In vitro toxicity of melamine against Tetrahymena pyriformis cells

Abstract

This study assessed the toxicity of melamine against the unicellular eukaryotic system of Tetrahymena (T.) pyriformis exposed to 0, 0.05, 0.25, 0.5, 2.5, and 5 mg/mL of melamine. Cell growth curves of different cultures, the half maximum inhibition concentration (IC(50)) value of melamine, and morphological changes in cells were obtained via optical and transmission electron microscopic observation. The effects of eleven melamine concentrations, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 mg/mL, on protein expression levels of T. pyriformis were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results showed an obvious inhibitory effect of melamine on the growth of eukaryotic cells. Cell growth dynamics indicated that the IC(50) value of melamine on T. pyriformis was 0.82 mg/mL. The cellular morphology was also affected in a concentration-dependent manner, with characteristics of atrophy or cell damage developing in the presence of melamine. The relative contents of the top four main proteins corresponding to peak mass-to-charge ratios (m/z) of 4466, m/z 6455, m/z 6514, and m/z 7772 in the MALDI-TOF-MS spectra were all found to be closely correlated with the melamine concentrations. In conclusion, exposure of eukaryotic cells to melamine could inhibit cell growth, cause changes in cytomorphology and even disturb the expression of proteins in a concentration-dependent manner. The described method of examining four sensitive proteins affected by melamine was also proposed to be used in a preliminary study to identify protein biomarkers in T. pyriformis.

Fig. 1

Growth dynamics of Tetrahymena (T.) pyriformis cells exposed to different concentrations of melamine. The presented cell concentration (D_x) was the mean of three replicates. Although the D_x of different cell cultures increased, cell growth was obviously inhibited in the presence of melamine. At various time spots, the D_x of each test culture was compared with that of the control culture using the Paired-Samples t test in the SPSS 16.0 software. The results showed that there were no significant differences between the test culture containing 0.05 mg/mL of melamine and the control group at any time point (p > 0.05). After 26 h of exposure to 0.25, 0.5, 2.5 and 5 mg/mL of melamine, the test cultures differed significantly from the control culture (p < 0.05).

Fig. 2

Inverted microscopic images of melamine-treated T. pyriformis cell cultures after 48 h of incubation. (A) Normal pear-like shaped T. pyriformis cells in the negative control culture. (B) Cellular shape and cell number were not obviously influenced by the presence of 0.05 mg/mL of melamine. (C) Cell number decreased evidently and cellular shape became irregular when the melamine concentration was 0.25 mg/mL. Characteristic cell atrophy appeared. (D) Cell number decreased drastically and many cells were damaged when the melamine concentration reached 2.5 mg/mL. ×40.

Fig. 3

Inverted microscopic images of melamine-treated T. pyriformis cells after 48 h of incubation. (A) A pear-like shaped normal T. pyriformis cell in the negative control culture. The cell membrane was integrated and its cytoplasm was clear and homogeneous. (B) Characteristic cell atrophy in the presence of 0.25 mg/mL of melamine. The cell membrane was damaged, and cytoplasm was not abundant. (C) T. pyriformis cells were severely atrophic when the melamine concentration was 2.5 mg/mL. ×200.

Fig. 4

Transmission electron microscopic images of T. pyriformis cells after 48 h of incubation. The concentration of melamine in culture medium A was 0 mg/mL, while it was 0.5 mg/mL in B and 2.5 mg/mL in C. In A, the plasma membrane of T. pyriformis cell was smooth and integrated, and the cytoplasm containing many large mucocysts was clear and homogeneous. Normal nucleoli distributed in the macronucleus region are shown in A, while thread-like nucleoli aggregates in the melamine-treated cell are shown in B. In C, the macronucleus region and mucocysts disappeared, and the previously homogeneous cytoplasm showed a character of porosity. MR: macronucleus region, n: nucleolus. Scale bars = 5 µm.

Fig. 5

MALDI-TOF-MS spectra of T. pyriformis intact proteins after 48 h of incubation. The concentration of melamine was 0 mg/mL (A), 3.0 mg/mL (B), and 4.5 mg/mL (C). Peaks at m/z 4466, m/z 6455, m/z 6514, and m/z 7772 in the MALDI-TOF-MS spectra corresponded to the four main proteins in T. pyriformis cell. Their relative intensities changed in response to gradual increases in the melamine concentrations.

Fig. 6

Alterations in the relative intensities of peaks m/z 4466, m/z 6455, m/z 6514, and m/z 7772 in the MALDI-TOF-MS spectra. The turning point of each of these polygonal lines was 3 mg/mL melamine. Peaks m/z 6455, m/z 6514 and m/z 7772 showed a first down-regulation and then up-regulation of their relative intensities before and after the turning point, whereas peak m/z 4466 showed a first up-regulation and then down-regulation of its relative intensity before and after the turning point.