The drug-resistance of malaria parasites is the main problem in the disease control. The huge Brazilian biodiversity promotes the search for new compounds, where the animal kingdom is proving to be a promising source of bioactive compounds. The main objective of this study was to evaluate the antiplasmodial and cytotoxic activity of the compounds obtained from the toad venoms of Brazilian Amazon. Toad venoms were collected from the secretion of Rhinella marina and Rhaebo guttatus in Mato Grosso State, Brazil. The powder was extracted at room temperature, yielding 2 extracts (RG and RM) and a substance ('1') identified as a bufadienolide, named telocinobufagin. Growth inhibition, intraerythrocytic development, and parasite morphology were evaluated in culture by microscopic observations of Giemsa-stained thin blood films. Cytotoxicity was determined against HepG2 and BGM cells by MTT and neutral red assays. The 2 extracts and the pure substance ('1') tested were active against chloroquine-resistant Plasmodium falciparum strain, demonstrating lower IC₅₀ values. In cytotoxic tests, the 2 extracts and substance '1' showed pronounced lethal effects on chloroquine-resistant P. faciparum strain and low cytotoxic effect, highlighting toad parotoid gland secretions as a promising source of novel lead antiplasmodial compounds.
Amphibian Venoms* ; Animals ; Biodiversity ; Brazil* ; Bufo marinus ; Malaria ; Neutral Red ; Parasites ; Plasmodium falciparum

BACKGROUND: The extract and hemiterpene glycosides of Ilex Rotunda Thunb exert antioxidant and anti-inflammatory effects. The effect of rotundarpene on apoptosis in neuronal cells caused by the 1-methyl-4-phenylpyridinium (MPP⁺) has not been reported previously. METHODS: Using differentiated PC12 cells and human neuroblastoma SH-SY5Y cells, we investigated the effect of rotundarpene on MPP⁺-caused apoptosis in relation to the cell death process. RESULTS: MPP⁺-induced cell death was identified using the MTT and neutral red uptake tests. Apoptosis was induced by eliciting decreases in the cytosolic levels of Bid and Bcl-2 proteins, increases in the cytosolic levels of Bax and p53, disruption of the mitochondrial transmembrane potential, and the release of cytochrome c and the activation of caspase-8, -9, and -3 in differentiated PC12 cells and SH-SY5Y cells. Treatment with rotundarpene reduced the MPP⁺-induced changes in the levels of apoptosis-regulated proteins, formation of reactive oxygen species, depletion and oxidation of glutathione, and cell death in both PC12 and SH-SY5Y cells. CONCLUSIONS: Rotundarpene may reduce MPP⁺-induced apoptosis in neuronal cells by suppressing the activation of the mitochondria-mediated pathway and the caspase-8 and Bid pathways. Rotundarpene appears to act by inhibiting the production of reactive oxygen species and by the depletion and oxidation of glutathione.
1-Methyl-4-phenylpyridinium ; Animals ; Apoptosis ; Caspase 8 ; Cell Death* ; Cytochromes c ; Cytosol ; Glutathione ; Glycosides ; Humans ; Ilex ; Membrane Potentials ; Neuroblastoma ; Neurons ; Neutral Red ; PC12 Cells ; Reactive Oxygen Species

BACKGROUND AND OBJECTIVES: Carcinogens result in the impairment of intercellular communication as well as intracellular communication of normal cells. Connexin (Cx) is a main constituent protein of gap junctions that let messengers such as ions communicate between cells. We evaluated the effect of carcinogen H2O2 on the expression of Cxs and gap junction intercellular communication (GJIC) in the human keratinocyte cell line (HaCaT) and analyzed the prevention effect of green tea extracts against H2O2. MATERIALS AND METHOD: We performed neutral red dye uptake tests to determine the optimal concentrations of H2O2, green tea extracts-epicatechin (EC) and epigallocatechin-3-gallate (EGCG) in this study. To analyze the expression change of Cxs, we performed RT-PCR, Western blot, and immunocytochemistry after 24-hour culture of HaCaT cells treated with agents. We also evaluated GJIC quantitatively using the 'scrape loading dye transfer (SLDT)' technique. RESULTS: Cx26, Cx30, Cx31, Cx43, but not Cx29 were expressed in the HaCaT cells. H2O2 (250 uM) down-regulated Cx26 and Cx43 proteins. In HaCaT cells treated with H2O2, EC (175 uM) up-regulated Cx26 and Cx43 proteins, but EGCG (50 uM) up-regulated only Cx43 protein. Immunocytochemistry showed the decreased expression and abnormal location of Cx26 and Cx43 under H2O2, and EC and EGCG (5 uM) inhibited the effect of H2O2, showing similar staining in the control. In SLDT, H2O2 down-regulated GJIC, while EC and EGCG significantly prevented HaCaT cells from the H2O2-induced, down-regulation of GJIC. CONCLUSION: The carcinogen, H2O2, inhibits GJIC in the keratinocyte cell line. Green tea extracts, such as EC and EGCG, prevent GJIC inhibition in the keratinocyte cell line treated with H2O2, suggesting they have a potential anti-cancer properties.
Blotting, Western ; Carcinogens ; Catechin ; Cell Line ; Connexin 43 ; Down-Regulation ; Gap Junctions ; Humans ; Immunohistochemistry ; Ions ; Keratinocytes ; Linear Energy Transfer ; Neutral Red ; Proteins ; Tea

BACKGROUND: The intrathecal grafting of adrenal chromaffin cells as a potential analgesic source, to delivery analgesic substances such as catecholamines and opioid peptides, is known to be effective at treating acute and chronic pain in several animal pain models. We tested whether the intrathecal implantation of encapsulated bovine chromaffin cells reduces cold allodynia in a rat model of neuropathic pain induced by chronic constriction injury of the sciatic nerve. METHODS: Bovine adrenal medullary chromaffin cells microencapsulated in sodium alginate-poly-l-lysin-alginate (APA) were implanted into the subarachnoid space of rats (n = 10) and foot cold sensitivity was investigated using an acetone test. At the end of the study, histology and capsule catecholamine production were evaluated. RESULTS: A significant reduction in cold allodynia was observed in animals implanted with chromaffin cells. In addition, the suppression of cold allodynia was reversed by naloxone. Abundant clusters of viable chromaffin cells stained with neutral red, were observed in the retrieved implants and after nicotine stimulation, and catecholamine was quantified. An ultrastructural study showed no fibrotic reaction against capsules, or disorganised capsules. CONCLUSIONS: These results suggest that intrathecal encapsulated chromaffin cells act as "mini pumps", which continuously deliver analgesic substances and produce analgesia in this chronic pain model of nerve injury-without immunosuppressant.
Acetone ; Analgesia ; Animals ; Capsules ; Catecholamines ; Chromaffin Cells* ; Chronic Pain ; Constriction ; Foot ; Hyperalgesia* ; Models, Animal ; Naloxone ; Neuralgia ; Neutral Red ; Nicotine ; Opioid Peptides ; Rats* ; Sciatic Nerve ; Sodium ; Spinal Cord* ; Subarachnoid Space ; Transplants

PURPOSE: Despite the significance of the p53 adenoviral vector in cancer gene therapy, an advanced strategy for the development of preferential tumor cell-specific delivery and the long-term persistent gene expression control of p53 are required. In this study, the time-course expression patterns of p53 and E6, on cervical cancer cells, were investigated to obtain a molecular level understanding of the cell-dependent tumor growth suppression effects of a recombinant adenovirus expressing p53, both in vitro and in vivo. MATERIALS AND METHODS: The expressions of p53 and E6 in CaSki, SiHa, HeLa, HeLaS3, C33A and HT3 cervical cancer cell lines were examined. After infection with AdCMVp53, the cell growth inhibition was studied via cell count, MTT and Neutral red assays. After transfecting the AdCMVp53 and AdCMVLacZ into the cancer cells-xenografted nude mice, the anti-tumor effects were investigated for one month. RESULTS: The p53 protein levels were more notably expressed in the CaSki and HeLa than in the SiHa and HeLaS3 On day 6, the p53 was only detected in the HeLaS3. In contrast, the p53 expression was highly maintained in the C33A and HT3. The E6 mRNA levels gradually decreased in only the CaSki and HeLa. The growth suppression effects also showed cell-dependent patterns, which were consistent with the reciprocal expression patterns of p53 and E6. After transfection of the AdCMVp53, into the CaSki- and SiHa-xenografted nude mice, the tumor size was remarkably decreased in the SiHa cells as compared to that in the AdCMVLacZ transfected mice, indicating cell-specific growth inhibition patterns. CONCLUSION: The adenovirus-mediated p53 gene transfection was very effective both in vitro and in vivo. Also, the anti-tumor effects were accomplished via the differential role of p53-specific apoptotic cell death, which was dependent on the cervical cancer cell line.
Adenoviridae* ; Animals ; Cell Count ; Cell Death ; Cell Line ; Gene Expression ; Genes, Neoplasm ; Genes, p53 ; Genetic Therapy ; Humans* ; Mice ; Mice, Nude ; Neutral Red ; RNA, Messenger ; Transfection ; Uterine Cervical Neoplasms*

OBJECTIVE: The purpose of this article is to estimate the anti-cancer effects of the major components of the green tea (polyphenols, catechin and EGCG) and the mechanism of EGCG on different cervical cancer cell lines. METHODS: Six cervical cancer cell lines (HeLa, HeLaS3, Caski, SiHa, HT3 and C33A) were treated with 20 microgram/ml green tea polyphenols (GTPs), 50 micrometer catechin and various concentrations of (-)-epigallo- catechin-3-gallate (EGCG). The viabilities were determined by trypan blue exclusion assay, neutral red assay and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. DNA fragmentation and nuclear condensation were used to see whether EGCG-induced anti-proliferation effect was due to apoptosis. RESULTS: Both GTPs, catechin and EGCG had growth inhibition effects on cervical cancer cell lines, but EGCG appeared to be the most effective. What's more, the sensitivity of each cell lines to EGCG was different. HT3 cells (HPV negative, mutant type p53) were most sensitive to EGCG (estimate IC50: 10 micrometer). Caski (HPV-16 positive, wild type p53) and HeLaS3 cells (HPV-18 positive, wild type p53) were less sensitive (estimate IC50: 35 and 70 micrometer respectively). EGCG-induced apoptosis can be seen in all the cell lines and it happened as early as 8 hours after EGCG treatment. CONCLUSION: Green tea or EGCG alone will be beneficial to the cervical cancer patients.
Apoptosis* ; Catechin ; Cell Line* ; Chemoprevention ; DNA Fragmentation ; Guanosine Triphosphate ; Humans ; Inhibitory Concentration 50 ; Neutral Red ; Polyphenols* ; Tea* ; Trypan Blue ; Uterine Cervical Neoplasms*

BACKGROUND: There is an increasing need for the development of in vitro models capable of substituting for animals in cutaneous irritancy studies. Until now, various culture models have been developed, including skin organ cultures, conventional and air-exposed cell cultures. The air-exposed culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum corneum layer in these cultures permits the application of potential irritants at the concentrations and formulations which are applied in vivo. Recently, a new human skin recombinant, made of human keratinocytes cultured on de-epidermized dermis with fibroblast-populated collagen matrix, has been developed and appears to represent a better skin equivalent model than previous models. OBJECTIVE: In the present study, monolayer-cultured human keratinocytes and the new human skin recombinants were evaluated for the test models of various skin irritants. METHODS: The extent of skin irritancy induced after application of 3 different irritants (sodium lauryl sulfate, methyl paraben, and polyethylene glycol-400) was evaluated on the basis of (1) MTT assay, (2) neutral red uptake assay, (3) LDH release, and (4) release of IL-1 alpha. In the human skin recombinants, morphological perturbations and changes in the expression of differentiation-specific protein markers (keratin 1, involucrin, filaggrin, and loricrin) were also evaluated. To determine the difference between in vivo and in vitro models for the detection of irritancy, a patch test was performed on 11 normal human volunteers with various concentrations of the different irritants RESULTS: The results of the present study show that irritant cytotoxicity correlates well with irritant concentration in both monolayer-cultured human keratinocytes and the new human skin recombinant. The new human skin recombinant is superior to monolayer culture as an in vitro model for skin irritancy screening in that the concentrations of test irritants are the same as in vivo. With the human skin recombinant, morphological changes were observed according to the irritant concentration. CONCLUSION: The new human skin recombinant can be used as an alternative to animals for skin irritancy screening.
Animals ; Cell Culture Techniques ; Collagen ; Dermis ; Epidermis ; Healthy Volunteers ; Humans* ; Interleukin-1alpha ; Irritants ; Keratinocytes ; Mass Screening ; Neutral Red ; Organ Culture Techniques ; Patch Tests ; Polyethylene ; Skin*

OBJECTIVES: The aim of this study is to find out the activity of autoproliferation of ratfibroblast exposed to crystalline silica and the role of mediators secreted from rat fibroblast. METHODS: The effect of alpha-quartz on production of growth factor (platelet-derived growth factor-AA and transforming growth factor beta)from rat fibroblasts were evaluated by ELISA and immunocytochemical analysis. Gene expression of these growth factors in rat fibrobast exposed to crystalline silica was evaluated by RT-PCR. Furthermore, fibroblast proliferation by culture supernatant of rat fibroblast was assayed by the neutral red test. RESULTS: The amounts of H2O2 and growth factors synthesized in rat fibroblasts were significantly increased by the stimulation of crystalline silica(alpha-quartz), which showed the dose-dependent manner to the concentration of alpha-quartz with the maximum response at the dosage of 100 microgram/cm2. The result of RT-PCR demonstrated that alpha-quartz induced gene expression of PDGF-AA and TGFbeta in rat fibroblast. We also found that supernatant of alpha-quartz-cocultured rat fibroblast induced a significant proliferation of fibroblast. CONCLUSION: Crystalline silica directly induce functional change in fibroblast such as increased release of reactive oxygen species and growth factors. The products of these functional change promote fibroblast proliferation via autocrine loop.
Animals ; Crystallins* ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts* ; Gene Expression ; Intercellular Signaling Peptides and Proteins ; Neutral Red ; Rats ; Reactive Oxygen Species ; Silicon Dioxide* ; Transforming Growth Factor beta ; Transforming Growth Factors

OBJECTIVES: This study was carried out to survey the prevalence of Helicobacter pylori infection and the incidence of vacuolating toxin producing H. pylori. A further aim of this study was to evaluate the quantitative assay for cell vacuolation on the basis of the rapid uptake of neutral red dye by vaculoes of the cells. METHODS: We studied the gastric biopsy specimens of patients with 154 cases of gastritis, 74 cases of gastric ulcer, and 167 cases of gastric cancer and in 44 cases of healthy persons. One of the biopsy specimen was placed into a CLOtest plate for rapid urease test and the other one of the biopsy spcimen was inoculated on Brain Heart Infusion blood agar for culture. The culture supernatant of isolated H. pylori was serially diluted with BHI broth. After 24 hour incubation of cultured RK-13 cells treated with the culture supernatant of H. pylori, cytoplasmic vacuolation of the cells were observed microscopically. RESULTS: The positivity of urease test and the rate of isolation of H. pylori from urease positive gastric biopsy materials were 34.1% and 93.3% in healthy person, 55.8% and 70.9% in gastritis, 60.8% and 71.1% in gastric ulcer, and 56.3% and 96.8% in gastric cancer. The isolation rate of H. pylori from patients between 20 and 39 years old was 16.8%, for patients between 40 and 59 years old it was 51.9%, and for patients above 60 years old it was 31.2%. The isolation rate of the vacuolating toxin producing H. pylori from gastric biopsy specimens was 66.7% in a healthy person, 76.6% in gastritis, 79.4% in gastric ulcer, and 80% in gastric cancer. CONCLUSIONS: The isolation rate of H. pylori from the patients with gastric diseases is higher than the rate of H. pylori from healthy persons, but the isolation rate of the vacuolating toxin producing H. pylori is not different between the patients with gastric diseases and healthy persons. The titers of vacuolating toxin produced by some H. pylori isolated from the patients with gastric diseases are higher than those from healthy persons.
Adult ; Agar ; Biopsy ; Brain ; Cytoplasm ; Gastritis ; Heart ; Helicobacter pylori* ; Helicobacter* ; Humans ; Incidence* ; Middle Aged ; Neutral Red ; Prevalence ; Stomach Diseases* ; Stomach Neoplasms ; Stomach Ulcer ; Urease

BACKGROUND: Paraquat is a widely used herbicide, known to cause lethal toxicity in humans. Most studies about paraquat have concentrated on systemic toxicity, however several cases of paraquat-induced dermatitis have been reported. OBJECTIVE: The purpose of this study was to confirm the cutaneous toxic effect of paraquat and to select potential antidotes in paraquat-induced dermatitis. METHODS: Keratinocyte toxicity due to paraquat and the toxicity reduction capacity of several drugs were investigated in eitro. Topical effects of these drugs on paraquat-induced dermatitis in guinea pig skin was also investigated. RESULTS: Over 50% of keratinocytes failed to survive at a concentration of 2X10-4M paraquat by a neutral red uptake assay. Skin irritation by paraquat was observed at 2% concentration by non-invasive methods as well as a skin biopsy. Dexamethasone, glutathione and tocopherol showed some capacity to reduce paraquat-induced keratinocyte toxicity in vitro. Only dexamethasone, however, showed a reduction of cutaneous blood flow volume and dermal inflammatory cell infiltration in the guinea pig study. CONCLUSION: This result indicates the possible in eitro protective effect of paraquat toxicity in glutathione and tocopherol. Dexamethasone was capable of reducing paraquat-induced cytotoxicity and dermatitis both in vitro and in vivo.
Animals ; Antidotes* ; Biopsy ; Dermatitis ; Dexamethasone ; Glutathione ; Guinea Pigs ; Humans ; In Vitro Techniques ; Keratinocytes ; Neutral Red ; Paraquat* ; Skin* ; Tocopherols