Liver DNA Fragmentation Research Articles

Molecular basis of pathophysiology of Indian childhood cirrhosis: role of nuclear copper accumulation in liver.

Indian Childhood Cirrhosis (ICC) is a disease of abnormal copper metabolism commonly characterized by swelling and degeneration of liver cells along with the presence of orcein staining deposits of copper. Hepatic copper content of ICC patients was about 43 fold higher than those of control subjects. The data on sub-cellular distribution of copper revealed massive accumulation of Copper (73%) of total cell copper) in the nuclear fraction (455 micrograms Cu/g tissue nuclei). On further distribution of copper in nuclear fraction, the enrichment of copper in heterochromatin and euchromatin of ICC nuclei was found to be 48 and 15 fold higher over control fractions respectively. The ultra-violet spectra of heterochromatin and euchromatin isolated from ICC nuclear fraction showed a broad absorption maxima as compared to controls. Further, A260/A280 ratio was markedly lower in heterochromatin and euchromatin of ICC liver in comparison to controls. An antioxidant enzyme, catalase activity was also significantly reduced in ICC liver as compared to control. Further, DNA fragmentation studies indicated that there was significantly increased DNA fragmentation in ICC liver. Collectively, these findings suggest that massive accumulation of copper in nucleus and decrease in catalase activity was associated with DNA fragmentation in hepatocyte of ICC disease.

Evaluation of DNA-Damaging, Clastogenic, and Promoting Activities of Metoclopramide and Procainamide in Rats

The DNA-damaging and clastogenic activities of metoclopramide (MCA) and procainamide (PCA), two substituted benzamides not systematically tested for genotoxicity before clinical use, were investigated in rats given a single high oral dose (500 mg/kg) of these drugs. Neither MCA nor PCA induced DNA fragmentation in liver, kidney, gastric mucosa, spleen, and bone marrow, as detected by the alkaline elution technique. Moreover, neither drug increased the frequency of micronucleated hepatocytes and the frequency of micronucleated polychromatic erythrocytes in the bone marrow of partially hepatectomized rats. However, in rats initiated with N-nitrosodiethylamine and given water containing 0.125% MCA for 14 successive days a clear-cut and statistically significant increase in the number and size of liver gamma-glutamyltranspeptidase-positive foci and basophilic foci, which are consistent with potential promoting activity, was observed. Under the same experimental conditions the effect of PCA was markedly lower, only limited to a modest increase of the number and area of gamma-glutamyltranspeptidase-positive foci.

Cinnamaldehyde-induced micronuclei in rodent liver

Cinnamaldehyde, a widely used flavoring agent, has so far been subjected to a limited range of genotoxicity tests, mainly carried out in vitro, which produced contradictory results. Therefore we have examined cinnamaldehyde using additional in vivo genotoxicity end-points. In Sprague-Dawley rats, a single oral dose equal to 1 2 LD 50 did not induce DNA fragmentation in liver and gastric mucosa as evaluated by the alkaline elution technique, increased the frequency of micronucleated hepatocytes but not of bone marrow micronucleated polychromatic erythrocytes, and gave rise to a significantly higher incidence of total nuclear anomalies but not of micronucleated cells in forestomach mucosa. In Swiss mice, the equitoxic dose of cinnamaldehyde caused the same elastogenic effect in the liver, whilst a negative response was observed in both bone marrow and forestomach mucosa. Finally, in rats initiated with N-nitrosodiethylamine the administration of 500 mg/kg/day cinnamaldehyde for 14 successive days produced a modest but statistically significant increase of the average diameter and area of γ-glutamyltranspeptidase-positive foci that, together with changes observed in other parameters, might be considered indicative of a potential promoting activity. Taken as a whole, these findings confirm that high doses of cinnamaldehyde may induce genetic alterations at the chromosomal level, and suggest that the liver is the preferential target of its undesirable effects.

Genotoxic Activity of 1,3-Dichloropropene in a Battery of in Vivo Short-Term Tests

The genotoxic activity of 1,3-dichloropropene, which has been classified as possibly carcinogenic to humans, was investigated in rats given high single doses of this chloroolefin. A dose-related amount of DNA fragmentation was observed at doses ranging from 62.5 to 250 mg/kg in liver and gastric mucosa, both of which are targets of DCP carcinogenic activity, as well as in the kidney. The frequency of DNA breaks, that were to a large extent repaired within 24 hr, was higher after po than after ip administration in the liver, while the converse occurred in the kidney. Any evidence of DNA fragmentation was, in contrast, absent in lung, bone marrow, and brain which are not sites of DCP-induced tumor development. A role of cytochrome P450 in the activation of DCP is suggested by the lower degree of liver DNA fragmentation observed in rats pretreated with methoxsalen. DCP produced a dose-dependent reduction of the liver GSH level, an effect that presumably hinders its detoxification and thus favors its DNA-damaging activity. In contrast with the satisfactory prediction of DCP carcinogenic activity provided by the results of the in vivo DNA damage/alkaline elution assay, neither the in vivo rat hepatocyte DNA repair assay nor the micronucleus assay, carried out on bone marrow, spleen, and liver cells of partially hepatectomized rats, supplied any evidence of DCP genotoxicity.

Lack of evidence of omeprazole genotoxicity in Sprague-Dawley rats.

Omeprazole is a proton pump inhibitor of increasingly wide use in the treatment of peptic ulcers. Although omeprazole has been subjected to an extensive range of genotoxicity tests, which have all been concluded as negative, the ability of this compound to interact with DNA and elicit unscheduled DNA synthesis in the rat gastric mucosa has been the subject of debate. Therefore, we have examined omeprazole using other genotoxicity end-points. In female Sprague-Dawley rats, the administration by the oral route of 100 mg/kg, either as neutral (pH 7.0) suspension or as suspension acidified to pH 1.5, which favours its transformation into the active form of sulphenamide, did not induce DNA fragmentation in gastric mucosa and liver, as detected by the alkaline elution technique. In the same experimental conditions, a frequency of total nuclear anomalies (micronuclei, pyknosis and karyorrhexis) that was significantly higher than in controls was detected with both types of suspension in forestomach and descending colon mucosa. However, in both tissues this higher frequency of nuclear anomalies was mostly due to pyknosis and karyorrhexis, which may be the outcome of a non-genotoxic effect, whereas there was no significant increase in the number of micronucleated cells, and this suggests the absence of clastogenic activity. Finally, in rats initiated with N-nitrosodiethylamine, the oral administration of 100 mg/kg omeprazole for 14 successive days produced a modest but statistically significant increase of liver gamma-glutamyltranspeptidase positive foci, which is consistent with a potential promoting activity.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA fragmentation by 2-nitropropane in rat tissues, and effects of the modulation of biotransformation processes

The occurrence and persistence of DNA fragmentation, as detected by the alkaline elution technique, have been studied in rats treated with single oral doses of the hepatocarcinogen 2-nitropropane (2-NP). A progressive increase of liver DNA fragmentation was observed at doses ranging from 0.5 to 8 mmol/kg; single strand breaks reached the maximum frequency 6 h after administration, and were partially reduced after 36 h. In contrast, DNA fragmentation was absent in lung, kidney, bone marrow and brain of rats given 8 mmol/kg. The role of cytochrome P-450 in the activation of 2-NP is indicated by the increase of liver DNA damage in rats pretreated with phenobarbital or β-naphtoflavone, and by its reduction produced by methoxsalen. Both administration of GSH and GSH depletion did not result in clearcut modifications of the genotoxic effect of 2-NP for the liver.

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

Chlordiazepoxide (CDE) reacts in acidic conditions with NaNO 2 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 NaNO 2. 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.

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.

Absence of Liver DNA Fragmentation in Rats Treated with High Oral Doses of 32 Benzodiazepine Drugs

Absence of Liver DNA Fragmentation in Rats Treated with High Oral Doses of 32 Benzodiazepine Drugs

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.

Optical isomers of the hepatocarcinogenic β-blocker zami 1305: Influence on nucleic acids synthesis and DNA integrity

The influence on nucleic acids synthesis and DNA integrity of the D-isomer and of the DL-racemic form of the oncogenic β-blocker 1-(2-nitro-3-methyl-phenoxy)-3- tert-butylamino-propan-2-ol (ZAMI 1305) and of the non-oncogenic β-blocker propranolol was tested in vitro and in vivo. Both D- and DL-ZAMI 1305, when added in vitro to nuclei isolated from rat liver, cause inhibition of DNA and RNA synthesis and DNA fragmentation, as evaluated by alkaline sucrose gradient analysis, in a similar dose-dependent fashion. D- and DL-ZAMI 1305 also inhibit to a similar extent the activity of DNA polymerase α and β from regenerating rat liver. When administered in vivo to female rats both D and DL-ZAMI 1305 cause a dose-dependent fragmentation of liver DNA. The D-isomer and DL-racemic form of the non-oncogenic β-blocker propranolol inhibit DNA and RNA synthesis and cause DNA fragmentation when added in vitro to isolated liver nuclei, being instead without effect when administered in vivo.

Possible participation of Ca 2+, Mg 2+-dependent endonuclease in liver DNA fragmentation after N-methyl- N-nitrosourea treatment

We examined the fragmentation of DNA treated with N-methyl- N-nitrosourea under conditions in which Ca 2+, Mg 2+-dependent endonuclease is active. The molecular mass of DNA found in mouse liver slices treated with methylnitrosurea in the presence of Ca 2+ plus Mg 2+ was 4 · 10 5 Da. Similar results were obtained with a reconstituted system containing partially purified Ca 2+, Mg 2+-dependent endonuclease and methylnitrosurea-treated DNA. The enzyme extensively cleaved methylnitrosurea-treated DNA, compared with non-treated DNA. The methylnitrosurea-treated nuclear proteins obtained from mouse liver nuclei had no effect on the DNA fragmentation by the enzyme. Using closed-circular DNA treated with methylnitrosurea, the enzyme produced single-strand cuts in the DNA, as was seen in non-treated, closed-circular DNA, however, the rate of hydrolysis was increased. Ca 2+, Mg 2+-dependent endonuclease thus warrants further investigation, with regard to the precise mechanism of extensive degradation of DNA in cells treated with carcinogenic alkylating agents.

Induction of presumed somatic gene mutations in mice by 2-naphthylamine

2-Naphthylamine (2-NA) is a bladder carcinogen in humans and also a proven carcinogen in mouse, hamster, dog, monkey (IARC Monograph, 1974) and rat (Radomski, 1979). It was selected as one of the well-studied carcinogens for the National Cancer Institute collaborative studies on mutagenesis as a prescreen for chemical carcinogens (Poirier and Weisburger, 1979) and for similar programmes in Japan (Kawachi et al., 1980) and the United Kingdom (Purchase et al., 1976), as well as for the extensive international Collaborative Programme for the Evaluation of Short-Term Tests for Carcinogenicity (de Serres and Ashby, 1981). After a critical appraisal of test results and test procedures it was concluded, in the Collaborative Programme, that 2-NA exhibits mutagenicity in the microbial assays and the mammalian systems, in vitro, other than that with specific loci in CHO cells. The yeast assays yielded controversial results: 4 assays indicated mutagenicity, 4 gave negative responses. In the assays in vivo, 2-NA was positive in the sex-linked recessive lethal assay with Drosophila melanogaster, but failed to induce sisterchromatid exchanges in the mouse. In the micronucleus test and sperm-abnormality assay with mice the results were inconclusive (Ashby, 1981). Few other data exist about the mutagenic effects of 2-NA in whole organisms: it induces DNA fragmentation in rat or mouse liver after treatment in vivo (Parodi et al., 1981; 1982) and is mutagenic in Drosophila when male larvae are treated (Vogel et al., 1983), but it induces neither chromosome aberrations in bone-marrow cells of rats nor mutations in the silk-worm (Kawachi et al., 1980). The genetic effects of naphthylamines have been reviewed by Mayer (1982).

Genotoxic effects in rodents given high oral doses of ranitidine and sodium nitrite.

The possible intragastric nitrosation of ranitidine to genotoxic derivatives has been investigated in rats and mice given, by gavage, high single doses of this histamine H2 receptor antagonist along with NaNO2. Liver DNA fragmentation, as revealed in rats by both DNA alkaline elution and DNA alkaline denaturation followed by hydroxylapatite chromatography, was found to be dependent either on the molar ratio drug/nitrite or on the gastric pH. It occurred only with doses of 175 mg/kg ranitidine HCl + 80 mg/kg NaNO2 (molar ratio 1:2.32) or 350 mg/kg ranitidine HCl + 80 mg/kg NaNO2 (molar ratio 1:1.16) and concurrent reduction of gastric pH from 5.5 to 2-3 (produced by prolonged fasting). A further reduction of pH elicited by histamine injection increased the amount of DNA damage. DNA fragmentation in gastric mucosa showed a similar dependence on both pH and ranitidine/NaNO2 ratio, but was more marked than in liver. Simultaneous administration of ascorbic acid reduced the damage of gastric DNA. Oral administration of 175 mg/kg ranitidine HCl + 80 mg/kg NaNO2 in fasted and histamine-injected mice induced a modest but statistically significant increase in the frequency of sister chromatid exchanges in bone marrow cells.

DNA-damaging activity in vivo and bacterial mutagenicity of sixteen aromatic amines and azo-derivatives, as related quantitatively to their carcinogenicity.

Sixteen aromatic amines and azo-derivatives were studied. They were: benzidine; 2-acetylaminofluorene; 3'-methyl-p-dimethylaminobenzene; o-aminoazo-toluene; p-dimethylaminoazobenzene; 2,4-diamino-toluene; 4,4'-oxydianiline; 2,4-diaminoanisole; 4,4'-methylenedianiline; 2-naphthylamine; Auramine O; Rhodamine B; Ponceau MX; 1-naphthylamine; p-aminoazobenzene and aniline. The compounds were examined for their capability to induce alkaline DNA fragmentation in rat liver after treatment in vivo, for their mutagenicity in the Salmonella strains TA 98 and TA 100, for their acute toxicity and for their carcinogenicity in mice and rats. For each parameter a quantitative potency index was established, and the correlation existing amongst the different parameters investigated. Only mutagenicity in the strain TA 98 was slightly correlated with carcinogenic potency (r = 0.408). DNA fragmentation and toxicity were not correlated with carcinogenicity. A significant correlation was found between DNA fragmentation and toxicity (r = 0.539). No correlation was found between DNA fragmentation and mutagenicity. The lack of correlation between DNA fragmentation and carcinogenicity is in contrast with previous results obtained with a family of hydrazine derivatives (12) and a group of nitrosocompounds (22). For these two groups of chemicals correlation between DNA fragmentation and carcinogenicity existed, but not between carcinogenicity and mutagenicity in the Ames' test. It is suggested that short term tests can perform very differently for different classes of chemicals.

Persistence and accumulation of (potential) single-strand breaks in liver DNA of rats treated with ethyl methanesulphonate

In vivo alkylation of DNA leads to DNA fragmentation in alkaline sucrose gradients. In a previous paper (Chem.-Biol. Interact., 19 (1977) 111) we presented evidence that, depending on the experimental conditions, a major fraction of the single-strand breaks observed might be derived from alkalilabile alkylphosphotriesters. Using alkaline gradients the present paper shows that injection of ethyl methanesulphonate (EMS) into Sprague—Dawley female rats results in significantly increased liver DNA fragmentation up to at least 56 days after injection. Accumulation of single-strand breaks was indicated by experiments in which at 6 days after the last of a series of 5 weekly EMS injections (5 × 110 mg/kg) 11.4 breaks/10 9 Dalton were found, being 3 times more than the number of breaks observed at 6 days after a single injection of 110 mg/kg EMS (3.8 breaks/10 9 Dalton). In animals treated with methyl methanesulphonate (MMS) single-strand breaks were observed at 4 h, 1 day and 2 days, but not at 6 days after injection (40 mg/kg). Repeated weekly injections of MMS (5 × 40 mg/kg) did not result in increased numbers of breaks when compared with animals receiving a single injection of this agent (1 × 40 mg/kg; animals were killed 1 day after (the last) injection). It is suggested that MMS-induced breaks are derived, either on the gradient or in situ, from apurinic sites, whereas persistent EMS-induced breaks reflect the presence of ethylphosphotriesters. The results are discussed in relation to the lacking capacity of EMS to induce foci of precancerous lesions in rat liver and the non-hepatocarcinogenic properties of both MMS and EMS.

Inhibition of Dimethylnitrosamine-Induced Strand Breaks in Liver DNA and Liver Cell Necrosis by Diethyldithiocarbamate 2

Diethyldithiocarbamate (DEDTC) prevented dimethylnitrosamine (DMN)-induced strand breaks in liver DNA and liver cell necrosis in male Wistar rats. In contrast, DEDTC did not inhibit the fragmentation of liver DNA caused by several other chemical carcinogens (N-hydroxy-2-acetylaminofluorene, 3-hydroxyxanthine, aflatoxin B1, N-acetoxy-2-acetylaminofluorene, methyl methanesulfonate, methylnitrosourea, and methylazoxy-methanol acetate), whether or not they required metabolic activation. Aminoacetonitrile exerted an action similar to that of DEDTC. The inhibitory effect was transitory, lasting at least for 4 hours, and protection for longer than 4 hours required multiple administrations of DEDTC. DEDTC also inhibited the serum clearance of DMN, methylation of liver DNA, and oxidative demethylation of DMN in the in vitro hepatic microsomal system prepared from either male Wistar rats or from hamsters. Interference of the metabolism of DMN appeared to be the mechanism by which DEDTC arrested DMN-induced biochemical and biologic effects.

Inhibition of diethylnitrosamine-induced strand breaks in liver DNA by disulfiram.

Disulfiram (DSF) delayed the appearance of diethylnitrosamine (DEN)-induced strand breaks in liver DNA of rats. The fragmentation of liver DNA produced by DEN was studied 4 and 24 h after administration of the carcinogen on alkaline sucrose gradients. A single dose of 500 mg/kg DSF given 1 h prior to carcinogen treatment delayed for at least 4 h the DEN--induced strand breaks in liver DNA. A single DSF pretreatment, however, did not protect against carcinogen-induced strand breaks when observed 24 h after DEN injection. It is possible that continuous administration of DSF might inhibit the DEN-produced damage of the genetic material.

In vivo repair of rat liver DNA damaged by 3-hydroxy-xanthine

Using alkaline and neutral sucrose gradients, it was observed that administration of the hepatocarcinogen, 3-hydroxyxanthine, to rats, resulted in the slower sedimentation of liver DNA in alkaline and neutral sucrose gradients. Slower sedimentation of liver DNA in alkaline sucrose gradients was apparent within 4 h following the administration of the carcinogen at a dose of 50 mug/g body weight. The fragmentation of liver DNA was progressive with an increase in the dose. Such fragmentation caused by a dose of 100 mug/g was largely repaired by 24 h following the administration of the carcinogen. Fragmentation and repair of liver DNA with a dose of 100 mug or more/g body weight was also seen using neutral sucrose gradients. In neutral sucrose gradients significant repair of the fragmented DNA was seen by 24 h after carcinogen. Liver DNA from rats given 3-hydroxyxanthine (100 mug/g for 4 h) or calf thymus DNA incubated in vitro with this carcinogen did not show any change in their melting profile.

Rapid repair of hepatic DNA damage induced by camptothecin in the intact rat

Administration of camptothecin (7,14 or 28 mg per 100 g body wt.) to rats intraperitoneally, induced single strand breaks in hepatic DNA. Fragmentation of liver DNA on alkaline sucrose gradients was noted within 5 minutes and reached a maximum by 30 to 60 minutes after the administration of the drug. By about 4 hours, the DNA damage was almost completely repaired. However, using neutral sucrose gradients, significant fragmentation of DNA could not be seen. The rapid repair of the liver DNA in vivo induced by camptothecin, a cancer chemotherapeutic agent, is interesting in view of the long delay in the repair of the DNA damage induced by several chemical carcinogens in an intact animal (9,10).