Characterization of individual bile acids in vivo utilizing a novel low bile acid mouse model.

Bile acids (BAs) are signaling molecules synthesized in the liver initially by CYP7A1 and CYP27A1 in the classical and alternative pathways, respectively. BAs are essential for cholesterol clearance, intestinal absorption of lipids, and endogenous modulators of farnesoid x receptor (FXR). FXR is critical in maintaining BA homeostasis and gut-liver crosstalk. Complex reactions in vivo and the lack of suitable animal models impede our understanding of the functions of individual BAs. In this study, we characterized the in vivo effects of three-day feeding of cholic acid (CA), deoxycholic acid (DCA), or ursodeoxycholic acid (UDCA) at physiological/non-hepatotoxic concentrations in a novel low-BA mouse model (Cyp7a1-/-/Cyp27a1-/-, DKO). Liver injury, BA levels and composition and BA signaling by the FXR-fibroblast growth factor 15 (FGF15) axis were determined. Overall, higher basal inflammation and altered lipid metabolism in DKO mice might be associated with low BAs. CA, DCA, and UDCA feeding activated FXR signals with tissue specificity. Dietary CA and DCA similarly altered tissue BA profiles to be less hydrophobic, while UDCA promoted a more hydrophobic tissue BA pool with the profiles shifted toward non-12α-OH BAs and secondary BAs. However, UDCA did not offer any overt protective effects as expected. These findings allow us to determine the precise effects of individual BAs in vivo on BA-FXR signaling and overall BA homeostasis in liver physiology and pathologies.

Metabolomics study reveals increased deoxycholic acid contributes to deoxynivalenol-mediated intestinal barrier injury.

AIMS: Deoxynivalenol (DON), namely vomitoxin, is one of the most prevalent fungal toxins in cereal crops worldwide. However, the underlying toxic mechanisms of DON remain largely unknown. MAIN METHODS: DON exposure-caused changes in the murine plasma metabolome and gut microbiome were investigated by an LC-MS/MS-based nontargeted metabolomics approach and sequencing of 16S rRNA in fecal samples, respectively. Cellular models were then used to validate the findings from the metabolomics study. KEY FINDINGS: DON exposure increased intestinal barrier permeability evidenced by its-mediated decrease in colonic Claudin 5 and E-cadherin, as well as increases in colonic Ifn-γ, Cxcl9, Cxcl10, and Cxcr3. Furthermore, DON exposure resulted in a significant increase in murine plasma levels of deoxycholic acid (DCA). Also, DON exposure led to gut microbiota dysbiosis, which was associated with DON exposure-caused increase in plasma DCA. In addition, we found not only DON but also DCA dose-dependently caused a significant increase in the levels of IFN-γ, CXCL9, CXCL10, and/or CXCR3, as well as a significant decrease in the expression levels of Claudin 5 and/or E-cadherin in the human colonic epithelial cells (NCM460). SIGNIFICANCE: DON-mediated increase in DCA contributes to DON-caused intestinal injury. DCA may be a potential therapeutic target for DON enterotoxicity.

Deoxycholic acid induces gastric intestinal metaplasia by activating STAT3 signaling and disturbing gastric bile acids metabolism and microbiota.

Intestinal metaplasia (IM) is the inevitable precancerous stage to develop intestinal-type gastric cancer (GC). Deoxycholic acid (DCA) is the main bile acid (BA) component of duodenogastric reflux and has shown an increased concentration during the transition from chronic gastritis to IM associated with continued STAT3 activation. However, the mechanisms underlying how DCA facilitates IM in the gastric epithelium need exploration. We evaluated IM and bile reflux in corpus tissues from 161 subjects undergoing GC screening. Cell survival and proliferation, proinflammatory cytokine expression and TGR5/STAT3/KLF5 axis activity were measured in normal human gastric cells, cancer cells, and organoid lines derived from C57BL/6, FVB/N and insulin-gastrin (INS-GAS) mice treated with DCA. The effects of DCA on IM development were determined in INS-GAS mice with long-term DCA supplementation, after which the gastric bacterial and BA metabolic profiles were measured by 16S rRNA gene sequencing and LC-MS. We revealed a BA-triggered TGR5/STAT3/KLF5 pathway in human gastric IM tissues. In gastric epithelial cells, DCA promoted proliferation and apoptotic resistance, upregulated proinflammatory cytokines and IM markers, and facilitated STAT3 phosphorylation, nuclear accumulation and DNA binding to the KLF5 promoter. DCA triggered STAT3 signaling and the downstream IM marker KLF5 in mouse gastric organoids in vitro and in vivo. In INS-GAS mice, DCA promoted the accumulation of serum total BAs and accelerated the stepwise development of gastric IM and dysplasia. DCA induced gastric environmental alterations involving abnormal BA metabolism and microbial dysbiosis, in which the Gemmobacter and Lactobacillus genera were specifically enriched. Lactobacillus genus enrichment was positively correlated with increased levels of GCA, CA, T-α-MCA, TCA and ß-MCA in DCA-administrated INS-GAS mice. DCA promotes nuclear STAT3 phosphorylation, which mediates KLF5 upregulation associated with gastric inflammation and IM development. DCA disturbs the gastric microbiome and BA metabolism homeostasis during IM induction.

Adverse effects of deoxycholic acid in submandibular glands, submental, inguinal and subplantar regions: a study in rats.

OBJECTIVE: We aimed to evaluate the effects of the deoxycholic acid (DCA) in the submental and subplantar regions of rats, and to histologically analyze the changes caused in the submandibular glands, soft tissues of the paw, and inguinal adipose tissue. MATERIAL AND METHODS: Sixty male Wistar rats were divided into DCA and control (CG) groups. DCA was injected in the submental, inguinal, and subplantar regions, and saline was injected in the CG. The animals were euthanized after 24 h and at 7 and 21 days. RESULTS: The DCA group showed edema in the submental region in 24 h and in the paw in all experimental times. In the paw there were also erythema and ulceration in 7 days, and alopecia after 21 days. At 21 days, a few animals also showed erythema and ulceration in paw; however, there was no significant difference from CG. Histological analysis of the paw showed an intense inflammatory process, with a predominance of neutrophils, lymphocytes, and plasma cells in 24 h and 7 days. In the adipose tissue, we observed loss of architecture and inflammatory infiltrate, followed with a lower number of adipose cells, and at 21 days, fibroplasia. In the submandibular glands we observed inflammatory infiltration, loss of tissue architecture, and fibrosis. CONCLUSIONS: DCA produces a significant inflammatory process in the structures. It can cause skin ulcerations and, in salivary glands, it causes loss of tissue architecture and fibrosis. CLINICAL RELEVANCE: There has been growing increase in the use of DCA for aesthetic purposes by health care providers. Due to the presence of important anatomical structures in the submental region, constant vigilance is required to report new adverse effects.

Antipruritic Effect of Nalbuphine, a Kappa Opioid Receptor Agonist, in Mice: A Pan Antipruritic.

Antipruritic effects of kappa opioid receptor (KOR) agonists have been shown in rodent models of acute and chronic scratching (itchlike behavior). Three KOR agonists, nalfurafine, difelikefalin, and nalbuphine, are in clinical studies for antipruritic effects in chronic itch of systemic and skin diseases. Nalfurafine (in Japan) and difelikefalin (in the USA) were approved to be used in the treatment of chronic itch in hemodialysis patients. The FDA-approved nalbuphine has been used in clinic for over 40 years, and it is the only narcotic agonist that is not scheduled. We aimed to study (a) antiscratch activity of nalbuphine against TAT-HIV-1 protein (controls HIV transcription)-, deoxycholic acid (DCA, bile acid)-, and chloroquine (CQ)-induced scratching in a mouse model of acute itch; and (b) whether the effect of nalbuphine is produced via KORs. First, dose-responses were developed for pruritogens. Mice were pretreated with nalbuphine (0.3-10 mg/kg) and then a submaximal dose of pruritogens were administered and the number of scratching bouts was counted. To study if the antiscratch effect of nalbuphine is produced via KOR, we used KOR knock out mice and pharmacologic inhibition of KORs using nor-binaltorphimine, a KOR antagonist. For this aim, we used CQ as a pruritogen. We found that: (a) TAT-HIV-1 protein elicits scratching in a dose-dependent manner; (b) nalbuphine inhibits scratching induced by TAT-HIV-1, DCA, and CQ dose-dependently; and (c) nalbuphine inhibits scratching induced by CQ through KORs. In conclusion, nalbuphine inhibits scratching elicited by multiple pruritogens.

Effect of amphotericin B-deoxycholate (Fungizone) on the mitochondria of Wistar rats' renal proximal tubules cells.

Amphotericin B-deoxycholate (Fungizone [FZ]) is a widely used potent antimycotic drug in spite of its nephrotoxic effect via different mechanisms. The effect of FZ on renal cell bioenergetics is not clear. The current study evaluated the effect of FZ on the bioenergetics of albino rats' isolated renal proximal tubule cells (PTCs). The cytotoxic effect of FZ on the isolated renal cells was assessed by MTT and lactate dehydrogenase (LDH) assays. The effect of FZ on the PTCs uptake (OAT1 and OCT2) and efflux (P-gp and MRP2) transporters was evaluated. Then, the effect of FZ on mitochondria was assessed by studying complexes I-IV activities, lactate assay, oxygen consumption rates (OCR), and western blotting for all mitochondrial complexes. Moreover, the effect of FZ on mitochondrial membrane fluidity (MMF) and fatty acids composition was evaluated. Additionally, the protective effect of coenzyme q10 was studied. Outcomes showed that FZ was cytotoxic to the PTCs in a concentration and time-dependent patterns. FZ significantly inhibited the studied uptake and efflux tubular transporters with inhibition of the mitochondrial complexes activities and parallel increase in lactate production and decrease in OCRs. Finally, FZ significantly reduced the expression of all mitochondrial complexes in addition to significant increase in MMF and MMFA concentration. Coenzyme Q10 was found to significantly decrease FZ-induced cytotoxicity and transporters impairment in the PTC. FZ significantly inhibits bioenergetics of PTC, which may stimulate the cascade of cell death and clinical nephrotoxicity.

Deoxycholic Acid-Induced Gut Dysbiosis Disrupts Bile Acid Enterohepatic Circulation and Promotes Intestinal Inflammation.

BACKGROUND: A Western diet is a risk factor for the development of inflammatory bowel disease (IBD). High levels of fecal deoxycholic acid (DCA) in response to a Western diet contribute to bowel inflammatory injury. However, the mechanism of DCA in the natural course of IBD development remains unanswered. AIMS: The aim of this study is to investigate the effect of DCA on the induction of gut dysbiosis and its roles in the development of intestinal inflammation. METHODS: Wild-type C57BL/6J mice were fed an AIN-93G diet, either supplemented with or without 0.2% DCA, and killed at 24 weeks. Distal ileum and colon tissues were assessed by histopathological analysis. Hepatic and ileal gene expression was examined by qPCR, and the gut microbiota was analyzed by high-throughput 16S rRNA gene sequencing. HPLC-MS was used for fecal bile acid quantification. RESULTS: Mice fed the DCA-supplemented diet developed focal areas of ileal and colonic inflammation, accompanied by alteration of the composition of the intestinal microbiota and accumulation of fecal bile acids. DCA-induced dysbiosis decreased the deconjugation of bile acids, and this regulation was associated with the repressed expression of target genes in the enterohepatic farnesoid X receptor-fibroblast growth factor (FXR-FGF15) axis, leading to upregulation of hepatic de novo bile acid synthesis. CONCLUSIONS: These results suggest that DCA-induced gut dysbiosis may act as a key etiologic factor in intestinal inflammation, associated with bile acid metabolic disturbance and downregulation of the FXR-FGF15 axis.

Orbital Hemorrhagic Necrosis, Globe Rupture, and Death From Intraorbital Injection of 1% Sodium Deoxycholate in a Murine Model.

PURPOSE: Deoxycholic acid (DCA) 1% is an injectable detergent indicated for submental fat reduction, although clinically it is being injected off-label for orbital fat prolapse. It is known to cause severe inflammation, local nerve dysfunction, and tissue necrosis, all of which could be catastrophic in the orbit and periocular region. This study evaluated the effects of periocular DCA on orbital and ocular adnexal tissues in a murine model. METHODS: Mice were treated via split-face intraorbital injections, subcutaneous injections, and topical cornea application with DCA versus phosphate-buffered saline. Whole heads were fixed, decalcified, and sectioned for orbital histology after 1-7 days. Matched pairs of human globes and mouse globes were immersed in either phosphate-buffered saline or 1% DCA for 72 hours. RESULTS: Six of 11 mice receiving intraorbital DCA injections died within minutes. Surviving mice developed severe orbital inflammatory necrosis. All orbits injected with phosphate-buffered saline were clinically and histologically normal. Six mice were treated with lower concentrations of DCA and all developed variable amounts of orbital inflammation, hemorrhage, and globe necrosis. Mice receiving subcutaneous DCA injection to the lower eyelid showed inflammatory necrosis, edema, and lid malposition. Topical application of DCA to mouse corneas caused no external or histologic changes. Human and mouse globes immersed ex vivo in DCA developed corneal edema and cataract formation without observable scleral changes. CONCLUSION: Intraorbital and periocular injection of DCA can cause devastating complications in a murine model, and significant caution is advised for off-label use in the periocular region.

Chronic High-Fat Diet Induces Early Barrett's Esophagus in Mice through Lipidome Remodeling.

Esophageal adenocarcinoma (EAC) incidence has been rapidly increasing, potentially associated with the prevalence of the risk factors gastroesophageal reflux disease (GERD), obesity, high-fat diet (HFD), and the precursor condition Barrett's esophagus (BE). EAC development occurs over several years, with stepwise changes of the squamous esophageal epithelium, through cardiac metaplasia, to BE, and then EAC. To establish the roles of GERD and HFD in initiating BE, we developed a dietary intervention model in C57/BL6 mice using experimental HFD and GERD (0.2% deoxycholic acid, DCA, in drinking water), and then analyzed the gastroesophageal junction tissue lipidome and microbiome to reveal potential mechanisms. Chronic (9 months) HFD alone induced esophageal inflammation and metaplasia, the first steps in BE/EAC pathogenesis. While 0.2% deoxycholic acid (DCA) alone had no effect on esophageal morphology, it synergized with HFD to increase inflammation severity and metaplasia length, potentially via increased microbiome diversity. Furthermore, we identify a tissue lipid signature for inflammation and metaplasia, which is characterized by elevated very-long-chain ceramides and reduced lysophospholipids. In summary, we report a non-transgenic mouse model, and a tissue lipid signature for early BE. Validation of the lipid signature in human patient cohorts could pave the way for specific dietary strategies to reduce the risk of BE in high-risk individuals.

In Barrett's epithelial cells, weakly acidic bile salt solutions cause oxidative DNA damage with response and repair mediated by p38.

The frequency of esophageal adenocarcinoma is rising despite widespread use of proton pump inhibitors (PPIs), which heal reflux esophagitis but do not prevent reflux of weakly acidic gastric juice and bile in Barrett's esophagus patients. We aimed to determine if weakly acidic (pH 5.5) bile salt medium (WABM) causes DNA damage in Barrett's cells. Because p53 is inactivated frequently in Barrett's esophagus and p38 can assume p53 functions, we explored p38's role in DNA damage response and repair. We exposed Barrett's cells with or without p53 knockdown to WABM, and evaluated DNA damage, its response and repair, and whether these effects are p38 dependent. We also measured phospho-p38 in biopsies of Barrett's metaplasia exposed to deoxycholic acid (DCA). WABM caused phospho-H2AX increases that were blocked by a reactive oxygen species (ROS) scavenger. WABM increased phospho-p38 and reduced bromodeoxyuridine incorporation (an index of S phase entry). Repair of WABM-induced DNA damage proceeded through p38-mediated base excision repair (BER) associated with reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease I (Ref-1/APE1). Cells treated with WABM supplemented with ursodeoxycholic acid (UDCA) exhibited enhanced p38-mediated responses to DNA damage. All of these effects were observed in p53-intact and p53-deficient Barrett's cells. In patients, esophageal DCA perfusion significantly increased phospho-p38 in Barrett's metaplasia. WABM exposure generates ROS, causing oxidative DNA damage in Barrett's cells, a mechanism possibly underlying the rising frequency of esophageal adenocarcinoma despite PPI usage. p38 plays a central role in oxidative DNA damage response and Ref-1/APE1-associated BER, suggesting potential chemopreventive roles for agents like UDCA that increase p38 activity in Barrett's esophagus.NEW & NOTEWORTHY We found that weakly acidic bile salt solutions, with compositions similar to the refluxed gastric juice of gastroesophageal reflux disease patients on proton pump inhibitors, cause oxidative DNA damage in Barrett's metaplasia that could contribute to the development of esophageal adenocarcinoma. We also have elucidated a critical role for p38 in Barrett's metaplasia in its response to and repair of oxidative DNA damage, suggesting a potential chemopreventive role for agents like ursodeoxycholic acid that increase p38 activity in Barrett's esophagus.

N-acetylcysteine reduces amphotericin B deoxycholate nephrotoxicity and improves the outcome of murine cryptococcosis.

Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis.

Biodistribution and histopathology studies of amphotericin B sodium deoxycholate sulfate formulation following intratracheal instillation in rat models.

Aerosol inhalation of amphotericin B (AmB) can be a clinically compliant way to administer the drug directly to the pulmonary route for treatment as well as prophylaxis of invasive pulmonary aspergillosis (IPA). We report aerosol formulation of AmB using sodium deoxycholate sulfate (SDCS), a lipid carrier synthesized in-house using natural precursor deoxycholic acid. In vitro toxicity was determined by MTT assay. Biodistribution and histopathology in rats were evaluated in targeted organs including the lungs, kidneys, spleen, and liver. No toxicity was observed when lung and kidney cells treated with AmB-SDCS formulations up to 8 µg/mL and minimal toxicity at higher concentration 16 µg/mL, while the Fungizone®-like formulation induced toxicity to lung and kidney cells with viability decreasing from 86 to 41% and 100 to 49%, respectively, when compared with an equivalent concentration of AmB-SDCS. Renal and hepatic markers were raised for Fungizone®-like formulation-treated rats but not for AmB-SDCS formulations following 7 days of regular dosing by intratracheal instillation. AmB concentrations were highest in the lungs (5.4-8.3 µg/g) which were well above minimum inhibitory concentration (MIC) of all Aspergillus species. Plasma concentration was also above MIC (> 2 µg/mL) for all AmB-SDCS formulations in comparison with Fungizone®-like formulation. No evidence of abnormal histopathology was observed in the lungs, liver, spleen, and kidneys for all AmB-SDCS formulations but was observed for the group treated with Fungizone®-like formulation. It is concluded that AmB-SDCS formulations can be efficiently administered via intratracheal instillation with no evidence of toxicity and may find great value in the treatment as well as prophylaxis of IPA through inhalation route.

Evaluation of the promiscuous component of several bacterial export pumps TolC as a biomarker for toxic pollutants in feedstuffs.

A significant risk to the food chain is the presence of noxious pollutants in the feeds of animals whose products are used in human nutrition. Consequently, analytical methods and biosensors have been developed to detect these types of contaminates in feeds. Here we have evaluated whether the expression of TolC, a promiscuous component of several ATP-dependent efflux pumps in E. coli, up-regulated in response to chemical stress, could be a useful biomarker for this aim. Changes in TolC expression in response to toxic compounds, with different abilities to induce DNA damage, were determined using two E. coli strains with (DH5α) and without (BL21(DE3)) inactivating mutation in RecA gene. Deoxycholic acid and potassium dichromate up-regulated TolC in both strains. In contrast, cisplatin-induced TolC up-regulation was abolished in the absence of a functional RecA. When the effect of several insecticides, herbicides, antibiotics and common soil pollutants on TolC expression was analyzed, a relationship between toxicity and their ability to up-regulate TolC was observed. However, this was not a general event because the insecticide α-cipermetrin induced a reduction in cell viability, which was not accompanied by TolC up-regulation. In contrast, the soil pollutant benzene was able to stimulate TolC expression at non-toxic concentrations. When this test was used to analyze aqueous extracts from different feedstuffs, up-regulation of TolC was found in the absence of cell toxicity and was even accompanied by enhanced cell viability. In conclusion, TolC expression is partly dependent on the integrity of the RecA/LexaA system. Although toxic compounds up-regulate TolC in a dose-dependent manner, this response is also activated by non-toxic agents. Thus, owing to its poor specificity regardless of its sensitivity, the use of TolC up-regulation in E. coli to detect the presence of toxic pollutants in conventional and unconventional sources of nutrients for ruminant feeding requires supplementary biomarkers.

Continuum of Host-Gut Microbial Co-metabolism: Host CYP3A4/3A7 are Responsible for Tertiary Oxidations of Deoxycholate Species.

The gut microbiota modifies endogenous primary bile acids (BAs) to produce exogenous secondary BAs, which may be further metabolized by cytochrome P450 enzymes (P450s). Our primary aim was to examine how the host adapts to the stress of microbe-derived secondary BAs by P450-mediated oxidative modifications on the steroid nucleus. Five unconjugated tri-hydroxyl BAs that were structurally and/or biologically associated with deoxycholate (DCA) were determined in human biologic samples by liquid chromatography-tandem mass spectrometry in combination with enzyme-digestion techniques. They were identified as DCA-19-ol, DCA-6ß-ol, DCA-5ß-ol, DCA-6α-ol, DCA-1ß-ol, and DCA-4ß-ol based on matching in-laboratory synthesized standards. Metabolic inhibition assays in human liver microsomes and recombinant P450 assays revealed that CYP3A4 and CYP3A7 were responsible for the regioselective oxidations of both DCA and its conjugated forms, glycodeoxycholate (GDCA) and taurodeoxycholate (TDCA). The modification of secondary BAs to tertiary BAs defines a host liver (primary BAs)-gut microbiota (secondary BAs)-host liver (tertiary BAs) axis. The regioselective oxidations of DCA, GDCA, and TDCA by CYP3A4 and CYP3A7 may help eliminate host-toxic DCA species. The 19- and 4ß-hydroxylation of DCA species demonstrated outstanding CYP3A7 selectivity and may be useful as indicators of CYP3A7 activity.

Deoxycholic acid disrupts the intestinal mucosal barrier and promotes intestinal tumorigenesis.

High-fat diet, which leads to an increased level of deoxycholic acid (DCA) in the intestine, is a major environmental factor in the development of colorectal cancer (CRC). However, evidence relating to bile acids and intestinal tumorigenesis remains unclear. In this study, we investigated the effects of DCA on the intestinal mucosal barrier and its impact on the development of CRC. Here we showed that DCA disrupted cell monolayer integrity and increased proinflammatory cytokine production in intestinal cancer and precancerous cell lines (Caco-2 and IMCE). Apcmin/+ mice receiving DCA increased the number and size of intestinal adenomas and promoted the adenoma-adenocarcinoma sequence. Importantly, DCA induced the activation of the NLRP3 inflammasome, increased the production of inflammatory cytokines, and led to intestinal low grade inflammation. A reduction of tight junction protein zonula occludens 1 (ZO-1) and the number of intestinal cells including goblet cells and Paneth cells was also observed after DCA treatment. Moreover, DCA significantly reduced the level of secretory immunoglobulin A (sIgA), and promoted the polarization of M2 macrophages in the intestine of Apcmin/+ mice. In conclusion, these data suggested that DCA induced intestinal low grade inflammation and disrupted the mucosal physical and functional barriers, aggravating intestinal tumorigenesis.

Multiple perspectives of qingkailing injection-fraction-single compound in revealing the hepatotoxicity of baicalin and hyodeoxycholic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: The complexity of ingredients in traditional Chinese medical formulas and the limited consideration of toxicological responses are fundamental issues that hamper prognostic information of drug quality control. MATERIALS AND METHODS: A multidisciplinary approach for quality control of Qingkailing injection (QKL) regarding drug induced liver toxicity was described for the first time. High content image analysis (HCA) was combined with reverse-phase chromatographic separation and high-resolution MS detection technologies to provide the dynamic responses of drug induced HepG2 cell injury. Firstly, a simple and rapid method for simultaneous qualification and quantification of 21 major constituents in QKL was established and validated using ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap), which were operated in full MS/dd-MS2 mode and thus simultaneously acquired quantitative high resolution (HR) full scan data and confirmatory HR MS2 data. Secondly, repeated semi-preparation HPLC was applied to obtain four fractions (F1-F4) for HCS analysis. Finally, potential hepatotoxicity was determined by five hepatotoxicity biomarkers, including cell loss, DNA condense, glutathione (GSH) depletion, reactive oxygen species (ROS) formation, and mitochondria membrane potential (MMP) depolarization. RESULTS: The detection in polarity switching mode empowered the coverage of comprehensive constituents with different chemical properties. Satisfactory linearity precisions, repeatability, stability, and recovery were achieved. QKL injection significantly induced HepG2 cell injury above the concentration of 1.25% (v/v). Meanwhile, flavone glycosides (F3) and stinasterols (F4) fractions exhibited hepatotoxicity above 75µg/mL and 50µg/mL, respectively. Still further, baicalin originated from F3 significantly caused cell loss and glutathione (GSH) depletion. In parallel, hyodeoxycholic acid from F4 induced cell loss, nucleus condense, and GSH reduction as well. CONCLUSIONS: Our work provides multiple perspectives based on injection-fractions-single compound format to improve QKL pharmacovigilance through revealing the potential hepatotoxic material basis. Additionally, our study provides an integrating paradigm for the comprehensive and systematic quality control of traditional Chinese medical formulas.

A cell impedance-based real-time in vitro assay to assess the toxicity of amphotericin B formulations.

Aerosolized liposomal amphotericin B (L-AmB) has been investigated as prophylaxis against invasive aspergillosis. However, the clinical results are controversial and some trials suggest that toxicity could be a limitation for wider use. Our aim was to assess the dynamics of cell toxicity induced in a human alveolar epithelial cell line (A549) after exposure to L-AmB (50 to 400µg/ml) or amphotericin B deoxycholate (D-AmB; 50 to 200µg/ml) by monitoring real-time A549 cell viability using an impedance-based technology. Results were expressed as cell index values integrating cell adhesion, proliferation, and survival. In parallel, the gene expression of proinflammatory cytokines was quantified at 6 and 24h after drug addition by real-time RT-PCR on cell lysates. No sustained reduction of cell indexes was observed with L-AmB or empty liposomes, even at 400µg/ml. Only the highest concentration tested of L-AmB (400µg/ml) yielded transient significant 6-fold and 4-fold induction of TNF-α and IL-8 mRNAs, respectively. In contrast, D-AmB induced a decrease in cell indexes and only the 50µg/ml concentration of D-AmB was followed by cell recovery, higher concentrations leading to cell death. Significant 4-fold, 7-fold and 3-fold inductions of TNF-α, IL-8 and IL-33 mRNAs were also observed at 6h with 50µg/ml of D-AmB. In conclusion, continuous cell impedance measurement showed no toxicity on overall cellular behavior although a slight proinflammatory cytokine expression is possible after L-AmB challenge. Real-time kinetics of cell impedance is an interesting tool for initial screening of cell toxicity.

Assessment of in vitro antifungal efficacy and in vivo toxicity of Amphotericin B-loaded PLGA and PLGA-PEG blend nanoparticles.

Amphotericin B (AmB) is widely applied in treatment of systemic fungal infections. However, the emergence of severe adverse effects, such as nephrotoxicity, hepatotoxicity and hemolytic anemia, can limit its clinical use. Poly(lactide-co-glycolide) (PLGA) or poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) blend nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. Nanoparticles were characterized by particle size, polydispersity index, Fourier transform infrared spectroscopy, differential scanning calorimetry and X-ray diffraction analyses. The antifungal activity was evaluated against strains of Candida albicans and Cryptococcus neoformans. Toxicity was evaluated by hemolysis assay and after 7 days treatment in rats. Mean nanoparticle size was below 200nm with low polydispersity and AmB encapsulation efficiency higher than 90%. Nanoencapsulation resulted in AmB amorphization and no chemical interaction between drug and polymer. In C. albicans, minimum inhibitory concentration (MIC) of AmB-loaded PLGA-PEG nanoparticles was 2-fold higher than free AmB or marketed deoxycholate AmB (AmB-D), while MIC of AmB-loaded PLGA nanoparticles was 10-fold higher than AmB-loaded PLGA-PEG. In C. neoformans, the efficacy of AmB-loaded PLGA nanoparticles was comparable to free AmB, while AmB-loaded PLGA-PEG nanoparticles and AmB-D did not present MIC in tested concentration range. Nanoparticles inhibited the AmB-induced hemolysis. After 7 days treatment in rats, histologic examination revealed AmB-D treatment presented initial liver damage, while AmB-loaded nanoparticles did not present any hepatic cellular alteration. Kidney alteration was not observed in all treatments. Thus, PLGA and PLGA-PEG nanoparticles are promising carriers for AmB delivery with potential application in antifungal therapy.

Pre-formulation and systematic evaluation of amino acid assisted permeability of insulin across in vitro buccal cell layers.

The aim of this work was to investigate alternative safe and effective permeation enhancers for buccal peptide delivery. Basic amino acids improved insulin solubility in water while 200 and 400 µg/mL lysine significantly increased insulin solubility in HBSS. Permeability data showed a significant improvement in insulin permeation especially for 10 µg/mL of lysine (p < 0.05) and 10 µg/mL histidine (p < 0.001), 100 µg/mL of glutamic acid (p < 0.05) and 200 µg/mL of glutamic acid and aspartic acid (p < 0.001) without affecting cell integrity; in contrast to sodium deoxycholate which enhanced insulin permeability but was toxic to the cells. It was hypothesized that both amino acids and insulin were ionised at buccal cavity pH and able to form stable ion pairs which penetrated the cells as one entity; while possibly triggering amino acid nutrient transporters on cell surfaces. Evidence of these transport mechanisms was seen with reduction of insulin transport at suboptimal temperatures as well as with basal-to-apical vectoral transport, and confocal imaging of transcellular insulin transport. These results obtained for insulin are the first indication of a possible amino acid mediated transport of insulin via formation of insulin-amino acid neutral complexes by the ion pairing mechanism.