Integrated analysis of gut metabolome, microbiome, and exfoliome data in an equine model of intestinal injury.

BACKGROUND: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions. METHODS: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches. RESULTS: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa. CONCLUSIONS: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract.

Investigating the Effect of an Anti-Inflammatory Drug in Determining NURR1 Expression and Thus Exploring the Progression of Parkinson's Disease.

Nonsteroidal anti-inflammatory drugs are the most widely used drugs for Parkinson's disease (PD), of which ibuprofen shows positive effects in suppressing symptoms; however, the associated risk needs to be addressed in different pathological stages. Initially, we developed an initial and advanced stage of the Parkinson disease mouse model by intraperitoneal injection of MPTP (20 mg/kg; 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine) for 10 and 20 days, respectively. Subsequently, ibuprofen treatment was administered for 2 months, and a pole test, rotarod test, histology, immunohistochemistry, and western blotting were performed to determine neuronal motor function. Histological analysis for 10 days after mice were injected with MPTP showed the onset of neurodegeneration and cell aggregation, indicating the initial stages of Parkinson's disease. Advanced Parkinson's disease was marked by Lewy body formation after another 10 days of MPTP injection. Neurodegeneration reverted after ibuprofen therapy in initial Parkinson's disease but not in advanced Parkinson's disease. The pole and rotarod tests confirmed that motor activity in the initial Parkinson disease with ibuprofen treatment recovered (p<0.01). However, no improvement was observed in the ibuprofen-treated mice with advanced disease mice. Interestingly, ibuprofen treatment resulted in a significant improvement (p<0.01) in NURR1 (Nuclear receptor-related 1) expression in mice with early PD, but no substantial improvement was observed in its expression in mice with advanced PD. Our findings indicate that NURR1 exerts anti-inflammatory and neuroprotective effects. Overall, NURR1 contributed to the effects of ibuprofen on PD at different pathological stages.

Adenomyotic Lesions Are Induced in the Mouse Uterus after Exposure to NSAID and EE2 Mixtures at Environmental Doses.

The aim of this study was to assess the long-term effect of exposure to environmentally relevant doses of non-steroidal anti-inflammatory drugs (NSAIDs; ibuprofen, and diclofenac) and 17ß-ethinylestradiol (EE2) on the mouse uterus. NSAID-EE2 mixtures were administered in the drinking water from gestational day 8 until 8 weeks post-birth (i.e., during embryo development, lactation, puberty, and sexual maturity). The incidence of adenomyosis lesions (presence of endometrial glands in the inner myometrium) increased up to 60% in the uterus of 8-week-old exposed females (F1) and to 85% in F2 females (exposed father). Histological analysis revealed aberrant proliferation and apoptosis, vacuolization of epithelial cells, and increased incidence of abnormal glands in the luminal and glandular epithelium in F1 and F2 uteri. Moreover, myofibroblast proportion (alpha-smooth muscle actin (α-SMA) expression analysis) and collagen expression (Picrosirius red stain; a fibrosis hallmark) were increased in F1 and F2 endometrium. Connexin-43 was aberrantly distributed in the endometrial stroma and glands of F1 and F2 uteri. Conversely, uterine 17ß-estradiol and progesterone levels were not affected in F1 and F2 females. These findings demonstrated that in mice, chronic exposure to NSAID and EE2 mixtures at environmental doses intergenerationally affects uterine physiology, particularly the endometrium. It may serve as a model to study the pathophysiology of human adenomyosis.

Ocular surface parameter changes in the untreated fellow eye after unilateral cataract surgery with short-term administration of anti-inflammatory eye drops.

This study aimed to investigate the changes in clinical parameters of dry eye disease and meibomian gland dysfunction in both the operated and untreated fellow eyes of patients who underwent unilateral cataract surgery with the short-term administration of anti-inflammatory eye drops in the surgical eye. The medical charts of 57 consecutive patients who underwent unilateral cataract surgery and received 1% prednisolone acetate and non-steroidal anti-inflammatory drug (NSAID, 0.1% bromfenac sodium) eye drops were reviewed. The preoperative ocular surface disease index questionnaire score (38.9 ± 20.5) decreased significantly to 15.2 ± 16.4 at post-surgical 1 week and further to 12.8 ± 11.4 after 1 month. Although meibum quality grade increased and corneal sensitivity decreased at 1 week in operated eyes, corneal erosion scores and Sjogren's International Collaborative Clinical Alliance ocular staining scores even improved over a month in the untreated fellow eyes. The tear matrix metalloproteinase (MMP)-9 grade decreased in both operated eyes and untreated fellow eyes after 1 month from surgery. In conclusion, the short-term topical anti-inflammatory treatment using steroid and NSAID eye drops in the operated eye after cataract surgery decreased subjective ocular surface discomfort and improved ocular surface staining scores and tear MMP-9 expression in the untreated fellow eyes.

A premature stop codon in the CYP2C19 gene may explain the unexpected sensitivity of vultures to diclofenac toxicity.

The unintended environmental exposure of vultures to diclofenac has resulted in the deaths of millions of old-world vultures on the Asian subcontinent. While toxicity has been since associated with a long half-life of elimination and zero order metabolism, the actual constraint in biotransformation is yet to be clarified. For this study we evaluated if the evident zero order metabolism could be due to defects in the CYP2C9/2C19 enzyme system. For this, using whole genome sequencing and de-novo transcriptome alignment, the vulture CYP2C19 open reading frame was identified through Splign analysis. The result sequence analysis revealed the presence of a premature stop codon on intron 7 of the identified open reading frame. Even if the stop codon was not present, amino acid residue analysis tended to suggest that the enzyme would be lower in activity than the equivalent human enzyme, with differences present at sites 105, 286 and 289. The defect was also conserved across the eight non-related vultures tested. From these results, we conclude that the sensitivity of the old-world vultures to diclofenac is due to the non-expression of a viable CYP2C19 enzyme system. This is not too dissimilar to the effects seen in certain people with a similar defective enzyme.

[Development of Novel COX-2 Imaging Agents for the Diagnosis of Brain Lesions].

Cyclooxygenase-2 (COX-2) has attracted attention as a biomarker for neurodegenerative brain diseases. The aim of this study was to develop a COX-2 imaging agent for positron emission tomography (PET) that binds to and emits radiation from COX-2 in the central nervous system to diagnose brain lesions related to COX-2. To this end, the development of PET imaging probes by derivatizing non-steroidal anti-inflammatory drugs that bind to COX-2 was investigated. Herein, we present the findings of a series of studies on indomethacin and nimesulide derivatives. All five 11C-labeled indomethacin derivatives showed low brain uptake and were rapidly metabolized in vivo, indicating that they are inadequate COX-2 imaging agents. However, the evaluation of 11C-labeled indomethacin derivatives revealed an inverse relationship between the amount taken up by the brain and the lipophilicity of the compound, and that P-glycoprotein (P-gp) may be responsible for the low brain uptake of 11C-labeled indomethacin derivatives. To overcome the problems associated with 11C-labeled indomethacin derivatives, nimesulide was selected as a novel COX-2 imaging agent. Although the nimesulide derivatives were less lipophilic and unaffected by P-gp, all three 11C-labeled nimesulide derivatives showed low brain uptake and were rapidly metabolized. However, the 11C-labeled nimesulide derivatives were partially useful as brain-targeted COX-2 imaging agents because they bound specifically to COX-2 in the brain of mice and successfully imaged the regional brain distribution associated with COX-2. In the development of COX-2 imaging agents, in vivo stability of the compounds is a future objective.

Innovative insight into the mechanism of enantioselective skin permeation for chiral drugs.

OBJECTIVES: A profound comprehension of the molecular mechanisms underpinning the enantioselective transdermal permeation of chiral drugs is critical in the design and assessment of transdermal preparations. The primary objective of this study is to investigate the distinct skin permeation behaviors exhibited by enantiomers of non-steroidal anti-inflammatory drugs (NSAIDs) and elucidate the intricate molecular mechanism at play. METHODS: In vitro and in vivo transdermal permeation studies of chiral NSAIDs were performed using transdermal patch and solution system. Chiral interaction between NSAIDs enantiomers and synthesized chiral ceramide present in the skin was characterized to clarify the different transdermal behaviors. RESULTS: The S-enantiomers of NSAIDs exhibited higher permeability through the skin than R-enantiomer in vitro (1.5-fold) and in vivo (2.0-fold), which was attributed to a stronger interaction between S-enantiomer and ceramide caused by more favorable spatial conformations. S-enantiomer required lower activation energy (24.4 kJ/mol) and Gibbs energy (43.3 kJ/mol), which was favorable in forming the H-bond with ceramide in the skin, resulting in more permeation. CONCLUSION: This research furnished an innovative comprehension of the molecular underpinnings governing the enantioselective permeation of drug enantiomers through the skin, fostering the minimization of undesired enantiomer ingestion (distomers) and amplifying therapeutic efficiency.

Urolithin A conjugation with NSAIDs inhibits its glucuronidation and maintains improvement of Caco-2 monolayers' barrier function.

Urolithin A (UA) is an ellagitannin-derived postbiotic metabolite which emerged as a promising health-boosting agent, promoting mitophagy, improving skeletal muscle function, and suppressing the inflammatory response. However, phase II intestinal metabolism severely limits its biopotency, leading to the formation of nonactive glucuronides. To address this constraint, a set of new UA derivatives (UADs), conjugated with nonsteroidal anti-inflammatory drugs (NSAIDs), was synthesized. The bioavailability and inhibitory activity of UADs against UA-glucuronidation were evaluated using differentiated Caco-2 cell monolayers. Parallelly, after the administration of tested substances, the transepithelial electrical resistance (TEER) of the cell monolayers was continuously monitored using the CellZscope device. Though investigated UADs did not penetrate Caco-2 monolayers, all of them significantly suppressed the glucuronidation rate of UA, while conjugates with diclofenac increased the concentration of free molecule on the basolateral side. Moreover, esters of UA with diclofenac (DicloUA) and aspirin (AspUA) positively influenced cell membrane integrity. Western blot analysis revealed that some UADs, including DicloUA, increased the expression of pore-sealing tight junction proteins and decreased the level of pore-forming claudin-2, which may contribute to their beneficial activity towards the barrier function. To provide comprehensive insight into the mechanism of action of DicloUA, Caco-2 cells were subjected to transcriptomic analysis. Next-generation sequencing (NGS) uncovered substantial changes in the expression of genes involved, for instance, in multivesicular body organization and zinc ion homeostasis. The results presented in this study offer new perspectives on the beneficial effects of modifying UA's structure on its intestinal metabolism and bioactivity in vitro.

The molecular basis of dapsone activation of CYP2C9-catalyzed nonsteroidal anti-inflammatory drug oxidation.

Positive heterotropic cooperativity, or "activation," results in an instantaneous increase in enzyme activity in the absence of an increase in protein expression. Thus, cytochrome P450 (CYP) enzyme activation presents as a potential drug-drug interaction mechanism. It has been demonstrated previously that dapsone activates the CYP2C9-catalyzed oxidation of a number of nonsteroidal anti-inflammatory drugs in vitro. Here, we conducted molecular dynamics simulations (MDS) together with enzyme kinetic investigations and site-directed mutagenesis to elucidate the molecular basis of the activation of CYP2C9-catalyzed S-flurbiprofen 4'-hydroxylation and S-naproxen O-demethylation by dapsone. Supplementation of incubations of recombinant CYP2C9 with dapsone increased the catalytic efficiency of flurbiprofen and naproxen oxidation by 2.3- and 16.5-fold, respectively. MDS demonstrated that activation arises predominantly from aromatic interactions between the substrate, dapsone, and the phenyl rings of Phe114 and Phe476 within a common binding domain of the CYP2C9 active site, rather than involvement of a distinct effector site. Mutagenesis of Phe114 and Phe476 abrogated flurbiprofen and naproxen oxidation, and MDS and kinetic studies with the CYP2C9 mutants further identified a pivotal role of Phe476 in dapsone activation. MDS additionally showed that aromatic stacking interactions between two molecules of naproxen are necessary for binding in a catalytically favorable orientation. In contrast to flurbiprofen and naproxen, dapsone did not activate the 4'-hydroxylation of diclofenac, suggesting that the CYP2C9 active site favors cooperative binding of nonsteroidal anti-inflammatory drugs with a planar or near-planar geometry. More generally, the work confirms the utility of MDS for investigating ligand binding in CYP enzymes.

Interleukin-13 Mediates Non-Steroidal Anti-Inflammatory-Drug-Induced Small Intestinal Mucosal Injury with Ulceration.

Non-steroidal anti-inflammatory drugs (NSAIDs), which are antipyretics and analgesics, cause gastrointestinal disorders, such as inflammation and ulcers. To prescribe NSAIDs more safely, it is important to clarify the mechanism of NSAID-induced gastrointestinal mucosal injury. However, there is a paucity of studies on small intestinal mucosal damage by NSAIDs, and it is currently unknown whether inflammation and ulceration also occur in the small intestine, and whether mediators are involved in the mechanism of injury. Therefore, in this study, we created an animal model in which small intestinal mucosal injury was induced using NSAIDs (indomethacin; IDM). Focusing on the dynamics of immune regulatory factors related to the injury, we aimed to elucidate the pathophysiological mechanism involved. We analyzed the pathological changes in the small intestine, the expression of immunoregulatory factors (cytokines), and identified cytokine secretion and expression cells from isolated lamina propria mononuclear cells (LPMCs). Ulcers were formed in the small intestine by administering IDM. Although the mRNA expression levels of IL-1ß, IL-6, and TNFα were decreased on day 7 after IDM administration, IL-13 mRNA levels increased from day 3 after IDM administration and remained high even on day 7. The IL-13 mRNA expression and the secretion of IL-13 were increased in small intestinal LPMCs isolated from the IDM-treated group. In addition, we confirmed that IL-13 was expressed in CD4-positive T cells. These results provided new evidence that IL-13 production from CD4-positive T cells in the lamina propria of the small intestine contributes to NSAID-induced mucosal injury.

Acetylsalicylic Acid Supplementation Affects the Neurochemical Phenotyping of Porcine Duodenal Neurons.

Aspirin (ASA) is a popular nonsteroidal anti-inflammatory drug (NSAID), which exerts its therapeutic properties through the inhibition of cyclooxygenase (COX) isoform 2 (COX-2), while the inhibition of COX-1 by ASA results in the formation of gastrointestinal side effects. Due to the fact that the enteric nervous system (ENS) is involved in the regulation of digestive functions both in physiological and pathological states, the aim of this study was to determine the influence of ASA on the neurochemical profile of enteric neurons in the porcine duodenum. Our research, conducted using the double immunofluorescence technique, proved an increase in the expression of selected enteric neurotransmitters in the duodenum as a result of ASA treatment. The mechanisms of the visualized changes are not entirely clear but are probably related to the enteric adaptation to inflammatory conditions resulting from aspirin supplementation. A detailed understanding of the role of the ENS in the development of drug-induced inflammation will contribute to the establishment of new strategies for the treatment of NSAID-induced lesions.

Effects of non-steroidal anti-inflammatory drug (ibuprofen) in low and high dose on stemness and biological characteristics of human dental pulp-derived mesenchymal stem cells.

PURPOSE: The effect of ibuprofen, an NSAID, on biological characteristics such as proliferation, viability, DNA damage and cell cycle in dental pulp derived stem cells (DPSCs) can be important for regenerative medicine. Our aim is to investigate how low and high doses of ibuprofen affect stem cell characteristics in DPSCs. MATERIALS AND METHODS: DPSCs were isolated from human teeth and characterized by flow cytometry and differentiation tests. Low dose (0.1 mmol/L) and high dose (3 mmol/L) ibuprofen were administered to DPSCs. Surface markers between groups were analyzed by immunofluorescence staining. Membrane depolarization, DNA damage, viability and cell cycle analysis were performed between groups using biological activity test kits. Cellular proliferation was measured by the MTT and cell count kit. Statistical analyzes were performed using GraphPad Prism software. RESULTS: High dose ibuprofen significantly increased CD44 and CD73 expression in DPSCs. High-dose ibuprofen significantly reduced mitochondrial membrane depolarization in DPSCs. It was determined that DNA damage in DPSCs decreased significantly with high dose ibuprofen. Parallel to this, cell viability increased significantly in the ibuprofen applied groups. High-dose ibuprofen was found to increase mitotic activity in DPSCs. Proliferation in DPSCs increased in parallel with the increase in mitosis stage because of high-dose ibuprofen administration compared to the control and low-dose ibuprofen groups. Our proliferation findings appeared to support cell cycle analyses. CONCLUSION: High dose ibuprofen improved the immunophenotypes and biological activities of DPSCs. The combination of ibuprofen in the use of DPSCs in regenerative medicine can make stem cell therapy more effective.

Characterising diflunisal as a transthyretin kinetic stabilizer at relevant concentrations in human plasma using subunit exchange.

Transthyretin (TTR) dissociation is the rate limiting step for both aggregation and subunit exchange. Kinetic stabilisers, small molecules that bind to the native tetrameric structure of TTR, slow TTR dissociation and inhibit aggregation. One such stabiliser is the non-steroidal anti-inflammatory drug (NSAID), diflunisal, which has been repurposed to treat TTR polyneuropathy. Previously, we compared the efficacy of diflunisal, tafamidis, tolcapone, and AG10 as kinetic stabilisers for transthyretin. However, we could not meaningfully compare diflunisal because we were unsure of its plasma concentration after long-term oral dosing. Herein, we report the diflunisal plasma concentrations measured by extraction, reversed phase HPLC separation, and fluorescence detection after long-term 250 mg BID oral dosing in two groups: a placebo-controlled diflunisal clinical trial group and an open-label Japanese polyneuropathy treatment cohort. The measured mean diflunisal plasma concentration from both groups was 282.2 µM ± 143.7 µM (mean ± standard deviation). Thus, quantification of TTR kinetic stabilisation using subunit exchange was carried out at 100, 200, 300, and 400 µM diflunisal concentrations, all observed in patients after 250 mg BID oral dosing. A 250 µM diflunisal plasma concentration reduced the wild-type TTR dissociation rate in plasma by 95%, which is sufficient to stop transthyretin aggregation, consistent with the clinical efficacy of diflunisal for ameliorating transthyretin polyneuropathy.

Grape Pomace Polyphenols as a Source of Compounds for Management of Oxidative Stress and Inflammation-A Possible Alternative for Non-Steroidal Anti-Inflammatory Drugs?

Flavonoids, stilbenes, lignans, and phenolic acids, classes of polyphenols found in grape pomace (GP), were investigated as an important alternative source for active substances that could be used in the management of oxidative stress and inflammation. The benefic antioxidant and anti-inflammatory actions of GP are presented in the literature, but they are derived from a large variety of experimental in vitro and in vivo settings. In these in vitro works, the decrease in reactive oxygen species, malondialdehyde, and thiobarbituric acid reactive substances levels and the increase in glutathione levels show the antioxidant effects. The inhibition of nuclear factor kappa B and prostaglandin E2 inflammatory pathways and the decrease of some inflammatory markers such as interleukin-8 (IL-8) demonstrate the anti-inflammatory actions of GP polyphenols. The in vivo studies further confirmed the antioxidant (increase in catalase, superoxide dismutase and glutathione peroxidase levels and a stimulation of endothelial nitric oxide synthase -eNOS gene expression) and anti-inflammatory (inhibition of IL-1𝛼, IL-1ß, IL-6, interferon-𝛾, TNF-α and C-reactive protein release) activities. Grape pomace as a whole extract, but also different individual polyphenols that are contained in GP can modulate the endogenous pathway responsible in reducing oxidative stress and chronic inflammation. The present review analyzed the effects of GP in oxidative stress and inflammation, suggesting that it could become a valuable therapeutic candidate capable to reduce the aforementioned pathological processes. Grape pomace extract could become an adjuvant treatment in the attempt to reduce the side effects of the classical anti-inflammatory medication like non-steroidal anti-inflammatory drugs (NSAIDs).

NSAIDs affect dendritic cell cytokine production.

BACKGROUND: Immunotherapy is now considered as the new pillar in treatment of cancer patients. Dendritic cells (DCs) play an essential role in stimulating anti-tumor immune responses, as they are capable of cross-presenting exogenous tumor antigens in MHCI complexes to activate naïve CD8+ T cells. Analgesics, like non-steroid anti-inflammatory drugs (NSAIDs), are frequently given to cancer patients to help relieve pain, however little is known about their impact on DC function. METHODS: Here, we investigated the effect of the NSAIDs diclofenac, ibuprofen and celecoxib on the three key processes of DCs required for proper CD8+ cytotoxic T cell induction: antigen cross-presentation, co-stimulatory marker expression, and cytokine production. RESULTS: Our results show that TLR-induced pro- and anti-inflammatory cytokine excretion by human monocyte derived and murine bone-marrow derived DCs is diminished after NSAID exposure. CONCLUSIONS: These results indicate that various NSAIDs can affect DC function and warrant further investigation into the impact of NSAIDs on DC priming of T cells and cancer immunotherapy efficacy.

Transplantation of adipose-derived mesenchymal stem cells ameliorates acute hepatic injury caused by nonsteroidal anti-inflammatory drug diclofenac sodium in female rats.

BACKGROUND: Although the beneficial role of adipose-derived mesenchymal stem cells (AD-MSCs) in acute liver injury has been addressed by numerous studies employing different liver injury inducers, the role of rat AD-MSCs (rAD-MSCs) in diclofenac sodium (DIC) - induced acute liver injury has not yet been clarified. OBJECTIVE: This study aimed to investigate whether rat adipose- rAD-MSCs injected intraperitoneal could restore the DIC-induced hepatoxicity. METHODS: Hepatotoxicity was induced by DIC in a dose-based manner, after which intraperitoneal injection of rAD-MSCs was performed. RESULTS: Here, the transplanted cells migrated to the injured liver, and this was evidenced by detecting the specific SRY in the liver samples. After administering DIC, a significant decrease in body weight, survival rate, serum proteins, antioxidants, anti-apoptotic gene expression, and certain growth factors, whereas hepatic-specific markers, pro-inflammatory mediators, and oxidative, pro-apoptotic, and ER-stress markers were elevated. These adverse effects were significantly recovered after engraftment with rAD-MSCs. This was evidenced by enhanced survival and body weight, improved globulin and albumin values, increased expression of SOD, GPx, BCL-2, VEGF, and FGF-basic expression, and decreased serum ALT, AST, ALP, and total bilirubin. rAD-MSCs also reduced liver cell damage by suppressing the expression of MDA, IL-1B, IL-6, BAX, JNK, GRP78/BiP, CHOP, XBP-1, and cleaved caspase 3/7. Degenerative hepatic changes and multifocal areas of fatty change within liver cells were observed in DIC-received groups. These changes were improved with the transplantation of rAD-MSCs. CONCLUSIONS: We could conclude that targeted AD-MSCs could be applied to reduce hepatic toxicity caused by NSAIDs (DIC).

Non-steroidal anti-inflammatory drugs caused an outbreak of inflammation and oxidative stress with changes in the gut microbiota in rainbow trout (Oncorhynchus mykiss).

One of the main contributors to pharmaceutical pollution of surface waters are non-steroidal anti-inflammatory drugs (NSAIDs) that contaminate the food chain and affect non-target water species. As there are not many studies focusing on toxic effects of NSAIDs on freshwater fish species and specially effects after dietary exposure, we selected rainbow trout (Oncorhynchus mykiss) as the ideal model to examine the impact of two NSAIDs - diclofenac (DCF) and ibuprofen (IBP). The aim of our study was to test toxicity of environmentally relevant concentrations of these drugs together with exposure doses of 100× higher, including their mixture; and to deepen knowledge about the mechanism of toxicity of these drugs. This study revealed kidneys as the most affected organ with hyalinosis, an increase in oxidative stress markers, and changes in gene expression of heat shock protein 70 to be signs of renal toxicity. Furthermore, hepatotoxicity was confirmed by histopathological analysis (i.e. dystrophy, congestion, and inflammatory cell increase), change in biochemical markers, increase in heat shock protein 70 mRNA, and by oxidative stress analysis. The gills were locally deformed and showed signs of inflammatory processes and necrotic areas. Given the increase in oxidative stress markers and heat shock protein 70 mRNA, severe impairment of oxygen transport may be one of the toxic pathways of NSAIDs. Regarding the microbiota, an overgrowth of Gram-positive species was detected; in particular, significant dysbiosis in the Fusobacteria/Firmicutes ratio was observed. In conclusion, the changes observed after dietary exposure to NSAIDs can influence the organism homeostasis, induce ROS production, potentiate inflammations, and cause gut dysbiosis. Even the environmentally relevant concentration of NSAIDs pose a risk to the aquatic ecosystem as it changed O. mykiss health parameters and we assume that the toxicity of NSAIDs manifests itself at the level of mitochondria and proteins.

Optimization of topical formulations using a combination of in vitro methods to quantify the transdermal passive diffusion of drugs.

This paper describes a new approach to the early-stage optimization of topical products and selection of lead formulation candidates. It demonstrates the application of open flow microperfusion in vitro in conjunction with the Franz diffusion cell to compare time-resolved, 24-hour profiles of diclofenac passive diffusion through all skin layers (including the skin barrier, dermis, and subcutis) resulting from nine topical formulations of different composition. The technique was successfully validated for in vitro sampling of diclofenac in interstitial fluid. A multi-compartmental model integrating the two datasets was analyzed and revealed that the passive diffusion of diclofenac through the dermis and subcutis does not correlate with its diffusion through the skin barrier and cannot be predicted using Franz diffusion cell data alone. The combined application of the two techniques provides a new, convenient tool for product development and selection enabling the comparison of topical formulation candidates and their impact on drug delivery through all skin layers. This approach can also generate the experimental data required to improve the robustness of mechanistic PBPK models, and when combined with clinical sampling via open flow microperfusion - for the development of better in vivo-in vitro correlative models.

Relationship between the risk of idiosyncratic drug toxicity and formation and degradation profiles of acyl-glucuronide metabolites of nonsteroidal anti-inflammatory drugs in rat liver microsomes.

Acyl glucuronides (AGs) are considered to cause idiosyncratic drug toxicity (IDT), and evaluating the chemical instability of AGs may be useful for predicting the IDT risk of novel drug candidates. However, AGs show variations in their chemical instability, degree of formation, and enzymatic hydrolysis. Therefore, we evaluated the degree of AG formation, enzymatic hydrolysis, and chemical instability in liver microsomes and their relationship with IDT risk. Nonsteroidal anti-inflammatory drugs (NSAIDs) were classified into three categories in terms of their IDT risk as parent drugs: safe (SA), warning (WA), and withdrawn (WDN). To evaluate the enzymatic and non-enzymatic degradation of AG, the parent drugs were incubated with rat liver microsomes in the absence or presence of AG hydrolase inhibitors. The degree of AG formation and disappearance was considered as the rate constant. For all NSAIDs investigated, the number of AGs formed notably increased following addition of AG hydrolase inhibitors. Particularly, AG was produced by WDN drugs at a lower level than that produced by WA and SA drugs in the absence of AG hydrolase inhibitors but was significantly increased after adding AG hydrolase inhibitors. The rate constants of AG formation and non-enzymatic AG disappearance did not significantly differ among the WDN, WA, and SA drugs, whereas the rate constant of enzymatic AG disappearance of WDN drugs tended to be higher than those of WA and SA drugs. In conclusion, we evaluated the enzymatic degradation and chemical instability of AG by simultaneously producing it in liver microsomes. This method enables evaluation of AG degradation without preparing AG. Moreover, we determined the relationship between enzymatic AG degradation in rat liver microsomes and IDT risk.

SNX10-mediated degradation of LAMP2A by NSAIDs inhibits chaperone-mediated autophagy and induces hepatic lipid accumulation.

Rationale: While some non-steroidal anti-inflammatory drugs (NSAIDs) are reported to induce hepatic steatosis, the molecular mechanisms are poorly understood. This study presented the mechanism by which NSAIDs induce hepatic lipid accumulation. Methods: Mouse primary hepatocytes and HepG2 cells were used to examine the underlying mechanism of NSAID-induced hepatic steatosis. Lipid accumulation was measured using Nile-red assay and BODIPY 493/503. The activity of chaperone-mediated autophagy (CMA) was determined by western blotting, qRT-PCR, and confocal imaging. The effect of NSAID on CMA inhibition was evaluated in vivo using diclofenac and CMA activator (AR7) administered mice. Results: All tested NSAIDs in this study accumulated neutral lipids in hepatocytes, diclofenac having demonstrated the most potency in that regard. Diclofenac-induced lipid accumulation was confirmed in both mouse primary hepatocytes and the liver of mice. NSAIDs inhibited CMA, as reflected by the decreased expression of lysosome-associated membrane glycoprotein 2 isoform A (LAMP2A) protein, the increased expression of CMA substrate proteins such as PLIN2, and the decreased activity of photoactivatable KFERQ-PAmCherry reporter. Reactivation of CMA by treatment with AR7 or overexpression of LAMP2A inhibited diclofenac-induced lipid accumulation and hepatotoxicity. Upregulation of sorting nexin 10 (SNX10) via the CHOP-dependent endoplasmic reticulum stress response and thus maturation of cathepsin A (CTSA) was shown to be responsible for the lysosomal degradation of LAMP2A by diclofenac. Conclusion: We demonstrated that NSAIDs induced SNX10- and CTSA-dependent degradation of LAMP2A, thereby leading to the suppression of CMA. In turn, impaired CMA failed to degrade PLIN2 and disrupted cellular lipid homeostasis, thus leading to NSAID-induced steatosis and hepatotoxicity.