Synthetic cannabinoids developed by chemical modification are believed to bind to cannabinoid receptors and cause neurological effects similar to cannabis; however, their effects on drug metabolizing enzymes are unknown. This study aimed to elucidate the effect of synthetic cannabinoids on cytochrome P450 1A activity. Naphthoylindole, a basic structure of the major synthetic cannabinoids, strongly inhibited CYP1A activity in a competitive manner; the apparent Ki value was 0.40 μM. The N-Alkylated derivatives of naphthoylindole, MAM-2201 and JWH-019, also inhibited CYP1A activity in a concentration-dependent manner; however, their inhibitory effects were weaker than naphthoylindole. An adamantylamidoindole derivative, STS-135, showed inhibition of CYP1A activity in a concentrationdependent manner, but the adamantoylindole derivatives, AB-001 and AM-1248, did not. A tetramethylcyclopropanoylindole derivative, UR-144, showed a weak inhibition of CYP1A activity at high concentrations. These results suggest that synthetic cannabinoids and their basic molecules are capable of inhibiting CYP1A enzymatic activity.
While photodynamic therapy (PDT) is an effective treatment for glioma, induction of apoptotic cell death of glioma cells is important for ensuring efficacy and safety of PDT treatment in glioma patients, as necrotic cell death can induce late appearance of obstacles in treatment. Here, we investigated the relationship between type of cell death and PDT treatment conditions involved in laser and photosensitizer dosage in human glioblastoma T98G cells. Photosensitizer talaporfin sodium-mediated PDT (NPe6-PDT) treatment induced laser and NPe6 dose-dependent cell death in T98G cells, whereas almost all cells pretreated with NPe6 at ≥ 30 µg/mL were killed by laser irradiation, regardless of laser dose. Morphological analysis showed that combination of high doses of NPe6 and laser irradiation changes the dominant cell death process from apoptosis to necrosis. Biochemical analysis (detection of caspase-3 activity and staining of cell surface-exposed phosphatidylserine) also showed that increasing laser dose changes the type of cell death from apoptotic to necrotic cell death after high-dose treatment with NPe6. Lactate dehydrogenase leakage assay demonstrated that a laser dose of 5 J/cm2 induced less leakage than 30 J/cm2. Our results suggested that type of glioma cell death in NPe6-PDT changed with fluctuations in laser and NPe6 dose, and that combination of 30 µg/mL NPe6 with 5 J/cm2 laser is the best treatment condition for inducing an increase in apoptotic cells while keeping rate of necrotic cell death low in this in vitro study.
The purpose of this study was to evaluate behavioral responses and biochemical changes induced by the extensively used pesticide cypermethrin (CYP) in liver, gills, brain and muscle tissues of mahseer (Tor putitora) fry. Behavioral changes in fish after exposure to an acute concentration of CYP involved jumping, abrupt swimming, loss of balance and equilibrium, increased surface activity and air gulping. These changes were more prominent with the passage of time. After a longer period of exposure, the fish became sluggish and before dying occasionally became motionless and sometimes showed a vertical position. Internal hemorrhage was also obvious. CYP exposure resulted in a significant decrease in total protein content in different tissues while antioxidant enzymes, catalase (CAT), peroxidase (POD) and glutathione reductase (GR) showed a time-dependent increasing trend in their activities in liver, brain, gills and muscle tissues. Similarly, lipid peroxidation (LPO) level also increased with time in different tissues of CYP-exposed fish. The results of the present study revealed that CYP is toxic to the mahseer Tor putitora. Therefore, its indiscriminate use can contribute in decreasing the population of mahseer in natural water bodies.
Recent studies have shown that epigenetic alterations correlate with carcinogenesis in various tissues. Identification of these alterations might help characterize the early stages of carcinogenesis. We comprehensively analyzed DNA methylation and gene expression in livers obtained from rats exposed to nitrosodiethylamine (DEN) followed by a promoter of hepatic carcinogenesis, phenobarbital (PB). The combination of DEN and PB induced marked increases in number and area of glutathione S-transferase-placental form (GST-P)-positive foci in the liver. In the liver of rats that received 30 mg/kg of DEN, pathway analysis revealed alterations of common genes in terms of gene expression and DNA methylation, and that these alterations were related to immune responses. Hierarchical clustering analysis of the expression of common genes from public data obtained through the Toxicogenomics Project-Genomics Assisted Toxicity Evaluation system (TG-GATEs) showed that carcinogenic compounds clustered together. MBD-seq and GeneChip analysis indicated that major histocompatibility complex class Ib gene RT1-CE5, which has an important role in antigen presentation, was hypomethylated around the promoter region and specifically induced in the livers of DEN-treated rats. Further, immunohistochemical analysis indicated that the co-localization of GST-P and protein homologous to RT1-CE5 was present at the foci of some regions. These results suggest that common genes were altered in terms of both DNA methylation and expression in livers, with preneoplastic foci indicating carcinogenic potential, and that immune responses are involved in early carcinogenesis. In conclusion, the present study identified a specific profile of DNA methylation and gene expression in livers with preneoplastic foci. Early epigenetic perturbations of immune responses might correlate with the early stages of hepatocarcinogenesis.
The present report was designed to determine the antigenotoxic capacity of beta-caryophyllene (BC) on the damage induced by benzo(a)pyrene (BaP) in mouse. We found no genotoxic potential of BC, and a significant inhibitory effect on the number of sister-chromatid exchanges (SCE) and chromosomal aberrations induced by BaP. The three tested doses of the agent (20, 200, and 2,000 mg/kg) produced a dose-dependent decrease of the two evaluated cytogenetic parameters. In comparison with the effect induced by BaP, the best inhibitory effect (about 80%) was obtained with the high tested dose of BC considering the two evaluated parameters. Other aim of the study was to explore whether in this effect participated the BC antioxidant capacity and/or its effect as inducer of GST activity. We found a dose-dependent decrease induced by BC in regard to both the oxidation of lipids and proteins produced by BaP.In the case of GST, when BC was administered alone we found a mean increase of 64% of the enzyme activity, respect to the control level, and when BC was administered in mice treated with BaP the increase obtained with the high dose of BC reached 27%. Therefore, our data established no in vivo genotoxicity by BC, and a significant antigenotoxic potential of the compound, which may be related with its capacity to block the molecular oxidation and to stimulate the GST activity.
Cadmium-induced cell death is associated with endoplasmic reticulum (ER) stress. We previously found that inhibition of FBXO6 expression, which is a ubiquitin ligase involved in ER-associated protein degradation (ERAD), induces high sensitivity to cadmium in HEK293 cells. However, the precise role of FBXO6 in ER stress remains unexplored. In this study, we investigated the role of FBXO6 in cadmium-induced ER stress in HEK293 cells. Our results showed that the cadmium-induced increase in expression of the ER stress marker proteins, BiP and CHOP, was further enhanced by inhibiting FBXO6 expression. Cadmium-induced c-Jun phosphorylation was also markedly increased by inhibition of FBXO6 expression. However, this c-Jun phosphorylation was almost entirely abolished by inhibition of c-Jun N-terminal kinase 1 (JNK1) expression. The level of high cadmium sensitivity induced by inhibition of FBXO6 expression was markedly lower in the JNK1-ablated cells than in the control cells. In addition, cadmium elevated the cellular level of ERAD substrate proteins, and this elevation was further enhanced by inhibiting FBXO6 expression. These results suggest that FBXO6 might inhibit cadmium-induced ER stress by functioning as a ubiquitin ligase in the ERAD system, thereby attenuating the cell death induced by subsequent JNK1 activation.
The role of thyroid hormones in gonad development remains incompletely understood. We examined the dose-related effects of perinatal hypothyroidism induced by a reversible goitrogen, 6-propyl-2-thiouracil (PTU), on reproductive development in male rat offspring. Timed-pregnant Sprague-Dawley rats were orally administered PTU (0, 0.5, 1.0, or 2.0 mg/kg/day) by gavage from gestational day 15 through postnatal day 20. We observed a significant dose-dependent decrease in body weight in offspring with PTU exposure up to 13 weeks of age, but body weight became comparable among groups by 26 weeks of age. Testicular weight tended to be lower up to 7 weeks but was higher after 13 weeks of age. Epididymis weight was not different among the groups at any age. Plasma concentrations of thyroxine and triiodothyronine in the PTU groups were significantly lower at 3 weeks of age but recovered to normal levels by 26 weeks of age. No dose-related trend in plasma testosterone concentrations was found. Seminiferous tubules were larger at 13 and 26 weeks of age with PTU exposure. The number of Sertoli cells was significantly higher from 3 through 26 weeks of age. The number of Leydig cells was significantly lower up to 7 weeks of age but was comparable among groups from 13 weeks of age onwards. Thus, transient gestational and lactational thyroid hormone suppression induced small testes in early life but led to paradoxical dose-dependent testicular enlargement in adults as indicated partly by larger seminiferous tubules with numerous Sertoli cells in male rat offspring.
The trichothecene deoxynivalenol (DON), a foodborne mycotoxin found in grain-based foods, has been associated with human and animal food poisoning. Although induction of anorexia has been described as a hallmark of DON-induced toxicity in many animal species, the mechanistic basis for this adverse effect is not fully understood. The purpose of this research was to determine the role of two proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in DON-induced anorexia. In a nocturnal mouse food consumption model, DON-induced anorectic response occurred at 1 hr and lasted up to 6 hr. Similar anorectic effects were observed following acute administration of exogenous TNF-α and IL-1β. Oral exposure to DON at 5 mg/kg bw stimulated splenic and hepatic mRNA and plasma protein elevations of TNF-α and IL-1β that corresponded to anorexia induction. Pretreatment with the TNF-α receptor (TNFR) antagonist R-7050 dose-dependently attenuated both TNF-α- and DON-induced anorexia. While, the type 1 IL-1 receptor (IL-1R1) antagonist IL-1RA dose-dependently attenuated both IL-1β- and DON-induced anorexia. Taken together, the results suggest that both TNF-α and IL-1β play contributory role in anorexia induction following oral exposure to DON.
Patients with diabetes mellitus, particularly those with cardiovascular complications, have increased risk of mortality when subject to anesthetics and surgery, compared with non-diabetic patients. Anesthetics may exert pressure on the cardiovascular system of diabetic patients, directly or by aggravating pre-existing cardiovascular complications. Advanced glycation end products (AGEs) are extremely accumulated in diabetes mellitus, and are confirmed to play an important role in the pathogenesis of diabetic microvascular and macrovascular complications. The purpose of the present study was to investigate the regulatory role of etomidate, which is widely used as intravenous general anesthetics, on the viability and apoptosis of human endothelial Eahy926 cells, in the presence of AGEs. The results demonstrated that etomidate and Glu-BSA (one type of AGE) synergistically reduced the human endothelial Eahy926 cell viability and induced cell apoptosis. In addition, western blot assay of apoptosis-associated molecules indicated that both agents synergistically upregulated the cytochrome c release, activated the apoptosis executor, caspase 3, and promoted the poly-ADP-ribose polymerase (PARP) lysis. Further results confirmed that the two agents synergistically promoted oxidative stress by decreasing mitochondrial respiratory chain complex IV and mitochondrial membrane potential (MMP), while upregulating reactive oxygen species (ROS) and mitochondrial superoxide. In conclusion, the results presented in this study offer a novel insight into the mechanisms of endothelial cell apoptosis in response to etomidate in the presence of AGEs. These results suggest that oxidative stress has important role in the synergistic promotion of apoptosis by etomidate and AGEs in endothelial Eahy926 cells.
The effects of five types of metal nanoparticles, gold (Au), silver (Ag), platinum (Pt), Au-polyvinylpyrrolidone (PVP) colloid, and Pt-PVP colloid, and two types of hydrophilic carbon black on cell behavior were examined. Stable nanoparticle dispersions were prepared and applied to the culture medium of human keratinocyte (HaCaT) and human lung carcinoma (A549) cells for 6 and 24 hr. Then, the mitochondrial activity (MTT assay) and the induction of cellular oxidative stress were examined. The exposure to Au and Ag decreased mitochondrial activity. The exposure to Pt nanoparticles induced an increase in the intracellular reactive oxygen species (ROS) level. In contrast, Au-PVP, Pt-PVP, and hydrophilic carbon black did not exhibit any effects. The observed increase in the ROS level induced by the Pt nanoparticles in this study contradicted our previous findings, in which Pt did not produce chemically reactive molecules. Some nanoparticle dispersions included chemicals as the dispersant, which is used in industrial applications. In some cases, the dispersing agent may have caused some cellular effects. Adsorption of agents on the surface of the nanoparticles may be an important factor here. Hence, the cellular effects of industrial nanoparticles should be evaluated carefully.
Virus infection has been shown to accelerate atherosclerosis. Serological studies indicate a link between the Herpes simplex virus (HSV) infection and atherosclerosis, which is initiated and progression of which is promoted by such factors as oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction. In order to recognize the direct role of HSV-2 in endothelial dysfunction, the present study investigated the infection of HSV-2 in endothelial ECV304 cells and the induction of cell apoptosis in the presence of ox-LDL. We firstly examined the HSV-2 infection by immunohistochemical assay for viral gB protein, quantitative PCR for viral ICP4 mRNA, or via virus growth determination. Then we investigated the regulation of HSV-2 infection on the cell viability and apoptosis, in the absence or presence of ox-LDL. In addition, we analyzed the apoptosis-associated molecules in the HSV-2-infected ECV304 cells. The results demonstrated that HSV-2 infected endothelial ECV304 cells and replicated efficiently, and the virus infection significantly reduced the cell viability and significantly induced cell apoptosis; particularly, cell viability reduction and cell apoptosis induction were aggravated by the ox-LDL presence. Moreover, the western blot assay confirmed the apoptosis induction; there was a significantly high level of released cytochrome c, activated caspase 3 and lyzed Poly (ADP-ribose) polymerase (PARP) by the activated caspase 3 in the HSV-2-infected ECV304 cells, particularly the cells subject to ox-LDL. Thus, we confirmed that HSV-2 infected endothelial ECV304 cells, induced cell apoptosis, which was aggravated by ox-LDL.
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