The influence of different fixatives and a tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on the induction of so-called dark cells in mouse epidermis. A light microscopical study

Epidermal "dark cells" (DC) are believed to play a specific role in the so-called promotion phase of experimental skin carcinogenesis. They are recognized by their morphological features both at the light and the electron microscopical level. The possible effects of fixation on the morphology of epidermal cells and hence on the number of DC have not yet been thoroughly studied. In the present light microscopical study we used a semiquantitative method together with simple cell counting to evaluate the influence of fixation on the specific cellular morphology which is traditionally used to determine the number of DC. The use of cacodylate vehicled prefixatives, either formaldehyde or glutaraldehyde, led to a higher incidence of DC, and furthermore both to an increased width of the intercellular spaces (ICS) and a more heavy staining of the keratinocytes than when s-collidine vehicled glutaraldehyde was used. Differences in yield of DC solely due to the prefixative itself (formaldehyde or glutaraldehyde) were not detected. Exposure to TPA or the use of a hyperosmolal prefixative vehicle both yielded higher DC numbers than did controls or conventional prefixative vehicles, respectively. After prefixation with hyperosmolal vehicles, however, TPA treatment did not induce higher DC yield than in a control series. Phenomena usually accompanying exposure to TPA, such as intercellular oedema (widening of the ICS) and cytoplasmic vacuolization, varied in parallel to the number of DC. Hence, there is reason to believe that the induction of epidermal DC is mainly associated with volume reduction of keratinocytes. Such shrinkage may be due to the cytotoxic properties of TPA and degenerative phenomena appearing during tissue processing.     

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on newly synthesized proteins in mouse epidermis

We have analyzed the effects of treatment of mouse epidermis with the potent tumor promoter TPA on the profile of newly synthesized proteins. TPA was applied to the skin of the intact mouse, and either 3 or 24 hr later skin fragments were pulse-labeled in vitro with ³⁵S-methionine for 4 hr. The epidermal proteins were extracted and separated by two-dimensional gel electrophoresis. Over 200 individual proteins were resolved in acidic gels. At least 10 of these showed major (by a factor of 5 or more) increases or decreases in response to TPA; eight of these appear to be keratin proteins. Two-dimensional gel profiles of basic proteins synthesized by mouse epidermis resolved over 100 individual proteins. Only one of these showed a significant change in response to TPA. This 41 kd protein increased more than 100-fold within 24 hr after the application of TPA. Treatment of mouse skin with mezerein, a plant diterpene structurally related to TPA, produces an almost identical change in the pattern of proteins produced. Four agents that induce hyperplasia but are not potent tumor promoters, ethylphenylpropiolate, acetic acid, turpentine oil and the Ca⁺⁺ ionophore A23187, modulate the synthesis of only three of the keratin proteins. Thus the changes in protein profiles induced by TPA and mezerein are not simply the consequence of hyperplasia. In addition, application to mouse skin of a glucocorticoid that is a potent inhibitor of tumor promotion inhibits most of the changes in protein profiles induced by TPA. Taken together, these results indicate that TPA and mezerein induce early and marked changes in the profile of specific epidermal proteins. It seems likely that some of these changes are directly related to the process of tumor promotion.     

12-0-Tetradecanoylphorbol-13-acetate induced alterations in mouse epidermal 3-hydroxy-3-methylglutaryl CoA reductase

Topical application of the tumor promoter 12-0-tetradecanoylphorbol-13-acetase to the mouse skin resulted in a significant initial increase (at 2 h) in the activity of epidermal hydroxymethylglutaryl CoA reductase followed by a significant decrease by 24 h. Phorbol and phorbol diacetate, which do not cause hyperplasia or promote skin tumor formation, has no effect on this enzyme. Ethylphenylpropiolate, a strong inducer of hyperplasia but a non-tumor promoter, also showed no effect on the reductase activity. Topical application of the products of hydroxymethylgutaryl CoA reductase viz. mevalanolactone or isopentenyl adenosine caused a decrease while cholesterol produced no change in hydroxymethylglutaryl CoA reductase activity. It is speculated that isopentenyladenosine or a closely related product, formed by the initial increase in hydroxymethylglutaryl CoA reductase activity following TPA application, might exert feedback inhibition.     

12-O-tetradecanoylphorbol-13-acetate induces selective desquamation of superficial cells in rat urinary bladder epithelium

Summary 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to affect the proliferation and/or differentiation of several types of cells. We injected TPA directly into the lumen of rat bladder to determine, using scanning and transmission electron microscopy, its effects on the bladder epithelium in vivo. At 1 h after TPA injection (1g/ml), the superficial cells of the epithelium had changed their morphology, and large spherical vacuoles occupied their cytoplasm. In some areas, the underlying intermediate cells were exposed by the desquamation of the superficial cells. During the next few hours, TPA was excreted from the bladder lumen by voluntary micturition, but the desquamation of the superficial cells proceeded further. All the superficial cells were lost from the luminal surface by 24 h after TPA injection. The changes noted were specific for the superficial cells and were not observed in the intermediate or basal cells. After 24h, part of the epithelium had a three-layer structure, indicating that regeneration was taking place. These results demonstrate that TPA selectively affects and desquamates superficial cells in a short period of time. This experimental system may be useful for studying in vivo cell proliferation and/or differentiation.     

Tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) can reduce the Ca-transporting ability of Ca-ionophores in T lymphocytes: the involvement of intracellular heavy metal ions

12-0-tetradecanoyl-phorbol-13-acetate (TPA) can significantly reduce the Ca-ionophore-induced rise in the intracellular calcium concentration (Cai) of T lymphocytes measured by quin2 or fura-2 fluorescence. This counteraction of TPA is maximal at a preincubation of 90 min at TPA concentrations higher than 20 nM. 45Ca uptake and efflux measurements directly indicate that TPA does not activate the calcium extrusion systems in thymocytes but impairs the Ca-transporting ability of Ca-ionophores. TPA causes no immobilization of the Ca-ionophores as it is demonstrated by the lack of significant changes in fluorescence and fluorescence polarisation of A23187 during TPA incubation. Similarly the energy transfer between the Tyr, Try groups of membrane proteins and A23187 shows no significant difference in control and TPA treated thymocytes. This indicates that A23187 is not in a membrane protein-bound form after TPA preincubation. The intracellular heavy metal chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) restores the ionophoretic ability of Ca-ionophores in TPA pretreated cells to the control level. Diacyl-glycerols also impair the Ca-transporting ability of Ca-ionophores. TPEN prevents this effect as well. These findings suggest that TPA and diacyl-glycerols may cause an increase in the availability of intracellular heavy metal ions. Our results may reflect a new, physiologically important mechanism of the action of diacyl-glycerols and phorbol esters.     

Antitumour activity of crocetin in accordance to tumor incidence, antioxidant status, drug metabolizing enzymes and histopathological studies

Lung cancer is the leading cause of cancer related mortality worldwide. Crocetin, saffron plant derivative known to play a role in cancer chemoprevention. In the present study the effects of crocetin was tested against lung cancer-bearing mice in both pre-initiation and post-initiation periods. Healthy male Swiss albino mice (6–8 weeks old) were used throughout the study. Experiment was designed with the treatment regimen of crocetin [20 mg/kg body weight dissolved in dimethyl sulphoxide (DMSO)] for 4 weeks before (pre-initiation) and from 12^th week after Benzo(a) pyrene B(a)p (50 mg/kg body weight) induced lung carcinoma(post-initation). The level of lipid peroxidation (LPO) and marker enzymes markedly increased in carcinogen administered animals, which was brought back to near normal by crocetin treatment. The activities of the enzymic antioxidants and glutathione metabolizing enzymes were decreased in B(a)p induced animals and increased upon drug treatment. Crocetin profoundly reverted back the pathological changes observed in cancerous animals. From the results crocetin proves to scavenge free radical and plays an important role in cellular function. Tumor incidence and histopathological studies proves crocetin is a potent antitumour agent.     

Effects of a tumour promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on expression of differentiated phenotype in the chick retinal pigmented epithelial cells and on their interactions with the native basement membrane and with artificial substrata

Chick retinal pigmented epithelial (RPE) cells grown in vitro on basement membrane matrices from the Engelbreth-Holm-Swarm tumour (BM-matrigel) do not spread, and they maintain their differentiated phenotype, most notably the heavy pigmentation. Maintenance of the differentiated phenotype by RPE cells on BM-matrigel is promoted not only by the biochemical composition of the gel but also by its mechanical properties, i.e., its low rigidity prevents cell spreading. In this report, RPE cells on BM-matrigel were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to promote the transformed phenotype and diminish cell traction. In contrast to most cell types TPA treatment induced RPE cells to increase their spread area. TPA promoted RPE cell spreading on BM-matrigel and changed the spatial organization of actin and actin-associated proteins in the cytoskeleton-ECM linkage complexes, uncoupling actin from its extracellular counterpart. TPA did not affect other components of the cytoskeleton in RPE cells. TPA also affected labile adhesions i.e., focal contacts and adherens junctions in statu nascendi, but preformed, stable adherens junctions were resistant to TPA. TPA enhanced proliferation, blocked melanogenesis and thus inhibited differentiation of RPE cells grown on either artificial substrata or their natural basement membrane.     

The tumor promoting phorbol diester, 12-0-tetradecanoylphorbol-13-acetate (TPA) increases glyoxalase I and decreases glyoxalase II activity in human polymorphonuclear leukocytes

Glyoxalase I and II catalyze the formation and breakdown of S-lactoylglutathione respectively. Recent studies have implicated this com-pound as a possible mediator of immune and inflammatory responses. Incubation of human polymorphonuclear leukocytes with the tumor promoter, 12-0-tetradecanoylphorbol-13-acetate has been found to affect the activities of both glyoxalase enzymes in an interrelated manner. The diester either increases the activity of glyoxalase I or decreases the activity of glyoxalase II or has both effects. It is suggested that a subsequent increase in S-lactoylglutathione might mediate some or all of the effects of the phorbol diesters.     

苯并(a)芘对鲫鱼肝脏EROD活性的影响

研究了典型多环芳烃类有机污染物苯并(a)芘(BaP)对鲫鱼(Carassius auratus)肝脏7-乙氧基-3-异吩唑酮-脱乙基酶(EROD)活性的影响。结果表明,注射后96 h,10和100 mg.kg-1处理组肝脏EROD活性被明显诱导,分别为对照组的2.3(P<0.05)和3.1倍(P<0.01)。肝脏EROD活性随着时间延长继续升高,至注射后14 d,1 mg.kg-1处理组鲫鱼肝脏EROD活性为对照的3.0倍(P<0.001),而100 mg.kg-1处理组则高达5.8倍(P<0.001)。鲫鱼肝脏EROD活性可作为反映BaP暴露水平的生物标志物。BaP对鲫鱼的最低效应浓度为1 mg.kg-1(鱼体重)。     

Copper thionein in the kidneys of copper-poisoned sheep

A study has been made of the distribution of copper in the kidneys of copper-poisoned sheep, containing up to 240 μg copper/g fresh cortex. About 64% of the copper in the cortex was present in the cytosol and 75% of this occurred in a form with molecular weight of approx. 12 000. This was partially purified by gel filtration on Sephadex G-75 and Bio-Gel P.10 and ion exchange chromatography on DEAE Sephadex A-25 to give three sub-fractions, which also contained zinc. The amino acid composition, copper content and chromatographic behaviour of these proteins indicated that they were copper-thioneins.No significant amounts of the proteins were detected in the plasma or erythrocytes of the copper-poisoned sheep when they were undergoing the haemolytic crisis typical of this syndrome. It is concluded that metallothionein constitutes the major copper-binding protein in the kidneys of copper-poisoned sheep. However the rapid accumulation of the protein in the kidney, and the development of kidney damage, are unlikely to have arisen from the release of the intact copper-protein from the liver and its transport via the blood to the kidneys.     

Restoration by parathyroid hormone and dibutyryl cyclic AMP of expression of the differentiated phenotype of chondrocytes inhibited by a tumor promoter, 12-0-tetradecanoylphorbol-13-acetate

12-0-Tetradecanoylphorbol-13-acetate (TPA) inhibited expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture. TPA transformed typical polygonal chondrocytes into multilayered, fibroblastic cells and also inhibited the rate of [35S]sulfate incorporation into glycosaminoglycan (GAG), a differentiated phenotype of chondrocytes. These changes were apparent within 24 h and reached a plateau at 48 h after the addition of TPA. Phorbol didecanoate and phorbol dibenzoate also inhibited sulfation of GAG, even though the effect was weaker than that of TPA. Phorbol diacetate and 4-0-methyl TPA did not inhibit sulfation of GAG. Addition of parathyroid hormone (PTH) or dibutyryl cyclic AMP simultaneously with TPA overcame the inhibition caused by TPA. PTH and dibutyryl cyclic AMP also reversed the inhibition and stimulated expression of the differentiated phenotype of chondrocytes even in de-differentiated cells which had been pretreated for 3 days with TPA. These findings suggest that cyclic AMP plays an important role in the restoration of the differentiated phenotype of chondrocytes in TPA-treated chondrocytes, and that the TPA-treated cells retain some of the differentiated phenotype of the original cells, such as responsiveness to PTH.     

Redox Properties Of Phenols,Their Relationships To Singlet Oxygen Quenching And To Their Inhibitory Effects On Benzo(A)Pyrene-Induced Neoplasia

The inhibitory effects of synthetic phenolic compounds on benzo(a)pyrene-induced neoplasia of the mouse forestomach have been measured by Wattenberg et al⁶ The efficiency of this inhibition has been estimated for each phenol, using R, the ratio of the number of tumors per mouse in the protected group over the number of tumours per mouse in the control group. We have observed a linear correlation between the chemoprotection efficiency R and the logarithm of the rate of quenching of singlet oxygen. k. by this family of phenols, log k being itself correlated with the one-electron oxidation potential of the phenols. These correlations suggest a charge transfer mechanism for the inhibition of neoplasia induced hy benzo(a)py-rene. The correlations described provide a theoretical basis for scaling the inhibitors of mutagenicity induced by polycyclic aromatic compounds in terms of their oxidation potentials     

Redox Properties Of Phenols, Their Relationships To Singlet Oxygen Quenching And To Their Inhibitory Effects On Benzo(A)Pyrene-Induced Neoplasia

The inhibitory effects of synthetic phenolic compounds on benzo(a)pyrene-induced neoplasia of the mouse forestomach have been measured by Wattenberg et al⁶ The efficiency of this inhibition has been estimated for each phenol, using R, the ratio of the number of tumors per mouse in the protected group over the number of tumours per mouse in the control group. We have observed a linear correlation between the chemoprotection efficiency R and the logarithm of the rate of quenching of singlet oxygen. k. by this family of phenols, log k being itself correlated with the one-electron oxidation potential of the phenols. These correlations suggest a charge transfer mechanism for the inhibition of neoplasia induced hy benzo(a)py-rene. The correlations described provide a theoretical basis for scaling the inhibitors of mutagenicity induced by polycyclic aromatic compounds in terms of their oxidation potentials     

Effects of Benzo(a)pyrene on Seabass (Dicentrarchus labrax L.): Biomarkers,Growth and Behavior

The main purpose of this study was to investigate the effects of benzo(a)pyrene (BaP) on seabass (Dicentrarchus labrax) juveniles using parameters at different levels of biological organization. Liver antioxidant status, BaP biotransformation and accumulation, growth, and behavior were determined in juveniles after 28 d exposure to BaP (1–16 μ g/l). Liver ethoxyresorufin O-deethylase increased in seabass exposed to 1–8 μ g/l of BaP. Liver glutathione S-transferases and catalase activities were significantly increased at 4 and 8 μ g/l, but a slight decrease was observed at the highest concentrations tested. Bile BaP metabolites were significantly different from the control group at 1 and 16 μ g/l BaP. Liver BaP metabolites and lipid peroxidation significantly increased at 8 and 16 μ g/l BaP. These results suggest that BaP metabolites' accumulation induces oxidative damage in seabass liver. Body weight and length increase were significantly reduced in fish exposed to BaP, with LOECs of 16 and 4 μ g/l, respectively. Food intake and swimming velocity were significantly decreased after exposure to BaP, with LOEC values of 16 and 8 μ g/l, respectively. Results suggest that at concentrations of BaP equal or higher than 8 μ g/l, the detoxification capacity decreases, an accumulation of liver BaP metabolites occurs causing lipid peroxidation, affecting growth and swimming capability of fish.     

Metabolism of Benzo(a)pyrene by Human Epithelial and Fibroblastic Cells: Metabolite Patterns and DNA Adduct Formation

We demonstrate in cell culture that mammary epithelial cells from normal human breast specimens metabolize benzo(a)pyrene (BaP) and form adducts with the bases of their DNA more readily and at lower concentrations of BaP than do fibroblasts from the same specimens. BaP metabolism and adduct formation was determined in the same incubations with epithelial cells grown out in early passage from each of three specimens and with fibroblasts from one of these specimens. The metabolite pattern of the epithelial cells was indicative of preferential formation of 7, 8-dihydrodiol-9, 10-dihydroepoxybenzo(a)pyrene the ultimate carcinogen. In contrast, fibroblasts formed mainly mono- and dihydroxide derivatives of BaP. The metabolite pattern from epithelial cells was compatible with the ease in which adducts between DNA and the diolepoxide of benzo(a)pyrene were formed. These results provide evidence that chemical carcinogens should be considered as possible factors in the induction of breast cancer in women.     

Effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate on peroxisomal activities and enzyme activities involved in lipid metabolism in rat liver

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on hepatic lipids and key enzymes involved in esterification, hydrolysis and oxidation of long-chain fatty acids at increasing doses were investigated in rats. TPA administration tended to decrease the mitochondrial activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase. The microsomal palmitoyl-CoA synthetase activity was increased. TPA administration was also associated with a dose-dependent increase of glycerophosphate acyltransferase activity both in the mitochondrial and microsomal fractions in particular. The data are consistent with a decreased catabolism of long-chain fatty acids at the mitochondrial level, and an increased capacity for esterification of fatty acids in the microsomal fraction. Peroxisomal beta-oxidation was increased about 2-fold in the peroxisome-enriched fraction of TPA-treated rats while the catalase and urate oxidase activities were only marginally affected. TPA administration revealed elevated capacity for hydrolysis of palmitoyl-CoA and palmitoyl-L-carnitine in the microsomal fraction. Neither increased cytosolic palmitoyl-CoA hydrolase activity nor increased hydroxylation of lauric acid nor changes of the hepatic content of cytochrome P-450 isoenzymic forms were observed in the TPA-treated animals. There was no induction of the protein content of the bifunctional enoyl-CoA hydratase. Thus, TPA behaves more like choline-deficient diet and ethionine treatment than well-known peroxisome proliferators. It seems possible that TPA selectively stimulated the peroxisomal activities, i.e., peroxisomal beta-oxidation rather than evoking a peroxisome proliferation capacity.     

A simple assay of aryl hydrocarbon hydroxylase in cultured human lymphocytes

A novel technique is described for assay of aryl hydrocarbon hydroxylase in cultured human lymphocytes. The sensitivity is equal to that of previous methods, but this method requires fewer manipulations. One million lymphocytes are incubated for one hour with 2 micrograms of benzo(a)pyrene in a glass cuvette. The reaction is stopped by addition of neutral formalin and the cell suspension is alkalinized with NaOH. Fluorescence intensity of the suspension is measured with excitation at 465 nm and emission at 520 nm.     

Biodegradation of benzo(a)pyrene by a newly isolated Fusarium sp

Benzo(a)pyrene (BaP) is a five-ring polycyclic aromatic hydrocarbon produced by the incomplete combustion of organic materials. It is one of the priority pollutants listed by the US Environmental Protection Agency. This study describes a fungal isolate that is able to biodegrade benzo(a)pyrene. The filamentous fungus, isolated from leaves of Pterocarpus macrocarpus Kurz., was identified as a Fusarium sp. (strain E033). Fusarium sp. E033 was able to survive in the presence of benzo(a)pyrene concentrations up to 1.2 mM (300 mg L(-1)). Biodegradation experiments using 0.4 mM (100 mg L(-1)) benzo(a)pyrene demonstrated that Fusarium sp. E033 was able to degrade 65-70% of the initial benzo(a)pyrene provided, and two transformation products, a dihydroxy dihydro-benzo(a)pyrene and a benzo(a)pyrene-quinone, were detected within 30 days of incubation at 32 degrees C. The factors affecting biodegradation efficiency were also investigated. While increasing aeration promoted better fungal growth and benzo(a)pyrene biodegradation, increasing the glucose concentration from 5 to 50 mM had an adverse effect on biodegradation. Ethanol and methanol, provided at 5 mM to increase benzo(a)pyrene water solubility, increased the fungal biomass yield but did not promote degradation. The Fusarium sp. E033 isolated in this study can tolerate and degrade relatively high concentrations of benzo(a)pyrene, suggesting its potential application in benzo(a)pyrene bioremediation.