Effects of the Amino Acid Constituents of Microcystin Variants on Cytotoxicity to Primary Cultured Rat Hepatocytes

Microcystins, which are cyclic heptapeptides produced by some cyanobacterial species from algal blooms, strongly inhibit serine/threonine protein phosphatase and are known as hepatotoxins. Microcystins have many structural variations, yet insufficient information is available on the differences in the cytotoxic potentials among the structural variants. In this study, the cytotoxicities of 16 microcystin variants at concentrations of 0.03–10 μg/mL to primary cultured rat hepatocytes were determined by measuring cellular ATP content, and subsequently determined by their 50% inhibitory concentration (IC50). Differences in the amino acid constituents were associated with differences in cytotoxic potential. [d-Asp3, Z-Dhb7] microcystin-LR exhibited the strongest cytotoxicity at IC50 of 0.053 μg/mL among the microcystin variants tested. Furthermore, [d-Asp3, Z-Dhb7] microcystin-HtyR was also highly cytotoxic. These results suggest that both d-Asp and Z-Dhb residues are important in determining the cytotoxic potential of microcystin variants.


Results
We evaluated the cytotoxicities of 16 MC variants. Their structures are shown in Figure 1. Figure 3 and R 2 positions, respectively, compared to MC-LR, and their cytotoxic activities were found to be higher than that of MC-LR.
[D-Asp 3 , Z-Dhb 7 ] MC-HtyR, which has a methyl group at the R 4 position, exhibited significantly high cytotoxic activity, compared to [D-Asp 3 , E-Dhb 7 ] MC-HtyR, which has a methyl group at the R 5 position (Figures 1 and 3).
MC-RR was less cytotoxic than both MC-LR and -YR (Table 1)

Discussion
We evaluated the cytotoxicities of MC variants isolated from some cyanobacteria to primary cultured rat hepatocytes. Accumulation of toxicity information for MC variants is absolutely essential to conduct the risk assessment or management of them. Therefore, toxic properties of 16 MV variants indicated in this study would be helpful basic information for the risk assessment or management of many MC variants in environmental water.
In this study, IC 50 of MC-LR to primary rat hepatocytes was 0.8 μg/mL, however earlier study was reported that IC 50 of MC-LR to the same cells was 48 ng/mL [42]. The MTT assay used in the earlier study and the assay which measure the cellular ATP contents (ATP assay) show occasional different IC 50 with certain differences of cell lines, exposure chemicals, number of cells etc. [43,44]. Therefore, the difference of IC 50 might be attributed to these assay methods to evaluate the cell viability. Moreover, in this study, we used the freeze-thawed cells and seeded 2.5-fold of cell number compared to the cell number in the earlier study. These differences might have caused the reduction of the cytotoxicity. We think that it is important to evaluate the result taking the difference of the experimental methods into consideration in the risk assessment or management.
Lack of a methyl group at either the R 3 or R 2 position of Dha 7 or D-Asp 3 , resulted in enhanced cytotoxic activities of MC-LR, -YR, and -RR. In particular, for MC-RR, the lack of a methyl group at the R 3 position might be responsible for the slight enhancement of cytotoxic activity, compared to demethylation at the R 2 position (Figure 4).
It has been reported that [Asp 3 , Dhb 7 ] MC-RR exhibits higher cytotoxicity than MC-RR [45,46]. In this study, D-Asp 3 and E-Dhb 7 or D-Asp 3 and Z-Dhb 7 on MC-LR and -HtyR also induced higher cytotoxic activities than D-Asp 3 or Dha 7 on MC-LR and -HtyR. These results suggest that substitution of the methyl group at the R 4 or R 5 positions is associated with cytotoxic potential, rather than the lack of a methyl group at the R 3 or R 2 positions. In fact, [D-Asp 3 , Z-Dhb 7 ] MC-LR exhibited the strongest cytotoxic activity among the 16

variants, and [D-Asp 3 , Z-Dhb 7 ] MC-HtyR also exhibited high cytotoxicity. A comparison of the IC 50 values among [D-Asp 3 , Z-Dhb 7 ] and [D-Asp 3 , E-Dhb 7 ] on MC-LR
and -HtyR suggests that the presence of a methyl group at the R 4 position caused stronger effects on cytotoxic activity than the presence of a methyl group at the R 5 position (Table 1). Although the D-Asp 3 residue may be essential for the enhancement of cytotoxicity, the R 4 methyl group of the Z-Dhb 7 residue may also play a key role in the cytotoxic activities of MCs such as the Adda moiety.
Distinct properties of MC variants in terms of uptake, cytotoxicity, protein phosphatase 1 and 2A inhibition, were reported in other organisms cells, brain and neuron cells of mice or Caco-2 cells from human [37][38][39]47]. It is thought that MC variants with the same hydrophobic properties show the same permeabilities to the cell membrane. Vesterkvist et al. suggested that the hydrophobic MC has pronounced cytotoxic potentials in Caco-2 cells [38]. Since [D-Asp 3 , Z-Dhb 7 ]-MC-LR and -HtyR are geometric isomers of [D-Asp 3 , E-Dhb 7 ]-MC-LR and -HtyR, respectively, they have same hydrophobic property. However, the Z geometric isomers had more potent cytotoxic activities than the E geometric isomers in primary cultured rat hepatocytes in our study. Therefore, at R 4 and R 5 position of 7th amino acid (Mdha, Dha or Dhb), the conformational property would be an important factor for cytotoxicity to rat hepatocytes. Differences in the response of the proteins on cell surface or inside the cell, which are caused by differences of the conformational property, might determine the toxic potentials. While the protein phosphatase inhibition activities of MCs were reported in various studies, it was reported that the activity with MC variants did not correlate with their toxic potential [38,46]. There is little information of cellular uptake of MC variants in each organ. More information on membrane permeability, cellular uptake in various organs or species, interaction with target proteins etc. is needed to fully understand different toxic potentials of MC variants.

MC Variants
The 16 MC variants were separated and purified by high performance liquid chromatography. The details of the separation procedure have been described in a previous report [48].

Cells
Primary cultured rat hepatocytes, which maintain both phase I and II metabolic activity as well as uptake transporter activity, were isolated from Sprague Dawley. The cells were purchased from Biopredic International (Rennes, France) and were stored at −135 °C until further use. Thawing medium, seeding medium, and long-term culture medium were purchased from Biopredic International (Rennes, France). The primary cultured rat hepatocytes were thawed in a water bath at 37 °C. The cells were poured into 30 mL of pre-warmed thawing medium. The cell suspension was centrifuged at 900 rpm (160 × g) for 1 min. The supernatant was removed and the cell pellets were re-suspended in 2 mL of the seeding medium. The cells were seeded in 96-well white plates at 0.3 × 10 6 cells/mL in 0.1 mL/well of the seeding medium and incubated at 37 °C in 5% CO 2 . The following day, the cells were exposed to sixteen MC variants with the long-term culture medium.

Cytotoxicity Assay
MC variants were dissolved in a mixture of DMSO:water = 9:1 (v/v) to make 1.0 mg/mL stock solutions taking the solubility of MC variants to DMSO into consideration. Stock solutions were diluted to 3-1000 μg/mL with DMSO, and subsequently diluted to final concentrations of 0.03-10 μg/mL in the medium. 1% DMSO solution was diluted 100-fold with the medium, and the resulting solution was used as a negative control. Cells were seeded in 96-well white plates and pre-cultured at 37 °C in 5% CO 2 overnight. Cells were then exposed to various concentrations of MC variants in the long-term culture medium for 72 h. Cytotoxicity was evaluated using the Cell Titer-Glo Luminescent Cell Viability Assay Kit (Promega Corporation, Madison, WI, USA), based on cellular ATP content. After 10 min of incubation with an equal volume of the Cell Titer-Glo reagent at room temperature, luminescence was measured for 1 s/well. The luminescence intensity of each well treated with an MC variant was determined, relative to the control, and those concentrations of the MC variants that showed 50% cell viability (IC 50 ) were calculated. MC-LR was used as a positive reference substance for every assay.

Statistical Analysis
All experiments values were expressed as the mean ± standard deviations (SD) of three independent experiments. The results were analyzed statistically with the Student's t-test. The test was conducted to verify the difference between each group exposed to MC variants and the control. Differences with p < 0.05 were considered statistical significant.

Conclusions
We have determined the cytotoxic potentials of 16 types of MC variants primary culture rat hepatocytes. MC variants containing D-Asp 3 and Dha 7 and/or Dhb 7 residues were found to exhibit stronger cytotoxic activities than their corresponding normal MC variants. The Z-Dhb 7 residue of the MC variants is important for their cytotoxic potential. Toxic properties of 16 MC variants indicated in this study would help accumulation of toxicity basic information on various MCs, and eventually contribute to the risk assessment and/or management of environmental water.