PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice.

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa2 (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

Inhibition of Connexin 36 attenuates HMGB1-mediated depressive-like behaviors induced by chronic unpredictable mild stress.

BACKGROUND: High mobility group box 1 (HMGB1) released by neurons and microglia was demonstrated to be an important mediator in depressive-like behaviors induced by chronic unpredictable mild stress (CUMS), which could lead to the imbalance of two different metabolic approaches in kynurenine pathway (KP), thus enhancing glutamate transmission and exacerbating depressive-like behaviors. Evidence showed that HMGB1 signaling might be regulated by Connexin (Cx) 36 in inflammatory diseases of central nervous system (CNS). Our study aimed to further explore the role of Cx36 in depressive-like behaviors and its relationship with HMGB1. METHODS: After 4-week chronic stress, behavioral tests were conducted to evaluate depressive-like behaviors, including sucrose preference test (SPT), tail suspension test (TST), forced swimming test (FST), and open field test (OFT). Western blot analysis and immunofluorescence staining were used to observe the expression and location of Cx36. Enzyme-linked immunosorbent assay (ELISA) was adopted to detect the concentrations of inflammatory cytokines. And the excitability and inward currents of hippocampal neurons were recorded by whole-cell patch clamping. RESULTS: The expression of Cx36 was significantly increased in hippocampal neurons of mice exposed to CUMS, while treatment with glycyrrhizinic acid (GZA) or quinine could both down-regulate Cx36 and alleviate depressive-like behaviors. The proinflammatory cytokines like HMGB1, tumor necrosis factor alpha (TNF-α), and interleukin-1ß (IL-1ß) were all elevated by CUMS, and application of GZA and quinine could decrease them. In addition, the enhanced excitability and inward currents of hippocampal neurons induced by lipopolysaccharide (LPS) could be reduced by either GZA or quinine. CONCLUSIONS: Inhibition of Cx36 in hippocampal neurons might attenuates HMGB1-mediated depressive-like behaviors induced by CUMS through down-regulation of the proinflammatory cytokines and reduction of the excitability and intracellular ion overload.

Erlotinib treatment induces cytochrome P450 3A activity in non-small cell lung cancer patients.

AIMS: Erlotinib is a tyrosine kinase inhibitor used in the treatment of non-small cell lung cancer highly metabolized by the cytochrome P450 (CYP) 3A. Hence, CYP3A4 activity might be a useful predictor of erlotinib pharmacokinetics in personalized medicine. The effect of erlotinib on CYP3A activity was therefore studied in non-small cell lung cancer patients. METHODS: The study included 32 patients scheduled for erlotinib monotherapy. CYP3A activity was assessed using quinine as a probe before and during erlotinib treatment. Plasma from blood samples drawn 16 hours post quinine administration were analysed using HPLC with fluorescence detection to determine the quinine/3-OH-quinine ratio. RESULTS: Matched samples, available from 13 patients, showed an induction of CYP3A activity (P = 0.003, Wilcoxon's signed rank test) after 2 months of treatment. The quinine/3-OH-quinine ratio decreased from 20.2 (± 13.4) at baseline to 11.0 (± 4.34). Single-point samples, available from 19 patients, supported the decrease in ratio (P = 0.007, Mann-Whitney U-test). Generally, females had a higher CYP3A activity both at baseline and after two months of treatment. Statistical analysis by gender also showed significant increase in CYP3A activity (males, n = 10, P = 0.001, and females, n = 22, P = 0.001). CONCLUSIONS: An induction of CYP3A activity was observed after 2 months of erlotinib treatment which was also seen when subdividing based on gender. It could be important to take this into consideration for patients co-administering other CYP3A-metabolizing drugs during erlotinib treatment and also makes it difficult to use baseline CYP3A activity to predict erlotinib pharmacokinetics.

Hen protein-derived peptides as the blockers of human bitter taste receptors T2R4, T2R7 and T2R14.

Bitter sensation is mediated by various bitter taste receptors (T2Rs), thus T2R antagonists are actively explored. Our objective was to look for novel T2R blockers in hen protein hydrolysate (HPH). We screened the least bitter HPH fractions using electronic tongue, and analyzed their peptide sequences and calcium mobilization in HEK293T cells expressing T2Rs. The results showed that the HPH fractions with higher bitterness intensity had higher hydrophobicity, more hydrophobic amino acids, and more positively charged peptides, but fewer known umami peptides. The peptide fractions from the least bitter HPH fraction significantly inhibited quinine bitterness (P < 0.05), and also significantly inhibited quinine- or diphenhydramine-dependent calcium mobilization of HEK293T cells expressing human T2R4, T2R7, or T2R14 (P < 0.05). Among them, the first eluted (least bitter) peptide fraction showed the strongest bitter-inhibitory effect. In conclusion, HPH peptides are the blockers of T2R4, T2R7, and T2R14.

Beef Protein-Derived Peptides as Bitter Taste Receptor T2R4 Blockers.

The aim of this work was to determine the T2R4 bitter taste receptor-blocking ability of enzymatic beef protein hydrolysates and identified peptide sequences. Beef protein was hydrolyzed with each of six commercial enzymes (alcalase, chymotrypsin, trypsin, pepsin, flavourzyme, and thermoase). Electronic tongue measurements showed that the hydrolysates had significantly ( p < 0.05) lower bitter scores than quinine. Addition of the hydrolysates to quinine led to reduced bitterness intensity of quinine with trypsin and pepsin hydrolysates being the most effective. Addition of the hydrolysates to HEK293T cells that heterologously express one of the bitter taste receptors (T2R4) showed alcalase, thermoase, pepsin, and trypsin hydrolysates as the most effective in reducing calcium mobilization. Eight peptides that were identified from the alcalase and chymotrypsin hydrolysates also suppressed quinine-dependent calcium release from T2R4 with AGDDAPRAVF and ETSARHL being the most effective. We conclude that short peptide lengths or the presence of multiple serine residues may not be desirable structural requirements for blocking quinine-dependent T2R4 activation.

Rituximab inhibits Kv1.3 channels in human B lymphoma cells via activation of FcγRIIB receptors.

Kv1.3 channels play an important role in modulating lymphocyte proliferation and apoptosis. We hypothesized that Kv1.3 channels in B lymphocytes might be regulated by rituximab, an antibody to CD20, a drug for treatments of B-cell lymphomas and autoimmune diseases. Using both whole-cell and cell-attached patch-clamp techniques, we found that rituximab inhibited Kv1.3 channels in Daudi human B lymphoma cells by promoting the channel inactivation at a concentration which was much greater than that required for activation of CD20. The effect of rituximab on Kv1.3 channels was abolished after selective blockade of FcγRIIB receptors with anti-FcγRIIB antibody. Western blot experiments showed that Daudi B cells expressed both Kv1.3 channel and the low affinity Fc receptor, FcγRIIB, which could be activated by the Fc region of rituximab. In contrast, normal lymphocytes expressed less Kv1.3 channels with faster inactivation. Confocal microscopy and flow cytometry data showed that rituximab induced apoptosis of Daudi B cells and that the effect was attenuated by blockade of FcγRIIB receptors and partially mimicked by inhibition of Kv1.3 channels. These results suggest that in addition to previously described complement-dependent cytotoxicity, rituximab also induces apoptosis of malignant B lymphocyte by stimulating FcγRIIB receptors and inhibiting Kv1.3 channels.

Dissecting the components of quinine accumulation in Plasmodium falciparum.

Although quinine, the active ingredient of chinchona bark, has been used in the treatment of malaria for several centuries, there is little information regarding the interactions of this drug with the human malaria parasite Plasmodium falciparum. To better understand quinine's mode of action and the mechanism underpinning reduced responsiveness, we have investigated the factors that contribute to quinine accumulation by parasites that differ in their susceptibility to quinine. Interestingly, passive distribution, in accordance with the intracellular pH gradients, and intracellular binding could account for only a small fraction of the high amount of quinine accumulated by the parasites investigated. The results of trans-stimulation kinetics suggest that high accumulation of quinine is brought about by a carrier-mediated import system. This import system seems to be weakened in parasites with reduced quinine susceptibility. Other data show that polymorphisms within PfCRT are causatively linked with an increased verapamil-sensitive quinine efflux that, depending on the genetic background, resulted in reduced quinine accumulation. The polymorphisms within PfMDR1 investigated did not affect quinine accumulation. Our data are consistent with the model that several factors, including acidotropic trapping, binding to intracellular sites and carrier-mediated import and export transport systems, contribute to steady-state intracellular quinine accumulation.

Phenotype-genotype variability in the human CYP3A locus as assessed by the probe drug quinine and analyses of variant CYP3A4 alleles.

The human cytochrome P450 3A (CYP3A) enzymes, which metabolize 50% of currently used therapeutic drugs, exhibit great interindividual differences in activity that have a major impact on drug treatment outcome, but hitherto no genetic background importantly contributing to this variation has been identified. In this study we show that CYP3A4 mRNA and hnRNA contents with a few exceptions vary in parallel in human liver, suggesting that mechanisms affecting CYP3A4 transcription, such as promoter polymorphisms, are relevant for interindividual differences in CYP3A4 expression. Tanzanian (n=143) healthy volunteers were phenotyped using quinine as a CYP3A probe and the results were used for association studies with CYP3A4 genotypes. Carriers of CYP3A4*1B had a significantly lower activity than those with CYP3A4*1 whereas no differences were seen for five other SNPs investigated. Nuclear proteins from the B16A2 hepatoma cells were found to bind with less affinity to the CYP3A4*1B element around -392 bp as compared to CYP3A4*1. The data indicate the existence of a genetic CYP3A4 polymorphism with functional importance for interindividual differences in enzyme expression.

Stereoselective transport of hydrophilic quaternary drugs by human MDR1 and rat Mdr1b P-glycoproteins.

1. The present study was performed to evaluate and compare the ability of human MDR1-, and rat Mdr1b- and Mdr2-P-glycoproteins to transport hydrophilic monoquaternary drugs. Transport studies were performed with plasma membrane vesicles isolated from MDR1-, Mdr1b-, or Mdr2-overexpressing insect cells. 2. As model substrates we used the N-methylated derivatives of the diastereomers quinidine and quinine, the monoquaternary compounds N-methylquinidine and N-methylquinine. Vincristine, an established MDR1 substrate, was used as a reference. 3. We observed ATP-dependent uptake of all drugs studied into MDR1- and Mdr1b-expressing vesicles. Mdr2 was not able to transport these compounds. MDR1- and Mdr1b-mediated transport was saturable, and could be inhibited by various drugs, including PSC-833. 4. For both MDR1 and Mdr1b the V(max)/K(m) ratios (or clearance) of N-methylquinidine were greater than those determined for N-methylquinine. This stereoselective difference was also evident from differential inhibitory studies with the two isomers. 5. Comparison of normalized clearance indicated that human MDR1 was more effective in transporting the tested substrates than rat Mdr1b. 6. In conclusion, our results demonstrate that MDR1 and Mdr1b, but not Mdr2, are able to transport the monoquaternary model drugs; both MDR1 and Mdr1b display stereospecificity for these cations; and indicate human MDR1 is more efficient in transporting these cations than its rat orthologue Mdr1b.

Amlodipine promotes kinin-mediated nitric oxide production in coronary microvessels of failing human hearts.

Recently, we found that amlodipine can release nitric oxide (NO) from canine coronary microvessels, which raises the question of whether amlodipine can also promote coronary NO production in failing human hearts. The goal of this study was to define the effect of amlodipine on NO production in failing human hearts and to determine the role of kinins in the control of NO production induced by amlodipine. Six explanted human hearts with end-stage heart failure were obtained immediately at transplant surgery. Coronary microvessels were isolated as previously described, and nitrite, the stable metabolite of NO in aqueous solution, was measured using the Griess Reaction. Amlodipine (10(-10) to 10(-5) mol/L) significantly increased nitrite production in coronary microvessels in a dose-dependent manner. The increase in nitrite in response to the highest dose of amlodipine (79%) was similar in magnitude to either that of the angiotensin-converting enzyme inhibitor ramiprilat (74%) or the neutral endopeptidase inhibitors phosphoramidon (61%) and thiorphan (72%). Interestingly, the increase in nitrite production induced by amlodipine was entirely abolished by N(omega)-nitro-L-arginine methyl ester and also HOE-140 (a bradykinin-2 antagonist) and dichloroisocoumarin (a serine protease inhibitor that blocks kallikrein activity). These results indicate that amlodipine can promote coronary NO production in failing human hearts and that this effect is dependent on a kinin-mediated mechanism.

Influence of substrate structure on substrate binding to the renal organic cation/H+ exchanger.

The carrier-mediated exchange of H+ for organic cations ("OC/H+ exchange") is the active step in OC secretion in renal proximal tubules. Although hydrophobicity is known to be an important criterion for binding of substrates to this transporter, the degree to which steric parameters of substrate structure influence binding to the exchanger is unclear. We examined this issue by measuring the inhibition of OC/H+ exchange produced by a group of quaternary ammonium compounds which share a common structural motif: an N1-pyridinium residue. Activity of the OC/H+ exchanger was determined by measuring transport of [14C]tetraethylammonium (TEA) in brush-border membrane vesicles (BBMV) from rabbit renal cortex. Transport was measured in the presence of a pH gradient (pHin 6.0; pHout 7.5) to maximize TEA/H+ exchange. Apparent inhibitory constants (Ki values) for each test agent were measured. The test agents included 4-phenylpyridiniums and 3-phenylpyridiniums, quinoliniums and acridiniums. The planar structure of these compounds permits a direct test of whether the presence of planar hydrophobic mass in different orientations relative to the pyridinium motif exerts a systematic effect on substrate binding to the OC/H+ exchanger. The hydrophobicity of each group of compounds was systematically varied by addition of different substituents at the quaternary nitrogen. Whereas decreases in Ki proved to be proportional to hydrophobicity, the position of the phenyl-ring substituent(s) had no effect on substrate interaction with the exchanger. The results led to the development of a preliminary quantitative structure-activity relationship (QSAR) correlating substrate hydrophobicity and substrate binding to the OC/H+ exchanger. This QSAR was used to predict the binding of 1-methyl-4-phenylpyridinium (MPP+), (+) and (-)nicotine, (+) and (-)ephedrine, quinine and quinidine to the OC/H+ exchanger. Molecular graphics representation of the 3D structures of the test agents was used to develop a working model of a hydrophobic, planar receptor surface on the OC/H+ exchanger against which substrates are suggested to interact during binding. Development of the QSAR and receptor surface model open the way to quantitative tests of the specific physical and structural determinants of substrate selectivity by the renal OC/H+ exchanger.

Metabolism of quinine in man: identification of a major metabolite, and effects of smoking and rifampicin pretreatment.

Our previous studies have shown that cigarette smoking and rifampicin pretreatment enhance the elimination of quinine, metabolism assumed, by analogy with quinidine, to be carried out by CYP3A (P450IIIA). This finding is unexpected since it has been shown that smoking induces the CYP1A rather than the CYP3A enzyme family, suggesting that the metabolism of quinine may be catalysed by CYP1A. Therefore, we conducted this study to identify possible quinine metabolites in human urine and to determine which metabolic pathway is induced by cigarette smoking and rifampicin pretreatment. A specific HPLC procedure was employed to identify metabolites of quinine in urine samples collected from healthy volunteers after an oral dose of 600 mg quinine sulphate. The results showed that there were at least seven possible metabolites of quinine detected in human urine. Three of these were identified as 2'-oxoquininone, quinine glucuronide and 3-hydroxyquinine. The 3-hydroxyquinine appeared to be a major metabolite of quinine in urine samples from every subject who took an oral dose of quinine. Although cigarette smoking and rifampicin pretreatment were shown to cause a marked increase in the elimination of quinine there were no significant changes in the formation of 3-hydroxyquinine as measured in the urine samples. This suggests that the effects of smoking and rifampicin are more complicated than we expected and require further investigation.

Drug distribution and binding to muscle in rat.

Drug binding to rat skeletal muscle homogenate was studied using quinidine and quinine as basic drugs, and furosemide and phenylbutazone as anionic drugs. Characteristically different binding fashions were observed among basic and anionic drugs. Quinidine and quinine which are stereoisomers bound to muscle not depending on drug concentration and showed similar binding ratios, while furosemide and phenylbutazone bound to muscle depending on drug concentration. Quinidine and quinine bound mainly to 1000 x g precipitates of muscle homogenate while furosemide and phenylbutazone bound exclusively to cytosol fraction. Binding to 1000 x g precipitates was not explained by binding to actin and myosin alone, while the second protein fraction eluted from cytosol by Sephadex G-75 gel filtration was found to have binding properties for furosemide.

In vitro cell perfusion. I. System for continuous observation of cells and analysis of perfusion fluid suitable for isolated mast cell and macrophage studies.

A system is described in which cells are perfused continuously with appropriate medium while simultaneously recording any change in cell morphology together with assay of released products (enzymes and mediators) following immunologic or non-immunologic perturbation. The released products in the perfusate are monitored continuously by automated fluorimetric assay procedures. The system is particularly applicable to investigate or toxicological studies of isolated mast cells or cultured macrophages.

Observations of gastric mucosal blood flow using 99Tcm in rat and man.

The 99Tcm clearance technique is shown to be a useful method of assessing gastric function. It is easily carried out and is relatively non-toxic--the dose of isotope could be reduced to 100 muCi or less without sacrificing the accuracy of the investigation. Although the results are preliminary and the number of cases investigated so far is small, they suggest that the measurement of pertechnetate clearance is directly related to gastric mucosal blood flow; it is a useful parameter of gastric function and may well prove to be a more accurate discriminant in cases of peptic ulceration than the conventional measurement of gastric acid secretion.