The cytotoxicity of gamma-L-glutaminyl-4-hydroxybenzene for cells that contain tyrosinase, a study of melanocytes in the hair follicle of the mouse.

The s.c. administration of gamma-L-glutaminyl-4-hydroxybenzene (GHB) to neonatal black mice produced a prompt, generalized, and selective swelling and lysis of the melanocytes of the hair follicles. The findings indicate that this cytotoxic effect was dependent upon the intracellular activation of GHB by tyrosinase. Supportive of this conclusion were: (a) an absence of comparable cytological alterations in adjacent keratinocytes; (b) a lack of response by melanocytes of albino mice; and (c) patterns of deficient pigmentation produced by GHB in juvenile black mice, suggesting that susceptible follicles were those in the tyrosinase-producing growth phase. The administration of GHB also induced condensation, or "apoptosis," of individual follicular keratinocytes of both black and albino mice and in the melanocytes in the latter. This response was apparently independent of tyrosinase. It was transitory and without appreciable effect on hair growth. The findings further characterize the selective cytolytic properties of GHB for mammalian cells that possess tyrosinase and suggest a potential for this natural compound as a chemotherapeutic agent against melanocarcinoma.

gamma-L-Glutaminyl-4-hydroxybenzene, an inducer of cryptobiosis in Agaricus bisporus and a source of specific metabolic inhibitors for melanogenic cells.

A stable phenol, gamma-L-glutaminyl-4-hydroxybenzene (GHB), is oxidized by tyrosinase in the gill tissues of the mushroom Agaricus bisporus to a quinone and a second oxidation product which together suppress mitochondrial energy production and the synthesis of proteins and nucleic acids in the zygote, thus establishing dormancy in the spores. Brief incubation of cultured murine L1210 leukemia and B-16 melanoma cells with muM concentrations of the purified quinone notably prolonged survival times or blocked tumor growth in histocompatible mice inoculated i.p. with high concentrations of the exposed cells. The instability of the quinone precluded in vivo administration. The short incubation of cultured B-16 melanoma cells with mM concentrations of GHB markedly prolonged survival times or abolished tumor growth in histocompatible C57BL/6J mice inoculated i.p. with 5 X 10(6) exposed cells. This response did not occur with L1210 leukemia cells, which lack the enzyme tyrosinase. The survival times of mice bearing B-16 melanoma, but not of those with L1210 leukemia, were slightly prolonged by a single injection and were significantly extended by daily i.p. injections of GHB. Normal C57BL/6J mice, given GHB i.p. as single or multiple 400-mg/kg doses, manifested no systemic toxicity but showed depigmentation of the hair after 2 to 3 weeks. These studies provide evidence that GHB exerts cytotoxicity specifically for cells that by their content of tyrosinase convert the phenol to the quinone. This targeted response minimizes systemic toxocity and underscores the potential therapeutic application of this agent to melanocarcinoma.

Cytostatic, cytocidal and potential antitumor properties of a class of quinoid compounds, initiators of the dormant state in the spores of Agaricus bisporus.

Evidence indicates that dormancy is initiated in the spores of Agaricus bisporus by two quinoid compounds that appear in the zygote during the prodromal period of sporulation. Both are derivatives of a phenol, gamma-L-glutaminyl-4-hydroxybenzene. When purified, these quinoids specifically inhibit mitochondrial respiratory enzymes and protein synthesis in the mushroom and have comparable effects with rat liver mitochondria and ribosomes, with intact bacteria, and with bacterial ribosomes and RNA polymerase in vitro. Five species of mouse ascites tumor cells showed prompt and marked inhibitions of nucleic acid and protein synthesis when millimolar concentrations of these quinoids were added to the tissue culture medium of the tumor cells. Only a small percentage of the cells was killed immediately, as judged by trypan blue uptake. When large numbers of exposed BP8 sarcoma and EL4 leukemic cells were reinjected intraperitoneally into histocompatible mice, the survival times of these animals were notably prolonged beyond those of animals injected with tumor cells that had not been exposed to these inhibitors. In a dose-dependent manner, increasing concentrations of inhibitors produced proportionate increments in survival time, while higher concentrations totally abolished tumor cell growth. The findings indicate that these simple quinoid compounds, which initiate the dormant state in spores, produce a cytostatic state in mammalian tumor cells and thus potentially have strong antitumor properties (Am J Pathol 78:33-48, 1975).

Bacteriocidal properties of a class of quinoid compounds related to sporulation in the mushroom, Agaricus bisporus.

A small phenolic compound, gamma-L-glutamyl-4-hydroxybenzene, and several quinoid derivatives appear in the gill tissues of the mushroom, Agaricus bisporus, during the prodromal period of sporulation. These quinoids markedly inhibit respiratory enzymes and protein synthesis. The temporal relationship of their appearance to sporulaton and their properties as metabolic inhibitors indicate that they initiate and maintain the dormant or cryptobiotic state of the spore. In very low concentrations, these quinoids show significant bacteriocidal action against a variety of microorganisms. The therapeutic potential of these antibiotic properties is considered.

Concerning the role of mitochondria in cryptobiosis.

The metabolic characteristics of the mitochondria of Agaricus bisporus are altered in the zygote by specific inhibitors that permit them to retain structural integrity in the dormant spore and enable them to initiate energy production, with apparent protein synthesis and replication during the initial phase of germination. The insensitivity of the earliest events of germination to selective cytoplasmic and nuclear inhibitors characterizes this as a transient period of unusual mitochondrial autonomy. To define the intrinsic metabolic potentials of the organelle and its role in cryptobiosis, mitochondria were fractionated aseptically from presporulating zygotes and were placed in dialysis chambers surrounded by nutrient media at 15 C. For periods through 48 hours, the isolated mitochondria manifested the capacity to incorporate labeled amino acids linearly into proteins and retained stable electrophoretic protein profiles for more than 5 days. They maintained fine structural integrity for at least 10 days, some developed septational membranes, and they increased numerically. These metabolic activities were dependent upon a nutrient substrate.