The micronucleus test and erythropoiesis: effects of cyclic adenosine monophosphate (cAMP) on micronucleus formation

The aim of this study was to determine the mechanism of the rodent bone marrow micronucleus test in relation to erythropoiesis. We have previously reported that an acceleration of erythropoiesis increases the frequency of micronucleated polychromatic erythrocytes (MPCE) induced by mutagens. The blood plasma erythropoietin level increased after the injection of N6-2-O-dibutyladenosine-3',5'-cyclic monophosphate into adenosine 3',5'-cyclic monophosphate (cAMP) at a dose of 500 mg/kg. A peak of erythropoietin induction was observed 3 h after the injection of cAMP. cAMP itself did not induce any micronuclei in erythroblasts of BALB/c mice. So, the frequency of MPCE did not increase after injection of cAMP. The highest frequency of MPCE and the dose-response relationship between the cAMP doses and micronucleus frequency were observed 30 h after injection of mitomycin C (MMC) in mice which had been administered cAMP 24 h previously. The highest effect of cAMP on the increase of MPCE was observed when cAMP was given 24 h before MMC injection, thus indicating that accelerating the multiplication of erythroblasts increases the frequency of MPCE induced by mutagens. The induction of MPCE in the bone marrow by three other chemicals (carboquone, 5-fluorouracil, and vincristine) also increased after pretreatment with cAMP. Our results suggest that the increase of MPCE induced by mutagens can be amplified following the acceleration of erythropoiesis by pretreatment with cAMP.     

Cytogenetic biomonitoring carried out in a village (Dolon) adjacent to the Semipalatinsk nuclear weapon test site

The Semipalatinsk region (Kazakhstan Republic) has been affected by extensive radioactive contamination due to more than 450 nuclear tests of which almost 100 were exploded in the atmosphere. The present results refer to cytogenetic assessments in a study cohort of the population of Dolon, a settlement located on the NE boundary of the nuclear weapon test site, which was exposed to elevated doses of ionising radiation primarily due to the first Soviet nuclear test in 1949. Conventional cytogenetic analyses were carried out on 21 blood samples from individuals (more than 50 years old) living in Dolon since the very beginning of nuclear testing. A matched control group included 20 individuals living in non-contaminated areas. Higher frequencies of chromosome aberrations were found in the Dolon cohort compared to the control group, even though they remain within the range of the background levels reported for large normal human population studies on elderly individuals. Received: 24 May 2000 / Accepted: 1 November 2000     

Cytogenetic effects of diethylstilbestrol-diphosphate (DES-dp) on mouse bone marrow monitored by the micronucleus test.

6 dosages of diethylstilbestrol-diphosphate (DES-dp), ranging from 0.01 to 500 mg per kg of body weight were compared to saline and phosphate buffered saline (negative controls) and two dosages of cyclophosphamide (positive control) in the micronucleus test with 115 ICR mice. DES-dp failed to generate a significant increase in micronucleated polychromatic erythrocytes over negative controls. Cyclophosphamide produced a dose-related increase in micronuclei similar to previously published reports. Iit was therefore determined that the micronucleus test did not detect the types of chromosomal changes known to be generated by DES-dp and DES.     

Comparison of single and multiple treatment regimens in the mouse bone marrow micronucleus assay for hydroquinone (HQ) and cyclophosphamide (CP)

Three experiments are reported as a contribution to the validation of the multiple treatment protocol presently under discussion. (1) In the single treatment experiment with hydroquinone (HQ) the time of maximum micronucleus response was determined to be 24 h with a dose of 75 mg/kg given intraperitoneally. In the subsequent dose-response study at the 24-h interval the lowest positive dose was 50 mg/kg of HQ (P less than 0.01). The increase in micronucleus frequencies was non-linear. (2) Daily treatments with 15 or 75 mg/kg of HQ and bone marrow sampling on days 2-4 after the start of treatment resulted in an increased micronucleus yield for the lower dose (P less than 0.05) and a decrease in micronucleus frequencies at the higher dose (P less than 0.01) with increasing numbers of treatments. At the same time no change in the composition of the erythrocyte population was observed. (3) With 25 mg/kg of cyclophosphamide (CP) the micronucleus yields increased from 1 to 3 daily doses but dropped significantly from 3 to 4 daily doses (P less than 0.01). The polychromatic to normochromatic erythrocyte ratio was significantly decreased after single (P less than 0.01) and marginally lower than the control value after 4 daily treatments with CP. The present data suggest that the response of the bone marrow micronucleus assay to multiple treatments may depend on the dose employed and may differ from chemical to chemical. The specific clastogenic action or other cellular effects of a chemical may influence the micronucleus yields of an individual chemical and it may be difficult to generate a strict protocol recommendation.     

Evaluation of the genotoxic potential of some microbial volatile organic compounds (MVOC) with the comet assay,the micronucleus assay and the HPRT gene mutation assay

Microbial volatile organic compounds (MVOC), metabolites of fungi detected in indoor moulds and in working places in compost facilities are considered as a potential health hazard. Their toxicological relevance, however, is largely unknown and data are rare. The aim of this study was to evaluate in vitro the genotoxic, clastogenic and mutagenic potential of same typical MVOC. For the study of DNA damage human lung carcinoma epithelial A549 cells, V79 Chinese hamster fibroblasts and human peripheral blood cells were exposed and subjected to the alkaline comet assay (single cell gel test). Taking the Chinese hamster V79 cell line as a target clastogenic effects were studied by the micronucleus test and mutagenic effects by the hypoxanthine-guanine-phosphoribosyl transferase gene mutation test (HPRT test). The cytogenic effects of MVOC were assessed by a clonogenic assay using the A549 cell line. The alkylating agent methyl methanesulfonate (MMS) was taken as a positive control. The results indicate that MVOC induced DNA damage is only seen in conditions in which also cytotoxic effects are observed. Clastogenic and mutagenic effects could not be detected.     

Influence of age and sex on the spontaneous DNA damage detected by micronucleus test and comet assay in mice peripheral blood cells

We have investigated the normal variations in basal DNA damage detected by Comet assay in leukocytes and micronucleated erythrocytes (MNE) using the Micronucleus test (MN) in peripheral blood cells from 45 female and male mice from different age groups (newborns, 3.5, 12, and 104 weeks) to clarify age and sex-related changes. Comparison of basal DNA damage detected by Comet assay showed significantly increased values in 104 weeks old mice in relation to the other ages (P < or = 0.01), and newborn mice showed higher values in MNE frequency when compared to all the other groups (P < or = 0.01). A positive correlation was observed between Damage Frequency (r =0.382, P = 0.010) and Damage Index (r = 0.640, P < 0.001) and age. Age was also correlated with the ratio of polychromatic erythrocytes/normachromatic erythrocytes (PCE/NCE) (r = -0.473, P = 0.001), and the MNE frequency was positively correlated with the ratio of PCE/NCE (r = 0.454, P = 0.002). These results suggest an age-related slow down of DNA repair efficiency of DNA damage and/or DNA damage accumulation. Furthermore, data on the spontaneous MNE frequency indicate that the reticuloendothelial system matures with age, and there is a close relationship between erythropoiesis and micronucleus induction in erythrocytes. The influence of sex in the parameters analyzed was less clear. In conclusion, age seems to influence in basal DNA damage and should be considered in genotoxicity studies using mice. Finally, comparisons between assays must be made with care when different cells are compared (e.g. leukocytes and erythrocytes), as found with the Comet assay and MN test.     

Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay)

The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity.     

Evaluation of the genotoxic potential of some microbial volatile organic compounds (MVOC) with the comet assay, the micronucleus assay and the HPRT gene mutation assay.

Microbial volatile organic compounds (MVOC), metabolites of fungi detected in indoor moulds and in working places in compost facilities are considered as a potential health hazard. Their toxicological relevance, however, is largely unknown and data are rare. The aim of this study was to evaluate in vitro the genotoxic, clastogenic and mutagenic potential of same typical MVOC. For the study of DNA damage human lung carcinoma epithelial A549 cells, V79 Chinese hamster fibroblasts and human peripheral blood cells were exposed and subjected to the alkaline comet assay (single cell gel test). Taking the Chinese hamster V79 cell line as a target clastogenic effects were studied by the micronucleus test and mutagenic effects by the hypoxanthine-guanine-phosphoribosyl transferase gene mutation test (HPRT test). The cytogenic effects of MVOC were assessed by a clonogenic assay using the A549 cell line. The alkylating agent methyl methanesulfonate (MMS) was taken as a positive control. The results indicate that MVOC induced DNA damage is only seen in conditions in which also cytotoxic effects are observed. Clastogenic and mutagenic effects could not be detected.     

Evaluation of genotoxicity using the micronucleus assay and nuclear abnormalities in the tropical sea fish Bathygobius soporator (Valenciennes, 1837) (Teleostei, Gobiidae)

The micronucleus and nuclear abnormalities assays have been used increasingly to evaluate genotoxicity of many compounds in polluted aquatic ecossystems. The aim of this study is to verify the efficiency of the micronucleus assay and nuclear abnormality assay in field and laboratory work, when using erythrocytes of the tropical marine fish Bathygobius soporator as genotoxicity biomarkers. Gill peripheral blood samples were obtained from specimens of Bathygobius soporator. In order to investigate the frequencies of micronuclei and to assess the sensitivity of species, the results were compared with samples taken at the reference site and maintained in the laboratory, and fish treated with cyclophosphamide. The micronucleus assay was efficient in demonstrating field pollution and reproducing results in the labotatory. There were significant higher frequencies of micronuclei in two sites subject to discharge of urban and industrial effluents. The nuclear abnormality assay did not appear to be an efficient tool for genotoxicity evaluation when compared with field samples taken at a reference site in laboratory, with a positive control.     

Caiman latirostris (broad-snouted caiman) as a sentinel organism for genotoxic monitoring: basal values determination of micronucleus and comet assay.

Caiman latirostris is one of the two crocodilian species that inhabit Argentina. In this country, as a consequence of agricultural frontiers expansion during the last years, many areas of the geographic distribution of the broad snouted caiman overlap with regions of intensive agricultural activity. Contaminants released to the environment may induce genetic alterations in wildlife, which could lead to mutations and/or carcinogenesis. Up to the moment, no studies had been made concerning the possibbility to apply biomarkers of genotoxic evaluation in C. latirostris. The aim of this study was to adapt two widely used genotoxic techniques, the comet assay and the micronucleus test, for their application in C. latirostris and to determine the baseline values in this species, in order to establish its suitability as a sentinel organism for future genotoxic monitoring of environmental pollutants. A total of 41 juvenile caimans of 4 months old (FMO) and 10 months old (TMO) were used. Genotoxic techniques were applied on peripheral blood erythrocytes introducing the necessary modifications required by the material, which are presented here. Our results show that baseline values of DNA damage are quite stable among juvenile caimans (MN: FMO animals 0.87+/-0.74 and TMO animals 1.04+/-0.92; DI: FMO animals 103.40+/-3.36 and TMO animals 120.08+/-11.33), being independent of the nest of origin, sex and size of the animals and confirm the potential value of both short term tests as accurate screening tools for the evaluation of genotoxic agents in C. latirostris. This is the first reference to the application of genotoxic techniques on C. latirostris and the second in crocodilians. Data provided here will be useful for future studies involving the biomonitoring of natural regions where C. latirostris occurs, employing this species as a sentinel organism for genotoxic assessment of environmental pollutants.     

Differential sensitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay.

Both the V79 and CHO cell lines are routinely used in the in vitro MN screening assay for the detection of possible genotoxicants. The CHO cell line is the predominant cell line currently used in the genetic toxicology testing industry. However, some laboratories routinely utilize the V79 cell line since the in vitro MN screening assay was initially developed using V79 cells. Our laboratory has historically used the CHO cell line. Therefore, our laboratory was interested in comparing the two cell lines with regard to possible similarities or differences in MN induction sensitivity after exposure to cyclophosphamide (CPA) and mitomycin C (MMC), the two standard positive control chemicals routinely used in this assay. Three exposure conditions in the presence of CPA and MMC were examined in both cell lines. Replicate cultures of CHO cells in McCoy's 5A and V79 cells in both McCoy's 5A and E-MEM were established and treated with 5 microg CPA/ml (4h exposure with S9), 0.5 microg MMC (4h exposure without S9) and 0.5 microg MMC (24h exposure without S9). A total of 400 cytochalasin B-blocked binucleated cells and 200 consecutive cells were analyzed from each culture for MN and cell cycle kinetics, respectively. Analysis of the data demonstrated that CHO cells were up to approximately five-fold more sensitive to the induction of CPA- and MMC-induced MN than V79 cells. Both cell lines exhibited similar average generation times among identical exposure groups. Therefore, the difference in MN sensitivity cannot be attributed to possible differences in cell cycle kinetics and is possibly related to inherent cellular differences in the processing of and/or repair of CPA- and MMC-induced damage by V79 and CHO cells.     

Activity of a nitroalkene derivative, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene, in the Salmonella/microsome assay and the mouse bone marrow micronucleus test

Mutagenicity of a substituted nitroalkene, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene (BNF) was tested in the Salmonella/microsome assay using the strains TA 98, TA 100 and TA 100NR (nitroreductase deficient). BNF was a direct mutagen in TA 98 and TA 100; the response was lowered when exogenous metabolic activation (S9) was used. A further decrease in mutagenicity was observed in strain TA 100NR, as compared to the parental TA 100, which showed the involvement of nitroreduction in the overall response elicited by BNF. The micronucleus assay was carried out in Swiss male mice which were given a single i.p. dose of 10–20 mg/kg of BNF dissolved in peanut oil, bone marrow being sampled 24 and 48 h later. The micronucleated polychromatic erythrocyte counts (MNPCE) showed a weak response in the dose range of 10–17.5 mg/kg at the second sampling (48 h) and a significant rise for 20 mg/kg at 24 and 48 h.     

Applicability and robustness of the hen's egg test for analysis of micronucleus induction (HET-MN): results from an inter-laboratory trial

The hen's egg test for analysis of micronucleus formation (HET-MN) was developed several years ago to provide an alternative test system to the in vivo micronucleus test. In order to assess its applicability and robustness, a study was carried out at the University of Osnabrueck (lab A) and at the laboratories of Henkel AG & Co. KGaA (lab B). Following transfer of the method to lab B, a range of test substances that had been pre-tested at lab A, were tested at Henkel: the genotoxins cyclophosphamide, dimethylbenz(a)anthracene, methotrexate, acrylamide, azorubin, N-nitroso-dimethylamine and the non-genotoxins, orange G and isopropyl myristate. In a second phase, additional compounds with known in vivo properties were examined in both labs: the non-genotoxin, ampicillin, the "irrelevant" positives, isophorone and 2,4-dichlorophenol ("irrelevant" means positive in standard in vitro tests, but negative in vivo), the clastogen p-chloroaniline, and the aneugens carbendazim and vinorelbine. All substances were correctly predicted in both labs with respect to their in vivo genotoxic properties, indicating that the HET-MN may have an improved predictivity compared with current standard in vitro test systems. The results support the promising role of the HET-MN assay as a supplement to existing test batteries.     

Evaluation of genotoxicity, cytotoxicity and cytostasis in human lymphocytes exposed to patulin by using the cytokinesis-block micronucleus cytome (CBMN cyt) assay

Patulin (PAT) is a fungal secondary metabolite commonly present in apples and apple products. In the present study, PAT was evaluated for its genotoxic, cytotoxic and cytostatic effects to human peripheral blood lymphocytes by using the cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Lymphocyte cultures were treated with PAT at the following concentrations, 0.1, 0.3, 0.5, 1.0, 2.5, 5.0, and 7.5 μM, as well as 0.5 μM mitomycin c (MMC) as a positive control and dimethyl sulfoxide (DMSO) as a vehicle control. PAT was found to induce nucleoplasmic bridges (NPBs) at 5.0 and 7.5 μM concentrations (P?<?0.05), apoptotic cells at 0.1, 1.0, 5.0 μM (P?<?0.05), 7.5 μM concentrations (P?<?0.01) and necrotic cells at 0.3 and 2.5 μM (P?<?0.05), 5.0 and 7.5 μM (P?<?0.01) concentrations in human lymphocytes. The 2.5, 5.0, and 7.5 μM PAT concentrations also led to a clear decrease in the nuclear division index (NDI) (P?<?0.05). PAT caused a significant dose-dependent increase in the number cells of NPBs, in the frequency of apoptotic and necrotic cells, and a significant dose-dependent decrease in the NDI values in lymphocytes. These results indicate that PAT at high concentrations is genotoxic, cytotoxic and cytostatic in cultured human lymphocytes.     

Caiman latirostris (broad-snouted caiman) as a sentinel organism for genotoxic monitoring: Basal values determination of micronucleus and comet assay

Caiman latirostris is one of the two crocodilian species that inhabit Argentina. In this country, as a consequence of agricultural frontiers expansion during the last years, many areas of the geographic distribution of the broad snouted caiman overlap with regions of intensive agricultural activity. Contaminants released to the environment may induce genetic alterations in wildlife, which could lead to mutations and/or carcinogenesis. Up to the moment, no studies had been made concerning the possibbility to apply biomarkers of genotoxic evaluation in C. latirostris.The aim of this study was to adapt two widely used genotoxic techniques, the comet assay and the micronucleus test, for their application in C. latirostris and to determine the baseline values in this species, in order to establish its suitability as a sentinel organism for future genotoxic monitoring of environmental pollutants.A total of 41 juvenile caimans of 4 months old (FMO) and 10 months old (TMO) were used. Genotoxic techniques were applied on peripheral blood erythrocytes introducing the necessary modifications required by the material, which are presented here.Our results show that baseline values of DNA damage are quite stable among juvenile caimans (MN: FMO animals 0.87 ± 0.74 and TMO animals 1.04 ± 0.92; DI: FMO animals 103.40 ± 3.36 and TMO animals 120.08 ± 11.33), being independent of the nest of origin, sex and size of the animals and confirm the potential value of both short term tests as accurate screening tools for the evaluation of genotoxic agents in C. latirostris. This is the first reference to the application of genotoxic techniques on C. latirostris and the second in crocodilians.Data provided here will be useful for future studies involving the biomonitoring of natural regions where C. latirostris occurs, employing this species as a sentinel organism for genotoxic assessment of environmental pollutants.     

Bacterial expression of the shrimp molt-inhibiting hormone (MIH): antibody production, immunocytochemical study and biological assay

Molting in shrimp is controlled by the molt-inhibiting hormone (MIH) and ecdysone. MIH inhibits the synthesis of ecdysone in the Y-organ, resulting in molt suppression; it is a neuropeptide member belonging to the eyestalk CHH/MIH/GIH family. The cloning of MIH (formerly MIH-like) of the shrimp Metapenaeus ensis has been reported in a previous study. To obtain a large quantity of fusion protein for antibody production and biological assay, the cDNA encoding the shrimp MIH was inserted into the pRSET bacterial expression vector. His-tagged fusion protein was produced and purified by an Ni2+-charged affinity column. Polyclonal antibody to rMIH was subsequently obtained by immunizing rabbits with purified recombinant proteins. Results from Western blot analysis indicated that the antibody was specific. Furthermore, results from immunocytochemical analysis showed that specific cells in three different clusters of the X-organ, the sinus gland and the axonal tract of the eyestalk contain MIH. To test for the molt-inhibiting activity of rMIH, shrimp at intermolt stage were injected with rMIH and the molt cycle duration of the injected shrimp was monitored. A significant increase in molt cycle duration was recorded for the shrimp injected with the recombinant protein.     

Effects of physical and chemical restraint on intravenous glucose tolerance test in crested black macaques (Macaca nigra)

The effects of physical and chemical restraint on glucose clearance and insulin secretion were evaluated during intravenous glucose tolerance testing in Macaca nigra. Conscious monkeys placed in plexiglas cylindrical restraining devices (CRD) appeared relaxed, but glucose clearance and insulin secretion were impaired. A combination of midazolam with ketamine, compared to ketamine alone, did not cause detectable changes in the intravenous glucose tolerance tests; midazolam also reduced adverse reactions to ketamine and extended the duration of anesthesia. The cylindrical restraining device can be convenient for examining monkeys, but it is limited by its adverse affects on metabolic and hormonal measurements in intravenous glucose-tolerance tests. Chemical restraint using ketamine with midazolam was more effective than ketamine alone.     

Monoclonal antibody based dot-enzyme linked immunosorbent assay (Dot-ELISA) and agar gel precipitation test (AGPT) for identification of Newcastle disease virus (NDV)

Two mouse monoclonal antibodies (MAbs), viz. 2B7 and 2 D10 raised against haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus (NDV) were used to identify several other field isolates and vaccine strains of NDV. These MAbs reacted specifically with all the NDV strains/isolates in Dot-ELISA whereas, only MAb 2D10 reacted with all the NDV strains/isolates in agar gel precipitation test. These two tests employing the MAbs were standardised for rapid diagnosis and identification of NDV.     

Comparison of metabolic and hormonal effects of calcitonin and somatostatin (SRIF) in the course of oral glucose tolerance test (OGTT)

A comparison is presented of the effect of two therapeutic doses of synthetic somatostatin (250 and 500 micrograms) and salmon calcitonin (50 and 100 U) on the blood levels of sugar, insulin (IRI), somatotropin (HGH) and cortisol in healthy volunteers following peroral administration of 75 g of glucose. Calcitonin was responsible for a significant change in glycaemia as well as IRI levels: following a retarded enhancement glycaemia as well as insulinaemia through out the first 15-30 minutes of OGTT, increased levels of both indicators were persistent at minute 120 and 180, so that the course of both curves was almost parallel. The effect was similar after SRIF had been administered, with the exception of insulin secretion being more pronounced, so that at a later stage of OGTT no hyperinsulinaemia was seen. The HGH levels tended to decrease due to both hormones, the tendency being more marked after SRIF, though statistically insignificant. There was a marked difference between the hormones as regards their effect on adrenocortical secretion. While the latter was constantly stimulated throughout OGTT under calcitonin infusion, the influence of SRIF was not significant. The metabolic and hormonal changes were found after both a lower and higher dose of both hormones, the only differences being that the inhibitory effect on the initial increase in glycaemia following a lower dose of SRIF was of no statistical significance. Hence, the metabolic and hormonal effects of calcitonin and SRIF in an acute experiment display many similarities, however, they do differ in some aspects; these effects do not depend on the doses demonstrated for both lower and higher doses of the above hormones.