Application of cell-based assays for toxicity characterization of complex wastewater matrices: Possible applications in wastewater recycle and reuse.

Exposure to pre-concentrated inlet or outlet STP wastewater extracts at different concentrations (0.001% to 1%) induced dose-dependent toxicity in MCF-7 cells, whereas drinking water extracts did not induce cytotoxicity in cells treated. GC-MS analysis revealed the occurrence of xenobiotic compounds (Benzene, Phthalate, etc.) in inlet/outlet wastewater extracts. Cells exposed to inlet/outlet extract showed elevated levels of reactive oxygen species (ROS: inlet: 186.58%, p<0.05, outlet, 147.8%, p<0.01) and loss of mitochondrial membrane potential (Δψm: inlet, 74.91%, p<0.01; outlet, 86.70%, p<0.05) compared to the control. These concentrations induced DNA damage (Tail length: inlet: 34.4%, p<0.05, outlet, 26.7%, p<0.05) in treated cells compared to the control (Tail length: 7.5%). Cell cycle analysis displayed drastic reduction in the G1 phase in treated cells (inlet, G1:45.0%; outlet, G1:58.3%) compared to the control (G1:67.3%). Treated cells showed 45.18% and 28.0% apoptosis compared to the control (1.2%). Drinking water extracts did not show any significant alterations with respect to ROS, Δψm, DNA damage, cell cycle and apoptosis compared to the control. Genes involved in cell cycle and apoptosis were found to be differentially expressed in cells exposed to inlet/outlet extracts. Herein, we propose cell-based toxicity assays to evaluate the efficacies of wastewater treatment and recycling processes.

Atrazine exposure causes mitochondrial toxicity in liver and muscle cell lines.

OBJECTIVE: Chronic exposure to atrazine and other pesticides is reported to cause metabolic disorders, yet information on effects of atrazine on expression of genes relevant to mitochondrial function is largely missing. In the present study, therefore, we investigated the expression of a battery of nuclear- and mitochondrial-encoded genes involved in oxidative phosphorylation (OXPHOS) in human liver (HepG2) and rat muscle (L6) cell lines due to short-term atrazine exposure. MATERIALS AND METHODS: We have determined the EC50 values of atrazine for cytotoxicity and mitochondrial toxicity (mitotoxicity) in terms of adenosine triphosphate (ATP) content in HepG2 and L6 cells. Further, the mRNA expression of nuclear- and mitochondrial-encoded genes was analyzed using quantitative real-time polymerase chain reaction. RESULTS: The EC50 value of atrazine for mitotoxicity in HepG2 and L6 cells was found to be about 0.162 and 0.089 mM, respectively. Mitochondrial toxicity was indicated by reduction in ATP content following atrazine exposure. Atrazine exposure resulted in down-regulation of many OXPHOS subunits expression and affected biogenesis factors' expression. Most prominently, superoxide dismutase (SOD) and sirtuin 3 (SIRT3) expressions were up-regulated in HepG2 cells, whereas SIRT3 expression was alleviated in L6 cells, without significant changes in SOD levels. Mitochondrial transcription factor A (TFAM) and SIRT1 expression were significantly down-regulated in both cell lines. CONCLUSION: Results suggest that TFAM and SIRT1 could be involved in atrazine-induced mitochondrial dysfunction, and further studies can be taken up to understand the mechanism of mitochondrial toxicity. Further study can also be taken up to explore the possibility of target genes as biomarkers of pesticide toxicity.

Proteomic identification of pterostilbene-mediated anticancer activities in HepG2 cells.

In the present study, we attempt to shed light on the underlying molecular mechanism of the anticancer activity of pterostilbene (PTS) in HepG2 cells through the proteomic approach. PTS was found to induce apoptosis by altering the expression of apoptotic genes and the G2/M phase of cell cycle arrest. Further, the 2-DE map showed the expression of 72 differentially regulated proteins in PTS-treated HepG2 cells, of which 8 spots with >2 fold up- or down-regulated level were identified by MALDI-TOF analysis, which has a regulatory role in apoptosis. These findings for the first time offer valuable insights into the mechanism of apoptotis by PTS in HepG2 cells.

Carbonic anhydrase mediated carbon dioxide sequestration: promises, challenges and future prospects.

Anthropogenic activities have substantially increased the level of greenhouse gases (GHGs) in the atmosphere and are contributing significantly to the global warming. Carbon dioxide (CO2 ) is one of the major GHGs which plays a key role in the climate change. Various approaches and methodologies are under investigation to address CO2 capture and sequestration worldwide. Carbonic anhydrase (CA) mediated CO2 sequestration is one of the promising options. Therefore, the present review elaborates recent developments in CA, its immobilization and bioreactor methodologies towards CO2 sequestration using the CA enzyme. The promises and challenges associated with the efficient utilization of CA for CO2 sequestration and scale up from flask to lab-scale bioreactor are critically discussed. Finally, the current review also recommends the possible future needs and directions to utilize CA for CO2 sequestration.

Beta caryophyllene and caryophyllene oxide, isolated from Aegle marmelos, as the potent anti-inflammatory agents against lymphoma and neuroblastoma cells.

Aegle marmelos (Indian Bael) is a tree which belongs to the family of Rutaceae. It holds a prominent position in both Indian medicine and Indian culture. We have screened various fractions of Aegle marmelos extracts for their anticancer properties using in vitro cell models. Gas chromatography-Mass spectrometry (GC-MS) was employed to analyze the biomolecules present in the Aegle marmelos extract. Jurkat and human neuroblastoma (IMR-32) cells were treated with different concentrations of the fractionated Aegle marmelos extracts. Flow cytometric analysis revealed that optimal concentration (50 µg/ml) of beta caryophyllene and caryophyllene oxide fractions of Aegle marmelos extract can induce apoptosis in Jurkat cell line. cDNA expression profiling of pro-apoptotic and anti-apoptotic genes was carried out using real time PCR (RT-PCR). Down-regulation of anti-apoptotic genes (bcl-2, mdm2, cox2 and cmyb) and up-regulation of pro-apoptotic genes (bax, bak1, caspase-8, caspase-9 and ATM) in Jurkat and IMR-32 cells treated with the beta caryophyllene and caryophyllene oxide fractions of Aegle marmelos extract revealed the insights of the downstream apoptotic mechanism. Furthermore, in-silico approach was employed to understand the upstream target involved in the induction of apoptosis by the beta caryophyllene and caryophyllene oxide fractions of Aegle marmelos extract. Herein, we report that beta caryophyllene and caryophyllene oxide isolated from Aegle marmelos can act as potent anti-inflammatory agents and modulators of a newly established therapeutic target, 15-lipoxygenase (15-LOX). Beta caryophyllene and caryophyllene oxide can induce apoptosis in lymphoma and neuroblastoma cells via modulation of 15-LOX (up-stream target) followed by the down-regulation of anti-apoptotic and up-regulation of pro-apoptotic genes.

Role of Carum copticum seeds in modulating chromium-induced toxicity on human bronchial epithelial cells and human peripheral blood lymphocytes.

Carum copticum seeds are well known for ailment of various diseases since ancient times. The present study pertains to investigate modulatory effects of methanolic extract of C. copticum seeds (MCE) against hexavalent chromium induced cytotoxicity, genotoxicity, apoptosis and oxidative stress on human bronchial epithelial cells (BEAS-2B) and isolated human peripheral blood lymphocyte (PBL) in vitro. Treatment of BEAS-2B and PBL with MCE prior to potassium dichromate (K(2)Cr(2)O(7)) treatment exhibited an increase in cell viability and decrease of DNA damage as compared to K(2)Cr(2)O(7) treatment alone, as evaluated by WST-8 and Comet assay respectively. Further, MCE administration 1h prior to graded doses of K(2)Cr(2)O(7) significantly decreased reactive oxygen species (ROS) level, increased the mitochondrial membrane potential, reduced apoptosis and caspase 3 activity. MCE also ameliorated K(2)Cr(2)O(7) induced decrease in superoxide dismutase (SOD), glutathione peroxidase (GPx) antioxidant enzyme levels in BEAS-2B and PBL cells accompanied by reduction in lipid peroxides with maximum effect at 50 µg/ml. Thus, this study provides strong evidence to support the beneficial effect of MCE in preventing Cr(VI) induced toxicity in BEAS-2B and PBL cells.

Bio-mitigation of CO(2), calcite formation and simultaneous biodiesel precursors production using Chlorella sp.

In this study, an attempt was made to use micro-algal system for the production of biodiesel precursors and simultaneous CO(2) mitigation. Chlorella sp. was found to have a higher growth rate as compared to the other algal species tested namely Chlamydomonas sp. and Synnecococcus sp. At different CO(2) concentrations (0.03%, 3%, 10% and 15%), the lipid productivity was 23.0, 20.0 and 27.3mg/L/d respectively. Calcite produced was characterized using FT-IR, SEM and XRD. The FAME in crude biofuel was analyzed by GC-FID that found to contain palmitic acid (C16:0), docosapentaenoic acid (C22:5) and docosahexaenoic acid (C22:6). The calorific value of Chlorella sp. was found to be 29kJ/g which is higher than values reported for fresh water microalgae making it a potential candidate to be used as an alternate fuel.

Antigenotoxic potential of Gymnema montanum leaves on DNA damage in human peripheral blood lymphocytes and HL-60 cell line.

In this study we have evaluated the genoprotective effect of the ethanol extract of Gymnema montanum (GLEt) leaves in human peripheral blood lymphocytes and HL-60 cell line in vitro using the comet assay. DNA damage was induced by treating the cells with H(2)O(2) and methyl methane sulphonate (MMS). GLEt treatment effectively protected the lymphocytes and HL-60 cell line from H(2)O(2)-induced oxidative DNA damage in a dose-dependent manner whereas it was not effective against alkylative DNA damage caused by MMS. The global percent repair efficiency also showed that both pre- and post- GLEt treatment provided effective protection against H(2)O(2) induced DNA damage but not as effective against MMS. At 200 microg ml(-1) level, its repair capacity against H(2)O(2) induced DNA damage was comparable to that of vitamin-C (100 microM). Furthermore, exposure to GLEt reduced the formation of apoptotic cells caused by H(2)O(2), which was demonstrated by the decreased sub-G1-DNA content in cell cycle analysis and apoptotic frequencies of lymphocytes in an annexin-V binding assay. In addition, GLEt was found to have effective peroxide scavenging ability in dose-dependent manner. The protective efficiency of the extract was found to be directly proportional to its total phenolic content. The present study indicates that G. montanum leaves are a significant source of phytochemicals with antigenotoxic and antioxidant activity, and thus has potential therapeutic use.

Exploratory studies on incineration of carbaryl waste.

A hazardous waste stream, generated during the manufacture of carbaryl (1-naphthyl-n-methylcarbamate), an insecticide, was explored for assessing its suitability for incineration. The physico-chemical characteristics such as proximate analysis (moisture, volatile matter, fixed carbon and ash), ultimate analysis (carbon, hydrogen, nitrogen, sulphur and oxygen), calorific value and the heavy metal content of the waste indicated that carbaryl waste was suitable for incineration. The incineration experiments were conducted in a bench-scale (25 mm ID, 450 mm long) quartz reactor at various operating temperatures (800 to 1200 degrees C) at a fixed gas-phase residence time of two seconds. Results of the experiments showed that carbaryl waste could be effectively incinerated at a temperature of 1200 degrees C with a gas-phase residence time of two seconds. The destruction and removal efficiency (DE) at these operating conditions was > 99.99% for carbaryl, which was monitored as a principal organic hazardous compound (POHC). The ash generated at these operating conditions was subjected to the Toxicity Characteristic Leaching Procedure (TCLP) and was found to be non-toxic in nature.

Modulatory effect of distillate of Ocimum sanctum leaf extract (Tulsi) on human lymphocytes against genotoxicants.

OBJECTIVE: To study the modulatory effect of distillate of Ocimum sanctum (traditionally known as Tulsi) leaf extract (DTLE) on genotoxicants. METHODS: In the present investigation, we studied the antigenotoxic and anticlastogenic effect of distillate of Tulsi leaf extract on (i) human polymorphonuclear leukocytes by evaluating the DNA strand break without metabolic activation against mitomycin C (MMC) and hexavalent chromium (Cr+6) and (ii) human peripheral lymphocytes (in vitro) with or without metabolic activation against mitomycin C (MMC), hexavalent chromium (Cr+6) and B[a]P by evaluating chromosomal aberration (CA) and micronucleus assay (MN). Three different doses of DTLE, 50 microL/mL, 100 microL/mL, and 200 microL/mL were selected on the basis of cytotoxicity assay and used for studying DNA strand break, chromosomal aberration and micronucleus emergence. The following positive controls were used for inducing genotoxicity and clastogenicity: MMC (0.29 micromol/L) for DNA strand break, chromosomal aberration and 0.51 micromol/L for micronucleus assay; Potassium dichromate (Cr+6) 600 micromol/L for DNA strand break and 5 micromol/L for chromosomal aberration and micronucleus assay; Benzo[a]pyrene (30 micromol/L) for chromosomal aberration and 40 micromol/L for micronucleus assay. The active ingredients present in the distillate of Tulsi leaf extract were identified by HPLC and LC-MS. RESULTS: Mitomycin C (MMC) and hexavalent chromium (Cr+6) induced statistically significant DNA strand break of respectively 69% and 71% (P<0.001) as revealed by fluorometric analysis of DNA unwinding. Furthermore, the damage could be protected with DTLE (50 microL/mL, 100 microL/mL, and 200 microL/mL) on simultaneous treatment. Chromosomal aberration and micronucleus formation induced by MMC, Cr+6 and B[a]P were significantly protected (P<0.001) by DTLE with and without metabolic activation. CONCLUSION: Distillate of Tulsi leaf extract possesses antioxidants contributed mainly by eugenol, luteolin and apigenin as identified by LC-MS. These active ingredients may have the protective effect against genotoxicants.

The Genotoxicity of Priority Polycyclic Aromatic Hydrocarbons (PAHs) Containing Sludge Samples.

ABSTRACT In this research work we developed in vitro tests utilizing mammalian cell cultures, which can rapidly assess effect of exposure of oily sludge-derived chemicals on human and ecological health. Many of these are hazardous to health and environment due to their toxicity and/or accumulation potential in sediments as well as in organisms. Petroleum refinery and petrochemical industry-derived oily sludges contain toxic polycyclic aromatic hydrocarbons (PAHs), some of which are lipophilic in nature. Risk assessment of environmental samples suffers from inadequate availability of toxicity data, lack of knowledge about behavior of genotoxic substances in complex matrices, paucity of information on synergistic and antagonistic interactions of mixture of components, etc.; the literature describing the behavior of genotoxic substances in complex mixtures is sparse and sometimes contradictory. The present study aims at assessing the genotoxic potential of oily sludges collected from an integrated petroleum refinery and petrochemical industry located in the southwestern part of India and a petrochemical industry located in the western part of India using a battery of genotoxicity assays such as DNA damage/strand break, chromosomal aberration, p(53) protein induction, and apoptosis in CHO-K1 cell culture system. Exposure with different dose levels of sludge extracts (25, 50, 100 muL) in CHO-K1 cells could cause statistically significant level of (P < 0.001) DNA damage, chromosomal aberration, p(53) protein induction, and apoptosis in comparison to negative control treatment groups, and the genotoxicity was attributed to PAHs present in the sludge as identified by GC-MS. This implies that the sludges are genotoxic in nature in mammalian cells tested, and the exposure to these may pose a potential genotoxic risk to human beings.

Anticlastogenic effect of redistilled cow's urine distillate in human peripheral lymphocytes challenged with manganese dioxide and hexavalent chromium.

OBJECTIVE: To study the anticlastogenic effect of redistilled cow's urine distillate (RCUD) in human peripheral lymphocytes (HLC) challenged with manganese dioxide and hexavalent chromium. METHODS: The anticlastogenic activity of redistilled cow's urine distillate was studied in human polymorphonuclear leukocytes (HPNLs) and human peripheral lymphocytes in vitro challenged with manganese dioxide and hexavalent chromium as established genotoxicants and clastogens which could cause induction of DNA strand break, chromosomal aberration and micronucleus. Three different levels of RCUD: 1 microL/mL, 50 microL/mL and 100 microL/mL, were used in the study. RESULTS: Manganese dioxide and hexavalent chromium caused statistically significant DNA strand break, chromosomal aberration and micronucleus formation, which could be protected by redistilled cow's urine distillate. CONCLUSION: The redistilled cow's urine distillate posseses strong antigenotoxic and anticlastogenic properties against HPNLs and HLC treated with Cr+6 and MnO2. This property is mainly due to the antioxidants present in RCUD.