Cytotoxicity in freshwater fishes induced by industrial effluents and dyes is a global issue. Trypan blue dye has many applications in different sectors, including laboratories and industries. This study determines to detect the cytotoxic effects of trypan blue dye in vivo. The objective of this study was to estimate the sub-lethal effects of azodye in fish. Cirrhinus mrigala, a freshwater fish, was exposed to three different grading concentrations of dye 5 mg/L, 10 mg/L, and 20 mg/L in a glass aquarium. Significant (p trypan blue dye, fishes were dissected to remove liver and kidney tissues. Histopathological assessments determined hepatotoxicity and nephrotoxicity induced by trypan blue through the paraffin wax method. This dye induces mild alterations in the liver such as congestion, hemolysis, dilated sinusoids, ruptured hepatocytes, vacuolization, edema of hepatocytes, necrosis, degeneration, aggregation, and inflammation. This dye not only alters liver tissue, also induces an acute level of tissue alterations in the kidneys, such as degeneration of epithelial cells of renal tubules, shrinkage of the glomerulus, congestion, reduced lumen, degeneration of glomerulus, absence of space of bowmen, glomerulonephritis, necrosis in hematopoietic interstitial tissues and glomerulus, reduced lumen, vacuolar degeneration of renal tubules, increased per tubular space. The current study concludes that trypan blue dye released even in small amounts is found to be associated with a high incidence of cytotoxicity. Such tissue alterations in this species could be used as biomarkers for azo dyes.
Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k1', value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ∼ 22 kJ mol(-1), while energy for laccase inactivation was 18 kJ mol(-1). Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT = 40 K) as opposed to fungal biomass (ΔT = 15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.
We previously reported that Modified Field Stain (MF) can be used as a rapid stain for diagnosis. In the present study we extend the observation to include the stain as an alternative method to assess viability of the cells.
The incidence of Acanthamoeba keratitis has been increasing since the previous decades, especially among contact lens users. This infection is majorly caused by the use of ineffective contact lens disinfecting solution. Thus, this study was conducted to evaluate the in vitro effects of multi-purpose disinfecting solutions (MPDS) against Acanthamoeba trophozoites and cysts. Acanthamoeba genotype T4 isolated from contact lens paraphernalia and an environmental strains were propagated for trophozoite or cyst-containing culture and adjusted in final concentration of 1 × 105 cells/ml. Amoebicidal and cysticidal assays were conducted by incubating trophozoites and cysts with OPTI-FREE® Express®, ReNu® Fresh™, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive according to the manufacturer's minimum recommended disinfectant time (MMRDT) for up to 12 h at 30 ⁰C. Trypan blue hemocytometer-based microscopic counts determined amoebicidal and cysticidal effects. The viability of Acanthamoeba trophozoites and cysts was confirmed by re-inoculated them in the 1.5% non-nutrient agar plates. It was found that none of the MPDS showed amoebicidal and cysticidal effects during the MMRDT. However, OPTI-FREE® Express® demonstrated a significant differences in average cell reduction for both stages within MMRDT. When subjected to 12 h exposure, both OPTI-FREE® Express® and ReNu® Fresh™ led to significant reduction in the number of trophozoite and cyst cells. Notably, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive did appreciably improve the solution effectiveness towards trophozoite cells when incubated for 12 h. All MPDS were largely ineffective, with 100% survival of all isolates at MMRDT, while OPTI-FREE® Express® showed limited amoebicidal activity against the contact lens paraphernalia isolate, however, it was more against the environmental strains after 12 h incubation time. The commercially available MPDS employed in this research offered minimal effectiveness against the protozoa despite the contact time. Improvement or development of new solution should consider the adjustment of the appropriate disinfectant concentration, adequate exposure time or the incorporation of novel chemical elements, which are effective against Acanthamoeba for accelerated disinfecting and more reduction of potential exposure of contact lens users to Acanthamoeba keratitis.
Acanthamoeba keratitis is a serious infection with blinding consequences and often associated with contact lens wear. Early diagnosis, followed by aggressive topical application of drugs, is a prerequisite in successful treatment, but even then prognosis remains poor. Several drugs have shown promise, including chlorhexidine gluconate; however, host cell toxicity at physiologically relevant concentrations remains a challenge. Nanoparticles, subcolloidal structures ranging in size from 10 to 100 nm, are effective drug carriers for enhancing drug potency. The overall aim of the present study was to determine whether conjugation with gold nanoparticles enhances the antiacanthamoebic potential of chlorhexidine. Gold-conjugated chlorhexidine nanoparticles were synthesized. Briefly, gold solution was mixed with chlorhexidine and reduced by adding sodium borohydride, resulting in an intense deep red color, indicative of colloidal gold-conjugated chlorhexidine nanoparticles. The synthesis was confirmed using UV-visible spectrophotometry that shows a plasmon resonance peak of 500 to 550 nm, indicative of gold nanoparticles. Further characterization using matrix-assisted laser desorption ionization-mass spectrometry showed a gold-conjugated chlorhexidine complex at m/z 699 ranging in size from 20 to 100 nm, as determined using atomic force microscopy. To determine the amoebicidal and amoebistatic effects, amoebae were incubated with gold-conjugated chlorhexidine nanoparticles. For controls, amoebae also were incubated with gold and silver nanoparticles alone, chlorhexidine alone, neomycin-conjugated nanoparticles, and neomycin alone. The findings showed that gold-conjugated chlorhexidine nanoparticles exhibited significant amoebicidal and amoebistatic effects at 5 μM. Amoebicidal effects were observed by parasite viability testing using a Trypan blue exclusion assay and flow-cytometric analysis using propidium iodide, while amoebistatic effects were observed using growth assays. In contrast, chlorhexidine alone, at a similar concentration, showed limited effects. Notably, neomycin alone or conjugated with nanoparticles did not show amoebicidal or amoebistatic effects. Pretreatment of A. castellanii with gold-conjugated chlorhexidine nanoparticles reduced amoeba-mediated host cell cytotoxicity from 90% to 40% at 5 μM. In contrast, chlorhexidine alone, at similar concentrations, had no protective effects for the host cells. Similarly, amoebae treated with neomycin alone or neomycin-conjugated nanoparticles showed no protective effects. Overall, these findings suggest that gold-conjugated chlorhexidine nanoparticles hold promise in the improved treatment of A. castellanii keratitis.
Introduction: Amniotic fluid (AF) consists of heterogenous population of cells with high diagnostic
and therapeutic values. The study of rat amniotic fluid cells is very limited, despite the extensive use
of this animal model in biomedical research. Primary culture of rat AF cells, especially from full term pregnancies has not been well established. Here we attempt to determine the suitable medium in
culturing rat AF cells that would enhance the cell viability, growth rate and heterogeneity. Methods:
The cell viability, growth rate and heterogeneity of rat AF cells were compared upon culturing the
primary cells in two different media; Amniomax or RPMI. Cell viability study was carried out using
trypan blue staining, while the growth rate was monitored based on the time required to passage the cells (population doubling time in hour). The heterogeneity of cells was examined based on the morphology of the cells. Statistical analysis was performed using t-test. Results: Amniomax was observed to provide a better culture condition in culturing rat AF cells as the cells are more viable, grow faster and more heterogenous as compared to the cells grown in RPMI. Conclusion: Amniomax is a more suitable medium for high quality and viability of full term rat AF cell culture, as compared to RPMI. Thus, warranting propagation of more rat AF cells for biomedical research.
Introduction: Curcumin, a natural compound present in turmeric (Curcuma longa) has been known to possess both anti-inflammatory and antioxidant effects. Objectives: The objectives of the study were to evaluate the cytotoxic activities and to determine the mode of cell death induced by curcumin towards the human mammary carcinoma cells (MDAMB-231). Methodology: Cytotoxicity of curcumin and its effect on cell viability were determined by using MTT assay and trypan blue dye exclusion method, respectively. The mode of cell death was detected by viewing under a light microscope and through DNA fragmentation analysis. Results and discussion: Curcumin was cytotoxic to MDA-MB-231 cells with the IC50 of 17.25 ì g/ml. Cell viability treatment using curcumin at concentrations of 30 ì g/ml and 10 ì g/ml was significantly (p
A key issue in the treatment of acute myeloid leukemia (AML) is the development of drug resistance to chemotherapeutic agents. Overexpression of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein, is associated with tumor progression and drug resistance in leukemia and several cancers. The purpose of this study was to investigate the effect of specific Mcl-1 small interference RNA (siRNA) on the proliferation and chemosensitivity of U-937 AML cell to etoposide. The siRNA transfection was conducted using Lipofectamine 2000. Quantitative real-time RT-PCR (qRT-PCR) and Western blot analysis were employed to measure the expression levels of mRNA and protein, respectively. To evaluate tumor cell growth after siRNA transfection, Trypan blue exclusion assay was conducted. The cytotoxic effects of siRNA and etoposide were determined using MTT assay on their own and in combination. DNA-histone ELISA and annexin-V/FITC assays were performed to study the apoptosis. Mcl-1 siRNA transfection significantly blocked the expression of Mcl-1 mRNA and protein in a time-dependent manner, leading to a strong growth inhibition and enhanced apoptosis (P less than 0.05). Furthermore, pretreatment with Mcl-1 siRNA, synergistically enhanced the cytotoxic and apoptotic effects of etoposide (P less than 0.05). Our results demonstrated that Mcl-1 plays a fundamental role in the survival and resistance of U-937 cells to etoposide. Therefore, Mcl-1 can be considered an attractive target in gene therapy of AML patients and siRNA-mediated silencing of this gene may be a novel strategy in AML treatment.
Naegleria fowleri (N. fowleri) causes primary amoebic meningoencephalitis (PAM) which almost always results in death. N. fowleri is also known as "brain-eating amoeba" due to its literal infestation of the brain leading to an inflammatory response in the brain tissues. Currently, there is no single drug that is available to treat PAM, and most treatments are combinations of antifungal, anticancer, and anti-inflammatory drugs. Recently nanotechnology has gained attention in chemotherapeutic research converging on drug delivery, while oleic acid (OA) has shown positive effects on the human immune system and inflammatory processes. In continuation of our recent research in which we reported the effects of oleic acid conjugated with silver nanoparticles (OA-AgNPs) against free-living amoeba Acanthamoeba castellanii, in this report, we show their antiamoebic effects against N. fowleri. OA alone and its nanoconjugates were tested against the amoeba by using amoebicidal and host cell cytopathogenicity assays. Trypan blue exclusion assay was used to determine cell viability. The results revealed that OA-AgNPs exhibited significantly enhanced antiamoebic effects (P < 0.05) against N. fowleri as compared to OA alone. Evidently, lactate dehydrogenase release shows reduced N. fowleri-mediated host cell cytotoxicity. Based on our study, we anticipate that further studies on OA-AgNPs could potentially provide an alternative treatment of PAM.
Probiotics are live microorganisms and when consumed in adequate amounts will confer health benefit on the host. Probiotic effects of Lactic Acid Bacteria (LAB) have been reported extensively, which rely generally on the viability of LAB cells. However, we have reported extensively the prominent probiotic effects of cell less postbiotics metabolites produced by various strains of Lactobacillus plantarum isolated from Malaysian foods on rats, poultry and pigs. L. plantarum is a major species of LAB. Despite the emerging evidence of anticancer properties of LAB, very limited information is available on the cytotoxic and antiproliferative activities of cytobiotic metabolites produced by LAB. Recently, we have documented the selective antiproliferative and cytotoxicity of cytobiotic produced by six strains of L. plantarum on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. Haemolytic assay was used to determine the toxicity of cytobiotic using human and various animal red blood cells. The cytotoxicity mode was subsequently determined for selected UL4 cytobiotic on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic ob-servation using AO/PI dye reagents and flow cytometric analyses. The selective cytotoxicity effect on various cancel cells that occurred in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells will be discussed in the presentation. Moreover, the antiproliferative effects and induction of late apoptosis effects against selected malignant cancer cells will be discussed further in the presentation. This report reveals the vast potential of cytobiotics produced by L. plantarum strains as functional supplement and as an adjunctive treatment for cancer.
Background: Imatinib mesylate, a tyrosine kinase inhibitor specifically targeting the BCR/ABL fusion protein, induces hematological remission in patients with chronic myeloid leukemia (CML). However, the majority of CML patients treated with imatinib develop resistance with prolonged therapy. Dendrophthoe pentandra (L.) Miq. is a Malaysian mistletoe species that has been used as a traditional treatment for several ailments such as smallpox, ulcers, and cancers. Methods: We developed a resistant cell line (designated as K562R) by long-term co-culture of a BCR/ ABL positive CML cell line, K562, with imatinib mesylate. We then investigated the anti-proliferative effects of D. pentandra methanol extract on parental K562 and resistant K562R cells. Trypan blue exclusion assays were performed to determine the IC50 concentration; apoptosis and cell cycle analysis were conducted by flow cytometry. Results: D. pentandra extract had greater anti-proliferative effects towards K562R (IC50= 192 μg/mL) compared to K562 (500 μg/ mL) cells. Upon treatment with D. pentandra extract at the IC50. concentration: K562 but not K562R demonstrated increase in apoptosis and cell cycle arrest in the G2/M phase. Conclusion: D. pentandra methanol extract exerts potent anti-proliferative effect on BCR/ABL positive K562 cells.
The positive response to tamoxifen in ERa-positive breast cancer patients is usually of a short duration as many
of the patients eventually develop resistance. Our preliminary results show that aloe emodin extracted from
the leaves of the Aloe barbadensis Miller demonstrated a cytotoxicity that is selective to ERa-positive breast
cancer cells (MCF-7), but not to ERa-negative breast cancer cells (MDA-MB-231) and to the control cells (MCF-
10A). The objective of this study was to test the hypothesis that aloe emodin may enhance the response of
MCF-7 cells to treatment with tamoxifen. MCF-7 cells were treated with aloe emodin alone, tamoxifen alone
or a combination of emodin and tamoxifen, at their respective IC50 concentrations and at different time points
of 24 hours, 48 hours and 72 hours. The respective IC50s were the concentrations of aloe emodin and tamoxifen
required to achieve 50% inhibition of the cells in the study. Cell viability and apoptosis were determined using
trypan blue exclusion and DNA fragmentation assays, respectively. The involvement of RAS/MEKs/ERKs genes
of MAPK signalling pathways with aloe emodin was determined using QuantiGene 2.0 Plex assay. Data was
evaluated using the one-way ANOVA test. Our findings showed that aloe emodin enhanced the cytotoxicity of
tamoxifen on MCF-7 cells through apoptosis by downregulation of MEK1/2 genes. Our research may provide a
rational basis for further in vivo studies to verify the efficacy of a combination of aloe emodin and tamoxifen
on the viability of ERa-positive-breast cancer cells.
Immunomodulators are agents that are able to stimulate or inhibit the immune response. The leaf extracts from Potentilla indica and Dendrophthoe pentandra were analyzed in vitro for immunomodulatory activity and an MTT colorimetric assay was conducted to determine the proliferation of mice splenocytes and thymocytes. A bromodeoxyuridine assay was performed to analyze DNA synthesis and the Trypan blue exclusion method was conducted to evaluate the changes in total cell population. The results indicated that treatment with P. indica and D. pentandra produced a time- and dose-dependent increase in cell viability and proliferation. Following 72 h of treatment with P. indica and D. pentandra, thymocyte proliferation was augmented by 18 and 41%, respectively and splenocyte proliferation increased by 35 and 42%, respectively, when compared with untreated cells. The present study demonstrated that these extracts may act as potential immunostimulants and, thus, represent an alternative source of immunomodulatory compounds for the treatment of human immune-mediated diseases.
In recent biomedical research, the area of cancer and infectious diseases has a leading position in the utilization of medicinal plants as a source of drug discovery. Malaysia has a diversity and a large number of underutilized fruits that are rich in phenolic compounds. Artoarpus altilis consider an underutilized fruit that is rich in phenolic compounds. Methanol extracts of A. altilis have been previously found to contain a high content of antioxidant phytochemicals. The purpose of the study was to evaluate the cytotoxicity and toxicological effect of methanol fruit extracts against MCF-7 cells. To determine the least concentration that might kill or suppress the growth of the cancer cells was in a concentration-dependent manner approach. The variation in the cytotoxic activity among the extracts was indicated by determining the IC50 of each extract against cells at 72 h. The IC50 of the samples was measured using a trypan blue exclusion assay. The methanol extract of the pulp part showed the least inhibition concentration of 15.40±0.91 μg/mL on MCF-7 cells. In the study, the molecular mechanism of methanol extracts-induced apoptosis and cell cycle arrested in human cancer cells were investigated in a time-dependent-manners approach by using flow cytometry. The treated cells were stained with nexin to detect early and late apoptosis and with propidium iodide (PI) for cell cycle arrest associated with the DNA fragmentation, various cell arrests occurred at G1/S, S, and G2/M phases. Lastly, the gene expression analysis by (RT-qPCR) method was carried out by analyzing the expression of the gene of interest for the quantification of mRNA levels. Results after cells treated with IC50 were revealed by upregulating anti-apoptotic genes/downregulated of pro-apoptotic BCL-2 gene expressions were triggered the treated cells into CASPASE-3, intrinsic and extrinsic pathways. These findings suggest that the methanol extracts of three parts of A. altilis fruit have potential anticancer activity against MCF-7 cells mainly the pulp part of the fruit.
The photodegradation of various dyes in aqueous solution was studied. Experiments were carried out using glass coated titanium dioxide thin film as photocatalyst. Photodegradation processes of methylene blue (MB), methyl orange (MO), indigo carmine (IC), chicago sky blue 6B (CSB), and mixed dye (MD, mixture of the four mentioned single dye) were reported. As each photodegradation system is pH dependent, the photodegradation experiment was carried out in each dye photodegradation reactive pH range at approximately 28 degrees C. The dyes removal efficiency was studied and compared using UV-vis spectrophotometer analysis. The total removal of each dye was: methylene blue (90.3%), methyl orange (98.5%), indigo carmine (92.4%), chicago sky blue 6B (60.3%), and mixed dyes (70.1%), respectively. The characteristic of the photocatalyst was investigated using X-ray diffractometer (XRD). The amount of each dye intermediate produced in the photodegradation process was also determined with the help of total organic carbon (TOC) analysis.