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Tyrosinase is known to be a key enzyme in melanin biosynthesis, involved in determining the color of mammalian skin and hair. Various dermatological disorders, such as melasama, age spots, and sites of actinic damage, arise from the accumulation of an excessive level of epidermal pigmentation. The inadequacy of current therapies to treat these conditions as well as high cytotoxicity and mutagenicity, poor skin penetration, and low stability of formulations led us to seek new whitening agents to meet the medical requirements for depigmenting agents. The inhibitory effect of licorice extract on tyrosinase activity was higher than that expected from the level of glabridin in the extract. This led us to test for other components that may contribute to this strong inhibitory activity. Results indicated that glabrene and isoliquiritigenin (2',4',4-trihydroxychalcone) in the licorice extract can inhibit both mono- and diphenolase tyrosinase activities. The IC(50) values for glabrene and isoliquiritigenin were 3.5 and 8.1 microM, respectively, when tyrosine was used as substrate. The effects of glabrene and isoliquiritigenin on tyrosinase activity were dose-dependent and correlated to their ability to inhibit melanin formation in melanocytes. This is the first study indicating that glabrene and isoliquiritigenin exert varying degrees of inhibition on tyrosinase-dependent melanin biosynthesis, suggesting that isoflavenes and chalcones may serve as candidates for skin-lightening agents.

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To investigate the fate of nitric oxide (NO) synthesized by activated macrophages, the concentrations of NO and its principal reaction products, nitrite (NO2-) and nitrate (NO3-), were measured as a function of time in suspension cultures of RAW264.7 macrophages attached to microcarrier beads. Synthesis of NO became evident 2-5 h after stimulation of the cells, and steady concentrations of NO were achieved after about 9 h. The appearance of NO in the extracellular fluid coincided with the appearance of NO2- and NO3-, which were formed thereafter at approximately equal and constant rates. Using a kinetic model based on rate constants measured previously in cell-free systems, only half of the NO2- formed could be accounted for by the reaction of NO with O2. It is known that NO reacts with superoxide (O2.) to give peroxynitrite and that NO also reacts with peroxynitrite to yield NO2-, so that the latter reaction may explain the "excess" NO2- formation. Adding superoxide dismutase to the medium markedly reduced the ratio of NO3- to NO2-, consistent with the hypothesis that NO3- in the medium results primarily from the extracellular reaction of NO with O2-.. The addition of morpholine, a model amine, resulted in formation of N-nitrosomorpholine, concurrent with the other products. Measured rates of nitrosomorpholine formation were 6-fold lower than predictions based on kinetics in simple solutions, suggesting that in the cell culture system there were additional reactions that lowered the concentration of nitrous anhydride, the principal nitrosating agent formed from NO and O2.

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Inflammatory cells such as phagocytes, neutrophils, and macrophages have been implicated in the pathogenesis of several forms of clinical and experimental tumor development. It is hypothesized that this process is mediated by the production of reactive species including NO., O2.-, H2O2, and ONOO- which inflict DNA damage. In this study, the role of NO. in combination with oxygen radicals in DNA damage was investigated. DNA deamination (xanthine) and oxidation [5-(hydroxymethyl)uracil (5HMU), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPY-G), and 8-oxoguanine (8oxoG)] products were identified in the DNA of macrophages (RAW264.7) activated with Escherichia coli lipopolysaccharide (LPS) and mouse gamma-interferon (INF-gamma). The formation of these products was inhibited by N-methyl-L-arginine (NMA), a nitric oxide synthase inhibitor. NMA inhibited only the production of nitric oxide and had no effect on superoxide production. These results demonstrate that NO. plays a dual role in damaging the DNA of activated macrophages. Autoxidation of NO. leads to nitrosating species which cause deamination of bases. Reaction of NO. with O2.- leads to DNA oxidative damage due to the formation of peroxynitrite which may have HO.-like oxidizing potential. Another possible mechanism of oxidative damage by NO. could be the mobilization of free iron by NO. which could ultimately cause Fenton-type reactions. Therefore, nitric oxide not only leads to deamination of DNA bases but is also an obligatory factor in oxidative damage to DNA.