Cellular toxicity induced by different pH levels on the R3230AC rat mammary tumour cell line. An in vitro model for investigation of the tumour destructive properties of electrochemical treatment of tumours.
BACKGROUND
The aim of this study was to evaluate the cellular toxicity of different pH levels on the R3230AC mammary tumour cell line (clone-D) in vitro and to determine in what way the pH affects the tumour cells. The results could be used to interpret the cell damaging effects seen in electrochemical treatment of tumours (EChT), where pH alteration in tissue is the major event.


METHODS
Tumour cells were treated with pH 3.5, 5, 7, 9, 10 and 11 for 10, 20 or 30 min, respectively, followed by studies with the viability assay 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (methyltetrazolium (MTT)), morphological observation in phase contrast microscope (PCM) and light microscope, nucleotide analogue incorporation (BrdU; 5-Brdmo-2'-deoxyuridine), Caspase-3 activity measurement and detection of DNA fragmentation by an agarose gel electrophoresis.


RESULTS
In the viability assay, it was found that different pH levels had cytotoxic effects; these effects were dependent on the pH value and on the time of exposure at a given pH. Morphologically, cells in pH 3.5 and 5 had shrunk, were rounded and had condensed chromatin, whereas prominent cell swelling and nuclear expansion were seen in the pH 9- and 10-treated cells. Gross cytolysis was found in pH 11. A BrdU incorporation assay indicated that proliferation rate is inhibited markedly both with decreasing and increasing pH. Significant Caspase-3 activity was found in pH 3.5 and 5 groups. Caspase-3 levels for the alkaline exposure were equal or below the normal control. DNA ladder formation, a characteristic of apoptosis, was only visualised in the treatment of pH 3.5 for 30 min.


CONCLUSIONS
pH changes inhibit cell proliferation and decrease cell viability. The pathway of killing tumour cell in low pH probably has at least two directions: apoptosis and cell necrosis, whereas high pH results in only cell necrosis. The study suggests that low pH environment can induce apoptosis in unphysiological condition comparable with tissue pH at EChT. In addition, it seems that R3230AC mammary tumour cells are more tolerant to high pH than to acidic changes. This supports the theory that anodic EChT should be more efficient than cathodic.
von Euler
Henrik
H
Department of Small Animal Clinical Sciences, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), P.O. Box 7037, SE-750 07 Uppsala, Sweden. henrik.von.euler@kirmed.slu.se
Söderstedt
Agneta
A
Thörne
Anders
A
Olsson
Jerker M
JM
Yongqing
Guo
G
D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D053938:DNA Fragmentation / Q000502:physiology; D004563:Electrochemistry; D004587:Electrophoresis, Agar Gel; D006863:Hydrogen-Ion Concentration; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology / Q000473:pathology*; D051381:Rats; D014407:Tumor Cells, Cultured
C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases