Oncology/endocrine
DNA Damaging Effects of the Dyes Used in Sentinel Node Biopsy: Possible Implications for Clinical Practice

https://doi.org/10.1016/j.jss.2008.07.039Get rights and content

Objective

This study investigates whether methylene blue (MB), patent blue V (PBV), and indigo carmine (IDC) commonly used in sentinel node biopsy cause DNA damage to breast epithelial cells in vitro.

Methods

MCF-7 and HB-2 cells were exposed for 5 minutes to the above dyes at the same concentrations used in clinical practice. Following exposure, the comet assay was performed to detect DNA damage. The enzyme, Fapy-DNA glycosylase (FpG) was incorporated to enable the detection of additional oxidative damage.

Results

Both PBV and MB stimulated DNA strand breaks in both MCF-7 and HB2 cell lines (P < 0.05). Levels were elevated over 3-fold (P < 0.05) in MCF-7 and HB2 cells treated with 2.5% PBV and 1% MB, compared with untreated control cells. In contrast, IDC did not stimulate DNA strand break damage at clinically relevant concentrations in either cell line. Addition of Fapy-DNA glycosylase enzyme also revealed significantly (P < 0.05) increased levels of oxidative DNA lesions (ODL) in MCF-7 cells treated with PBV (17.6% ODL) compared with control cells (5.9% ODL).

Conclusions

This study shows, for the first time, that certain dyes (MB and PBV) commonly used in SLNB have genotoxic effects on breast cells at clinically relevant concentrations in vitro. In vivo studies are now warranted to assess and minimize DNA damage caused by these dyes during SLNB.

Introduction

Sentinel lymph node biopsy (SLNB) is a relatively new procedure used to surgically stage the axillary lymph node status in breast cancer patients. Identification of sentinel lymph nodes (SLN) is aided by injection of dye and/or radioisotope labeled tracer into the breast before surgery to highlight lymphatic drainage [1]. The dyes utilized for this procedure vary between countries. Patent blue V (PBV) is the gold standard in the UK and Europe whereas its isomer isosulfan blue is mainly used in the USA 2. Many centers in the UK, USA, and the Middle East are increasingly utilizing methylene blue (MB) for SLNB as it is a cheaper alternative that is readily available [2, 3, 4, 5]. Indigo carmine (IDC) is also used in the Far East for this procedure with good localization rates reported [6].

The genome of eukaryotic cells is constantly being subjected to various endogenous and exogenous attacks that can result in DNA damage [7]. DNA damage is known to play an important role in mutagenesis and carcinogenesis [8]. Previously published data have demonstrated that chromoendoscopy procedures utilizing MB in the oesophagus and colon result in DNA damage to associated epithelium [9, 10] and occurs as a result of photoactivation of MB. With the lack of information on dyes used in SLNB, particularly PBV, we undertook this study to assess the potential genotoxic effect of PBV, MB, and IDC on both benign and malignant breast cells.

Using the alkaline comet assay, we report elevated DNA damage levels in breast cells following treatment with clinically relevant concentrations of PBV and MB, in contrast with IDC, which does not induce DNA damage.

Section snippets

Cell Culture and Dye Exposure

The cell lines used for the study were the MCF-7 (malignant breast cell line cultured from metastatic pleural effusion [11]) and HB-2 cell line (benign immortalized epithelial breast cell using simian virus 40 [12]). Cells were cultured in six well plates until approximately 60% confluence was achieved. MCF-7 were maintained in RPMI-1640 media containing L-glutamine (Invitrogen, Paisley, UK) and 5% (vol/vol) heat treated fetal calf serum (FCS; Harlan Sera-Lab, Belton, UK). DMEM media

Results

MCF-7 cells were used to compare the amount of DNA damage in cells treated with different concentrations of dyes compared with controls. Both PBV and MB showed evidence of DNA damage at clinically relevant concentrations. Levels of DNA damage were increased over 3.5-fold in cells treated with 2.5% PBV (13.6%) compared with control cells (3.66%) (P < 0.05) (Fig. 1A). Levels of DNA damage remained significantly elevated even when the concentration of PBV was reduced 25-fold to 0.1%. Similar to

Discussion

The results demonstrate that the gold standard dye used for SLNB in the UK and other parts of Europe, namely PBV, and also MB, cause DNA damage in vitro. This is primarily the induction of strand breaks, but there was some evidence for raised levels of FpG sensitive sites, indicating additional oxidative damage, with the use of PBV. IDC is less commonly used for SLNB, but this dye did not show evidence of DNA damage at the concentrations used in clinical practice.

The importance of these results

Acknowledgments

The authors thank Dr. J. Olliver for her help in the comet assays and the Breast Cancer Research Awareness Group (BCRAG) for financial support.

References (24)

  • T. Aihara et al.

    Dye-guided sentinel node biopsy revisited; validation and observational study from a single institute

    Breast Cancer

    (2003)
  • R. De Bont et al.

    Endogenous DNA damage in humans: A review of quantitative data

    Mutagenesis

    (2004)
  • Cited by (0)

    View full text