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A probe for intracellular concentrations of drugs: delayed fluorescence from acridine orange
P Wardman1, M F Dennis, J White
1Cancer Research Campaign, Mount Vernon Hospital, Northwood, Middx., U.K.
Abstract:
The aim of this work is to develop fluorescent probes that will indicate effective concentrations of therapeutic agents, or endogenous protectors, at important cellular sites. Acridine orange associates with nucleic acids and emits a 'delayed' fluorescence signal. This signal is quenched by oxidants such as oxygen, nitroaryl radiosensitizers, adriamycin and mitomycin-c, and reductants such as thiols, ascorbate and other radioprotectors. The quenching of the acridine orange delayed fluorescence reflects the effective concentration of these therapeutically-important oxidants and reductants near DNA. The relative concentration of basic radiosensitizers such as pimonidazole (Ro 03-8799) near the DNA is greater than that of misonidazole. Thiols quench the delayed fluorescence signal according to the degree of ionization of the thiol function; this may model the reactivity of thiols with guanine radical sites in DNA. Ascorbate and aminopyrine do not quench the delayed fluorescence from cells stained with acridine orange as these compounds are taken up by cells very inefficiently.
Insights
This study introduces acridine orange as a fluorescent probe to measure cellular concentrations of therapeutic agents. Its delayed fluorescence signal indicates effective levels of oxidants and reductants near DNA.
Area of Science:
- Biochemistry
- Cell Biology
- Medical Imaging
Background:
- Therapeutic agents require precise concentration monitoring at cellular sites for efficacy.
- Fluorescent probes offer a method for real-time cellular analysis.
- Acridine orange is a known nucleic acid stain with unique fluorescent properties.
Purpose of the Study:
- To develop acridine orange as a fluorescent probe for indicating effective concentrations of therapeutic agents.
- To investigate the quenching of acridine orange's delayed fluorescence by various cellular compounds.
- To correlate fluorescence quenching with the cellular concentration of oxidants and reductants.
Main Methods:
- Utilizing acridine orange as a fluorescent probe in cellular assays.
- Measuring the delayed fluorescence signal emitted by acridine orange.
- Assessing the quenching effect of oxidants (e.g., oxygen, adriamycin) and reductants (e.g., thiols, ascorbate) on the fluorescence signal.
- Comparing the cellular distribution of radiosensitizers like pimonidazole and misonidazole.
Main Results:
- Acridine orange's delayed fluorescence is quenched by therapeutically relevant oxidants and reductants.
- The degree of fluorescence quenching reflects the effective concentration of these agents near DNA.
- The cellular concentration of pimonidazole near DNA was found to be higher than misonidazole.
- Thiol ionization degree influences fluorescence quenching, potentially modeling DNA radical site interactions.
Conclusions:
- Acridine orange serves as a valuable fluorescent probe for assessing cellular concentrations of therapeutic agents.
- The quenching mechanism provides insights into the interaction of oxidants and reductants with cellular components, particularly DNA.
- This method can differentiate the cellular uptake and distribution of different therapeutic agents.