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ABSTRACT: With the aim to analyze the ontogeny of the mouse endothelium, monoclonal antibody (mAb) MEC 13.3 was used for the immunohistochemical staining of frozen sections of different stages of mouse embryo development. This mAb specifically recognizes membrane reinforcement of endothelial cells (EC) from mouse blood vessels, indicating the expression of a molecule related to the murine form of PECAM-1/CD31. The present study reports the expression of the murine PECAM-1/CD31 antigen, observed with the peroxidase-antiperoxidase technique, in a single cell type, with a typical non-differentiated morphology at an early stage of mouse postimplantation embryos. A progressive increase in the number of this cell type was observed in the early stages of murine development, but few were detected at mature stages. On the other hand, EC in days 9.5, 14.5 and 19.5 postcoitum embryos were also recognized by the same mAb MEC 13.3 allowing the recognition of a cell type related directly or indirectly to vascular network development.
Cellular and molecular biology 06/1998; 44(3):537-41. · 0.98 Impact Factor
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ABSTRACT: In syngeneic mice, the H5V polyoma middle-T oncogene-transformed endothelioma cell line induces Kaposi's sarcoma-like cavernous haemangiomas that regress transiently, probably because of an anti-tumour immune response, but eventually grow progressively and kill the host. To evaluate the generation of tumour-specific cytotoxic T lymphocytes (CTLs), spleen cells of tumour-bearing mice were restimulated with irradiated H5V cells in mixed leucocyte-tumour cell cultures. Tumour-specific CTLs were demonstrable only when low numbers of H5V stimulator cells were used (<1 H5V cell per 50 splenocytes). We found that H5V cells secrete immunosuppressive mediators because CTL generation was blocked when H5V cells culture supernatants were added to allogeneic mixed leucocyte cultures. As numerous tumour-derived immunosuppressive mediators may interfere with interleukin 12 (IL-12) production, we tested whether IL-12 treatment of the tumour-bearing mice would augment their immune response and thus suppress tumour growth. Indeed, IL-12 inhibited tumour growth and prevented mortality, but did not increase anti-H5V CTL generation either in vitro or in vivo. Moreover, the anti-tumour activity in IL-12-treated mice was abrogated by anti-interferon (IFN)-gamma monoclonal antibody (MAb) co-administration. These results strongly suggest that the anti-tumour effect of IL-12 is principally mediated by IFN-gamma release that in turn blocks H5V cell proliferation and induces the release of factors that suppress angiogenesis.
British Journal of Cancer 02/1998; 77(4):656-62. · 5.04 Impact Factor
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ABSTRACT: In order to clearly visualize blood vessels, the monoclonal antibody (mAb) MEC 13.3 was used for an immunohistochemical staining on frozen sections of different mice mammary tumors. MEC 13.3 mAb is specific for endothelial cells (ECs) of mouse blood vessels and recognizes a molecule related to the murine form of CD31/PECAM. This mAb with immunoenzymatic technique or immunofluorescent labelling, was found to be a useful tool to quantify tumor neovascularization. Specifically, membrane reinforcement could be observed in vessel ECs, indicating the expression of CD31/ PECAM in their surface. The staining of ECs from tumors and from normal tissues was also compared. In this work, the use of MEC13.3 mAb is reported to recognize mice mammary tumor ECs as a useful tool to identify neovascularization. It would also be helpful for research on the origin and function of vascular endothelium in murine tumor experimental models.
Biocell: official journal of the Sociedades Latinoamericanas de Microscopía Electronica ... et. al 05/1997; 21(1):39-46. · 0.63 Impact Factor
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ABSTRACT: A retroviral construct encoding polyoma middle-sized T antigen was used to generate transformed endothelial cell lines from heart (H5V), brain (B9V), and whole-embryo (E10V) of C57BL/6 mice. When injected into syngeneic recipients, H5V and the less studied B9V and E10V cells caused vascular tumors which, depending on the number of cells inoculated, regressed or progressed, leading to death of the host. When H5V cells were injected into immunodeficient mice, tumors were observed with inocula which did not form lesions in immunocompetent recipients and regression did not occur. Treatment with anti-LFA-1, anti-Thy-1.2, and anti-CD8 antibodies abolished rejection; anti-CD4 was a somewhat less effective inhibitor of resistance. Animals with progressive tumors exhibited secondary lesions in various organs with prominent skin involvement in nude mice. Histologically, the tumors had the appearance of a hemangioma, with areas resembling Kaposi sarcoma. Cells lining vascular lacunae had the morphological features of injected H5V cells. The lesions were characterized by prominent neovascularization and mononuclear cell infiltration. Southern blot hybridization analysis revealed that approximately 5% of the cells in the tumor mass were transplanted H5V cells. Thus, the H5V transformed endothelial line causes vascular lesions that are sustained to a large extent by recruitment of host cells and manifests full malignant behavior only in immunocompromised hosts. The hypothesis of a tumor sustained by a minute proportion of transformed cells, which recruit host elements and express full malignant behavior only in immunodeficient hosts, would account for several features of some vascular neoplasms in man.
Proceedings of the National Academy of Sciences 08/1994; 91(15):7291-5. · 9.68 Impact Factor
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ABSTRACT: 1. Some studies of cyclophosphamide (CP) and its metabolite acrolein in chick embryo liver were carried out in order to investigate the mechanism of the porphyrinogenic action of CP. 2. In vitro and in vivo studies revealed that CP induced but did not activate delta-aminolaevulinic acid (ALA) synthase. 3. Pretreatments with phenobarbital (PB) or SKF-525A did not modify ALA-synthase induction produced by CP. 4. Acrolein administration neither induced ALA-synthase activity nor increased cytochrome P-450 content or led to hepatic porphyrin accumulation. 5. Time course induction of cytochrome P-450 content after administration of CP or PB was similar. 6. The results obtained would indicate that CP is a strong inducer of both ALA-synthase activity and microsomal cytochrome P-450 content in liver of 17 day-old chick embryos and that its porphyrinogenic activity is not mediated by its metabolite acrolein.
Comparative Biochemistry and Physiology Part C Comparative Pharmacology and Toxicology 06/1992; 102(1):143-8.
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ABSTRACT: The present study was undertaken to explore the effect of the presence of hepatic tumors induced by diethylinitrosamine (DENA) on the metabolic heme pathway, and to assess whether these tumors can modify the response of rats to the porphyrinogenic drug hexachlorobenzene (HCB) and whether the above mentioned effects occur to a greater extent in females than males. The results obtained showed that: a) Females were more susceptible to the hepatocarcinogenicity of DENA than males. b) Female normal and DENA treated rats were more susceptible than male rats to the porphyrinogenicity of HCB. c) The presence of hepatic DENA induced tumors could diminish basal hepatic ferrochelatase activity. d) Hepatic tumors could modify the response of animals to a porphyrinogenic drug such as HCB. Thus, both female and male DENA/HBC rats accumulated more porphyrins and showed a lower delta-aminolevulinate synthase and uroporphyrinogen I synthase induction than HCB rats. e) The heme pathway was functional in DENA induced tumors in both male and female rats but they were little affected by HCB.
Tumori 11/1991; 77(5):379-84. · 0.86 Impact Factor
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ABSTRACT: The response of female BDVI rats bearing diethylnitrosamine(DENA)-induced hepatic tumors to the porphyrinogenic action of hexachlorobenzene (HCB) was studied. (1) The heme pathway operates in these tumors but they were less affected by HCB than the liver. (2) Tumors did not accumulate porphyrins although the surrounding liver accumulated more porphyrins than livers treated with HCB. (3) DENA/HCB livers which developed a well defined tumor showed slightly less porphyrinogen carboxylyase inhibition and delta-aminolaevulinate synthase induction than HCB rats. (4) The results of the present work suggest that endogenously formed porphyrins would be unable to be accumulated by DENA-induced tumors when the tumoral development precedes the onset of the porphyria.
Cancer Letters 08/1991; 58(3):225-32. · 4.24 Impact Factor
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ABSTRACT: The response of animals bearing N-nitroso-N-methylurea (NMU)-induced mammary tumors to the porphyrinogenic action of hexachlorobenzene (HCB) was studied. delta-Aminolevulinic acid (ALA), porphobilinogen and porphyrins in urine, ALA-synthase and porphyrinogen carboxylase activities and porphyrin content in liver and tumor were measured. The results obtained indicate that the metabolic heme pathway operates in mammary tumors but tumor response to HCB treatment could not be detected. HCB administration produced an earlier and greater hepatic porphyria in tumor-bearing rats than in healthy rats suggesting that the presence of tumors exacerbates the action of HCB.
Cancer Letters 12/1990; 55(1):67-73. · 4.24 Impact Factor
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ABSTRACT: 1. The present work undertakes a comparative study on the hexachlorobenzene (HCB) porphyria induction in female rats of Wistar and CHBBTHOM strains. The purpose was to characterize the CHBBTHOM strain with respect to the haem metabolic pathway, its regulatory mechanisms and its response to foreign drugs. 2. After 7 weeks of treatment it was observed that the hepatic porphyrins increased 140 times, ALA-synthase 4 times and PCL was 73% inhibited in the Wistar strain. 3. On the other hand the animals of CHBBTHOM strain showed lesser alteration on these parameters; hepatic porphyrins increased only 3-fold, ALA-synthase 1.7-fold and PLC was only 22% inhibited. 4. Total iron liver content was nearly equal in both strains of rats. 5. The results obtained would indicate that the lower susceptibility of the CHBBTHOM strain to acquire porphyria does not seem to be due to either: (1) congenital alterations of any parameters of the haem metabolic pathway, since the behaviour of normal animals from both strains was similar; or (2) a lower hepatic iron content in such animals. 6. These findings would suggest that the differential response to HCB to this strain would be looked for in another metabolic pathway, such as that involved in the metabolization process of the toxic.
International Journal of Biochemistry 02/1989; 21(4):377-81.
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ABSTRACT: 1. The porphyrinogenic ability of several antineoplastics used in the therapy of the different cancers was evaluated. The action of cyclophosphamide, busulfan and 5-fluorouracil on the amount and nature of the accumulated hepatic porphyrins and on the activity of delta-aminolaevulinate synthase (ALA-S), were estimated at different doses and times of drug treatment in 17-day-old chick embryos. 2. It was observed that cyclophosphamide produces a significant increase in the accumulation of hepatic porphyrins at different doses as well as in the activity of the ALA-S, at all the incubation times. Cyclophosphamide alters the pattern of porphyrins accumulated in the liver, where a coproporphyrin: protoporphyrin ratio higher than in the controls can be observed. 3. Busulfan increased the hepatic porphyrins accumulated in the liver but to a lesser degree than cyclophosphamide. 4. 5-Fluorouracil did not modify the hepatic porphyrin content when it was administered at doses up to 40 mg/embryo. 5. When the embryos were injected with busulfan or 5-fluorouracil no significant differences were observed in the activity of ALA-S up to 11 hr of incubation. 6. These results indicate that cyclophosphamide has a remarkable porphyrinogenic capacity in chick embryo while busulfan, notwithstanding the fact that it alters the haem pathway, it does so to a degree that does not impair the regulation of ALA-S activity. Fluorouracil seems to be non porphyrinogenic in this system, up to 40 mg/embryo.
International Journal of Biochemistry 02/1988; 20(9):1015-20.
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ABSTRACT: The purpose of the present work is to investigate the ability of desferrioxamine (DF) as an iron chelator to revert or decrease a severe experimental porphyria induced by hexachlorobenzene (HCB) in rats; DF treatment started after 17 weeks of HCB intoxication and was continued until the 27th week. The urinary excretions of -aminolevulinic acid (ALA), porphobilinogen and porphyrins were weekly quantitated. At the end of the experiment the animals were sacrificed and hepatic porphyrins, ALA-synthase and porphyrinogen carboxy-lyase activities were determined. The results obtained indicated that, under the present conditions, the administered iron chelator does not improve the disturbance promoted by HCB on the haem pathway. These results were compared with those obtained when the DF was given simultaneously with HCB from the beginning of fungicide administration. In this last situation the chelator was able to delay and diminish the porphyrinogenic effect of HCB.
Acta physiologica et pharmacologica latinoamericana: organo de la Asociación Latinoamericana de Ciencias Fisiológicas y de la Asociación Latinoamericana de Farmacología 02/1987; 37(4):541-54.
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ABSTRACT: This study aimed to confirm the presence of an inhibitor in the hexachlorobenzene-porphyric liver that is able to decrease the normal activity of porphyrinogen carboxy-lyase (PCL), to determine whether any relation exists between the degree of hexachlorobenzene-induced porphyria and the ability of a porphyric liver preparation to reduce the enzyme activity of normal liver and to seek extraction methods in order to characterize the inhibitor by gas-liquid chromatography. A perfused liver supernatant (11,000 X g) filtered through a Sephadex G-25 column and heated for 5 min at 100 degrees C was used as the inhibitor source. The results show that the inhibitor was eluted together with a protein peak by gel filtration, the inhibitor was thermostable, the extent of the inhibitory effect reached by this preparation increased with the degree of porphyria, ether and toluene extracts from both heated and non-heated porphyric liver preparations also exhibited an inhibitory effect on the normal activity of PCL and the degree of inhibition depended on the amount of the preparation added. Therefore, there is an inhibitor of PCL activity in the hexachlorobenzene-porphyric liver, the concentration of which increases as the degree of porphyria increases. This inhibitor is soluble in organic solvents and is presently being characterized by gas-liquid chromatography.
IARC scientific publications 02/1986;
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ABSTRACT: This study was designed to determine whether iron contents are altered in hexachlorobenzene (HCB)-induced porphyria, and whether there is any relation between these alterations and the effects of the drug on several enzymes of the haem pathway. To this end, the effect of HCB administration on total, non-haem and haem iron levels was studied. Further, the effects of the addition of: both heated and non-heated HCB-porphyric liver preparations, iron as sulfate, ferritin and haemin and alpha alpha'-bipyridyl and 8-hydroxyquinoline were studied on the following enzymes: delta-aminolaevulinic acid synthase, delta-aminolaevulinic acid dehydratase, porphobilinogenase and porphyrinogen carboxy-lyase. Total and non-haem iron levels increased significantly as a result of HCB intoxication, but there was a non-significant decrease in haem iron content. The increased iron levels did not appear to be directly involved in the increased activities of delta-aminolaevulinic acid synthase and delta-aminolaevulinic acid dehydratase observed in HCB-induced porphyria, since it was not possible to detect any activation in heating and crossed assays nor by the addition of inorganic iron, protein-iron or haemin. Results from heating and crossed assays suggested the existence of an activator for porphobilinogenase and an inhibitor for porphyrinogen carboxy-lyase, while results obtained with chelating agents suggested that iron could partly account for the activation of porphobilinogenase. Iron was not directly involved with the decreased activity of porphyrinogen carboxy-lyase, since neither iron chelators nor different types of iron produced physiologically significant effects.
IARC scientific publications 02/1986;
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ABSTRACT: The effect of desferrioxamine on hexachlorobenzene (HCB)-induced porphyria was studied in female rats in order to investigate the role of iron in the development of this porphyria. Repeated injections of desferrioxamine delayed and remarkably diminished the urinary excretion of precursors and porphyrins and the accumulation of porphyrins in the liver. These effects were produced because the desferrioxamine attenuated the alterations produced by HCB in two key enzymes: porphyrinogen carboxy-lyase and delta-aminolaevulinic acid synthase. The effect of desferrioxamine on both enzymes was also studied in vitro. This work showed that iron plays an important role in the onset of HCB-induced porphyria and supplied information on the mechanism of action. A common iron-involving mechanism for the production of porphyria by different chlorinated compounds is suggested.
IARC scientific publications 02/1986;
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ABSTRACT: Studies were carried out to elucidate if in the hexachlorobenzene (HCB) porphyria, total, nonhaem and haem iron contents in liver are altered and if any relation exists between these alterations and the hepatic porphyrinogen carboxy-lyase (PCL) decrease in rats treated with the drug. It was observed that in porphyric livers total and non-haem iron levels increased significantly as a consequence of HCB intoxication while this treatment produced a non significant decrease in the haem iron content. Enzymic preparations of porphyric livers filtered through Sephadex G-25 columns which separate the free iron and that has a content of iron-protein greater than those in normals, exhibited a strong inhibition of PCL. Chelating agents, alpha' alpha' bipyridyl and 8-hydroxyquinoline do not revert such inhibition. The effect in vitro of ferritin, haemin and inorganic iron at different concentrations on normal PCL activity was also assayed. So, it could be observed that inorganic iron and haemin produce slight inhibition of PCL when added in concentrations higher than those corresponding to a porphyric liver (0.08 mM and 10(-6) M, respectively, as mean in the incubation media). So, they have not physiological significance. Ferritin does not modify the decarboxylation process. From these results it arises that iron does not play a direct role in the decrease of PCL activity in the experimental porphyria by HCB, not being the inhibitor made evident by heating assays. Iron could perhaps stimulate the metabolization of HCB, giving rise to active metabolite.
Acta physiologica et pharmacologica latinoamericana: organo de la Asociación Latinoamericana de Ciencias Fisiológicas y de la Asociación Latinoamericana de Farmacología 02/1985; 35(4):481-91.
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ABSTRACT: The effect of a pretreatment with phenobarbitone (PB) on the porphyrinogenic action exerted by hexachlorobenzene (HCB) was examined in female rats. Kinetic studies of enzyme function after HCB poisoning showed that porphyrinogen carboxy-lyase was the only enzyme of haem biosynthesis that markedly lowered its activity. Both stages of uroporphyrinogen (UPG) III decarboxylation were decreased. This enzyme, together with UPG I synthase (increased levels) were the first enzymes altered. Subsequently, an increase in delta-aminolaevulinate (AmLev) synthase and ferrochelatase was detected; AmLev dehydratase was the last to increase. On long-term exposure, PB alone did not modify the basal values of haem intermediates; only the content of cytochrome P-450 increased. All the enzyme activities studied showed no significant changes, except ferrochelatase, which increased. With both drugs the metabolic impairment promoted by HCB was accelerated and enhanced by prior PB treatment leading to the onset of an earlier and stronger porphyria. A more noticeable accumulation and excretion of higher carboxylated porphyrins and precursors was more promptly observed as a consequence of the early porphyrinogen carboxy-lyase blockade and the concomitant induction of AmLev synthase. Although the enzymic activities of both AmLev dehydratase and ferrochelatase were enhanced, this response differed in time. For UPG I synthase this pretreatment elicited lower values than those found in the HCB group. Cytochrome P-450 contents were immediately and slightly enhanced by all the drugs, but the values for the combined treatment were the lowest. Of the several hypotheses that could explain the action of HCB on the haem pathway, our results would suggest that the porphyrinogenic action of HCB is mediated by some of its metabolic products.
Biochemical Journal 04/1984; 218(3):753-63. · 4.90 Impact Factor
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ABSTRACT: The present work tries to elucidate if the strong decrease of porphyrinogen carboxy-lyase (PCL) observed in the experimental porphyria caused by hexachlorobenzene (HCB) is due to the presence of an inhibitor or to a modification of the protein structure of the enzyme. For this purpose: a) cross-assays and heating ones were performed in order to look for the existence of a compound, that present in porphyric animals, would be responsible for the decrease of PCL activity found in them; b) the effect in vitro of HCB, HCB metabolites and other related compounds was studied to find the inhibitor of PCL activity and to look for a relation structure-inhibitory effect; c) hepatic PCL from porphyric and normal rats was purified, and enzyme properties were comparatively studied looking for structural differences between the enzymes obtained from both animal lots. The results indicate that: a) the heat deproteinized porphyric liver preparation produces an inhibition on the normal preparation, but it is smaller than the decrease produced in vivo by the HCB on this enzyme activity, thus other reasons may explain this behavior of PCL in intoxicated animals; b) the HCB had no effect on PCL activity, the phenolic compounds exhibited inhibitions of variable extent that were increased by the presence of electrophilic substituents on the benzenic ring. Pentachlorophenol, the main HCB metabolite, produced inhibition in the in vitro assays, but at doses that were not physiologically significant; thus, it seems not to be the inhibitor found in the heating assays; c) purification of 110 times for the hepatic PCL of both porphyric and normal rats was obtained. Incubation conditions, the effect of salts and chelating agents, the chromatographic behavior in DEAE-cellulose and Sephadex G-100 columns, were comparatively studied with both enzymatic preparations. The effect of sodium diethyldithiocarbamate, sodium pyrophosphate, dithiothreitol, temperature, pH and O2, as well as the chromatographic behavior, would suggest that structural differences in the PCL of porphyric animals may exist; the presence of a thermostable inhibitor could also contribute to the decrease of PCL activity due to HCB.
Acta physiologica et pharmacologica latinoamericana: organo de la Asociación Latinoamericana de Ciencias Fisiológicas y de la Asociación Latinoamericana de Farmacología 02/1984; 34(4):393-407.
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ABSTRACT: Red cell porphyrinogen carboxy-lyase activity was measured using uroporphyrinogen III as substrate in 18 normal persons, 7 male patients with porphyria cutanea tarda, 3 female patients with erythropoietic protoporphyria and 2 female patients with variegate porphyria. The mean values obtained in normal subjects were 0.151 nmol of uroporphyrinogen disappeared in 30 min per mg of protein, and 0.038 nmol of coproporphyrinogen formed in 30 min per mg of protein. We have not been able to detect significant differences between males and females. In porphyria cutanea tarda the enzyme activity was the same as in normal subjects considering either substrate disappearance or end product formation. The differences were not significant at the p less than 0.05 level. Patients with variegata porphyria also exhibited normal erythrocyte porphyrinogen carboxy-lyase activity. The enzyme activity of erythrocytes from patients with erythropoietic protoporphyria was higher than in normals; mean values for specific activities being 0.204 nmol of uroporphyrinogen disappeared, and 0.071 nmol of coproporphyrinogen formed. The significance of the results with respect to the chemical picture of different porphyrias is discussed.
Clinica Chimica Acta 01/1981; 108(3):447-56. · 2.54 Impact Factor
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International Journal of Biochemistry 02/1980; 12(5-6):1027-32.
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International Journal of Biochemistry 02/1980; 12(5-6):1039-44.
