Lead metal biosorption and isotherms studies by metal-resistant Bacillus strain MRS-2 bacterium.

In this study, lead (Pb) biosorption studies in aqueous solution were performed with metal-resistant Bacillus strain MRS-2 (ATCC 55674) bacterium which was previously isolated from wastewater plant. It showed minimum inhibition concentration of 300 ppm Pb on the nutrient agar plates. Pb biosorption using MRS-2 bacteria was investigated under different parameters such as pH, temperature, biomass dosage, initial Pb concentration, contact time, and type of biomass by batch experiments. Pb concentration was analyzed through Inductively coupled plasma-optical emission spectrometry. The rate of biosorption (Q) and Pb biosorption capacity (qe ) were calculated for above mentioned parameters. It was observed that Pb precipitates by itself from the solution at pH 2 and 8 or above without bacteria and precipitation did not increase even in the presence of bacteria. The results showed that the highest biosorption rate and biosorption capacity (mg/g) were observed at pH 7, 25°C, 2-h contact time with live bacteria. The highest biosorption rate was observed at 1.5 g/L biomass dose and 5 ppm initial Pb concentration, whereas the highest Pb biosorption capacity was observed at 0.25 g/L biomass dose and 12.5 ppm initial Pb concentration. It was observed that Pb biosorption by live bacteria occurred through adsorption on cell surface. In this study, the biosorption isotherm analysis favored the Langmuir isotherm model indicating monolayer biosorption. This Bacillus strain showed higher Pb biosorption capacity than most of the previously reported Bacillus strains. In conclusion, this study indicates that the Bacillus MRS-2 strain can be used to remove Pb from industrial wastewaters in an ecofriendly approach.

Identification of 7,8-dihydroxy-3-phenylcoumarin as a reversible monoamine oxidase enzyme inhibitor.

We herein report the biological evaluation of 3-arylcoumarin derivatives (3a-l) as potential human monoamine oxidase-A and -B (hMAO-A and hMAO-B) inhibitors. The result indicated that 7,8-dihydroxy-3-(4-nitrophenyl)coumarin (3j) was most effective against MAO-A (inhibition concentration [IC50 ] = 6.46 ± 0.02 µM) and MAO-B (IC50 = 3.8 ± 0.3 µM) enzymes than other synthesized compounds and reference compounds (pargyline and moclobemide). Furthermore, compound (3j) showed (a) nonselectivity against hMAO enzymes, (b) reversible hMAO enzymes inhibition, and (c) neuroprotection against H2 O2 -treated human neuroblastoma (N2a) cells. Finally, a molecular modeling study revealed that the hMAO enzymes inhibitory activity of the compound (3j) may be due to the orientation where the nitro (NO2 ) group lies deep into the receptor and the phenyl ring directed toward flavin adenosine dinucleotide via hydrogen bond interaction, and possible π-π interaction with various important residues. Thus, the results of the present study demonstrate that compound (3j) can be considered as a promising scaffold for the development of hMAO-A and hMAO-B inhibitors.

Skeletal Muscle Protein Composition Adaptations to 10 Weeks of High-Load Resistance Training in Previously-Trained Males.

While high-load resistance training increases muscle hypertrophy, the intramuscular protein responses to this form of training remains largely unknown. In the current study, recreationally resistance-trained college-aged males (N = 15; mean ± SD: 23 ± 3 years old, 6 ± 5 years training) performed full-body, low-volume, high-load [68-90% of one repetition maximum (1RM)] resistance training over 10 weeks. Back squat strength testing, body composition testing, and a vastus lateralis biopsy were performed before (PRE) and 72 h after the 10-week training program (POST). Fiber type-specific cross-sectional area (fCSA), myofibrillar protein concentrations, sarcoplasmic protein concentrations, myosin heavy chain and actin protein abundances, and muscle tissue percent fluid were analyzed. The abundances of individual sarcoplasmic proteins in 10 of the 15 participants were also assessed using proteomics. Significant increases (p < 0.05) in type II fCSA and back squat strength occurred with training, although whole-body fat-free mass paradoxically decreased (p = 0.026). No changes in sarcoplasmic protein concentrations or muscle tissue percent fluid were observed. Myosin heavy chain protein abundance trended downward (-2.9 ± 5.8%, p = 0.069) and actin protein abundance decreased (-3.2 ± 5.3%, p = 0.034) with training. Proteomics indicated only 13 sarcoplasmic proteins were altered with training (12 up-regulated, 1 down-regulated, p < 0.05). Bioinformatics indicated no signaling pathways were affected, and proteins involved with metabolism (e.g., ATP-PCr, glycolysis, TCA cycle, or beta-oxidation) were not affected. These data comprehensively describe intramuscular protein adaptations that occur following 10 weeks of high-load resistance training. Although previous data from our laboratory suggests high-volume resistance training enhances the ATP-PCr and glycolytic pathways, we observed different changes in metabolism-related proteins in the current study with high-load training.

An optimized procedure for isolation of rodent and human skeletal muscle sarcoplasmic and myofibrillar proteins.

Several published protocols exist for isolating contractile or myofibrillar (MF) proteins from skeletal muscle, however, achieving complete resuspension of the myofibril pellet can be technically challenging. We performed several previously published MF isolation methods with the intent of determining which method was most suitable for MF protein isolation and solubilization. Here, we provide an optimized protocol to isolate sarcoplasmic and solubilized MF protein fractions from mammalian skeletal muscle suitable for several downstream assays.

Skeletal Muscle Myofibrillar Protein Abundance Is Higher in Resistance-Trained Men, and Aging in the Absence of Training May Have an Opposite Effect.

Resistance training generally increases skeletal muscle hypertrophy, whereas aging is associated with a loss in muscle mass. Interestingly, select studies suggest that aging, as well as resistance training, may lead to a reduction in the abundance of skeletal muscle myofibrillar (or contractile) protein (per mg tissue). Proteomic interrogations have also demonstrated that aging, as well as weeks to months of resistance training, lead to appreciable alterations in the muscle proteome. Given this evidence, the purpose of this small pilot study was to examine total myofibrillar as well as total sarcoplasmic protein concentrations (per mg wet muscle) from the vastus lateralis muscle of males who were younger and resistance-trained (denoted as YT, n = 6, 25 ± 4 years old, 10 ± 3 self-reported years of training), younger and untrained (denoted as YU, n = 6, 21 ± 1 years old), and older and untrained (denoted as OU, n = 6, 62 ± 8 years old). The relative abundances of actin and myosin heavy chain (per mg tissue) were also examined using SDS-PAGE and Coomassie staining, and shotgun proteomics was used to interrogate the abundances of individual sarcoplasmic and myofibrillar proteins between cohorts. Whole-body fat-free mass (YT > YU = OU), VL thickness (YT > YU = OU), and leg extensor peak torque (YT > YU = OU) differed between groups (p < 0.05). Total myofibrillar protein concentrations were greater in YT versus OU (p = 0.005), but were not different between YT versus YU (p = 0.325). The abundances of actin and myosin heavy chain were greater in YT versus YU (p < 0.05) and OU (p < 0.001). Total sarcoplasmic protein concentrations were not different between groups. While proteomics indicated that marginal differences existed for individual myofibrillar and sarcoplasmic proteins between YT versus other groups, age-related differences were more prominent for myofibrillar proteins (YT = YU > OU, p < 0.05: 7 proteins; OU > YT = YU, p < 0.05: 11 proteins) and sarcoplasmic proteins (YT = YU > OU, p < 0.05: 8 proteins; OU > YT&YU, p < 0.05: 29 proteins). In summary, our data suggest that modest (~9%) myofibrillar protein packing (on a per mg muscle basis) was evident in the YT group. This study also provides further evidence to suggest that notable skeletal muscle proteome differences exist between younger and older humans. However, given that our n-sizes are low, these results only provide a preliminary phenotyping of the reported protein and proteomic variables.

Muscle fiber hypertrophy in response to 6 weeks of high-volume resistance training in trained young men is largely attributed to sarcoplasmic hypertrophy.

Cellular adaptations that occur during skeletal muscle hypertrophy in response to high-volume resistance training are not well-characterized. Therefore, we sought to explore how actin, myosin, sarcoplasmic protein, mitochondrial, and glycogen concentrations were altered in individuals that exhibited mean skeletal muscle fiber cross-sectional area (fCSA) hypertrophy following 6 weeks of high-volume resistance training. Thirty previously resistance-trained, college-aged males (mean ± standard deviation: 21±2 years, 5±3 training years) had vastus lateralis (VL) muscle biopsies obtained prior to training (PRE), at week 3 (W3), and at week 6 (W6). Muscle tissue from 15 subjects exhibiting PRE to W6 VL mean fCSA increases ranging from 320-1600 µm2 was further interrogated using various biochemical and histological assays as well as proteomic analysis. Seven of these individuals donated a VL biopsy after refraining from training 8 days following the last training session (W7) to determine how deloading affected biomarkers. The 15 fCSA hypertrophic responders experienced a +23% increase in mean fCSA from PRE to W6 (p<0.001) and, while muscle glycogen concentrations remained unaltered, citrate synthase activity levels decreased by 24% (p<0.001) suggesting mitochondrial volume decreased. Interestingly, repeated measures ANOVAs indicated that p-values approached statistical significance for both myosin and actin (p = 0.052 and p = 0.055, respectively), and forced post hoc tests indicated concentrations for both proteins decreased ~30% from PRE to W6 (p<0.05 for each target). Phalloidin-actin staining similarly revealed actin concentrations per fiber decreased from PRE to W6. Proteomic analysis of the sarcoplasmic fraction from PRE to W6 indicated 40 proteins were up-regulated (p<0.05), KEGG analysis indicated that the glycolysis/gluconeogenesis pathway was upregulated (FDR sig. <0.001), and DAVID indicated that the following functionally-annotated pathways were upregulated (FDR value <0.05): a) glycolysis (8 proteins), b) acetylation (23 proteins), c) gluconeogenesis (5 proteins) and d) cytoplasm (20 proteins). At W7, sarcoplasmic protein concentrations remained higher than PRE (+66%, p<0.05), and both actin and myosin concentrations remained lower than PRE (~-50%, p<0.05). These data suggest that short-term high-volume resistance training may: a) reduce muscle fiber actin and myosin protein concentrations in spite of increasing fCSA, and b) promote sarcoplasmic expansion coincident with a coordinated up-regulation of sarcoplasmic proteins involved in glycolysis and other metabolic processes related to ATP generation. Interestingly, these effects seem to persist up to 8 days following training.

7,8-Dihydroxy-3-arylcoumarin Induces Cell Death Through S-Phase Arrest in MDA-MB-231 Breast Cancer Cells.

BACKGROUND/AIM: Coumarins remain one of the most versatile classes of compounds for anticancer drug design and discovery. The present study aimed to evaluate the in vitro cytotoxic activity of 7,8-Dihydroxy-3-arylcoumarin derivatives (7a-i) in A549, MDA-MB-231and PC-3 cancer cell lines. MATERIALS AND METHODS: Cell viability, cell-cycle progression and regulatory protein expression were evaluated using crystal violet dye-binding assay, flow cytometry and western blot analysis. RESULTS: 7,8-Diacetoxy-3-(4-nitrophenyl)coumarin (7h) showed the highest cytotoxic activity with CC50 of 7.51±0.07 µM in MDA-MB-231 cell line. The mechanism of cytotoxic action indicated that 7h caused significant (p<0.05) MDA-MB-231 cells arrest in the S phase as well as moderate cells arrest in the G2/M phase; confirmed by up-regulation of cyclins A/B1, p21 and CDKs 4/6, and down-regulation of cyclin E2 and CDK2 regulatory proteins. CONCLUSION: These results suggest that 7h could serve as a valuable template for the development of novel synthetic compounds for breast cancer treatment.

Comparative whole genome transcriptome analysis and fenugreek leaf extract modulation on cadmium­induced toxicity in liver cells.

Cadmium (Cd), an economically valuable metal, is widely used in various industrial processes. Although it is of economic value, it is hazardous to human health. Cd accumulates in vital organs where it causes various diseases. Natural compounds with chelating or antioxidant properties have been tested to reduce the toxic effect of Cd. The anti­oxidant, anti­diabetic and hypocholesterolemic properties of fenugreek (Trigonella foenum-graecum) leaves make it a candidate for investigation as protective agent against Cd­induced toxicity. In the present study, the protective effects of fenugreek leaf extract (FLE) on cell viability, morphology, and whole genomic transcription in cadmium chloride (CdCl2)­treated rat liver cells were analyzed. The cells were treated with 25 µM CdCl2 alone, or co­treated with 5 µg/ml FLE for 48 h. The co­treated cells were pretreated with FLE for 2 or 4 h, followed by CdCl2 treatment. Genomic transcription analysis was performed in the CdCl2­treated cells following treatment for 6 h. The CdCl2 caused a significant decrease in viability (35.8±4.1%) and morphological distortion of the cells, compared with the untreated control cells; whereas 4 h pretreatment with FLE (5 µg/ml) reversed the Cd­induced morphology alteration and increased the cell viability to 102±3.8%. Genomic transcription analysis of the CdCl2 only­treated cells showed 61 upregulated and 124 downregulated genes, compared with 180 upregulated and 162 downregulated genes in the FLE pretreated cells. Furthermore, 37 and 26% of the affected total genomic genes in the CdCl2 only­treated cells were involved in binding and catalytic activities, respectively, whereas 50 and 20% of the genes in the FLE pretreated cells were involved in binding and catalytic activities, respectively. In conclusion, these results suggested that genome transcriptome modulation may be important in the protective effect of FLE against Cd­induced toxicity in normal rat liver cells.

Modulation of cytokines and chemokines expression by NAC in cadmium chloride treated human lung cells.

Cadmium (Cd), is one of the most hazardous metals found in the environment. Cd exposure through inhalation has been linked to various diseases in lungs. It was shown that Cd induces proinflammatory cytokines through oxidative stress mechanism. In this report, we studied the immunomodulatory effect of a well known antioxidant, N-acetylcysteine (NAC) on cadmium chloride (CdCl2 ) treated human lung A549 cells through human cytokine array 6. The lung cells were treated with 0 or 75 µM CdCl2 alone, 2.5 mM NAC alone, or co-treated with 2.5 mM NAC and 75 µM CdCl2 for 24 h. The viability of cells was measured by crystal violet dye. The array results were validated by human IL-1alpha enzyme- linked immunosorbent assay (ELISA) kit. The viability of the 75 µM CdCl2 alone treated cells was decreased to 44.5%, while the viability of the co-treated cells with 2.5 mM NAC was increased to 84.1% in comparison with untreated cells. In the cell lysate of CdCl2 alone treated cells, 19 and 8 cytokines were up and down-regulated, while in the medium 15 and 3 cytokines were up and downregulated in comparison with the untreated cells. In the co-treated cells, all these cytokines expression was modulated by the NAC treatment. The IL-1α ELISA result showed the same pattern of cytokine expression as the cytokine array. This study clearly showed the modulatory effect of NAC on cytokines and chemokines expression in CdCl2- treated cells and suggests the use of NAC as protective agent against cadmium toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1612-1619, 2016.

Effect of cadmium on the expression levels of interleukin-1α and interleukin-10 cytokines in human lung cells.

Cadmium is an environmentally hazardous metal, which causes toxicity in humans. Inhalation of cigarette smoke and industrial fumes containing cadmium are sources of cadmium exposure. It is responsible for the malfunction of various organs, leading to disease particularly in the lungs, liver and kidneys. In the present study, the effect of cadmium chloride (CdCl2) on cell viability, and the expression levels of interleukin (IL)­1α and IL­10 cytokines at various concentrations and incubation durations were assessed in MRC­9 human normal lung and A549 human lung cancer cells to elucidate the mechanism of cadmium toxicity. Cell viability was measured using a crystal violet dye binding assay. The expression levels of the cytokines were measured by cytokine specific enzyme­linked immunosorbent assay kits. The viability assay results revealed higher sensitivity of the A549 lung cancer cells to CdCl2 compared with the normal MRC­9 lung cells. In the normal MRC­9 lung cells, higher expression levels of the cytokines were observed at the lowest CdCl2 concentration at a shorter exposure time compared with the lung cancer cells. Higher levels of the cytokines were observed in the A549 lung cancer cells at all other times and concentrations compared with the MRC­9 cells, indicating higher levels of inflammation. The cytokine levels were reduced at higher CdCl2 concentrations and longer exposure durations, demonstrating the toxic effect of cadmium. The results indicated that CdCl2 affected the expression levels of the cytokines and led to cytotoxicity in human lung cells, and suggested that compounds which reduce inflammation may prevent cadmium toxicity.

Synthesis and in vitro evaluation of 3-(4-nitrophenyl)coumarin derivatives in tumor cell lines.

Coumarins are naturally-occurring compounds that have attracted considerable interest due to their numerous biological activities depending on their pattern of substitution on the coumarin molecule. In this present investigation, we synthesized 3-(4-nitrophenyl)coumarin derivatives (9a-e) and evaluated their in vitro cytotoxic effect on human lung (A549), breast (MDA-MB-231) and prostate (PC3) cancer cell lines for 48 h using crystal violet dye binding assay. Cytotoxic effects of the most active compound on normal human lung (MRC-9) and breast (MCF-10A) cell lines, cell cycle analysis using flow cytometry and mitochondrial membrane potential (MMP) using Tetramethyl Rhodamine Methyl Ester (TMRM; rhodamine-123) fluorescent dye were also examined. Among the compounds that were evaluated, 9c showed cytotoxic effect (active), caused significant cells arrest (p<0.05) in G0/G1 and S phases of cell cycle and loss of MMP in A459, MDA-MB-231 and PC3 cell lines. Additionally, the cytotoxic effect of 9c was compared to reference drugs (Coumarin and Docetaxel) for comparative study. These results further demonstrate that acetoxy group at C-7 and C-8 positions of 9c are responsible for the observed cytotoxic effect in these cancer cell lines.

Chemoprotective effect of monoisoamyl 2, 3-dimercaptosuccinate (MiADMS) on cytokines expression in cadmium chloride treated human lung cells.

Cadmium is commercially profitable element, but it causes toxicity in humans and animals leading to diseases in various organs. The main route of cadmium exposure to humans is through inhalation. Lungs respond to insult through secretion of cytokines. In this study, the chemoprotective effect of monoisoamyl 2, 3-dimercaptosuccinate (MiADMS) was evaluated on viability and cytokines expression in CdCl2 treated human lung A549 cells by cytokine array. Cells were treated with 0, 50, 75, and 100 µM CdCl2 alone, 300 µM MiADMS alone, and co-treated with 300 µM MiADMS and 75 µM CdCl2 for 24 h. The viability was measured by crystal violet dye. The level of cytokines in the cells' lysate and cell culture medium was measured using Ray Biotech's Human Cytokine Array 6 in control cells, 75 µM CdCl2 alone and MiADMS co-treated cells. Array results were validated by ELISA kit. The CdCl2 caused a dose dependent decrease in cell viability, while MiADMS co-treatment resulted in a significant increase in viability of CdCl2 treated cells. Morphology of the cells treated with CdCl2 was destroyed, while MiADMS restored the lost shape in CdCl2 treated cells. In addition, the cells co-treated with MiADMS and CdCl2 showed modulation of cytokines expression in comparison to the CdCl2 alone treated cells. The ELISA results showed the similar pattern of cytokine expression as Human Cytokine Array and validated the array results. These results clearly show the chemoprotective effect of MiADMS and suggest that MiADMS can be used as antidote at moderate dose against CdCl2 toxicity.

Cytotoxic activity of N, N'-Bis (2-hydroxybenzyl) ethylenediamine derivatives in human cancer cell lines.

BACKGROUND: Compounds containing ethylenediamine (-NCH2CH2N-) moiety are known to exhibit antimicrobial, -fungal, -bacterial, -tuberculosis and -cancer activities. MATERIALS AND METHODS: In the present study, we evaluated the in vitro cytotoxic activity of N,N'-bis(2-hydroxybenzyl)- (6), N,N'-bis(5-bromo-2-hydroxybenzyl)- (7) and N,N'-bis(5-chloro-2-hydroxybenzyl) (8)- ethylenediamine dihydrochlorides; and N,N'-bis(2-hydroxybenzyl)- (9), N,N'-bis(5-bromo-2-hydroxybenzyl)- (10) and N,N'-bis(5-chloro-2-hydroxybenzyl) (11)- ethylenediamine toward human lung (A549), breast (MDA-MB-231) and prostate (PC3) cancer cell lines after 24-h treatment using crystal violet dye binding assay. Effects on the cell cycle the using flow cytometry, and mitochondrial membrane potential using rhodamine-123 florescent dye were also evaluated. RESULTS: Compounds 7 and 8 exhibit cytotoxic activity, causing cell arrest at different phases of the cell cycle and loss of mitochondrial membrane potential in the above cancer cell lines. CONCLUSION: These findings clearly demonstrate, to our knowledge for the first time, that ethylenediamine dihydrochloride salts-compounds 7 and 8-exhibit concentration-dependent cytotoxic activity towards A549, MDA-MB-231 and PC3 cancer cell lines, which may serve as a basis for future work on novel therapeutic agents.

Coumarin-based benzopyranone derivatives induced apoptosis in human lung (A549) cancer cells.

In the present investigation, we report on the possible underlying mechanism for the cytotoxicity of compounds: 3-(4-(2-(dimethylamino)ethoxy)-phenyl)-7-methoxy-4-phenyl coumarin 5, 3-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-7-methoxy-4-phenylcoumarin 6, and 3-(4-(2-(diethylamino) ethoxy)phenyl)-7-methoxy-4-methylcoumarin 7 in the human lung (A549) cancer cell line, using Ray Biotech's Human Apoptosis Arrays and apoptotic protein antibodies. Apoptosis array results showed differential apoptotic proteins expression in the extracts of cells treated with compounds 5-7. Western blotting demonstrated that compound 5 induced apoptosis and caused cell death in the A549 cell line via an increase (up-regulation) in Bax protein expression (pro-apoptotic pathway) and a slight decrease (down-regulation) in Bcl-2 protein expression (anti-apoptotic pathway) after 6 h of treatment. These observations may provide valuable information on the mechanism by which these coumarin-based benzopyranone derivatives induce cytotoxicity in the human lung (A549) cancer cell line.

Mitigative action of monoisoamyl-2,3-dimercaptosuccinate (MiADMS) against cadmium-induced damage in cultured rat normal liver cells.

Cadmium is non-essential, carcinogenic and multitarget pollutant in the environment. Monoisoamyl-2,3-dimercaptosuccinate (MiADMS) is an ester of dimercaptosuccinic acid that acts as an antioxidant and chelator. Therefore, the mitigative action of MiADMS on viability, morphology, antioxidative enzymes and cell cycle were studied on rat liver cells treated with cadmium chloride (CdCl2). The cells were treated with 150 µM CdCl2 alone or cotreated with 300 µM MiADMS (concurrently, 2 h or 4 h post-CdCl2 treatment) for 24 h. The viability of cells treated with CdCl2 alone was decreased in comparison to the control cells. Cotreatment with MiADMS resulted in an increase in cell viability in comparison to the CdCl2 alone treated cells. The CdCl2 treatment altered the morphological shape of the cells, while cotreatment with MiADMS restored the shape. Antioxidative enzymes activities were decreased in the cells treated with CdCl2 alone, while MiADMS cotreatment resulted in an increase in enzyme activities. The CdCl2 arrested the cells in S phase of the cell cycle. Cotreatment with MiADMS alleviated cell cycle arrest by shifting to G1 phase. These results clearly show the mitigative action of MiADMS on CdCl2 toxicity and may suggest that MiADMS can be used as an antidote against cadmium.

Cytotoxic activity of new acetoxycoumarin derivatives in cancer cell lines.

BACKGROUND: Coumarin and their derivatives are important and useful compounds with diverse pharmacological properties. In the present study, we evaluated the in vitro cytotoxic activity of new acetoxycoumarin derivatives: 4-(7-methoxy-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (1), 4-(1-methyl-3-oxo-3H-benzo[f]chromen-2-yl)phenyl acetate (2), 4-(6-propionamido-4-methyl-2-oxo-2H-chromen-3-yl) phenyl acetate (3), 4-(7-acetoxy-2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (4), 4-(2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (5), 4-(6-bromo-2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (6), 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (7), 4-(6,8-dibromo-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (8) against A549 human lung cancer, CRL 1548 rat liver cancer and CRL 1439 normal rat liver cells. MATERIALS AND METHODS: The cytotoxic activity was evaluated by crystal violet dye-binding assay. The effect of compounds 5 and 7 on different phases of the cell cycle was determined using flow cytometry. RESULTS: In the A549 lung cancer cell line, the 50% lethal dose (LD50) values for compounds 1-4, 6 and 8 were found to be >100 µM while those for 5 and 7 were 89.3 and 48.1 µM, respectively after 48 h treatment. In the CRL 1548 liver cancer cell line, only compound 7 showed toxicity, with an LD50 of 45.1 µM. Compounds 5 and 7 caused different cell phase arrest in lung and liver cancer cell lines. CONCLUSION: The results indicate that 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (7) had the highest cytotoxic activity in all of the examined cell lines.

Protective effects of N-acetylcysteine against cadmium-induced damage in cultured rat normal liver cells.

In this study, the protective effects of N-acetylcysteine (NAC), a precursor of reduced glutathione, were studied by measuring the viability, the levels of antioxidant enzymes, and by analyzing the cell cycle in cadmium (Cd)-treated rat liver cells. The cells were treated with 150 µM CdCl2 alone or co-treated with 150 µM CdCl2 and 5 mM NAC (2 h pre-, simultaneous or 2 h post-treatment) for 24 h. The viability of the cells treated with 150 µM CdCl2 alone decreased to 40.1%, while that of the cells co-treated with 5 mM NAC (pre-, simultaneous and post-treatment) significantly increased to 83.7, 86.2 and 83.7%, respectively in comparison to the control cells (100%). The catalase enzyme level decreased to undetectable level in the cells treated with CdCl2 alone, while it significantly increased in the co-treated cells (pre-, simultaneous and post-treatment) to 40.1, 34.3 and 13.2%, respectively. In the cells treated with CdCl2 alone, the glutathione peroxidase enzyme level decreased to 78.3%, while it increased in the co-treated cells (pre-, simultaneous, and post-treatment) to 84.5, 83.3 and 87.9%, respectively. The glutathione reductase enzyme level decreased to 56.1% in the cells treated with cadmium alone, but significantly increased in the cells co treated with NAC (pre-, simultaneous and post-treatment) to 79.5, 78.5 and 78.2%, respectively. Cd caused cell cycle arrest at the S and G2/M phases. The co-treatment with NAC inhibited cell cycle arrest by shifting the cells to the G1 phase. These results clearly show the protective effects of NAC against Cd-induced damage in rat liver cells.

In vitro cytotoxicity of benzopyranone derivatives with basic side chain against human lung cell lines.

BACKGROUND: Coumarins belong to an important group of useful drugs with diverse pharmacological properties. In the present study, the in vitro cytotoxicity of new coumarin-based benzopyranone derivatives containing diethylaminoethoxy (5), dimethylaminoethoxy (6), morpholinoethoxy (7), piperidinylethoxy (8) and pyrrolidinylethoxyl (9) amino side chain against human carcinoma (A549) and normal (LL47) lung cell lines was evaluated. MATERIALS AND METHODS: The cytotoxicity was evaluated by crystal violet dye binding assay. The effect of compound 9 on different phases of the cell cycle was determined using flow cytometry. RESULTS: In A549 cells, the 50% lethal dose (LD(50)) for compounds 5-9 were found to be 7.08, 5.0, 34.2, 8.33 and 5.83 µM, respectively, while in LL47 cells, the LD(50) values were found to be 16.7, 20.4, 34.6, 15.4 and 8.75 µM, respectively after 48 h treatment. Cell cycle data indicated that A549 cells were arrested at different phases depending on the concentration. CONCLUSION: Compounds 5-9 showed anticancer activity against lung cancer cell lines, while compound 6 showed highly selective anticancer activity.

Cytotoxicity and stress gene microarray analysis in cadmium-exposed CRL-1439 normal rat liver cells.

Cadmium is a biologically non-essential divalent hazardous metal. Previous studies demonstrated that cadmium toxic effect was caused by reactive oxygen species. Since gene expression is influenced by the presence of these reactive oxygen species, the association between metal intoxication and gene expression has recently become a major focus of research. We examined the effect of cadmium chloride on cell viability at 4, 8 and 24 h. Our results indicate that cadmium chloride did not alter cell viability at 4 or 8 h, but decreased the viability in a dose-dependent manner (p>0.01) at 24 h. Using DNA microarray, we studied the profile of stress gene expression in rat primary hepatocytes treated with cadmium for different time periods using a 100 microM cadmium chloride concentration. Microarray analysis indicated that cadmium treatment caused different patterns of gene expression profiles at each time point of incubation. Of the 207 stress genes on the microarray, only 32 genes were regulated. Since microarrays were hybridized by radioactive cDNA which was less sensitive than fluorescent-labeled cDNA, an experimental/control ratio >1.3 or <0.7 (30% increase or decrease) was taken as significant up- or down-regulation. Exposure of cells to cadmium for 4 h resulted in the expression of three up-regulated genes and six down-regulated genes. Longer exposure to cadmium for 8 h resulted in an increase in up-regulated genes to six and down-regulated genes to 14. After 24 h of cadmium exposure, 15 genes were down-regulated and six genes were up-regulated. Our findings suggest that the cells maintained complete viability up to 8 h with cadmium due to expression of various heat shock proteins and stress response proteins like heme oxygenase. Longer exposure periods, due to the down-regulation of the basic cell function proteins and cell-cycle regulating proteins, led to toxicity in cells and eventually to cell death.

Comparative evaluation of cytotoxicity of cadmium in rat liver cells cultured in serum-containing medium and commercially available serum-free medium.

Cadmium (Cd) is an industrial pollutant and carcinogenic metal. Most in vitro Cd toxicity studies have been carried out in various cell lines cultured in 10% fetal bovine serum (FBS) containing medium. In this report, we compared the toxic effect of Cd (0-300 microM) on cell growth, total RNA, total proteins, and antioxidant enzymes in rat normal liver cells cultured in medium with 10% FBS or commercially available serum-free medium for 4 or 8 hours. With Cd concentration at above 100 microM, the total levels of RNA, protein and cell growth decreased in serum-containing medium, while their levels increased in serum-free medium compared to the controls. The glutathione peroxidase and glutathione reductase levels were lower in serum-free medium than in serum-containing medium, indicating less oxidative stress in cells grown in serum-free medium. These results clearly suggest that Cd showed higher toxicity to liver cells grown in serum-containing medium in comparison to commercially available serum-free medium. It is speculated that albumin and other substances present in commercial serum-free medium chelated Cd and thereby protected these cells against Cd toxicity. Even under in vivo conditions, cadmium enters into various organs after passing through blood which contains serum. Based on these studies, it appears that media containing serum may be ideal for in vivo toxicity correlation studies with animal cells.