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Biomedical subjects

P Carlo

Publications and source records attributed to P Carlo.

At least 37 records · Page 2Linked to original sources
PubMed1984

Phase transitions in nuclei and chromatin. Is nuclear volume controlled by the chromatin or by the nuclear matrix?

Changes in the volume of rat liver nuclei have been monitored as a function of modifications in ionic environment (from 0 to 20 mM), temperature (from 4 to 37 degrees C), and pH (from 1 to 8). An abrupt reduction of nuclear volume occurred with increasing ion concentration, this contraction being more pronounced with bivalent (either Ca2+ or Mg2+) than with monovalent (either Na+ or K+) cations. The lowering of pH produced a similar effect. Parallel changes in chromatin structure took place at the same time as phase-like transitions. Atomic absorption spectroscopy allowed determination of free and nuclei-bound ions, pointing to the presence of a sizeable number of free binding sites for chromatin-DNA even within intact nuclei. DNA-phosphate sites appear to be neutralized by ions strictly according to the size of the electric charge and polyelectrolyte theory. Partial digestion (by micrococcal nuclease) or simple breaks (by chemical carcinogens) of the chromatin-DNA fiber caused respectively elimination or reduction of the abrupt volume changes in the intact nuclei. The apparent role of chromatin structure versus nuclear matrix in determining the shape and volume of intact nuclei is briefly discussed.

Animals↗
PubMed1984

The usefulness of viscometric analysis for detecting minimal levels DNA fragmentation.

Basic requirements for a method used to study the genotoxic potential of xenobiotic agents in the intact animal are sensitivity and capability of detecting the effect of the compound under test in various tissues. A new viscometric technique, which has been found capable of measuring DNA damage in liver, kidney, and lung of rats treated with small single doses of 10 chemical carcinogens, seems to possess such requirements. Single-strand breaks and probably other types of lesions indeed cause changes in DNA supercoiling which can be sensitively measured by monitoring time-dependent changes of DNA viscosity. The main advantage of this technique is that clear-cut modifications of viscometric parameters can be obtained with doses of various carcinogens markedly lower than those found to be the minimal effective ones in other commonly employed short-term in vivo tests. The importance of studying the genotoxic effects of pharmacologically meaningful doses is discussed.

Animals↗
PubMed1983

Quaternary and quinternary structures of native chromatin DNA in liver nuclei: differential scanning calorimetry.

Differential scanning calorimetry of chromatin isolated from rat liver cells revealed three discrete thermal transitions whose temperatures and melting enthalpies depend on ionic strength in the range 0 to 600 millimolar NaCl. Intact nuclei showed a fourth thermal transition at a lower temperature and different melting enthalpies for the other three transitions still present at temperatures similar to those obtained in isolated chromatin. The data are discussed in terms of the tertiary, quaternary, and quinternary structures of chromatin DNA.

Animals↗
PubMed1983

Viscometric analysis of DNA damage in kidney and lung following exposure of rats to small doses of chemical carcinogens.

A new viscometric technique has been used to detect DNA damage in kidney and lung of rats treated with six chemical carcinogens. In alkaline conditions (pH 12.5) the reduced viscosity (eta red) of kidney and lung DNA from control rats increased slowly with time reaching a maximum, (eta red)max, after 9-12 h. Carcinogens, by inducing DNA strand breaks either chemically or indirectly by excision repair or during incubation in alkali, cause a reduction of DNA supercoiling which can be sensitively measured by monitoring changes in viscosity. Computerized analysis of time-viscosity curves showed that a statistically significant reduction of the time required for eta red to reach 95% of its maximum value (t-95) was induced by the following single i.p. doses: N-nitrosodimethylamine (DMN), kidney 0.07 mg/kg, lung 0.28 mg/kg; N-nitrosodiethylamine (DEN), kidney 3.2 mg/kg, lung 12.8 mg/kg; N-nitroso-N-methylurea, kidney and lung 0.5 mg/kg; 1,2-dimethylhydrazine (DMH), kidney 1 mg/kg, lung 16 mg/kg; 4-nitroquinoline-1-oxide (NQO), kidney 2.5 mg/kg, lung 0.63 mg/kg; 2-acetylaminofluorene, kidney and lung 12.5 mg/kg. The decrease of t-95 was constantly dose-related. The comparison with data previously obtained from liver demonstrates that DMN, DEN, DMH and NQO caused the greatest amount of DNA damage in the organ most susceptible to tumor induction. Viscosity changes elicited by DMN, DEN and DMH are quantitatively well correlated with the extent of DNA alkylation.

Animals↗
PubMed1983

Viscometric detection of liver DNA fragmentation in rats treated with minimal doses of chemical carcinogens.

A new technique, using an oscillating viscometer capable of measuring changes of DNA reduced viscosity (eta red), has been used to detect DNA damage in liver of rats treated with various chemical carcinogens. In denaturing conditions (pH 12.5), the eta red of liver DNA from control rats increased slowly with time, reaching a maximum, (eta red)max, after 10 to 13 hr. Single i.p. doses of N-nitrosodimethylamine (0.07 mg/kg), N-nitrosodiethylamine (0.2 mg/kg), N-nitroso-N-methylurea (0.5 mg/kg), 1,2-dimethylhydrazine (0.06 mg/kg), procarbazine (1 mg/kg), methyl methanesulfonate (8 mg/kg), and N-diazoacetylglycine amide (3.7 mg/kg) induced a statistically significant reduction of the time (t95) required for eta red to reach its maximal value. A dose-dependent decrease of t95 was observed for dosages markedly lower than those found to be effective in eliciting DNA fragmentation by the use of alkaline elution or alkaline sucrose gradient sedimentation. 2-Acetylaminofluorene (12.5 mg/kg) and 4-nitroquinoline 1-oxide (10 mg/kg) caused a clear-cut increase of (eta red)max. 7,12-Dimethylbenz(a)anthracene (10 mg/kg) markedly prolonged t95. This viscometric assay of in vivo DNA damage allows a reliable assessment of DNA lesions induced by doses of chemical carcinogens sufficiently small not to produce significant alterations in the pharmacokinetic behavior of these compounds.

Animals↗
PubMed1982

Early effects of chemical carcinogens as compared to induced cell proliferation. II. Automated image analysis.

Static automated image analysis was applied to study early variations of chromatin structure in Feulgen-stained liver nuclei from rats injected i.p. with a single dose of dimethylnitrosamine (DMNA), a well known hepatocarcinogen. An increase of nuclear area and a correspondent decrease of average optical density (integrated optical density/area) was observed, as compared with controls, in nuclei from rats treated with 5.4 mg/kg of DMNA. These findings, which were comparable with those induced by partial hepatectomy, indicate the existence in DMNA-treated cells of a chromatin DNA relaxation similar to the G0-G1 transition previously described for human diploid fibroblasts stimulated to proliferate. Because similar results were independently obtained by flow microfluorimetry, it seems reasonable to hypothesize that chromatin decondensation could be a prerequisite for cancer induction.

Animals↗
PubMed1982

Absence of DNA damage in liver of rats given high doses of cimetidine and sodium nitrite.

A correlation between cimetidine and gastric cancer has been suggested. Nitrosation of cimetidine in the presence of nitrite and HCl and mutagenic activity as well as DNA damage in mammalian cells displayed by nitrosocimetidine, as these phenomena occur in vitro, were the supporting hypothesis. Previous studies have shown that liver DNA damage was a well correlated index of potential carcinogenic activity of N-nitroso compounds and that such a damage was found after long-term simultaneous oral administration of aminopyrine and nitrite in rats. In this work, liver DNA fragmentation was investigated by three different techniques: DNA alkaline elution, DNA alkaline denaturation followed by hydroxylapatite chromatography and a new viscometric method markedly more sensitive than the above mentioned ones in detecting DNA damage. Evidence of DNA damage was not gained in any of the groups of rats treated with high single or successive oral daily doses of cimetidine (250 mg/kg) along with nitrite (80 mg/kg) in approximately equimolar amounts. Cimetidine and nitrite given alone were also ineffective. The lowering of gastric pH, obtained with fasting and histamine administration before giving cimetidine and nitrite combination in a single dose, did not favor the induction of liver DNA fragmentation neither in the above condition nor even when the amount of cimetidine was lowered to 125 mg/kg in order to obtain an approximately 2-fold molar amount of nitrite.

Animals↗
PubMed1981

[Effect of treatment with Triton X-100 on DNA integrity and DNA:RNA: protein ratio of rat liver nuclei].

A procedure is described that gives clean nuclei with intact DNA from a rat liver cell suspension. Cytoplasmic contamination is removed by successive treatments with a non ionic detergent, Triton X-100 (0.75%, v/v). We found that with ratios DNA:RNA: protein of 1:0.09:3.29 (2 Triton X-100 steps) the integrity of DNA is preserved: further decrease in RNA and protein content (3 Triton X-100 steps) causes DNA breakage, probably because of extraction of nuclear proteins. In order to estimate DNA integrity, its viscosity was determined by the use of a new oscillating crucible viscometer; this method makes possible the evaluation of extremely small levels of DNA damage as that induced by 0.067 mg/kg of DMNA injected i.p.

Animals↗
PubMed1981

[Staining with naphthol yellow S. 1. Evaluation of the Keq of the protein-dye binding reaction].

A histochemical technique, using acid dye NY-S, for quantitative determination of proteins was tested using cultured cells fixed in a fixative mixture of methanol, acetic acid, formalin (85:5:10, v/v) and stained with increasing ratios of [Dye-]/[R-HN+3] for times up to 30 min. We attempted to evaluate the Keq of the binding reaction of NY-S to proteins. Using a Microphotometer Leitz M PV 2; we have not found the saturation level. We think that this is due either to the presence of secondary binding sites between dye and proteins and stacking phenomena of the dye, or to factors affecting measurements.

Animals↗
PubMed1981

[Staining with naphthol yellow S. 2. Study of the reversibility of the protein-dye binding reaction].

In the present work we studied the reversibility of the binding reaction of Naphthol Yellow S to proteins using a Testing Automatic System Leitz. We presently think that the NY-S staining procedure, can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins only under standardized conditions: the ratio R = [Dye-] / [R-NH+3] must be more greater than 10; for staining periods shorter than 45 min, the slides, at equilibrium, must be mounted in dyeing solution; for quantitative determinations using irreversible bound dye, the staining period must be significantly longer than 45 min (see above) and differentiated in running water for several hours.

Animals↗
PubMed1981

[Viscosimetric evaluation of DNA fragmentation induced in vivo by alkylating agents].

A sensitive viscosimetric approach for measuring DNA single-strand breaks induced by low doses of alkylating agents is presented. The assay is based on the breaks-induced increasing rate of strand separation of rat liver DNA in alkaline solution (pH 12,5; 22 C). Because of the strong dependence of viscosity on DNA unwinding, measurement of time-course of viscosity increase can be used to detect small breakage of DNA. The kinetics of DNA denaturation has been also studied under two different alkaline conditions.

Alkylating Agents↗
PubMed1979

DNA damage and repair induced by diazoacetyl derivatives of amino acids with different mechanism of cytotoxicity. Correlations with mutagenicity and carcinogenicity.

Eight synthetic N-diazoacetyl amino acids, prepared by inserting a diazoacetyl group onto the alpha-nitrogen of a natural amino acid, and two natural diazoazetyl amino acids, azaserine (9-diazoacetyl-L-serine) and DON (6-diazo-5-oxo-L-norleucine), have been studied by autoradiography for their capacity to induce DNA repair synthesis in mouse cells cultivated "in vitro". Dose-dependent unscheduled DNA synthesis was present in cells treated with the eight N-diazoacetyl derivatives, and was absent in cells exposed to approximately equitoxic concentrations of azaserine and DON. Azaserine and DON, unlike N-diazoacetyl derivatives, did not alkylate gamma-(4-nitrobenzyl) pyridine at an appreciable extent. When DNA damage (single stranded breaks or weak points in alkali) was measured by the sensitive technique of alkaline elution, DGA was found about 4 times as potent as azaserine and about 12 times as DON on a molar basis, but about 800 and 17,000 times as potent as azaserine and DON respectively by extrapolating to equitoxic concentrations. Carcinogenicity and mutagenicity seem to follow mainly the capability of inducing DNA damage.

Alkylation↗
PubMed1978

DNA repair synthesis in primary cultures of kidneys from BALB/c and C3H mice treated with dimethylnitrosamine.

A combined 'in vivo--in vitro' autoradiographic method was employed to examine the DNA repair induced in the kidney by a single dose of dimethylnitrosamine (DMNA). Unscheduled DNA synthesis was found to be dose-dependent in primary kidney cultures of DMNA-treated mice, and practically not detectable in controls. Its amount was positively correlated with the different susceptibility of C3H and BALB/c mice to kidney tumor induction by DMNA. This experimental model appears sensitive and able to provide repeatable results. It may be useful to detect the organotropic activity of a carcinogen toward the kidney.

Animals↗