mRNA Levels Of Apoptosis-related Genes Research Articles

Multi-walled carbon nanotube induced liver injuries possibly by promoting endoplasmic reticulum stress in Cyprinus carpio

The mass production and discharge of carbon nanotubes (CNTs) to the water environment are of great concern since they threaten the health of organisms in the aquatic ecosystem. CNTs induce multi-organ injuries in fish, but limited literature is available regarding the mechanisms involved. In the present study, juvenile common carp (Cyprinus carpio) were exposed to multi-walled carbon nanotubes (MWCNTs) (0.25 mg L−1 and 2.5 mg L−1) for four weeks. MWCNTs caused dose-dependent alterations in the pathological morphology of liver tissues. Ultrastructural changes manifested as nuclear deformation, chromatin condensation, endoplasmic reticulum (ER) disorderly arrangement, mitochondria vacuolation, and mitochondrial membrane destruction. TUNEL analysis indicated that the apoptosis rate in hepatocytes markedly increased upon exposure to MWCNTs. Moreover, the apoptosis was confirmed by significant upregulation of mRNA levels of apoptosis-related genes (Bcl-2, XBP1, Bax, and caspase3) in MWCNTs-exposure groups, except for Bcl-2 expression which was not significantly changed in HSC groups (2.5 mg L−1 MWCNTs). Furthermore, real-time PCR assay indicated the increased expression of ER stress (ERS) marker genes (GRP78, PERK, and eIF2α) in the exposure groups compared to the control groups, suggesting that the PERK/eIF2α signaling pathway involved in the injuries of the liver tissue. Overall, the results above indicate that MWCNTs induce ERS by activating the PERK/eIF2α pathway in the liver of common carp, and resulted in the initiation of apoptosis procedure.

Environmentally relevant concentrations of organic (benzophenone-3) and inorganic (titanium dioxide nanoparticles) UV filters co-exposure induced neurodevelopmental toxicity in zebrafish

UV filters, widely used in personal care products, are ubiquitous environmental pollutants detected and pose a significant public health concern. Benzophenone-3 (BP3) and titanium dioxide nanoparticles (nano-TiO2) are the predominant organic and inorganic UV filters in environmental media. However, few studies have explored the combined developmental neurotoxic (DNT) effects and the underlying mechanisms when co-exposed to BP3 and nano-TiO2. In the present study, zebrafish (Danio rerio) embryos were exposed to environmentally relevant concentrations of BP3 (10 μg/L), nano-TiO2 (100 μg/L), and mixtures starting from 6 h post fertilization (hpf), respectively. Developmental indicators and motor behaviors were investigated at various developmental stages. Our results showed that BP3 alone or co-exposed with nano-TiO2 increased spontaneous movement at 24 hpf, co-exposure decreased touch response at 30 hpf and hatching rate at 60 hpf. Consistent with these motor deficits, co-exposure to BP3 and nano-TiO2 inhibited relative axon length of primary motor neuron during the early developmental stages, disturbed the expression of axonal growth-related genes at 30 and 48 hpf, increased cell apoptosis on the head region and mRNA levels of apoptosis-related genes, and also increased reactive oxygen species (ROS) levels in zebrafish, suggesting the functional relevance of structural changes. Taken together, our findings demonstrated that BP3 alone or in combination with nano-TiO2 at environmentally relevant concentrations induced evident neurotoxic effects on the developing embryos in zebrafish.

Vitamin D3 and Salinomycin synergy in MCF-7 cells cause cell death via endoplasmic reticulum stress in monolayer and 3D cell culture

1α, 25, dihydroxyvitamin D3 (1,25D), the active form of vitamin D3, has antitumor properties in several cancer cell lines in vitro. Salinomycin (Sal) has anticancer activity against cancer cell lines. This study aims to examine the cytotoxic and antiproliferative effect of Sal associated with 1,25D on MCF-7 breast carcinoma cell line cultured in monolayer (2D) and three-dimensional models (mammospheres). We also aim to evaluate the molecular mechanism of Sal and 1,25D-mediated effects. We report that Sal and 1,25D act synergistically in MCF-7 mammospheres and monolayer causing G1 cell cycle arrest, reduction of mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) overproduction with a long-lasting cytotoxic response represented by clonogenic and mammosphere assay. We observed the induction of cell death by apoptosis with upregulation in mRNA levels of apoptosis-related genes (CASP7, CASP9, and BBC3). Extensive cytoplasmic vacuolization, a morphological characteristic found in paraptosis, was also seen and could be triggered by endoplasmic reticulum stress (ER) as we found transcriptional upregulation of genes related to ER stress (ATF6, GADD153, GADD45G, EIF2AK3, and HSPA5). Overall, Sal and 1,25D act synergistically, inhibiting cell proliferation by activating simultaneously multiple death pathways and may be a novel and promising luminal A breast cancer therapy strategy.

UV-filter octyl methoxycinnamate causes reproductive toxicity associated with germline apoptosis and vitellogenin decrease in Caenorhabditis elegans

Octyl methoxycinnamate (OMC) is a common UV filter found in personal care products such as sunscreen and cosmetics. However, OMC's presence in wastewater has raised concerns that it could potentially pollute aquatic ecosystems because of its limited biodegradability and its estrogenic disrupting properties. In this study, we investigated the environmental toxicity of OMC and its potential biomarkers using the nematode Caenorhabditis elegans. Our results showed that body length, eggs in utero, and total brood size decreased with increasing dose (experimental concentrations=0, 1, 5, 10, 100, 500μM for body length and eggs in utero, and 0, 5, 10μM for total brood size) in C. elegans after L1 larval stage (the first larval stage for 0 - 12 hours post-hatching) larval stage exposure to OMC. The minimum effective concentrations were 1, 5, and 10μM, respectively. Modeling results demonstrated that the threshold concentration of OMC inducing 10% inhibited eggs in utero was 0.33μM (95.11μg/L). Furthermore, germline apoptosis was induced in 10μM OMC-treated worms (experimental concentrations=0, 5, 10μM). Decreased mRNA levels of vitellogenin-related genes (vit-2 and vit-6) and increased mRNA levels of apoptosis-related genes (egl-1 and ced-3) were observed in 10μM OMC-treated C. elegans (experimental concentrations=0, 10μM), suggesting that reproductive toxicity was associated with decreased vitellogenin levels and germline apoptosis. In summary, our study shows that OMC is reproductively toxic and leads to reduced egg formation and decreased brood size in C. elegans by reducing vitellogenin levels and promoting germline apoptosis.

Tripterygium wilfordii multiglycoside-induced hepatotoxicity via inflammation and apoptosis in zebrafish.

Tripterygium wilfordii multiglycoside (GTW) is a commonly used compound for the treatment of rheumatoid arthritis (RA) and immune diseases in clinical practice. However, it can induce liver injury and the mechanism of hepatotoxicity is still not clear. This study was designed to investigate GTW-induced hepatotoxicity in zebrafish larvae and explore the mechanism involved. The 72 hpf (hours post fertilization) zebrafish larvae were administered with different concentrations of GTW for three days and their mortality, malformation rate, morphological changes in the liver, transaminase levels, and histopathological changes in the liver of zebrafish larvae were detected. The reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the levels of microRNA-122 (miR-122) and genes related to inflammation, apoptosis, cell proliferation and liver function. The results showed that GTW increased the mortality of zebrafish larvae, while significant malformations and liver damage occurred. The main manifestations were elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), significant liver atrophy, vacuoles in liver tissue, sparse cytoplasm, and unclear hepatocyte contours. RT-PCR results showed that the expression of miR-122 significantly decreased by GTW; the mRNA levels of inflammation-related genes il1β, il6, tnfα, il10, cox2 and ptges significantly increased; the mRNA level of tgfβ significantly decreased; the mRNA levels of apoptosis-related genes, caspase-8 and caspase-9, significantly increased; the mRNA level of bcl2 significantly decreased; the mRNA levels of cell proliferation-related genes, top2α and uhrf1, significantly reduced; the mRNA levels of liver function-related genes, alr and cyp3c1, significantly increased; and the mRNA level of cyp3a65 significantly decreased. In zebrafish, GTW can cause increased inflammation, enhanced apoptosis, decreased cell proliferation, and abnormal expression of liver function-related genes, leading to abnormal liver structure and function and resulting in hepatotoxicity.

Chlorothalonil induces the intestinal epithelial barrier dysfunction in Caco-2 cell-based in vitro monolayer model by activating MAPK pathway.

The widespread use of chlorothalonil (CTL) has caused environmental residues and food contamination. Although the intestinal epithelial barrier (IEB) is directly involved in the metabolism and transportation of various exogenous compounds, there are few studies on the toxic effects of these compounds on the structure and function of IEB. The disassembly of tight junction (TJ) is a major cause of intestinal barrier dysfunction under exogenous compounds intake, but the precise mechanisms are not well understood. Here, we used Caco-2 cell monolayers as an in vitro model of human IEB to evaluate the toxicity of CTL exposure on the structure and function of IEB. Results showed that CTL exposure increased the paracellular permeability of the monolayers and downregulated mRNA levels of the TJ genes (ZO-1, OCLN, and CLDN1), polarity marker gene (SI), and anti-apoptosis gene (BCL-2) but upregulated the mRNA levels of apoptosis-related genes, including BAD, BAX, CASP3, and CASP8. Western blot analysis and immunofluorescence assay results showed the decreased levels and disrupted distribution of TJ protein network, including ZO-1 and CLDN1 in CTL-exposed IEB. In addition, the accumulation of intracellular reactive oxygen species, decreased mitochondrial membrane potential, and increased active CASP3 expression were observed in treated IEB. The result of TUNEL assay further confirmed the occurrence of cell apoptosis after CTL exposure. In addition, the phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38, was increased in CTL-exposed IEB. In summary, our results demonstrated that CTL exposure induced IEB dysfunction in Caco-2 cell monolayers by activating the mitogen-activated protein kinase pathway.

Chronic Maternal Tobacco Smoke Exposure and/or Alpha-Lipoic Acid Treatment Causes Long-Term Deterioration of Testis and Sexual Behavior in Adult Male Rats

BackgroundTobacco use during pregnancy is known to have several negative effects on the offspring's reproductive health in the long term. The use of alpha-lipoic acid (ALA) as a dietary supplement during pregnancy has increased greatly in recent years and has been known to have positive effects on various pregnancy outcomes including miscarriage, diabetic embryopathy, preterm delivery, and congenital malformations. AimTo evaluate the effects of tobacco smoke exposure (TSE) on sexual behavior, reproductive parameters, and testicles in adult male rats and to reveal the possible role of ALA administration on these parameters. MethodsPregnant rats (n = 7 per group) were treated with tobacco smoke (TS), ALA (20 mg/kg), and TS + ALA for a total of 11 weeks. The following parameters were compared with 8 control rats: puberty parameters, sexual behavior; levels of serum gonadotropins and testosterone, total antioxidant status, and total oxidant status; the expression of the apoptotic protease-activating factor-1 and caspase 9 mRNA levels in the testis; and assessment of immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay of testis. Main Outcome MeasureSexual behavior, changes in puberty parameters, and hormonal and genetic alterations were the outcomes analyzed in this study. ResultsMaternal TSE caused a significant decrease in the number of intromissions compared to the control group. Similarly, ALA decreased erectile function in sexual behavior by decreasing the number of intromissions and intromission ratio in the ALA group compared to the control group. In addition, TSE and ALA treatment caused an impairment of some consummatory sexual behaviors. Also, in parallel with this inhibitory effect, the age of pubertal onset was significantly delayed in the TS + ALA group compared to other groups. Also, histopathological changes in testicular tissue, oxidative stress markers, apoptotic index, and mRNA levels of apoptosis-related genes increased in all treatment groups. Clinical ImplicationsThe use of ALA and/or tobacco products during pregnancy may adversely affect the reproductive health of male newborns in the long term. Strengths & LimitationsTo the best of our knowledge, this study is the first to show the effects of maternal ALA treatment and/or TSE on the sexual behavior and reproductive parameters in male rats; however, the study is based on an animal model, and the present findings partially reflect the characteristics of human sexual behavior. ConclusionMaternal TSE and/or ALA treatment may impair sexual behavior in adulthood in male rats because of testicular damage caused by oxidative stress during gonadal development.Yardimci A, Akkoc RF, Tektemur A, et al. Chronic Maternal Tobacco Smoke Exposure and/or Alpha-Lipoic Acid Treatment Causes Long-Term Deterioration of Testis and Sexual Behavior in Adult Male Rats. J Sex Med 2020;17:1835–1847.

Neurodevelopmental toxicity assessments of alkyl phenanthrene and Dechlorane Plus co-exposure in zebrafish

Alkyl phenanthrene (A-Phen) and Dechlorane Plus (DP) are ubiquitous environmental pollutants that widely co-exist in the environment. It has been established that both A-Phen and DP elicit neurotoxicity, but the potential interactive toxicity of these contaminants is not well-known. To determine whether a mixture of A-Phen and DP would exhibit interactive effects on neurodevelopment, we co-exposed 3-methylphenanthrene (3-MP), a representative of A-Phen, with DP. Our results illustrated that exposure to 5 or 20 μg/L 3-MP alone or in combination with 60 μg/L DP caused neurobehavioral anomalies in zebrafish. In accordance with the behavioral deficits, 3-MP alone or co-exposed with DP significantly decreased axonal growth of secondary motoneurons, altered intracellular Ca2+ homeostasis and induced cell apoptosis in the muscle of zebrafish. Additionally, 3-MP alone or co-exposed with DP significantly increased reactive oxygen species (ROS) and the mRNA levels of apoptosis-related genes. These findings indicate that 3-MP alone or co-exposed with DP induces neurobehavioral deficits through the combined effects on neuronal connectivity and muscle function. Chemical analysis revealed significant increases in 3-MP and DP bioaccumulation in zebrafish co-exposed with 3-MP and DP. Elevated bioaccumulation resulting from mixture exposure may represent a significant contribution of the synergistic effects observed in combined chemical exposure.

Laminarin enhances the quality of aged pig oocytes by reducing oxidative stress.

Laminarin (LAM) is a β-glucan oligomer known to possess biological activities such as anticancer and antioxidant effects. This study explored the influence of LAM supplementation on in vitro aged porcine oocytes and the underlying mechanisms behind this influence. We found that LAM delayed the aging process and improved the quality of aged oocytes. LAM supplementation enhanced the subsequent developmental competence of aged oocytes during the in vitro aging process. The blastocyst formation rate was significantly increased in aged oocytes treated with 20 µg/ml LAM compared to non-treated aged oocytes (45.3% vs. 28.7%, P < 0.01). The mRNA levels of apoptosis-related genes, B cell lymphoma-2-associated X protein (Bax) and Caspase-3, were significantly lower in blastocysts derived from the LAM-treated aged oocytes during the in vitro aging process. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased in aged oocytes following LAM treatment. Mitochondrial membrane potential was increased, and the activities of caspase-3 and cathepsin B were significantly reduced in the LAM-treated aged oocytes compared with the non-treated aged oocytes. Taken together, these results suggest that LAM is beneficial for delaying the aging process in porcine oocytes.

Effect of ethanol extract of HPRS, a Traditional Chinese Medicine formula, on HCT116 cell Line

OBJECTIVEIn this study, we investigated the effects of ethanol extract of Hupiruisu Fang (HPRS), a formula of Traditional Chinese Medicine, on HCT116 cell line. METHODSMKN45, A549, Hela and HCT116 cells were treated with ethanol extract of HPRS alone or the extract plus 5-Fluorouracil (5-FU) for 48 h, and then the cell viabilities were measured using CCK-8 Kit. The early apoptosis rate and total apoptosis rate in both HCT116 cells were evaluated by flow cytometry. The mRNA levels of apoptosis-related genes including caspase-3, caspase-8, Bcl2 and Bax were detected by real-time quantitative polymerase chain reaction. Lastly, the protein activities and expressions of those apoptosis related genes were observed for further verifying the pro-apoptosis of the extract of HPRS. RESULTSEthanol extract of HPRS could significantly induce apoptosis in HCT116 cell line. Synergistic analysis revealed that the extract exhibited a significant effect upon 5-FU-associated cytotoxicity in the cell line. CONCLUSIONThe ethanol extract of HPRS plus 5-FU might have the potential to improve the treatment of colorectal cancer.

Blood cardioplegia: Difference between young and old rat hearts

Objective: We compared the hemodynamic performance, protein expression, phosphorylation and mRNA levels of apoptosis-related genes in young and old rat hearts after application of Buckberg's blood cardioplegia (BCP) to evaluate differences between the age groups regarding postischemic myocardial function and cellular survival.

Chemopreventive effects of Ginkgo biloba extract in estrogen-negative human breast cancer cells

Excessive level of estrogen is considered as a main cause of breast cancer, therefore, many studies have focused on estrogen receptor (ER)-positive breast cancer, even though ER-negative cancer has a poor prognosis than ER-positive breast cancer. We evaluated the anti-cancer effects of Ginkgo biloba extract (GBE) in estrogen-independent breast cancer. GBE has been traditionally used as a platelet activating factor, a circulatory stimulant, a tonic, and anti-asthmatic drug, and anti-cancer agent.However, anti-cancer effects of GBE on ER-negative breast cancer have not been proved yet. In this study, we tested chemotherapeutic potential of GBE in the MDA-MB-231 (ER-negative) human breast cancer cell line.Our results showed that cytotoxicity effects of GBE in MDA-MB-231 lead to DNA fragmentation at high concentrations (500 and 1,000μg/ml). Caspase-3 was significantly activated and mRNA levels of apoptosis-related genes (Bcl-2 and Bax) were altered. These results indicate that GBE induces apoptosis in MDA-MB-231 cells. It is presumed that GBE has chemopreventive effects in ER-independent breast cancer through anti-proliferation and apoptosis-inducing activities.

Marijuana smoke condensate induces p53-mediated apoptosis in human lung epithelial cells

Since the largely abused worldwide used of marijuana, there have been many ongoing debates regarding the adverse health effects of marijuana smoking. Marijuana smoking was recently proved to cause pulmonary toxicity by inducing genotoxic effects or generating reactive oxygen species. Because p53, a tumor suppressor gene, has an important pathophysiologic role in the regulation of lung epithelial cell DNA damage responses, we hypothesized that p53 may be involved in the oxidative stress-mediated apoptosis induced by marijuana smoking. First, we confirmed that marijuana smoke condensate (MSC) induces oxidative stress in BEAS-2B cells. We observed that reactive oxygen species (ROS) generation was increased by MSC in the DCFH-DA assay. Also, antioxidant enzyme (superoxide dismutase, catalase) activity and their mRNA expressions were up-regulated by MSC. Second, we investigated p53 involvement in the MSC-induced apoptotic pathway in BEAS-2B cells. The results showed that MSC increased caspase-3 activation and DNA fragmentation as markers of apoptosis. In addition, the mRNA levels of apoptosis-related genes (p53 and Bax) were increased by MSC and phospho-p53, along with the increase of Bax protein expression by MSC. Apoptosis and apoptosis-related gene expression were partially blocked by an inhibitor of p53-dependent transcriptional activation (pifithrin-α). The results indicate that p53 plays a role in MSC-induced apoptosis. Taken together, the findings of the present study suggest that MSC partially induces p53-mediated apoptosis through ROS generation in human lung epithelial cells and this may have broader implications for our understanding of pulmonary diseases.

Stimulatory Effects of Thyroid Hormone on Brain Angiogenesis in vivo and in vitro

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day 21 (P21) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an altered expression of vascular endothelial growth factor A (Vegfa) and basic fibroblast growth factor (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain-derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfa upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing factor to the neurologic deficits induced by thyroid hormone deficiency.

Transcriptional regulation of TNF family receptors and Bcl-2 family by chemotherapeutic agents in murine CT26 cells.

Various chemotherapeutic agents have been shown to sensitize cancer cells to members of the tumor necrosis factor (TNF) family. However, it is unclear whether sensitization by chemotherapeutic agents involves the transcriptional regulation of apoptosis-related genes. In this study, we investigated mRNA regulation of TNF family receptors and Bcl-2 family members after treating the murine colon cancer cell line, CT26, with various apoptosis inducers. We found that treatment with cycloheximide, a protein synthesis inhibitor, remarkably increased CD40 mRNA levels by semi-quantitative RT-PCR. Other protein synthesis inhibitors, such as anisomycin and emetine, also enhanced CD40 mRNA expression, which was significantly blocked by a NF-kappaB antagonist and a p38 MAP kinase antagonist. After treatment with cycloheximide, and further cultivation in fresh medium, CD40 protein levels were found to increase by flow cytometry. Additionally, we found that cycloheximide treatment appeared to downregulate the Bcl-xL mRNA level but not the Bax mRNA level by RNase protection assay. Because the upregulation of CD40 mRNA and the downregulation of Bcl-xL correlated with CT26 cell death, our results suggest that chemotherapeutic agents, including cycloheximide, may exert their synergistic effects on the TNF family treatment of cancer cells by regulating the mRNA levels of apoptosis-related genes.