Photoinduced reaction of dimethylnitrosamine with DNA and polynucleotides.
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Biomedical subjects
Publications and source records attributed to S Grilli.
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This article presents results of the first Italian quality control program for determining the estradiol receptor on lypophilized guinea pig and calf uteri. Despite considerable variability in quantitative terms, the results concur in ability to define samples as positive or negative for receptor content. One of the parameters that most strongly influences accuracy of determination of receptor concentration is protein assay. The evaluation of several lyophilized preparations at scalar concentrations permitted identification, by linear regression, for each laboratory of the systematic and non-systematic variables. More comparable results will be forthcoming when a standardized methodology program has been fully adopted.
An immunocytochemical method for oestrogen receptor (ER) analysis of breast carcinomas is described and compared with the dextran-coated charcoal (DCC) assay and a cytochemical method. The method described is superior to the purely cytochemical, especially in terms of sensitivity, demonstration of nuclear receptors and correlation with the DCC assay. The DCC assay is reliable except in the case of poorly cellular tumours: in these the immunocytochemical method is far superior, being independent of tumor cellularity. With this new method, lobular carcinomas are shown to be ER-rich compared with their ductal counterparts, a conclusion currently disputed, largely because methods employed mostly fail to take account of tumour cellularity. A combination of the DCC assay and the immunocytochemical method here described gives the maximum information about the ER-status of a particular carcinoma.
The cytoplasmic concentrations of ER, AR, PR, and GR were determined in 124 specimens of normal and abnormal endometrium and other uterine human tissues by the DCC technique. In the endometrial carcinoma group, we observed that pretreatment with MAP leads to low cellularity, higher amount of AR, lower amounts of detectable ER, GR, and PR: the last receptor was almost always absent. A positive correlation between ER presence and tumor grade of differentiation was found in endometrial tumors from hormone-untreated patients. With the value of 142 fmol/mg DNA as the cut off point between high and low binding capacity, the frequency of the single receptors within the hormone-untreated cancer group ranged from 61% to 88%; ER and PR were simultaneously present in 55% of cases (they are tightly correlated in the different biopsies with respect to frequency and amount); ER-AR-PR were present in 45% and all the four receptors in 40% of cases. Slightly higher values were found in normal endometrium collected from hormone-untreated patients.
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Occurrence of steroid hormone receptor has been evaluated in 30 prostatic cancer-bearing patients: 5 alpha-dihydrotestosterone receptor (DHTR) occurrence was correlated with both tumor grade of differentiation and clinical response to hormone therapy.
Using the value of 0.24 fmoles/microgram DNA as breaking point between high and low binding capacities, we quantified receptors for 17 beta-estradiol (ER), 5 alpha-dihydrotestosterone (DHTR), progesterone (PR) and cortisol (CR) in normal and neoplastic human uterine tissues. Concerning receptors occurrence, significant relationships were observed between ER and PR, ER and DHTR, and DHTR and PR. A direct correlation between the presence of ER and tumor grading was found: PR was less frequent in grade II and absent in grade III endometrial carcinoma, however this was not a significant correlation. In endometrial carcinoma at least 1 of the receptors was detected in 67-91% of the cases, 3 receptors (ER, DHTR, PR) in 56%, and all 4 receptors in 45%. The simultaneous detection of multiple receptors could play an important role in determining hormone response.
DNA repair time-course was studied after injury by N-methyl-N-nitrosourea (MNU) in rat liver cells of animals of different ages and in fetuses using hydroxyurea (HU) as inhibitor of scheduled DNA synthesis. DNA repair was a rapid phenomenon, more so in young adults than in newborns, and was not detectable in fetuses. A correlation seems to exist among organ sensitivity to carcinogen, age of animal and DNA repair.
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7-Methylguanine is the main compound resulting from in vivo interaction between dimethylnitrosamine (DMNA) and nucleic acids, detected after strong acid hydrolysis. However, enzymic and alkaline hydrolysis of nucleic acids leads to quantitative liberation of methylamine. Methylamine isolated from liver nucleic acids of 15N-DMNA-treated rats has molecular weight of 31, thus demonstrating that DMNA-nitrogen is not involved in the binding.
The effect of N-hydroxyurethan (HUR) on DNA synthesis has been tested both in vivo on various tissues and in vitro on concanavalin A (ConA)-stimulated rat thymocytes and compared with the action of urethan and hydroxyurea. HUR suppresses scheduled DNA synthesis, except that of non-stimulated spleen cells in vitro. The inhibition is efficient and rapid and takes place immediately if the drug is administered at the peak of the S-phase. UR inhibits DNA synthesis in vitro only at much higher doses and with different time course. It is effective or slightly effective if it is administered at the peak of the S-phase. A conversion of urethan into HUR the latter depressing DNA synthesis could partly explain the differences observed. No toxicity was found after treatment with drugs at the concentration employed. Finally, the relationships between drug doses and cell responses have been particularly observed in vivo.
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Determinations of specific cytoplasmic receptors for 17 beta-estradiol (E), 5 alpha-dihydrotestosterone (DHT) and progesterone (P) in normal and abnormal endometrium are reported. The standardization of methodology with particular emphasis on specificity trials is outlined. Receptors were present in all but one case, a moderately differentiated endometrial adenocarcinoma. Generally speaking, steroid and peptide hormone plasma content in patients with malignant conditions were at the lower limit values of normal, except for follicle-stimulating hormone which had values significantly higher than normal. The question of E competition with DHT in binding DHT-receptor and the therapeutic implications of P-receptor estimation are discussed.
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Radioactive alkylated bases, ribose or phosphate, were never found either in acid and alkaline hydrolysates of polyribonucleotides or in alkaline hydrolysates of DNA after incubation with 14C-dimethylnitrosamine (DMNA) in a microsomal system. Two radioactive compounds, which were co-chromatographed with methylamine and N-methylhydrazine, respectively, on column, paper, and thin-layer, were always detected. They differed from the compound derived from 7-methylguanosine after the alkali-mediated fission of the imidazole ring in its molecule. The in vitro system employed well represents the in vivo situation (7-methylguanine which is liberated from DNA after acid hydrolysis); however, it has given results which do not agree with the generally-accepted mechanism of DMNA alkylation at the N-7 position of guanine.
Urethan, in a single dose of 1 mg/g body weight, exerts a strong inhibitory effect on DNA synthesis in lymphoid organs and bone marrow of rat. The inhibition observed in spleen and thymus is longer lasting than that exerted on bone marrow or regenerating liver, demonstrating a marked sensitivity of lymphoid cells to the drug. This effect can explain the rapid reduction of weight and cell number in thymus and spleen under urethan treatment, in absence of any lymphocytolytic action. The different effect of urethan on the different subpopulations of lymphoid cells therefore appears to be due to their kinetics rather than to a specific sensitivity of some of them.
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