Egr1 Gene Expression as a Potential Biomarker for In Vitro Prediction of Ocular Toxicity

Animal models are used for preclinical toxicity studies, and the need for in vitro alternative methods has been strongly raised. Our study aims to elucidate the potential mechanism of change in EGR1 expression under situations of toxic injury and to develop an Egr1 promoter–luciferase gene reporter assay for an in vitro alternative method for toxicity prediction in drug discovery. We first found an increase in early growth response-1 (EGR1) mRNA/protein expressions in the liver and kidney of cisplatin-treated injured rats. Additionally, the EGR1 protein level was also elevated under situations of ocular injury after sodium lauryl sulfate (SLS) eye drops. These in vivo observations on injury-related EGR1 induction were confirmed by in vitro studies, where human corneal epithelial cells were treated with representative irritants (SLS and benzalkonium chloride) and 17 chemicals having different UN GHS irritant categories. Additionally, our results suggest the involvement of ERK, JNK, p38 MAPK pathways in EGR1 elevation in response to gamma-butyrolactone-induced injury. As EGR1 is considered to be a pivotal factor in proliferation and regeneration, siRNA-mediated knockdown of Egr1 promoted cytotoxic potential through a delay of injury-related recovery. More importantly, the elevation of promoter activities was observed by various irritants in cells transfected with Egr1 promoter-reporter vector. In conclusion, Egr1 can be a potential biomarker in a promoter-reporter system to improve the accuracy of in vitro predictions for ocular irritation.

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Hepato- and Nephroprotective Effects of the Polyphenol Concentrate From Cabernet Sauvignon Grape Varieties Cultivated in Kazakhstan in the Experimental Model Of CCL4-Induced Toxicity

INTERNATIONAL JOURNAL OF TOXICOLOGICAL AND PHARMACOLOGICAL RESEARCH ◽

10.25258/ijtpr.v9i03.9068 ◽

2017 ◽

Vol 9 (03) ◽

Author(s):

Nurgozhin T. ◽

Sergazy S. H. ◽

Adilgozhina G. ◽

Gulyayev A. ◽

Shulgau Z. ◽

...

Keyword(s):

Carbon Tetrachloride ◽

Experimental Model ◽

Lethal Dose ◽

Radical Scavenging ◽

Cabernet Sauvignon ◽

Intragastric Administration ◽

Liver And Kidney ◽

Antioxidant Stress

Objective:This study investigates the hepatoprotective effect and the antioxidant role of polyphenol concentrate in the experimental model of carbon tetrachloride (CCl4) induced toxicity. Methods: Antioxidant activity of Cabernet Sauvignon grape polyphenol were evaluated by radical scavenging of 1,1-diphenyl-2-picryl hydrazyl radical (DPPH), 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS.+). In addition, the effects of polyphenol concentrate on the survival of Wistar rats in the toxicity model, was also investigated. The polyphenol concentrate was administered for 5 five days prior to injection of carbon tetrachloride in a sub-lethal dose of 300 mg/kg of animal body weight in order to perform histological examinations of the liver and kidney, and detect the levels of AST, ALT and bilirubin. Results: Administration of polyphenol concentrate increased animal survival in the experimental model. Moreover, the intragastric administration of polyphenol concentrate prior to the initiation of the experimental model of toxicity, which was caused by a sub-lethal CCl4 dose, reduced morphological injuries in the liver and kidney, decreased the AST and ALT levels of the blood serum. Discussion and conclusion: Our data demonstrate that polyphenol concentrate possesses an antioxidant potential both in vitro and in vivo by reducing antioxidant stress that was caused by CCl4 administration into rats.

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A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test

Alternatives to Laboratory Animals ◽

10.1177/026119299802600511 ◽

1998 ◽

Vol 26 (5) ◽

pp. 679-708 ◽

Cited By ~ 5

Author(s):

Horst Spielmann ◽

Michael Balls ◽

Jack Dupuis ◽

Wolfgang J. W. Pape ◽

Odile de Silva ◽

...

Keyword(s):

Neutral Red ◽

Optimum Concentration ◽

Alternative Methods ◽

Uv Filter ◽

Eu Directive ◽

Optimum Concentration Range ◽

The Impact ◽

Positive Results

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1μg/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100μg/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1μg/ml and 10μg/ml, and that false positive results can be obtained at concentrations greater than 100μg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100μg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.

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Kynurenic Acid Accelerates Healing of Corneal Epithelium In Vitro and In Vivo

Pharmaceuticals ◽

10.3390/ph14080753 ◽

2021 ◽

Vol 14 (8) ◽

pp. 753

Author(s):

Anna Matysik-Woźniak ◽

Waldemar A. Turski ◽

Monika Turska ◽

Roman Paduch ◽

Mirosław Łańcut ◽

...

Keyword(s):

Epithelial Cell ◽

Corneal Epithelium ◽

Kynurenic Acid ◽

Pharmaceutical Products ◽

Corneal Epithelial Cell ◽

Eye Drops ◽

Conjunctival Epithelium ◽

Corneal Erosion

Kynurenic acid (KYNA) is an endogenous compound with a multidirectional effect. It possesses antiapoptotic, anti-inflammatory, and antioxidative properties that may be beneficial in the treatment of corneal injuries. Moreover, KYNA has been used successfully to improve the healing outcome of skin wounds. The aim of the present study is to evaluate the effects of KYNA on corneal and conjunctival cells in vitro and the re-epithelization of corneal erosion in rabbits in vivo. Normal human corneal epithelial cell (10.014 pRSV-T) and conjunctival epithelial cell (HC0597) lines were used. Cellular metabolism, cell viability, transwell migration, and the secretion of IL-1β, IL-6, and IL-10 were determined. In rabbits, after corneal de-epithelization, eye drops containing 0.002% and 1% KYNA were applied five times a day until full recovery. KYNA decreased metabolism but did not affect the proliferation of the corneal epithelium. It decreased both the metabolism and proliferation of conjunctival epithelium. KYNA enhanced the migration of corneal but not conjunctival epithelial cells. KYNA reduced the secretion of IL-1β and IL-6 from the corneal epithelium, leaving IL-10 secretion unaffected. The release of all studied cytokines from the conjunctival epithelium exposed to KYNA was unchanged. KYNA at higher concentration accelerated the healing of the corneal epithelium. These favorable properties of KYNA suggest that KYNA containing topical pharmaceutical products can be used in the treatment of ocular surface diseases.

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Ophthalmic Drug Dosage Forms: Characterisation and Research Methods

The Scientific World JOURNAL ◽

10.1155/2014/861904 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 87

Author(s):

Przemysław Baranowski ◽

Bożena Karolewicz ◽

Maciej Gajda ◽

Janusz Pluta

Keyword(s):

Defense Mechanisms ◽

Drug Application ◽

Drug Dosage ◽

Eye Drops ◽

Eye Tissues ◽

Ophthalmic Drug ◽

Application Frequency

This paper describes hitherto developed drug forms for topical ocular administration, that is, eye drops, ointments,in situgels, inserts, multicompartment drug delivery systems, and ophthalmic drug forms with bioadhesive properties. Heretofore, many studies have demonstrated that new and more complex ophthalmic drug forms exhibit advantage over traditional ones and are able to increase the bioavailability of the active substance by, among others, reducing the susceptibility of drug forms to defense mechanisms of the human eye, extending contact time of drug with the cornea, increasing the penetration through the complex anatomical structure of the eye, and providing controlled release of drugs into the eye tissues, which allows reducing the drug application frequency. The rest of the paper describes recommendedin vitroandin vivostudies to be performed for various ophthalmic drugs forms in order to assess whether the form is acceptable from the perspective of desired properties and patient’s compliance.

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ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1α-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02229-6 ◽

2022 ◽

Vol 41 (1) ◽

Author(s):

Shuohui Dong ◽

Shuo Liang ◽

Zhiqiang Cheng ◽

Xiang Zhang ◽

Li Luo ◽

...

Keyword(s):

Colorectal Cancer ◽

Glucose Metabolism ◽

Pentose Phosphate ◽

Metabolic Reprogramming ◽

Cell Models ◽

Primary Tumors ◽

Pharmacological Inhibition ◽

Potential Biomarker

Abstract Background Acquired resistance of 5-fluorouracil (5-FU) remains a clinical challenge in colorectal cancer (CRC), and efforts to develop targeted agents to reduce resistance have not yielded success. Metabolic reprogramming is a key cancer hallmark and confers several tumor phenotypes including chemoresistance. Glucose metabolic reprogramming events of 5-FU resistance in CRC has not been evaluated, and whether abnormal glucose metabolism could impart 5-FU resistance in CRC is also poorly defined. Methods Three separate acquired 5-FU resistance CRC cell line models were generated, and glucose metabolism was assessed by measuring glucose and lactate utilization, RNA and protein expressions of glucose metabolism-related enzymes and changes of intermediate metabolites of glucose metabolite pool. The protein levels of hypoxia inducible factor 1α (HIF-1α) in primary tumors and circulating tumor cells of CRC patients were detected by immunohistochemistry and immunofluorescence. Stable HIF1A knockdown in cell models was established with a lentiviral system. The influence of both HIF1A gene knockdown and pharmacological inhibition on 5-FU resistance in CRC was evaluated in cell models in vivo and in vitro. Results The abnormality of glucose metabolism in 5-FU-resistant CRC were described in detail. The enhanced glycolysis and pentose phosphate pathway in CRC were associated with increased HIF-1α expression. HIF-1α-induced glucose metabolic reprogramming imparted 5-FU resistance in CRC. HIF-1α showed enhanced expression in 5-FU-resistant CRC cell lines and clinical specimens, and increased HIF-1α levels were associated with failure of fluorouracil analog-based chemotherapy in CRC patients and poor survival. Upregulation of HIF-1α in 5-FU-resistant CRC occurred through non-oxygen-dependent mechanisms of reactive oxygen species-mediated activation of PI3K/Akt signaling and aberrant activation of β-catenin in the nucleus. Both HIF-1α gene knock-down and pharmacological inhibition restored the sensitivity of CRC to 5-FU. Conclusions HIF-1α is a potential biomarker for 5-FU-resistant CRC, and targeting HIF-1a in combination with 5-FU may represent an effective therapeutic strategy in 5-FU-resistant CRC.

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IN VITRO AND IN VIVO EVALUATION OF 2-CHLOROE THYLN ITROSOUREA DERIVATIVES AS ANTITUMOR AGENTS

Experimental Oncology ◽

10.31768/2312-8852.2015.37(1):23-29 ◽

2015 ◽

Vol 37 (1) ◽

pp. 23-29

Author(s):

A Sen ◽

K K Goswami ◽

A Mallick ◽

A K Saxena ◽

U Sanyal ◽

...

Keyword(s):

T Cells ◽

Antitumor Agents ◽

Malignant Tumors ◽

Optimum Dose ◽

Mouse Tumor ◽

Drug Induced ◽

In Vivo Evaluation ◽

Liver And Kidney

Aim: To evaluate potential of Naphthal-NU, Napro-NU and 5-Nitro-naphthal-NU, 2-chloroethylnitrosourea compounds with substituted naphthalimide in the pre-clinical studies. Materials and Methods: In vitro cytotoxicity of three nitrosoureas was determined in human and mouse tumor cell lines by MTT assays. In vivo anti-tumor potential was evaluated in Sarcoma-180 (S-180) and Ehrlich’s carcinoma (EC) solid tumors. Apoptosis in S-180 cells was analyzed by using Annexin V-Propidium Iodide (PI). Histological analysis of liver and kidney was performed at optimum dose (50 mg/kg). Expression status of CD4+, CD8+ and CD25+ cells in treated mouse were also examined. Results: Significant tumor growth retardation by the compounds was noted in early and advanced disease groups, as the life span of drug treated mice increased considerably. Drug induced killing was observed by induction of apoptosis. Naphthal-NU and 5-Nitro-naphthal-NU were effective to normalize the tumor induced structural abnormalities of liver and kidney. The compounds have no immunotoxic effect on CD4+ and CD8+ T cells and down regulate CD4+CD25+ regulatory T cells. Conclusion: Overall data holds promise for the antitumor activity with lower toxicity of the compounds that can be utilized for the treatment of human malignant tumors.

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AMPHOTERICIN B TOPICAL EXTEMPORANEOUS PREPARATIONS FOR THE TREATMENT OF NON-DERMATOPHYTIC ONYCHOMYCOSIS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2020.v13i12.39645 ◽

2020 ◽

pp. 135-139

Author(s):

KOMESMUNEEBORIRAK PHOJANA ◽

WERAWATGANONE PORNPEN ◽

MUANGSIRI WALAISIRI

Keyword(s):

Amphotericin B ◽

Chemical Stability ◽

High Performance ◽

Sodium Lauryl Sulfate ◽

Physical Stability ◽

Lauryl Sulfate ◽

Physical And Chemical ◽

Injection Product

Objective: At present, the nail preparation to cure onychomycosis, caused by non-dermatophyte molds, is not commercially available in Thailand. The physical and chemical stability of amphotericin B (AmB) extemporaneous preparations in the presence of 30% dimethyl sulfoxide (DMSO) and their in vitro nail permeation was evaluated. Methods: AmB extemporaneous preparations in the presence of 30% DMSO were prepared from a commercial sterile injection product, and cream or hydrophilic ointment. Physical stability was tested at 30°C for 2 months, or using 6 heating-cooling cycles. The chemical stability and in vitro nail permeation of AmB content were analyzed using high-performance liquid chromatography (HPLC). In vitro nail permeation was performed by applying 3.5 mg/mm2 of the tested formulation on nail clippings for 5 consecutive days. Results: The AmB cream and ointment extemporaneous preparations containing 30% DMSO, a permeation enhancer, were homogeneous and pale yellow to yellow cream or ointment. The AmB ointment was stable for up to 60 days. The ointment preparation allows in vitro penetration through nails up to 14.17 μg/cm2. The ointment preparation allows significantly better penetration through than the cream preparation due to the presence of DMSO, sodium lauryl sulfate (SLS), and water in the ointment preparation. Conclusion: The AmB extemporaneous ointment was successfully compounded from a commercial sterile injection product with a beyond-use date of 60 days. The ointment preparation is currently under further investigation for in vivo efficacy.

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Preparation and Evaluation of Topically Applied Azithromycin Based on Sodium Hyaluronate in Treatment of Conjunctivitis

Pharmaceutics ◽

10.3390/pharmaceutics11040183 ◽

2019 ◽

Vol 11 (4) ◽

pp. 183

Author(s):

Chen ◽

Yin ◽

Ma ◽

Tu ◽

Shen

Keyword(s):

Acute Toxicity ◽

Hydroxypropyl Methylcellulose ◽

Sodium Hyaluronate ◽

Drug Formulation ◽

Resonance Spectroscopy ◽

Eye Drops ◽

Scanning Calorimetry ◽

Eye Irritation

Azithromycin (AZI) eye drops containing sodium hyaluronate (SH) were developed to improve the bioavailability of AZI. Interaction between AZI and SH in the AZI-SH formulation was investigated by differential scanning calorimetry, X-ray diffraction, and 1H-nuclear magnetic resonance spectroscopy analyses. Moreover, advantages of using SH as an excipient were investigated by comparing physiological properties and pharmacokinetic behaviors of SH-containing AZI eye drops with that of hydroxypropyl methylcellulose (HPMC)-containing formulation. In addition, safety of the developed AZI-SH eye drops was evaluated by in vitro 3-(4,5-dimethyl-2-Thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay (MTT assay) and neutral red uptake assay as well as in vivo eye irritation test and acute toxicity test. The results indicated that AZI formed a complex with SH under a slightly acidic condition. The area under the curve (AUC) of AZI in SH-containing formulation was 1.58-fold higher (P<0.01) than that in HPMC-containing formulation due to the interaction between the amine group of AZI and the carboxyl group of SH, despite of the higher viscosity of HPMC-containing formulation. Safety evaluation showed that AZI-SH eye drops caused no obvious eye irritation and acute toxicity. In conclusion, the developed SH-containing AZI formulation possessing advantages of longer retention time and higher drug availability was a promising drug formulation for topical ocular therapy.

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Coxsackievirus B3 Responds to Polyamine Depletion via Enhancement of 2A and 3C Protease Activity

Viruses ◽

10.3390/v11050403 ◽

2019 ◽

Vol 11 (5) ◽

pp. 403 ◽

Cited By ~ 8

Author(s):

Courtney N. Dial ◽

Patrick M. Tate ◽

Thomas M. Kicmal ◽

Bryan C. Mounce

Keyword(s):

Protease Activity ◽

Rna Virus ◽

Coxsackievirus B3 ◽

Eukaryotic Cells ◽

Luciferase Reporter ◽

Reporter System ◽

3C Protease ◽

Picornavirus Infection

Polyamines are small positively-charged molecules abundant in eukaryotic cells that are crucial to RNA virus replication. In eukaryotic cells, polyamines facilitate processes such as transcription, translation, and DNA replication, and viruses similarly rely on polyamines to facilitate transcription and translation. Whether polyamines function at additional stages in viral replication remains poorly understood. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to polyamine depletion both in vitro and in vivo; however, precisely how polyamine function in picornavirus infection has not been described. Here, we describe CVB3 mutants that arise with passage in polyamine-depleted conditions. We observe mutations in the 2A and 3C proteases, and we find that these mutant proteases confer resistance to polyamine depletion. Using a split luciferase reporter system to measure protease activity, we determined that polyamines facilitate viral protease activity. We further observe that the 2A and 3C protease mutations enhance reporter protease activity in polyamine-depleted conditions. Finally, we find that these mutations promote cleavage of cellular eIF4G during infection of polyamine-depleted cells. In sum, our results suggest that polyamines are crucial to protease function during picornavirus infection. Further, these data highlight viral proteases as potential antiviral targets and highlight how CVB3 may overcome polyamine-depleting antiviral therapies.

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Circular RNA_CNST Promotes the Tumorigenesis of Osteosarcoma Cells by Sponging miR-421

Cell Transplantation ◽

10.1177/0963689720926147 ◽

2020 ◽

Vol 29 ◽

pp. 096368972092614

Author(s):

Ji-Hai Wang ◽

Xue-Jian Wu ◽

Yong-Zhuang Duan ◽

Feng Li

Keyword(s):

Colony Formation ◽

Specific Binding ◽

Tumor Model ◽

Gene Expression Omnibus ◽

Pcr Analysis ◽

Circular Rnas ◽

Xenograft Tumor ◽

Potential Biomarker

Circular RNAs (circRNAs) act crucial roles in the progression of multiple malignancies including osteosarcoma (OS). But, the underlying mechanisms by which hsa_circ_0017311 (circCNST) contributes to the tumorigenesis of OS remain poorly understood. Our present study aimed to explore the role and mechanisms of circCNST in OS tumorigenesis. The differentially expressed circRNAs were identified by the Gene Expression Omnibus database. The association of circCNST with clinicopathological features and prognosis in patients with OS was analyzed by RNA fluorescence in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (PCR) analysis. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation assays, and a xenograft tumor model were conducted to assess the role of circCNST in OS cells in vitro and in vivo. CircCNST-specific binding with miR-421 was confirmed by FISH, luciferase gene report, and RNA immunoprecipitation assays. As a result, we found that the expression levels of circCNST were dramatically increased in OS tissues and cell lines as compared with the adjacent normal tissues, and it was associated with tumor size and poor survival in OS patients. Knockdown of circCNST repressed cell viability, colony formation, and xenograft tumor growth, while restored expression of circCNST reversed these effects. Furthermore, circCNST was colocalized with miR-421 in the cytoplasm and acted as a sponge of miR-421, which attenuated circCNST-induced proliferation-promoting effects in OS cells by targeting SLC25A3. In conclusion, our findings demonstrate that circCNST promotes the tumorigenesis of OS cells by sponging miR-421, and provides a potential biomarker for patients with OS.

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