Myeloperoxidase-mediated bioactivation of 5-hydroxythiabendazole: A possible mechanism of thiabendazole toxicity

Thiabendazole (TBZ), an antihelminthic and antifungal agent, is associated with a host of adverse effects including nephrotoxicity, hepatotoxicity, and teratogenicity. Bioactivation of the primary metabolite of TBZ, 5-hydroxythiabendazole, has been proposed to yield a reactive intermediate. Here we show that this reactive intermediate can be catalyzed by myeloperoxidase (MPO), a neutrophil-bourne peroxidase. Using a cell viability endpoint, we examined the toxicity of TBZ, 5OH-TBZ, and MPO-generated metabolites in cell-based models including primary rat proximal tubule epithelial cells, NRK-52E rat proximal tubule cells, and H9C2 rat myocardial cells. Timecourse experiments with MPO showed complete turnover of 5OH-TBZ within 15 min and a dramatic leftward shift in dose-response curves after 12 h. After a 24 h exposure in vitro, the LC₅₀ of this reactive intermediate was 23.3 ± 0.2 μM reduced from greater than 200 μM from 5OH-TBZ alone, an approximately 10-fold decrease. LC₅₀ values were equal in all cell types used. Comparison of lactate dehydrogenase leakage and caspase 3/7 activity revealed that cell death caused by the reactive intermediate is primarily associated with necrosis rather than apoptosis. This toxicity can be completely rescued via incubation with rutin, an inhibitor of MPO. These results suggest that MPO-mediated biotransformation of 5OH-TBZ yields a reactive intermediate which may play a role in TBZ-induced toxicity.     

Structure of 3-Acetyl-5-fluorouracil (5-FU): Implication for Its Rearrangements During Hydrolysis and upon Heating

Single-crystal X-ray diffraction data show that the 3-acetyl group in l,3-diacetyl-5-FU (FU = fluorouracil) is perpendicular to the plane of the 5-FU ring, while the 1-acetyl group is coplanar with the ring. Analyses of ¹H NMR and IR spectra provide evidence that the 1-and 3-acyl groups are in different electronic environments, which is consistent with the X-ray diffraction structure. 3-Acetyl-5-FU is thermally unstable, giving mainly l-acetyl-5-FU (80%) and 5-FU (20%) upon heating. The hydrolysis of 3-acyl derivatives of 5-FU showed a biexponential relationship between In concentration and time which had not been previously observed. The behavior of 3-acetyl-5-FU during hydrolysis can be explained by postulating its initial rapid equilibrium with an intermediate, 2-acetyl-5-FU, which subsequently hydrolyzes to 5-FU or rearranges to l-acetyl-5-FU, which hydrolyzes to 5-FU. The 2-acetyl intermediate was trapped by its reaction with formaldehyde. The formaldehyde adducts of the symmetrical 2-acetyl intermediate rearranged to yield equal amounts of 1- and 3-acetyloxymethyl-5-FU.     

Antioxidant and prooxidant effects of quercetin on glyceraldehyde-3-phosphate dehydrogenase.

Anti- and prooxidant properties of quercetin under different conditions were investigated using glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme containing essential cysteine residues. Quercetin was shown to produce hydrogen peroxide in aqueous solutions at pH 7.5, this resulting in the oxidation of the cysteine residues of the enzyme. Quercetin significantly increased oxidation of GAPDH observed in the presence of ferrous ions, particularly when FeSO(4) was added to the solution containing GAPDH and quercetin. The results suggest the formation of hydroxyl radical in the case of the addition of FeSO(4) to a quercetin solution. At the same time, quercetin protects GAPDH from oxidation in the presence of ascorbate and Fe(3+). In the absence of metals, quercetin protects SH-groups of GAPDH from oxidation by the superoxide anion generated by the system containing xanthine/xanthine oxidase.     

Toward a biologically based dose-response model for developmental toxicity of 5-fluorouracil in the rat: a mathematical construct.

Biologically based dose-response (BBDR) models comprise one way to incorporate mechanistic information into a dose-response assessment to be used for risk assessments. The chemotherapeutic drug 5-fluorouracil (5-FU) has been used as a prototypic compound for the construction of a BBDR model for developmental toxicity. Previous work has provided data and a general mechanistic framework for the developmental toxicity of 5-FU when it was administered to pregnant rats subcutaneously on gestation day 14. In this paper, a mathematical model relating maternally administered treatment with 5-FU to embryonal thymidylate synthetase inhibition and thymidylate synthetase inhibition to various measures of deoxyribonucleotide triphosphate (dNTP) pool perturbation is developed, and parameters are estimated using the data collected. The strategy used was to develop semi-empirical submodels for each of the intervening steps, and to estimate model parameters from previously described data. The models developed predict that there is no practical threshold for dNTP pool perturbation; that is, even minimal doses of 5-FU should result in some perturbation of dNTP pools. In particular, the relationship between dNTP pool perturbation and fetal weight deficit suggests that if there is a biological threshold for the effect of 5-FU on fetal weight, the responsible repair or compensation mechanism must be downstream of dNTP pool perturbation, and saturable at 5-FU doses lower than 10 mg/kg (the lowest dose examined for developmental effects in these studies).     

Modulation of 5-fluorouracil cytotoxicity through thymidylate synthase and NF-kappaB down-regulation and its application on the radiolabelled iododeoxyuridine therapy on human hepatoma cell

The inhibition of thymidylate synthase (TS) by 5-fluorouracil (5-FU) was known to increase the incorporation of radiolabelled iododeoxyuridine (IdUrd) into DNA. The relatively non-toxic compounds such as thiol-containing antioxidant pyrrolidinodithiocarbamte (PDTC) or aromatic fatty acid phenylbutyrate (PB) had been reported to enhance the cytotoxic efficacy of 5-FU. We designed a novel strategy through triplet combination of PB, PDTC and 5-FU to increase the radiolabelled IdUrd uptake and investigated the underlying mechanisms. The growth inhibition and [(125)I]IdUrd-DNA incorporation by PB, PDTC, 5-FU in different combinations were tested on parent or p21(Waf1) transfected Hep3B cells. The combination of PB and PDTC was more effective in enhancing 5-FU cytotoxicity than either drug alone. The combination of PB/PDTC and 5-FU blocked cells in S-phase and resulted in 8.5-fold increase of radiolabelled IdUrd-DNA incorporation. The transfection of p21(Waf1) did not change the general pattern of enhancement. Intriguingly, the combination of PB and PDTC effectively down-regulated NF-kappaB and TS and prevented their up-regulation from 5-FU treatment than either drug alone through a p21(Waf1)-independent mechanism. Based on this strategy, the 3-drug combination offered potential for improved radiolabelled IdUrd molecular radiotherapy for hepatoma treatment.     

8-OH-DPAT as a 5-HT7 agonist: phase shifts of the circadian biological clock through increases in cAMP production

Neurons in the suprachiasmatic nucleus (SCN), the site of the endogenous biological clock in mammals, fire spontaneously, peaking in firing rate near ZT6 or at the midpoint of the light phase in a 12:12 light-dark cycle. In rat hypothalamic slices, tissue incubations with drugs can produce a shift in this daily rhythm, the magnitude of which is dependent upon dose and the time of treatment. Previous work with 8-OH-DPAT had noted its ability to produce a phase advance, an earlier occurrence of the peak in neuronal firing, when applied at ZT6. Activation of 5-HT7 receptors was thought to be responsible for the shift, despite the clear preference of 8-OH-DPAT for 5-HT1A sites in terms of receptor binding affinity. In the present work, the actions of 8-OH-DPAT in SCN slices were confirmed and expanded to include additional dose-response and antagonist treatments. By itself, 8-OH-DPAT produced a concentration-dependent phase advance that was sensitive to co-application with 5-HT7 antagonists (ritanserin, mesulergine, SB-269970), but not to 5-HT1A antagonists (WAY-100,635, UH-301). Assignment of the receptor mechanisms for the antagonists employed was accomplished in experiments measuring binding affinities and the generation of cAMP, the latter monitored in a HEK-293 cell line expressing the r5-HT7 receptor and in tissue derived from rat SCN. The results indicate that the increases observed in cAMP levels are small but appear to be sufficient to produce a pharmacological resetting of the clock pacemaker. By aiding in the identification of the 5-HT receptor subtype responsible for the observed phase shifts and cAMP changes, 8-OH-DPAT represents an important pharmacological tool for 5-HT7 receptor activation, essentially broadening its role as the prototypical 5-HT1A agonist to one combining these two receptor activities.     

Specific expression of an A-kinase anchoring protein subtype,AKAP-150, and specific regulatory mechanism for Na(+),K(+)-ATPase via protein kinase A in the parotid gland among the three major salivary glands of the rat

We have examined the expression of A-kinase anchoring protein (AKAP) in the three major salivary glands, i.e. the parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG), of the rat to elucidate the functional relevance between saliva secretion and Na(+),K(+)-ATPase regulation by protein kinase A (PKA)-dependent phosphorylation, since an AKAP subtype, AKAP-150, is known to be involved in the regulation of the ATPase in PG. Although AKAP-150 and its mRNA were clearly detected in the PG, they were hardly detectable in either the SMG or SLG. The membrane-bound form of the RII regulatory subunit of PKA, an index for the total amount of AKAP subtypes and therefore of the anchored PKA holoenzyme, was also undetectable in membranes from the SMG and SLG but was found in the PG; though a substantial and comparable amount of Na(+),K(+)-ATPase was present in all of these membrane preparations. Incubation with [gamma-32P]ATP revealed that Na(+),K(+)-ATPase in the PG membranes was quickly phosphorylated upon the addition of cAMP, whereas the ATPases in the membranes from SMG and SLG were not; though they were readily and equally phosphorylated by the exogenously added PKA catalytic subunit. AKAP-150 in the basolateral membranes of PG acinar cells was co-immunoprecipitated with RII by an anti-RII antiserum; and AKAP-150 and Na(+),K(+)-ATPase were immunohistochemically co-localized predominantly on the basolateral membranes, suggesting a possibility that the ATPase might directly interact with the AKAP to form an ATPase/AKAP/PKA complex or associate with the AKAP, such association being mediated via some scaffolding molecule. Expression of AKAP-150 and quick down-regulation of Na(+),K(+)-ATPase by AKAP-anchored PKA in response to cAMP elevation are characteristics specific to PG among the three major salivary glands, suggesting the presence of PG-specific regulatory mechanisms for saliva production/secretion.     

Synergistic effect of 5-fluorouracil and N-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in a human mammary carcinoma

The biological effects of $\text{N-(}\text{phosphonacetyl}\text{)-}\text{l}\text{-}\text{aspartate}$ (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10^?7 to 1.5 × 10^?5M) and PALA (6.0 × 10^?5 to 3.6 × 10^?3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [³H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [¹⁴C]deoxyguanosine and [³H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10^?5M PALA and 1 × 10^?6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10^?6M [³H]-5-FU into poly(A)RNA in comparison to [³H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10^?5 M PALA and 1 × 10^?6 M [³H]-5-FU resulted in a 285 per cent increase in the incorporation of [³H]-5-FU into non-poly(A)RNA. The incorporation of either [¹⁴C]deoxyguanosine or [³H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [³H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.     

Methyl-beta-cyclodextrin enhances the susceptibility of human breast cancer cells to carboplatin and 5-fluorouracil: involvement of Akt, NF-kappaB and Bcl-2

The response rates of extensively used chemotherapeutic drugs, carboplatin (Carb) or 5-fluorouracil (5-FU) are relatively disappointing because of considerable side effects associated with their high-dose regimen. In the present study, we determined whether treatment with a cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), enhances the weak efficacy of low doses of Carb or 5-FU in human breast cancer cells. Data demonstrate that pretreatment with MCD significantly potentiates the cytotoxic activity of Carb and 5-FU in both MCF-7 and MDA-MB-231. Furthermore, we explored the molecular basis of enhanced cytotoxicity, and our data revealed that low-dose treatment with these drugs in MCD pretreated cells exhibited significantly decreased Akt phosphorylation, NF-kappaB activity and down-regulation in expression of anti-apoptotic protein Bcl-2. In addition, MCD pretreated cells demonstrated an increased intracellular drug accumulation as compared to cells treated with drugs alone. Taken together, our data provide the basis for potential therapeutic application of MCD in combination with other conventional cytotoxic drugs to facilitate reduction of drug dosage that offers a better chemotherapeutic approach with low toxicity.     

Tumor necrosis factor alpha partially contributes to lipopolysaccharide-induced downregulation of CYP3A in fetal liver: its repression by a low dose LPS pretreatment

With embryonic development, fetal hepatocytes gradually express various types of cytochromes P450 (CYPs) that play a key role in the detoxification of xenobiotics. In the present study, we showed that maternal lipopolysaccharide (LPS) exposure downregulated cyp3a11 mRNA and CYP3A protein in fetal liver. The increased level of TNF-alpha protein in fetal liver, transferred from either the maternal circulation or amniotic fluid, seems to be associated with LPS-induced downregulation of cyp3a11 mRNA and CYP3A protein in fetal liver. Interestingly, a low dose LPS (10mug/kg) pretreatment attenuated LPS-induced downregulation of cyp3a11 mRNA and CYP3A protein in fetal liver. Correspondingly, a low dose LPS pretreatment attenuated LPS-induced downregulation of pregnane X receptor (pxr) in fetal liver. Additional experiment showed that a low dose LPS pretreatment decreased the level of TNF-alpha in maternal serum and amniotic fluid and counteracted LPS-induced expression of TNF-alpha mRNA in maternal liver and placenta. Although a low dose LPS pretreatment alleviated LPS-induced increase in TNF-alpha in fetal liver, it had little effect on TNF-alpha mRNA in fetal liver. These results suggest that a low dose LPS pretreatment protects fetuses against LPS-induced downregulation of hepatic cyp3a11 and pxr expression through the repression of maternally sourced TNF-alpha production.     

Melatonin differentially modulates the expression and function of the hMT1 and hMT2 melatonin receptors upon prolonged withdrawal

Melatonin is synthesized and released following a circadian rhythm and reaches its highest blood levels during the night. It relays signals of darkness to target tissues involved in regulating circadian and seasonal rhythms. Here, we report the expression of human melatonin receptors type 1 and 2 (hMT(1) and hMT(2), respectively) in Chinese hamster ovary (CHO) cells following exposure to melatonin treatments mimicking the amplitude (400 pM) and duration (8 hr) of the nightly melatonin peak and upon withdrawal. Exposure of CHO-MT(1) cells to melatonin (400 pM) for 0.5, 1, 2, 4, and 8 hr significantly increased specific 2-[125I]iodomelatonin (500 pM) binding to hMT(1) melatonin receptors upon 16-hr withdrawal. However, the same treatment did not affect the expression of hMT(2) melatonin receptors. The increase in specific 2-[125I]iodomelatonin (500 pM) binding (162+/-29%, N=3, P<0.05) 16 hr after melatonin withdrawal was parallel to increases in hMT(1) melatonin receptor mRNA (231+/-33%, N=4, P<0.05). This effect was due to an increase in the total number of hMT(1) receptors [B(max) 833+/-97 fmol/mg protein (N=3), control; 1449+/-41 fmol/mg protein (N=3), treated], with no change in binding affinity. The melatonin-mediated increase in MT(1) melatonin receptor expression upon withdrawal was not mediated through either a direct effect of the hormone in the promoter's vector or in the rate of mRNA degradation. In conclusion, melatonin differentially regulates the expression of its own receptors, which may have important implications in the transduction of dark signals in vivo.     

Effects of Salviae Mitiorrhizae and Cortex Moutan extract on the rat heart after myocardial infarction: a proteomic study

In this study, we characterized the therapeutical effects of Salviae Mitiorrhizae (Danshen) and Cortex Moutan (Danpi) extract (SDD) on Sprague-Dawley rats subjected to coronary artery ligation, and applied proteomic approach to investigate its potential mechanism of action. The chemical composition of SDD was investigated by HPLC/MS(n) analysis. Measurement for serum levels of creatine kinase (CK), creatine kinase-MB (CK-MB), nitrite and histological study for infarct area of heart were performed. Moreover, protein abundance profiles of myocardium were compared by two-dimensional gel electrophoresis and altered proteins were identified by MALDI-TOF-MS. The results showed SDD significantly decreased CK, CK-MB concentration in serum and infarct area of heart, while increased the release of nitrite in rats with coronary occlusion. Increased concentration of ATP and total adenine nucleotide indicated the energy metabolism has been improved in ischemic heart induced by SDD. Proteomic data revealed that 23 proteins associated with energy metabolism, oxidative stress and cytoskeleton were modulated in SDD treated rats.     

Anti-cancer action of 4-iodo-3-nitrobenzamide in combination with buthionine sulfoximine: inactivation of poly(ADP-ribose) polymerase and tumor glycolysis and the appearance of a poly(ADP-ribose) polymerase protease

E-ras 20 tumorigenic malignant cells and CV-1 non-tumorigenic cells were treated with a drug combination of 4-iodo-3-nitrobenzamide (INO(2)BA) and buthionine sulfoximine (BSO). Growth inhibition of E-ras 20 cells by INO(2)BA was augmented 4-fold when cellular GSH content was diminished by BSO, but the growth rate of CV-1 cells was not affected by the drug combination. Analyses of the intracellular fate of the prodrug INO(2)BA revealed that in E-ras 20 cells about 50% of the intracellular reduced drug was covalently protein-bound, and this binding was dependent upon BSO, whereas in CV-1 cells BSO did not influence protein binding. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the protein that covalently binds the reduction product of INO(2)BA, which is 4-iodo-3-nitrosobenzamide. Since only the enzymatically reduced drug INOBA bound covalently to GAPDH, the BSO-dependent covalent protein-drug association indicated an apparent nitro-reductase activity present in E-ras 20 cells, but not in CV-1 cells, explaining the selective toxicity. Covalent binding of INOBA to GAPDH inactivated this enzyme in vitro; INO(2)BA+BSO also inactivated cellular glycolysis in E-ras 20 cells because it provided the precursor to the inhibitory species: INOBA. Another event that occurred in INO(2)BA+BSO-treated E-ras 20 cells was the progressive appearance of a poly(ADP-ribose) polymerase protease. This enzyme was partially purified and characterized by the polypeptide degradation product generated from PARP I, which exhibited a 50kDa mass. This pattern of proteolysis of PARP I is consistent with a drug-induced necrotic cell killing pathway.     

Tissue dependent differences in G-protein coupled receptor kinases associated with 5-HT4 receptor desensitization in the rat gastro-intestinal tract

Desensitization of 5-HT₄ receptors is regulated by G-protein coupled receptor kinases (GRKs). However, the specific GRK(s) that regulates the desensitization of 5-HT₄ receptors in the in vivo setting is unknown. We investigated the in situ expression of 5-HT₄ receptors and the GRKs in the rat gastrointestinal tract using immunohistochemistry and their interaction using coimmunoprecipitation. 5-HT₄ receptors were expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, myenteric plexus, circular muscle, submucosal plexus and muscularis mucosae of both the proximal and distal colon. GRK2 was expressed in longitudinal muscle and occasionally in myenteric plexus whilst GRK5 showed limited expression in the nerve endings of the myenteric plexus and submucosal plexus of the colon. GRK3 was expressed in the tunica muscularis mucosae of the oesophagus, circular muscle, submucosal plexus and muscularis mucosae of the colon. GRK6 was expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, circular muscle, and muscularis mucosae of the colon. Stimulation of tunica muscularis mucosae of the oesophagus and distal colon using the 5-HT₄ receptor agonist, tegaserod, followed by analysis of the 5-HT₄ receptor antibody immunoprecipitate, revealed the coimmunoprecipitation of GRK6 with 5-HT₄ receptors in the tunica muscularis mucosae of oesophagus while GRK2 and GRK6 were coimmunoprecipitated with 5-HT₄ receptors in the distal colon. This study indicates that GRK6 may be involved in the regulation of the desensitization of 5-HT₄ receptors in the rat oesophagus whilst GRK2 and GRK6 may be involved in regulation of the desensitization of 5-HT₄ receptors in the distal colon.     

Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 μM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 μM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to β-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.     

Comparative effects of the metabolic inhibitors 2,4-dinitrophenol and iodoacetate on mouse neuroblastoma cells in vitro

The toxic effects of two metabolic inhibitors, dinitrophenol and iodoacetic acid, were compared. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed to different concentrations of the toxic compounds for 24, 48 and 72 h to study basal toxicity effects (cell proliferation by quantification of total protein content (PR) and relative neutral red uptake (RNRU) by lysosomes). The following biochemical indicators assessed in the in vitro test system were: cytosolic phosphofructokinase (PFK) and enolase (ENL) activities in glycolysis; mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle; lysosomal β-galactosidase (GAL) activity; and neuronal acetylcholinesterase (AChE) activity. The effects of the two metabolic inhibitors on the various indicators differed. Iodoacetic acid was found to be far more toxic than dinitrophenol to neuroblastoma cell proliferation at 24 h exposure. Though 2,4-dinitrophenol and iodoacetic acid both inhibited cell proliferation of the neuroblastoma cells, their effects on the other endpoints were opposite. Dinitrophenol was a general activator of the metabolism, particularly affecting lysosomal function. Iodoacetic acid did not significantly alter general metabolism, but considerably modified lysosomal function and AChE activity. The modification of lysosomal function of Neuro-2a cells by the two compounds was quite different: dinitrophenol increased RNRU and GAL activity, and iodoacetic acid decreased both parameters.     

Positive allosteric modulators at GABAB receptors exert intrinsic actions and enhance the influence of baclofen on light-induced phase shifts of hamster circadian activity rhythms

Light-induced phase shifts of hamster circadian activity rhythms are modulated by GABA_B receptors. Recently, positive allosteric modulators (PAM)s at GABA_B receptors were described, but it is not known whether they affect light-induced entrainment of circadian rhythms. Therefore, we studied the effects of two GABA_B PAMs, GS39783 and RacBHFF, upon light-induced phase advances and delays of hamster circadian wheel-running activity rhythms. Wheel running activity was recorded for Syrian hamsters maintained in constant darkness. Drugs administered intraperitoneally were evaluated for their ability to modulate a light-induced shift of the circadian activity rhythm. Baclofen (3.75-15 mg/kg) dose-dependently inhibited both light-induced phase advances and delays of hamster wheel running rhythms, and its actions were blocked by the selective GABA_B antagonist, SCH50911 (5 mg/kg). Neither GS39783 (3-30 mg/kg) nor RacBHFF (0.63-10 mg/kg) affected phase advances when injected alone, but both GS39783 (3 mg/kg) and RacBHFF (10 mg/kg) augmented the inhibitory effect of baclofen (5 mg/kg). At doses above 3 mg/kg, GS39783 and RacBHFF significantly inhibited phase delays alone, consistent with the notion of “agonist-allosteric” properties. GS39783 (0.5 mg/kg), but not RacBHFF (10 mg/kg), augmented the inhibitory action of baclofen on phase delays. These data are consistent with the possibility that GS39783 and RacBHFF act as PAMs at GABA_B receptors inhibiting light-induced phase advances, yet that they also posses “allosteric agonist” actions at the (presumably separate) population of GABA_B receptors modulating light-induced phase delays. GABA_B receptors clearly warrant further investigation as agents for modulation of circadian dysfunction associated with CNS disorders such as depression.     

Monitoring Cyp2b10 mRNA expression at cessation of 2-year carcinogenesis bioassay in mouse liver provides evidence for a carcinogenic mechanism devoid of human relevance: the dalcetrapib experience

Introduction

Dalcetrapib is a cholesteryl ester transfer protein (CETP) modulator in clinical assessment for cardiovascular outcome benefits. In compliance with regulatory requirements, dalcetrapib was evaluated in rodent 2-year carcinogenesis bioassays. In the mouse bioassay, male mice demonstrated increased liver weight and statistically increased incidences of hepatocellular adenoma/carcinoma. Hepatic cytochrome p450 (Cyp) 2b10 mRNA induction and increased Cyp2b10 enzyme activity signify activation of hepatic nuclear receptor constitutive androstane receptor (CAR), a widely established promoter of rodent-specific hepatic tumors. We therefore monitored hepatic Cyp2b10 mRNA and its enzyme activity in a subset of dalcetrapib-treated male mice from the bioassay.

Methods

Liver samples were obtained from ~ 1/3 of male mice from each dose group including vehicle-controls (mean and earliest study day of death 678 and 459 respectively). Quantitative real time PCR (qRT-PCR) was performed to determine Cyp2b10 mRNA expression and Cyp1a-, Cyp2b10- and Cyp3a-selective activities were monitored.

Results

Cyp2b10 mRNA was strongly induced by dalcetrapib with an expected wide inter-individual variation (5-1421-fold). Group average fold-induction versus vehicle-controls showed a dose-related increase from 48-fold (250 mg/kg/day) to 160-fold (750 mg/kg/day), which declined slightly at 2000 mg/kg/day (97-fold). Cyp enzyme activities showed approximate doubling of total Cyp P450 content per milligram protein and a 9-fold increase in Cyp2b10-selective pentoxyresorufin O-dealkylase activity (750 mg/kg/day).

Discussion

These data from hepatic Cyp2b10 monitoring are strongly suggestive of CAR activation by dalcetrapib, a mechanism devoid of relevance towards hepatocarcinogenesis in humans; results show feasibility of Cyp2b10 as a surrogate marker for this mechanism at cessation of a carcinogenesis bioassay.