Use of tissue culture to investigate the effect of methylation on methylene blue cytotoxicity

Abstract

Animal tissue culture has been in existence for over a century and, for much of that time, has played a subsidiary role to that of other life sciences. However, the discipline has expanded rapidly over recent years and is now firmly established as a science in its own right. The main purpose of this study was to research the historical and modern development of animal tissue culture and to gain practical expertise in some of the basic techniques which are employed in a working tissue culture laboratory.

Tissue culture techniques were used to study the effects of substitution on the efficacies of three well known, commercially available dyes, methylene blue, toluidine blue-O and Victoria blue-BO. These dyes, exploited traditionally in industry for their powers as photosensitizers, have now attracted interest as possible agents in clinical photodynamic therapy (PDT). PDT is a novel treatment for both microbial and malignant disease. It combines the application of a photosensitizing drug with red light irradiation, in the presence of molecular oxygen, to induce a cytotoxic response in target cells.

The major proportion of the study examined the effects of methylation on the photocytotoxicity and dark toxicity of the phenothiazinium dyes, methylene blue and toluidine blue-O, against the mammary tumour cell line, EMT-6. The toxicities of the dyes and several derivatives were assayed using standard tissue culture techniques and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT') assay. Both I-methyl methylene blue and 1 ,9-dimethyl methylene blue exhibited increased photocytotoxicity, with concomitant higher dark toxicity, compared to methylene blue. A similar trend was found in the toluidine blue series, but with some variation. It would appear therefore, that, in terms of a clinical application, the methylated derivatives may hold greater promise than the parent compound itself.

In addition, the effect of amino substitution on the cellular uptake of the triarylmethane, Victoria blue-BO, was examined, also in the EMT-6 cell line. Victoria blue-BO and its derivative, MOVB, were extracted from cells using methanol and their intracellular concentrations determined spectrophotometrically at 612 nm and 622 nm respectively. Amino substitution reduced cellular uptake at lower concentrations, but at concentrations above 2.5 uM, uptake of MOVB was similar to that of the parent compound.

These results are important, but it must be remembered that, although animal tissue culture is a valuable tool for preliminary testing, cultured cells are not representative of similar cells in vivo, and cannot replace whole animal testing of potential drugs.