ß-Carotene absorbed 2 equimolar amounts of N0 2 accompanying the complete destruction of ß-carotene. Electron spin resonance study using 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-l-oxyl revealed that no significant amounts of NO were released by the interaction. Nitrogen atoms derived from N0 2 were tightly bound to the ß-carotene molecules. Destruction of ß-carotene was inhibited little by a-tocopherol and polyunsaturated fatty esters, and slightly by ascorbyl palmitate, indicating that ß-carotene was a more effective scavenger of N0 2 . ONOOH/ONOO -and 3-morpholinosydononimine similarly destroyed ß-carotene. The results suggest that ß-carotene contributes to the prevention of cytotoxicity and genotoxicity of N0 2 and ONOOH/ONOO" derived from NO.

The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants ofSaccharomyces cerevisiaecells overexpressing either the endogenous multidrug efflux pump Pdr5 or itsCandida albicansorthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.

Whether dithiocarbamate-Fe(II) complexes used in the electron spin resonance (ESR) studies are specific for the detection of NO was investigated. It was found that the typical ESR signals of dithiocarbamate-Fe(II)-NO spin adducts appeared in the interaction of N-methyl-D-glucaminedithiocarbamate (MGD)-, bis(hydroxyethyl) dithiocarbamate (HED)- and diethyldithiocarbamate (DED)-Fe(II) complexes with the NO-derived nitrogen oxides, nitrogen dioxide (NO2) and nitrite ion (NO2-) in a prolonged incubation. Under the conditions of prolonged incubation, appearance of the ESR signals of the dithiocarbamate-Fe(II)-NO spin adducts may indicate the presence of not only NO but NO2 and NO2-.

Peroxynitrous acid synthesized by reaction of hydrogen peroxide and nitrite and generated from 3-morpholinosydononimine (SIN-1) induced cellular DNA breaking of human promyelocytic leukemia HL-60 cells in phosphate buffer (pH 7.5) as assessed by alkaline single cell gel electrophoresis (comet) assay and quantification of comet types. Ascorbate and Trolox inhibited cellular DNA breaking induced by peroxynitrous acid, but the concentrations of these antioxidants required for effective inhibition was about 50-fold higher than that of peroxynitrous acid. beta-Carotene protected DNA breaking by peroxynitrous acid in 20% tetrahydrofuran-phosphate buffer (pH 7.5) much more effectively than ascorbate and Trolox. The concentrations of beta-carotene required for effective inhibition was lower than the concentration of peroxynitrous acid.

In the NO2-exposure of tyrosine in 70% dioxane/phosphate buffer (pH 7.4), beta-carotene enhanced the degradation of tyrosine and/or 3-nitrotyrosine produced, whereas alpha-tocopherol and ascorbyl palmitate inhibited the transformation of tyrosine into 3-nitrotyrosine. Generation of certain active species in the interaction of beta-carotene with NO2 was suggested. Ascorbyl palmitate effectively and alpha-tocopherol slightly inhibited the transformation of tyrosine in the NO2-exposure in the presence of beta-carotene. In the reaction of tyrosine with ONOO-/ONOOH, beta-carotene enhanced the degradation of 3-nitrotyrosine produced suggesting generation of certain active species, whereas alpha-tocopherol and ascorbyl palmitate completely suppressed the transformation of tyrosine into 3-nitrotyrosine.