Analytically pure melamine (C₃H₆N₆), sucrose, D-glucose, NaCl, Na₂HPO₄·2H₂O, KH₂PO₄ and KCl were obtained from the Beijing Chemical Reagent Co. (China). Tryptone and yeast extract were purchased from Oxoid (UK). Analytically pure formaldehyde solution was obtained from the Shantou Xilong Chemical Factory (China). Anhydrous ethanol and HPLC grade acetone was purchased from Sinopharm Chemical Reagent (China). α-cyano-4-hydroxycinnamic acid (CHCA), trifluoroacetic acid (TFA) and formic acid solution were obtained from Sigma-Aldrich (USA). HPLC grade Acetonitrile was obtained from SK Chemicals (Korea). Osmic acid, HPLC grade acetone, uranyl acetate dehydrate puriss. p.a., lead (II) citrate tridydrate, 25% (wt.) glutaraldehyde solution, and a ProteoMass Protein MALDI-MS calibration kit were obtained from Sigma-Aldrich (USA). Epoxy resin Spurr was purchased from Electron Microscopy Science (USA). Milli-Q Academic ultrapure water (Millipore, USA) was used throughout the experiment. In addition, 0.1 mol/L phosphate buffer solution (pH 7.4) was prepared by adding 80 g NaCl, 11.5 g Na₂HPO₄·2H₂O, 2 g KH₂PO₄, and 2 g KCl to 1 L of double distilled water.

T. pyriformis was initially grown as a pure culture at 28℃ in basic liquid culture medium containing 1% (w/v) tryptone, 0.1% (w/v) yeast extract, and 0.2% (w/v) D-glucose without shaking for 24 h in darkness.

A series of test culture media were prepared by adding 0.001, 0.005, 0.01, 0.05, and 0.1 g of melamine to 20 mL of basic liquid culture medium to give melamine concentrations of 0.05, 0.25, 0.5, 2.5, and 5 mg/mL, respectively. Two replicas of each test culture medium were prepared. In addition, three negative control culture media containing no melamine were prepared. All flasks were autoclaved at 103 kPa and 121℃ for 20 min.

All procedures were conducted using the upper layer of the culture solution. Briefly, 200 µL of the original T. pyriformis 24 h culture solution was added to each flask (Time T₀). All cell cultures were then incubated at 28℃ in the dark without shaking. Cell counts were conducted microscopically by using an Olympus CX21 microscope (Japan) and a hemocytometer. Each time, 192 µL of cell culture solution was mixed with 8 µL of formaldehyde solution to fix the cells. At time T₀, the initial cell concentration (D₀) of each cell culture was 1 × 10⁴ cells/mL. Cell counts were then conducted every 4 h successively for 52 h.

A plot of log (D_x/D₀) against incubation time (h) yielded linear equations that were used to evaluate the generation times of the control cell culture (GTC) and of each test cell culture (GTT). When the cell concentration doubled, i.e. D_x/D₀ = 2 and log (D_x/D₀) = 0.301, the GTCs and GTTs were calculated from these equations. Consequently, the relative generation time (RGT) was calculated using the ratio RGT (%) = [(GTT - GTC)/GTC] × 100. Finally, a plot of RGT (%) versus the melamine concentrations was presented as a linear regression equation, from which the IC₅₀ value was determined when the RGT (%) was 50 [7].

The growth dynamics of the populations were evaluated by plotting the incubation time versus the D_x/D₀ ratios, where D_x and D₀ were the cell concentration at time T₀ + x h and time T₀, respectively. The populations were characterized by their generation time, which is the time required for the D_x/D₀ ratio to increase from one to two [7]. The relative toxicity of the melamine is generally quantified by determining the IC₅₀, which is the concentration required to induce a 50% increase in RGT (i.e. GTT is prolonged to 1.5 GTC). If the toxicity of melamine is concentration-dependent, the RGT values could be calculated by monadic linear regression analysis. Data are fitted to the following model: RGT (%) = a X + b [7], where X represents the IC₅₀ value of melamine when RGT (%) is 50.

The cellular morphology at different stages of growth was observed by using an inverted microscope (IX71; Olympus, Japan).

After 48 h of incubation, the flask containing the cell culture was shaken slightly to give a cell suspension solution. After being allowed to stand for 10 min, 5 mL of the cell suspension were removed from near the surface layer of the culture medium using a pipette and placed into a centrifuge tube. The cells were then harvested by centrifugation at 7,000 rpm for 2 min at room temperature. The supernatant was discarded immediately after the centrifugation, after which 450 µL of ultrapure water was added to the tube to disperse the aggregated cells. Next, 50 µL of 25% (w/v) glutaraldehyde solution was added to the solution, to fix the samples, and the mixture was stored at 4℃ overnight. The mixture was then centrifuged at 7,000 rpm for 2 min at 4℃, after which the supernatant was immediately discarded. Next, 500 µL of 0.1 mol/L phosphate buffer solution (pH 7.4) was added to the centrifuge tube to rinse the cells three times. The sample was then centrifuged at 7,000 rpm for 2 min at 4℃, after which the supernatant was discarded and 500 µL of 1% osmic acid solution was added. The mixture was then centrifuged at 7,000 rpm for 2 min at 4℃, after which the supernatant was discarded and 500 µL of 0.1 mol/L phosphate buffer solution (pH 7.4) was added to rinse the precipitates three times. The specimen was then harvested by centrifugation at 7,000 rpm for 2 min at 4℃. The samples were then dehydrated by gradient elution using 30%, 50%, 70%, 80%, 90% and 100% (v/v) acetone in sequence. For each concentration of acetone, 500 µL of the acetone solution was added to the precipitates and then removed after 5 min. Next, the samples were subjected to the epoxy resin coating technique. Briefly, the specimen and the liquid resin with sclerosing agent were mixed thoroughly, allowing the specimen to become embedded in the liquid mixture. The liquid resin was then allowed to harden into a transparent solid. Finally, the naturally solidified resin containing the TEM specimen was divided into ultrathin sections using the ultramicrotome (Ultracut UC6i; Leica, Germany), and then placed on the sample stage of the TEM (JEM-1230; JEOL, Japan) for observation.

A new series of culture media were prepared by adding 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 g of melamine to 20 mL of refresh basic liquid culture medium to give final concentrations of 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 mg/mL, respectively. All flasks were then autoclaved at 103 kPa and 121℃ for 20 min, after which they were allowed to cool. Next, 200 µL of original T. pyriformis culture solution was added to each flask and incubated at 28℃ in the dark without shaking.

After 48 h of incubation, 10 mL of the upper layer of the cell culture solution was withdrawn from each flask and centrifuged at 7,000 rpm for 10 min at 4℃. The supernatant was then discarded, after which 30 µL of water plus 90 µL of alcohol were added. The samples were then centrifuged again at 10,000 rpm for 2 min at 4℃, after which the supernatant was discarded and 50 µL of 70% formic acid solution plus 50 µL of acetonitrile were added to the precipitate. The samples were then immediately placed at -70℃ for 30 min, after which they were subject to 10 min of ultrasonication and then placed at -70℃ again for 30 min. This freezing and thawing procedure was conducted three times.

Following centrifugation at 10,000 rpm for 2 min at 4℃, 0.5 µL of the supernatant was spotted directly onto one well of the stainless MALDI plate and allowed to air dry. Next, 0.5 µL of the matrix saturated solution was immediately added to the dried sample spot. Fresh matrix solution containing 10 mg/mL CHCA in a 50% acetonitrile/0.1% TFA water solution was prepared daily.

All MS experiments were conducted using a 4,700 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, USA) equipped with an Nd: YAG laser (355 nm). Mass spectra were obtained in the positive ion linear mode with a 740 ns delay time at an acceleration voltage of 20 kV. The bin size was 10 ns, and the input bandwidth was 25 MHz. Each mass spectrum was the sum of 60 sub-spectra and 25 shots were averaged automatically per sub-spectrum. The instrument was externally calibrated using a standard bovine insulin solution.

Melamine was found to have a concentration-dependent inhibitory effect on T. pyriformis. Specifically, as the melamine concentration increased, the growth of T. pyriformis was inhibited and its generation time was prolonged accordingly.

Time courses of the cell concentration (cells/mL) are shown in Fig. 1. The presence of 0.05 mg/mL of melamine in the culture medium did not have a visible effect on cell growth, whereas increasing amounts of melamine in the culture medium resulted in greater inhibition of cell growth. After 20 h of incubation, the concentrations of different test cultures began to show noticeable differences, which were used to correlate the cell concentration with melamine concentrations.

A logarithmic plot of (D_x/D₀) against incubation time was used to evaluate the GTCs and GTTs by linear equations (Table 1). Based on the control culture (y = 0.1144 + 0.0325x), the GTC was determined to be 5.7 h in this study, while the test cultures had longer generation times. The presence of melamine caused a concentration-dependent decrease in population growth.

RGT (%) was then calculated from the GTC and GTTs data (Table 1). A plot of RGT (%) versus the concentrations provided the following equation, y = -13.91 + 79.184x, with a relation coefficient of 0.9645. When the RGT (%) was 50, the IC₅₀ value of melamine was determined to be 0.82 mg/mL.

Optical microscopy revealed a normal pear-like shape of T. pyriformis (Figs. 2A and 3A). Under the experimental conditions, the T. pyriformis cell shape was not visibly influenced by the presence of 0.05 mg/mL melamine (Fig. 2B), whereas cells exposed to 0.25 mg/mL melamine showed characteristics of atrophy or deformation. An obvious decrease in cell number was also observed (Figs. 2C and 3B). T. pyriformis cells could not maintain a normal pear-like shape when the melamine concentration reached 2.5 mg/mL (Fig. 3C), and many of them were damaged (Fig. 2D).

Electron microscopic imaging of T. pyriformis revealed alterations of the intracellular structure. When the cell was in a normal condition, its plasma membrane covered the entire organism and was smooth and integrated. In the cell, the cytoplasm was clear and homogeneous, and large mucocysts that were oblong in shape and contained mucoid material appeared to play a role in the endocytic uptake of nutrients (Fig. 4A). However, when T. pyriformis cells were grown in culture medium containing 0.5 mg/mL of melamine, the plasma membrane became folded, and the number of mucocysts decreased dramatically (Fig. 4B). In addition, T. pyriformis had only one spherical, polyploidy macronucleus, about 10 µm in diameter, which was covered by a nuclear membrane to form a closed region (Fig. 4A). Furthermore, the chromatin granules, which are composed of DNA and proteins, were visible as small dark particles in the macronucleus region (Fig. 4). In the presence of 0.5 mg/mL of melamine, the nucleoli of T. pyriformis cells aggregated to form denser and larger fusion bodies associated with thread-like elements (Fig. 4B). When the concentration of melamine in the culture medium reached 2.5 mg/mL, the nuclear membrane was damaged and the macronucleus region of the configuration disappeared from the T. pyriformis cells (Fig. 4C).

After 48 h of incubation, T. pyriformis was in the middle of its logarithmic growth phase (Fig. 1). The control culture and test cultures were analyzed simultaneously by MALDI-TOF-MS. When the concentrations of melamine in the culture medium changed, the MALDI-TOF-MS spectra of T. pyriformis differed accordingly (Figs. 5A-C), indicating that melamine influenced the protein expression of T. pyriformis cells.

Melamine showed different effects on different proteins (Fig. 6). Initially, the relative intensity of m/z 4466 increased in response to the increasing concentrations of melamine up to 3 mg/mL, after which it decreased. This pattern of alterations in relative peak intensity was opposite in the case of the peaks at m/z 6455, m/z 6514 and m/z 7772 (Fig. 6). Taken together, these findings indicate that 3 mg/mL of melamine was a joint turning point that caused a reversal in the protein expression levels.