Chemicals and reagents
Betulin (≥ 98 % purity), Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium (Eagle, EBSS), foetal bovine serum, antibiotic-antimycotic solution, nonessential amino acids (NEAAs), glutamine, Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red based in vitro toxicology assay kit (TOX4), lactic dehydrogenase based in vitro toxicology assay kit (TOX7) and sulforhodamine B based in vitro toxicology assay kit (TOX6) were purchased from Sigma-Aldrich, Poland. Dimethyl sulfoxide (DMSO) was purchased from Chempur, Poland.
Compound preparation
A stock solution of betulin was prepared in DMSO at a concentration of 2500 µg/mL, and working solutions were prepared in cell maintenance media (serum-free) at final concentrations of 0 (control cells), 0.244, 0.488, 0.976, 1.95, 3.9, 7.8, 15.625 and 31.25 µg/mL immediately before use in cell culture.
Cell lines
NIH/3T3 (ATCC CRL-1658) mouse embryonic fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM glutamine, 10 % foetal bovine serum and a 1 % antibiotic-antimycotic solution at 37 °C in a humidified atmosphere with 5 % CO2.
BF-2 (ATCC CCL-91) bluegill caudal trunk fibroblasts were grown in minimum essential medium (Eagle, EBSS) supplemented with 1 % nonessential amino acids (NEAAs), 10 % foetal bovine serum and 1 % antibiotic-antimycotic solution at 23 °C in a humidified atmosphere with 5 % CO2.
NIH/3T3 and BF-2 cells were seeded in 96-well plates at a density of 1 × 104 or 2 × 104 cells per well, respectively. After 24 h of incubation at an appropriate temperature, cells were exposed to betulin at the concentrations mentioned above for 24, 48 and 72 h. Control (untreated) cells served as the negative control and a reference point. Triton X-100 treated cells served as the positive internal control in MTT, NRU and LDH assays. Due to the specific character of SRB assay, there was no positive control in this test. After 24, 48 and 72 h of treatment, the four different cytotoxicity assays were performed. All experiments were repeated three times.
Cytotoxicity assays
Four in vitro cytotoxicity colorimetric assays were conducted to identify possible mechanisms of betulin cytotoxicity, namely, the MTT reduction assay, the neutral red uptake assay (NRU), the lactate dehydrogenase leakage assay (LDH) and the sulforhodamine B assay (SRB).
MTT assay
The MTT assay is used to estimate mitochondrial damage, since it determines the activity of mitochondrial enzymes. These enzymes reduce the water-soluble tetrazolium dye MTT to insoluble purple formazan, which accumulates inside viable cells [29].
The MTT assay was conducted using the protocol described by Mosmann [29], with some modifications described in our previous article [30]. The results are reported as the percentage of control (nontreated) cell viability.
The MTT assay was used to determine the cytotoxicity of both betulin and the vehicle (DMSO).
NRU assay
The NRU assay evaluates lysosomal membrane integrity. It measures neutral red dye accumulation within lysosomes of viable cells. This phenomenon depends on the capacity of viable cells to maintain a pH gradient; hence, dead cells are unable to retain the dye [31].
The NRU assay was performed using a commercially available kit (TOX4) (Sigma-Aldrich, Poland) according to the manufacturer’s protocol. The full description of the test procedure was also provided in a published article [30]. The results obtained are reported as the percentage of control (nontreated) cell viability.
LDH assay
The LDH assay measures leakage of the stable cytosolic enzyme lactate dehydrogenase into the culture medium, which is an indicator of irreversible cell membrane damage and cell death [32].
The LDH assay was performed using a commercially available kit (TOX7) (Sigma-Aldrich, Poland) according to the manufacturer’s protocol. The full description of the test procedure was also reported previously [30]. The results obtained are reported as the percentage of LDH leakage to the culture media in control (nontreated) cells.
SRB assay
The sulforhodamine B colorimetric assay measures the cellular protein content, and the assay results are proportional to the number of cells. This assay has been used to determine the cell density; however, it does not distinguish between living and dead cells [33].
The SRB assay was performed using a commercially available kit (TOX6) (Sigma-Aldrich, Poland) according to the manufacturer’s protocol. In this assay, SRB binds to protein components of trichloroacetic acid-fixed cells, and the protein-bound dye is extracted with Tris. The results obtained are presented as the percentage of the total cellular protein content in control (nontreated) cells.
Statistical analysis
All experiments were repeated three times. The results are presented as the mean values ± SD (standard deviation). Statistical analyses were performed using GraphPad Prism software (GraphPad Software, USA). Data were subjected to one-way analysis of variance (ANOVA). Dunnett’s posttest was used to determine differences between control and betulin-exposed cells at the same time points. Differences between the two cell lines at the same time points were determined using Student’s t-test. The 50 % cytotoxic concentrations (CC50, defined as the concentration of the compound that reduced cell viability by 50 %) were calculated using the Quest Graph™ ED50 Calculator (AAT Bioquest, Inc.) [34].