Postnatal Smoke Exposure Further Increases the Hepatic Nicotine Metabolism in Prenatally Smoke Exposed Male Offspring and Is Linked with Aberrant Cyp2a5 Methylation.

Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.

Intracellular RIG-I Signaling Regulates TLR4-Independent Endothelial Inflammatory Responses to Endotoxin.

Sepsis is a systemic inflammatory response to infections associated with organ failure that is the most frequent cause of death in hospitalized patients. Exaggerated endothelial activation, altered blood flow, vascular leakage, and other disturbances synergistically contribute to sepsis-induced organ failure. The underlying signaling events associated with endothelial proinflammatory activation are not well understood, yet they likely consist of molecular pathways that act in an endothelium-specific manner. We found that LPS, a critical factor in the pathogenesis of sepsis, is internalized by endothelial cells, leading to intracellular signaling without the need for priming as found recently in immune cells. By identifying a novel role for retinoic acid-inducible gene-I (RIG-I) as a central regulator of endothelial activation functioning independent of TLR4, we provide evidence that the current paradigm of TLR4 solely being responsible for LPS-mediated endothelial responses is incomplete. RIG-I, as well as the adaptor protein mitochondrial antiviral signaling protein, regulates NF-κB-mediated induction of adhesion molecules and proinflammatory cytokine expression in response to LPS. Our findings provide essential new insights into the proinflammatory signaling pathways in endothelial cells and suggest that combined endothelial-specific inhibition of RIG-I and TLR4 will provide protection from aberrant endothelial responses associated with sepsis.

Rat precision-cut liver slices predict drug-induced cholestatic injury.

Drug-induced cholestasis (DIC) is one of the leading manifestations of drug-induced liver injury (DILI). As the underlying mechanisms for DIC are not fully known and specific and predictive biomarkers and pre-clinical models are lacking, the occurrence of DIC is often only reported when the drug has been approved for registration. Therefore, appropriate models that predict the cholestatic potential of drug candidates and/or provide insight into the mechanism of DIC are highly needed. We investigated the application of rat precision-cut liver slices (PCLS) to predict DIC, using several biomarkers of cholestasis: hepatocyte viability, intracellular accumulation of total as well as individual bile acids and changes in the expression of genes known to play a role in cholestasis. Rat PCLS exposed to the cholestatic drugs chlorpromazine, cyclosporine A and glibenclamide for 48 h in the presence of a 60 µM physiological bile acid (BA) mix reflected various changes associated with cholestasis, such as decrease in hepatocyte viability, accumulation and changes in the composition of BA and changes in the gene expression of Fxr, Bsep and Ntcp. The toxicity of the drugs was correlated with the accumulation of BA, and especially DCA and CDCA and their conjugates, but to a different extent for different drugs, indicating that BA toxicity is not the only cause for the toxicity of cholestatic drugs. Moreover, our study supports the use of several biomarkers to test drugs for DIC. In conclusion, our results indicate that PCLS may represent a physiological and valuable model to identify cholestatic drugs and provide insight into the mechanisms underlying DIC.

P-gp activity and inhibition in the different regions of human intestine ex vivo.

Although intestinal P-glycoprotein (P-gp) has been extensively studied in vitro and in animals, its activity and the consequences of P-gp inhibition for drug disposition and toxicity in humans are still difficult to accurately extrapolate from these studies. Moreover, existing in vitro models do not take into consideration that the intestine is heterogeneous with respect to P-gp expression. Recently, we reported rat precision-cut intestinal slices (PCIS) as a physiological ex vivo model to study the regional gradient of P-gp activity and inhibition. Here we extended the application of PCIS to the human intestine. For this purpose rhodamine 123 (R123) accumulation in the presence or absence of the P-gp inhibitors verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 was measured in PCIS of human duodenum, jejunum, ileum and colon. R123 accumulation in the presence of the P-gp inhibitors appeared to be most enhanced in the ileum compared to the other regions. Moreover, the regional differences in accumulation are in line with published differences in abundance of P-gp. The rank order of the potency of the P-gp inhibitors, reflected by their IC50 , was comparable to that in rat PCIS. However, the increase in accumulation of the P-gp substrate R123 by the inhibitors was larger in human ileum PCIS than in rat PCIS, indicating species difference in P-gp abundance. These data show that human PCIS are an appropriate ex vivo model to study the activity of intestinal P-gp and predict the inhibitory effect of drugs and of transporter-mediated drug-drug interactions in the human intestine. Copyright © 2016 John Wiley & Sons, Ltd.

The Consequence of Drug-Drug Interactions Influencing the Interplay between P-Glycoprotein and Cytochrome P450 3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices.

P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites.

Diclofenac toxicity in human intestine ex vivo is not related to the formation of intestinal metabolites.

The use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is associated with a high prevalence of gastrointestinal side effects. In vivo studies in rodents suggested that reactive metabolites of DCF produced by the liver or the intestine might be responsible for this toxicity. In the present study, precision-cut intestinal slices (PCIS) prepared from the jejunum of 18 human donors were used as an ex vivo model to investigate whether DCF intestinal metabolites are responsible for its intestinal toxicity in man. PCIS were incubated with a concentration range of DCF (0-600 µM) up to 24 h. DCF (≥400 µM) caused direct toxicity to the intestine as demonstrated by ATP depletion, morphological damage, caspase 3 activation, and lactate dehydrogenase leakage. Three main metabolites produced by PCIS (4'-hydroxy DCF, 5-hydroxy DCF, and DCF acyl glucuronide) were detected by HPLC. Protein adducts were detected by immunohistochemical staining and showed correlation with the intestinal metabolites. DCF induced similar toxicity to each of the samples regardless of the variation in metabolism among them. Less metabolites were produced by slices incubated with 400 µM DCF than with 100 µM DCF. The addition of the metabolic inhibitors such as ketoconazole, cimetidine, or borneol decreased the metabolite formation but increased the toxicity. The results suggest that DCF can induce intestinal toxicity in human PCIS directly at therapeutically relevant concentrations, independent of the reactive metabolites 4'-OH DCF, 5-OH DCF, or diclofenac acylglucuronide produced by the liver or formed in the intestine.

Unraveling Hematotoxicity of α-Amanitin in Cultured Hematopoietic Cells.

Amanita phalloides poisonings account for the majority of fatal mushroom poisonings. Recently, we identified hematotoxicity as a relevant aspect of Amanita poisonings. In this study, we investigated the effects of the main toxins of Amanita phalloides, α- and ß-amanitin, on hematopoietic cell viability in vitro. Hematopoietic cell lines were exposed to α-amanitin or ß-amanitin for up to 72 h with or without the pan-caspase inhibitor Z-VAD(OH)-FMK, antidotes N-acetylcysteine, silibinin, and benzylpenicillin, and organic anion-transporting polypeptide 1B3 (OATP1B3) inhibitors rifampicin and cyclosporin. Cell viability was established by trypan blue exclusion, annexin V staining, and a MTS assay. Caspase-3/7 activity was determined with Caspase-Glo assay, and cleaved caspase-3 was quantified by Western analysis. Cell number and colony-forming units were quantified after exposure to α-amanitin in primary CD34+ hematopoietic stem cells. In all cell lines, α-amanitin concentration-dependently decreased viability and mitochondrial activity. ß-Amanitin was less toxic, but still significantly reduced viability. α-Amanitin increased caspase-3/7 activity by 2.8-fold and cleaved caspase-3 by 2.3-fold. Z-VAD(OH)-FMK significantly reduced α-amanitin-induced toxicity. In CD34+ stem cells, α-amanitin decreased the number of colonies and cells. The antidotes and OATP1B3 inhibitors did not reverse α-amanitin-induced toxicity. In conclusion, α-amanitin induces apoptosis in hematopoietic cells via a caspase-dependent mechanism.

Nanoparticle formulation of a poorly soluble cannabinoid receptor 1 antagonist improves absorption by rat and human intestine.

The inclusion of nanoparticles dispersed in a hydrophilic matrix is one of the formulation strategies to improve the bioavailability of orally administered Biopharmaceutics Classification System (BCS) class II and IV drugs by increasing their dissolution rate in the intestine. To confirm that the increased dissolution rate results in increased bioavailability, in vitro and in vivo animal experiments are performed, however, translation to the human situation is hazardous. In this study, we used a range of in vitro and ex vivo methods, including methods applying human tissue, to predict the in vivo oral bioavailability of a model BCS class II CB-1 antagonist, formulated as a nanoparticle solid dispersion. The enhanced dissolution rate from the nanoparticle formulation resulted in an increased metabolite formation in both rat and human precision-cut intestinal slices, suggesting increased uptake and intracellular drug concentration in the enterocytes. In Ussing chamber experiments with human tissue, both the metabolite formation and apical efflux of the metabolite were increased for the nanoparticulate solid dispersion compared with a physical mixture, in line with the results in intestinal slices. The pharmacokinetics of the different formulations was studied in rats in vivo. The nanoparticle formulation indeed improved the absorption of the cannabinoid receptor 1 (CB-1) antagonist and the delivery into the brain compared with the physical mixture. In conclusion, the combined approach provides a valuable set of tools to investigate the effects of formulation on the absorption of poorly soluble compounds in human intestine and may provide relevant information on the oral bioavailability in humans early in the development process.

Evaluation of the intestinal toxicity and transport of xenobiotics utilizing precision-cut slices.

1.The precision-cut intestinal slice (PCIS) technology is a relatively new addition to the battery of in vitro assays for evaluation of xenobiotic toxicity, metabolism, and transport. 2.The intestine is an important target for drug-induced toxicity due to its high exposure after oral administration. Therefore, the prediction of drug-induced intestinal side effects remains a significant safety issue in pharmaceutical development. Although animal experiments have been proven useful, species differences and the requirement for reduction of animal use warrant the development of in vitro methods which can apply human tissue. 3.The enterocytes lining the villi express high activities of enzymes and transporters involved in drug disposition. They vary highly in activities: along the length of the intestine and along the villi, gradients of expression levels of the enzymes and proteins exist, which necessitates an in vitro model that can reflect the different regions of the intestine. 4.In this chapter, the application of PCIS in studies on transport and toxicity of xenobiotics is reviewed. PCIS can be prepared from each region of the intestine and from various species in a similar manner, and the results published so far indicate that they represent a promising model to evaluate intestinal toxicity and transport.

Cryopreservation of precision-cut tissue slices.

1.Cryopreservation of precision-cut tissue slices (PCTS) would have many advantages for drug development and would encourage more extensive use of the PCTS preparation. 2.Three methods have been studied to date: slow freezing, fast freezing, and vitrification. 3.Slow freezing can be very effective for some PCTS but is devastating to rat liver PCTS. Fast freezing can be successful for rat liver PCTS but is devastating to renal PCTS and has given inconsistent results even for rat liver PCTS. Vitrification has been effective for some slice systems but less effective for rat liver PCTS. Rat liver PCTS appear to be particularly difficult to cryopreserve well. 4.The general cryobiological principles of slow freezing, rapid freezing, and vitrification are reviewed. The empirical literature on the cryopreservation of PCTS has not taken sufficient account of these principles, and may, for example, include the effects of easily preventable osmotic injury. 5.More attention is needed to the effects of cryopreservation on specific cell types within PCTS and to the general integrity and viability of cryopreserved PCTS. Drug metabolism as a sole endpoint of study can be highly misleading. 6.Better application of cryobiological principles may enable improved results in the future.

Transport of the coumarin metabolite 7-hydroxycoumarin glucuronide is mediated via multidrug resistance-associated proteins 3 and 4.

Coumarin (1,2-benzopyrone) is a natural compound that has been used as a fragrance in the food and perfume industry and could have therapeutic usefulness in the treatment of lymphedema and different types of cancer. Several previous pharmacokinetic studies of coumarin have been performed in humans, which revealed extensive first-pass metabolism of the compound. 7-Hydroxycoumarin (7-HC) and its glucuronide (7-HC-G) are the main metabolites formed in humans, and via this route, 80 to 90% of the absorbed coumarin is excreted into urine, mainly as 7-HC-G. Active transport processes play a role in the urinary excretion of 7-HC-G; however, until now, the transporters involved remained to be elucidated. In this study, we investigated whether the efflux transporters multidrug resistance-associated proteins (MRP)1-4, breast cancer resistance protein, or P-glycoprotein play a role in 7-HC and 7-HC-G transport. For this purpose, we measured uptake of the metabolites into membrane vesicles overexpressing these transporters. Our results showed that 7-HC is not transported by any of the efflux transporters tested, whereas 7-HC-G was a substrate of MRP3 and MRP4. These results are in line with the pharmacokinetic profile of coumarin and suggest that MRP3 and MRP4 are the main transporters involved in the excretion of the coumarin metabolite 7-HC-G from liver and kidney.

Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices.

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time. With this method, CPA concentration versus time profiles were varied using various commercially available CPA mixtures [VMP, VM3, M22, and modified M22 (mM22)]. The viability of PCLS was determined after CPA loading and unloading and subsequent incubation during 3h at 37°C. Despite the reduction of osmotic stress, the viability of slices did not improve with gradual loading and unloading and remained considerably lower than that of untreated slices. The toxicity of the three CPA solutions did not correlate with either their potential osmotic effects or their total concentrations, and did not change strongly with exposure time in 100% CPA. The most likely explanation for these observations is that PCLS are not very sensitive to osmotic changes of the magnitude imposed in our study, and chemical toxicity of the CPA solutions is the main barrier to be overcome. The chemical toxicity of the CPAs used in this study probably originates from a source other than the total concentration of the solutions. The presented gradient method using the specially designed chamber is more time and cost effective than the step-wise method and can be universally applied to efficiently evaluate different CPA solutions.

On-line HPLC analysis system for metabolism and inhibition studies in precision-cut liver slices.

A novel approach for on-line monitoring of drug metabolism in continuously perifused, precision-cut liver slices (PCLS) in a microfluidic system has been developed using high-performance liquid chromatography with UV detection (HPLC-UV). In this approach, PCLS are incubated in a microfluidic device made of poly(dimethylsiloxane) (PDMS) by continuous, single-pass perifusion with fresh medium. Two syringe pumps are incorporated into the system to infuse substrates or inhibitors at varying concentrations into the perfusion medium just before the chip entrance. The medium containing the metabolites produced by the PCLS is directed toward an injection loop. Once filled, the content of this injection loop is automatically injected onto an HPLC for analysis. The on-line analysis of metabolites was tested by using the substrate, 7-hydroxycoumarin (7-HC). Rapid switching between substrate and solvent control was possible, and a direct metabolic response of the liver slice to perifusion with substrate was detected. Very stable phase II metabolism over a period of 24 h was observed. The inhibitory effect of phloxine B on the formation of 7-hydroxycoumarin glucuronide (phase II product of 7-HC) was also investigated. Phloxine B was injected into the incubation medium in increasing concentrations varying from 0 to 200 µM. The results showed a concentration-dependent inhibition of 7-HC glucuronide formation and allowed the calculation of an IC50 value (concentration in which 50% of the enzyme is inhibited) of ∼85 µM using one single liver slice. On-line detection was also shown to be advantageous for the detection of unstable metabolites. This was demonstrated by determination of the metabolites of the drug diclofenac. The reactive metabolite, acyl glucuronide, was detected at relatively high concentrations which remained very constant over a period of 4 h. In contrast, only low and decreasing amounts of diclofenac acyl glucuronide could be measured in the conventional well-plate incubation system. The advantages of this novel on-line analysis system for PCLS include the capability to obtain direct information about tissue function, assess the concentration dependence of drug-drug interactions in one single slice, and detect unstable metabolites. The system also enables fast analysis without the need to store samples, thus eliminating the associated freeze-thaw problems, and allows the simultaneous analysis of multiple metabolites.

Rifampicin Induces Gene, Protein, and Activity of P-Glycoprotein (ABCB1) in Human Precision-Cut Intestinal Slices.

P-glycoprotein (ABCB1), an ATP-binding cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions. Drug-mediated induction of intestinal ABCB1 is a clinically relevant phenomenon associated with significantly decreased drug bioavailability. Currently, there are no well-established human models for evaluating its induction, so drug regulatory authorities provide no recommendations for in vitro/ex vivo testing drugs' ABCB1-inducing activity. Human precision-cut intestinal slices (hPCISs) contain cells in their natural environment and express physiological levels of nuclear factors required for ABCB1 induction. We found that hPCISs incubated in William's Medium E for 48 h maintained intact morphology, ATP content, and ABCB1 efflux activity. Here, we asked whether rifampicin (a model ligand of pregnane X receptor, PXR), at 30 µM, induces functional expression of ABCB1 in hPCISs over 24- and 48-h incubation (the time to allow complete induction to occur). Rifampicin significantly increased gene expression, protein levels, and efflux activity of ABCB1. Moreover, we described dynamic changes in ABCB1 transcript levels in hPCISs over 48 h incubation. We also observed that peaks of induction are achieved among donors at different times, and the extent of ABCB1 gene induction is proportional to PXR mRNA levels in the intestine. In conclusion, we showed that hPCISs incubated in conditions comparable to those used for inhibition studies can be used to evaluate drugs' ABCB1-inducing potency in the human intestine. Thus, hPCISs may be valuable experimental tools that can be prospectively used in complex experimental evaluation of drug-drug interactions.

Carbosilane Dendrimers Loaded with siRNA Targeting Nrf2 as a Tool to Overcome Cisplatin Chemoresistance in Bladder Cancer Cells.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as the master regulator of antioxidant and cytoprotective gene expressions. Moreover, it plays a pivotal role in cancer progression. Nrf2 mediates the adaptive response which contributes to the resistance to chemotherapeutic pro-oxidant drugs, such as cisplatin (CDDP), in various tumors, including bladder cancers. For this reason, Nrf2 could be a promising target to overcome chemoresistance. There are several known Nrf2 pharmacological inhibitors; however, most of them are not specific. The use of a specific small interfering RNA (siRNA) targeting the Nrf2 gene (siNrf2) loaded into nanovehicles is an attractive alternative, since it can increase specificity. This study aimed to evaluate the biological activity of siNrf2 loaded on guanidine-terminated carbosilane dendrimers (GCDs) in overcoming CDDP resistance in bladder cancer cells with a high level of Nrf2. Parameters such as viability, proliferation, apoptosis, migration, and oxidative stress level were taken into account. Results demonstrated that siNrf2-GCD treatment sensitized CDDP-resistant cells to CDDP treatment. Moreover, data obtained by treating the non-cancerous human kidney HK-2 cell line strongly suggest a good safety profile of the carbosilane dendrimers loaded with siNrf2. In conclusion, we suggest that siNrf2-GCD is a promising drug delivery system for gene therapy to be used in vivo; and it may represent an important tool in the therapy of CDDP-resistant cancer.

Glutathione-responsive cyclodextrin-nanosponges as drug delivery systems for doxorubicin: Evaluation of toxicity and transport mechanisms in the liver.

The potential mammalian hepatotoxicity of a new class of GSH-responsive cyclodextrin-based nanosponges loaded with the anticancer drug doxorubicin (Dox-GSH-NS) was investigated. Previous studies showed that these nanosponges can release medicaments preferentially in cells having high GSH content, a common feature of chemoresistant cells, and showed enhanced anti-tumoral activity compared to free Dox in vitro and in vivo in cells with high GSH content. Following these promising results, we investigated here the Dox-GSH-NS hepatotoxicity in human HepG2 cells (in vitro) and in the organotypic cultures of rat precision-cut liver slices (PCLS, ex vivo), while their accumulation in rat liver was assessed in vivo. Moreover, the transport in Dox uptake, as well as its efflux, was studied in vitro. Overall, benefiting of the integration of different investigational models, a good safety profile of Dox-GSH-NSs was evidenced, and their hepatotoxicity resulted to be comparable with respect to free Dox both in vitro and ex vivo. Furthermore, in vivo studies showed that the hepatic accumulation of the Dox loaded in the NS is comparable with respect to the free drug. In addition, Dox-GSH-NSs are taken up by active mechanisms, and can escape the efflux drug pump, thus, contributing to overcoming drug resistance.

Prenatal smoke exposure induces persistent Cyp2a5 methylation and increases nicotine metabolism in the liver of neonatal and adult male offspring.

Prenatal smoke exposure (PSE) is a risk factor for nicotine dependence. One susceptibility gene for nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher activity of the CYP2A6 enzyme is associated with nicotine dependence, but no research has addressed the PSE effects on the CYP2A6 gene or its mouse homologue Cyp2a5. We hypothesized that PSE affects Cyp2a5 promoter methylation, Cyp2a5 mRNA levels, and nicotine metabolism in offspring. We used a smoke-exposed pregnant mouse model. RNA, DNA, and microsomal protein were isolated from liver tissue of foetal, neonatal, and adult offspring. Enzyme activity, Cyp2a5 mRNA levels, and Cyp2a5 methylation status of six CpG sites within the promoter region were analysed via HPLC, RT-PCR, and bisulphite pyrosequencing. Our data show that PSE induced higher cotinine levels in livers of male neonatal and adult offspring compared to controls. PSE-induced cotinine levels in neonates correlated with Cyp2a5 mRNA expression and promoter methylation at CpG-7 and CpG+45. PSE increased methylation in almost all CpG sites in foetal offspring, and this effect persisted at CpG-74 in male neonatal and adult offspring. Our results indicate that male offspring of mothers which were exposed to cigarette smoke during pregnancy have a higher hepatic nicotine metabolism, which could be regulated by DNA methylation. Given the detected persistence into adulthood, extrapolation to the human situation suggests that sons born from smoking mothers could be more susceptible to nicotine dependence later in life.

Shock-induced stress induces loss of microvascular endothelial Tie2 in the kidney which is not associated with reduced glomerular barrier function.

Both hemorrhagic shock and endotoxemia induce a pronounced vascular activation in the kidney which coincides with albuminuria and glomerular barrier dysfunction. We hypothesized that changes in Tie2, a vascular restricted receptor tyrosine kinase shown to control microvascular integrity and endothelial inflammation, underlie this loss of glomerular barrier function. In healthy murine and human kidney, Tie2 is heterogeneously expressed in all microvascular beds, although to different extents. In mice subjected to hemorrhagic and septic shock, Tie2 mRNA and protein were rapidly, and temporarily, lost from the renal microvasculature, and normalized within 24 h after initiation of the shock insult. The loss of Tie2 protein could not be attributed to shedding as both in mice and healthy volunteers subjected to endotoxemia, sTie2 levels in the systemic circulation did not change. In an attempt to identify the molecular control of Tie2, we activated glomerular endothelial cell cultures and human kidney slices in vitro with LPS or TNF-alpha, but did not observe a change in Tie2 mRNA levels. In parallel to the loss of Tie2 in vivo, an overt influx of neutrophils in the glomerular compartment, which coincided with proteinuria, was seen. As neutrophil-endothelial cell interactions may play a role in endothelial adaptation to shock, and these effects cannot be mimicked in vitro, we depleted neutrophils before shock induction. While this neutrophil depletion abolished proteinuria, Tie2 was not rescued, implying that Tie2 may not be a major factor controlling maintenance of the glomerular filtration barrier in this model.

Ex vivo toxicological evaluation of experimental anticancer gold(i) complexes with lansoprazole-type ligands.

Gold-based compounds are of great interest in the field of medicinal chemistry as novel therapeutic (anticancer) agents due to their peculiar reactivity and mechanisms of action with respect to organic drugs. Despite their promising pharmacological properties, the possible toxic effects of gold compounds need to be carefully evaluated in order to optimize their design and applicability. This study reports on the potential toxicity of three experimental gold-based anticancer compounds featuring lansoprazole ligands (1-3) studied in an ex vivo model, using rat precision cut kidney and liver slices (PCKS and PCLS, respectively). The results showed a different toxicity profile for the tested compounds, with the neutral complex 2 being the least toxic, even less toxic than cisplatin, followed by the cationic complex 1. The dinuclear cationic gold complex 3 was the most toxic in both liver and kidney slices. This result correlated with the metal uptake of the different compounds assessed by ICP-MS, where complex 3 showed the highest accumulation of gold in liver and kidney slices. Interestingly compound 1 showed the highest selectivity towards cancer cells compared to the healthy tissues. Histomorphology evaluation showed a similar pattern for all three Au(i) complexes, where the distal tubular cells suffered the most extensive damage, in contrast to the damage in the proximal tubules induced by cisplatin. The binding of representative gold compounds with the model ubiquitin was also studied by ESI-MS, showing that after 24 h incubation only 'naked' Au ions were bound to the protein following ligands' loss. The mRNA expression of stress response genes appeared to be similar for both evaluated organs, suggesting oxidative stress as the possible mechanism of toxicity. The obtained results open new perspectives towards the design and testing of bifunctional gold complexes with chemotherapeutic applications.

Growth factors of stem cell niche extend the life-span of precision-cut intestinal slices in culture: A proof-of-concept study.

Precision-cut intestinal slices (PCIS) is an ex vivo culture technique that found its applications in toxicology, drug transport and drug metabolism testing, as well as in fibrosis research. The main limiting factor of PCIS as experimental model is the relatively short viability of tissue slices. Here, we describe a strategy for extending the life-span of PCIS during culture using medium that is routinely used for growing intestinal organoids. Mouse and rat PCIS cultured in standard medium progressively showed low ATP/protein content and severe tissue degradation, indicating loss of tissue viability. In turn, organoid medium, containing epithelial growth factor (EGF), Noggin and R-spondin, maintained significantly higher ATP/protein levels and better preserved intestinal architecture of mouse PCIS at 96 h. In contrast, organoid medium that additionally contained Wnt, had a clear positive effect on the ATP content of rat PCIS during 24 h of culture, but not on slice histomorphology. Our proof-of-concept study provides early evidence that employing organoid medium for PCIS culture improved tissue viability during extended incubation. Enabling lasting PCIS cultures will greatly widen their range of applications in predicting long-term intestinal toxicity of xenobiotics and elucidating their mechanism of action, among others.