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R Montesano

Publications and source records attributed to R Montesano.

At least 217 records · Page 12Linked to original sources
PubMed1983

Differential filipin labeling of the luminal membranes lining the pancreatic acinus.

The cytochemical labeling of cholesterol by filipin in plasma membranes of the exocrine pancreas has shown a difference in the reactivity to filipin between the acinar and centro-acinar cells. The acinar cell has a poorly labeled luminal membrane but numerous filipin-cholesterol complexes on the basolateral membrane. In contrast, the centro-acinar cell shows a comparable degree of filipin labeling on both luminal and basolateral membrane. This difference in the distribution of filipin labeling suggests underlying differences in the organization of the respective membranes, probably related to their specific functions.

Animals↗
PubMed1983

Stability and capacity of dimethylnitrosamine-induced O6-methylguanine repair system in rat liver.

Repeated administration of dimethylnitrosamine (DMN) (2 mg/kg for 3 weeks) to BD IV rats results in an increased capacity for the liver to repair O6-methylguanine in DNA, whereas other DNA adducts (7-methylguanine and 3-methyladenine) are not affected. In the experiments reported here, data on the rapidity of action of the enhanced system, its capacity after different challenging doses of [14C]DMN (0.2, 2.0, and 20 mg/kg), and the persistence after cessation of the DMN pretreatment are described. The results show that, after a dose of [14C]DMN (2.0 mg/kg), the increased activity acts very rapidly (10 min) repairing O6-methylguanine as soon as it is formed and that, by 2 hr, 65% of the O6-methylguanine generated in liver DNA has already been removed. Very little removal of O6-methylguanine occurs within this time in control rats not receiving any DMN pretreatment. The DMN-induced repair activity is of limited capacity, since its effect can be detected after DNA damage induced by 0.2 or 2.0 mg of [14C]DMN per kg, whereas this activity has little impact on the O6-methylguanine generated in liver DNA by 20 mg of [14C]DMN per kg. Upon cessation of the DMN pretreatment, the enhanced repair activity, as determined also by the in vitro activity of the O6-methyltransferase, decays slowly, but after 25 days, the repair activity is still higher than control values. No correlation was observed between increased [3H]thymidine incorporation in liver DNA and increased O6-methylguanine repair, indicating that liver cell proliferation is not necessarily coupled with an elevated methyltransferase level.

Alkylation↗
PubMed1983

Carcinogenicity of single doses of N-nitroso-N-methylurea and N-nitroso-N-ethylurea in Syrian golden hamsters and the persistence of alkylated purines in the DNA of various tissues.

The carcinogenicity of N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) has been determined in adult male Syrian golden hamsters following a single i.p. injection or two-thirds of the acute 50% lethal dose, or 30 and 60 mg/kg, respectively. The principal site of action of these agents was the forestomach, squamous cell papillomas of this organ developing in 53 and 61% of the animals receiving the higher doses of NMU and N-nitroso-N-ethylurea, respectively. NMU also induced a low incidence of liver tumors (17%). Very few tumors were seen at other sites. The formation and removal of alkylated purines in DNA was measured in various tissues up to 50 hr after administration of [14C]NMU. Methylation products were detected in all tissues examined, the level in liver being somewhat higher than in other tissues. The removal of 7-methylguanine and 3-methyladenine from DNA occurred at approximately similar rates in all tissues examined, indicating no substantial differences in N-glycosylase activities. Removal of the promutagenic DNA lesion O6-methylguanine varied considerably from tissue to tissue; very little occurred in brain or kidney, while up to 36 and 32% were lost from DNA of intestine and testes, respectively. In the liver, there were relatively small changes in O6-methylguanine levels up to 24 hr; but by 50 hr, 38% had been removed. The persistence of O6-methylguanine relative to 7-methylguanine was highest in the DNA of the brain and intestine and lowest in that of the liver. These results indicate that in this experimental system, the formation and persistence of O6-methylguanine in DNA is insufficient alone to account for the organotropic effect of NMU.

Alkylation↗
PubMed1982

Removal of O6-methylguanine from DNA by human liver fractions.

In in vitro assays using methylated DNAs as substrates, human liver fractions were shown to be able to catalyze the removal of O6-methylguanine. The amount of removal was proportional to the amount of protein added, and the loss of O6-methylguanine occurred with stoichiometric formation of guanine in the DNA and S-methylcysteine in protein. This indicates that human liver contains a protein similar to that previously found in bacteria exposed to alkylating agents. This protein acts as a transmethylase, transferring the intact methyl group from O6-methylguanine in DNA to a cysteine residue on that protein. A similar activity is present in rodent liver, but it was found that human liver was about 10 times more active in carrying out this reaction. In contrast, there was no difference between the human and rat liver extracts in catalyzing the loss of another methylation product, 7-methylguanine, from alkylated DNA. The liver is the organ most likely to be alkylated after exposure to exogenous potential alkylating agents such as dimethylnitrosamine. The present results show that human liver has a significant capacity to repair O6-methylguanine in DNA, which has been implicated as a critical product in carcinogenesis and mutagenesis.

Animals↗
PubMed1982

Benzo[a]pyrene metabolites: formation in rat liver cell-culture lines, binding to macromolecules, and mutagenesis in V79 hamster cells.

Benzo(a]pyrene was metabolized in liver cell lines derived from BC-IV and BD-IV rats which included several chemically-transformed lines (IAR-6-1; IAR-19; IAR-28), one spontaneous transformant (IAR-27) as well as one non-malignant line (IAR-20). Cultures were treated with tritiated benzo[a]pyrene over a 5-day period. The cells and medium were extracted with ethyl acetate and the distribution between organic-soluble and water-soluble metabolites determined. Organic-soluble metabolites consisting of dihydrodiols, phenols and quinones were determined by high-pressure liquid chromatography, and macromolecular binding of BP to each cell line was measured over a 24-h period. Comparisons between binding and overall metabolism were not directly proportional in these liver cell lines. However, there was a positive correlation for benzo[a]pyrene mutagenesis in the V-79 hamster cell assay with 8-azaguanine as a marker when the cell lines with the highest (IAR-20) and lowest (IAR-27) metabolic competence were used as activating cell layers.

Animals↗
PubMed1982

Patterns of filipin-sterol complex distribution in intact erythrocytes and intramembrane particle-aggregated ghost membranes.

When fixed erythrocytes are exposed to the cholesterol probe, filipin, and freeze-fractured, their membrane is labeled with only a limited number of typical, 25 nm diameter filipin-sterol complexes. In addition, the membrane contains many less distinct filipin-induced perturbations that give it an overall rippled appearance. In contrast, red cell ghosts subjected to conditions that result in an aggregation of their intramembrane particles have large particle-free membrane domains which are completely filled with 25 nm filipin-sterol complexes. These results suggest that some constraint exists in the intact erythrocyte membrane which restricts the formation of typical filipin-sterol complexes.

Animals↗
PubMed1982

Cell shape and organization of cytoskeleton and surface fibronectin in non-tumorigenic and tumorigenic rat liver cultures.

Morphological changes associated with neoplastic transformation of epithelial cells were studied in a series of IAR cell lines derived from rat liver. The series included three independently obtained, non-tumorigenic lines and five derived, tumorigenic lines. The morphology of cell surfaces was observed by scanning electron microscopy; the distribution of actin, tubulin and fibronectin was determined by indirect immunofluorescence. All the non-tumorigenic lines had a typical epithelioid morphology: isolated cells of these lines spread on the substratum had a discoid shape and contained circular, marginal bundles of microfilaments and microtubules. In denser areas, the cells formed monolayered sheets with characteristic marginal bundles of microfilaments near the free edges. Decreased spreading of isolated cells on the substratum was the characteristic feature that distinguished tumorigenic lines from their non-tumorigenic parent lines. In particular a decrease in the size of the ring-like, peripheral lamella and its disintegration into several discrete lamellar zones were often observed; as a result, the cell shape was altered from discoid to polygonal or elongated. The altered distribution of microfilament bundles and microtubules was characteristic in elongated cells; the pattern of the cytoskeletal elements of these cells resembled that of polarized fibroblasts. Complete disappearance of microfilament bundles was observed in cells of only one tumorigenic line. Various degrees of disorganization of monolayered cell sheets were observed in tumorigenic cultures, accompanied by an altered distribution of microfilament bundles. The alterations in the fibronectin-containing structures were more complex: there were often fewer fibronectin "spots' and fibrils at the lower surfaces of cells of tumorigenic cultures as compared with those of non-tumorigenic ones; there were more fibrils in dense cultures of certain lines but fewer in others. It is concluded that alterations in the ability to spread on the substratum and to form cell-cell contacts are common features of morphologically transformed fibroblastic and epithelial cultures. However, the actual changes in the cytoskeletal structures that accompany these alterations are different in transformed cultures of various tissue types.

Animals↗
PubMed1982

An overview of phthalate ester carcinogenicity testing results: the past.

This paper presents a short review of the available carcinogenicity tests which have been carried out on various phthalate esters prior to the availability of the National Cancer Institute bioassay studies. All of these studies suffer from diverse limitations in design or reporting when compared to more recent experimental protocols and reports. There are three papers related to di(2-ethylhexyl) phthalate, one to di-n-butyl phthalate, two to butyl benzyl phthalate, one to ethyl phthalyl ethyl glycolate nd another to butyl phthalylbutyl glycolate (1).

Animals↗
PubMed1982

Detection of aflatoxins and related metabolites by radioimmunoassay.

Rabbit (outbred albino New Zealand White) antisera have been raised against aflatoxin B1 (AFB1) using as immunogen conjugates in which the hapten was coupled to bovine serum albumin (BSA) either through C1 or C8 with the oxime and the dichloride derivatives of AFB1 as intermediates. Radioimmunoassay (RIA), with [3H]AFB1 as tracer, showed that the antiserum prepared with the conjugate in which BSA was coupled to the AFB1 oxime derivative was highly specific to AFB1, whereas the antiserum raised against the conjugate in which BSA was coupled to AFB1 through the AFB1Cl2 derivative (C-antiserum) cross-reacted to a large extent with other aflatoxins, including aflatoxin M1, an important urinary metabolite of AFB1 in several species including humans. AFB1-related metabolites in the urine of inbred BD IV adult male rats given AFB1 orally at doses from 600 pmol to 385 nmol can easily be followed over 9 days by RIA in which the "cross-reactive" C-antiserum is used. This suggests that similar methodologies could be used for the monitoring of human exposure to AFB1.

Aflatoxin B1↗
PubMed1981

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells.

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.

Animals↗
PubMed1981

[Ultrastructural visualization of binding and internalization of cholera and tetanuts toxins].

In monolayer cultures of a liver line, a population of non-coated membrane microinvaginations is preferentially involved in both the initial binding and subsequent internalization of colloidal gold-labelled cholera and tetanus toxins. These two toxins thus appear to be internalized via another pathway than most other polypeptide ligands studied, which enter cells via coated plasma membrane invaginations.

Animals↗
PubMed1981

Alkylation of DNA and tissue specificity in nitrosamine carcinogenesis.

A peculiarity of nitrosamines is the high degree of cell and organ specificity in inducing tumors. There is substantial evidence that the initiation of the carcinogenesis process by carcinogens of this group is linked to the metabolic competence of the target tissue or cell to convert these carcinogens into mutagenic metabolites and to the binding of those metabolites to cellular DNA. Alkylation occurs in the DNA at the N-1, N-3, and N-7 positions of adenine; the N-3, N-7, and O6 of guanine; the N-3, and O2 of cytosine; and the N-3, O4, and O2 of thymine; and the phosphate groups. The initial proportion of each DNA adduct depends upon the alkylating agent used. The various DNA adducts are lost to a variable extent from DNA in vivo by spontaneous release of bases and/or by specific DNA repair processes. Studies conducted in vitro and vivo indicate that alkylation at the oxygen atoms of DNA bases is more critical than alkylation at other positions in the mutagenesis and carcinogenesis induced by N-nitroso compounds. In particular, tissues in which tumors occur more frequently after a pulse dose of nitrosamine are those in which O6-alkylguanine persists longest in DNA, presumably resulting in an increased probability that a miscoding event (mutation) will take place during DNA synthesis. The more rapid removal of O6-methylguanine from the DNA of liver (as compared with extrahepatic tissues) of rats has been associated with the absence of tumor production in this organ by a single dose of dimethylnitrosamine; however, a significant incidence of liver tumors is observed if the same dose is given 24 hr after partial hepatectomy, and tumors are induced by such a dose of dimethylnitrosamine in the liver of hamsters, which has a low capacity to remove O6-methylguanine from its DNA. These data also indicate that the rate of disappearance of 7-methylguanine from the liver or extrahepatic tissues is independent of the dose of dimethylnitrosamine; whereas O6-methylguanine is lost from DNA more rapidly after a low dose of this nitrosamine. It has been shown that in liver the removal of O6-methylguanine but not other DNA adducts, from DNA can be affected by pretreating the animals with N-nitroso compounds. The modulation of DNA repair processes observed after a single dose and after chronic treatment with nitrosamines is discussed in relation to the tissue-specific carcinogenic effect of this group of carcinogens.

Alkylation↗
PubMed1981

Mutagenicity assays of estrogenic hormones in mammalian cells.

In view of the extensive use of estrogenic hormones by the human population, either as therapeutic agents, or in the composition of contraceptive pills, it is important to investigate more thoroughly the adverse biological effects of synthetic hormones. Diethylstilbestrol, ethynylestradiol, estradiol-17beta and estrone were chosen for our experiments. Evidence of carcinogenicity in rodents has been reported for each of these compounds, but so far only few studies have been carried out in vitro. Because it has been shown that isolated liver cells in suspension are able efficiently to metabolize steroid hormones, we have tested these chemicals in V79 cells with a cell-mediated system using primary hepatocytes from male and female rats as the metabolic layer. The incubation in the presence of the chemical to be tested was carried out at concentrations ranging from 25 to 100 microM, for 48 h before plating the V79 cells to score for mutagenicity or toxicity. In the absence of hepatocytes, the 4 estrogenic hormones were very toxic, but not mutagenic. The co-cultivation of V79 cells with primary hepatocytes decreased the toxic effect induced by the sex hormones, except in the case of ethynylestradiol. However, no mutation, determined as 8-azaguanine- or ouabain-resistance, was induced under these conditions by any of the hormones tested. The lack of mutagenic activity of these hormones in our assay had been confirmed by the use of primary liver cells that originated from a rat treated with Aroclor, an inducer of drug-metabolizing enzymes.

Animals↗