C. Bolognesi

Improved microfluorometric DNA determination in biological material using 33258 Hoechst

Abstract A simple and rapid microfluorometric method is described for the determination of DNA in submicrogram quantities using 33258 Hoechst fluorochrome. A high degree of reproducibility was obtained using calf thymus and phage DNA, mouse liver chromatin, and HeLa cells homogenate preparations. None or very little interference by the routinely used preparation reagents or by the cellular components was found. Compared to other commonly used procedures this innovative and versatile technique can be conveniently applied to DNA microdetermination for the high sensibility/reproducibility ratio and can also be used without the need of previous purification steps.

Evaluation of damage to DNA after in vivo exposure to different classes of chemicals

Swiss CD1 male mice were injected intraperitoneally (i.p.) with compounds belonging to different chemical classes in order to investigate the influence of those chemicals on the in vivo production of single-strand DNA-breaks in liver and kidney. DNA damage was evaluated by the alkaline elution technique coupled with a microfluorometric method for DNA assay. An increased elution rate was obtained after in vivo treatment with known carcinogens. Our results are in good agreement with the data obtained by long-term carcinogenicity tests and less with those obtained by mutagenicity assays.

DNA damage induced in vivo in various tissues by nitrochlorobenzene derivatives

Mono-, di-, and trinitrochlorobenzenes were injected i.p. into albino Swiss CD1 mice. Their effects were evaluated, in brain, liver and kidney, as single-strand DNA breaks. DNA damage was recognizable 4 h after administration in vivo, and its increment seemed to be related to the number of nitro groups contained in the chlorobenzene molecule. The simple and accurate microfluorometric procedure for DNA assay associated to the alkaline elution technique improved the application in vivo, avoiding the radiolabeling of DNA.

Standardization of the alkaline elution procedure using X-ray-damaged nuclear DNA

Structural modifications of DNA were induced by X-irradiation of crude hepatic nuclei at various dose ranges to standardize DNA damage evaluated by the alkaline elution technique. This quantitative assay can be used as reference for DNA damage induced by the in vivo administration of mutagens and/or carcinogens involved in the environment.

Alkylation of γ-(4-nitrobenzyl)pyridine by four N-diazoacetyl derivatives of amino acids. Lack of fine correlation with the unscheduled DNA synthesis induced in mouse cells “In vitro”

Summary Four N-diazoacetyl derivatives of amino acids, N-diazoacetylglycine amide (DGA), N-diazoacetylglycine methylamide (DGMA), N-diazoacetylglycine hydrazide (DGI), and N-diazoacetyl-D,L-alanine ethyl ester (DAE), two of them, DGA and DGI, known to be carcinogens and mutagens, were quantitatively assayed for both alkylation of the acceptor γ-(4-nitrobenzyl)pyridine and autoradiographic DNA repair induced in mouse cell cultures. All four compounds were positive for the above mentioned effects but there was no fine quantitative correlation between them.

A new method to reveal the genotoxic effects of N-nitrosodimethylamine in pregnant mice

DNA damage and repair in kidney and liver of mouse fetuses exposed to selected doses of N-nitrosodimethylamine (NDMA) (CAS No. 62.75.9) were studied using the alkaline elution technique. CD1 female mice (15 days pregnant) were treated i.p. with 2 and 10 mg/kg b.w. of NDMA; a slight increase in DNA damage was observed in their fetuses compared to untreated controls. A 2-fold higher extent of DNA damage was induced when mice were treated by intrafetal injections of a rat S9 activating fraction (S9) immediately before exposure to the same dose of NDMA by transplacental means. The DNA-strand breaks disappeared as a function of time in animals treated with NDMA alone. In contrast, a significant persistence of DNA damage was detected in the liver and lung of fetuses which were treated with S9 and NDMA in sequence. These experiments demonstrate the metabolic immaturity of unborn mice as far as the carcinogenic activation of NDMA is concerned and show the high susceptibility of fetal tissues to DNA-damaging agents. The alkaline elution applied in vivo by the transplacental route combined with the intrafetal injection of an exogenous activating microsomal fraction allow to extend our knowledge on the interaction of metabolism-dependent chemicals with fetal tissues.

Effects of the Cobalt Ion on the in Vitro Growth of Mouse Fetus Fibroblasts.

The effect of cobalt II (CoCl2.6 H2O) on growth and plating efficiency of an established line of BALB/c mouse embryo cells was examined. The degree of inhibition of cell proliferation was dependent on both cobaltous ion dose and duration of treatment. Plating efficiency was enhanced by low doses and/or short time of exposure, but was reduced by treatment with higher doses or of longer duration.