Inhibition of UVB-induced skin phototoxicity by a grape seed extract as modulator of nitrosative stress,ERK/NF-kB signaling pathway and apoptosis,in SKH-1 mice

Molecular mechanisms concerning the modulation of nitrosative stress, signal transduction and proliferation/apoptosis by a grape seed extract, Burgund Mare variety (BM), in SKH-1 mice exposed to UVB, were investigated. The animals were irradiated with single and multiple doses of UVB in 10 consecutive days. In each experiment were used five groups of animals: control, vehicle, UVB irradiated, vehicle + UVB, BM + UVB. The extract was applied topically, 30 min before each UVB exposure, in a dose of 4 mg total polyphenols/cm². BM remarkably inhibited UVB-induced activation of inducible nitric oxide synthase (iNOS) and therefore generation of nitric oxide (NO) and nitrotyrosine, in a UVB single dose regimen. BM also suppressed NF-kB activation by UVB but did not affect the activity of total ERK 1/2. In multiple UVB irradiations, BM increased NO formation and total ERK 1/2 activity and reduced iNOS activity and nitrotyrosine levels, inhibited cell proliferation, diminished p53 and caspase-3 immunoreactivities and increased the percentage of Bcl-2 positive cells. We concluded that BM modulates the apoptotic response of SKH-1 mice skin in UVB irradiation by the inhibition of p53, caspase-3, Bax/Bcl-2 and proliferating cell nuclear antigen expressions, as well as by reducing the activation of iNOS and NF-kB.     

Histopathological,cytotoxicological, and genotoxic effects of the semi-synthetic compound dillapiole n-butyl ether in Balb/C mice

ABSTRACT

Dillapiole n-butyl ether is a substance derived from dillapiole, which exhibits potential insecticidal effects on Aedes aegypti, the principal vector of the Dengue fever, Zika, and Chikungunya viruses, as well as Aedes albopictus, a vector of Dengue fever. As these mosquitoes are resistant to synthetic insecticides, dillapiole n-butyl ether may represent a valuable, plant-based alternative for their control. Dillapiole n-butyl ether has insecticidal and genotoxic effects on A. aegypti and A. albopictus, as shown by the reduction in clutch size and egg viability, and increased mortality rates, as well as a high frequency of micronuclei and chromosomal aberrations. However, the potential cytotoxic and genotoxic effects of this substance in mammals are still unknown. In Balb/C mice, structural changes were detected in hepatic, renal, and cardiac tissues, which were directly proportional to the concentration of the dose applied, in both genders. The induction of genotoxic, mutagenic, and cytotoxic effects was also observed at the highest concentrations (150 and 328 mg/kg). Further research will be necessary to better characterize the potential genotoxicity of this substance at lower concentrations, for the evaluation of the potential health risks related to its presence in environmental features, such as drinking water.     

Nuclear translocation of nuclear factor kappa B is regulated by G protein signaling pathway in arsenite‐induced apoptosis in HBE cell line

Arsenite is a certainly apoptosis inducer in various cell types. However, the detailed mechanism underlying how arsenite trigger apoptosis remains elusive. In this study, using human bronchial epithelial cell as a culture system, we demonstrated that arsenite‐induced nuclear translocation of nuclear factor kappa B (NF‐κB) resulted in the release of cytochrome c, the modulation of Fas and FasL, caspase activation, and ultimately leading to cell apoptosis. Importantly, we showed for the first time that the NF‐κB‐mediated apoptosis induced by arsenite was regulated by G protein‐adenylate cyclase (AC)‐cyclic adenosine monophosphate (cAMP)‐protein kinase A (PKA) pathway. Inhibition of this classical G protein signaling pathway by a typical PKA inhibitor, H‐89, caused the inactivation of NF‐κB, the depletion of caspase‐3, 8 and 9 activities, and thus reducing the level of cell apoptosis. Taken together, our results indicate that arsenite is able to trigger cell apoptosis in human bronchial epithelial cells through the nuclear translocation of NF‐κB, which can be modulated by G protein signaling pathway. These findings further suggest that inhibition of G protein‐mediated pathway by specific inhibitors may be a potential strategy for the prevention of arsenite toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1819–1833, 2016.     

Delphinidin induces apoptosis and inhibits epithelial‐to‐mesenchymal transition via the ERK/p38 MAPK‐signaling pathway in human osteosarcoma cell lines

Delphinidin is major anthocyanidin that is extracted from many pigmented fruits and vegetables. This substance has anti‐oxidant, anti‐inflammatory, anti‐angiogenic, and anti‐cancer properties. In addition, delphinidin strongly suppresses the migration and invasion of various cancer cells during tumorigenesis. Although delphinidin has anti‐cancer effects, little is known about its functional roles in osteosarcoma (OS). For these reasons, we have demonstrated the effects of delphinidin on OS cell lines. The effects of delphinidin on cell viability and growth of OS cells were assessed using the MTT assay and colony formation assays. Hoechst staining indicated that the delphinidin‐treated OS cells were undergoing apoptosis. Flow cytometry, confocal microscopy, and a western blot analysis also indicated evidence of apoptosis. Inhibition of cell migration and invasion was found to be associated with epithelial‐to‐mesenchymal transition (EMT), observed by using a wound healing assay, an invasion assay, and a western blot analysis. Furthermore, delphinidin treatment resulted in a profound reduction of phosphorylated forms of ERK and p38. These findings demonstrate that delphinidin treatment suppressed EMT through the mitogen‐activated protein kinase (MAPK) signaling pathway in OS cell lines. Taken together, our results suggest that delphinidin strongly inhibits cell proliferation and induces apoptosis. Delphinidin treatment also suppresses cell migration and prevents EMT via the MAPK‐signaling pathway in OS cell lines. For these reasons, delphinidin has anti‐cancer effects and can suppress metastasis in OS cell lines, and it might be worth using as an OS therapeutic agent.     

Dose-dependent effects of berberine on cell cycle pause and apoptosis in Balb/c 3T3 cells

In determining the morphological appearance of Balb/c 3T3 cells from berberine-treated (100 and 200 g/ml) cultures by light microscopy demonstrated that the high berberine concentration (200 g/ml) treatment was associated with the accumulation of numerous apoptotic cells, as identified by condensed nuclei and decrease in cell size. On the other hand, accumulation of cells in G₂/M phase instead of induction of apoptosis was observed after 48–72 h of 100 g/ml berberine treatment. Berberine was found mainly in cytoplasm during berberine-induced (100 g/ml) cell cycle G₂/M arrest, while it was highly concentrated in nuclei in the induction of apoptosis under high dose of berberine (200 g/ml) treatment. Further addition of berberine (100–200 g/ml) had little effect on the induction of apoptosis in the cells that had already been exposed to 100 g/ml of berberine for 48 h. Our results suggest that there may exist in Balb/c 3T3 cells an important threshold for regulation of cell cycle pause and induction of apoptosis, that is dose-dependent.     

Cadmium-induced germline apoptosis in Caenorhabditis elegans: the roles of HUS1, p53, and MAPK signaling pathways.

The transition metal cadmium (Cd) has been shown to induce apoptosis in a variety of cell lines and tissues. Caspase activation of the tumor suppressor gene p53 and mitogen-activated protein kinase (MAPK) signaling cascades have been reported to be involved in Cd-induced apoptosis. However, the underlying pathways of Cd-induced apoptosis have not been clearly elucidated in the in vivo systems, primarily for the lack of appropriate animal models. The nematode Caenorhabditis elegans has been shown to be a good model to study basic biological processes, including apoptosis. In this study, we used the mutated alleles of C. elegans homologs of known mammalian genes that are involved in regulation of apoptosis. Sublethal doses of Cd exposure increased C. elegans germline apoptosis in a dose- and time-dependent manner. The loss-of-function mutations of DNA damage response (DDR) genes HUS1 and p53 exhibited significant increase in germline apoptosis under Cd exposure, and the depletion of p53 antagonist ABL1 significantly enhanced apoptosis. Cd-induced apoptosis was blocked in the loss-of-function alleles of both c-Jun N-terminal kinase (JNK) and p38 MAPK cascades, which behaved normally under gamma-irradiation. Our findings implicate that both JNK and p38 MAPK cascades participate in Cd-induced apoptosis. Together, the results of this study suggest the nonessential roles of the DDR genes hus1 and p53 in Cd-induced germline apoptosis and that the apoptosis occurs through the ASK1/2-MKK7-JNK and ASK1/2-MKK3/6-p38 signaling pathways in a caspase-dependent manner. Finally, our study demonstrates that C. elegans is a mammalian in vivo substitute model to study the mechanisms of Cd-induced apoptosis.     

Effect of polypeptides in bee venom on growth inhibition and apoptosis induction of the human hepatoma cell line SMMC-7721 in-vitro and Balb/c nude mice in-vivo

Polypeptides in bee venom (PBV) produced a significant growth inhibition against SMMC-7721 human hepatoma cell line. Analysis of the mechanisms of cell death indicated that PBV induced an apoptotic cell death. SMMC-7721 cells exposed to PBV (10.0 microg mL(-1)) produced an insignificant morphological change. Analysis of the cytotoxicity with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay confirmed that the cytotoxic effects of PBV were dose- and timedependent. The result of Ki67 immunohistochemistry demonstrated that the proliferation of SMMC-7721 cells treated with PBV (10.0 mug mL(-1)) was inhibited. The apoptotic cell death was then confirmed by annexin V, propidium iodide staining and DNA fragmentation analysis. In in-vivo experiments, treatment with PBV (1.5 or 3 mg kg(-1)) resulted in a significant retardation of SMMC-7721 cell growth in Balb/c nude mice. These findings suggested that PBV could be used as a chemotherapeutic agent against tumours.     

Diterpenoid C of Radix Curcumae: An inhibitor of proliferation and inducer of apoptosis in human colon adenocarcinoma cells acting via inhibiting MAPK signaling pathway

Context: Radix Curcumae is a traditional Chinese medicine that possesses antitumor properties, from which a new compound, diterpenoid C, was previously isolated and characterized.

Objective: In this study, using human colon adenocarcinoma SW620 cells, we further investigated the antitumor effects of diterpenoid C and the underlying mechanisms.

Materials and methods: Cell proliferation was assessed with the MTT assay. Cell apoptosis and cell-cycle progression were analyzed with flow cytometry. The expression of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and their phosphorylated forms, as well as caspase-3 protein levels were examined with Western blots.

Results: Diterpenoid C could inhibit the proliferation of SW620 cells in a dose- and time-dependent manner. The median inhibitory concentration (IC₅₀) at 24, 48, and 72?h were 28.31, 15.58, and 6.14?μg/ml, respectively. The inhibition of proliferation was found to be statistically significant as compared with the well-established drugs 5-fluorouracil (5-Fu) and oxaliplatin (L-OHP) (p?Discussion and conclusion: Diterpenoid C inhibits proliferation and induces apoptosis of cancer cells by suppressing the MAPK signaling pathway and inducing apoptotic factor caspase-3. Our results suggest that this novel compound might become a potent chemotherapeutic agent for the treatment of colon cancer and further studies are warranted.     

依托咪酯通过调控MAPK/ERK信号通路对阿尔茨海默病小鼠认知功能和氧化应激的影响

目的　探究依托咪酯通过调控丝裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶（ERK）信号通路对阿尔茨海默病小鼠认知功能和氧化应激的影响。方法　本研究院于2020年5月至2021年9月进行。将50只APP/PS1转基因小鼠分为模型组,依托咪酯低、中、高剂量组,阳性对照组,10只SPF级C57BL/6小鼠作为对照组,其中依托咪酯低、中、高剂量组分别给予1.0、2.5、5.0 mg/kg依托咪酯灌胃,阳性对照组给予10 mg/kg盐酸多奈哌齐灌胃,模型组、对照组给予等体积生理盐水灌胃,1次/ d,灌胃30 d。采用Morris水迷宫实验观察小鼠学习、记忆能力,并测定小鼠神经损伤评分、脑组织含水量;蛋白免疫印迹法（Western blot）检测小鼠脑组织肿瘤坏死因子（TNF）-α、白细胞介素（IL）-10、IL-1β、MAPK/ERK信号通路相关蛋白表达;酶联免疫吸附法（ELISA）检测小鼠脑组织超氧化物歧化酶（SOD）活性、过氧化氢酶（CAT）活性、谷胱甘肽过氧化物酶（GSH-Px）活性、丙二醛（MDA）含量。计量资料多组间比较采用单因素方差分析,进一步组内间比较用LSD-t检验。结果　与对照组相比,模型组的逃避潜伏期均明显延长,跨平台次数、平台区停留距离、SOD活性、CAT活性、GSH-Px活性均降低,神经损伤评分、脑组织含水量、MDA含量均增加,上调TNF-α、IL-10、IL-1β蛋白表达,下调磷酸化细胞外信号调节激酶（p-ERK）1/2、p-p38蛋白表达（均P<0.05）;与模型组比较,依托咪酯低、中、高剂量组的逃避潜伏期均明显缩短,跨平台次数、平台区停留距离、SOD活性、CAT活性、GSH-Px活性均升高,神经损伤评分、脑组织含水量、MDA含量均降低,并下调TNF-α、IL-10、IL-1β蛋白表达,上调p-ERK1/2、p-p38蛋白表达（均P<0.05）。结论　依托咪酯可能通过增加MAPK/ERK信号通路改善阿尔茨海默病小鼠认知功能,并减轻小鼠脑组织氧化应激和炎性反应。     

Acute toxicity assessment of choline by inhalation, intraperitoneal and oral routes in Balb/c mice

Studies suggest that choline has potential to be used as a dietary supplement and a drug for immune inflammatory diseases like asthma and rhinitis. But there are apprehensions regarding adverse effects of choline when given orally in high doses. To address this knowledge gap, toxicity assessment of choline chloride was carried out by intranasal (i.n.), oral and intraperitoneal (i.p.) routes in Balb/c mice for 28 days. Body weight, food and water consumption of mice were recorded daily. Hematology and clinical chemistry were assessed to check hepatocellular functions and morphological alterations of the cells. Splenocyte counts were analysed for evaluating cellular immunity. Liver function test was performed by assaying different enzyme systems in serum such as, urea, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Body weight, food and water consumption did not differ between mice treated with choline and the saline control group. Hematologic and biochemical variables were not affected with any increase in serum toxicity marker enzymes indicating normal liver functioning. Choline administration did not affect total cholesterol and high density lipoprotein levels as compared to their respective controls. Urea and blood urea nitrogen levels in choline treated mice were not different than controls. Creatinine level was, however, higher than control in i.p. treatment group, but other parameters were normal. In conclusion, the repeated consumption of choline chloride via i.n. and oral or i.p. routes did not cause toxicity in mice in the toxicological endpoints examined.     

Effect of daidzein on anxiety, social behavior and spatial learning in male Balb/cJ mice

Daidzein is an abundant isoflavone present in soy. It is unique as it can be further metabolized into equol, a compound with greater estrogenic activity than other isoflavones. The potential benefit of daidzein in the prevention of various cancers and cardiovascular diseases has drawn attention to this molecule and isoflavone consumption is increasing around the world. However, it remains unclear whether daidzein affects locomotor activity, anxiety, social behavior or spatial memory. Here we report the results from a range of tests designed to assess anxiety, social behavior and spatial learning and memory in male Balb/cJ mice following consumption of daidzein for 30 days. We found that daidzein treatment significantly increased locomotor activity in the open field, elevated plus-maze and Morris water-maze tests; resulted in increased amicable behavior, decreased aggression and decreased sexual behavior during social interaction tests; and resulted in an anxiolytic effect amongst treated males. We found no effect of daidzein consumption on the acquisition and retrieval of spatial memory. Our results suggest that long-term consumption of daidzein may produce significant effects on locomotor activity, mood and social behavior without significant effects on learning and memory.     

Methanolic bark extract of Acacia catechu ameliorates benzo(a)pyrene induced lung toxicity by abrogation of oxidative stress,inflammation, and apoptosis in mice

Benzo(a)pyrene [B(a)P] is a well‐known carcinogen present in the environment. In this study, we evaluated the protective potential of methanolic bark extract of Acacia catechu Willd. (MEBA) against the lung toxicity induced by B(a)P in Swiss albino mice. To determine the protective efficacy of MEBA, it was orally administered to the mice at two doses (200 and 400 mg/kg body weight) once daily for 7 days. Mice were also exposed (orally) to B(a)P at a dose of 125 mg/kg body weight on 7th day. Administration of B(a)P increased the activities of toxicity markers such as LDH, LPO, and XO with a subsequent decrease in the activities of tissue anti‐oxidant armory (CAT, SOD, GST, GPx, GR, QR, and GSH). It also caused activation of the apoptotic and inflammatory pathway by upregulation of TNF‐α, NF‐kB, COX‐2, p53, bax, caspase‐3, and downregulating Bcl‐2. Pretreatment with MEBA at two different doses (200 and 400 mg/kg body weight) significantly ameliorates B(a)P‐induced increased toxicity markers and activities of detoxifying enzymes along with the levels of glutathione content. It also significantly attenuated expression of apoptotic and inflammatory markers in the lungs. Histological results further confirmed the protective role of MEBA against B(a)P‐induced lung toxicity. The results indicate that MEBA may be beneficial in ameliorating the B(a)P‐induced oxidative stress, inflammation, and apoptosis in the lungs of mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1566–1577, 2017.     

DNA damage, redox changes, and associated stress-inducible signaling events underlying the apoptosis and cytotoxicity in murine alveolar macrophage cell line MH-S by methanol-extracted Stachybotrys chartarum toxins

Spore-extracted toxins of the indoor mold Stachybotrys chartarum (SC) caused cytotoxicity (release of lactate dehydrogenase), inhibition of cell proliferation, and cell death in murine alveolar macrophage cell line MH-S in a dose- and time-dependent manner. Apoptotic cell death, confirmed based on morphological changes, DNA ladder formation, and caspase 3/7 activation, was detectable as early as at 3 h during treatment with a toxin concentration of 1 spore equivalent/macrophage and was preceded by DNA damage beginning at 15 min, as evidenced by DNA comet formation in single cell gel electrophoresis assay. The apoptotic dose of SC toxins did not induce detectable nitric oxide and pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) but showed exacerbated cytotoxicity in presence of a non-apoptotic dose of the known pro-inflammatory agent LPS (10 ng/ml). Intracellular reduced glutathione (GSH) level showed a significant decrease beginning at 9 h of the toxin treatment whereas oxidized glutathione (GSSG) showed a corresponding significant increase, indicating a delayed onset of oxidative stress in the apoptosis process. The toxin-treated macrophages accumulated p53, an indicator of DNA damage response, and showed activation of the stress-inducible MAP kinases, JNK, and p38, in a time-dependent manner. Chemical blocking of either p38 or p53 inhibited in part the SC toxin-induced apoptosis whereas blocking of JNK did not show any such effect. This study constitutes the first report on induction of DNA damage and associated p53 activation by SC toxins, and demonstrates the involvement of p38- and p53-mediated signaling events in SC toxin-induced apoptosis of alveolar macrophages.     

Cadmium-induced apoptosis in the BJAB human B cell line: involvement of PKC/ERK1/2/JNK signaling pathways in HO-1 expression

Heme oxygenase-1 (HO-1, EC 1.14.99.3) is a key enzyme in the cellular response to tissue injury and oxidative stress. It oxidizes heme, a pro-oxidant and toxic species, to biliverdin, CO, and free iron. Cytoprotection during the heat shock response is a complex phenomenon involving multiple inducible mechanisms. Several important pathways involving serine/threonine kinases mediate the induction of HO-1 in response to external stimuli.     

Acute and subacute (20-d) oral dose toxicity study of modified fluoroquinolone compound 6C in BALB/c mice

Introduction: Antibiotics are compounds used to treat inflammation; fluoroquinolones are antibiotics used in resistant cases. Objective: The purpose of this study was to investigate acute and subacute toxicity for a new modified flouroquinolone compound (MFC 6C) – a broad spectrum antibiotic which was invented at Faculty of Pharmacy in University of Jordan – using BALB/c mice. Materials and methods: In the pilot study (8 groups; 1 Male:1 Female/group), MFC 6C was administered to these mice at dose levels of 500, 600, 700, 1000, 1200, 1600, 3000 and 5000?mg/kg/d. In the subacute study (5 groups; 5 Males:5 Females/group), MFC 6C was administered for two groups at dose levels of 500, 250?mg/kg/d for 20?d by oral gavage; other groups were control groups. Results: Before the acute study, a pilot study was conducted (on 8 separate days) and no mortality was found even at 5000?mg/kg; therefore, LD₅₀ was found to be >5000?mg/kg and no further acute effects need to be investigated; so MFC 6C is slightly toxic. The biochemical study revealed that, in subacute toxicity study (20 consecutive days), MFC 6C 500?mg/kg caused a decrease in male and female mice blood serum SGOT [p?=?0.0189 (for males), 0.0309 (for females)] and a decrease in male mice blood serum CPK level (p?=?0.023). MFC 6C (250?mg/kg) caused a significant decrease of male mice blood serum sugar (p?=?0.04278) and CPK level (p?=?0.005). The histopathological study revealed that, in subacute toxicity study (20 consecutive days), MFC 6C (500?mg/kg) caused; periportal lymphocytic inflammation (male, 60%; female 40%), lymphoid follicle (female, 60%), neutrophilic aggregation and mitotic activity (female, 40%) in the liver. Moreover, it caused interstitial lymphocytic inflammation (male 60%; female 20%) in the kidney. Other changes like follicular hyperplasia (male 40%) were observed in the spleen. It also caused neutrophilic aggregation (male 40%) in the heart. Also, congestion and macrophages were observed. Changes like lymphocytic infiltration (male 20%; female 20%), congestion (male, 20%) and pleural mesothelial hyperplasia (female, 20%) were found in the lungs. MFC 6C (250?mg/kg), in subacute study, caused; lobular lymphocytic infiltration (male 100%; female 100%), portal lymphocytic inflammation (male 40%; female 40%), granuloma and extramedullary hematopoeisis (male 20%; female 20%), apoptotic bodies and plasma collection in the liver. On the other hand, it caused; reactive lymphoid hyperplasia (male 20%; female 20%) in the spleen. Fibrinous pericarditis (male 40%; female 40%), pericardial mesothelial hyperplasia and degenerated myofibers (male 20%; female 20%) were observed in the heart. Parenchymal lymphocytic infiltration was observed in the lungs (male 40%; female 40%). While no changes occurred in testis at the dose (250?mg/kg). No observed effect level (NOEL) was 125?mg/kg/d for 20-d subacute toxicity study. Conclusion: MFC 6C may suppress the function and\or morphology of the body organs.     

Human epithelial carcinoma cytotoxicity and inhibition of DMBA/TPA induced squamous cell carcinoma in Balb/c mice by Acacia catechu heartwood

Objectives Acacia catechu heartwood contains significant amounts of polyphenolic compounds that exhibit powerful antioxidant activity. The purpose of this study was to evaluate the cytotoxicity of A. catechu heartwood extracts in a human epithelial carcinoma cell line (A431) and antitumour activity against DMBA/TPA induced squamous cell carcinoma in Balb/c mice. Methods Various extracts, including aqueous, ethyl acetate, chloroform and n‐hexane, were tested for cytotoxic properties on a human epithelial carcinoma cell line (A431) by using MTT, sulforhodamine B and lactate dehydrogenase leakage assays. The standardized A. catechu heartwood aqueous extract (AQCE) was further evaluated for antitumour activity against 7,12‐dimethylbenz[a]anthracene (DMBA)/12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) induced skin carcinoma in Balb/c mice. Key findings The results showed that administration of AQCE showed a dose‐dependent growth inhibition response, with an IC50 value of 78.56 µg/ml. Tumour incidence was significantly decreased (P < 0.001) to 30% with AQCE compared with 100% in the DMBA/TPA group. The AQCE was also found to significantly upregulate different antioxidant enzymes in skin and liver tissue. Conclusions The results suggest that AQCE may exert its chemopreventive activity by acting as an antioxidant.