Chemical-toxicological insights and process comparison for estrogenic activity mitigation in municipal wastewater treatment plants.

Efforts in water ecosystem conservation require an understanding of causative factors and removal efficacies associated with mixture toxicity during wastewater treatment. This study conducts a comprehensive investigation into the interplay between wastewater estrogenic activity and 30 estrogen-like endocrine disrupting chemicals (EEDCs) across 12 municipal wastewater treatment plants (WWTPs) spanning four seasons in China. Results reveal substantial estrogenic activity in all WWTPs and potential endocrine-disrupting risks in over 37.5 % of final effluent samples, with heightened effects during colder seasons. While phthalates are the predominant EEDCs (concentrations ranging from 86.39 %) for both estrogenic activity and major EEDCs (phthalates and estrogens), with the secondary and tertiary treatment segments contributing 88.59 ± 8.12 % and 11.41 ± 8.12 %, respectively. Among various secondary treatment processes, the anaerobic/anoxic/oxic-membrane bioreactor (A/A/O-MBR) excels in removing both estrogenic activity and EEDCs. In tertiary treatment, removal efficiencies increase with the inclusion of components involving physical, chemical, and biological removal principles. Furthermore, correlation and multiple liner regression analysis establish a significant (p < 0.05) positive association between solid retention time (SRT) and removal efficiencies of estrogenic activity and EEDCs within WWTPs. This study provides valuable insights from the perspective of prioritizing key pollutants, the necessity of integrating more efficient secondary and tertiary treatment processes, along with adjustments to operational parameters like SRT, to mitigate estrogenic activity in municipal WWTPs. This contribution aids in managing endocrine-disrupting risks in wastewater as part of ecological conservation efforts.

Characterization of cadmium biosorption by inactive biomass of two cadmium-tolerant endophytic bacteria Microbacterium sp. D2-2 and Bacillus sp. C9-3.

In this study, two cadmium-tolerant endophytic bacteria (Microbacterium sp. D2-2 and Bacillus sp. C9-3) were employed as biosorbents to remove Cd(II) from aqueous solutions. The influence of initial pH, initial Cd(II) concentration, adsorbent biomass, temperature and contact time on Cd(II) removal were investigated. Results showed that the Langmuir isotherms were found to best fit the equilibrium data, and the maximum biosorption capacities were found to be 222.22 and 163.93 mg/g at a solution pH of 5.0 for Microbacterium sp. D2-2 and Bacillus sp. C9-3, respectively. The biosorption kinetics followed well pseudo-second-order kinetics. Fourier transform infrared spectroscopic analysis suggested that the hydroxyl, carboxyl, carbonyl and amino groups on Microbacterium sp. D2-2 and Bacillus sp. C9-3 biomass were the main binding sites for Cd(II). The results presented in this study showed that Microbacterium sp. D2-2 and Bacillus sp. C9-3 are potential and promising adsorbents for the effective removal of Cd(II) from aqueous solutions.

Metagenomic analysis revealed the sulfur- and iron- oxidation capabilities of heterotrophic denitrifying sludge.

Heterotrophic denitrification is widely applied in wastewater treatment processes to remove nitrate. However, the ability of the heterotrophic denitrifying sludge to use inorganic matter as electron donors to perform autotrophic denitrification has rarely been investigated. In this study, we enriched heterotrophic denitrifying sludge and demonstrated its sulfur- and iron- oxidizing abilities and denitrification performance with batch experiments. Based on high-throughput sequencing of 16S rRNA genes, high diversity and abundance of sulfur-oxidizing bacteria (SOB) (e.g., Sulfuritalea, Thiobacillus, and Thiothrix) and iron (II)-oxidizing bacteria (FeOB) (e.g., Azospira and Thiobacillus) were observed. Metagenomic sequencing and genome binning results further suggested that the SOB in the heterotrophic denitrifying sludge were mainly Alphaproteobacteria and Betaproteobacteria instead of Gammaproteobacteria and Epsilonproteobacteria. The similarities of potential iron-oxidizing genes with known sequences were very low (32-51%), indicating potentially novel FeOB species in this system. The findings of this study suggested that the heterotrophic denitrifying sludge harbors diverse mixotrophic denitrifying bacterial species, and based on this finding, we proposed that organic carbon and inorganic electron donors (e.g., sulfur, thiosulfate, and iron) could be jointly used in engineering practices according to the quality and quantity of wastewater to balance the cost and efficiency of the denitrification process.

Structural and functional alterations of gut microbiome in mice induced by chronic cadmium exposure.

Mammalian gut microbiome is readily affected by acute or subchronic cadmium (Cd) intoxication, but it susceptibility following chronic Cd exposure at environmentally-relevant levels remains unknown. This study comprehensively assessed the effects of Cd exposure at doses of 10 and 50 ppm in drinking water for 20 weeks on gut microbiome in mice. Results showed that the Cd exposure induced alterations in gut morphology with potentially increased gut permeability and inflammation. These changes were accompanied by marked perturbation of gut microbiota characterized by significantly decreased gut microbial richness and lowered abundance of short chain fatty acid (SCFA)-producing bacteria, resulting in reduced SCFAs production in the gut. Moreover, the Cd exposure caused substantial metabolic functional changes of the gut microbiome, with significant inhibitions on gene pathways associated with metabolism of amino acid, carbohydrate, and energy, as well as promotions on metabolic pathways such as glutathione metabolism and aminobenzoate degradation. Our findings provide new insights into the hazards assessment of environmental Cd exposure towards gut microbiome.

Effects of chronic cadmium exposure at food limitation-relevant levels on energy metabolism in mice.

Cadmium (Cd) exposure has been implicated in the perturbation of energy metabolism and the development of cardiometabolic disease, but disease predisposition from chronic low-dose Cd exposure remains unclear. This study employed a mouse model to investigate the toxic effects of chronic Cd exposure at food limitation-relevant levels on energy metabolism and the associated liver and gut microbiome functions. Results showed that the Cd exposure induced the perturbation of energy metabolism in mice, evidenced by the alteration of various metabolites associated with the phosphorogen (adenosine triphosphate-creatine phosphate) system, tricarboxylic acid cycle, and lipid metabolism, as well as the increase of the cardiometabolic risk factor, triglyceride. Moreover, both liver and gut microbiome underwent marked structural/histological and functional alterations, prone to the onset of cardiometabolic disease following the Cd exposure. Certain hepatic transcription factors and gut microbes, specifically PPARα, SREBP1c, HNF4A and the Clostridiales_vadinBB60_group, were identified to be highly correlated with altered urinary metabolites, revealing potential toxicological interactions between the liver and gut microbiome, and energy metabolism. Our findings provide new insights into the progression of metabolic diseases induced by Cd exposure. We also propose a stricter Cd limitation in future food safety standards.

Inhibition of Mitochondrial Fatty Acid Oxidation Contributes to Development of Nonalcoholic Fatty Liver Disease Induced by Environmental Cadmium Exposure.

Cadmium (Cd) is one of the most prevalent toxic metal pollutants widely distributed in water and soil environments. Epidemiological studies have shown that exposure to Cd is implicated in the prevalence of nonalcoholic fatty liver disease (NAFLD) in middle-aged human population, but biological evidence is lacking and its toxicological mechanism remains unclear for the disease predisposition from environmental Cd exposure. In this study, we established a chronic Cd-exposure mouse model mimicking the liver Cd deposition in middle-aged human population to determine whether the environmental Cd exposure can induce NAFLD. Results showed that hepatic Cd burden at levels of 0.95 and 6.04 µg/g wet weight resulting from 20-week Cd exposure at different doses induced NAFLD and nonalcoholic steatohepatitis-like phenotypes in mice, respectively. The Cd exposure caused marked hepatic mitochondrial dysfunction and fatty acid oxidation deficiency, along with significant suppression of sirtuin 1 (SIRT1) signaling pathway in the liver. In vitro study confirmed that Cd evidently inhibited the mitochondrial fatty acid oxidation in hepatocytes and that SIRT1 signaling was potentially involved in the process. Our findings suggest that exposure to environmental Cd is a tangible risk factor for NAFLD, and the induced public health risks deserve greater attention.

Comparative analysis of toxicity reduction of wastewater in twelve industrial park wastewater treatment plants based on battery of toxicity assays.

Wastewater treatment plants (WWTPs) in industrial parks provide centralized treatment for industrial and domestic wastewater. However, the information on toxicity reduction of wastewater and its correlation with treatment process in industrial park is limited. This study compared the toxicity reduction of wastewater in 12 industrial park WWTPs based on battery of toxicity assays. Nine toxic endpoints involving microorganism, phytoplankton, zooplankton, plant and human cell lines were applied. All the influents of WWTPs induced high toxicities, which were significantly reduced after the treatments from 7 of the studied WWTPs. However, the effluents of five WWTPs induced higher toxicity in one or more toxic endpoints compared to the influents. This study also found that most of anaerobic-anoxic-oxic (A2/O)-based processes had good removal efficiency of wastewater toxicity, while the sequencing batch reactor (SBR)-based processes had the lowest removal efficiency. Moreover, low correlation coefficients were obtained among all toxic endpoints, indicating that battery of toxicity assays was necessary to completely characterize the toxicity and risk of wastewater in industrial parks. This study shed new lights to the toxicity reduction of wastewater and its correlation with treatment process, which is very useful for the design, management and operation of WWTPs in industrial parks.

Comprehensive insights into the key components of bacterial assemblages in pharmaceutical wastewater treatment plants.

Due to complexity and variety of pharmaceutical wastewater composition, little is known as for functionally important microflora of pharmaceutical wastewater treatment plants (pWWTPs). We compared bacterial composition and diversity of pWWTPs (27 sludge samples collected from 12 full-scale pWWTPs) with those of other industrial (iWWTPs) (27 samples) and municipal wastewater treatment plants (mWWTPs) (27 samples) through meta-analysis based on 16S rRNA gene amplicon sequencing, and identified putatively important organisms and their ecological correlations. Non-metric multidimensional scaling indicated that the pWWTPs, iWWTPs and mWWTPs showed distinctive differences in bacterial community composition (P < 1e-04), and the pWWTPs had significantly lower bacterial diversity than the mWWTPs (P < 1e-06). Thermotogae and Synergistetes phyla only strictly dominated in the pWWTPs, and 26, 30 and 6 specific genera were identified in the pWWTPs, mWWTPs and iWWTPs, respectively. Totally, 15 and 1300 OTUs were identified as core and occasional groups, representing 23.2% and 66.2% of the total read abundance of the pWWTPs, respectively. Permutational multivariate analysis of variance revealed that the bacterial components were clearly clustered corresponding to the types of pharmaceutical wastewater, and a total of 129 local specific OTUs were identified in the pWWTPs, among which anticancer antibiotics pWWTPs had the highest number of specific OTUs (40 ones). Co-occurrence network revealed that the species dominating in the same type of pWWTPs tended to co-occur much more frequently than theoretical random expectation. The results may extend our knowledge regarding the ecological status and correlation of the key microflora in pWWTPs.

Rapid and complete dehalogenation of halonitromethanes in simulated gastrointestinal tract and its influence on toxicity.

Halonitromethanes (HNMs) as one typical class of nitrogenous disinfection byproducts in drinking water and wastewater are receiving attentions due to their high toxicity. This study applied a simulator of the human gastrointestinal tract to determine the dehalogenation processes of trichloronitromethane, bromonitromethane and bromochloronitromethane for the first time. Influence of digestion process of HNMs on gut microbiota and hepatotoxicity was further analyzed. Results showed that the three HNMs were rapidly and completely dehalogenated in the gastrointestinal tract, especially in the stomach (2 h retention Time) and small intestine (4 h retention Time). Mucin, cysteine, pancreatin and bile salts in the digestive juice played major roles in the dehalogenation process. HNMs and their dehalogenation products in the resulting fluids of stomach induced the highest toxicity followed by those in intestine and colon, exhibiting dose-dependent effects. Although most HNMs were degraded in the stomach and small intestine, residual HNMs entered into colon changed the microbial community. Abundance of several genera, such as Bacteroides, Lachnospiraceae_unassigned and Lactobacillus had high correlation with exposure concentration of HNMs. This study sheds new light on dehalogenation and toxic processes of HNMs by oral exposure, which provides basic data for their human health risk assessment.

Cytotoxicity and Efflux Pump Inhibition Induced by Molybdenum Disulfide and Boron Nitride Nanomaterials with Sheetlike Structure.

Sheetlike molybdenum disulfide (MoS2) and boron nitride (BN) nanomaterials have attracted attention in the past few years due to their unique material properties. However, information on adverse effects and their underlying mechanisms for sheetlike MoS2 and BN nanomaterials is rare. In this study, cytotoxicities of sheetlike MoS2 and BN nanomaterials on human hepatoma HepG2 cells were systematically investigated at different toxic end points. Results showed that MoS2 and BN nanomaterials decreased cell viability at 30 µg/mL and induced adverse effects on intracellular ROS generation (≥2 µg/mL), mitochondrial depolarization (≥4 µg/mL), and membrane integrity (≥8 µg/mL for MoS2 and ≥2 µg/mL for BN). Furthermore, this study first found that low exposure concentrations (0.2-2 µg/mL) of MoS2 and BN nanomaterials could increase plasma membrane fluidity and inhibit transmembrane ATP binding cassette (ABC) efflux transporter activity, which make both nanomaterials act as a chemosensitizer (increasing arsenic toxicity). Damage to plasma membrane and release of soluble Mo or B species might be two reasons that both nanomaterials inhibit efflux pump activities. This study provides a systematic understanding of the cytotoxicity of sheetlike MoS2 and BN nanomaterials at different exposure levels, which is important for their safe use.

High Levels of Antibiotic Resistance Genes and Their Correlations with Bacterial Community and Mobile Genetic Elements in Pharmaceutical Wastewater Treatment Bioreactors.

To understand the diversity and abundance of antibiotic resistance genes (ARGs) in pharmaceutical wastewater treatment bioreactors, the ARGs in sludge from two full-scale pharmaceutical wastewater treatment plants (PWWTPs) were investigated and compared with sludge samples from three sewage treatment plants (STPs) using metagenomic approach. The results showed that the ARG abundances in PWWTP sludge ranged from 54.7 to 585.0 ppm, which were higher than those in STP sludge (27.2 to 86.4 ppm). Moreover, the diversity of ARGs in PWWTP aerobic sludge (153 subtypes) was higher than that in STP aerobic sludge (118 subtypes). In addition, it was found that the profiles of ARGs in PWWTP aerobic sludge were similar to those in STP aerobic sludge but different from those in PWWTP anaerobic sludge, suggesting that dissolve oxygen (DO) could be one of the important factors affecting the profiles of ARGs. In PWWTP aerobic sludge, aminoglycoside, sulfonamide and multidrug resistance genes were frequently detected. While, tetracycline, macrolide-lincosamide-streptogramin and polypeptide resistance genes were abundantly present in PWWTP anaerobic sludge. Furthermore, we investigated the microbial community and the correlation between microbial community and ARGs in PWWTP sludge. And, significant correlations between ARG types and seven bacterial genera were found. In addition, the mobile genetic elements (MGEs) were also examined and correlations between the ARGs and MGEs in PWWTP sludge were observed. Collectively, our results suggested that the microbial community and MGEs, which could be affected by DO, might be the main factors shaping the profiles of ARGs in PWWTP sludge.

Arsenic Metabolism and Toxicity Influenced by Ferric Iron in Simulated Gastrointestinal Tract and the Roles of Gut Microbiota.

Iron (Fe) is a common trace element in drinking water. However, little is known about how environmental concentrations of Fe affect the metabolism and toxicity of arsenic (As) in drinking water. In this study, influence of Fe at drinking water-related concentrations (0.1, 0.3, and 3 mg Fe (total)/L) on As metabolism and toxicity, and the roles of gut microbiota during this process were investigated by using in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME). Results showed that Fe had ability to decrease bioaccessible As by coflocculation in small intestine. 0.1 and 0.3 mg/L Fe significantly increased As methylation in simulated transverse and descending colon. Gut microbiota played an important role in alteration of As species, and Fe could affect As metabolism by changing the gut microbiota. Bacteroides, Clostridium, Alistipes, and Bilophila had As resistance and potential ability to methylate As. Cytotoxicity assays of effluents from simulated colons showed that the low levels of Fe decreased As toxicity on human hepatoma cell line HepG2, which might be due to the increase of methylated As. When assessing the health risk of As in drinking water, the residual Fe should be considered.

Comparison of Cytotoxicity and Inhibition of Membrane ABC Transporters Induced by MWCNTs with Different Length and Functional Groups.

Experimental studies indicate that multiwalled carbon nanotubes (MWCNTs) have the potential to induce cytotoxicity. However, the reports are often inconsistent and even contradictory. Additionally, adverse effects of MWCNTs at low concentration are not well understood. In this study, we systemically compared adverse effects of six MWCNTs including pristine MWCNTs, hydroxyl-MWCNTs and carboxyl-MWCNTs of two different lengths (0.5-2 µm and 10-30 µm) on human hepatoma cell line HepG2. Results showed that MWCNTs induced cytotoxicity by increasing reactive oxygen species (ROS) generation and damaging cell function. Pristine short MWCNTs induced higher cytotoxicity than pristine long MWCNTs. Functionalization increased cytotoxicity of long MWCNTs, but reduced cytotoxicity of short MWCNTs. Further, our results indicated that the six MWCNTs, at nontoxic concentration, might not be environmentally safe as they inhibited ABC transporters' efflux capabilities. This inhibition was observed even at very low concentrations, which were 40-1000 times lower than their effective concentrations on cytotoxicity. The inhibition of ABC transporters significantly increased cytotoxicity of arsenic, a known substrate of ABC transporters, indicating a chemosensitizing effect of MWCNTs. Plasma membrane damage was likely the mechanism by which the six MWCNTs inhibited ABC transporter activity. This study provides insight into risk assessments of low levels of MWCNTs in the environment.

Low levels of graphene and graphene oxide inhibit cellular xenobiotic defense system mediated by efflux transporters.

Low levels of graphene and graphene oxide (GO) are considered to be environmentally safe. In this study, we analyzed the potential effects of graphene and GO at relatively low concentrations on cellular xenobiotic defense system mediated by efflux transporters. The results showed that graphene (<0.5 µg/mL) and GO (<20 µg/mL) did not decrease cell viability, generate reactive oxygen species, or disrupt mitochondrial function. However, graphene and GO at the nontoxic concentrations could increase calcein-AM (CAM, an indicator of membrane ATP-binding cassette (ABC) transporter) activity) accumulation, indicating inhibition of ABC transporters' efflux capabilities. This inhibition was observed even at 0.005 µg/mL graphene and 0.05 µg/mL GO, which are 100 times and 400 times lower than their lowest toxic concentration from cytotoxicity experiments, respectively. The inhibition of ABC transporters significantly increased the toxicity of paraquat and arsenic, known substrates of ABC transporters. The inhibition of ABC transporters was found to be based on graphene and GO damaging the plasma membrane structure and fluidity, thus altering functions of transmembrane ABC transporters. This study demonstrates that low levels of graphene and GO are not environmentally safe since they can significantly make cell more susceptible to other xenobiotics, and this chemosensitizing activity should be considered in the risk assessment of graphene and GO.

Metagenomic profiles and antibiotic resistance genes in gut microbiota of mice exposed to arsenic and iron.

Iron (Fe) has been widely applied to treat arsenic (As)-contaminated water, and Fe could influence bioavailability and toxicity of As. However, little is known about the impact of As and/or Fe on gut microbiota, which plays important roles in host health. In this study, high-throughput sequencing and quantitative real time PCR were applied to analyze the impact of As and Fe on mouse gut microbiota. Co-exposure of As and Fe mitigated effects on microbial community to a certain extent. Correlation analysis showed the shifts in gut microbiota caused by As and/or Fe exposure might be important reason of changes in metabolic profiles of mouse. For antibiotic resistance genes (ARGs), co-exposure of As and Fe increased types and abundance of ARGs. But for high abundance ARGs, such as tetQ, tetO and tetM, co-exposure of As and Fe mitigated effects on their abundances compared to exposure to As and Fe alone. No obvious relationship between ARGs and mobile genetic elements were found. The changes in ARGs caused by metal exposure might be due to the alteration of gut microbial diversity. Our results show that changes of gut microbial community caused by As and/or Fe can influence host metabolisms and abundances of ARGs in gut, indicating that changes of gut microbiota should be considered during the risk assessment of As and/or Fe.

Metagenomic analysis reveals significant changes of microbial compositions and protective functions during drinking water treatment.

The metagenomic approach was applied to characterize variations of microbial structure and functions in raw (RW) and treated water (TW) in a drinking water treatment plant (DWTP) at Pearl River Delta, China. Microbial structure was significantly influenced by the treatment processes, shifting from Gammaproteobacteria and Betaproteobacteria in RW to Alphaproteobacteria in TW. Further functional analysis indicated the basic metabolic functions of microorganisms in TW did not vary considerably. However, protective functions, i.e. glutathione synthesis genes in 'oxidative stress' and 'detoxification' subsystems, significantly increased, revealing the surviving bacteria may have higher chlorine resistance. Similar results were also found in glutathione metabolism pathway, which identified the major reaction for glutathione synthesis and supported more genes for glutathione metabolism existed in TW. This metagenomic study largely enhanced our knowledge about the influences of treatment processes, especially chlorination, on bacterial community structure and protective functions (e.g. glutathione metabolism) in ecosystems of DWTPs.

Promotion of melanoma cell invasion and tumor metastasis by microcystin-LR via phosphatidylinositol 3-kinase/AKT pathway.

Recently, we have indicated that microcystin-LR, a cyanobacterial toxin produced in eutrophic lakes or reservoirs, can increase invasive ability of melanoma MDA-MB-435 cells; however, the stimulatory effect needs identification by in vivo experiment and the related molecular mechanism is poorly understood. In this study, in vitro and in vivo experiments were conducted to investigate the effect of microcystin-LR on invasion and metastasis of human melanoma cells, and the underlying molecular mechanism was also explored. MDA-MB-435 xenograft model assay showed that oral administration of nude mice with microcystin-LR at 0.001-0.1 mg/kg/d posed no significant effect on tumor weight. Histological examination demonstrated that microcystin-LR could promote lung metastasis, which is confirmed by Matrigel chamber assay suggesting that microcystin-LR treatment at 25 nM can increase the invasiveness of MDA-MB-435 cells. In vitro and in vivo experiments consistently showed that microcystin-LR exposure increased mRNA and protein levels of matrix metalloproteinases (MMP-2/-9) by activating phosphatidylinositol 3-kinase (PI3-K)/AKT. Additionally, microcystin-LR treatment at low doses (≤25 nM) decreased lipid phosphatase PTEN expression, and the microcystin-induced invasiveness enhancement and MMP-2/-9 overexpression were reversed by the PI3-K/AKT chemical inhibitor LY294002 and AKT siRNA, indicating that microcystin-LR promotes invasion and metastasis of MDA-MB-435 cells via the PI3-K/AKT pathway.

Metagenomic analysis of cadmium and copper resistance genes in activated sludge of a tannery wastewater treatment plant.

In order to comprehensively characterize the copper and cadmium resistance in activated sludge of a tannery wastewater treatment plant, a resistance protein database of the two heavy metals was manually created by retrieving annotated sequences and related information from the public databases and published literatures. The metagenomic DNA was extracted from the activated sludge for Illumina high-throughput sequencing, and the obtained 11,973,394 clean reads (1.61 Gb) were compared against the established databases using BLAST tool. Annotations of the BLAST hits showed that 222 reads (0.019 per thousand) and 197 reads (0.016 per thousand) were identified as copper and cadmium resistance genes, respectively. Among the identified cadmium resistance genes, czcA encoding cobalt-zinc-cadmium resistance protein had the highest abundance (83 reads, 0.0069 per thousand), which was further confirmed by annotation of the open reading frames predicted with the assembly contigs. Among the copper resistance genes, copA (66 reads, 0.0055 per thousand) was most abundant, followed by copK and cusR. Alignment against the Clusters of Orthologous Groups (COG) database also suggested that 87.26% of the matched reads were grouped in COG0474 (cation transport ATPase). This study may be practically helpful for exploring various functional genes in the environment using high-throughput sequencing and bioinformatics methods.

Microcystin-LR promotes melanoma cell invasion and enhances matrix metalloproteinase-2/-9 expression mediated by NF-κB activation.

This study aimed to explore the molecular mechanisms behind the stimulation effects of microcystin-LR (a well-known cyanobacterial toxin produced in eutrophic lakes or reservoirs) on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Boyden chamber assay showed that microcystin-LR exposure (>12.5 nM) evidently enhanced the invasion ability of the melanoma cells (MDA-MB-435). Tumor Metastasis PCR Array demonstrated that 24 h microcystin-LR treatment (25 nM) caused overexpression of eight genes involved in tumor metastasis, including MMP-2, MMP-9, and MMP-13. Quantitative real-time PCR, Western blotting and gelatin zymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in the cells after microcystin-LR exposure (P < 0.05 each). Immunofluorescence assay and electrophoretic mobility shift assay revealed that microcystin-LR could activate nuclear factor kappaB (NF-κB) by accelerating NF-κB translocation into the nucleus and enhancing NF-κB binding ability. Furthermore, addition of NF-κB inhibitor in culture medium could suppress the invasiveness enhancement and MMP-2/-9 overexpression. This study indicates that microcystin-LR can act as a NF-κB activator to promote MMP-2/-9 expression and melanoma cell invasion, which deserves more environmental health concerns.

Effects of microcystin-LR exposure on matrix metalloproteinase-2/-9 expression and cancer cell migration.

This study assessed the effects of microcystin-LR (MC-LR) exposure on matrix metalloproteinases (MMPs) expression and cancer cell migration. After male mice were orally administered with different concentrations of MC-LR for 270 d, histopathologic observation revealed an obvious hepatic lymphocyte infiltration or fatty degeneration. Immunohistochemical staining and enzyme-linked immunosorbent assay demonstrated that MC-LR treatment (even at 1 nM) caused up-regulated expressions of hepatic MMP-2/-9. Quantitative reverse-transcriptase PCR showed that the exposure to 80 nM MC-LR induced an increase of MMP-2/-9 mRNA levels by 1.0 and 1.9 fold. Breast cancer cells (MDA-MB-435s) were also cultured with MC-LR solutions and a wound healing assay demonstrated that MC-LR posed a time/dose-dependent stimulation effect on migration of the cancer cells. Gelatin electrophoresis and quantitative PCR showed significant increases in cellular MMP-2/-9 expressions after MC-LR exposure. This study indicated that chronic exposure to MC-LR could alter MMP-2/-9 expressions and stimulate cancer cell migration.