Evaluation of microbial diversity of three recreational water bodies using 16S rRNA metagenomic approach.

Surface water plays a significant role in world development by promoting economic growth and health benefits to humans and animals whose lives depend on good water quality in the ecosystem. Thus, this study investigated the differences in physical and chemical properties of surface water from two lakes (Lakes Jackson and Talquin) and a pond (Pedrick Pond). Also, the influence of environmental factors on the microbial communities that live within the water environment was examined. Genomic DNA was extracted from the water samples collected and 16S rRNA sequencing method was employed to characterize the microbial community compositions across the three locations. The results obtained suggest that the water sources met the recommended recreational water quality criteria standard for clean water. Overall, Proteobacteria, Actinobacteria, Cyanobacteria, Bacteroidetes were the main bacterial phyla present in the communities, while Archaea was mainly dominated by Euryachaeota. Pressure, conductivity, temperature, dissolved oxygen (DO), and pH accounted for 74.2% of the variation in the distribution of the microbial community in the three locations (P < 0.05), while 58.2% of the variation in the microbial community distribution was accounted for by pressure and conductivity. The high temperature observed in the Pedrick Pond correlated with the distribution of genera hgcl_clades and Legionella. While in Lake Talquin, water conductivity was significantly associated with the abundance of Cyanobium_PCC_6307, Sediminibacterium, and Conexibacter. The results from this study indicate that the microbial communities in the two lakes are different from the pond and all the environmental variables accounted for a significant portion of the total variation, but pressure, conductivity, and temperature are more important factors due to significant correlation with the distribution of the microbial communities.

Diallyl disulfide attenuation effect on transcriptome in rat liver cells against cadmium chloride toxicity.

In this report, liver cells were treated with cadmium chloride (CdCl2 ) and diallyl disulfide (DADS), a major compound from garlic to attenuate the toxic effect of Cd on transcriptome. The viability of Cd treated cells was reduced to 19.9% ± 2.4% in comparison to the untreated cells, whereas the viability of DADS pretreated cells was increased to 48.6% ± 2%. The attenuation effect of DADS was studied at shorter period (6 hours). Transcriptome analysis of CdCl2 alone treated cells resulted in 2119 and 982 (up and down) regulated genes (≥ 2 or ≤ 2-fold), whereas pretreated cells with DADS resulted in 2597 and 1784 genes. These genes were known to function in many important biological processes. Affymetrix array analysis was validated by the pathway specific PCR array that exhibited the same trend of expression. The current study clearly shows the DADS attenuation effect on transcriptome in CdCl2 -treated rat liver cells.

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.

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.

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.

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.

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.

Mechanism of DNA damage by cadmium and interplay of antioxidant enzymes and agents.

Cadmium is an environmental toxicant, which causes cancer in different organs. It was found that it damages DNA in the various tissues and cultured cell lines. To investigate the mechanism of DNA damage, we have studied the effect of cadmium-induced DNA damage in plasmid pBR322 DNA, and the possible ameliorative effects of antioxidative agents under in vitro conditions. It was observed that cadmium alone did not cause DNA damage. However, it caused DNA damage in the presence of hydrogen peroxide, in a dose dependent manner, because of production of hydroxyl radicals. Findings from this study show the conversion of covalently closed circular double-stranded pBR 322 DNA to the open circular and linear forms of DNA when treated with 10 muM cadmium and various concentrations of H(2)O(2). The conversion was due to nicking in DNA strands. The observed rate of DNA strand breakage was dependent on H(2)O(2) concentration, temperature, and time. Metallothionein I failed to prevent cadmium-induced DNA nicking in the presence of H(2)O(2). Of the two antioxidant enzymes (catalase and superoxide dismutase) studied, only catalase conferred significant (50-60%) protection. EDTA and DMSO exhibited protection similar to catalase, while mannitol showed only about 20% protection against DNA damage. Ethyl alcohol failed to ameliorate cadmium-induced DNA strands break. From this study, it is plausible to infer that cadmium in the presence of hydrogen peroxide causes DNA damage probably by the formation of hydroxyl ions. These results may indicate that cadmium in vivo could play a major role in the DNA damage induced by oxidative stress.

Effect of cadmium-induced oxidative stress on antioxidative enzymes in mitochondria and cytoplasm of CRL-1439 rat liver cells.

Cadmium affects human health through occupational and environmental exposure. In this report, we present the response of mitochondrial and cytoplasmic antioxidant enzymes of CRL-1439 cells exposed to different concentrations (0-150 microM) of CdCl2 for 24 h at 37 degrees C. Exposure of liver cells to 50 microM CdCl2 increased mitochondrial catalase and glutathione reductase (GR) activities more than the cytoplasmic enzymes. Although the mitochondrial selenium-dependent glutathione peroxidase (Se-GPx) showed less enzymatic activity than the cytoplasmic enzyme, the mitochondrial selenium-independent glutathione peroxidase (non-Se-GPx) showed a slight increase in activity over its cytoplasmic counterpart compared to untreated controls. With 100 microM CdCl2, catalase maintained an increase in specific activity in mitochondria over the cytoplasmic enzyme compared to the controls. The level of GR was higher in the cytoplasm than in the mitochondria. However, the activity of Se-GPx and non-Se-GPx decreased slightly in the mitochondria compared to their cytoplasmic counterparts. Exposure of cells to 150 microM CdCl2 decreased all antioxidant enzyme activities compared to the 100 microM CdCl2-treated samples due to toxic effect. Each antioxidant enzyme exhibited its own pattern of activation or inhibition upon exposure to different concentrations of cadmium, with more oxidative stress observed in the mitochondria.