Urinary 6β-hydroxycortisol: a validated test for evaluating drug induction or drug inhibition mediated through CYP3A in humans and in animals

6beta-Hydroxycortisol (6beta-OHF) urinary excretion has, for a long time, been considered a marker of drug induction and, more recently, of drug inhibition in humans and in laboratory animals, but its specificity is still under debate. In this work, we review 277 papers devoted to 6beta-OHF urinary excretion. We have evaluated factors that could modify 6beta-OHF excretion and, thus, could explain contradictory results. We have examined the effect of the analytical techniques on physiological values. Intra- and inter-individual variability and the effect of circadian rhythms on urinary excretion of 6beta-OHF as well as cortisol and 17-hydroxycorticosteroids have been evaluated. We also give an overview of drugs that induce, inhibit or have no effect on 6beta-OHF. For inducing and inhibiting drugs, we calculated the ranges of variation of 6beta-OHF excretion from the results indicated in the different papers. This work was done for well-known inducers, such as anticonvulsants, but also for other inducing or inhibiting drugs found in the literature. The time-course of variation in 6beta-OHF excretion when different drugs are co-administered was also investigated. The potential relationship between cytochrome P(450) 3A4 (CYP3A4) polymorphism and 6beta-OHF excretion was studied. Finally, the interest of 6beta-OHF urinary excretion was compared with that of other tests proposed to measure CYP3A4 activity. This review demonstrates that 6beta-OHF urinary excretion is a good test to evaluate drug-metabolising enzyme inducing or inhibiting properties of drugs when the subjects are their own controls, but this test is not reliable enough to measure actual CYP3A4 activity.     

Effects of the neuroleptic α-flupenthixol on latent inhibition in aversively- and appetitively-motivated paradigms: evidence for dopamine-reinforcer interactions

Three experiments examined the influence of the dopamine (DA) D₁/D₂ receptor antagonist -flupenthixol on the latent inhibition (LI) effect. LI is a phenomenon which is manifest when non-reinforced pre-exposure to a stimulus retards subsequent conditioning to that stimulus, and has been proposed as an animal model of the selective attentional processes that are disrupted in acute schizophrenia. Experiment 1 extended previous findings that neuroleptics enhance the LI effect in conditioned suppression paradigms in rats to -flupenthixol (0.23 mg/kg). Experiment 2 demonstrated that this enhancement of the LI effect was also seen in a parallel appetitively-motivated conditioning paradigm at the same dose. In both Experiment 1 and Experiment 2, the enhancement of the LI effect by -flupenthixol appeared to be accompanied by a decrease in the impact of the reinforcer (be it appetitive or aversive). Experiment 3 investigated the possible role of the reinforcer in the effect of -flupenthixol on the LI effect in the aversive, conditioned suppression paradigm by increasing the intensity of footshock in rats treated with -flupenthixol. Increasing the intensity of the footshock completely abolished the enhancement of LI found following injection of -flupenthixol, a result which could not be attributed to a floor effect. The results provide no support for interpretations of the influence of DA manipulations on the LI effect that draw parallels with deficits in selective attention observed in acute schizophrenia.     

Presynaptic β2-adrenoceptors on the sympathetic nerve fibres of the human saphenous vein: no evidence for involvement in adrenaline-mediated positive feedback loop regulating noradrenergic transmission

Summary Spirally cut strips of human saphenous veins preincubated with ³H-noradrenaline were superfused in the presence of corticosterone and, unless stated otherwise, of cocaine or desipramine. Tritium overflow was stimulated electrically (2 Hz). Adrenaline (in the presence of rauwolscine), isoprenaline and the preferential ₂-adrenoceptor agonist procaterol concentration-dependently increased the electrically evoked tritium overflow. Prenalterol, a -adrenoceptor agonist with moderate preference for ₁-adrenoceptors, was ineffective. The concentration-response curve of isoprenaline was shifted to the right by the nonselective -adrenoceptor antagonist propranolol and by the preferential ₂-adrenoceptor antagonist ICI 118,551, but was not affected by the ₁-selective antagonist atenolol. In experiments on strips preexposed to adrenaline 10 nmol/l (i. e. a concentration higher than that which normally occurs in vivo) for 32 min in the absence of cocaine or desipramine, the electrically evoked ³H overflow was not affected 12 and 44 min after withdrawal of adrenaline, irrespective of whether propranolol was absent or present in the superfusion fluid. — In veins incubated with ³H-adrenaline, a considerable amount of the radioactivity was accumulated. During subsequent superfusion with ³H-adrenaline-free solution, electrical stimulation induced tritium overflow in a tetrodotoxin-sensitive manner. Propranolol failed to modify the evoked tritium overflow. — It is concluded that the sympathetic nerve fibres of the human saphenous vein are endowed with facilitatory presynaptic ₂-adrenoceptors. These receptors do not seem to play a substantial role in a local adrenaline (previously taken up)-mediated positive feedback loop regulating noradrenergic transmission, at least under the present in vitro conditions.This study was supported by a grant of the Deutsche Forschungsgemeinschaft Send offprint requests to M. Göthert     

Hesperidin attenuates learning and memory deficits in APP/PS1 mice through activation of Akt/Nrf2 signaling and inhibition of RAGE/NF-κB signaling

Numerous studies have demonstrated that oxidative stress and inflammation play a pivotal role in the pathophysiology of Alzheimer disease (AD). Hesperidin (HP) has various pharmacological effects including anti-oxidative, anti-inflammatory and neuroprotective properties. In this study, APP/PS1 mice were used to evaluate the neuroprotective effects of HP. We reported that intragastric administration of HP (40 mg/kg) for 90 days significantly attenuated cognitive impairment in APP/PS1 mice. HP treatment suppressed oxidative stress by reducing the levels of ROS, LPO, protein carbonyl and 8-OHdG and increasing the activity of HO-1, SOD, catalase, and GSH-Px. HP treatment also inhibited inflammation by decreasing the levels of TNF-α, C-reactive protein and MCP-1 and reducing the activity of NF-κB. Moreover, HP could reverse the decreased phosphorylation of Akt, the decreased phosphorylation of GSK-3β, the lessened Nrf2 and the reduced expression of HO-1. HP could also inhibit the increased the RAGE expression, the enhanced phosphorylation of IκBα, and the augmented nuclear translocation of NF-κB/p65 in cortex of APP/PS1 mice. Taken together, HP suppresses oxidative stress and inflammation via activation of Akt/Nrf2 signaling and inhibition of RAGE/NF-κB signaling and further confers neuroprotection.     

Knockdown of TMEM45A overcomes multidrug resistance and epithelial–mesenchymal transition in human colorectal cancer cells through inhibition of TGF-β signalling pathway

Colorectal cancer (CRC), a leading cause of cancer death, has recently been known as the most prevalent malignancy worldwide. Although chemotherapy is an important therapeutic option for CRC patients, multidrug resistance (MDR) still remains a major cause of chemotherapy failure. Transmembrane protein 45A (TMEM45A) has been found highly expressed in various cancers, and is also proposed as an interesting biomarker for chemoresistance. However, the association between TMEM45A and MDR in CRC remains unclear. This study aimed to investigate the key role of TMEM45A in CRC by knockdown of its expression in 5-FU-resistant CRC cells (HCT-8/5-FU and SW480/5-FU) and their parental cells (HCT-8 and SW480). Data showed that TMEM45A was significantly up-regulated in HCT-8/5-FU and SW480/5-FU cells in comparison with their parental HCT-8 and SW480 cells. Knockdown of TMEM45A enhanced 5-FU sensitivity and 5-FU-induced apoptosis in HCT-8/5-FU and SW480/5-FU cells. It was also found that inhibition of TMEM45A increased the intracellular accumulation of Rhodamine-123 and down-regulated the expression of MDR1 in HCT-8/5-FU and SW480/5-FU cells. In addition, knockdown of TMEM45A suppressed migration and invasion of HCT-8/5-FU and SW480/5-FU cells. Furthermore, knockdown of TMEM45A not only attenuated MDR-enhanced epithelial–mesenchymal transition (EMT), but also suppressed MDR-enhanced activation of the TGF-β signalling pathway in HCT-8/5-FU and SW480/5-FU cells. Taken together, our study suggests that knockdown of TMEM45A can effectively overcome MDR and inhibit EMT via suppression of the TGF-β signalling pathway in human CRC cells, and that targeting TMEM45A will be a potential strategy in the treatment of MDR in CRC.     

Opposite modulation of noradrenaline and ATP release in guinea-pig vas deferens through prejunctional β-adrenoceptors: evidence for the β2 subtype

Activation of prejunctional -adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of -adrenoceptor involved.In [³H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1–100 nM), salbutamol (0.01–1 M) and terbutaline (0.1–10 M) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the ₂-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 M). Isoprenaline (1–100 nM), salbutamol (0.01–1 M) and terbutaline (0.1–10 M) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin-and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained contraction elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the ₁-selective antagonist 1-[2-((3carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM).The results confirm the opposite -adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional -adrenoceptor is of the ₂ subtype.     

3-Deoxysappanchalcone inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 expression in human keratinocytes through activated protein-1 inhibition and nuclear factor-kappa B DNA binding activity

Tumor necrosis factor α (TNF-α), which is a primary cytokine responsible for inflammatory responses in skin, induces the synthesis of matrix metalloproteinase-9 (MMP-9), which causes skin aging. The protective effects of 3-deoxysappanchalcone against TNF-α-induced damage was investigated using human skin keratinocytes. The results showed that 3-deoxysappanchalcone inhibited MMP-9 expression at the protein and mRNA level, by blocking the activation of activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). Taken together, the inhibitory activity of 3-deoxysappanchalcone on MMP-9 expression and production in TNF-α-treated cells was found to be mediated by the suppression of AP-1 and NF-κB activation.     

Hypothesis-based weight of evidence: A tool for evaluating and communicating uncertainties and inconsistencies in the large body of evidence in proposing a carcinogenic mode of action—naphthalene as an example

Human health risk assessment consists of bringing to bear a large body of in vitro, animal, and epidemiologic studies on the question of whether environmental exposures to a substance are a potential risk to humans. The body of scientific information is typically less than definitive and often contains apparent contradictions. Often various possible conclusions about potential human risks may be drawn from the data and these may vary from very strong to tenuous. The task, therefore, is to communicate the uncertainties in the inferences from the data effectively, giving proper consideration to contrary data and alternative scientifically plausible interpretations. We propose an approach, Hypothesis-Based Weight of Evidence (HBWoE), to organize, evaluate, and communicate the large body of available relevant data on a given chemical, using naphthalene as an example. The goal for our use of the term “weight of evidence” (WoE) is broad in that we express the relative degrees of credence that should be placed in alternative possible interpretations of the naphthalene data and hypothesized carcinogenic modes of action (MoAs), expressed in a way that shows how such credence is tied to specific scientific interpretations, considering consistencies, inconsistencies, and contradictions within the data set.     

The lectin-like domain of TNF protects from listeriolysin-induced hyperpermeability in human pulmonary microvascular endothelial cells — A crucial role for protein kinase C-α inhibition

Listeriosis can lead to potentially lethal pulmonary complications in newborns and immune compromised patients, characterized by extensive permeability edema. Listeriolysin (LLO), the main virulence factor of Listeria monocytogenes, induces a dose-dependent hyperpermeability in monolayers of human lung microvascular endothelial cells in vitro. The permeability increasing activity of LLO, which is accompanied by an increased reactive oxygen species (ROS) generation, RhoA activation and myosin light chain (MLC) phosphorylation, can be completely inhibited by the protein kinase C (PKC) α/β inhibitor GÖ6976, indicating a crucial role for PKC in the induction of barrier dysfunction. The TNF-derived TIP peptide, which mimics the lectin-like domain of the cytokine, blunts LLO-induced hyperpermeability in vitro, upon inhibiting LLO-induced protein kinase C-α activation, ROS generation and MLC phosphorylation and upon restoring the RhoA/Rac 1 balance. These results indicate that the lectin-like domain of TNF has a potential therapeutic value in protecting from LLO-induced pulmonary endothelial hyperpermeability.     

The lifetime feeding study in mice and rats — An examination of its validity as a bioassay for human carcinogens

Currently used designs for carcinogenic bioassay (lifetime feeding studies in mice or rats using maximum tolerated doses of the test compound) are examined to see if they meet the requirements of a bioassay, using the results of 170 compounds reported on as of June, 1980, by the National Cancer Institute. It is concluded that the lifetime feeding study has never been subjected to proper validation as an assay for human carcinogens. When an attempt is made to validate it on the basis of these reported studies and those in the literature, it appears to lack acceptable specificity and sensitivity. It is suggested that a drastically different design is needed and that such redesigning of the assay will require proper validation.     

Correlation between serology and nucleic acid amplification test in blood donors who are reactive for hepatitis B virus,hepatitis C and human immunodeficiency virus and evaluation of the epidemiological profile of infected people in blood centers in the State of Paraná

ObjectivesTo compare the results of conventional serological tests and molecular technology (NAT, Nucleic Acid Amplification Test), identify donors in the diagnostic window period, and determine the prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) among the samples of blood donors blocked by serological screening.MethodsA retrospective cross-sectional study was carried out by analyzing blood donor information contained in the database of 20 blood centers in Paraná, from January 2018 to December 2019.ResultsA total of 1,496 blood bags were reactive for HBV, HCV, or HIV in serological and/or NAT tests. The 20th Regional Health (RH) Unit had the greatest number of unfit individuals with altered screening for the three infections, with a prevalence of 0.70%. The lowest number of blocked blood donors occurred in the 15th RH, with a prevalence of 0.08%. The highest prevalence of HBV occurred in the 8th RH, with a reagent serology of 0.34% and a positive NAT of 0.17%. For HCV, the prevalence for reagent serology was 0.28%, while that for NAT was 0.02%, which occurred in the 20th RH. For HIV and for NAT, the prevalence of blood donors with positive serology occurred in the 20th RH, at 0.25% and 0.04%, retrospectively. The 13th RH had the highest prevalence of HIV in relation to NAT, that is, conventional serology in concomitance with NAT technology, at 0.07%. During the 2-year period, only 1 reactive donor in the 9th was found for NAT (HBV), in a diagnostic window.ConclusionIn Paraná’s blood centers, the inability to donate due to HBV, HCV, and HIV, occurred mainly in initial donors, men, those with >8 years of education, aged 16–45 years, married, and O positive. The most affected regions were located in the west and northwest of Paraná. Most of the results showed a discrepancy between the methodologies used.     

Mixed-effect circadian rhythm model for human erythrocyte acetylcholinesterase activity—application to the proof of concept of cholinesterase inhibition by acorn extract in healthy subjects with galantamine as positive control

Purpose  

The aim of this study was to develop a non-linear mixed effect circadian rhythm model of acetylcholinesterase (AChE) activity variation and to evaluate the inhibitory effect of acorn extract (2 g) and galantamine (16 mg), used as positive control, on human AChE in red blood cells (RBC).     

Pertussis toxin suppresses carbachol-evoked cardiodepression but does not modify cardiostimulation mediated through β1- and putative β4-adrenoceptors in mouse left atria: no evidence for β2- and β3-adrenoceptor function

Activation of beta1-, beta2-, beta 3- and putative beta4-adrenoceptors modifies cardiac function. These receptors are usually coupled to Gs protein, but beta2- and beta3-adrenoceptors could also couple to Gi/o proteins. The mouse heart is used increasingly for studies of genetically disrupted or overexpressed proteins, including beta-adrenoceptor subtypes. We therefore investigated in contracting mouse left atria (2 Hz, 37 degrees C) if inactivation of Gi/o proteins with pertussis toxin modifies or uncovers effects mediated through beta-adrenoceptor subtypes. The negative inotropic effects of carbachol in atria exposed to catecholamine or high calcium (6.8 mmol/l) were assumed to be mediated through activation of muscarinic receptors coupled to Gi/o. We report conditions under which incubation of left atria with 200 ng/ml pertussis toxin for 24 h nearly abolished the carbachol responses. Although it has been reported that muscarinic receptor-mediated cardiodepression has an obligatory contribution of nitric oxide, the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (0.1-1 mmol/l) did not modify the negative inotropic effects of carbachol, inconsistent with an involvement of nitric oxide. The positive inotropic effects of (-)-noradrenaline and (-)-adrenaline, mediated through beta1-adrenoceptors, were not affected by pertussis toxin. (-)-Adrenaline did not cause positive inotropic effects attributable to beta2-adrenoceptor-mediation, in the presence of CGP 20712A (300 nmol/l) to block beta1-adrenoceptors, in control atria or atria pretreated with pertussis toxin. The positive inotropic effects of (-)-CGP 12177 (1 micromol/l), a compound with agonist activity at the putative beta4-adrenoceptor, were unaffected by pertussis toxin. The beta3-adrenoceptor-selective agonist BRL 37344 (1 micromol/l), in the presence of (-)-propranolol (200 nmol/l), did not cause positive or negative inotropic effects in control and pertussis toxin-treated atria. In left atria obtained from mice injected with 150 microg/kg i.p. pertussis toxin which abolished carbachol-evoked cardiode-pression, the positive inotropic effects of (-)-adrenaline were antagonised by CGP 20712A. The beta2-adrenoceptor-selective antagonist ICI 118551 (50 nmol/l) did not cause additional blockade of the effects of high (-)-adrenaline concentrations in the presence of CGP 20712A, ruling out the involvement of beta2-adrenoceptors. The results with intraparenteral PTX validate our in vitro PTX method. We conclude that inhibition of murine Gi/o proteins does not alter atrial positive inotropic effects mediated through beta1- and putative beta4-adrenoceptors and does not reveal functional beta2- and beta3-adrenoceptors.     

Synergistic induction of CCL5, CXCL9 and CXCL10 by IFN-γ and NLRs ligands on human fibroblast-like synoviocytes—A potential immunopathological mechanism for joint inflammation in rheumatoid arthritis

Interferon-γ (IFN-γ) is traditionally regarded as a proinflammatory cytokine by virtue of its strong macrophage activating potential and its association with Th1 driven immune responses. NOD1 and NOD2 are cytoplasmic receptors that can initiate the initial immune response by sensing bacterial components or danger signals. In this study, we investigated the immunopathological roles of IFN-γ and NOD1, 2 ligands iE-DAP/MDP on the activation of fibroblast-like synoviocytes (FLS) in RA. FLS constitutively express functional NOD1 and NOD2, and the gene and protein expression of NOD1 and NOD2 could be enhanced by the treatment with IFN-γ. The synergistic effect was observed in the combined treatment of IFN-γ and NOD1 ligand iE-DAP or NOD2 ligand MDP on the release of CCL5, CXCL9 and CXCL10 from FLS, and its effect was in a dose-dependent manner. The co-stimulation which IFN-γ combined with iE-DAP/MDP could abolish the inhibition of CXCL8 level by IFN-γ alone. Further investigations showed that synergistic effects on the production of CCL5, CXCL9 and CXCL10 in FLS stimulated by IFN-γ and iE-DAP/MDP were differentially regulated by intracellular activation of NF-κB, p38MAPK and ERK pathways. In conclusion, our data confirmed the inflammatory effect of IFN-γ and iE-DAP/MDP on human FLS for the first time and therefore provided a new insight into the IFN-γ combined with NOD1 or NOD2 activated immunopathological mechanisms mediated by distinct intracellular signal transduction in joint inflammation of RA.     

A comparison of the actions of noradrenaline and UK-14,304 in the longitudinal smooth muscle of the rat isolated portal vein — no evidence for a population of post-junctional alpha2-adrenoceptors

Summary The pharmacological characteristics of post-junctional alpha-adrenoceptors mediating contractions of the longitudinal smooth muscle of the rat isolated portal vein have been examined. Responses to the noncumulative addition of either noradrenaline, or the selective alpha₂-adrenoceptor agonist UK-14,304, were equally sensitive to a low concentration (0.005 mol/l) of prazosin. Idazoxan (0.1 mol/l–0.5 mol/l), a selective alpha₂-adrenoceptor antagonist, was less potent than prazosin against both agonists. The combination of 0.1 mol/l idazoxan and 0.125 pmol/l prazosin failed to produce a greater inhibition of responses to UK-14,304 than 0.125 mol/l prazosin alone. A study involving the effect of various antagonists against a single concentration producing a submaximal response to UK-14,304, provided evidence for a prazosin-resistant component of responses which was sensitive to phentolamine. This component could, therefore, be attributed to an alpha-adrenoceptor. However, this particular response could not be ascribed to stimulation of an alpha₂-subtype since the selective alpha, -adrenoceptor antagonist, corynanthine, produced greater inhibition than the selective alpha₂-adrenoceptor diastereoisomer rauwolscine.A preliminary account of these results was presented at The Joint Meeting of the French and German Pharmacological and Toxicological Societies, Freiburg, September 1983 (Wilson et al. 1983)