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Mutation Research Aug 2013A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of...
A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of hepatocarcinogens at relatively low doses, as indicated by dose-dependent micronucleus induction. Because the adult rat liver is known to have a long life-span, this desirable property of the assay will be an advantage in detecting micronucleated hepatocytes (MNHEPs) that have persisted for long periods in the liver following repeated dosing. However, no data directly supporting the underlying mechanisms have been published to date. In the present study, we verified the mechanisms by means of pulse-labeling of micronucleated hepatocytes with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The rodent hepatocarcinogen diethylnitrosamine (DEN) was repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old) for up to 2 weeks, and EdU was injected intraperitoneally on days 1, 7, or 14. Hepatocytes were isolated by use of a non-perfusion technique at 24h, 1 week, or 2 weeks after EdU injection and analyzed for EdU incorporation and micronucleus formation. The results of our study confirmed that MNHEPs labeled with EdU on the first day of DEN administration persisted until 2 weeks post-administration in the rat livers. However, the frequency of MHNEPs among EdU-labeled hepatocytes decreased over time. In addition, the number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive cells in the liver tissue increased, suggesting selective removal of micronucleated cells. Theoretical calculation of the cumulative MNHEP frequency on each of the days on which DEN was administered, taking into account the rate of loss, came out closer to the actual value observed in the liver micronucleus test. Taken together, these results indicate that although micronucleated cells induced in rat livers by administration of the genotoxic hepatocarcinogen DEN undergo selective removal, they persist for a long time in a certain proportion, and repeated administration results in their accumulation and increased frequency.
Topics: Administration, Oral; Animals; Cell Separation; Deoxyuridine; Diethylnitrosamine; Dose-Response Relationship, Drug; Hepatocytes; In Situ Nick-End Labeling; Injections, Intraperitoneal; Liver; Male; Micronucleus Tests; Models, Genetic; Mutagens; Rats; Time Factors
PubMed: 23830928
DOI: 10.1016/j.mrgentox.2013.03.012 -
Drug Metabolism Reviews Feb 1999
Review
Topics: Biotransformation; Carcinogens; Diethylnitrosamine; Humans; Hydroxylation; Oxidation-Reduction
PubMed: 10065371
DOI: 10.1081/dmr-100101913 -
A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals.Cancer Science Jan 2003A reliable medium-term bioassay system for rapid detection of carcinogenic potential of chemicals in the human environment has been developed. The 8-week-protocol... (Review)
Review
A reliable medium-term bioassay system for rapid detection of carcinogenic potential of chemicals in the human environment has been developed. The 8-week-protocol consists of 2 stages; male F344 rats are given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg) for initiation of liver carcinogenesis, followed by a 6-week test chemical treatment starting 2 weeks thereafter. Test chemicals are usually given in the diet or the drinking water and in the 2nd week of test chemical treatment, all rats are subjected to two-thirds partial hepatectomy in order to induce regenerative cell replication. The end-point marker is the glutathione S-transferase placental form (GST-P)-positive hepatic focus, the numbers and sizes of which are analyzed using an image-analyzer and expressed as values per unit liver section (1 cm2). When the yield of GST-P-positive foci is significantly enhanced (P<0.05) over the control value, a chemical is judged to possess carcinogenic or promotion potential for the liver. Among 313 chemicals already tested in this system in our laboratory, 30/31 (97%) mutagenic hepatocarcinogens and 29/33 (88%) non-mutagenic hepatocarcinogens gave positive results. Ten out of 43 (23%) agents known to be carcinogenic in organs other than the liver were also positive. It is particularly important that only one of 48 non-carcinogens gave a very weak positive result, so that the system has a very low false-positivity rate. It is now well documented that the assay system is highly effective for detecting hepatocarcinogens, bridging the gap between traditional long-term carcinogenicity tests and short-term screening assays. At the Fourth International Conference on Harmonization, our medium-term liver bioassay based on an initiation and promotion protocol was recommended in the guidelines as an acceptable alternative to the long-term rodent carcinogenicity test.
Topics: Administration, Oral; Animals; Anticarcinogenic Agents; Biological Assay; Biomarkers, Tumor; Carcinogenicity Tests; Carcinogens; Cocarcinogenesis; Diethylnitrosamine; Dose-Response Relationship, Drug; Drug Interactions; False Positive Reactions; Glutathione Transferase; Hepatectomy; Hepatocytes; Image Processing, Computer-Assisted; Injections, Intraperitoneal; Liver; Liver Neoplasms, Experimental; Liver Regeneration; Male; Rats; Rats, Inbred F344
PubMed: 12708466
DOI: 10.1111/j.1349-7006.2003.tb01343.x -
Biochemical and Biophysical Research... Apr 1981
Topics: Adenosine Triphosphatases; Animals; Cells, Cultured; Diethylnitrosamine; Female; Ferritins; Fluorescent Antibody Technique; Liver; Liver Neoplasms, Experimental; Nitrosamines; Rats
PubMed: 6454416
DOI: 10.1016/0006-291x(81)91245-6 -
European Journal of Cancer & Clinical... Jul 1988When rats are fed diethylnitrosamine (10 mg/kg/day), no O6-ethylguanine is found in liver DNA after 2 weeks, but a considerable amount accumulates after 4 weeks. On the...
When rats are fed diethylnitrosamine (10 mg/kg/day), no O6-ethylguanine is found in liver DNA after 2 weeks, but a considerable amount accumulates after 4 weeks. On the other hand, a 2-week feeding of diethylnitrosamine is not sufficient to induce liver cancers, whereas a 4-week treatment leads to hepatocarcinomas in 50% of the animals. Administration of phenobarbital (75 mg/kg/day) together with diethylnitrosamine during 4 weeks prevents the formation of liver cancers. It also prevents accumulation of O6-ethylguanine in liver DNA. Phenobarbital does not change the amount of O6-ethylguanine repair activity present in liver chromatin after 2 or 4 weeks of treatment with diethylnitrosamine. It is thus concluded that, by inducing the development of the endoplasmic reticulum, phenobarbital decreases the equilibrium concentration of the ultimate carcinogen derived from this indirect alkylating agent.
Topics: Animals; DNA; DNA Repair; Diethylnitrosamine; Guanine; Liver; Male; Phenobarbital; Rats; Rats, Inbred Strains
PubMed: 3416896
DOI: 10.1016/0277-5379(88)90117-4 -
Carcinogenesis Jun 1995Avian embryos (turkey) were exposed to diethylnitrosamine in ovo. On the first day of incubation doses of 0.5-5.0 mg/egg were injected into the white of the fertilized...
Avian embryos (turkey) were exposed to diethylnitrosamine in ovo. On the first day of incubation doses of 0.5-5.0 mg/egg were injected into the white of the fertilized egg. The experiment was terminated 4 days before hatching. Livers were removed and prepared for subsequent histological examination. In haematoxylin and eosin stained sections the areas of hepatocyte nuclear profiles were measured by semi-automatic image analysis. In liver samples of diethylnitrosamine-exposed embryos hepatocyte nuclei of more than twice the size of normal hepatocyte nuclei were found. The incidence of the enlarged nuclei was clearly dose dependent. An increase in the size of hepatocyte nuclei was observed after low doses of diethylnitrosamine that did not induce common signs of non-specific toxic effects, e.g. cell death, fat vacuoles or loss of glycogen. The slope of the dose-response curve was rather steep. A 10-fold increase in the dose of the carcinogen resulted in a 100-fold increase in the incidence of enlarged hepatocyte nuclei. In combination with preneoplastic foci of altered hepatocytes, the quantification of nuclear enlargement can provide a valuable complementary parameter for the evaluation of carcinogen-induced effects in ovo.
Topics: Animals; Cell Nucleus; Diethylnitrosamine; Dose-Response Relationship, Drug; Liver; Precancerous Conditions; Turkeys
PubMed: 7788854
DOI: 10.1093/carcin/16.6.1351 -
Xenobiotica; the Fate of Foreign... Dec 1985The acute toxicities of dimethylnitrosamine and diethylnitrosamine have been evaluated in adult male crayfish Austropotamobius pallipes; LD50 values are 2250 mg/kg and...
The acute toxicities of dimethylnitrosamine and diethylnitrosamine have been evaluated in adult male crayfish Austropotamobius pallipes; LD50 values are 2250 mg/kg and 230 mg/kg, respectively. Toxicokinetic studies of 14C-dimethylnitrosamine and 14C-diethylnitrosamine in crayfish, administered by i.v. injection, show high concentrations of 14C in abdominal muscle and hepatopancreas. Excretion is greater with dimethylnitrosamine, and retention in the tissues, especially the hepatopancreas, is greater with diethylnitrosamine. Metabolites identified in excreta include monomethylnitrosamine from dimethylnitrosamine, and hydroxyethyl-ethyl-, bishydroxyethyl- and carboxyethyl-ethylnitrosamine from diethylnitrosamine.
Topics: Animals; Astacoidea; Diethylnitrosamine; Dimethylnitrosamine; Kinetics; Liver; Male; Mass Spectrometry; Muscles; Pancreas
PubMed: 4090529
DOI: 10.3109/00498258509049104 -
Mutation Research. Genetic Toxicology... Apr 2018Regulatory guidance documents stress the value of assessing multiple tissues and the most appropriate endpoints when evaluating chemicals for in vivo genotoxic...
Regulatory guidance documents stress the value of assessing multiple tissues and the most appropriate endpoints when evaluating chemicals for in vivo genotoxic potential. However, conducting several independent studies to consider multiple endpoints and/or tissue compartments is resource intensive. Furthermore, conventional approaches for scoring genotoxicity endpoints are slow, tedious, and less objective than what would be considered ideal. In an effort to address these issues with current practices, we attempted to i) employ flow cytometry-based methods to score liver micronuclei, blood micronuclei, and blood Pig-a gene mutation, and ii) integrate the endpoints into a common general toxicology study design-the rat 28-day repeat dose study. A proof-of-principle experiment was performed with 6-week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) for 28 consecutive days. One day later blood was collected for micronucleated reticulocyte (MN-RET) and Pig-a mutation assays, and liver tissue was obtained for micronucleated hepatocyte (MNHEP) scoring. MN-RET frequencies were not affected by DEN exposure, and mean Pig-a mutant cell frequencies were only slightly elevated. On the other hand, % MNHEP showed marked, dose-related increases (2.2, 7.2, and 9.1 mean fold-increase for 5, 10, 15 mg DEN/kg/day, respectively). Concurrent with MNHEP analyses, assessments of Ki-67-positive events and the proportion of 8n nuclei provided evidence for treatment-related changes to hepatocyte proliferation. Collectively, these results reinforce the importance of evaluating chemicals' genotoxic potential in liver in addition to hematopoietic cells, and suggest that several automated measurements can be successfully integrated into repeat-dose studies for higher efficiencies and better utilization of fewer animals.
Topics: Animals; Diethylnitrosamine; Dose-Response Relationship, Drug; Flow Cytometry; Hepatocytes; Male; Membrane Proteins; Micronucleus Tests; Mutagenicity Tests; Mutation; Rats, Sprague-Dawley; Reticulocytes
PubMed: 29555062
DOI: 10.1016/j.mrgentox.2018.02.005 -
Biochimica Et Biophysica Acta Oct 2000Vanadium (V) has recently been found to possess potent anti-neoplastic activity in rat hepatocarcinogenesis. Recent studies have suggested that the active metabolite of...
Inhibition of diethylnitrosamine-induced rat liver chromosomal aberrations and DNA-strand breaks by synergistic supplementation of vanadium and 1alpha,25-dihydroxyvitamin D(3).
Vanadium (V) has recently been found to possess potent anti-neoplastic activity in rat hepatocarcinogenesis. Recent studies have suggested that the active metabolite of vitamin D(3), 1alpha, 25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], can inhibit growth and/or induce differentiation of a variety of cell types. In the present study, attempts have been made to investigate the combination effects on chromosomal aberrations (CAs) and DNA-strand breaks during the early preneoplastic stage of diethylnitrosamine (DEN)-induced rat liver carcinogenesis in male Sprague-Dawley rats. V (0.5 ppm ad libitum) and/or 1,25(OH)(2)D(3) (0.3 microg/0.1 ml propylene glycol per os twice weekly) either alone or in combination were given to DEN-treated and control rats 4 weeks prior to DEN injection. Under these experimental conditions it was observed that, when given in combination, V and 1,25(OH)(2)D(3) offered maximum protection against DEN-induced structural aberrations 96 h (66.7%, P<0.05), 15 days (44.9%, P<0.005) and 30 days (63.8%, P<0.001) after DEN injection. Synergistic supplementation of both V and 1, 25(OH)(2)D(3) 4 weeks before DEN injection was found to offer significant (64.1%, P<0.001) protection against generation of single-strand breaks when compared with the DEN control. Thus, the combination effect of V, an essential trace element, and of 1, 25(OH)(2)D(3), a dietary micronutrient, appears beneficial in preventing genetic damage in liver cells upon alkylation induced by DEN.
Topics: Animals; Antineoplastic Agents; Calcitriol; Chromosome Aberrations; DNA Damage; Diethylnitrosamine; Drug Synergism; Liver; Liver Neoplasms, Experimental; Male; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Vanadium
PubMed: 11040452
DOI: 10.1016/s0925-4439(00)00053-3 -
Asian Pacific Journal of Cancer... 2006Phenobarbital (PB), a rodent non-genotoxic carcinogen, showed hormesis, biphasic effects on rat liver carcinogenesis. To test the hypothesis that the hormesis earlier...
Phenobarbital (PB), a rodent non-genotoxic carcinogen, showed hormesis, biphasic effects on rat liver carcinogenesis. To test the hypothesis that the hormesis earlier observed for PB induced hepatocarcinogenesis might also exist in the TGF-alpha transgenic mice model, one which is highly susceptible to carcinogenesis, the carcinogenic or promotion effects of a wide range of phenobarbital (PB) concentrations were investigated. Two weeks after a single i.p. dose of 5 mg /kg bw of diethylnitrosamine (DEN) to 15 day old mice, animals were treated with diet containing PB at doses of 0, 2, 15 or 500 ppm. The incidence and multiplicity of tumors, including hepatocellular adenomas and carcinomas, were significantly increased by the high dose of PB, but no significant difference among the groups receiving 2 and 15 ppm for liver tumors when compared to DEN alone group. The proliferating cell nuclear antigen indices for liver tumors and surrounding hepatocytes in high dose PB treated mice were significantly increased, but no change was noted at the lower doses. The total cytochrome P450 content in the liver was also elevated by 500 ppm of PB, while hepatic 8-OHdG levels demonstrated no significant change. In conclusion, PB at high dose enhances DEN-induced hepatocarcinogenesis in TGF-alpha transgenic mice, but low doses lack any significant effects. One possible mechanism of phenobarbital carcinogenicity might be influenced by cytochrome P450 system exhibiting a strong promoting activity for liver of mice.
Topics: Animals; Anticonvulsants; Carcinogenicity Tests; Carcinogens; Diethylnitrosamine; Dose-Response Relationship, Drug; Liver Neoplasms; Mice; Mice, Transgenic; Phenobarbital; Transforming Growth Factor alpha
PubMed: 16839222
DOI: No ID Found