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Mammalian genotoxicity assessment of methylene blue in plasma: implications for virus inactivation
S J Wagner1, M A Cifone, H Murli
1Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland, USA.
Background:
The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems.
Study Design And Methods:
Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent.
Results:
Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x 10(-6).
Conclusion:
Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.
Insights
Methylene blue (MB) shows mutagenic effects in cultured mammalian cells but not in vivo. Further research is needed to assess the genotoxic potential of MB in virus inactivation procedures.
Area of Science:
- Toxicology
- Genetics
- Biomedical Science
Background:
- Virus inactivation procedures for blood products must balance viral risk against treatment risks.
- Methylene blue (MB), used in Europe for plasma virus inactivation, has shown mutagenic potential in bacterial systems.
- This study evaluates MB genotoxicity in mammalian models.
Purpose of the Study:
- To investigate the genotoxic potential of methylene blue (MB) in mammalian cells and in vivo.
- To assess if MB used for virus inactivation poses a genotoxic risk.
Main Methods:
- Methylene blue (MB) in heat-treated plasma was tested in an in vitro mouse lymphoma cell assay for gene mutation.
- The assay was conducted with and without rat liver S9 for metabolic activation.
- MB in heat-treated plasma was also evaluated using an in vivo mouse micronucleus assay.
Main Results:
- Methylene blue (MB) significantly increased mutation frequency in mouse lymphoma cells at concentrations of 10 µg/mL (with S9) and 30 µg/mL (without S9).
- In contrast, intravenous administration of MB at 62 mg/kg did not elevate micronucleus frequency in mouse bone marrow.
- These results indicate in vitro mutagenicity but no in vivo genotoxicity in the tested systems.
Conclusions:
- Methylene blue (MB) is mutagenic in cultured mammalian cells.
- Genotoxicity of MB does not appear to be expressed in vivo based on the mouse micronucleus assay.
- Further investigation is crucial to fully assess the in vivo genotoxic risk of intravenously administered MB, considering photoproducts and metabolic conversion.