Biological Evaluation of Boron for Health Science for Research Purpose
Louis Pasteur Center for Medical Research
Biological Evaluation: Cytotoxicity
Case Report 1
The cell proliferation/cytotoxicity effects of the carboranyl-fluorescein derivative 2 in a squamous cell carcinoma cell line (SCC-VII) was determined using WST-8 test, as shown in Figure 3. The IC50value of 1-methyl-o-carborane was 1 mM or 21 ppm 10B, reflecting moderate toxicity relative to that of BSH (28 mM) and BPA (7.9 mM)(reference-1). This is likely due to the increased boron content of fluorescein-tagged 1-methyl-o-carborane compared to BSH and BPA.
Figure 3. WT8-C cytotoxicity assay of compound2in SCC-VII.
Reference-1
Patel, H.; Takagaki, M.; Bode, B.P.; Snajdr, I.; Devangi, P.; Sharman, C.; Bux, M.; Bux, S.; Kotora, M.; Hosmane, N.S. Carborane-Appended Saccharides: Prime Candidates for Boron Neutron Capture Therapy (BNCT). Biochemical and Biophysical Journal of Neutron Capture Therapy & Cancer Treatments, 2013, 1(1), pp 15 – 21.
Case Report 2
Cytotoxicity studies:15J2a/b-OH and 15J2a/b-Cs
Toxicity
The murine squamous cell carcinoma cell line (SCCVII)were incubated in subconfluent condition in standard medium of MEM (Nissui, Tokyo, Japan) supplemented with 10% fetal cow serum, in 36℃5% carbon dioxide atmosphere for overnight.
The IC50(moles/liter), i.e. the concentration that inhibited the growth of SCC VII cells by 50% after 3 day of continuous exposure with agents, was determined via CellTiter 96®Aqueous One Solution Cell Proliferation Assay. SCCVII cells were incubated in a 96 well plate for 3 days in MEM containing various concentration of 15J2a/b-OH, 15J2a/b-Cs, and 10BSH.
In-vitro BNCT
For in-vitro BNCT study, aliquots of 15J2a/b-OH, 15J2a/b-Cs, and 10BPA were added for 3 hrs into the petridishes respectively for uptaking of tumor cells in dose-dependent manner (0.43 and 1.29 ppm 10B/15J2a/b-OH, 6 and 20 ppm 10B/15J2a/b-Cs, and 20 ppm 10B/10BPA)) and/or time-dependent manners. Boron-10 concentrations in mediums were confirmed by prompt g-rayspectroscopy. The tumor cells were washed in twice after boron loading. Aliquots containing 5×103cells/ml were pipetted into cylindrical Teflon tubes 1 cm in diameter and 3 cm high that did not generate any secondary radiation when subjected to thermal neutron. The inability of cells to adhere to Teflon allows precise quantitative manipulation of cells without trypsinization. The thermal neutron fluence was determined by averaging the activity of two gold films symmetrically attached to the Teflon tube surface along the thermal neutron axis. The thermal/epithermal neutron fluence ranged from 0 to 3.4×1012/ 6.1×1011n/cm2(absorbed equivalent dose <1.4 Gy). The g-ray dose was monitored by thermoluminescent dosimeters (TLDs) attached to the tubes and ranged from 0 to 0.6 Gy. Immediately after irradiation, 500 cells were seeded in 6-cm Petri dishes (Corning, NY) and incubated for 10 days in a humidified 36℃atmosphere of 98% air/5% carbon dioxide to allow colony formation. The colonies were fixed and stained with a 10% formaldehyde/1% toluidine blue solution and then counted microscopically.
Results
Carborane attached 5-Thio-D-Glucopyranose did not allow further experiments of in-vitro BNCT for their water insolubility and extremely toxic characters although the toxicity of carborane non-attached form of 5-Thio-D-Glucopyranose was biologically tolerable (Table 1).
15J2a/b-OH (carborane ribose) also hardly dissolved in water and/or MEM. However, fortunately, their IC50could be decreased in 78% (1-1.3/5.8=0.78), 5.8x10-2mM, but still fur beyond biological tolerance. There were no significant difference of toxicity between isomers.
Table 1: Cytotoxicity IC50: carborane non-attached form of 5-Thio-G-Glucopyranose = 2.4 mM, and carborane attached 5-Thio-D-Glucopyranose = 1.3x10-2mM(=1.4 ppm nB or 0.3 ppm 10B equivalent).
No any BNCT effect was obtained in maximum tolerant concentration (Fig.1).
Fortunately cytotoxicity of Cesium form of them (15J2a/b-Cs) slightly improved to 1.0x10-1mM, but still almost no accumulation into tumor cell. Differences among the surviving fractions were not significant (Fig.2).
Fig.1 Surviving fraction on 15J2a/b-OH. No significant difference between them and control fraction.
Fig. 2 Surviving fraction on 15J2a-CS (left) and 15J2b-Cs (right). No significant difference between control fractions. Small decrease of survival fractions on 15J2a/b-Cs might be toxic effect of the compounds.
Statistics
Triplicate of the cytotoxicity study and thein-vitroBNCT were carried out. Values were expressed as mean ±SE. Significant differences on the survival study were assessed by the student t-test. These statistical analysis was performed using Prism®5 of GraphPad Soft Ware Inc., CA, USA.
Conclusions
Carborane attached 5-Thio-D-Glucopyranose was extremely toxic like an insecticide. We concluded that carborane attached 5-Thio-D-Glucopyranose showed a little priority for further BNCT study. The mechanism of highly toxic character of this compound is very interesting and further investigation is warranted for explaining and improving this mechanism of its toxicity.
Water solubility could be extremely increased via conjugate with Cesium salt but still very toxic.
