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Application of advanced HepG2 3D cell model for studying genotoxic activity of cyanobacterial toxin cylindrospermopsin
2020
Hercog, Klara | Štampar, Martina | Štern, Alja | Filipič, Metka | Žegura, Bojana
Cylindrospermopsin (CYN) is an emerging cyanotoxin increasingly being found in freshwater cyanobacterial blooms worldwide. Humans and animals are exposed to CYN through the consumption of contaminated water and food as well as occupational and recreational water activities; therefore, it represents a potential health threat. It exhibits genotoxic effects in metabolically active test systems, thus it is considered as pro-genotoxic. In the present study, the advanced 3D cell model developed from human hepatocellular carcinoma (HepG2) cells was used for the evaluation of CYN cyto-/genotoxic activity. Spheroids were formed by forced floating method and were cultured for three days under static conditions prior to exposure to CYN (0.125, 0.25 and 0.5 μg/mL) for 72 h. CYN influence on spheroid growth was measured daily and cell survival was determined by MTS assay and live/dead staining. The influence on cell proliferation, cell cycle alterations and induction of DNA damage (γH2AX) was determined using flow cytometry. Further, the expression of selected genes (qPCR) involved in the metabolism of xenobiotics, proliferation, DNA damage response, apoptosis and oxidative stress was studied. Results revealed that CYN dose-dependently reduced the size of spheroids and affected cell division by arresting HepG2 cells in G1 phase of the cell cycle. No induction of DNA double strand breaks compared to control was determined at applied conditions. The analysis of gene expression revealed that CYN significantly deregulated genes encoding phase I (CYP1A1, CYP1A2, CYP3A4, ALDH3A) and II (NAT1, NAT2, SULT1B1, SULT1C2, UGT1A1, UGT2B7) enzymes as well as genes involved in cell proliferation (PCNA, TOP2α), apoptosis (BBC3) and DNA damage response (GADD45a, CDKN1A, ERCC4). The advanced 3D HepG2 cell model due to its more complex structure and improved cellular interactions provides more physiologically relevant information and more predictive data for human exposure, and can thus contribute to more reliable genotoxicity assessment of chemicals including cyanotoxins.
Показать больше [+] Меньше [-]Time-averaged concentrations are effective for predicting chronic toxicity of varying copper pulse exposures for two freshwater green algae species
2017
Angel, Brad M. | Simpson, Stuart L. | Granger, Ellissah | Goodwyn, Kathryn | Jolley, Dianne F.
Intermittent, fluctuating and pulsed contaminant discharges may result in organisms receiving highly variable contaminant exposures. This study investigated the effects of dissolved copper pulse concentration and exposure duration on the toxicity to two freshwater green algae species. The effects of single copper pulses of between 1 and 48 h duration and continuous exposures (72 h) on growth rate inhibition of Pseudokirchneriella subcapitata and Chlorella sp. were compared on a time-averaged concentration (TAC) basis. Relationships were then derived between the exposure concentration and duration required to elicit different levels of toxicity expressed as inhibition concentration (IC). Continuous exposure IC50's of 3.0 and 1.9 μg/L were measured on a TAC basis for P. subcapitata and Chlorella sp., respectively. Algal growth rates generally recovered to control levels within 24–48 h of the copper pulse removal, with some treatments exhibiting significantly (p < 0.05) higher rates of cell division than controls in this recovery period. For both algae, when exposed to treatments with equivalent TACs, the continuous exposure elicited similar or slightly greater growth rate inhibition than the pulsed exposures. To elicit equivalent inhibition, the exposure concentration increased as the exposure duration decreased, and power models fitted this relationship reasonably well for both species. Water quality guideline values (WQGVs) are predominantly derived using data from continuous exposure toxicity bioassays, despite intermittent contaminant exposures often occurring in aquatic systems. The results indicate the WQGV for copper may be relaxed for pulsed exposures by a factor less than or equivalent to the TAC and still achieve a protection to these sensitive algae species.
Показать больше [+] Меньше [-]Perfluorinated substances and telomeres in an Arctic seabird: Cross-sectional and longitudinal approaches
2017
Blévin, Pierre | Angelier, Frédéric | Tartu, Sabrina | Bustamante, Paco | Herzke, Dorte | Moe, Børge | Bech, Claus | Gabrielsen, Geir Wing | Bustnes, Jan Ove | Chastel, Olivier
Telomeres are non-coding DNA repeats located at the termini of eukaryotic chromosomes, regulated by dynamic processes balancing shortening and maintenance. Despite a mechanism to slow-down telomere shortening, cell division leads to progressive attrition of chromosomes, leading to the onset of cellular senescence or apoptosis. However, telomere restoration based on telomerase activity is the primary mechanism for telomere maintenance. Telomere length is associated to health and survival and can be impacted by a broad panel of environmental factors. However, the effect of contaminants on telomeres is poorly known for living organisms. The aim of this study was to investigate relationships between some poly- and perfluoroalkyl substances (PFASs), body condition and telomere length by using both a cross-sectional and longitudinal approach in adult breeding Black-legged kittiwakes (Rissa tridactyla) from Svalbard. First, we examined the associations between absolute telomere length and PFASs contamination in a given year (cross-sectional approach). Second, we investigated the relationships between telomere dynamics and PFASs contamination within a two years’ time frame (longitudinal approach). Our results did not show any significant relationships of PFASs and body condition with absolute telomere length in a given year. Surprisingly, we found a positive and significant relationship between PFASs and telomere dynamics in both sexes with elongated telomere in birds bearing the highest concentrations of PFASs. Our study underlines (i) the need to investigate PFAS effects on telomere dynamics with a longitudinal approach and (ii) a potential positive effect of these contaminants on telomere length, with the most contaminated birds showing the slowest rate of telomere shortening or even displaying elongated ones. Our study is the first to report a relationship between PFASs and telomere length in free-living vertebrates. A possible underlying mechanism and other potential confounding factors are discussed.
Показать больше [+] Меньше [-]Ecotoxicity and genotoxicity of cyclophosphamide, ifosfamide, their metabolites/transformation products and their mixtures
2016
Česen, Marjeta | Eleršek, Tina | Novak, Matjaž | Žegura, Bojana | Kosjek, Tina | Filipič, Metka | Heath, Ester
Cyclophosphamide (CP) and ifosfamide (IF) are commonly used cytostatic drugs that repress cell division by interaction with DNA. The present study investigates the ecotoxicity and genotoxicity of CP, IF, their human metabolites/transformation products (TPs) carboxy-cyclophosphamide (CPCOOH), keto-cyclophosphamide (ketoCP) and N-dechloroethyl-cyclophosphamide (NdCP) as individual compounds and as mixture. The two parent compounds (CP and IF), at concentrations up to 320 mg L−1, were non-toxic towards the alga Pseudokirchneriella subcapitata and cyanobacterium Synecococcus leopoliensis. Further ecotoxicity studies of metabolites/TPs and a mixture of parent compounds and metabolites/TPs performed in cyanobacteria S. leopoliensis, showed that only CPCOOH (EC50 = 17.1 mg L−1) was toxic. The measured toxicity (EC50 = 11.5 mg L−1) of the mixture was lower from the toxicity predicted by concentration addition model (EC50 = 21.1 mg L−1) indicating potentiating effects of the CPCOOH toxicity. The SOS/umuC assay with Salmonella typhimurium revealed genotoxic activity of CP, CPCOOH and the mixture in the presence of S9 metabolic activation. Only CPCOOH was genotoxic also in the absence of metabolic activation indicating that this compound is a direct acting genotoxin. This finding is of particular importance as in the environment such compounds can directly affect DNA of non-target organisms and also explains toxicity of CPCOOH against cyanobacteria S. leopoliensis. The degradation study with UV irradiation of samples containing CP and IF showed efficient degradation of both compounds and remained non-toxic towards S. leopoliensis, suggesting that no stable TPs with adverse effects were formed. To our knowledge, this is the first study describing the ecotoxicity and genotoxicity of the commonly used cytostatics CP and IF, their known metabolites/TPs and their mixture. The results indicate the importance of toxicological evaluation and monitoring of drug metabolites as they may be for certain aquatic species more hazardous than parent compounds.
Показать больше [+] Меньше [-]Shift of calcium-induced Microcystis aeruginosa colony formation mechanism: From cell adhesion to cell division
2022
Huang, Xuhui | Gu, Peng | Wu, Hanqi | Wang, Zhikai | Huang, Suzhen | Luo, Xingzhang | Zheng, Zheng
Colony formation is an essential stage of cyanobacterial blooms. High calcium concentration can promote Microcystis aeruginosa aggregation behavior, but the mechanism of colony formation caused by calcium has rarely been reported. In this study, high calcium-induced colony formation was identified as a shift from cell adhesion to cell division, rather than only cell adhesion as previously thought. Algae responded to this calcium-induced environmental pressure by aggregating and forming colonies. Algal cells initially secreted large quantities of extracellular polysaccharides (EPS) and rapidly aggregated by cell adhesion. The highest aggregation proportion was up to 68.93%. However, high calcium concentrations cannot completely inhibit algal cell growth, but only delay the algae into the rapid growth phase. With adaption to calcium and existing high EPS content, the daughter cells reduced EPS synthesis and the aggregation proportion decreased. The increasing growth rate was also responsible for the decreased xylose content in EPS. The mechanism of colony formation changed to cell division. The downregulation of genes related to EPS secretion also supported this hypothesis. Overall, these results can benefit for our understanding of cyanobacterial bloom formation.
Показать больше [+] Меньше [-]The toxicity of graphene oxide affected by algal physiological characteristics: A comparative study in cyanobacterial, green algae, diatom
2020
Yin, Jingyu | Fan, Wenhong | Du, Juan | Feng, Weiying | Dong, Zhaomin | Liu, Yingying | Zhou, Tingting
Though the main toxic mechanisms of graphene oxide (GO) to algae have been accepted as the shading effect, oxidative stress and mechanical damage, the effect of algal characteristics on these three mechanisms of GO toxicity have seldom been taken into consideration. In this study, we investigated GO toxicity to green algae (Chlorella vulgaris, Scenedesmus obliquus, Chlamydomonas reinhardtii), cyanobacteria (Microcystis aeruginosa) and diatoms (Cyclotella sp.). The aim was to assess how the physiological characteristics of algae affect the toxicity of GO. Results showed that 10 mg/L of GO significantly inhibited the growth of all tested algal types, while S. obliquus and C. reinhardtii were found to be the most susceptible and tolerant species, respectively. Then, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the physiological characteristics of the assessed algae. The presence of locomotive organelles, along with smaller and more spherical cells, was more likely to alleviate the shading effect. Variations in cell wall composition led to different extents of mechanical damage as shown by Cyclotella sp. silica frustules and S. obliquus autosporine division being prone to damage. Meanwhile, growth inhibition and cell division were significantly correlated with the oxidative stress and membrane permeability, suggesting the latter two indicators can effectively signal GO toxicity to algae. The findings of this study provide novel insights into the toxicity of graphene materials in aquatic environments.
Показать больше [+] Меньше [-]The mechanism of root growth inhibition by the endocrine disruptor bisphenol A (BPA)
2020
Bahmani, Ramin | Kim, DongGwan | Modareszadeh, Mahsa | Thompson, Drew | Park, Jeong Hoon | Yoo, Hye Hyun | Hwang, Seongbin
Bisphenol A (BPA) is a harmful environmental contaminant acting as an endocrine disruptor in animals, but it also affects growth and development in plants. Here, we have elucidated the functional mechanism of root growth inhibition by BPA in Arabidopsis thaliana using mutants, reporter lines and a pharmacological approach. In response to 10 ppm BPA, fresh weight and main root length were reduced, while auxin levels increased. BPA inhibited root growth by reducing root cell length in the elongation zone by suppressing expansin expression and by decreasing the length of the meristem zone by repressing cell division. The inhibition of cell elongation and cell division was attributed to the enhanced accumulation/redistribution of auxin in the elongation zone and meristem zone in response to BPA. Correspondingly, the expressions of most auxin biosynthesis and transporter genes were enhanced in roots by BPA. Taken together, it is assumed that the endocrine disruptor BPA inhibits primary root growth by inhibiting cell elongation and division through auxin accumulation/redistribution in Arabidopsis. This study will contribute to understanding how BPA affects growth and development in plants.
Показать больше [+] Меньше [-]Uptake, translocation and distribution of three veterinary antibiotics in Zea mays L
2019
Zhang, Cheng | Xue, Jianming | Cheng, Dengmiao | Feng, Yao | Liu, Yuanwang | Aly, Hesham M. | Li, Zhaojun
Frequently detected residuals of antibiotics in crops has drawn increasing attention from research community and the general public. This study was conducted under the controlled environmental conditions to investigate the uptake, translocation and distribution of three different veterinary antibiotics (VAs) in plants of Zea mays L. (maize, the third largest crop in the world, especially in China) and the associated mechanisms. The distribution color-maps of mixed-VAs showed that the highest RCF (root concentration factors) values of chlortetracycline (CTC) and sulfamethoxazole (SMZ) were found in the 0.5–2.0 mm zone (cell division zone), while the highest RCF value of sulfathiazole (ST) was in the 6.0–8.0 mm zone (elongation zone) of root tips (0.5–10.0 mm) after 120 h of exposure to VAs. The translocation factor (TF) of CTC was greater than 1.0, but the TFs of SMZ and ST were less than 1.0 under addition of single antibiotic. However, the TFs of three VAs were all greater than 1.0 at the end of exposure under addition of mixed-VAs. The dissipation of antibiotics by maize was also demonstrated by harvesting all plant parts in an enclosed system. The possible mechanisms for uptake and translocation of VAs in maize were investigated by adding multiple respiration inhibitors into the culture solution. The RCFs of VAs were suppressed heavily by salicylhydroxamic acid (SHAM) and sodium azide (NaN3), which indicates that the uptake of VAs was an active process. The results of TFs and stem concentration factors (SCFs) of CTC and SMZ in HgCl2 treatments revealed that the translocation of VAs was associated with the aquaporin activity in maize. The findings from this study will have significant implications for the management of crop food contamination by VAs and for the development of phytoremediation technology for antibiotics in the environment.
Показать больше [+] Меньше [-]Occurrence of the fungus mycotoxin, ustiloxin A, in surface waters of paddy fields in Enshi, Hubei, China, and toxicity in Tetrahymena thermophila
2019
Cheng, Shiyang | Liu, Hao | Sun, Qian | Kong, Ren | Letcher, Robert J. | Liu, Chunsheng
There has been an increasing incidence rate of rice false smut in global rice cultivation areas. However, there is a dearth of studies on the environmental concentrations and hazards of ustiloxin A (UA), which is the major mycotoxin produced by a pathogenic fungus of the rice false smut. Here, the concentrations of UA in the surface waters of two paddy fields located in Enshi city, Hubei province, China, were measured, and its toxicity in T. Thermophila was evaluated. This is the first study to detect UA in the surface waters of the two paddy fields, and the measured mean concentrations were 2.82 and 0.26 μg/L, respectively. Exposure to 2.19, 19.01 or 187.13 μg/L UA for 5 days significantly reduced the theoretical population and cell size of T. thermophila. Furthermore, treatment with 187.13 μg/L UA changed the percentages of T. thermophila cells in different cell-cycle stages, and with an increased malformation rate compared with the control, suggesting the disruption of the cell cycle. The expressions of 30 genes involved in the enriched proteasome pathway, 7 cyclin genes (cyc9, cyc10, cyc16, cyc22, cyc23, cyc26, cyc33) and 2 histone genes (mlh1 and hho1) were significantly down-regulated, which might be the modes of action responsible for the disruption of cell cycling due to UA exposure.
Показать больше [+] Меньше [-]Toxicity of perfluorooctane sulfonate and perfluorooctanoic acid to Escherichia coli: Membrane disruption, oxidative stress, and DNA damage induced cell inactivation and/or death
2016
Liu, Gesheng | Zhang, Shuai | Yang, Kun | Zhu, Lizhong | Lin, Daohui
Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are two widely used polyfluorinated compounds (PFCs) and are persistent in the environment. This study for the first time systematically investigated their toxicities and the underlying mechanisms to Escherichia coli. Much higher toxicity was observed for PFOA than PFOS, with the 3 h half growth inhibition concentrations (IC50) determined to be 10.6 ± 1.0 and 374 ± 3 mg L−1, respectively, while the bacterial accumulation of PFOS was much greater than that of PFOA. The PFC exposures disrupted cell membranes as evidenced by the dose-dependent variations of cell structures (by transmission electron microscopy observations), surface properties (electronegativity, hydrophobicity, and membrane fluidity), and membrane compositions (by gas chromatogram and Fourier transform infrared spectroscopy analyses). The increases in the contents of intracellular reactive oxygen species (ROS) and malondialdehyde and the activity of superoxide dismutase indicated the increment of oxidative stress induced by the PFCs in the bacterial cells. The fact that the cell growth inhibition was mitigated by the addition of ROS scavenger (N-acetyl cysteine) further evidenced the important role of oxidative damage in the toxicities of PFOS and PFOA. Eighteen genes involved in cell division, membrane instability, oxidative stress, and DNA damage of the exposed cells were up or down expressed, indicating the DNA damage by the PFCs. The toxicities of PFOS and PFOA to E. coli were therefore ascribed to the membrane disruption, oxidative stress, and DNA damage induced cell inactivation and/or death. The difference in the bactericidal effect between PFOS and PFOA was supposed to be related to their different dominating toxicity mechanisms, i.e., membrane disruption and oxidative damage, respectively. The outcomes will shed new light on the assessment of ecological effects of PFCs.
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