Abstract:Microcystins (MCs) are primarily hepatotoxins produced by cyanobacteria and are responsible for intoxication in humans and animals. There are many incidents of chronic exposure to MCs, which have been attributed to the inappropriate treatment of water supplies or contaminated food. Using RAW 264.7 macrophages, we showed the potency of microcystin-LR (MC-LR) to stimulate production of pro-inflammatory cytokines (tumor necrosis factor α and interleukin-6) as a consequence of fast nuclear factor κB and nitrogen-a… Show more
“…Increased phosphorylation was observed in the human HL7702 liver cell line after 48-h exposure to 10 µM MC-LR [72], but not in human HL1-hT1 adult liver stem cells exposed to 1 µM [56], despite toxin uptake was documented in both studies. Elevated P-ERK1/2 levels were additionally reported by Adamovsky et al [31] in murine RAW 264.7 macrophages after 30-min exposure to 1 µM MC-LR, but probably triggered via interactions of the toxin with membrane receptors, independently of MC-LR cellular uptake and inhibition of PPs.…”
Changes in ecological and environmental factors lead to an increased occurrence of cyanobacterial water blooms, while secondary metabolites-producing cyanobacteria pose a threat to both environmental and human health. Apart from oral and dermal exposure, humans may be exposed via inhalation and/or swallowing of contaminated water and aerosols. Although many studies deal with liver toxicity, less information about the effects in the respiratory system is available. We investigated the effects of a prevalent cyanotoxin, microcystin-LR (MC-LR), using respiratory system-relevant human bronchial epithelial (HBE) cells. The expression of specific organic-anion-transporting polypeptides was evaluated, and the western blot analysis revealed the formation and accumulation of MC-LR protein adducts in exposed cells. However, MC-LR up to 20 µM neither caused significant cytotoxic effects according to multiple viability endpoints after 48-h exposure, nor reduced impedance (cell layer integrity) over 96 h. Time-dependent increase of putative MC-LR adducts with protein phosphatases was not associated with activation of mitogen-activated protein kinases ERK1/2 and p38 during 48-h exposure in HBE cells. Future studies addressing human health risks associated with inhalation of toxic cyanobacteria and cyanotoxins should focus on complex environmental samples of cyanobacterial blooms and alterations of additional non-cytotoxic endpoints while adopting more advanced in vitro models.Keywords: microcystin-LR; human bronchial epithelial cells; in vitro; HBE1; 16HBE14o-, mitogen-activated protein kinase; cytotoxicity; OATP
Key Contribution:The study demonstrated the uptake of microcystin-LR into human bronchial epithelial cells HBE1 and 16HBE14o-. No significant changes in the cell viability, impedance (cell layer integrity) or activation of MAPKs ERK1/2 and p38 were detected in multiple exposure and concentration scenarios.
“…Increased phosphorylation was observed in the human HL7702 liver cell line after 48-h exposure to 10 µM MC-LR [72], but not in human HL1-hT1 adult liver stem cells exposed to 1 µM [56], despite toxin uptake was documented in both studies. Elevated P-ERK1/2 levels were additionally reported by Adamovsky et al [31] in murine RAW 264.7 macrophages after 30-min exposure to 1 µM MC-LR, but probably triggered via interactions of the toxin with membrane receptors, independently of MC-LR cellular uptake and inhibition of PPs.…”
Changes in ecological and environmental factors lead to an increased occurrence of cyanobacterial water blooms, while secondary metabolites-producing cyanobacteria pose a threat to both environmental and human health. Apart from oral and dermal exposure, humans may be exposed via inhalation and/or swallowing of contaminated water and aerosols. Although many studies deal with liver toxicity, less information about the effects in the respiratory system is available. We investigated the effects of a prevalent cyanotoxin, microcystin-LR (MC-LR), using respiratory system-relevant human bronchial epithelial (HBE) cells. The expression of specific organic-anion-transporting polypeptides was evaluated, and the western blot analysis revealed the formation and accumulation of MC-LR protein adducts in exposed cells. However, MC-LR up to 20 µM neither caused significant cytotoxic effects according to multiple viability endpoints after 48-h exposure, nor reduced impedance (cell layer integrity) over 96 h. Time-dependent increase of putative MC-LR adducts with protein phosphatases was not associated with activation of mitogen-activated protein kinases ERK1/2 and p38 during 48-h exposure in HBE cells. Future studies addressing human health risks associated with inhalation of toxic cyanobacteria and cyanotoxins should focus on complex environmental samples of cyanobacterial blooms and alterations of additional non-cytotoxic endpoints while adopting more advanced in vitro models.Keywords: microcystin-LR; human bronchial epithelial cells; in vitro; HBE1; 16HBE14o-, mitogen-activated protein kinase; cytotoxicity; OATP
Key Contribution:The study demonstrated the uptake of microcystin-LR into human bronchial epithelial cells HBE1 and 16HBE14o-. No significant changes in the cell viability, impedance (cell layer integrity) or activation of MAPKs ERK1/2 and p38 were detected in multiple exposure and concentration scenarios.
“…MC-LR at the lowest dose (1 ng/mL) increased the mRNA expression of IL-6, whereas the highest dose markedly diminished the expression of this cytokine. In contrast, the production of IL-6 in macrophages (RAW 264.7 cell line) was only slightly induced at the highest concentration (1000 nM) of MC-LR (Adamovsky et al, 2015). The evidence of disturbance of IL-6 potentially indicates the negative effect of MC-LR on the inflammatory reaction after a 6-h treatment.…”
Microcystins (MCs) are included in drinking water and a family of cyclic heptapeptide hepatotoxins that have been implicated in the impairment of liver function in various animals. There is scarce information on the effect of MCs on cytokines and apoptotic gene expression and on whether MCs can induce inflammation and apoptosis in avian hepatic tissue. This study investigated the expression of genes related to proinflammatory interleukins, apoptosis, and antioxidant function in chicken liver tissues cultured in the presence of different doses of microcystin-leucinearginine (MC-LR). Livers were collected from five hens and liver slices were placed in sterile tubes containing Dulbecco's medium supplemented with 0, 1, 10, or 100 ng/mL of MC-LR. After 6 h of cultivation, total RNA was extracted and quantitative PCR analysis was performed for interleukin genes , TNF sf15, an apoptotic gene (caspase-3), and genes involved in antioxidant function ([catalase [CAT ], glutathione peroxidase [GSH-PX ], and superoxide dismutase [SOD]). Liver tissues in each group were fixed for histopathology. MC-LR downregulated the mRNA levels of IL-1β, IL-8, and TNF sf15 as compared to the control (0 ng/mL) in dosedependent patterns; however, the differences were not significant. The expression of IL-6 in liver tissues exposed to 100 ng/mL of MC-LR was significantly (P<0.05) lower than that in tissues exposed to 1 ng/mL. In contrast, MC-LR upregulated the mRNA expression of caspase-3 and genes involved in antioxidant function in the liver tissues after 6 h, without the difference reaching statistical significance. Hepatocytes showed vacuolar degeneration and focal necrosis according to the dose of MC-LR. This study highlighted the risk of low doses of MC-LR in chicken liver. Moreover, MC-LR could modulate the transcriptional patterns of at least IL-6 in liver-tissue culture of chicken after 6 h of exposure.
“…Previous studies on MC‐LR (Adamovsky et al, ; Adegoke, Wang, Machebe, Adeniran, et al, ; Adegoke, Wang, Machebe, Wang, et al, ) indicated that MC‐LR elicit its toxic effects via TLR4 signaling pathways. Therefore, the TLR4 signaling pathway was made the target for amelioration and attenuation of MC‐LR toxicity in this study.…”
Section: Discussionmentioning
confidence: 98%
“…According to Huggins et al (), the TLRs, including TLR4, have been shown to play a crucial role in inflammatory diseases. MC‐LR has been reported to induce inflammation in bovine Sertoli cells (Adegoke, Wang, Machebe, Wang, et al, ), in human umbilical vein endothelial cells (Shi, Zhou, & Zhang, ) and activates TLRs, particularly TLR4 in the RAW 264.7 cell line (Adamovsky et al, ). Meanwhile, all TLR signaling pathways culminate in activation of the transcription factor nuclear factor‐kappaB (NF‐κB), which controls the expression of an array of inflammatory cytokine genes (Taro & Shizuo, ).…”
This study investigated the pharmacological inhibition of the toll‐like receptor 4 (TLR4) genes as a measure to attenuate microcystin‐LR (MC‐LR) reproductive toxicity. Bovine Sertoli cells were pretreated with TLR4‐IN‐C34 (C34) for 1 hour. Thereafter the pretreated and non‐pretreated Sertoli cells were cultured in medium containing 10% heat‐activated fetal bovine serum + 80 μg/L MC‐LR for 24 hours to assess the ability of TLR4‐IN‐C34 to attenuate the toxic effects of MC‐LR. The results showed that TLR4‐IN‐C34 inhibited MC‐LR‐induced mitochondria membrane damage, mitophagy and downregulation of blood‐testis barrier constituent proteins via TLR4/nuclear factor‐kappaB and mitochondria‐mediated apoptosis signaling pathway blockage. The downregulation of the mitochondria electron transport chain, energy production and DNA replication related genes (mt‐ND2, COX‐1, COX‐2, ACAT, mtTFA) and upregulation of inflammatory cytokines (interleukin [IL]‐6, tumor necrosis factor‐α, IL‐1β, interferon‐γ, IL‐4, IL‐10, IL‐13 and transforming growth factor β1) were modulated by TLR4‐IN‐C34. Taken together, we conclude that TLR4‐IN‐C34 inhibits MC‐LR‐related disruption of mitochondria membrane, mitophagy and downregulation of blood‐testis barrier proteins of the bovine Sertoli cell via cytochrome c release and TLR4 signaling blockage.
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