Photoaffinity labeling of the Sarcoma 180 cell surface by daunomycin.

We have used photoaffinity labeling to investigate the distribution and function of daunomycin binding sites in Sarcoma 180 cells. When native daunomycin is irradiated at 366 or 488 nm in the presence of cells, the drug is irreversibly incorporated into cellular molecules. The cellular acceptor for the photoincorporation cannot be extracted by chloroform-methanol nor can it be degraded by DNase. However, the drug acceptor is susceptible to trypsin digestion. These results show that the photoincorporation site is composed of protein but not of lipid or DNA. Furthermore, the fact that photoincorporation proceeds equally well at 0 degrees (where drug does not accumulate inside the cells) as compared to 37 degrees (where free drug concentrates in the cells) suggests that the labeling reaction occurs principally at the cell surface. The photolabeling process is not highly specific since it is not saturable at high drug concentrations and cannot be competed for by unlabeled daunomycin. When 2 X 10(5) daunomycin molecules are incorporated per Sarcoma 180 cell, the cells can still accumulate free drug. This result suggests that the photolabeling reaction does not occur at the drug transport locus. Photoincorporation of daunomycin also does not affect the viability of Sarcoma 180 cells, as judged by a cloning assay. Thus, there is probably no surface receptor for the drug which mediates cytotoxicity when occupied. This result is as expected from previous work predicting that the mechanism of daunomycin involves disruption of some generalized membrane property like fluidity. However, in a series of Sarcoma 180 sublines selected for increasing resistance to daunomycin, the photoincorporation increases in direct proportion to drug sensitivity. Consequently, daunomycin appears to be capable of photoaffinity labeling a cell surface protein which, although not directly involved in the mechanism of cytotoxicity is implicated in the expression of drug resistance.

Reduction of daunorubicin aqueous solutions by COO.- free radicals. Reactions of reduced transients with H2O2.

Daunorubicin aqueous solutions were reduced by COO- free radicals produced by gamma-radiolysis. This reaction leads to 7-deoxyaglycon daunomycinone. Added before irradiation, H2O2 oxidized hydroquinone daunorubicin giving back the drug directly and thus preventing C-O bond cleavage. The implications of this reaction on the mechanism of the reductive cleavage are discussed.

Photosensitized reactions of nucleic acids.

The main effects of near-ultraviolet and visible light on cellular DNA are reviewed with emphasis on base lesions, oligonucleotide single-strand breaks and DNA-protein cross-links. Model system photosensitization reactions of DNA are also discussed. This includes photodynamic effects, menadione-mediated photooxidation, photoionization of antibiotics, the photochemistry of 5-halogenopyrimidines and urocanic acid.

Enhanced response to daunomycin of normal, tumor and metastatic cell lines via drug photoactivation.

We have compared the cytotoxicity of daunomycin in vitro to highly differentiated normal epithelial cells (Fisher rat thyroid cells, FRTL-5) and to two neoplastic cell lines, a thyroid carcinoma (TK-6) and its lung metastasis (MPTK-6). Whereas the cell lines are equally sensitive to the drug in the dark, if irradiated during incubation with daunomycin (86 J/cm2 at 488 nm), they become more and differently sensitive. Namely, the drug doses producing 50% mortality decrease by factors of about 22, 28 and 16 for FRTL-5, TK-6 and MPTK-6 cell lines, respectively. This result correlates with differences in drug uptake and resistance observed in the normal and neoplastic cell lines.

Photoprotection of daunorubicin hydrochloride with sodium sulfite.

Protection of 10(-4) M daunorubicin hydrochloride (DNRUB) solutions against photolytic degradation was investigated using sodium sulfite (SS). Photodegradation of the drug under fluorescent light was found to be accelerated at higher pHs, and the photoprotecting ability of SS (1.9 x 10(-3) M) increased progressively with an increase in pH in the range of 3.7 to 8.2. Increase in ionic strength (mu) appeared to decrease the rate of degradation of the drug, in the presence or absence of SS, in acetate buffer (0.1 M) of pH 5.5. Photostability of DNRUB solutions was enhanced with an increase in SS concentration in pH 6.5 citrate buffer (0.1 M, mu = 0.5). The effect of various buffer species on the photostability of DNRUB was carried out using acetate, phosphate and citrate buffers. Divalent phosphate anion appeared to demonstrate the greatest catalyzing effect followed by trivalent citrate, combination of divalent and trivalent citrate (9:11 molar ratio), and monovalent acetate anion. Sodium sulfite was found to significantly diminish the catalyzing effect of all the buffers.

Pulse radiolysis study of daunorubicin redox reactions: redox cycles or glycosidic cleavage?

Two aspects of daunorubicin reactivity were investigated by pulse radiolysis. The reactions of O2 and O2- with the semiquinone and the hydroquinone transients of daunorubicin were determined and their rate constants measured. Although O2- can reduce the drug and its semiquinone form, it is a more powerful oxidant towards the two reduced transients. The hydroquinone daunorubicin glycosidic cleavage in aqueous solution was studied. Three intermediates were seen and characterized by their absorption spectra, their formation and decay kinetics. The competition between these two main processes was evaluated in the conditions of pulse radiolysis. Even under low O2 partial pressures the redox cycles are much more rapid than the glycosidic cleavage and a relatively high O2- steady state is settled. Biological implications are discussed.

Photoinduced reactions of anthraquinone antitumor agents with peptides and nucleic acid bases: an electron spin resonance and spin trapping study.

The photoexcitation (lambda = 313 +/- 10 nm) of adriamycin, daunomycin, and mitoxantrone in the presence of peptides or pyrimidine nucleic acid bases was investigated. In air-saturated and air-free solutions, peptides are decarboxylated by the photoexcited drug molecules. The decarboxylation reactions were shown to occur specifically at the C-terminal amino acid of the peptide. The decarboxylated peptide radicals were spin-trapped using 2-methyl-2-nitrosopropane (MNP) and identified by electron spin resonance (ESR). In air-free solutions, nucleic acid bases are oxidized by the photoexcited drug molecules predominantly generating C(5)-carbon-centered radicals in the pyrimidine rings of uracil, cytosine, and thymine. However, spin adducts of MNP and thymine were also obtained at the N(1) or N(3) positions of the pyrimidine ring. In air-saturated adriamycin and daunomycin solutions, the spin adducts of MNP with uracil or thymine are similar to those obtained following hydroxyl radical reactions with these pyrimidines. This suggests that in the presence of oxygen, the photoexcited adriamycin and daunomycin transfer an electron to oxygen generating the superoxide anion radicals (O2-.), which are precursors of hydroxyl radicals. O2-. was also formed when O2-saturated DNA solutions were photoirradiated (lambda = 313 +/- 10 and 438 +/- 10 nm) in the presence of adriamycin and daunomycin, indicating that the photodegradation of DNA in the presence of these drugs caused by hydroxyl radicals is mediated by dissolved oxygen.

Photodegradation of doxorubicin, daunorubicin and epirubicin measured by high-performance liquid chromatography.

The degradation kinetics of doxorubicin, daunorubicin and epirubicin in aqueous solution under fluorescent light and sunlight were studied using high-performance liquid chromatographic (HPLC) methods. The rates of photodegradation of all three drugs were similar, they were inversely proportional to the drug concentration and were accelerated by an increase in the pH of the vehicle. Photodegradation followed first-order kinetics. At concentrations greater than or equal to 500 micrograms/ml no special precautions appeared to be necessary to protect freshly prepared solutions of these agents from light. Photolysis was very rapid, however, at concentrations in the low microgram range therefore, when these solutions are used for in-vitro work or stability studies, they should be protected from light at all times. In addition, adsorptive losses, which may also be pronounced in low concentration solutions, should be prevented by storage in polypropylene containers.

One-electron reduction of daunorubicin intercalated in DNA or in a protein: a gamma radiolysis study.

The one-electron reduction of daunorubicin, an anthracycline antibiotic, intercalated in DNA or in the apoprotein of the riboflavin binding protein, was studied by gamma radiolysis. The two reduction mechanisms appear very similar to the one found for the non-intercalated drug. Hydrogen peroxide, which oxidizes non-intercalated hydroquinone daunorubicin with two electrons in one step (C. Houée-Levin, M. Gardès-Albert and C. Ferradin, FEBS lett., 173, 27-30, (1984], reacts with daunorubicin hydroquinone in DNA but not in the protein. It appears thus that the site accessibility to hydrogen peroxide in DNA is better than in the protein. Biological consequences are discussed.