism of the lactone form, indicating a major role of CYP2C76 for the difference in the metabolism of pitavastatin and possibly other drugs between monkeys and humans. The current investigation on the involvement of CYP2C76 in the metabolism of other drugs is expected to reveal further the further importance of this monkey-specific drug-metabolizing enzyme.

A role of ATP in nonadrenergic, noncholinergic (NANC) relaxations was examined in the Wistar rat jejunum. Electrical field stimulation (EFS) induced NANC relaxation of longitudinal muscle of the jejunal segments in a frequency-dependent manner. A purinoceptor antagonist, adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, 100 muM) inhibited the relaxation: relaxations induced by EFS at lower or higher frequencies were either completely or partially inhibited, respectively. After the jejunal segments had been desensitized to ATP, the relaxations were decreased to the same extent as those inhibited by A3P5PS. An inhibitor of small conductance Ca(2+)-activated K(+) channels (SK channels), apamin (100 nM), completely inhibited EFS-induced relaxations. Treatment of the segments with an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, thapsigargin (1 muM), significantly inhibited the relaxations. The exogenous ATP-induced relaxation of longitudinal muscle occurred with a concomitant decrease in intracellular Ca(2+) levels. Apamin and thapsigargin abolished these ATP-induced responses. A3P5PS significantly inhibited the inhibitory junction potentials which were induced in the longitudinal muscle cells. In addition, apamin significantly inhibited the hyperpolarization that was induced by exogenous ATP in the cells. These findings in the Wistar rat jejunum suggest that ATP participates in the NANC relaxation via activation of SK channels induced by Ca(2+) ions that are released from the thapsigargin-sensitive store site.

Aim: To investigate the possibility whether THPB-18 (l-12-shloroscoulerine) possesses the D1 agonist-D2 antagonist action on meso-accumbens-mPFC DA system., Methods: Single unit spontaneous firing activity was recorded in the nucleus accumbens (NAc) neurons of naive and unilateral-6-hydroxydopamine (6-OHDA)-lesioned Sprague-Dawley rats. The effects of drugs applied intravenously or iontophoretically were determined by the change of firing rates., Results: Under normal conditions, the systemic administration of THPB-18 produced a decrease-increase biphasic firing pattern in the NAc neurons during cumulative doses. High dose of THPB-18 was capable of reversing the inhibition induced by both D2 agonist LY171555 and D1/D2 agonist APO on NAc firing activity. Spiperone pretreatment could not block the high dose of THPB-18-induced firing rate increase, which was reversed by the D1 selective antagonist SCH23390. The tested NAc neurons were effectively inhibited by iontophoretically applied THPB-18 in 90 % of 6-OHDA-lesioned rats, while THPB-18 caused variable effects on the firing of NAc neurons in the neurons of unlesioned rats. The inhibitory effect of THPB-18 was blocked by iontophoretic application of SCH23390, but not D2 antagonist spiperone., Conclusion: Similar to L-stepholidine, THPB-18 also possesses the D1 agonistic-D2 antagonistic dual action on the VTA-NAc DA system.

Background: The effect of pitavastatin on the mRNA levels of apolipoprotein (apo) A-I, peroxisome proliferator-activated receptor alpha (PPARalpha), cholesterol 7alpha-hydroxylase (CYP7A1), and farnesoid X receptor (FXR) in HepG2 cells was examined to establish whether pitavastatin affects bile acid synthesis and if so, to determine a possible molecular mechanism., Methods and Results: HepG2 cells were cultured in serum-free Dulbecco's modified Eagle medium for 18 h before drug treatment. Total RNA was extracted at set times and mRNA levels were quantified by reverse transcription-real time polymerase chain reaction. Pitavastatin at 0.1, 1, 5, and 10 micromol/L increased the mRNA levels of apo A-I, PPARalpha, CYP7A1, and FXR in a dose-dependent manner. The mRNA levels of apo A-I, PPAR alpha, CYP7A1, and FXR similarly increased with increasing doses of pitavastatin. Coincubation of mevalonate (4 mmol/L) with pitavastatin (5 micromol/L) reversed the inductive effects of pitavastatin on the mRNA levels of these genes, indicating that the inductive effects of pitavastatin were related to its inhibition of HMG-CoA reductase., Conclusions: Pitavastatin increased the mRNA levels of CYP7A1 in HepG2 cells, suggesting that increased conversion of cholesterol to bile acids may be the mechanism for its potent low-density lipoprotein cholesterol-lowering effects.

The noncompetitive NMDA receptor antagonist MK801 (dizocilpine) produces behavioral stimulation mediated, in part, through indirect activation of the dopamine (DA) system. Previous reports indicate that D2/3 agonists inhibit MK801-induced stereotypies; however, it is unclear if these agonists also attenuate MK801-induced locomotion. As such, the ability of the D2/3 agonists, quinelorane and quinpirole, and the partial D3 agonist, BP897, to attenuate the locomotor activating effects of MK801 was examined in mice. MK801 (0.1-1.0 mg/kg) produced a biphasic effect on total distance traveled with the intermediate dose of 0.3 mg/kg producing the greatest stimulation. The increase in MK801-induced total distance traveled was attenuated by the coadministration of quinelorane and quinpirole at doses that alone had no effect on activity. Similarly, the partial D3 agonist, BP897, blocked the effects of MK801. The D3-preferring antagonist, nafadotride, reversed the attenuation of quinelorane and partially reversed the attenuation of quinpirole. The D2-preferring antagonist, eticlopride, reversed the attenuating effects of quinelorane, but was not effective against quinpirole. Nafadotride and eticlopride were ineffective against the attenuating effects of BP897 on MK801-induced locomotion. Because BP897 is a partial agonist it was tested against quinelorane/MK801 and quinpirole/MK801 combinations. BP897 reversed the attenuating effects of quinelorane, but not those of quinpirole on MK801's effects. These results demonstrate that the DA system, through D2/3 receptor activation, modulates the locomotor activating effects produced by noncompetitive NMDA receptor blockade.

This article reviews the precautions and adverse effects associated with vesnarinone use, and the potential mechanisms responsible for these complications as well as suggested treatment strategies. Vesnarinone, a quinolinone derivative, improves the haemodynamics and quality of life in patients with congestive heart failure (CHF); however, it is associated with the adverse effects of increased sudden cardiac death and neutropenia. These adverse effects have limited the application of vesnarinone to the general population but perhaps with continued research into vesnarinone-induced neutropenia and advances in arrhythmia management, the risk/ benefit ratio of vesnarinone may become favourable. For now, the use of vesnarinone should be limited to patients with CHF who have demonstrated a poor response to other cardiac medications and devices. These patients should be closely monitored for both cardiac and non-cardiac adverse effects.

A series of studies was conducted to determine the best animal model for human CYP and UGT activities. The investigation focused primarily on the interactions occurring in the CYP- or UGT-mediated metabolism of pitavastatin, and involved in vitro and in vivo experiments. We found that the best animal models for human CYP-mediated hydroxylation and UGT-mediated lactonization of pitavastatin were rats and dogs, respectively. In addition, a large difference in the metabolic properties of pitavastatin was found between monkeys and humans. In the presence of gemfibrozil, the CYP- or UGT-mediated metabolism of pitavastatin was inhibited in vitro. However, gemfibrozil treatment had no inhibitory effect on the AUC of pitavastatin and its lactone form in rats and dogs. We conclude that the plasma level of pitavastatin would not be increased by co-administration of gemfibrozil in humans.

To gain a better understanding of the metabolic stability and transport of pitavastatin (CAS 147526-32-7), a new and potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, experiments were conducted using human hepatocytes and oocytes of Xenopus laevis expressing human organic anion transporting polypeptide-2 (OATP2), respectively. Almost the entire radioactivity was from the unchanged substance or lactone form in human hepatocytes, and the cytochrome P450 (CYP)-mediated metabolism of pitavastatin was negligible. The results suggested that CYPs are not critically involved in determining the metabolic fate of pitavastatin. The hepatic uptake of pitavastatin reached saturation with a Km of 2.99 +/- 0.79 micromol/L. Also, the uptake of pitavastatin was mediated by OATP2 expressed in oocytes with a Km of 5.53 +/- 1.70 micromol/L. These results indicated that OATP2 plays a major role in the distribution of pitavastatin in liver. Furthermore, to elucidate the increase in the plasma concentration of pitavastatin in a clinical setting, the inhibitory effect of ciclosporin (cyclosporin A, CAS 59865-13-3) on the uptake of pitavastatin was examined. The uptake of pitavastatin was inhibited in the presence of cyclosporin A and the apparent IC50 value was 2.91 +/- 0.78 micromol/L. This result may at least partly explain the drug-drug interaction between pitavastatin and cyclosporin A. In conclusion, the characterization of transporters needs to be taken into account to avoid transporter-mediated drug-drug interaction.

Background: The objective of this study was to evaluate whether addition of milk to green, black and decaffeinated black teas alters their antimutagenic activity., Materials and Methods: Two model mutagens were used, the direct-acting N'-methyl-N'-nitro-nitrosoguanidine (MNNG) and the indirect-acting 2-amino-3-methylimidazo-[4,5-flquinoline (IQ), and their mutagenic activity was determined in the Ames test, in the presence of tea and milk/tea mixtures. Solids from the milk/tea mixtures were removed by centrifugation and the supernatant analysed by HPLC for individual catechins and gallic acid in green tea, and thearubigins, theaflavins and flavonol glycosides in black teas., Results: Addition of skimmed milk to all the teas failed to influence their antimutagenic activity towards the two mutagens. Addition of milk to green tea resulted primarily in loss of (-)-epigallocatechin gallate whereas in the black teas it primarily reduced theaflavins levels., Conclusion: The antimutagenic activity of green and black teas is not modulated by the presence of skimmed milk, even at high concentrations.

To assess the effect of dietary sucrose on liver cytochrome P450 1A content and activity, male F344 rat weanlings were randomized into two diet groups for a period of 90 days. One group was fed a diet containing 65% of total calories from sucrose (HSD) while the other was fed standard lab chow (0% sucrose). Microsomal fractions from each of 10 animals in each group were used in Western immunoblot, mutagenesis and 7-alkoxyresorufin-0-deethylase (AROD) assays. No statistically significant difference in the mean quantity of liver CYP 1A2 was detected by Western blot analysis, while a significant decrease in mean liver CYP 1A1 was observed in the rats fed the HSD. Liver microsomal-dependent mutagenesis of two heterocyclic amines (HCAs), 2-amino-3,8-dimethyl-imadazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), in Salminella typhimurium TA98 was decreased in animals on the HSD compared to those on the control diet by 33% (p < 0.001) and 25% (p < 0.001), respectively. In addition, rats on the HSD had significantly decreased ethoxyresorufin-0-deethylase (EROD) and methoxyresorufin-0-deethylase (MROD) activity over a range of substrate concentrations. These results show that a HSD alters hepatic CYP 1A content and activity and suggests that the metabolism of substrates for this P450 subfamily may be significantly altered.

The effects of the flavonoids quercetin, apigenin and chrysin (10 microM) on the genetic toxicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo[a]pyrene (BaP) was investigated at sub-cytotoxic concentrations in Chinese hamster V79 cells expressing human or rat cytochromes P450. In V79 r1A2-NH and V79 h1A1-MZ cells, none of the flavonoids increased DNA strand breaks (SB) (measured by the Comet assay) or produced detectable DNA adducts (measured by 32P-post-labelling). Neither IQ nor BaP produced DNA damage in the absence of expressed CYP1A2 or CYP1A1, respectively. DNA damage measured as SB and DNA adducts was detectable in V79 r1A2-NH cells expressing rat CYP1A2 when treated with IQ (2.5-50 microM) and this was inhibited by quercetin. Likewise, DNA damage (SB and DNA adducts) was elevated in V79 h1A1-MZ cells expressing human CYP1A1 when treated with BaP (0.1-0.5 microM) and this was inhibited by chrysin and apigenin, but not by quercetin. The specificity of CYP1A1 inhibition by chrysin and apigenin and CYP1A2 inhibition by quercetin was confirmed by ethylresorufin O-deethylase assay.

Background: The cause and pathogenesis of chronic urticaria are still poorly understood. IgE-independent reactions, are common in adult patients with chronic urticaria, who have daily spontaneous occurrence of weals. H(1)-receptor antagonists (antihistamines) are the major class of therapeutic agents used in the management of urticaria and angioedema. Nevertheless, chronic urticaria is often difficult to treat and may not be controlled by antihistamines alone. It has been postulated that mediators other than histamine, such as kinins, prostaglandin and leukotrienes, may be responsible for some of the symptoms in urticaria which are not controlled by antihistamines. In this study, which was randomized double-blind, placebo-controlled, we compare the clinical efficacy and safety of montelukast (MT) 10 mg given once a day and cetirizine (CET) 10 mg given once a day with placebo (PLA), in the treatment of patients with chronic urticaria who have positive challenge to acetylsalicylic acid (ASA) and/or food additives., Patients and Methods: A group of 51 patients, ranging in age from 15 to 71 years, with chronic urticaria and positive challenge to food additives and/or ASA, participated in this study for a period of 4 weeks, starting from a 3-day run-in. The assessment of the efficacy was based on scores of daily urticaria symptoms., Results: MT significantly increased the percentage of symptom-free days for hive and itch. Analysis of frequency distribution of urticaria scores for each symptom gave similar results (MT vs. CET and MT vs. PLA, P < 0.001). The interference with sleep due to their skin condition was also lower in the group treated with MT (P < 0.001). In addition, the median number of days without the rescue medication was significantly higher in the MT group (24 days) than both the CET and the PLA groups (18 days, P < 0.001, and 20 days, P < 0.001, respectively). Finally, a low incidence of adverse events was observed in this study., Conclusion: The results of this comparative study demonstrate that montelukast orally administered once a day is very effective for the treatment of cutaneous symptoms in patients with chronic urticaria due to food additives and/or ASA.

Opioids have been shown to produce both an early and delayed phase of cardioprotection; however, the signaling pathways involved, particularly in the delayed response, have not been well defined. Therefore, we investigated the potential of BW373U86 (BW), a potent delta opioid agonist, to produce delayed cardioprotection and characterized the role of opioid receptors and oxygen-derived free radicals (OFRs) in this delayed response. All rats underwent 30 min of ischemia followed by 2 h of reperfusion. The rats were divided into four groups. First, rats were pretreated with selective opioid receptor antagonists or the antioxidant, 2-mercaptopropionyl glycine (2-MPG), in the presence of BW and allowed to recover for 24 h before the ischemia-reperfusion protocol. Second, rats were pretreated with BW, allowed to recover for 24 h, and subsequently treated with either opioid antagonists or 2-MPG, 10 min prior to the ischemia-reperfusion protocol. Third, rats underwent ischemic preconditioning (IPC) (1x5 min occlusion) both with and without 2-MPG to determine the role of OFRs in acute cardioprotection. Fourth, rats were pretreated with TAN-67, an opioid agonist known to signal through the delta1 opioid receptor in the presence and absence of 2-MPG. Control rats were injected with saline and allowed to recover for 24 h. BW produced a bell-shaped dose-related reduction in infarct size with a maximal reduction observed at 0.1 mg/kg v control (16+/-3%v 60+/-3%, P<0.001). Surprisingly, the delayed protection induced by BW was only partially blocked by pretreatment with the delta1-selective antagonist, BNTX; however, it was completely blocked by pretreatment with 2-MPG (47+/-5%, P<0.001). Only naloxone given acutely inhibited the protective effects of BW; however, at the dose used, 2-MPG partially reduced the protective effect of acute IPC. TAN-67 (0.1 mg/kg) also produced a significant reduction in infarct size compared to control (18+/-4%v 60+/-3%, P<0.001). This protection was blocked by pretreatment with 2-MPG (42+/-4%, P<0.001). These data suggest that BW and TAN-67 mediate delayed cardioprotection via a free radical mechanism that appears to be only partially dependent on delta opioid receptor stimulation. Furthermore, it is the early burst in OFRs that is crucial to initiating the protective effect., (Copyright 2001 Academic Press.)

Background: Leukotrienes have been implicated in the selective infiltration of eosinophils into the bronchial mucosa in asthma., Objective: We studied whether eosinophil transmigration through cultured human umbilical vein endothelial cells (HUVECs) can be blocked by a specific cysteinyl LT1-receptor-antagonist., Methods: Unstimulated and stimulated eosinophils from patients with asthma and normal controls were subjected to confluent human umbilical vein endothelial cell (HUVEC) monolayers separating the upper and lower chamber of Transwell culture plates. Unstimulated eosinophils or cells pre-incubated in the presence of the eosinophil activating cytokines GM-CSF or IL-13 were placed in the upper chambers while PAF, a potent chemoattractant factor for eosinophils, was added to the lower chamber. Migration of eosinophils was quantified by a beta-glucuronidase assay., Results: The assumption that eosinophils express CysLT1 (cysteinyl-leukotriene 1)-receptors was based on our demonstration of mRNA-expression for the CysLT-1-receptor by polymerase chain reaction on purified eosinophils. The chemotactic response to PAF was significantly reduced when eosinophils were pre-incubated with montelukast for 15 min. When eosinophils were pre-incubated with GM-CSF and/or IL-13, the migratory response to PAF was also significantly reduced by montelukast., Conclusion: From these data we conclude that the specific cysteinyl LT1-receptor antagonist montelukast can inhibit PAF-induced eosinophil transmigration through cultured HUVEC monolayers.

Background: Leukotriene receptor antagonists have demonstrated clinical benefits in chronic asthma studies of up to 3 months in duration. The effects of these agents over extended periods of time have not been reported., Objective: To describe the long-term effect of oral montelukast, a potent and specific cysteinyl leukotriene receptor antagonist, compared with inhaled corticosteroids in both adult and paediatric patients with chronic asthma., Methods: Male and female patients with chronic, stable asthma (adults aged 15-85 years, children aged 6-14 years), who had completed double-blind, placebo-controlled clinical studies, participated in three extension studies with oral montelukast taken once daily (10 mg tablet for adults, 5 mg chewable tablet for paediatric patients) or inhaled corticosteroids (beclomethasone 200 microg twice daily for adults, beclomethasone 100 microg or equivalent three times daily for children). A double-blind adult extension study was 37 weeks in duration; open-label adult extension studies were 156 (adults) and 112 (paediatric) weeks in duration. A total of 436, 374, and 245 patients entered these extension studies, respectively., Results: Treatment with both montelukast and inhaled corticosteroids resulted in improvement in multiple parameters of asthma control. Improvements in daytime symptom scores were generally comparable among treatment groups. No tachyphylaxis to the effects of montelukast was evident. In the adult open-label study, however, the effect of beclomethasone on mean forced expiratory volume in 1 second (FEV1) gradually decreased from start of the study to the end of the follow-up treatment period., Conclusion: Both montelukast and inhaled corticosteroids were effective in controlling mild to moderate chronic asthma; the relative effectiveness of montelukast and beclomethasone were similar in open-label conditions. The hypothesis, that clinical practice conditions (e.g., adherence) may have a significant impact on the effectiveness of these therapies, should be tested in future clinical trials.

1. It has been suggested that osteopontin promotes the development of atherosclerosis, especially under diabetic conditions. 2. In the present study, we found that NK-104, a new potent synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, reduced osteopontin expression both at protein and mRNA levels in cultured rat aortic smooth muscle cells. 3. The inhibitory effect of NK-104 was almost completely reversed by mevalonate, suggesting that mevalonate or its metabolites play important roles in the regulation of osteopontin expression. 4. Furthermore, oral administration of NK-104 (3 mg kg(-1) day(-1) for 7 days) effectively suppressed abnormally upregulated expression of osteopontin mRNA in the aorta and kidney of streptozotocin-induced diabetic rats. 5. These data support a notion that NK-104 is a suitable drug for the treatment of diabetic patients with hypercholesterolaemia.

The present study was designed to investigate the effect of a selective GABA(B) receptor agonist baclofen on the pain-like nociceptive behavior (scratching, biting and licking) induced by intrathecal (i.t.) injection of N-methyl-D-aspartate (NMDA) or (+)TAN-67, the enantiomorphs of 2-methyl-4aalpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha-octahydro-quinolino[2,3,3g]isoquinoline (TAN-67), in the mouse. NMDA (0.05-0.2 microg/mouse) given i.t. immediately caused nociception in a dose-dependent manner. The nociception was significantly antagonized by i.t. co-injection with dizocilipine (0.1-1.0 microg/mouse), a non-competitive NMDA receptor antagonist. I.t. co-injection with baclofen (37.5-150 ng/mouse) significantly reduced the NMDA-induced nociceptive behavior in a dose-dependent fashion. The inhibition produced by baclofen was completely reversed by a selective GABA(B) receptor antagonist 2-hydroxysaclofen (0.15 and 0.3 microg/mouse). An i.t. injection of (+)TAN-67 at doses of 3.75-15 microg/mouse elicited a long-lasting and a dose-related nociception. The nociceptive behavior induced by (+)TAN-67 given i.t. was markedly suppressed by i.t. co-injection with baclofen (3-30 ng/mouse), and the inhibitory effect of baclofen was prevented by i.t. injection of 2-hydroxysaclofen (1 and 3 microg/ mouse). In addition, the (+)TAN-67-induced nociception was also attenuated by i.t. co-injection with dizocilipine (0.1-1.0 microg/mouse). These results suggest that spinal GABA(B) receptors may be implicated in the expression of nociception elicited by i.t. injection of either NMDA or (+)TAN-67 in the mouse.

Heterocyclic amines (HCA's) are promutagens produced by high temperature cooking of meat products and are activated by cytochrome P450 (CYP) lA2. Using Aroclor 1254 induced rat liver S9 we tested four citrus flavonoids diosmin, naringenin, naringin and rutin for their effects on the mutagenicity of HCA's MeIQx, Glu-P-1*, IQ and PhIP in Salmonella typhimurium TA98. The effects of the citrus flavonoids on CYPlA2 activity was determined by measuring demethylation of methoxyresorufin (MROD). MeIQx induced mutagenesis in S. typhimurium was significantly inhibited by all four flavonoids in a concentration dependent manner at 0.25, 0.5 and 1.0 mumole. Glu-P-1 induced mutagenesis was inhibited by rutin and naringenin. IQ induced mutagenesis was significantly inhibited by each flavonoid except diosmin at all three doses. With the exception of diosmin and naringin at 0.25 mumole all four flavonoids at all three doses significantly inhibited PhlP induced mutagenesis. The inhibition of MROD activity by the citrus flavonoids correlated best with the inhibition of MeIQx induced mutagenesis but also correlated with the inhibition of IQ induced mutagenesis except for diosmin and with the inhibition of PhlP induced mutagenesis except for the 0.25 mumole dose of diosmin and naringin. Our data suggest a chemopreventive potential for diosmin, naringin, naringenin and rutin towards CYPlA2 mediated mutagenesis of HCA's.

We examined the role of the sarcolemmal and mitochondrial ATP-sensitive potassium (K(ATP)) channel in a rat model of myocardial infarction after stimulation with the selective delta(1)-opioid receptor agonist TAN-67. Hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Infarct size was expressed as a percentage of the area at risk. TAN-67 significantly reduced infarct size/area at risk (29.6 +/- 3.3) versus control (63. 1 +/- 2.3). The sarcolemmal-selective K(ATP) channel antagonist HMR 1098, administered 10 min before TAN-67, did not significantly attenuate cardioprotection (26.0 +/- 7.3) at a dose (3 mg/kg) that had no effect in the absence of TAN-67 (56.3 +/- 4.3). Pretreatment with the mitochondrial selective antagonist 5-hydroxydecanoic acid (5-HD) 5 min before the 30-min occlusion completely abolished TAN-67-induced cardioprotection (54.3 +/- 2.7), but had no effect in the absence of TAN-67 (62.6 +/- 4.1), suggesting the involvement of the mitochondrial K(ATP) channel. Additionally, we examined the antiarrhythmic effects of TAN-67 in the presence or absence of 5-HD and HMR 1098 during 30 min of ischemia. Control animals had an average arrhythmia score of 10.40 +/- 2.41. TAN-67 significantly reduced the arrhythmia score during 30 min of ischemia (2.38 +/- 0. 85). 5-HD and HMR 1098 in the absence of TAN-67 produced an insignificant decrease in the arrhythmia score (8.80 +/- 2.56 and 4. 20 +/- 1.07, respectively). 5-HD administration before TAN-67 treatment abolished its antiarrhythmic effect (4.71 +/- 1.11). However, HMR 1098 did not abolish TAN-67-induced protection against arrhythmias (1.67 +/- 0.80). These data suggest that delta(1)-opioid receptor stimulation is cardioprotective against myocardial ischemia and sublethal arrhythmias and suggest a role for the mitochondrial K(ATP) channel in mediating these cardioprotective effects.

Vesnarinone, a synthetic quinolinone derivative used in the treatment of cardiac failure, exhibits immunomodulatory, anti-inflammatory, and cell growth regulatory properties. The mechanisms underlying these properties are not understood, but due to the critical role of nuclear transcription factor NF-kappa B in these responses, we hypothesized that vesnarinone must modulate NF-kappa B activation. We investigated the effect of vesnarinone on NF-kappa B activation induced by inflammatory agents. Vesnarinone blocked TNF-induced activation of NF-kappa B in a concentration- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of I kappa B alpha, an inhibitor of NF-kappa B. The effects of vesnarinone were not cell type specific, as it blocked TNF-induced NF-kappa B activation in a variety of cells. NF-kappa B-dependent reporter gene transcription activated by TNF was also suppressed by vesnarinone. The TNF-induced NF-kappa B activation cascade involving TNF receptor 1-TNF receptor associated death domain-TNF receptor associated factor 2 NF-kappa B-inducing kinase-IKK was interrupted at the TNF receptor associated factor 2 and NF-kappa B-inducing kinase sites by vesnarinone, thus suppressing NF-kappa B reporter gene expression. Vesnarinone also blocked NF-kappa B activation induced by several other inflammatory agents, inhibited the TNF-induced activation of transcription factor AP-1, and suppressed the TNF-induced activation of c-Jun N-terminal kinase and mitogen-activated protein kinase kinase. TNF-induced cytotoxicity, caspase activation, and lipid peroxidation were also abolished by vesnarinone. Overall, our results indicate that vesnarinone inhibits activation of NF-kappa B and AP-1 and their associated kinases. This may provide a molecular basis for vesnarinone's ability to suppress inflammation, immunomodulation, and growth regulation.

Biological actions of a novel non-peptide B2 receptor agonist, FR190997, were examined by comparing them with those of bradykinin. The paw edema was induced by subcutaneous injection of 30 microl of solution of bradykinin (0.3, 0.6, and 1.2 nmol) or FR190997 (0.1, 0.3, and 0.9 nmol) into the right hind paw of ICR male mice. Bradykinin caused a dose-dependent edema formation, which peaked at 15 min and ceased after 150 min. FR190997 also formed a dose-dependent edema, peaking at 15-30 min with a slight delay compared to bradykinin and this response continued over 200 min. The edema formed by bradykinin or FR190997 was inhibited by pretreatment with HOE140 (1 mg/kg) injected intraperitoneally 30 min before the injection of each agonist. A novel non-peptide B2 antagonist, FR173657 (30 mg/kg, i.p. 30 min before the agonist), also diminished these responses by bradykinin and FR190997 dose-dependently. Indomethacin (10 mg/kg, i.p. 30 min before) inhibited the response to FR190997, suggesting that release of prostaglandins induced by the B2 agonistic action might be involved in this inflammatory process induced by FR190997. The hypotensive action of FR190997 was also examined. Intravenously injected FR190997 caused the systemic hypotensive response in Sprague-Dawley male rats anesthetized with pentobarbital. The potency of FR190997 was weaker than that of bradykinin, when compared with the maximal hypotension. Duration of the hypotensive response of FR190997 was significantly longer than that of bradykinin. These results indicate that FR190997 has the B2 agonistic action similar to bradykinin and is also a good tool for in vivo examination of the B2 receptor.

Multiple drug resistance (MDR) mechanisms are known to limit the effectiveness of some cancer chemotherapies, probably through enhancing P-glycoprotein-mediated drug efflux from mammalian cells. Similar mechanisms appear to act in other organisms, including bacteria, and may affect not only the toxicity but also the mutagenicity of certain chemicals. At least in some experimental situations, MDR can be overcome through concomitant treatment of the cells with various types of inhibitors. Two MDR inhibitors, verapamil, a calcium channel blocker, and trifluoperazine, a calmodulin inhibitor, were assayed for their ability to modulate the potency of nine mutagens with varying mechanisms of action in various Salmonella typhimurium his- strains. Neither verapamil nor trifluoperazine affected the direct mutagenicity of sodium dichromate and 2-methoxy-6-chloro-9[3-(2-chloroethyl)amino-propyl-amino] dihydrochloride (ICR 191) or the S9-mediated mutagenicity of benzo[a]pyrene and 2-amino-3,4-dimethyl-amidazo[4,5-f]quinoline (MeIQ). Both modulators enhanced the direct mutagenicity of doxorubicin. Moreover, trifluoperazine sharply increased the S9-mediated mutagenicity of cyclophosphamide and 2-aminofluorene, while it consistently decreased the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The contrasting effect towards the aromatic amine 2-aminofluorene and the heterocyclic amine Trp-P-2, representative of important chemical families responsible for the bacterial mutagenicity of cigarette smoke, may explain the observed lack of influence of trifluoperazine on the mutagenicity of a cigarette smoke condensate. These observations extend the known range of chemical types whose mutagenicity can be modulated by inhibitors of MDR and suggest that there may be value in adding MDR inhibitors, especially trifluoperazine, to optimize the detection of mutagenicity by certain types of chemicals in the Salmonella/mammalian microsome mutagenicity test.

Possible mechanisms of antimutagenicity of various tea extracts (green, pouchong, oolong and black tea) toward 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo[a]pyrene (B[a]P) were investigated using a Salmonella/microsome assay. Tea extracts exhibited no inhibitory effects toward IQ and B[a]P in bio-antimutagenic assays, indicating that their antimutagenic activity is desmutagenic in nature. The mutagenicities of IQ and B[a]P decreased as the reaction periods of tea extracts with promutagens, S9 mix, or mutagen metabolites increased. The antimutagenicity of tea extracts toward IQ could be attributed (primarily) to an interaction between tea extracts and S9 mix. Apart from their interaction with S9 mix, tea extracts also exhibited antimutagenicity by markedly decreasing the mutagenicity of B[a]P metabolites. These results suggest that tea extracts: (1) inhibit the cytochrome P-450-mediated metabolism of IQ and B[a]P to their ultimate mutagenic metabolite form; and (2) interact with both promutagens and their metabolites in such a way as to reduce their mutagenic potentials. Therefore, the antimutagenic actions of tea extracts are due to a combination of the above distinctive mechanisms, and can vary with the type of mutagen under test.

The mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Salmonella typhimurium TA98 is inhibited by flavonoids with distinct structure-antimutagenicity relationships (Edenharder, R., I. von Petersdorff I. and R. Rauscher (1993). Antimutagenic effects of flavonoids, chalcones and structurally related compounds on the activity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and other heterocyclic amine mutagens from cooked food, Mutation Res., 287, 261-274). With respect to the mechanism(s) of antimutagenicity, the following results were obtained here. (1) 7-Methoxy- and 7-ethoxyresorufin-O-dealkylase activities in rat liver microsomes, linked to cytochrome P-450-dependent 1A1 and 1A2 monooxygenases catalyzing oxidation of IQ to N-hydroxy-IQ (N-OH-IQ), were effectively inhibited by 16 flavonoids (IC50: 0.4-9.8 microM). Flavones and flavonols are in general more potent enzyme inhibitors than flavanones, isoflavones, and chalcones. Among flavones the presence of hydroxyl or methoxyl groups resulted in minor changes only. However, among flavonols and flavanones the parent compounds exerted the strongest inhibitory effects, which decreased in dependence on number and position of hydroxyl functions. Contrary to the results obtained in the Salmonella assay in the tests with alkoxyresorufins no extraordinary counteracting effects of isoflavones, of hydroxyl groups at carbons 6 or 2' or of the elimination of ring B (benzylideneacetone) were detected. (2) No effects of flavonoids on NADPH-dependent cytochrome P-450 reductase activity could be detected. (3) The effects of 30 flavonoids on mutagenicity induced by N-OH-IQ in S. typhimurium TA98NR were again structure dependent. The most striking feature was the, in principle, reverse structure-antimutagenicity pattern as compared to IQ: non-polar compounds were inactive and a 50% inhibition was achieved only by some flavones and flavonols (IC50: 15.0-148 nmol/ml top agar). Within the flavone and flavonol subgroups inhibitory effects increased in dependence on number and position of hydroxyl functions. Isoflavones and flavanones, however, as well as glycosides, were inactive. Hydroxyl groups at carbons 7, 3', 4', and 5' generated antimutagenic compounds, a hydroxyl function at C5 was ineffective, but hydroxyls at C3 and 6 as well as methoxyl groups at C3' (isorhamnetin) or 4' (diosmetin) generated comutagenic compounds. 4. Cytosolic activation of IQ to mutagenic metabolites as determined by experiments with the hepatic S105 fraction comprises about 10% of the mutagenicity after activation by the combined microsomal and cytosolic fractions (S9). The pattern of inhibition as produced by 20 flavonoids was closely similar to that observed with the S9 fraction. 5. In various experiments designed for modulation of the mutagenic response, it could be shown that further mechanisms of flavonoid interaction with the overall mutagenic process may exist, such as interactions with biological membranes (luteolin, fisetin) and effects on fixation and expression of.DNA damage (flavone, fisetin).

The neuroprotective effects of U-101017, [7-chloro-5-[cis-3,5-dimethylpiperazine)carbonyl]-imidazole[1,5a]quinoli ne-3-carboxylate], a GABA(A) receptor partial agonist, were investigated in 3-acetylpyridine (3-AP) treated Wistar rats. A significant (P < 0.01) reduction in both cGMP and ATP in the cerebellum was observed at 96 h after treatment with 3-AP (500 micromol/kg i.p.). Oral administration of U-101017 before and after treatment with 3-AP significantly attenuated 3-AP-induced decreases in cGMP and ATP, and this effect was dose related. Consistent with the neurochemical effect, U-101017 prevented 3-AP-induced loss of motor coordination. Treatment with U-101017 partially, but significantly (P < 0.01) prevented the loss of inferior olivary neurons. U-101017 had no significant effect on body temperature. Thus, hypothermia was not involved in neuroprotective effects of U-101017. Co-administration of flumazenil with each treatment of U-101017 blocked the neuroprotective effect of U-101017, indicating that it mediated neuroprotection via the benzodiazepine binding sites on the GABA(A) receptor complex. Delayed administration of U-101017 at various time intervals after treatment with 3-AP demonstrated a significant neuroprotective effect even at 8 h, suggesting that this drug has a wide therapeutic window.

The antinociceptive potencies of the enantiomorphs of TAN-67 (2-methyl-4-alpha alpha-(3-hydroxyphenyl)-1,2,3,4,4 a 5, 12, 12 a alpha-octahydroquinolino[2,3,3,-g]isoquinoline), (-)-TAN-67 and (+)-TAN-67, given intracerebroventricularly (i.c.v.) on the antinociceptive response were studied in streptozotocin-induced diabetic mice using the tail-flick test. (-)-TAN-67 at doses of 3-10 micrograms given i.c.v. produced dose-dependent inhibition of the tail-flick response in both non-diabetic and diabetic mice. The antinociceptive effect of (-)-TAN-67 in the tail-flick test in diabetic mice was greater than that in non-diabetic mice. The antinociceptive effect of (-)-TAN-67 was not antagonized by pretreatment with either beta-funaltrexamine, a selective mu-opioid receptor antagonist, or nor-binaltorphimine, a selective kappa-opioid receptor antagonist. When 7-benzylidenenaltrexone, a selective delta 1-opioid receptor antagonist, was administered 10 min before treatment with (-)-TAN-67, the antinociceptive effect of (-)-TAN-67 was significantly antagonized. However, naltriben, a selective delta 2-opioid receptor antagonist, had no significant effect on the antinociceptive effect of (-)-TAN-67. On the other hand, in the tail-flick test. (+)-TAN-67 at doses of 3-30 micrograms given i.c.v. did not produce dose-dependent inhibition of the tail-flick response in either non-diabetic or diabetic mice. In conclusion, (-)-TAN-67, but not its enantiomer (+)-TAN-67, produced an antinociceptive effect through the activation of delta 1-opioid receptors.

Male F344 rats were exposed for 8 weeks to extracts of green tea (2% w/v) or black tea (1% w/v), or to 0.1% dietary indole-3-carbinol (I3C). In weeks 3 and 4 of the study, rats were given 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) every other day by oral gavage (50 mg/kg body wt) in order to induce aberrant crypt foci (ACF) in the colon. Compared with controls given IQ alone, all three inhibitors reduced the number of total aberrant crypts per colon, and green tea and I3C inhibited significantly the mean number of ACF (P < 0.05). Rats pre-treated with green tea, black tea, or I3C and given a single p.o. injection of 50 mg IQ/kg body wt 24-48 h before sacrifice had reduced levels of IQ-DNA adducts in the liver, and excreted lower amounts of IQ and other promutagens in the urine and feces. Inhibitors also reduced the excretion of IQ-sulfamate in the urine, but increased the relative amounts of IQ-5-O-sulfate and IQ-5-O-glucuronide. Western blotting together with assays for 7-ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase established that I3C preferentially induced cytochrome P4501A1 over 1A2, consistent with the altered profile of urinary metabolites. However, both teas caused slight induction of cytochrome P4501A2 versus 1A1, which would be predicted to enhance the activation of IQ. Thus, green tea and black tea are likely to protect against IQ-DNA adducts and ACF by mechanisms other than induction of cytochromes P450, such as inhibition of enzymes which activate IQ or the scavenging of reactive intermediates.

Grilled ground beef contains a number of heterocyclic amine carcinogens, such as 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), as well as anticarcinogenic conjugated linoleic acids (CLA). In the present study, CLA was administered to male F344 rats by gavage on alternating days in weeks 1-4, while IQ was given by gavage every other day in weeks 3 and 4 (100 mg/kg body wt). Rats were killed 6 h after the final carcinogen dose 16 in order to score colonic aberrant crypt foci (ACF). In the ACF study, CLA had no effect on the size of the foci, but inhibited significantly (P < 0.05) the number of ACF/colon, from 4.3 +/- 2.4 in controls to 1.1 +/- 1.3 in CLA-treated rats (mean +/- SD, n = 10). Rats given CLA also had significantly lower IQ-DNA adducts in the colon as determined by 32P-postlabeling analysis; relative adduct labeling levels (RAL x 10(7) for the major adduct were 9.13 +/- 2.6 in controls versus 5.42 +/- 1.8 in CLA-treated animals (P < 0.05). Mechanism studies indicated that CLA and other fatty acids interact with certain heterocyclic amines in a manner consistent with substrate-ligand binding. However, no such interaction occurred with IQ, and CLA failed to inhibit significantly the mutagenicity of N-hydroxy-IQ in the Salmonella assay. Liver microsomes from CLA-treated rats exhibited lower activities for dealkylation of 7-ethoxyresorufin and methoxyresorufin and activated IQ to DNA binding species less effectively than microsomes from control animals. Direct addition of CLA to the in vitro incubation inhibited IQ-DNA binding and was associated with increased recovery of unmetabolized parent compound. In the Salmonella assay, CLA inhibited the mutagenic activity of IQ in the presence of S9 or ram seminal vesicle microsomes. Collectively, these results support a mechanism involving inhibition of carcinogen activation by CLA, as opposed to direct interaction with the procarcinogen, scavenging of electrophiles or selective induction of phase I detoxification pathways.

This report describes a 1-year carcinogenicity bioassay in which male F344 rats received 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by gavage 2-3 times per week throughout the first 35 weeks of study, while 0.1% sodium/copper chlorophyllin (CHL) was given concomitantly in the drinking water. The carcinogen dose was calculated to provide an average exposure of 4.2 mg IQ/rat per day, equivalent to that reported in previous carcinogenicity study in which rats were given 0.03% IQ in the diet. Adjusted for length of follow-up, inhibition was highly significant for total tumor burden (P < 0.001), and for specific sites was significant in the small intestine and Zymbal's gland (P < or = 0.1), and in the liver (P < 0.01). In rats given CHL, the first onset of tumors in the Zymbal's gland was delayed from week 20 to 36, but was shortened from week 31 to 26 in the skin (P < 0.05). These results support a chemopreventive role for CHL in several of the major target organs for IQ tumorigenesis in the rat, but raise concerns over possible deleterious effects in the skin.

The antimutagenicity of 17 natural and synthetic anthraquinones was determined using Salmonella typhimurium TA98 against 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the presence of Aroclor 1254-induced rat hepatic S9. In general, the relationship between the chemical structures of anthraquinones and their antimutagenicity was found to contain one or more of the following features: (i) C9 carbonyl group, (ii) hydroxyl group at C1 and C4, (iii) C2 ethyl group, and (iv) C3 methyl group. The inhibitory effect of anthraquinones on 7-ethoxycoumarin O-deethylase (ECD) of Aroclor, 1254-induced hepatic microsomes was also examined. In addition, we studied the effect of anthraquinones on the metabolism of IQ by Aroclor 1254-induced microsomes using high-performance liquid chromatography. The antimutagenicity correlated with the inhibition of cytochrome P-450IA2-linked ECD activity in hepatic microsomes, and with the inhibition of N-hydroxy-IQ formation of IQ metabolism by hepatic microsomes. Moreover, we also examined the antimutagenicity of anthraquinones against synthetic N-hydroxy-IQ. Quinizarin and anthraflavic acid were shown to have more effect on the direct mutagenicity of N-hydroxy-IQ than that of the anthraquinones tested. This might explain why both anthraquinones showed higher antimutagenicity; although they inhibited ECD less. These results suggest that there exist at least two mechanisms of action in modifying roles of anthraquinones on the mutagenicity of IQ: (i) mediation through interaction with microsomal activating enzymes to inhibit the major active metabolite of N-hydroxy-IQ formation and (ii) direct interaction with the proximate metabolite of IQ, N-hydroxy-IQ, to block its attack on DNA.

The edible parts of 13 fruits--apples, apricots, bananas, blackberries, sweet cherries, red currants, white grapes, oranges, peaches, pears, plums, raspberries, and strawberries--and of 12 vegetables--asparagus, green beans, broccoli, brussels sprouts, red and white cabbage, carrots, cauliflower, onions, green peppers, spinach, and tomatoes--were squeezed in order to separate juices and residues. The residues were washed, lyophilized, and extracted sequentially with n-hexane, dichloromethane, acetone, and 2-propanol. Solvent extracted materials were tested in Salmonella typhymurium TA 98 for antimutagenicity against IQ and MeIQx. We found antimutagenic activities in 96% of the n-hexane extracts, 64% of the dichloromethane extracts, 44% of the acetone extracts, and 36% of the 2-propanol extracts. Since no or only minor differences were seen between the mutagens IQ and MeIQx investigations were continued with IQ only. Additional antimutagenic activities were detected in a total of 29.6% of extracts tested when an enzyme preparation with glycosidase-activities (fecalase) was included in the assay. These activities were found in originally inactive or less active dichloromethane, acetone, and 2-propanol extracts, and are therefore strongly suggestive for the liberation of antimutagenic aglycones from inactive glycosides. The existence of possibly a multitude of antimutagenic factors in fruits and vegetables was further substantiated by: (1) solvent partitioning of the n-hexane extracts of cauliflower, peaches, and spinach; (2) separation of the n-hexane and dichloromethane extracts of cauliflower, peaches, and spinach into acidid, neutral, and basic compounds; (3) chromatographic analysis of the n-hexane and dichloromethane extracts of spinach. Taken together, antimutagenic activities were present in 32 of 36 subfractions, corresponding to 88.9%. In the green vegetables beans, broccoli, and spinach the known antimutagen chlorophyll was proven to contribute considerably to antimutagenic potency. Other important contributions may be caused by various fibers: (I) antimutagenicity of fruit and vegetable solvent extracts was extensively heat stable; (II) heating surprisingly caused an increase of antimutagenic potencies or generated new antimutagenic activities in several solvent fractions, especially of broccoli, white and red cabbage. Indeed, mutagenicity induced by IQ was strongly reduced by lignin, weakly by alginic acid and pectin A, while cellulose, gum arabic, gum guar, and xylan were ineffective. With respect to the mechanisms of antimutagenicity binding of IQ by various fibers and inhibition of cytochrome P-450-dependent monooxygenases might be of major importance since no solvent fraction of any fruit or vegetable was able to reduce mutagenic activity induced by N-OH-IQ in S. typhimurium TA 98NR.

The antimutagenicity of dichloromethane extracts from eight amino acid/sugar model systems was determined using Salmonella typhimurium TA98 against 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) in the presence of Aroclor 1254-induced rat hepatic S9. The Maillard reaction products in the dichloromethane extracts were then quantified and qualified by capillary gas chromatography and gas chromatography-mass spectrometry, respectively. Pyrazines and furans were found to be the major Maillard reaction products yielded in the extracts. Moreover, the antimutagenicity of dichloromethane extracts correlated positively with the total amounts of pyrazines and furans. To elucidate the mechanism of antimutagenicity of dichloromethane extracts, the inhibitory effect of pyrazines on ethoxycoumarin deethylase activity in Aroclor 1254-induced hepatic microsomes was examined. We also studied the effects of pyrazines on IQ metabolism by Aroclor 1254-induced microsomes using high-performance liquid chromatography. The antimutagenicity of pyrazines correlated positively with both the inhibition of cytochrome P-450 IA2-linked ethoxycoumarin deethylase in hepatic microsomes and the inhibition of N-hydroxy-IQ formation from IQ metabolism by hepatic microsomes. Thus we concluded that pyrazines in dichloromethane extracts from eight amino acid/sugar model systems play an important role in the antimutagenicity of IQ. Moreover, we concluded that the modifying effect of pyrazines on the mutagenicity of IQ is mediated through interaction with microsomal activating enzymes to inhibit the major active metabolite in N-hydroxy-IQ formation.

The present study was undertaken in order to evaluate the in vitro and ex vivo antimutagenicity of anthracene against the food-borne carcinogen IQ (2-amino-3-methylimidazo[4,5-f]quinoline). Anthracene caused a marked, concentration-dependent decrease in the mutagenicity of IQ in the Ames test, whether hepatic S9 or isolated microsomes from Aroclor 1254-induced rats served as the activation systems. Anthracene gave rise to a concentration-dependent inhibition of the 0-deethylation of ethoxyresorufin, a diagnostic probe for CYP1A (cytochrome P450 family 1, subfamily A) activity, and of the metabolic activation of Glu-P-1 (2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, a diagnostic probe for CYP1A2. When microsomal metabolism of IQ was terminated by menadione, incorporation of anthracene into the incubation mixture once again inhibited the mutagenicity of IQ. All the above observations indicate that anthracene owes its antimutagenic response against IQ to: (a) inhibition of its CYP1A-mediated activation and (b) direct interaction between anthracene and the reactive intermediate(s) of IQ leading to their inactivation. Treatment of rats with anthracene did not greatly influence the ability of hepatic preparations to bioactivate IQ to mutagens. Similarly, administration of anthracene 2 h before sacrifice to Aroclor 1254-pretreated rats, did not modulate the hepatic activation of IQ. These findings demonstrate that the in vitro mechanisms of the antimutagenicity of anthracene are not operative in vivo, and further illustrate the inadequacy of in vitro studies, conducted in isolation, in predicting such effects.