Tumor-promoting cyanotoxin microcystin-LR does not induce procarcinogenic events in adult human liver stem cells

Microcystin-LR Does Not Alter Cell Survival and Intracellular Signaling in Human Bronchial Epithelial Cells

“…Nevertheless, the formation of MC-LR protein adducts in the exposed cells was qualitatively and quantitatively comparable between both HBE1 and 16HBE14o-cell lines, which indicates that other OATP isoforms than 1B1/1B3 (e.g., 3A1 or 4A1), or other membrane transporters or cellular mechanisms, could have been involved in the uptake of MC-LR by bronchial epithelial cells. This is consistent with a previous study, which reported formation of MC-LR protein adducts in immortalized adult human liver cells HL1-hT1 occurring independently on the expression or activity of OATP1B1/1B3, since HL1-hT1 cultures were found to express OATP2A1 and 3A1 only, and the putative MC-LR uptake was not affected by pharmacological inhibitors of OATP1B1/1B3 [56]. MC-LR-protein adducts accumulated in both HBE1 and 16HBE14o-cells included major bands with molecular weights around 35-37 kDa, i.e., corresponding to PP1/PP2A catalytic subunits, which represent primary intracellular targets known to covalently bind MC-LR [57].…”

“…For example, Wang et al [45] observed significantly increased ERK1/2 activity after 24-h exposure to 0.5 µM MC-LR in murine epithelial ATII cells; however, Wang et al [25] reported no significant changes in P-ERK1/2 following 24-h exposure to 1 µM MC-LR in human A549 lung cancer cells. 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.…”

“…Additionally, a time-dependent reduction in survival was observed in a series of murine RAW246.7 macrophage-like, BV-2 immortalized microglial, and N2a neuroblastoma-derived cells exposed to 10 µM MC-LR for 24-72 h [62]. However, 10 µM MC-LR did not decrease the viability of human adult liver stem cells HL1-hT1 [56,63], human hepatocellular carcinoma cells HepG2, human colorectal carcinoma cells Caco-2 or monkey kidney epithelial Vero-E6 line [64][65][66]. The data presented in the literature thus suggests that the cytotoxic effects of MC-LR are cell type-specific and possibly species-specific.…”

“…MC-LR-protein adducts accumulated in both HBE1 and 16HBE14o-cells included major bands with molecular weights around 35-37 kDa, i.e., corresponding to PP1/PP2A catalytic subunits, which represent primary intracellular targets known to covalently bind MC-LR [57]. Additional protein bands detected by anti-MC-LR antibody had molecular weights around 20 kDa, 30 kDa, and in the case of 16HBE14o-cells also 60 kDa, which might correspond to putative intracellular targets of MC-LR (e.g., 55 kDa ATP-synthase β subunit, [58]; 56 kDa aldehyde dehydrogenase 2 [59]; or 23 kDa Proteasome β2 subunit [60], or to yet-to-be-identified proteins [56]).…”

“…An assay combining three indicative dyes was adopted from Raska et al [56] to evaluate the disruption of various vital cellular processes: the plasma membrane integrity and esterase activity by 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), the metabolic reductive potential as resazurin reduction (Alamar blue ® assay), and neutral red uptake (NRU) by lysosomes [48,49]. Further details of the method and used materials are given in the Supplementary Materials (Section S1: Methods).…”