Simultaneous quantitative monitoring of four indicator contaminants of emerging concern (CEC) in different water sources of Central India using SPE/LC-(ESI)MS-MS.

Environmental occurrence of CECs poses a great threat to both aquatic life and human health. The aim of this study was to optimize and validate SPE/LC-(ESI)MS-MS method for simultaneous quantitative monitoring of two sub-classes of CECs (pharmaceuticals and hormones) and to estimate the concentrations of select CECs in environmental water samples. For all the tested analytes, recoveries in laboratory reagent water were greater than 81%. Average percent (relative standard deviation) RSD of the analytes in recovery, repeatability, and reproducibility experiments were ≤ 10%. Determination coefficients (r2) of primidone, diclofenac, testosterone, and progesterone were estimated to be 0.9979, 0.9972, 0.9968, and 0.9962, respectively. Limits of detection (LOD) for primidone, diclofenac, testosterone, and progesterone were 4.63 ng/L, 5.36 ng/L, 0.55 ng/L, and 0.88 ng/L, respectively. Limits of quantification (LOQ) for primidone, diclofenac, testosterone, and progesterone were 14.72 ng/L, 17.06 ng/L, 1.766 ng/L, and 2.813 ng/L, respectively. Average recoveries in environmental water and wastewater samples were greater than 74% and RSD were ≤ 7%. Trace levels (68.33-125.70 ng/L) of primidone were detected in four environmental water samples, whereas diclofenac was not detected in any of the tested sample. Trace levels of progesterone were observed in two environmental samples (16.64 -203.73 ng/L), whereas testosterone was detected in STP inlet sample (178.16 ng/L).

In vitro cytotoxicity of Gymnema montanum in human leukaemia HL-60 cells; induction of apoptosis by mitochondrial membrane potential collapse.

OBJECTIVES: Gymnema montanum Hook, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. Here, we report anti-cancer effects and molecular mechanisms of ethanolic extract of G. montanum (GLEt) on human leukaemia HL-60 cells, compared to peripheral blood mononuclear cells. MATERIALS AND METHODS: HL-60 cells were treated with different concentrations of GLEt (10-50 µg/ml) and cytotoxicity was assessed by MTT assay. Levels of lipid peroxidation, antioxidants, mitochondrial membrane potential and caspase-3 were measured. Further, apoptosis was studied using annexin-V staining and the cell cycle was analyzed by flow cytometry. RESULTS: GLEt had a potent cytotoxic effect on HL-60 cells (IC50 -20 µg/ml), yet was not toxic to normal peripheral blood mononuclear cells. Exposure of HL-60 cells to GLEt led to elevated levels of malonaldehyde formation, but to reduced glutathione, superoxide dismutase, catalase and glutathione peroxidase activities (P < 0.05). Induction of apoptosis was confirmed by observing annexin-V positive cells, associated with loss of mitochondrial membrane potential. Cell cycle arrest at G0/G1 was observed in GLEt-treated HL-60 cells, indicating its potential at inducing their apoptosis. CONCLUSIONS: Findings of the present study suggest that G. montanum induced apoptosis in the human leukaemic cancer cells, mediated by collapse of mitochondrial membrane potential, generation of reactive oxygen species and depletion of intracellular antioxidant potential.

Effect of thymol on peripheral blood mononuclear cell PBMC and acute promyelotic cancer cell line HL-60.

Thymol, a naturally occurring phenolic compound, has been known for its antioxidant, anti microbial, and anti inflammatory activity. Thymol has also been reported as anti-cancer agent, but its anti-cancer mechanism has not yet been fully elucidated. Thus, we aimed to investigate anticancer activity of thymol on HL-60 (acute promyelotic leukemia) cells. In our study, thymol demonstrated dose dependent cytotoxic effects on HL-60 cells after 24h of exposure. However, thymol did not show any cytotoxic effect in normal human PBMC. The cytotoxic effect of thymol on HL-60 cells appears to be associated with induction of cell cycle arrest at sub G0/G1 phase, and apoptotic cell death based on genomic DNA fragmentation pattern. Thymol also showed significant increase in production of reactive oxygen species (ROS) activity, increase in mitochondrial H(2)O(2) production and depolarization of mitochondrial membrane potential. On performing Western Blot analysis, thymol showed increase in Bax protein level with a concomitant decrease in Bcl2 protein expression in a dose dependent manner. Our study also showed activation of caspase -9, -8 and -3 and concomitant PARP cleavage, which is the hallmark of caspase-dependent apoptosis. Moreover, to rule out the involvement of other mechanisms in apoptosis induction by thymol, we also studied its effect on apoptosis inducing factor (AIF). Thymol induced AIF translocation from mitochondria to cytosol and to nucleus, thus indicating its ability to induce caspase independent apoptosis. We conclude that, thymol-induced apoptosis in HL-60 cells involves both caspase dependent and caspase independent pathways.

Genotoxicity of sludges, wastewater and effluents from three different industries.

Many surface waters in Europe, Asia and South America have been reported to be contaminated with genotoxic substances. Therefore, it is important to establish strategies for identification of the most critical sources. In this study, we used a battery of four genotoxicity assays namely chromosomal aberration, DNA strand break, DNA laddering and P53 accumulation tests in mononuclear blood cells. Before cleaning of wastewater high levels of genotoxic contamination could be observed. For instance, we observed an increase in chromosomal aberrations from 2.6 +/- 1.1 (aberrant cells in %; control), to 33.6 +/- 6.6 in a petrochemical plant, 29.4 +/- 3.3 in a petroleum refinery and 14.4 +/- 1.8 in a coke plant of steel industry. A good correlation between the four assays was found. The most sensitive and reproducible results were obtained with the chromosomal aberration assay. Interestingly, clear differences in the efficiency of wastewater cleaning in three different treatment plants were observed. The first and second treatment plants in petrochemical industry and coke plant of steel industry completely eliminated genotoxicity of the wastewater. However, the third plant in petroleum refinery could achieve a reduction in genotoxicity but significant genotoxic contaminations were still present. In conclusion, our battery of genotoxicity tests allows the identification of critical sources contributing to contamination of surface waters.

Decolorisation, biodegradation and detoxification of benzidine based azo dye.

The present study deals with the decolorisation, biodegradation and detoxification of Direct Black-38, a benzidine based azo dye, by a mixed microbial culture isolated from an aerobic bioreactor treating textile wastewater. The studies revealed a biotransformation of Direct Black-38 into benzidine and 4-aminobiphenyl followed by complete decolorisation and biodegradation of these toxic intermediates. From cytotoxicity studies, it was concluded that detoxification of the dye took place after degradation of the toxic intermediates by the culture.

Temperature-induced stress on growth and expression of hsp in freshwater alga Scenedesmus quadricauda.

OBJECTIVE: To investigate the impact of various levels of sublethal temperature (26 degrees C, 31 degrees C, 33 degrees C, 36 degrees C, and 39 degrees C) on growth and heat shock protein (hsp) expression in freshwater green alga Scenedesmus quadricauda. METHODS: Impact of selected levels of temperature on growth rate (based on optical density), population count, chlorophyll-a and biomass of the alga was evaluated in artificial growth medium for 19 days. To determine the induction of hsp in the alga, it was exposed to selected temperature levels for 3 h and further kept for 6 h at culturing condition at 26 degrees C. Induction of hsp was confirmed by immuno-detection followed by SDS-polyacrylamide gel electrophoresis. RESULTS: The selected growth parameters such as growth rate, population count, chlorophyll-a and biomass were reduced significantly (P < 0.001) at 39 degrees C. However, hsp 70 expression was observed only at 39 degrees C. CONCLUSION: Temperature up to 36 degrees C may be considered as the limit of safe exposure for thermal stress for the alga Scenedesmus quadricauda.

DNA damage caused by pesticide-contaminated soil.

OBJECTIVE: To determine the DNA damaging potential and the genotoxicity of individual compounds in pesticide contaminated soil. METHODS: In the present study, DNA damaging potential of pesticide-contaminated soil and the genotoxicity of individual compounds present in the soil were assessed using fluorimetric analysis of DNA unwinding assay. RESULTS: The contaminated soil sample showed 79% (P < 0.001) of DNA strand break, whereas technical grade of major carbaryl and alpha-naphthol constituents of the contaminated soil showed 64% (P < 0.01) and 60% (P < 0.02) damage respectively. CONCLUSION: Our results indicate that the toxicity caused by contaminated soil is mainly due to carbaryl and alpha-napthol, which are the major constituents of the soil sample analyzed by GC-MS.