A collaborative study was conducted to evaluate whether a liver micronucleus assay using four-week-old male F344 rats can be used to detect genotoxic rat hepatocarcinogens using double-dosing with a ...single-sampling 4 days after the second dose. The assay methods were thoroughly validated by the seven laboratories involved in the study. Seven chemicals, 2,4-diaminotoluene, diethyl nitrosamine,
p-dimethylaminoazobenzene, 1,2-dimethylhydrazine dihydrochloride, 2,4-dinitrotolunene, 2,6-dinitrotoluene and mitomycin C, known to produce positive responses in the single-dosing/triple-sampling method were selected for use in the present study, and each chemical was examined in two laboratories with the exception of 2,4-dinitrotolunene. Although several of the compounds were examined at lower doses for reasons of toxicity than in the single-dosing/triple-sampling method, all chemicals tested in the present study induced micronuclei in liver cells indicating a positive result. These findings suggest that the liver micronucleus assay can be used in young rats to detect genotoxic rat hepatocarcinogens using a double-dosing/single-sampling procedure. Further, the number of animals used in the liver micronucleus assay can be reduced by one-third to a half by using the double-dosing/single-sampling method. This reduction in animal numbers also has significant savings in time and resource for liver perfusion and hepatocyte isolation.
► The minor isomers and 2,4-DNT were not genotoxic in liver cells or peripheral blood. ► 2,6-DNT caused DNA damage in liver cells, indicating that 2,6-DNT is genotoxic. ► This supports work ...indicating that DNT carcinogenicity is attributable to 2,6-DNT.
Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as “likely to cause cancer in humans.”
In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two
in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer.
Cigarette smoking is associated with carcinogenesis owing to the mutagenic and genotoxic effects of cigarette smoke. The aim of this study was to evaluate the mutagenic and genotoxic effects of ...Korean cigarettes using in vitro assays.
We selected 2 types of cigarettes (TL and TW) as benchmark Korean cigarettes for this study, because they represent the greatest level of nicotine and tar contents among Korean cigarettes. Mutagenic potency was expressed as the number of revertants per μg of cigarette smoke condensate (CSC) total particulate matter whereas genotoxic potency was expressed as a concentration-dependent induction factor. The CSC was prepared by the International Organization for Standardization 3308 smoking method. CHO-K1 cells were used in vitro micronucleus (MNvit) and comet assays. Two strains of Salmonella typhimurium (Salmonella enterica subsp.enterica; TA98 and TA1537) were employed in Ames tests.
All CSCs showed mutagenicity in the TA98 and TA1537 strains. In addition, DNA damage and micronuclei formation were observed in the comet and MNvit assays owing to CSC exposure. The CSC from the 3R4F Kentucky reference (3R4F) cigarette produced the most severe mutagenic and genotoxic potencies, followed by the CSC from the TL cigarette, whereas the CSC from the TW cigarette produced the least severe mutagenic and genotoxic potencies.
The results of this study suggest that the mutagenic and genotoxic potencies of the TL and TW cigarettes were weaker than those of the 3R4F cigarette. Further study on standardized concepts of toxic equivalents for cigarettes needs to be conducted for more extensive use of in vitro tests.
Arsenic, a naturally occurring metalloid is a well-known water contaminant which causes a wide range of serious adverse health effects including cancer upon long-term exposure. Recent studies have ...shown high arsenic contamination in the ground water of North Eastern states of India including Southern Assam. Smokeless tobacco consumption locally known as "sadagura" is one of the most prevalent life style habit in southern Assam. The present study was undertaken in mice test system in vivo. Mice were exposed to smokeless tobacco (5 mg/kg body weight /day) and sodium arsenite (0.2 mg/kg body weight /day, 2 mg/kg body weight/day) independently and in combination for 90 days.
The results were compared with groups with only sodium arsenite exposure and groups which were exposed to only smokeless tobacco extract. Genotoxicity was evaluated by studying the incidence of micronucleated polychromatic erythrocytes from bone marrow. Both the tested doses of sodium arsenite induced statistically significant micronucleated polychromatic erythrocytes as compared to control group, however, sodium arsenite and smokeless tobacco extract could not increase the incidence of micronucleated polychromatic erythrocytes as compared to their individual counterparts when treated in combination in mice test system. Germ cell toxicity was evaluated by recording the sperm head abnormalities and total sperm count. Combined treatment of sodium arsenite and smokeless tobacco extract in lower dose induced a significant increase in sperm head abnormality as compared to only sodium arsenite and smokeless tobacco extract. Liver, kidney and intestine tissues were analyzed for various oxidative stress evaluations such as lipid peroxidation (MDA), Glutathione (GSH) and superoxide dismutase (SOD) assay. Sodium arsenite in combination with smokeless tobacco extract show higher genotoxic and germ cell toxic effects as compared to control but not when compared to their individual counterparts.
Impairment of the sperm head morphology by sodium arsenite and smokeless tobacco extract alone and in combination with lower dose of sodium arsenite could be oxidative stress mediated effects. Besides, combination treatment of both the agents may not produce additive effects related to micronucleated polychromatic erythrocytes induction and decline of total sperm count.
Current standards for radiological protection of the public have been uniformly established. However, individual differences in radiosensitivity are suggested to exist in human populations, which ...could be caused by nucleotide variants of DNA repair genes. In order to verify if such genetic variants are responsible for individual differences in radiosensitivity, they could be introduced into cultured human cells for evaluation. This strategy would make it possible to analyse the effect of candidate nucleotide variants on individual radiosensitivity, independent of the diverse genetic background. However, efficient gene targeting in cultured human cells is difficult due to the low frequency of homologous recombination (HR) repair. The development of artificial nucleases has enabled efficient HR-mediated genome editing to be performed in cultured human cells. A novel genome editing strategy, ‘transcription activator-like effector nuclease (TALEN)-mediated two-step single base pair editing’, has been developed, and this was used to introduce a nucleotide variant associated with a chromosomal instability syndrome bi-allelically into cultured human cells to demonstrate that it is the causative mutation. It is proposed that this editing technique will be useful to investigate individual radiosensitivity.
The aim of the present study was to perform cytogenetic analysis by means of a semi‑automated micronucleus‑centromere assay in lymphocytes from medical radiation workers. Two groups of workers ...receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere‑negative micronucleus (MNCM‑) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low‑dose' (0‑100 mGy) MNCM‑ dose‑response curve to evaluate the sensitivity and suitability of the micronucleus‑centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi‑automated scoring of MNCM‑ enables the detection of a dose of 50 mGy. The comparison of MNCM‑ yields in medical radiation workers and control individuals showed enhanced MNCM‑ scores in the medical radiation workers group (P=0.15). The highest MNCM‑ scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM‑ scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the highest occupational radiation burdens.
Ability of ethanol to produce chromosomal changes has been controversial in past many years; nevertheless many recent studies have shown that ethanol itself produces genotoxic effects like ...acetaldehyde. This study was carried out to evaluate the ability of ethanol and its metabolite acetaldehyde to induce chromosomal changes using
in vitro CBMN assay (Cytokinesis Blocked Micronucleus assay) in conjunction with immunofluorescent labeling of kinetochores. Kinetochore staining was used with a view to differentiate, between the genotoxic effects of both chemicals, and ascertain the mechanisms of genotoxicity induction by ethanol and acetaldehyde. Both ethanol and acetaldehyde produced statistically significant (
P
<
0.05) dose dependent increase in MN induction as compared with the controls over the dose range tested. Kinetochore analysis proved that the MN induced in ethanol were originated by an aneugenic mechanism, whereas in the case of acetaldehyde most of the MN had originated by a clastogenic mechanism. This not only confirms the ability of ethanol to produce DNA damage
in vitro but it also establishes the efficacy of CBMN assay to detect and differentiate between the genotoxic effects of different genotoxins. Here we report that ethanol is itself genotoxic, at least
in vitro, and produces genotoxic effects mainly through an aneugenic mechanism whereas its metabolite acetaldehyde is a clastogen.
Handling of chicken litter leads to exposure to toxic gases, endotoxins and airborne microorganisms. Aim of this study was to investigate if this results in acute cytotoxicity and to damage of the ...genetic material which is involved in the etiology of various diseases including cancer. Nuclear anomalies which reflect genotoxic and cytotoxic effects were monitored in exfoliated buccal and nasal cells which were collected from workers (n=25) of a power plant which processes chicken manure and from controls (n=21). Furthermore, biochemical parameters of the redox status (malondialdehyde, oxLDL and TEAC) and C-reactive protein (CRP) in plasma and the concentrations of toxic gases and endotoxins in the air were determined. No increase of anomalies which reflect chromosomal damage (micronuclei, binucleates, nuclear buds) but significantly higher rates of nuclear aberrations which are indicative for cytotoxicity (karyolysis, karyorrhexis, condensed chromatin) were found in the workers. These effects were in nasal cells more pronounced as in buccal cells. MDA, oxLDL and CRC levels were in both study groups similar. Chemical analyses show that the workers are exposed to high concentrations of NO and endotoxins, while the levels of NO2, NH3 and H2S were below the MAK levels. Taken together, the results show that anomalies that are due to cytotoxicity are increased in the workers and suggest that the exposure may lead to inflammations in the respiratory tract. However, the lack of induction of anomalies that reflect chromosomal damage indicate that no health effects will take place which are due to instability of the genetic material.
Microbiologically derived cyclodextrin glucanotransferase (CGTase) is used commercially as a processing agent in manufacture of food, pharmaceuticals, and cosmetics. Its toxic potential was evaluated ...in anticipation of use in the production of
-glycosyl isoquercitrin, a water-soluble form of quercetin.
Following OECD guidelines, CGTase, produced by
DK-1139, was evaluated in a genotoxicity battery consisting of a bacterial reverse mutation assay, an
micronucleus (MN) assay and MN and comet assays using B6C3F1 male and female mice. These same genotoxicity assays were also conducted for sodium sulfate, a contaminant of CGTase preparation. In a 90-day Sprague Dawley rat toxicity study, CGTase was administered by gavage in water at daily doses of 0, 250, 500, and 1000 mg/kg/day.
CGTase did not induce mutations with or without metabolic activation in the bacterial reverse mutation assay. Formation of micronuclei was not induced in either
or
MN assays with or without metabolic activation. No induction of DNA damage was detected in male or female mouse liver, stomach, or duodenum in the comet assay. Sodium sulfate also tested negative in these same genotoxicity assays. In the 90-day repeated dose rat study there were no treatment-related adverse clinical or pathological findings.
The genotoxicity assays and repeated dose toxicity study support the safe use of CGTase in production of
-glycosyl isoquercitrin.
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