High cytotoxicity of betulin towards fish and murine fibroblasts: Is betulin safe for nonneoplastic cells?

Abstract

Background Betulin, a natural pentacyclic triterpene with the lupane structure that is present in significant amounts in the outer bark of birch, is known for its broad array of biological and pharmacological properties. Betulin has attracted attention as a potential, natural-origin antimicrobial substance. The literature describes it as selectively toxic to neoplastic cells but safe for normal cells. The research aim was to evaluate the basal cytotoxicity of betulin towards fish (BF-2) and murine (NIH/3T3) fibroblasts. We used four colorimetric tests that provide a preliminary evaluation of possible mechanisms of the cytotoxicity of a compound to assess the degree of the toxicity of betulin after 24, 48 and 72 h of incubation with cells: the MTT assay (mitochondrial activity assessment), the NRU assay (lysosomal membrane integrity assessment), the LDH assay (cellular membrane integrity assessment) and the SRB assay (total cellular protein content determination). Results The results revealed an exceptionally high sensitivity of mitochondria to the effect of betulin, with the other endpoints being less sensitive. Although murine fibroblasts were more vulnerable to the toxic effect of betulin than fish fibroblasts, the betulin CC50 values for both cell lines were comparable with analogous IC50 values determined by other researchers in studies involving cancerous cells. Conclusions The results indicate the need to verify the claim about the selective toxicity of betulin towards malignant cells and to conduct safety/toxicity tests before any potential therapeutic use of betulin in veterinary medicine.

The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

“…The location of formazan formation and its intracellular transportation has remained controversial. While the role of mitochondria in MTT reduction [ 2 , 4 , 5 ] has been a justification for the common application of the assay to measure mitochondrial activity [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ], biochemical and microscopic studies have located formazan in various intracellular organelles. Intracellular formazan granules have been observed in the endoplasmic reticulum, cytosolic lipid droplets [ 13 , 14 ], plasma membranes [ 1 , 15 ], nucleus, and microsomes [ 5 ].…”

“…Several studies have revealed limitations of the MTT assay [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ] and various confounding factors that are needed to be considered in designing, performing, analyzing, and interpreting the assay results [ 5 , 9 , 10 , 11 , 12 , 13 , 14 ]. However, the MTT assay is still commonly used and interpreted, overlooking these limitations and escaping the necessary optimization steps.…”

Fructose‐Derived Levan Nanoparticles Protect Against Osteoarthritis by Directly Blocking CD44 Activation

“…[ 18,19 ] We evaluated the cytotoxicity of LevNPs and DMSO (control) against fibroblasts as reported previously. [ 20–22 ] As shown in Figure S3B, Supporting Information, 0.1% DMSO showed no cytotoxicity with over 90% cell viability and LevNPs with 0.06% residual DMSO also did not exhibit toxicity toward cells.…”

The Influence of Betulin and Its Derivatives EB5 and ECH147 on the Antioxidant Status of Human Renal Proximal Tubule Epithelial Cells

“…Studies conducted by Zehra et al on normal mouse fibroblasts (NIH-3T3) showed low toxicity of betulin [ 28 ]. On the other hand, studies by Małaczewska et al have shown that betulin is highly toxic to NIH-3T3, and these cells were more sensitive to betulin than fish fibroblasts BF-2 [ 29 ]. This study showed the different sensitivity of the cells, depending on the species origin.…”