E. Hecker

On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene: III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene☆☆☆

Abstract Previous studies have shown that the carcinogen N -hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N -acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H 2 O 2 . The free radical intermediate was detected by its ESR signal, and the yields of N -acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N -acetoxy- N -2-acetyl[2′- 3 H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N -guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N -acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.

On the metabolic activation of N-hydroxy-N-2-acethylamino-fluorene: II. Simultaneous formation of 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene from N-hydroxy-N-2-acetylaminofluorenevia a free radical intermediate☆☆☆

Abstract The proximate carcinogen N-hydroxy-N-2-acetylaminofluorene is readily converted by non-enzymatic one-electron oxidants (e.g. [Fe(CN)6]3−) into an unstable nitroxide radical. This radical undergoes disproportionation in organic or aqueous solutions to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene. The simultaneous formation of these two compounds from the nitroxide radical is of interest as a possible mechanism of metabolic activation in carcinogenesis by N-hydroxy-N-2-acetylaminofluorene. The same type of reaction was also observed with several analogous compounds, i.e.N-hydroxy-N-2-benzoylaminofluorene, N-hydroxy-N-2-tetradecanoylaminofluorene, and N-hydroxy-N-4-acetylaminotoluene. The chemical properties and time course of disintegration of the intermediate nitroxide radicals were investigated with spectroscopic and ESR measurements, and a possible mechanisms for the reaction is discussed.

On the active principles of Croton Oil

Resolution of fraction Bx from Croton Oil by Craig-distribution reveals 5 new irritant and cocarcinogenic compounds B3–B7. By physical methods such as mass-spectrometry and NMR-spectroscopy combined with chemical degradation these compounds are shown to be diesters of the polyfunctional Diterpen alcohol Phorbol C20H28O6 each with a short chain (acetic-, (+)-S-2-methylbutanoic-, tiglic acid) and a long chain fatty acid (octanoic-, decanoic-, dodecanoic acid). The new compounds are being characterized by their irritant and cocarcinogenic activity in mice. Die Auftrennung der früher beschriebenen Fraktion Bx aus Crotonöl durch Craig-Verteilung führt zu fünf bisher unbekannten entzündlichen und cocarcinogenen Wirkstoffen B3–B7, die rein dargestellt werden. Durch physikalische Methoden wie Massenspektrometrie und NMR-Spektroskopie kombiniert mit chemischen Abbauverfahren wird gezeigt, daß diese Wirkstoffe Diester des polyfunktionellen Diterpenalkohols Phorbol C20H28O6 mit jeweils einer kurzkettigen (Essig-, (+)-S-2-Methylbutter-, Tiglinsäure) und einer langkettigen Fettsäure (Capryl-, Caprin-, Laurinsäure) sind. Die neuen Wirkstoffe werden durch ihre entzündliche und cocarcinogene Wirkung an Mäusen Charakterisiert.

Two isozymes of PKC found in HL-60 cells show a difference in activation by the phorbol ester TPA

Cytosol from untreated cells and a detergent extract of the particulate fraction from TPA‐treated HL‐60 cells were analyzed for protein kinase C activity by consecutive column chromatography on Mono Q and hydroxyapatite. From both preparations two separate peaks of enzyme activity were obtained. The first peak, eluting at lower salt concentrations, is activated at lower TPA concentrations (3 × 10−9 M) than the other (10−7 M), which was eluted at higher salt concentrations.

Induction of epstein-barr virus early antigens by tumor promoters of the diterpene ester type in Raji cells and specific (receptor) binding as compared to irritant and promoting activities

SummarySixteen new diterpene esters (DTE) of the tigliane, ingenane, daphnane, and 1α-alkyldaphnane types were investigated in two in vitro assays: as inhibitors of specific binding of 3H-labeled 12-O-tetradecanoylphorbol 13-acetate (TPA) to protein kinase C in a receptor preparation from mouse brain, and as inducers of Epstein-Barr virus (EBV) early antigens in Raji cells. Inhibition of binding of [3H]TPA to the receptor preparation by tigliane and ingenane DTE correlates with irritant activity in vivo, while some daphnane and 1α-alkyldaphnane DTE inhibit binding of [3H]TPA in a less pronounced manner but still are very irritant. Tumor-promoting activity does not correlate consistently with the receptor-binding data. To test the hypothesis that early antigen induction in Raji cells by DTE is coupled to functional DTE receptors (protein kinase C), the latter were searched on these Raji cells by a ‘cold acetonefilter assay’ and shown to be present. The dependence of the early antigen induction rate on the concentration of the DTE tested was demonstrated. At a given concentration of DTE, differences in the induction rate between various DTE are seen. However, a clear quantiative correlation either between early antigen induction and receptor binding data in vitro, or early-antigen-inducing activity in vitro versus irritancy and tumor-promoting activity in vivo was not observed.

Phospholipids involved in specific binding of 12-O-(5-azido-2-nitrobenzoyl)phorbol-13-acetate to epidermal microsomes, a photolabeling study

The preparation of 12-O-(5-azido-2-nitro)benzoylphorbol-13-acetate (NABPA) is described. It is used as a photoaffinity probe to study the biochemical components involved in the specific binding of phorbol esters to an epidermal particulate fraction (microsomes) from NMRI mice: without irradiation NABPA binds in a saturable and high affinity manner (KD = 12 nM; Rt = 2.6 pmol/mg protein) to microsomes; after irradiation (at 350 nm) specific photolabeling (representing specific binding of NABPA) is found of phospholipids (phosphatidyl-serine (PS) and -ethanolamine(PE)), but not of protein. The results are discussed in the context of protein kinase C being a receptor for phorbol esters.

Toxicodynamics of tumour promoters of mouse skin. III: Specific binding of the tumour promoter thapsigargin as measured by the cold-acetone filter assay

SummaryA method is described for measuring rapid, specific, and saturable binding of the skin irritant and tumour-promoting secretagogue thapsigargin (sesquiterpene lactone) to the microsomal fraction from mouse brain. Employing the tritium-labelled compound its apparent dissociation constant,Kd, and the maximal amount of bindingBmax are shown to be 9.8 nM and 1.9 pmol/mg protein respectively. Such aKd for thapsigargin is similar to (a) its IC50 value for inhibiting Ca2+ uptake in the microsomal fraction from rat brain and (b) its EC50 values for inducing a rise in the cytoplasmic Ca2+ concentration of human platelets and histamine release from rat peritoneal mast cells. A positive correlation is found between the binding affinities of thapsigargin, thapsitranstagin, and trilobolide, their potencies as secretagogues and their lipophilicities. This correlation does not extend to the skin-irritant activities of the compounds thus emphasizing that their mechanism of action is unlike that of 12-O-tetradecanoylphorbol 13-acetate.

Toxicodynamics of tumour promoters of mouse skin

SummaryIn binding competition assays using a protein kinase C preparation from mouse brain (particulate fraction)3H-labelled 12-O-tetradecanoylphorbol-13-acetate (TPA), for a series of new diterpene esters (DTE) the relative binding affinity [rba=Kia(TPA)/Kia(DTE)] in relation to TPA was determined. A wide range of values was noticed, some of the DTE binding more strongly than TPA (rba >1), others binding less strongly than TPA (rba <1) In comparative terms, competition for specific binding sites appears to correlate better with irritant than with promoting acitvity of the DTE. Using mouse peritoneal neutrophils, binding of [3H]-TPA was determined by a modification of the “cold-acetone filter assay”; saturation of high-affinity sites (Kda=0.2 nM) was obtained at concentrations ≦ 1 nM, but there was also evidence for specific binding at “low-affinity” sites (Kda=26 nM). Induction of chemoluminescence in the presence of luminol in mouse peritoneal neutrophils with a set of DTE usually elecited two peaks; at concentrations ≧10 nM DTE a short-lived, “spike-like” response lasting only from 0 to about 5 min (phase A) ist followed by a “plateau” response from about 5–120 min (phase B). This latter phase of chemoluminescence stimulation with luminol correlated well with theirritant potential of the DTE used. The sequence of the two phases can be inverted partially by using first TPA at 2,5 nM followed by a quick concentration increase to 100 nM; this indicates two different concentration-dependent events. As regards the intensity of the chemoluminescent response, quantitative but not qualitative differences between DTE were observed, which show some correlation with strong and weak tumour-promoting activity. Inhibition studies suggest the involvement of the myeloperoxidase/H2O2/Cl− system in the luminogenic response; it is suggested that the release of hypochlorite or a closely related oxidant may be instrumental in tumour promotion.

NEW INSIGHTS INTO THE MOLECULAR STRUCTURE OF THE AGONISTS BINDING SITE OF PROTEIN KINASE C BY PSEUDORECEPTOR MODELING

Protein kinase C (PKC) comprises a family of isoenzymes with serine/threonine kinase activity similar in molecular size, structure and mechanism of activation. This enzyme family is an important part of one of the major signal transduction pathways regulating many intracellular processes such as modulation of gene expression, cell proliferation and differentiation. PKC was identified as the major cellular receptor for skin tumor promoting phorbol esters and certain other compounds exhibiting skin tumor promoting bioactivity. Moreover, PKC is a target for molecules with antineoplastic activity like bryostatin. Thus by various investigators it is considered as a potential target for discovery and development of new anticancer drugs. On the other hand for PKC so far no potent and isoenzyme selective activators or inhibitors are known, and a 3-D structure of the enzyme is not available. Therefore a new technique of molecular modeling was investigated to design the agonist binding site of PKC: ‘Pseudoreceptor modeling’. It is a comprehensive strategy in the design of potent, selective and novel ligands for unknown receptors (Vedani et al., 1993). The new approach is focused on the binding site of the receptor and allows the construction of hypothetical 3-D-models of binding pockets using the directionality of receptor-ligand interactions (hydrogen bonds, metal-ligand interaction, hydrophobic interactions). In the investigation described an ensemble of six ligands, binding all specifically to the regulatory domain of the enzyme and representing together a pharmacophore model for activation of PKC, was successfully used. The pseudoreceptor model of PKC will be presented. It may be used as a surrogate of the agonist binding site of PKC to guide the synthesis of new and selective PKC-agonists allowing further investigation of PKC-isoforms in the signal transduction pathway. In addition it may be of assistance in the development for new antineoplastic drugs.

EPR of Hydrophobic Interactions

Abstract Molecules distributing between the membrane and solution are exposed to several different binding regions of the membranes. Binding of molecules to membranes, glass surfaces and a protein was compared. The specific structural differences are equally pronounced in the first two systems, although the protein binding is less dependent on the structural differences of the hydrophilic part of the molecule.