Presence of carbazole and polyhalogenated carbazoles in human urine.

Exposure to carbazole (CZ) and polyhalogenated carbazoles (PHCZs) may pose a threat to human health, owing to their potential dioxin-like toxicity. Until now, the presence of these chemicals in the human urine from the general population is still unclear. Human urine samples (n = 210) were taken from the general population in Quzhou, China in this study, and were analyzed for CZ and 14 PHCZs. CZ and nine PHCZs were detected in collected human urine. CZ (detection frequency 100 %), 3-chlorocarbazole (3-CCZ; 88 %), 3,6-dichlorocarbzole (36-CCZ; 84 %), and 3-bromocarbazole (3-BCZ; 80 %) were more frequently detected. Among detected PHCZs, 3-CCZ (mean 0.49 ng/mL, < LOD-4.3 ng/mL) had comparatively higher urinary levels, followed by 3-BCZ (0.30 ng/L, < LOD-1.9 ng/mL) and 36-CCZ (0.20 ng/L, < LOD-1.4 ng/mL). Urinary concentrations of CZ in male participants (1.3 ± 0.26 ng/mL) were significantly (p < 0.05) higher than that in female participants (0.92 ± 0.24 ng/mL). No obvious trend in urinary concentrations with the age of participants was found for CZ and detected PHCZs. The mean daily excretion was found highest for CZ (31 ng/kg bw/day), followed by 3-CCZ (19 ng/kg bw/day) and 3-BCZ (8.5 ng/kg bw/day). This study provides the first data, to our knowledge, on the presence and levels of CZ and PHCZs in human urine, which is necessary for conducting the human exposure risk assessment.

Combined toxic effects of polystyrene microplastics and 3,6-dibromocarbazole on zebrafish (Danio rerio) embryos.

Microplastics (MPs) and polyhalogenated carbazoles (PHCZs) are widely detected in the aquatic environment, and their ecological risks have become a research focus. Although there is an extensive co-distribution of MPs and PHCZs, their combined toxicity to aquatic organisms is still unclear. This study investigated the toxic effects of polystyrene microplastics (PS-MPs) and 3,6-dibromocarbazole (3,6-DBCZ) on zebrafish embryos by individual/combined exposure. This study showed that individual or combined exposure of PS-MPs (10 mg/L) and 3,6-DBCZ (0.5 mg/L) could significantly increase the rate of zebrafish embryo deformity, whereas no significant effect was observed on mortality and hatching rate. Furthermore, exposure to 3,6-DBCZ or PS-MPs increased reactive oxygen species (ROS) levels in zebrafish embryos, and the resulting oxidative stress induced apoptosis. Comparably, the levels of oxidative stress and apoptosis in zebrafish embryos were significantly reduced with the combined exposure of 3,6-DBCZ and PS-MPs. These observations suggest that the combined exposure of 3,6-DBCZ and PS-MPs has an antagonistic effect on oxidative stress and apoptosis. Fluorescence PS-MPs tracing and 3,6-DBCZ enrichment analysis showed that, with the protection of chorion, the entry of PS-MPs (5 and 50 µm) into the embryonic stage (55 hpf) of zebrafish was prevented. Moreover, after exposure for 96-144 hpf, PS-MPs served as a carrier to promote the 3,6-DBCZ accumulation and its dioxin-like toxicity in zebrafish larvae through ingestion. Compared with 5-µm PS-MPs, 50-µm PS-MPs promoted higher accumulation and dioxin-like toxicity of 3,6-DBCZ in zebrafish larvae. These findings provide that MPs can be used as an important carrier of PHCZs, influencing their toxicity and bioaccumulation in the organisms.

Tissue-specific accumulation, depuration and histopathological effects of 3,6-dichlorocarbazole and 2,7-dibromocarbazole in adult zebrafish (Danio rerio).

Although polyhalogenated carbazoles have been detected with increasing frequency in aquatic ecosystems, their bioaccumulation in fish and corresponding pathological effects related to bioaccumulation are still unclear. Here, we investigated the tissue-specific accumulation, depuration, and histopathological effects of two typical PHCZs, 3,6-dichlorocarbazole (36-CCZ) and 2,7-dibromocarbazole (27-BCZ), in adult zebrafish at three levels (0, 0.15 µg/L (5 × environmentally relevant level), and 50 µg/L (1/10 LC50). The lowest concentrations of 36-CCZ (1.2 µg/g ww) and 27-BCZ (1.4 µg/g ww) were observed in muscle, and the greatest concentrations of 36-CCZ (3.6 µg/g ww) and 27-BCZ (4 µg/g ww) were detected in intestine among the tested tissues. BCFww of 36-CCZ and 27-BCZ in zebrafish ranged from 172.9 (muscle) to 606.6 (intestine) and 285.2 (muscle) to 987.5 (intestine), respectively, indicating that both 36-CCZ and 27-BCZ have high potential of bioaccumulation in aquatic system. The 0.15 µg/L level of 36-CCZ or 27-BCZ caused lipid accumulation in liver, while 50 µg/L of 36-CCZ or 27-BCZ induced liver lesions such as fibrous septa, cytolysis, and nuclear dissolution. Brain damage such as multinucleated cells and nuclear solidification were only observed at 50 µg/L of 27-BCZ. This study provided valuable information in assessing the health and ecological risks of 36-CCZ and 27-BCZ.

Antimicrobial Activity and Toxicity of Newly Synthesized 4-[4-(benzylamino)butoxy]-9H-carbazole Derivatives.

One of the main challenges of medicinal chemistry is the search for new substances with antimicrobial potential that could be used in the fight against pathogenic microorganisms. Therefore, the antimicrobial activity of newly synthesized compounds is still being investigated. Carbazole-containing compounds appear to be promising antibacterial, antifungal, and antiviral agents. The aim of this study was to examine the antimicrobial potential and toxicity of newly synthesized isomeric fluorinated 4-[4-(benzylamino)butoxy]-9H-carbazole derivatives. Their antimicrobial activity against bacteria and fungi was tested according to CLSI guidelines. Similarly to previously studied carbazole-containing compounds, the tested derivatives showed the ability to effectively inhibit the growth of Gram-positive bacteria. The addition of carbazole derivatives 2, 4, and 8 at the concentration of 16 µg/mL caused the inhibition of S. aureus growth by over 60%. The MIC value of compounds 2-5 and 7-10 was 32 µg/mL for Staphylococcus strains. Gram-negative strains of E. coli and P. aeruginosa were found to be more resistant to the tested carbazole derivatives. E. coli cells treated with compounds 3 and 8 at a concentration of 64 µg/mL resulted in a greater-than-40% reduction in bacterial growth. In the case of the P. aeruginosa strain, all compounds in the highest concentration that we tested limited growth by 35-42%. Moreover, an over-60% inhibition of fungal growth was observed in the cultures of C. albicans and A. flavus incubated with 64 µg/mL of compounds 2 or 7 and 1 or 4, respectively. The hemolysis of red blood cells after their incubation with the tested carbazole derivatives was in the range of 2-13%. In the case of human fibroblast cells, the toxicity of the tested compounds was higher. Derivative 1, functionalized with fluorine in position 2 and its hydrobromide, was the least toxic. The obtained results indicated the antimicrobial potential of the tested 4-[4-(benzylamino)butoxy]-9H-carbazole derivatives, especially against S. aureus strains; therefore, it is worth further modifying these structures, in order to enhance their activity against pathogenic microorganisms.

Pollution characteristics, distribution, and source analysis of carbazole and polyhalogenated carbazoles in coastal areas of Bohai Bay, China.

Polyhalogenated carbazoles (PHCZs) are a class of emerging environmental contaminants formed by the substitution of hydrogen on carbazole (CZ) benzene rings with halogens (Cl, Br, I) with potential dioxin-like toxicity, and they have been frequently detected in various environmental media and organisms recently. Nevertheless, co-research of CZ/PHCZs with PAHs is very limited. In addition, I-PHCZs, which are believed to be much more toxic than CZ, Cl-PHCZs and Br-PHCZs, have a few data in sediments previously. The concentration and distribution of CZ/PHCZs and PAHs were analyzed in 18 surface sediments of Bohai Bay, China. There is a significant correlation (R = 0.64, P＜0.05) between PHCZs and PAHs, and principal component analysis (PCA) also indicating that they may have a certain similarity in origin. Additionally, total CZ and PHCZs was up to 230.57 ng/g dw in the studied samples, which was approximately 1-2 orders of magnitude lower than PAHs and other common persistent organic pollutants (POPs). The compositions of the CZ/PHCZs in our study were dominated by CZ (2.74-18.28, median 2.92 ng/g dw), 3,6-dichlorocarbazole (n.d-6.78, median 0.97 ng/g dw) and 3,6-iodocarbazole (n.d-12.68, median 1.65 ng/g dw). Results of this study discovered the varying origins of CZ and PHCZs and/or a complexity of anthropogenic influences and natural sources processes, and revealed a wide distribution of CZ/PHCZs across the studied. Moreover, more attention should be paid by comparing CZ/PHCZs with other widely distributed POPs.

Acute Toxicity Study of 5, 11-Dihydroindolo [3, 2-ß]carbazole as per OECD Regulatory Guidelines.

BACKGROUND: 5,11-Dihydroindolo [3, 2-ß]carbazole is one of the phytoconstituent of the Arisaema genus, which might have various important biological activities. Recently, we have predicted the antiviral potential of this phytoconstituent against the Japanese Encephalitis virus (JEV). METHODS: Thus, in the current study, the acute toxicity profile of 5, 11-dihydroindolo [3, 2-ß]carbazole as per OECD regulatory guidelines in female Wistar rats was evaluated. RESULTS: We did not find any adverse effects, mortality, and altered behaviour in animals after administration of 5, 11-dihydroindolo [3, 2-ß] carbazole at a dose of 300 and 2000 mg/Kg. Furthermore, no significant changes in physiological and haematological parameters were observed. The histopathological study of vital organs also showed no significant changes compared to the control. CONCLUSION: Based on the findings of the current investigation, 5, 11-dihydroindolo [3, 2-ß]carbazole is a safe phytoconstituent of the Arisaema genus, which can be explored for various biological activities.

Polyhalogenated carbazoles induce hepatic metabolic disorders in mice via alteration in gut microbiota.

Polyhalogenated carbazoles (PHCZs) have been widely accepted as emerging pollutants, whereas their ecological and health risks remain uncertain. Herein, female and male Sprague-Dawley (SD) mice were treated with four typical PHCZs to investigate their negative consequences, along with alternations in gut microbiota to indicate underlying mechanisms. In female mice, the relative liver weight ratio increased after four PHCZs exposure; 2-bromocarbazole (2-BCZ) increased urine glucose level; 3-bromocarbazole (3-BCZ) decreased the glucose and total cholesterol levels; 3,6-dichlorocarbazole (3,6-DCCZ) decreased glucose level. The only disturbed biochemical index in male mice was the promoted alkaline phosphatase (ALP) level by 3,6-DCCZ. We also found that the differential blood biochemical indices were correlated with gut microbiota. 3-BCZ and 3,6-DCCZ altered Bacteroidetes and Proteobacteria phyla in female and male mice, which were correlated with metabolic disorders. Our findings demonstrated the correlation between PHCZs induced potential hepatotoxicity and metabolic disorders may be due to their dioxin-like potentials and endocrine disrupting activities, and the gender differences might result from their estrogenic activities. Overall, data presented here can help to evaluate the ecological and health risks of PHCZs and reveal the underlying mechanisms.

Polyhalogenated carbazoles (PHCZs) induce cardiotoxicity and behavioral changes in zebrafish at early developmental stages.

Polyhalogenated carbazoles (PHCZs) are widely present in the environment, and their health risks are of increasing concern. Available studies primarily confirm their dioxin-like toxicity mechanism based on biomarkers, such as aryl hydrocarbon receptor (AHR) and CYP1A1, while few studies have investigated their actual toxic effects at the level of individual organisms. In the present study, the developmental toxicity of two typical PHCZs with a high detection rate and high concentration in the environment (3,6-dichlorocarbazol (3,6-DCCZ) and 3,6-dibromocarbazole (3,6-DBCZ)) was investigated based on a fish embryo acute toxicity test (FET, zebrafish) and transcriptomics analysis. The 96 h LC50 values of 3,6-DCCZ and 3,6-DBCZ were 0.636 mg/L and 1.167 mg/L, respectively. Both tested PHCZs reduced the zebrafish heart rate and blocked heart looping at concentrations of 0.5 mg/L or higher. The swimming/escaping behavior of zebrafish larvae was more vulnerable to 3,6-DBCZ than 3,6-DCCZ. Transcriptomics assays showed that multiple pathways linked to organ development, immunization, metabolism and protein synthesis were disturbed in PHCZ-exposed fish, which might be the internal mechanism of the adverse effects. The present study provides evidence that PHCZs cause cardiac developmental toxicity and behavioral changes and improves our understanding of their health risks.

Successful management of severe carprofen toxicity with manual therapeutic plasma exchange in a dog.

OBJECTIVE: To report the use of manual therapeutic plasma exchange (TPE) in a dog with severe carprofen toxicity. SUMMARY: A 12-year-old neutered female Pembroke Welsh Corgi weighing 20 kg was evaluated after ingesting 223 mg/kg of carprofen. Emesis was attempted with apomorphine at the primary care veterinarian but was unsuccessful, and a dose of activated charcoal with sorbitol was administered. On presentation to the referral center, approximately 8 hours after ingestion, the dog's physical examination revealed mild abdominal discomfort but was otherwise unremarkable. Treatment consisted of a combination of supportive care including activated charcoal with sorbitol, cholestyramine, IV lipid emulsion, and manual TPE. Blood samples were collected prior to the initiation of manual TPE and at the completion of 12 exchange cycles. Carprofen levels were determined by high-pressure liquid chromatography. A 57% decrease in carprofen levels was achieved with the combination of activated charcoal, cholestyramine, IV lipid emulsion, and manual TPE. The dog did not develop organ dysfunction secondary to toxicity and was discharged 4 days after ingestion. NEW OR UNIQUE INFORMATION PROVIDED: This report describes the successful decrease of plasma carprofen in a dog with the combination of decontamination techniques and manual TPE. While TPE has been previously reported as a successful therapeutic in dogs with nonsteroidal anti-inflammatory toxicity, including carprofen, equipment and expertise of this platform is not readily available. Manual TPE is technically simple and can be performed in any hospital with a large blood centrifuge.

HIF-1alpha/VEGF pathway mediates 1,3,6,8-tetrabromo-9 H-carbazole-induced angiogenesis: a potential vascular toxicity of an emerging contaminant.

The dioxin-like substances polyhalogenated carbazoles (PHCZs) may trigger the aryl hydrocarbon receptor (AhR) signaling pathway. Although the crosstalk between AhR and the hypoxia inducible factor-1 (HIF-1) pathways is generally believed to occur, the exact mechanisms of the HIF-1 pathway in PHCZ toxicity have not been determined. We aimed to elucidate the effect of PHCZs on the HIF-1 pathway and its involvement in the regulation of target genes of HIF-1. Herein, we employed human HepG2 cells transiently transfected with a hypoxia response element (HRE) luciferase reporter to identify PHCZs that could influence HIF-1 pathway. We found that exposure to one of the four selected PHCZs, specifically 1,3,6,8-tetrabromo-9 H-carbazole (1368-BCZ), induced a significant enhancement of the activity of HRE activity. In silico data supported 1368-BCZ-induced HIF-1α activity preferentially. Moreover, 1368-BCZ significantly upregulated the expression of HIF-1 target genes, including endothelial growth factor (VEGF) and erythropoietin. Importantly, the stimulated secretion of VEGF by 1368-BCZ promoted the angiogenesis in human umbilical vein endothelial cells. Therefore, the present experimental and computational studies provide new and direct evidence of 1368-BCZ - HIF-1 interaction, which sheds light on the HIF-mediated cardiovascular toxicity and allows a knowledge-based risk assessment of emerging pollutants.

Benzoindoxazine derivatives containing carbazole for detection of CN- and its application in plant seed extracts and cell imaging.

Cyanide (CN-) is a highly toxic compound that exists in many substances and is harmful to the environment and human health. Therefore, it is of great significance to develop excellent CN- ion probes, especially solvent-induced on-off fluorescent probes. Based on the condensation reaction of indolo[2,1-b][1,3]oxazine molecules with aldehydes, probes (E)-13a-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)-14,14-dimethyl-10-nitro-13a,14-dihydro-8H-benzo[e]benzo[5,6][1,3]oxazino[3,2-a]indole (NCO) and (E)-13a-(2-(9-benzyl-9H-carbazol-3-yl)vinyl)-14,14-dimethyl-10-nitro-13a,14-dihydro-8H-benzo[e]benzo[5,6][1,3]oxazino[3,2-a]indole (NBO) were synthesized to detect CN-. Compared with other cyanogen ion probes, NCO and NBO have special carbazole ring structures and large conjugate systems. When CN- is added to the probe-detection solution, color changes that are visible to the naked eye can occur. The UV-vis spectrum test using differential spectroscopy shows that the probe (i) has excellent solvent-induced switching characteristics and stability (CH3OH-H2O) and (ii) high selectivity, anti-interference ability, and sensitivity for the detection of CN-. The fluorescence limit of detections (LODs) are 1.05 µM for NCO and 1.34 µM for NBO. The UV LODs are 0.83 µM for NCO and 0.87 µM for NBO. Fluorescence spectroscopy shows that the probe has remarkable fluorescence properties. Fluorescence titration experiments, liver cancer cell (Hep G2) imaging, and cytotoxicity experiments all show that the probes have high biocompatibility, low toxicity, high cell permeability, and high sensitivity for the detection of CN- in cells. In addition, NCO and NBO have been successfully used for the detection of cyanogenic glycosides in the seeds of ginkgo, crabapple, apple, and cherry. Test strips were fabricated to detect CN-. After adding CN-, the color of the test strip changed significantly-from brown to light yellow; thus, the test strips have a high application value in the fields of drug quality control, drug safety testing, and pharmacological research.

Toxic Effects of High-dose Meloxicam and Carprofen on Female CD1 Mice.

The nonsteroidal anti-inflammatory drugs meloxicam and carprofen are commonly used as analgesics in mice. The current recommended doses of meloxicam at 0.2-1.0 mg/kg once daily and carprofen at 5-10 mg/kg twice daily may not be adequate to provide analgesia in mice. Several studies have suggested that doses up to 20 mg/kg of meloxicam and carprofen are needed to provide analgesic efficacy. This study investigated the clinical safety of these higher doses of meloxicam and carprofen by evaluating their potential for renal and gastrointestinal toxicity. Female CD-1 mice were given 20 mg/kg of either meloxicam, carprofen, or an equivalent volume of saline subcutaneously once daily for 3 or 7 d. On day 4, mice treated for 3 d were euthanized, and on days 8 and 15, mice treated for 7 d were euthanized. Blood was collected by cardiocentesis for serum chemistry analysis. Feces was collected from the colon for fecal occult blood testing, and tissues were collected for histopathology. No clinically significant changes in serum chemistry profiles were found in the drug-treated mice at any time point as compared with the saline controls. Fecal occult blood and histologic evidence of gastritis was associated with meloxicam administration in mice evaluated at days 4 and 8. By day 15, there was no association with meloxicam treatment and the presence of fecal occult blood or gastritis. There was no association between fecal occult blood and gastritis in the carprofen or saline-treated mice regardless of the treatment durations. These findings suggest that 20 mg/kg of meloxicam in mice causes gastric toxicity when given for 3 or 7 d and should be used cautiously; however, carprofen at 20 mg/kg appears to have minimal toxic effects with regard to the parameters measured.

The Ability of AhR Ligands to Attenuate Delayed Type Hypersensitivity Reaction Is Associated With Alterations in the Gut Microbiota.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates T cell function. The aim of this study was to investigate the effects of AhR ligands, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), and 6-Formylindolo[3,2-b]carbazole (FICZ), on gut-associated microbiota and T cell responses during delayed-type hypersensitivity (DTH) reaction induced by methylated bovine serum albumin (mBSA) in a mouse model. Mice with DTH showed significant changes in gut microbiota including an increased abundance of Bacteroidetes and decreased Firmicutes at the phylum level. Also, there was a decrease in Clostridium cluster XIV and IV, which promote anti-inflammatory responses, and an increase in Prevotella copri that facilitates pro-inflammatory responses. Interestingly, treatment of mice with TCDD attenuated the DTH response, induced Tregs, suppressed Th17 cells in the mesenteric lymph nodes (MLNs), and reversed the gut microbiota composition toward normalcy. In contrast, FICZ exacerbated the DTH response, induced heightened Th17 cells, and failed to cause a major shift in gut microbiota. Furthermore, TCDD but not FICZ caused an increase in the levels of short-chain fatty acids (SCFA), n-butyric acid, and acetic acid. Administration of sodium butyrate into mice with DTH suppressed the response, increased Tregs, and reduced Th17 cells IL17. Butyrate also caused an increase in the abundance of Clostridium and a decrease in Prevotella. Lastly, TCDD, as well as butyrate but not FICZ, were able to inhibit proinflammatory Histone deacetylases (HDACs) class I and II. Together, our data suggest that AhR ligands, such as TCDD that suppress DTH response, may mediate this effect by reversing the gut dysbiosis induced during this inflammatory response, while FICZ may fail to suppress the DTH response because of its inability to overturn the dysbiosis.

Exploration of Benzo[b]carbazole-6,11-diones as anticancer agents: Synthesis and studies of hTopoIIα inhibition and apoptotic effects.

Two series of (hetero)arylamino-naphthoquinones and benzo-fused carbazolequinones were considered for study with the rationale that related structural motifs are present in numerous drugs, clinical trial agents, natural products and hTopoIIα inhibitors. Total 42 compounds were synthesized by reactions including dehydrogenative CN and Pd-catalyzed CC bond forming transformations. These compounds were screened against numerous cancer cells including highly metastatic one (MCF-7, MDA-MB-231, H-357 and HEK293T), and normal cells (MCF 10A). Some of the active compounds were evaluated for clonogenic cell survival and apoptotic effects in cancer cells (DAPI nuclear staining, Comet assay, Annexin-V-FITC/PI dual staining, flow cytometry, and western blot analysis with relevant proteins). All compounds were tested for hTopoIIα inhibitory activity. The investigated series compounds showed important properties like significant apoptotic antiproliferation in cancer cells with cell cycle arrest at S-phase and downregulation of NF- κß signaling cascade, relatively less cytotoxicity to normal cells, and hTopoIIα inhibition with more efficiency compared to an anticancer drug etoposide.

Ionic Carbazole-Based Water-Soluble Two-Photon Photoinitiator and the Fabrication of Biocompatible 3D Hydrogel Scaffold.

Two-photon polymerization of a three-dimensional (3D) hydrogel structure has been widely applied in biological tissue engineering. For improving the biocompatibility of hydrogel structures, a new kind of ionic carbazole water-soluble photoinitiator was prepared to realize the fabrication of a 3D hydrogel structure in aqueous phase. 3,6-Bis[2-(1-methyl-pyridinium)vinyl]-9-methyl-carbazole diiodide (BMVMC) and cucurbit[7]uril (CB7) have been employed to generate a complex with better water solubility by host-guest interactions. The binding ratio of the complex was demonstrated to be 1:1 through the characterization of isothermal titration calorimetry (ITC). The two-photon absorption (TPA) cross section of the complex increases to 2500 GM compared with the 750 GM of the BMVMC molecule. Then, an aqueous-phase photoresist was obtained using the CB7/BMVMC complex as the photoinitiator and poly(ethylene glycol) diacrylate (PEGda) as the hydrogel monomer. Two-photon fabrication capability in aqueous phase has been studied using the as-prepared photoresist. A low laser threshold of 3.7 mW as well as a high resolution of 180 nm are achieved. Benefiting from the fluorescence properties of the photoinitiator, we can achieve the confocal fluorescence images without any assistance of fluorescent probes. Subsequently, a 3D engineered hydrogel scaffold microstructure was fabricated by the two-photon polymerization technology, whose biocompatibility was demonstrated by culturing the structure with living cells of L929. The BMVMC-CB7 complex and the as-prepared photoresist are demonstrated to have good biocompatibility, which is prospective for further application in tissue engineering.

AhR Ligands Differentially Regulate miRNA-132 Which Targets HMGB1 and to Control the Differentiation of Tregs and Th-17 Cells During Delayed-Type Hypersensitivity Response.

Aryl hydrocarbon receptor (AhR), is a transcription factor and an environmental sensor that has been shown to regulate T cell differentiation. Interestingly, AhR ligands exert varying effects from suppression to exacerbation of inflammation through induction of Tregs and Th-17 cells, respectively. In the current study, we investigated whether the differential effects of AhR ligands on T cell differentiation are mediated by miRNA during delayed-type hypersensitivity (DTH) reaction against methylated Bovine Serum Albumin (mBSA). Treatment of C57BL/6 mice with TCDD attenuated mBSA-mediated DTH response, induced Tregs, decreased Th-17 cells, and caused upregulation of miRNA-132. TCDD caused an increase in several Treg subsets including inducible peripheral, natural thymic, and Th3 cells. Also, TCDD increased TGF-ß and Foxp3 expression. In contrast, treating mice with FICZ exacerbated the DTH response, induced inflammatory Th17 cells, induced IL-17, and RORγ. Analysis of miRNA profiles from draining lymph nodes showed that miR-132 was upregulated in the TCDD group and downregulated in the FICZ group. Transfection studies revealed that miRNA-132 targeted High Mobility Group Box 1 (HMGB1). Downregulation of HMGB1 caused an increase in FoxP3+ Treg differentiation and suppression of Th-17 cells while upregulation of HMGB1 caused opposite effects. Moreover, TCDD was less effective in suppressing DTH response and induction of Tregs in mice that were deficient in miR-132. In summary, this study demonstrates that TCDD and FICZ have divergent effects on DTH response and T cell differentiation, which is mediated through, at least in part, regulation of miRNA-132 that targets HMGB1.

Possible Roles of Proinflammatory Signaling in Keratinocytes Through Aryl Hydrocarbon Receptor Ligands for the Development of Squamous Cell Carcinoma.

Aryl hydrocarbon receptor (AhR) provides a deeper insight into the pathogenesis of cutaneous squamous cell carcinoma (cSCC). AhR ligands, such as 6-formylindolo[3,2-b] carbazole (FICZ), and 7,12-Dimethylbenz[a]anthracene (DMBA), constitute major substrates for the cytochrome P450 (CYP) family, and influence the expression of various cytokine genes, including IL-17 and IL-23-related genes via the AhR. On the other hand, proinflammatory cytokines could drive tumor progression through the TRAF-ERK5 signaling pathway in cSCC. From the above findings, we hypothesized that AhR ligands might enhance the mRNA expression of proinflammatory cytokines via the AhR, leading to the development of cSCC. The purpose of this study was to investigate (1) the immunomodulatory effects of FICZ and DMBA on normal human keratinocytes (NHKCs), focusing on IL-17, and related cytokines/chemokines (IL-23, IL-36γ, and CCL20), (2) the expression of these factors in AhR-dependent pathways using a two-stage chemically induced skin carcinogenesis mouse model, and (3) the expression of these factors in lesion-affected skin in cSCC. Both FICZ and DMBA augmented the expression of CYP1A1, p19, CCL20, and IL-36γ mRNA in NHKCs in vitro. Moreover, the mRNA expression of these proinflammatory factors, as well as IL-17, in mouse cSCC is significantly decreased in the AhR-(fl/fl) Krt5-(Cre) mice compared to wild type mice, leading to a decrease in the number of developed cSCC lesions. Furthermore, CCL20, IL-23, as well as IL-17, are detected in the lesion-affected skin of cSCC patients. Our study demonstrates a possible mechanism for the development of cSCC involving AhR-mediated signaling by epidermal keratinocytes and recruitment of Th17 cells.

Carbazole derivatives containing chalcone analogues targeting topoisomerase II inhibition: First principles characterization and QSAR modelling.

Recently, a series of carbazole derivatives containing chalcone analogues (CDCAs) were synthetized as potent anticancer agents and apoptosis inducers. These compounds target the inhibition of topoisomerase II and present cytotoxic activities. After comparison to experiment, we validated the use of B3LYP, a density functional theory-based approach, to describe the structure and molecular properties of the carbazole subunit and CDCAs compounds of interest. Then, we derived relationships between the chemical descriptors and activity of these carbazole derivatives using multi-parameter optimization and quantitative structure activity relationships (QSAR) approaches. For the QSAR studies, we used multiple linear regression and artificial neural network statistical modelling. Our predicted activities are in good agreement with the experimental ones. We found that the most important parameter influencing the activity of the considered compounds is the octanol-water partition coefficient, highlighting the importance of flexibility as a key molecular parameter to favor cell membrane crossing and enhance the action of these CDCAs against topoisomerase II. Our results provide useful guidelines for designing new oral active CDCAs medicaments for cytotoxic inhibition.

Gastrointestinal obstruction secondary to activated charcoal granule impaction in a dog.

OBJECTIVE: To describe a serious adverse event of gastrointestinal obstruction requiring surgery following routine administration of multiple doses of activated charcoal (AC) granules, which were prescribed for carprofen toxicosis. CASE SUMMARY: A 2-year-old female neutered Airedale Terrier presented for ingestion of 207 mg/kg of carprofen. Decontamination was initiated with apomorphine to induce emesis. Along with additional supportive care, the dog received an initial dose of 75 mL of AC suspension containing sorbitol by mouth (15.6 g of AC, or 0.6 g/kg), followed by 50 g of AC granules every 8 hours for 4 additional doses. While hospitalized, the dog experienced clinical signs, including vomiting and black diarrhea, as well as bloodwork changes including mild to moderate elevations in kidney and liver enzymes. Given clinical improvement after 72 hours of hospitalization, the patient was discharged for monitoring and ongoing care at home. Two days later, the patient presented again for nausea, dark diarrhea with frank blood, and panting. Abdominal ultrasound showed findings suspicious for partially obstructive foreign material or atypical impacted fecal material partially occluding the distal ileum. Despite medical management overnight, recheck ultrasound the following day demonstrated persistent obstruction with ileal foreign material. Exploratory laparotomy and enterotomy revealed moderate distension and obstruction of the distal ileum with black granular foreign material consistent with charcoal granules. The patient remained in hospital for supportive care for 4 days following the procedure, and all clinical signs were resolved at the time of discharge. NEW OR UNIQUE INFORMATION PROVIDED: This report documents a serious adverse event of gastrointestinal obstruction associated with routine multidose AC administration, which has been occasionally reported in people but not in dogs. The potential for this complication should be taken into account when prescribing multiple doses of AC granules.

Antikinetoplastid Activity of Indolocarbazoles from Streptomyces sanyensis.

Chagas disease and leishmaniasis are neglected tropical diseases caused by kinetoplastid parasites of Trypanosoma and Leishmania genera that affect poor and remote populations in developing countries. These parasites share similar complex life cycles and modes of infection. It has been demonstrated that the particular group of phosphorylating enzymes, protein kinases (PKs), are essential for the infective mechanisms and for parasite survival. The natural indolocarbazole staurosporine (STS, 1) has been extensively used as a PKC inhibitor and its antiparasitic effects described. In this research, we analyze the antikinetoplastid activities of three indolocarbazole (ICZs) alkaloids of the family of staurosporine STS, 2-4, and the commercial ICZs rebeccamycin (5), K252a (6), K252b (7), K252c (8), and arcyriaflavin A (9) in order to establish a plausive approach to the mode of action and to provide a preliminary qualitative structure-activity analysis. The most active compound was 7-oxostaurosporine (7OSTS, 2) that showed IC50 values of 3.58 ± 1.10; 0.56 ± 0.06 and 1.58 ± 0.52 µM against L. amazonensis; L. donovani and T. cruzi, and a Selectivity Index (CC50/IC50) of 52 against amastigotes of L. amazonensis compared to the J774A.1 cell line of mouse macrophages.