The serine/threonine protein phosphatase (PP) 2A inhibitor, microcystin-LR, selectively induces liver damage and promotes hepatocarcinogenesis. It is thought that microcystin-LR affects hepatocellular viability mainly through inhibition of PP2A, partially through PP1, and, in addition, by generation of reactive oxygen species (ROS). However, the molecular basis of the selective liver damage and the balance between cell death and survival remained unclear. We analyzed the cytotoxicity of low doses of microcystin-LR using HEK293 cells stably expressing the human hepatocyte uptake transporters, organic anion transporting polypeptide (OATP)1B1 (HEK293-OATP1B1 cells) and OATP1B3 (HEK293-OATP1B3 cells). HEK293-OATP1B1 (IC(50) 6.6nM) and HEK293-OATP1B3 cells (IC(50) 6.5nM) were equally very sensitive to microcystin-LR. In contrast, control-vector-transfected (HEK293-CV) cells were resistant to microcystin-LR. Using HEK293-OATP1B3 cells, the cytotoxicity was attenuated by substrates and inhibitors of OATP1B3, including bromosulfophthalein, rifampicin, and cyclosporin A. Microcystin-LR was transported into HEK293-OATP1B3 cells with 1.2 microM Km value, and its uptake was inhibited by above substances. Accumulation of microcystin-LR in the HEK293-OATP1B1 and HEK293-OATP1B3 cells was increased in a dose-dependent manner but not in HEK293-CV cells. Cellular serine/threonine PP activity of HEK293-OATP1B3 cells was decreased by microcystin-LR but not in HEK293-CV cells. Apoptotic changes were observed after incubation of the HEK293-OATP1B3 cells with microcystin-LR. We found by FACS analysis that microcystin-LR induced apoptosis but not necrosis in HEK293-OATP1B3 cells. Microcystin-LR activated several mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 through inhibition of PP2A. In addition, the cytotoxicity of microcystin-LR was attenuated by the inhibitors of MAPK pathways, including U0126, SP600125, and SB203580. The ROS scavenger N-acetyl-L-cysteine partially attenuated the cytotoxicity of microcystin-LR. Thus, the present study demonstrates that microcystin-LR induces apoptosis through activation of multiple MAPK pathways subsequent to its selective uptake via OATP1B1 and OATP1B3 and followed by inhibition of PP2A, in addition to the ROS generation which might contribute to apoptosis.

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl dulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.

Three liver hazards, two confirmed-hepatitis B virus (HBV) and aflatoxin (AFB), and one rarely studied in populations-microcystin (MC), simultaneously exist in tropical and humid areas; however, there are no epidemiological data on their risks in the same population. We conducted a community-based cross-sectional survey among 5493 adults in two rural towns and statistically analyzed the comparative and combinative effects of the three factors after detecting HBsAg and HBV DNA titers, determining estimated daily intakes (EDIs) of AFB1 and MC-LR and testing serum AST and ALT as liver injury markers for each participant. We observed a HBsAg(+) rate of 7.6%, a relatively high AFB1 exposure level (mean EDI = 471.30 ng/d), and a relatively low MC-LR exposure level (mean EDI = 228.25 ng/d). ORs for abnormal AST (2.42, 95%CI = 1.69-3.45) and ALT (2.87, 95%CI = 1.91-4.29) increased in HBV infections compared with HBV-unexposed participants but did not increase in participants with separate or combined exposure to AFB1 and MC-LR (EDIs ≥ mean). Meanwhile, after adjustment for confounding factors, means of AST and ALT and ORs of abnormal AST and ALT were successively elevated after exposure to HBV, HBV&AFB1 (or HBV&MC-LR), and HBV&AFB1&MC-LR, especially in the group with detectable HBV DNA (AST: OR = 11.38, 95%CI = 3.91-33.17; ALT: OR = 17.09, 95%CI = 5.36-54.53). Notably, ORs for abnormal AST and ALT in the HBV exposed group were not significantly different from those in HBV&AFB1 or in the HBV&MC-LR exposed group but were significantly higher in the HBV&AFB1&MC-LR exposed group (P = 0.029 and P = 0.037, respectively). Our study indicated that microcystin may have the potential to increase the risk of liver injury induced by combined exposure to HBV and aflatoxin. However, in consideration of the uncertainties in the detection of the toxins and evaluation of the EDIs, more epidemiological data are expected to determine the increasing toxic effects of microcystins.

Accumulated evidence indicates that rare variants exert a vital role on predisposition and progression of human diseases, which provides neoteric insights into disease etiology. In the current study, based on three independently retrospective studies of 5,016 lung cancer patients and 5,181 controls, we analyzed the associations between five rare polymorphisms (i.e., p.Glu116Lys, p.Asn118Ser, p.Arg138Cys, p.Ala195Thr and p.Leu259Phe) in MKK7 and lung cancer risk and prognosis. To decipher the precise mechanisms of MKK7 rare variants on lung cancer, a series of biological experiments was further performed. We found that the MKK7 p.Glu116Lys rare polymorphism was significantly associated with lung cancer risk, progression and prognosis. Compared with Glu/Glu common genotype, the 116Lys rare variants (Lys/Glu/+ Lys/Lys) presented an adverse effect on lung cancer susceptibility (odds ratio [OR] = 3.29, 95% confidence interval [CI] = 2.70–4.01). These rare variants strengthened patients’ clinical progression that patients with 116Lys variants had a significantly higher metastasis rate and advanced N, M stages at diagnosis. In addition, the patients with 116Lys variants also contributed to worse cancer prognosis than those carriers with Glu/Glu genotype (hazard ratio [HR] = 1.53, 95% CI = 1.32–1.78). Functional experiments further verified that the MKK7 p.116Lys variants altered the expression of several cancer-related genes and thus affected lung cancer cells proliferation, tumor growth and metastasis in vivo and in vitro. Taken together, our findings proposed that the MKK7 p.Glu116Lys rare polymorphism incurred a pernicious impact on lung cancer risk and prognosis through modulating expressions of a serial of cancer-related genes.

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