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E Hecker

Publications and source records attributed to E Hecker.

At least 19 recordsLinked to original sources
PubMed1992

Short-term assays for detection of conditional cancerogens. I. Construction of DR-CAT Raji cells and some of their characteristics as tester cells.

A number of agents including the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) (TPA) can induce an abortive virus cycle in the EBV-non-producer Burkitt's-lymphoma line Raji. Two distant regions, DL and DR, of the EBV genome with almost complete homology carry strong promoters which are induced in an abortive or lytic cycle and additionally function as lytic origins of viral DNA replication. To set up a system in which the activity of EBV-inducing agents can be measured in a quantitative and reproducible fashion, we generated a cell line which carries multiple copies of a DR-promoter chloramphenicol-acetyltransferase (CAT) construct on an episomal vector. CAT activity is low in untreated cells, but high upon treatment of the cells with various EBV-inducing agents. Combinations of different agents can produce an over-additive effect. The Raji-DR-CAT cell line may provide a simple quantitative and reproducible test system for EBV-inducing agents, especially for tumor promoters which activate protein kinases C.

Carcinogens↗
PubMed1992

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

A 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 binding Bmax are shown to be 9.8 nM and 1.9 pmol/mg protein respectively. Such a Kd 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.

Animals↗
PubMed1992

Spin-labeled phorbol esters and their interactions with cellular membranes--IV. Lipophilic binding and molecular orientation of spin-labeled phorbol-12,13-diesters in human erythrocyte membrane.

In human erythrocyte membranes, membrane binding of spin-labeled TPA-analogous phorbol (doxyl)esters [(n,m)PA] was investigated during measurement of the kinetics of the decay of their electron paramagnetic resonance signal by ascorbate reduction. In membrane-bound (n,m)PA the reduction rate was dependent of the position of doxyl in the aliphatic chain of their 12-O-acyl moiety. To describe quantitatively the reaction kinetics observed, two hypotheses (models) were developed and used. Model 1 is based on the assumption that ascorbate reduction takes place in the extracellular space. In this case the experimental data could be fitted by the partition and permeability coefficients of (n,m)PA determining model 1 only, if non-realistic values of these parameters were used. The more refined model 2, corresponding to a bilayer membrane structure, assumes the reduction to take place in the hydrophilic region of the membrane. Assuming a finite probability of finding the doxyl group within the hydrophilic membrane region, model 2 describes quantitatively the dependence of the reduction rate on the position of the doxyl in the aliphatic chain of the (n,m)PA used. From the validity of this model it may be postulated that the molecular orientation of TPA-analogous (n,m)PA in the bilayer membrane is determined by an anchoring of their lipophilic ester moiety in the lipophilic region of the membrane bilayer, thus locating the hydrophilic phorbol moiety within the hydrophilic region of the membrane. With regard to the well-known categories of non-specific versus specific binding of bioactive phorbol esters to protein kinase C/membrane complexes it is deduced that anchoring of (n,m)PA (and hence TPA) in the hydrophobic interior of the membrane structure may be the molecular equivalent of their non-specific binding.

Ascorbic Acid↗
PubMed1992

Spin-labeled phorbol esters and their interactions with cellular membranes--V. Electron paramagnetic resonance of spin-labeled phorbol-12,13-diesters bound to their receptors in mouse brain particulate fraction.

The relatively small concentrations required for in vivo bioactivity of diterpene ester skin irritants and promoters (approximately 10 nmol per animal; approximately 10 nM in cell cultures) has discouraged studies of EPR spectra of bioactive, TPA-analogous, spin-labeled phorbol-12,13-diesters [(n,m)PA] bound to their membrane receptors, protein kinases C (PKC). To meet the requirements of present EPR spectrometers, particulate fraction from mouse brain containing at least 25 x 10(-12) mol of receptors/mg protein (PKC species) were employed together with certain (n,m)PA selected to give an optimal ratio of specific to non-specific binding. For selection and optimization of experimental conditions, a theoretical model was developed that considers all characteristic parameters of the system. By fitting the model calculations to the experimental data of competitive agonist displacement from the particulate fraction of tritium-labeled TPA, the dissociation constants Kd for four selected (n,m)PA used as antagonists were determined. Optimal experimental conditions are met by (5,6)PA and by (5,8)PA, in that for both compounds the relative amount of displaced (n,m)PA is in accordance with the predictions derived from the model. Moreover, the model turned out also to be reliable for samples containing either small or large amounts of membranes. To obtain an EPR spectrum of an agonist bound to brain particulate fraction, the (5,6)PA was used. It shows a broad EPR spectrum typical for an immobilized molecule. The spectrum changes if an excess of TPA is added to the system; the slight differences in shape are due to displacement of (5,6)PA from specific receptor sites by non-labeled TPA and show up as a decreased central peak amplitude. This is the first time that the agonist/receptor interaction of a diterpene ester type irritant and tumor promoter has been demonstrated by direct spectroscopic measurement.

Animals↗
PubMed1992

A skin irritant phorbol ester from Euphorbia cooperi N E Br.

From the fresh latex of Euphorbia cooperi N E Br was isolated by partition and chromatographic methods, a diterpene ester 12-deoxyphorbol-16-isobutyrate-13-tigliate. The phorbol ester exhibited highly irritant activity on the mouse ear. Since skin irritancy is an indication of possible tumour promotion, the use of this plant as a medicine should be discouraged.

Animals↗
PubMed1991

Toxicodynamics of tumour promoters of mouse skin. II. Binding to protein kinase C of some new diterpene esters and induction of luminol-enhanced chemoluminescence in mouse peritoneal neutrophils.

In 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 greater than 1), others binding less strongly than TPA (rba less than 1) In comparative terms, competition for specific binding sites appears to correlate better with irritant than with promoting activity 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 less than or equal to 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 greater than or equal to 10 nM DTE a short-lived, "spike-like" response lasting only from 0 to about 5 min (phase A) its followed by a "plateau" response from about 5-120 min (phase B). This latter phase of chemoluminescence stimulation with luminol correlated well with the irritant 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.

Animals↗
PubMed1991

Biological assays for irritant, tumor-initiating and -promoting activities. III. Computer-assisted management and validation of biodata generated by standardized initiation/promotion protocols in skin of mice.

The initiation/promotion standard protocol 28 (protocol 28), developed and used previously as an experimental model to verify the cancerogenic process of initiation/promotion in mouse skin, was revised in three aspects: (a) statistically it was shown sufficient to use, per promoter dose group, 16 colony-outbred female NMRI mice: (b) by weekly individual records of tumor response (and health status) of each mouse in a dose group, cumulative tumor incidences (and mean and extreme body weights) are determined; from these data the collective records (tumor response, health status), the only data accessible from protocol 28, may be generated in addition; (c) the details of dose groups and all data on tumor response and health status are processed by computer using the program package PAPILLOM. The latter was developed specifically for this purpose, is written in the programming language APL and designed for easy handling by staff of animal laboratories. The program package calculates, from the individual records per promoter dose group, cumulative tumor incidences (and survival data) with confidence limits for any one exposure time, and the package may be linked to programs for statistical validations. In addition, from the collective records it calculates the tumor rates, tumor yields and survival rates for any one exposure time. These data, obtained by either of the standard protocols (16 or 28), are fully comparable. For pure compounds they may be used to calculate semiquantitative tumor-promoting potencies. These values for more than 80 polyfunctional diterpenes of the tigliane, ingenane and daphnane type, scattered in or calculated from previous papers, together with their irritancies, were compiled. Within recent years, computer-assisted standard protocol 16 has been used to handle and evaluate about 1000 promoter dose groups. Protocol 16 allows one to extract and utilize more and better toxicological information on tumor response and health status from any one dose group, utilizing significantly fewer experimental animals than required by protocol 28. Thus, the computer-assisted standard protocol 16 optimizes the utility of the experimental model of mouse skin for the amount, quality and management of experimental data as well as for the requirements of animal protection.

Animals↗
PubMed1991

Toxicokinetics of tumour promoters of mouse skin. II. Metabolism of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate in mouse skin and biological activities of metabolites.

The metabolism of the phorbol diester [20-3H]12-O-tetradecanoylphorbol-13-acetate [( 3H]TPA) was studied in the back skin of NMRI mice after topical administration of a single tumour-promoting dose, dp. Up to 72 h after administration most of the radioactivity recovered from the skin surface, and from the epidermis and dermis of the treated skin area was unchanged TPA, as determined by silica gel HPLC of extracts obtained from these skin fractions. The major TPA metabolite was less polar than TPA and chromatographed in the range of long-chain TPA-20-acylates. At 72 h, it accounted for about 25, 5 and 30% of total radio-activity extracted from skin surface, epidermis and dermis respectively. Of metabolites more polar than TPA, phorbol-13-acetate (PA) by far predominated over 12-O-tetradecanoylphorbol (TP) in both the epidermis and dermis, and by 72 h its relative amount was 3.9 and 2.4% in these skin fractions. Both phorbol monoesters, PA and TP, were not detected in skin surface extracts. In addition to metabolites, various autoxidation products of TPA were present in small amounts in the extracts from each of the skin fractions. The TPA-20-acylate fraction of metabolites isolated from the extracts of skin fractions at 24 h was separated further into the individual metabolites by the combined use of argentation and reversed-phase HPLC. These individual metabolites were identified by co-chromatography with authentic reference compounds. They were TPA-20-acylates carrying saturated fatty acids (16-26 carbon atoms), cis-mono-unsaturated fatty acids (16-24 carbon atoms), linoleic acid and arachidonic acid. The conjugation of TPA with long-chain fatty acids is the first example of a new route of xenobiotic metabolism in skin. When tested for irritant activity on the mouse ear, TPA-20-acylates were about one to two orders of magnitude less active than TPA. Similarly, when TPA-20-tetradecanoate was tested for tumour-promoting activity on the back skin of NMRI mice over a dose range of dp = 50-200 nmol applied twice weekly according to the computer-assisted standard protocol 16, it was found to be of only intermediate potency as compared to the highly potent tumour promoter TPA. The results of the present investigation indicate that metabolic conjugation of TPA with long-chain fatty acids to yield TPA-20-acylates is another pathway of metabolic deactivation of TPA, thus supporting the hypothesis that TPA itself is the ultimate tumour promoter in mouse skin.

Animals↗
PubMed1990

Comparative tumorigenicity of picene and dibenz[a,h]anthracene in the mouse.

The carcinogenic activity of the two polycyclic aromatic hydrocarbons (PAHs), picene (benzo[a]chrysene) and dibenz[a,h]anthracene (DBA), was determined in NMRI mice by five different experimental protocols in order to find out if picene is a carcinogen as predicted by recent quantum mechanical calculations in contrast to earlier observations which could not confirm any carcinogenic activity of picene. Single s.c. treatment of adult mice with picene or DBA (308 nmol/animal, each) led to the formation of fibrosarcomas in 63.3% of treated animals regardless of the PAH used. Chronic epicutaneous application of both PAHs (total dose 1.36 mumol) to the back of mice resulted in the development of papillomas with a tumor rate of 22% in the case of picene and of 32% in the case of DBA. When newborn mice were s.c. treated once on day 2 of their life with each of the two PAHs (400 nmol/animal), 27.8% of treated animals developed lung adenomas after 40 weeks in the case of picene compared to 92.1% in the case of DBA. Histopathological examination of the tumors in the three experimental models revealed no difference in the type of tumor between picene and DBA. Epicutaneous application of both PAHs (600 nmol/animal) followed by chronic treatment with 12-O-tetradecanoyl-phorbol-13-acetate for 24 weeks led to the formation of papillomas in 93% of animals treated with DBA while picene showed no tumorigenic activity at all. Initiation of tumorigenesis in the two-stage tumor model with 7,12-dimethylbenz[a]anthracene (1 mumol/animal) and chronic treatment with picene (total dose 4.8 mumol) for 24 weeks was equally ineffective in producing tumors in NMRI mice. This rare biological property of picene, which is a complete carcinogen, yet at most a very weak tumor initiator, is explained in terms of its inefficient biotransformation to mutagenic and carcinogenic metabolites as compared to the strong tumor initiator DBA.

Animals↗
PubMed1990

Toxicokinetics of tumor promoters of mouse skin. I. Metabolism of phorbol and ingenol esters by mouse liver microsomes.

The metabolism of ten selected phorbol esters and of one ingenol ester was investigated in mouse liver microsomes fortified with cofactors. The major metabolites were generated by an esterolytic activity in the microsomes. The following 12,13-diesters were cleaved readily to yield the 13-monoesters: 12-O-tetradecanoylphorbol-13-acetate, phorbol-12,13-dipropionate, bis(13-O-acetylphorbol)-12, 12'-tetradecanedioate, phorbol-12,13-dibenzoate; the monoesters 12-O-tetradecanoylphorbol and 3-O-tetradecanoylingenol yielded the corresponding parent alcohols. Other 12,13-di- or 13-monoesters such as 'inverse TPA', i.e. 12-O-acetylphorbol-13-tetradecanoate, phorbol-12,13-didecanoate, 12-O-retinoylphorbol-13-acetate, phorbol-13-decanoate and 12-O-tetradecanoyl-4 alpha-phorbol-13-acetate were either comparatively or completely resistant to esterolysis. The data indicate that metabolism of diterpene esters depends on the nature and position of the acyl moiety, as well as on the structure of the diterpene moiety. The esterolytic activity in mouse liver microsomes can be inhibited by i.p. administration of bis(4-nitrophenyl)phosphate to mice. Evidence for other relevant metabolic pathways besides esterolysis was lacking.

Animals↗
PubMed1990

[Analysis of data management in anesthesia from an ergonomic viewpoint].

In the perioperative phase the anesthetist has to manage an increasing amount of knowledge, information and data. Using a system-ergonomic approach we can define three types of data management (DM): Exploratory DM, Operative DM, Concluding DM. The preliminary examination of the patient is Exploratory DM. Data are collected and recorded. Here, a well structured form prevents things being forgotten, provides forgetting anything. Help from electronic devices is not available. Control of anaesthesia is based on Operative DM. The anesthetist is part of an ongoing process. He investigates and records a situation based on his knowledge and experience and a prompt reaction to untoward circumstances may be necessary. Today's workplace provides insufficient support for this task. Data presentation is unstructured and distributed around the workplace which produces potentially dangerous overloading in critical situations. It is necessary to view the work layout as an integrated whole. The data being displayed must be hierarchically structured and appropriate to the situation. Concluding DM involves summarising data and information on completion of a process in ways appropriate to specific purposes. With this the anesthetist completes an anaesthesia and transfers the patient to the next unit, e.g. to the recovery room. He has to fill in several forms for clinical and statistical reasons. Electronic aids are available only for parts of some tasks. The goal should be a multifunctional summary satisfactory for clinical and statistical purposes, most aspects of which are created automatically by a computer system.

Anesthesiology↗
PubMed1989

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 X 10(-9) M) than the other (10(-7) M), which was eluted at higher salt concentrations.

Animals↗
PubMed1989

Biological assays for irritant, tumor-initiating and tumor-promoting activities. II. Standardized initiation/promotion protocol and semiquantitative estimation of promoting (or initiating) potencies in skin of NMRI mice.

A versatile standardized protocol of initiation/promotion of skin is described using 7,12-dimethylbenz[a]anthracene as initiator and diterpene esters as promoters in 28 female NMRI mice per promoter (or initiator) dose group including appropriate positive and negative controls ("standardized initiation/promotion protocol 28" or "protocol 28"). To monitor the weekly tumor responses, the tumor rate Tr and the tumor yield Ty were recorded per dose group collectively as a measure of the tumor-promoting (or initiating) activity of individual compounds; similarly the general health status of the group was controlled twice weekly by collective weighing of the dose group and recorded daily by the survival rate Sr. Synoptic consideration of Tr, Ty and the promoter dose (or initiator dose) at a set time period of exposure was used for the semiquantitative estimation of the relative tumor-promoting (or initiating) potency, referring to a high-potency promoter (or initiator) as standard. The full scope of utilization of "protocol 28" to determine comparable individual promoting activities and -at 24 weeks of exposure--relative tumor-promoting potencies (rtpp24) is demonstrated in typical examples with 12-O-tetradecanoylphorbol 13-acetate (TPA) as well as various other phorbol esters as promoters. For estimation of promoting potencies TPA is rated a rtpp24 of + + + +. The other phorbol derivatives exhibit rtpp24 between 0 and + + + +. In this manner, "protocol 28" was verified in 1000 promoter dose groups (i.e. 28,000 mice) to determine tumor-promoting activities of individual diterpene esters and partly also their rtpp24. Grading of tumor-promoting potencies of diterpene esters in skin by rtpp24 proved useful for evaluating the relative environmental risk involved in exposure to various diterpene esters as well as in comparative mechanistic investigations including structure/activity relationships. As an example, the correlation of irritancy and tumor-promoting potency of certain diterpene esters in epithelial tissue, postulated previously from essentially qualitative data, is verified in semiquantitative terms by comparison--as a typical example--of the irritancy of phorbol esters on the ear and of the corresponding rtpp24 on the back skin of NMRI mice.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

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.

Sixteen new diterpene esters (DTE) of the tigliane, ingenane, daphnane, and 1 alpha-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 1 alpha-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 acetone-filter 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 quantitative 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.

Animals↗