Hepatotoxicity risk factors and acetaminophen dose adjustment, do prescribers give this issue adequate consideration? A French university hospital study.

BACKGROUND: The hepatotoxicity of acetaminophen is recognised worldwide. Unfavourable prognoses relating to overdose include liver transplantation and/or death. Several hepatotoxicity risk factors (HRFs) should motivate the adjustment of acetaminophen daily intake (to < 4 g/day): advanced age, weight < 50 kg, malnutrition, chronic alcoholism, chronic hepatitis B and C and HIV infection, severe chronic renal failure and hepatocellular insufficiency. METHOD: Over a 7-day period in Rennes University Hospital in December 2017, using DxCare® software, with an odds ratio estimation, we analysed all acetaminophen prescriptions, to assess to what extent the presence of HRFs altered the prescribers' choice of acetaminophen dose (< 4 g/day versus 4 g/day). RESULTS: Among 1842 patients, considering only the first acetaminophen prescription, 73.7% were on 4 g/day. Almost half this population had at least 1 HRF. Whereas around 80% of the prescriptions in the < 4 g/day group were for patients with at least 1 HFR, only 53% of the prescriptions in the 4 g/day group concerned patients without HFRs (p < 0.001). Age > 75 and low weight were associated with the prescriber's choice of dose. Neither chronic alcoholism nor hepatocellular insufficiency influenced the acetaminophen doses prescribed. CONCLUSION: Considering the widespread use of acetaminophen and its favourable safety profile compared with other analgesic drugs, it appears urgent to remind prescribers of the maximum daily dose recommendations for acetaminophen for patients with HRFs, especially those with chronic alcoholism and hepatocellular insufficiency.

Hypoxia interferes with aryl hydrocarbon receptor pathway in hCMEC/D3 human cerebral microvascular endothelial cells.

The expression of aryl hydrocarbon receptor (AhR) transcription factor was detected at transcript level in freshly isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line. Recent studies have demonstrated that AhR pathway is able to crosstalk with other pathways such as hypoxia signaling pathway. Therefore, we used the hCMEC/D3 cell line to investigate the potential crosstalk between AhR and hypoxia signaling pathways. First, we performed two different hypoxia-like procedures in hCMEC/D3 cells; namely, exposition of cells to 150 µM deferoxamine or to glucose and oxygen deprivation for 6 h. These two procedures led to hypoxia-inducible factor (HIF)-1α and HIF-2α proteins accumulation together with a significant induction of the two well-known hypoxia-inducible genes VEGF and GLUT-1. Both HIF-1α and -2α functionally mediated hypoxia response in the hCMEC/D3 cells. Then, we observed that a 6 h exposure to 25 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin, a strong AhR ligand, up-regulated CYP1A1 and CYP1B1 expression, and that this effect was AhR dependent. Regarding AhR and hypoxia crosstalk, our experiments revealed that an asymmetric interference between these two pathways effectively occurred in hCMEC/D3 cells: hypoxia pathway interfered with AhR signaling but not the other way around. We studied the putative crosstalk of AhR and hypoxia pathways in hCMEC/D3 human cerebral microvascular endothelial cells. While hypoxia decreased the expression of the two AhR target genes CYP1A1 and CYP1B1, AhR activation results in no change in hypoxia target gene expression. This is the first sign of AhR and hypoxia pathway crosstalk in an in vitro model of the human cerebral endothelium.

The Medicago truncatula hypermycorrhizal B9 mutant displays an altered response to phosphate and is more susceptible to Aphanomyces euteiches.

Inorganic phosphate (Pi) plays a key role in the development of arbuscular mycorrhizal (AM) symbiosis, which is favoured when Pi is limiting in the environment. We have characterized the Medicago truncatula hypermycorrhizal B9 mutant for its response to limiting (P/10) and replete (P2) Pi. On P2, mycorrhization was significantly higher in B9 plants than in wild-type (WT). The B9 mutant displayed hallmarks of Pi-limited plants, including higher levels of anthocyanins and lower concentrations of Pi in shoots than WT plants. Transcriptome analyses of roots of WT and B9 plants cultivated on P2 or on P/10 confirmed the Pi-limited profile of the mutant on P2 and highlighted its altered response to Pi on P/10. Furthermore, the B9 mutant displayed a higher expression of defence/stress-related genes and was more susceptible to infection by the root oomycete pathogen Aphanomyces euteiches than WT plants. We propose that the hypermycorrhizal phenotype of the B9 mutant is linked to its Pi-limited status favouring AM symbiosis in contrast to WT plants in Pi-replete conditions, and discuss the possible links between the altered response of the B9 mutant to Pi, mycorrhization and infection by A. euteiches.

Induction of P-glycoprotein and Bcrp at the rat blood-brain barrier following a subchronic morphine treatment is mediated through NMDA/COX-2 activation.

Subchronic morphine treatment induces P-glycoprotein (P-gp) up-regulation at the blood-brain barrier. This study investigates the rate and extent to which P-gp and breast cancer-resistance protein (Bcrp) increase at the rat blood-brain barrier following subchronic morphine treatment. Rats were given increasing doses of morphine (10-40 mg/kg) or saline i.p. twice daily for 5 days. The brain cortex large vessels and microvessels were then mechanical isolated 6, 9, 12, 24, and 36 h after the last injection. The gene and protein expression of P-gp and Bcrp in morphine-treated and control rats were compared by qRT-PCR and western blotting. The levels of Mdr1a and Bcrp mRNAs were not significantly modified 6 h post morphine, but the Mdr1a mRNA increased 1.4-fold and Bcrp mRNA 2.4-fold at 24 h. P-gp and Bcrp protein expression in brain microvessels was unchanged 6 h post morphine and increased 1.5-fold at 24 h. This effect was more pronounced in large vessels than in microvessels. However, extracellular morphine concentrations of 0.01-10 µM did not modify the expressions of the MDR1 and BCRP genes in hCMEC/D3 human endothelial brain cells in vitro. MK-801 (NMDA antagonist) and meloxicam (cyclo-oxygenase-2 inhibitor) given after morphine treatment completely blocked P-gp and Bcrp up-regulation. Interestingly, misoprostol and iloprost, two well-known agonists of prostaglandin E2 receptors induced both MDR1 and BCRP mRNA levels in hCMEC/D3. Thus, morphine does not directly stimulate P-gp and Bcrp expression by the brain endothelium, but glutamate released during morphine withdrawal may do so by activating the NMDA/cyclo-oxygenase-2 cascade.

Proteomic analysis of Medicago truncatula root plastids.

Despite the recognized importance of non-photosynthetic plastids in a wide array of plant processes, the root plastid proteome of soil-grown plants still remains to be explored. In this study, we used a protocol allowing the isolation of Medicago truncatula root plastids with sufficient protein recovery and purity for their subsequent in-depth analysis by nanoscale capillary LC-MS/MS. Besides providing the first picture of a root plastid proteome, the results obtained highlighted the identification of 266 protein candidates whose functional distribution mainly resembled that of wheat endosperm amyloplasts and tobacco proplastids together with displaying major differences to those reported for chloroplasts. Most of the identified proteins have a role in nucleic acid-related processes (16%), carbohydrate (15%) and nitrogen/sulphur (12%) metabolisms together with stress response mechanisms (10%). It is noteworthy that BLAST searches performed against the proteins reported in different plastidomes allowed detecting 30 putative root plastid proteins for which homologues were previously unsuspected as plastid-located, most of them displaying a common putative role in participating in the plant cell responses against abiotic and/or biotic stresses. Taken together, the data obtained provide new insights into the functioning of root plastids and reinforce the emerging idea for an important role of these organelles in sustaining plant defence reactions.

Rho-ROCK-dependent ezrin-radixin-moesin phosphorylation regulates Fas-mediated apoptosis in Jurkat cells.

Upon engagement by its ligand, the Fas receptor (CD95/APO-1) is oligomerized in a manner dependent on F-actin. It has been shown that ezrin, a member of the ERM (ezrin-radixin-moesin) protein family can link Fas to the actin cytoskeleton. We show herein that in Jurkat cells, not only ezrin but also moesin can associate with Fas. The same observation was made in activated human peripheral blood T cells. Fas/ezrin or moesin (E/M) association increases in Jurkat cells following Fas triggering and occurs concomitantly with the formation of SDS- and 2-ME-stable high molecular mass Fas aggregates. Ezrin and moesin have to be present together for the formation of Fas aggregates since down-regulation of either ezrin or moesin expression with small interfering RNAs completely inhibits Fas aggregate formation. Although FADD (Fas-associated death domain protein) and caspase-8 associate with Fas in the absence of E/M, subsequent events such as caspase-8 activation and sensitivity to apoptosis are decreased. During the course of Fas stimulation, ezrin and moesin become phosphorylated, respectively, on T567 and on T558. This phosphorylation is mediated by the kinase ROCK (Rho-associated coiled coil-containing protein kinase) I subsequently to Rho activation. Indeed, inhibition of either Rho or ROCK prevents ezrin and moesin phosphorylation, abrogates the formation of Fas aggregates, and interferes with caspase-8 activation. Thus, phosphorylation of E/M by ROCK is involved in the early steps of apoptotic signaling following Fas triggering and regulates apoptosis induction.

A case report of photodermatoses induced by the antifungal drug terbinafine.

Terbinafine is an antifungaldrug(inhibitor of ergosterol synthesis) known to induce skin reactions. A 58-year-old female was treated with terbinafine for onychomycosis. On the fifth day of treatment a skin rash emerged on her sun-exposed areas. Biological testing did not find any allergic signs. Other skin reactions are well known with terbinafine, but we depict here, a case of photosensitisation induced by treatment, generally unknown for this antifungal. This side effect can be prevented by medication reconciliation and pharmaceutical advice from the clinical pharmacist.

Induction of multidrug resistance-associated protein 3 expression by diesel exhaust particle extract in human bronchial epithelial BEAS-2B cells.

Diesel exhaust particles (DEPs) are common environmental air pollutants known to impair expression and activity of drug detoxifying proteins, including hepatic ATP-binding cassette (ABC) drug transporters. The present study was designed to determine whether organic DEP extract (DEPe) may also target ABC drug transporters in bronchial cells. DEPe (10 µg/mL) was demonstrated to induce mRNA and protein expression of the multidrug resistance-associated protein (MRP) 3 in cultured bronchial epithelial BEAS-2B cells, whereas mRNA levels of other MRPs, multidrug resistance gene 1 or breast cancer resistance protein were unchanged, reduced or not detected. DEPe also increased MRP3 mRNA expression in normal human bronchial epithelial cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway by AhR antagonist or AhR silencing, as well as the silencing of nuclear-factor-E2-related factor 2 (Nrf2) repressed DEPe-mediated MRP3 induction. This underlines the implication of the AhR and Nrf2 signaling cascades in DEPe-mediated MRP3 regulation. DEPe was additionally demonstrated to directly inhibit MRP activity in BEAS-2B cells, in a concentration-dependent manner. Taken together, these data indicate that DEPs may impair expression and activity of MRPs, notably MRP3, in human bronchial cells, which may have consequences in terms of lung barrier and toxicity for humans exposed to diesel pollution.

Overexposure to Bisphenol A and Its Chlorinated Derivatives of Patients with End-Stage Renal Disease during Online Hemodiafiltration.

The health safety conditions governing the practice of online hemodiafiltration (OL-HDF) do not yet incorporate the risks related to the presence of endocrine disruptors such as bisphenol A (BPA). The aim of this study was to assess, for the first time, the exposure to BPA but also to its chlorinated derivatives (ClxBPA) (100 times more estrogenic than BPA) during OL-HDF. We demonstrated that BPA is transmitted by the different medical devices used in OL-HDF: ultrafilters, dialysis concentrate cartridges (and not only dialyzers, as previously described). Moreover, BPA has been found in dialysis water as well as in ultrapure dialysate and replacement fluid due to contamination of water coming from municipal network. Indeed, due to contaminations provided by both ultrafilters and water, high levels of BPA were determined in the infused replacement fluid (1033 ng.L-1) from the beginning of the session. Thus, our results demonstrate that dialysis water must be considered as an important exposure source to endocrine disruptors, especially since other micropollutants such as ClxBPA have also been detected in dialysis fluids. While assessment of the impact of this exposure remains to be done, these new findings should be taken into account to assess exposure risks in end-stage renal disease patients.

Interactions of pesticides with membrane drug transporters: implications for toxicokinetics and toxicity.

INTRODUCTION: Drug transporters are now recognized as major actors of pharmacokinetics. They are also likely implicated in toxicokinetics and toxicology of environmental pollutants, notably pesticides, to which humans are widely exposed and which are known to exert various deleterious effects toward health. Interactions of pesticides with drug transporters are therefore important to consider. Areas covered: This review provides an overview of the interactions of pesticides with membrane drug transporters, i.e. inhibition of their activity, regulation of their expression, and handling of pesticides. Consequences for toxicokinetics and toxicity of pesticides are additionally summarized and discussed. Expert opinion: Some pesticides belonging to several chemical classes, such as organochlorine, pyrethroid, and organophosphorus pesticides, have been demonstrated to interact with various uptake and efflux drug transporters, including the efflux pump P-glycoprotein (P-gp) and the uptake organic cation transporters (OCTs). This provides proof of the concept that pesticide-transporter relationships merit attention. More extensive and systematic characterization of pesticide-transporter relationships, possibly through the use of in silico methods, is however likely required. In addition, consideration of transporter polymorphisms, pesticide mixture effects, and realistic pesticide concentrations reached in humans may help better define the in vivo relevance of pesticide-transporter interactions in terms of toxicokinetics and toxicity.

Involvement of a Rho-ROCK-JNK pathway in arsenic trioxide-induced apoptosis in chronic myelogenous leukemia cells.

The apoptotic signals activated by As(2)O(3) in the chronic myelogenous leukemia (CML) cell lines K562 and KCL22 were investigated. As(2)O(3) was found to induce apoptosis in these cells via the intrinsic pathway. As(2)O(3) also induced a sustained c-Jun NH2-terminal kinase (JNK) activation which preceded and was necessary for caspase-9 activation. We established that Rho and its effector, the kinase ROCK, are activated by As(2)O(3). Inhibition of either Rho or ROCK prevented JNK activation and protected against apoptosis. Thus, in CML cells, apoptosis induced by As(2)O(3) is mediated, at least in part, via a Rho-ROCK-JNK axis. These findings define a novel signaling pathway for As(2)O(3)-induced apoptosis.

Inhibition of Human Drug Transporter Activities by the Pyrethroid Pesticides Allethrin and Tetramethrin.

Pyrethroids are widely-used chemical insecticides, to which humans are commonly exposed, and known to alter functional expression of drug metabolizing enzymes. Limited data have additionally suggested that drug transporters, that constitute key-actors of the drug detoxification system, may also be targeted by pyrethroids. The present study was therefore designed to analyze the potential regulatory effects of these pesticides towards activities of main ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, using transporter-overexpressing cells. The pyrethroids allethrin and tetramethrin were found to inhibit various ABC and SLC drug transporters, including multidrug resistance-associated protein (MRP) 2, breast cancer resistance protein (BCRP), organic anion transporter polypeptide (OATP) 1B1, organic anion transporter (OAT) 3, multidrug and toxin extrusion transporter (MATE) 1, organic cation transporter (OCT) 1 and OCT2, with IC50 values however ranging from 2.6 µM (OCT1 inhibition by allethrin) to 77.6 µM (OAT3 inhibition by tetramethrin) and thus much higher than pyrethroid concentrations (in the nM range) reached in environmentally pyrethroid-exposed humans. By contrast, allethrin and tetramethrin cis-stimulated OATP2B1 activity and failed to alter activities of OATP1B3, OAT1 and MATE2-K, whereas P-glycoprotein activity was additionally moderately inhibited. Twelve other pyrethoids used at 100 µM did not block activities of the various investigated transporters, or only moderately inhibited some of them (inhibition by less than 50%). In silico analysis of structure-activity relationships next revealed that molecular parameters, including molecular weight and lipophilicity, are associated with transporter inhibition by allethrin/tetramethrin and successfully predicted transporter inhibition by the pyrethroids imiprothrin and prallethrin. Taken together, these data fully demonstrated that two pyrethoids, i.e., allethrin and tetramethrin, can act as regulators of the activity of various ABC and SLC drug transporters, but only when used at high and non-relevant concentrations, making unlikely any contribution of these transporter activity alterations to pyrethroid toxicity in environmentally exposed humans.

Aryl hydrocarbon receptor regulates CYP1B1 but not ABCB1 and ABCG2 in hCMEC/D3 human cerebral microvascular endothelial cells after TCDD exposure.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor activated by a variety of widespread persistent environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It can transactivate the expression of several target genes. Recently AhR transcripts were detected in isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line, an in vitro model of the human cerebral endothelium. To date AhR implication in the co-regulation of ABCB1, ABCG2 and CYP1B1 at human cerebral endothelium has not been addressed. Here we investigated whether AhR could co-regulate ABCB1, ABCG2 and CYP1B1 expressions in the hCMEC/D3 cell line. Exposure to TCDD induced a concentration-dependent increase in CYP1B1 expression. We demonstrated AhR involvement in the TCDD-mediated increase in CYP1B1 expression by using small interfering RNA against AhR. Western blotting analysis also revealed an increase in CYP1B1 protein expression following TCDD exposure in hCMEC/D3. Regarding ABCB1 and ABCG2, exposure to TCDD had no effect on their protein expressions and functional activities. In conclusion our data indicated a differential modulation of CYP1B1 and ABCB1/ABCG2 expressions in hCMEC/D3 cells following TCDD exposure.

Interplay of drug metabolizing CYP450 enzymes and ABC transporters in the blood-brain barrier.

The recent identification of drug-metabolizing enzymes cytochrome P450 (CYP) in the human blood-brain barrier (BBB) raises the question of whether these enzymes act in concert with ATP-binding cassette (ABC) transporters to limit the brain distributions of drugs. We recently demonstrated several CYP genes in freshly isolated human brain microvessels; the main isoforms expressed were CYP1B1 and CYP2U1. Many studies using different experimental approaches have revealed that P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and the multidrug resistance-associated protein 4 (MRP4, ABCC4) are the main ABC transporters in the human BBB. The first part of this review covers recent studies on the expression, regulation and function of CYP450 and ABC transporters in the rodent and human BBBs. The second part focuses on the possible interplay between some CYPs and certain ABC transporters at the BBB, which makes it a determining element of brain drug concentrations and thus of the effects of centrally acting drugs.

Aryl hydrocarbon receptor-dependent upregulation of Cyp1b1 by TCDD and diesel exhaust particles in rat brain microvessels.

BACKGROUND: AhR activates the transcription of several target genes including CYP1B1. Recently, we showed CYP1B1 as the major cytochrome P450 (CYP) enzyme expressed in human brain microvessels. Here, we studied the effect of AhR activation by environmental pollutants on the expression of Cyp1b1 in rat brain microvessels. METHODS: Expression of AhR and Cyp1b1 was detected in isolated rat brain microvessels. AhR was immunovisualised in brain microvessel endothelial cells. The effect of AhR ligands on Cyp1b1 expression was studied using isolated brain microvessels after ex vivo and/or in vivo exposure to TCDD, heavy hydrocarbons containing diesel exhaust particles (DEP) or Δ9-tetrahydrocannabinol (Δ9-THC). RESULTS: After ex vivo exposure to TCDD (a highly potent AhR ligand) for 3 h, Cyp1b1 expression was significantly increased by 2.3-fold in brain microvessels. A single i.p. dose of TCDD also increased Cyp1b1 transcripts (22-fold) and Cyp1b1 protein (2-fold) in rat brain microvessels at 72 h after TCDD. Likewise, DEP treatment (in vivo and ex vivo) strongly induced Cyp1b1 protein in brain microvessels. DEP-mediated Cyp1b1 induction was inhibited by actinomycin D, cycloheximide, or by an AhR antagonist. In contrast, a sub-chronic in vivo treatment with Δ9-THC once daily for 7 seven days had no effect on Cyp1b1 expression CONCLUSIONS: Our results show that TCDD and DEP strongly induced Cyp1b1 in rat brain microvessels, likely through AhR activation.