Dose-response curves for liver DNA fragmentation induced in rats by sixteen N-nitroso compounds as measured by viscometric and alkaline elution analyses.

A new viscometric technique, capable of detecting DNA strand breaks and alkali-labile sites by monitoring time-dependent changes of DNA-reduced viscosity, has been used to analyze dose-response curves for the induction of DNA damage in liver of rats treated with single p.o. doses of sixteen N-nitroso compounds. Statistically significant changes of DNA viscometric parameters, which are considered indicative of DNA fragmentation, were produced by N-nitrosodimethylamine (0.022 mg/kg), N-nitrosomethylethylamine (0.025 mg/kg), N-nitrosodiethylamine (0.067 mg/kg), N-nitrosodiethanolamine (1.03 mg/kg), N-nitrosodi-n-propylamine (0.31 mg/kg), N-nitrosodi-n-butylamine (0.083 mg/kg), N-nitroso-N-methylurea (0.56 mg/kg), N-nitroso-N-ethylurea (0.37 mg/kg), N-nitroso-N-butylurea (0.16 mg/kg), streptozotocin (20 mg/kg), N-nitrosomorpholine (0.4 mg/kg), N-nitrosopiperidine (2.22 mg/kg), N-nitrosopyrrolidine (5.0 mg/kg), 1-nitroso-2-imidazolidinone (0.31 mg/kg), and N-methyl-N'-nitro-N-nitrosoguanidine (5.57 mg/kg). The contemporary measurement of liver DNA fragmentation by the alkaline elution technique revealed that in our experimental conditions higher doses are needed to produce a statistically significant increase of DNA elution rate. This suggests that the viscometric method is capable of detecting smaller levels of N-nitroso compound-induced DNA fragmentation, but it does not exclude that the sensitivity of alkaline elution can be improved by appropriate modifications of the experimental procedure. With both techniques DNA damage was undetectable in liver of rats treated with 540 mg/kg of the non-hepatocarcinogen N-nitrosodiphenylamine. With the exception of N-nitrosodiethanolamine, that exhibited a plateau effect, all the other N-nitroso compounds examined displayed a linear dose-response curve over the entire wide range of doses tested. Consequently, a nonlinearity of the relationship between dose and tumor response cannot be attributed to a nonlinearity of the pharmacokinetic processes involved in the formation of DNA damage.

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.

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.

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.

DNA damage induced by nitrosated ranitidine in cultured mammalian cells.

Ranitidine, a new H-2 receptor antagonist more potent than cimetidine in inhibiting gastric secretion, reacted under acid conditions with a twofold molar amount of nitrite (a nitrite/ranitidine ratio about 1000 times that likely to occur in gastric juice of treated humans) yielding a nitroso derivative capable of inducing a dose-dependent DNA fragmentation in cultured Chinese hamster ovary cells.

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by the biotic ketoaldehyde methylglyoxal.

The number of sister-chromatid exchanges (SCEs) per metaphase was determined in Chinese hamster ovary cells after 16 h exposure to methylglyoxal (MG) concentrations ranging from 0.1 to 0.75 mM. MG produced an increase of SCE frequency that proved to be dose-dependent, and to reach a maximum of 2 X baseline at the highest nontoxic concentration (0.5 mM).

[An effective immunodepressive treatment for demonstrating in allogeneic mice the tumorigenic properties of cells grown in vitro (author's transl)]. / Un efficace trattamento immunodepressivo per la dimostrazione in topi allogenici della oncogenicità di cellule coltivate in vitro

Short intensive treatment with N-diazoacetylglycine amide (DGA) before the inoculum, followed by prolonged daily administration of L-asparaginase (Asnase), was tested for its ability to elicit tumorigenic properties of fibroblast-like cells cultured in vitro. With this treatment progressive tumor growth was obtained in allogeneic mice injected with cells of a transformed subline. Results show that combined use of DGA and Asnase affords a higher probability of proving in vivo the tumorigenic properties of injected cells than in newborm or X-irradiated recipients. Experimental data indicate that L-asparagine depletion does not inhibit the in vitro growth of fibroblast-like cells.

Effect of ten thiocompounds on rat liver DNA damage induced by a small dose of N-nitrosodimethylamine.

The use in a chemoprevention study of high doses of the genotoxic agent might result in erroneous information because of possible nonlinearity of pharmacokinetic processes and toxicity-induced derangement of physiological defense mechanisms. According to these premises ten thiocompounds, potentially active as inhibitors of metabolic activation and/or scavengers, were examined for their capability of reducing the frequency of liver DNA lesions induced by a very small dose of N-nitrosodimethylamine (NDMA). This was accomplished by means of a viscometric technique previously found suitable to detect a minimal amount of DNA fragmentation. Rats were injected i.p. or i.v. with 1 mmol/kg of thiocompound, 0.2 mg/kg NDMA given by gavage 1 h afterwards, and killed for DNA damage assessment 14 h later. Statistically significant changes of viscometric parameters, which are considered indicative of a protective activity, were produced by disulfiram (DSF), and to a lower extent by diethyldithiocarbamate (DEDTC). Any modification of NDMA-induced DNA damage was absent in rats pretreated with glutathione reduced form (GSH) and dimethyl sulfoxide (DMSO). Allyl disulfide (ADS), L-cysteine (CYS), N-acetylcysteine (NAC), alpha-mercaptopropionylglycine (MPG), ethylxanthic acid (PEX), and 2-mercaptoethane sulfonic acid (MESNA) increased in various degree the frequency of DNA-strand breaks. In subsequent experiments the protective activity of DSF was found to be dose-related, dependent on the time of administration, and greater by oral route. Taken as a whole, these results suggest that several putative anticarcinogens might be ineffective against the DNA-damage produced by the low doses encountered in human exposure.

Absence of liver DNA fragmentation in rats treated with high oral doses of 32 benzodiazepine drugs.

Literature data on mutagenic-carcinogenic activity of benzodiazepines are scarce, restricted to few of them, and contradictory. Consequently, in order to provide additional information for the assessment of the genotoxic risk connected with the use of this family of drugs, 32 benzodiazepines of various chemical structure have been tested for their capability to induce DNA damage in vivo, which is considered a sensitive index of potential mutagenic-carcinogenic activity. The frequency of DNA single-strand breaks and/or alkali-labile sites was checked in the liver of rats given orally a single dose (1 mmol/kg) or 15 successive daily doses (0.2 mmol/kg) by the use of a new viscometric technique capable of detecting one DNA lesion per 10(10) Da. Statistically significant changes of viscometric parameters indicative of liver DNA fragmentation were absent with all 32 benzodiazepines, after both acute and subacute treatments. Since the doses tested in rats were from 100 to more than 5000 times higher than doses usually administered to humans, these negative results are in favor of the absence of mutagenic-carcinogenic effects in patients taking benzodiazepines.

Viscometric detection of liver DNA fragmentation in rats treated with ten aromatic amines. Discrepancies with results provided by the alkaline elution technique.

A new viscometric technique, capable of detecting DNA strand breaks and alkali-labile sites by monitoring time-dependent changes of DNA reduced viscosity, has been used to evaluate DNA fragmentation in liver of rats treated with single i.p. doses of ten aromatic amines. Persistent and dose-dependent changes of DNA viscometric parameters, which are considered indicative of DNA fragmentation, were produced by six hepatocarcinogenic aromatic amines: 2-naphthylamine, benzidine, 2,4-diaminotoluene, auramine O, 4-aminoazobenzene, and 4-dimethylaminoazobenzene. In contrast, changes of liver DNA viscometric parameters were minimal and transient or practically absent in rats treated with aniline, 1-naphthylamine, 4,4'-oxydianiline and 2,4-diaminoanisole, all of which are devoid of hepatocarcinogenic activity. The comparison with data previously obtained with the alkaline elution technique demonstrates that the two methods can give different results, and that viscometrically-detected DNA damage is better correlated with carcinogenic activity than DNA damage detected by alkaline elution.

DNA damage induced in rats by oral administration of chlordiazepoxide plus sodium nitrite or of N-nitrosochlordiazepoxide.

Chlordiazepoxide (CDE) reacts in acidic conditions with NaNO2 yielding N-nitrosochlordiazepoxide (NO-CDE), previously shown to exert genotoxic effects in some in vitro systems. The possible intragastric nitrosation of CDE to NO-CDE has been investigated in rats given by gavage high single doses of this benzodiazepine along with NaNO2. Liver DNA fragmentation, as revealed by both DNA alkaline elution and a more sensitive viscometric method, was found to occur consistently and to be essentially independent of the molar ratio drug/nitrite or of gastric pH. The significant increase in the frequency of DNA lesions observed in rats treated for 15 successive days indicates that DNA repair did not keep pace with the accumulation of the damage. Oral administration of single doses of NO-CDE induced similar dose-dependent amounts of DNA fragmentation in liver, gastric mucosa, and brain. Due to the demonstrated absence of carcinogenic activity in rodents, the present results should be interpreted solely as indicating that NO-CDE is intrinsically capable of producing DNA lesions in vivo, an effect by itself not sufficient to induce tumor growth.

[Viscosimetric evaluation of DNA fragmentation induced in vivo by alkylating agents]. / Valutazione viscosimetrica della frammentazione del DNA indotta "in vivo" da agenti alchilanti.

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.

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.

Laser flow measurements of scattering and fluorescence from cell nuclei in the presence of increasing Mg++ concentrations.

The effects of Mg++ on the spatial organization of nuclei from rat hepatocytes are analyzed in the range 0-60 mM, in the presence of suitable concentrations of KCl to reproduce physiological conditions. It is shown that the scatter-signal distribution measured by means of a flow microfluorimeter is greatly affected by this range of Mg concentrations. By coupling this result to phase-contrast-automated image analysis, it is possible to identify a shrinking process induced by Mg++ in the range 0-2.5 mM, which reaches a plateau in the range 5-20 mM and is followed by a swelling process in the range 30-60 mM. The same Mg ranges are shown to affect the intercalation of the fluorochrome acridine orange into chromatin, suggesting that the shrinking-swelling phenomenon has also a molecular correspondence at the genome level. Possible implications in terms of the influence of Mg++ on the organization of chromatin inside intact cells are briefly discussed.

Higher sensitivity for the detection of chemically-induced DNA damage: role of DNA unfolding in determining alkaline elution rate.

CHO-K1 cells exposed to log-spaced concentrations of methyl methanesulfonate (MMS) and liver cells from rats treated with log-spaced single i.p. doses of N-nitrosodimethylamine (DMN) were examined for changes in the rate of DNA alkaline elution induced by incubation in high ionic strength non-denaturing (pH 10) lysing solution. While the elution rate of DNA from control cells was marginally modified, that of treated cells increased proportionally to the length (from 0.5 to 48 h) of incubation, without any significant reduction of DNA average mol. wt. These experiments were suggested and then explained by a recently described physico-chemical model which has shown that the changes in DNA elution profiles are strongly determined also by related changes in DNA chain flexibility and packing. These chemical-induced changes in DNA superpacking and the subsequent differential kinetics of DNA unfolding in the lysing solution are compatible with previous results by independent physical methods. This allows significantly higher sensitivity in the detection of chemically-induced DNA damage, now possible even for a quite low chemical concentration. At the same time, it may be inferred that any material capable of altering DNA superpacking--by damaging either DNA itself or any other chromosomal constituents, such as protein--may well give rise to an increased elution of DNA from the filter.

DNA-damaging activity of biotic and xenobiotic aldehydes in Chinese hamster ovary cells.

Alkaline elution was employed to study DNA damage in CHO-Kl cells treated with a series of biotic and xenobiotic aldehydes. DNA cross-linking was measured in terms of the reduction in the effect of methyl methanesulphonate on the kinetics of DNA elution and was observed in cells treated with formaldehyde, acetaldehyde, methylglyoxal and malonaldehyde. Propionaldehyde, valeraldehyde, hexanal and 4-hydroxynonenal produced DNA single-strand breaks, or lesions which were converted to breaks in alkali. Both types of DNA damage occurred in cells exposed to malealdehyde. These findings support the hypothesis of a carcinogenic effect of the aldehydic products (malonaldehyde, methylglyoxal, propionaldehyde, hexanal, 4-hydroxynonenal) released in biomembranes during lipid peroxidation.

A computerized system for acquiring DNA solution viscosity data.

A new computerized system for monitoring time-dependent viscosity changes of DNA solutions is reported. It provides an extremely sensitive measure of DNA lesions induced by chemical carcinogens. The described apparatus consists of some precision mechanical modules, derived by an early manual version of the system, and of some new electronic parts as optical sensors, a microprocessor-based unit and a personal computer. The new system allows the analysis of more than one specimen at the same time. Compared with the early manual method of measuring, this computerized system, by removing subjectivity and a certain number of casual and systematic errors that might occur with the operator manual intervention, makes possible the evaluation of the resolving power of the viscometers themselves in detecting viscosity changes.

Formation of DNA-damaging nitroso compounds by interaction of drugs with nitrite. A preliminary screening for detecting potentially hazardous drugs.

Fifty-seven theoretically nitrosatable widely used drugs that are commonly administered orally have been screened to determine the formation of nitroso compounds by drug-nitrite interaction and to evaluate the genotoxicity of their nitrosation products against Chinese hamster ovary (CHO) cells, measured as DNA-damaging potency by the alkaline elution technique. The drug (0.1 mmol) was reacted with NaNO2 (0.4 mmol) at pH 3-3.5 for 1 h. Nitroso compounds were present in varying yield in the nitrosation mixture of 47 drugs. Twenty-two drugs formed direct-acting nitroso compounds capable of producing DNA fragmentation, i.e., a statistically significant (p less than 0.01) increase in the elution rate of CHO cell DNA. On a molar basis, their DNA-damaging potency varied over a 570-fold range, with 12 exhibiting greater potency than that of N-nitroso-N-methylurea.