Yield Of Micronuclei Research Articles

Sex differences in constructing dose-response calibration curves for micronuclei using cytokinesis-blocked micronucleus assay for radiation biological dosimetry in the Iranian population

Biological dosimetry, using chromosome damage biomarkers, can be considered as a key measure for radiation overexposure assessment. Therefore, for accurate dose estimation through biodosimetry, it's imperative for each biological dosimetry laboratory to establish its own specific dose-response calibration curve. In this research, the cytokinesis-blocked micronucleus (CBMN) assay was utilized to determine the frequencies of micronuclei (MN) per binucleated cell in human peripheral blood lymphocytes exposed to x-ray radiation using a LINAC (6 MV) at doses up to 4 Gy. The aim was to establish an in vitro dose-response calibration curve in our laboratory for the Iranian demographic by analyzing blood samples of ten participants (5 males and 5 females) through CABAS and Dose Estimate software. Our findings indicate an over-dispersion of the Poisson distribution in the pooled data across both sexes, coupled with a linear-quadratic increase in MN yield with dose which was particularly pronounced in the female group. The constructed dose-response curves for micronuclei yield are represented by equations Y= (0.0102 ± 0.0016) +(0.0296 ± 0.0054) D+(0.0232 ± 0.0017) D2 and Y= (0.0084 ± 0.0010) +(0.0212 ± 0.0034) D+(0.0160 ± 0.0011) D2 for the female groups, respectively. The alpha and beta coefficients, derived from the Dose Estimate software for each male and female group, were closely comparable with those obtained from the CABAS program and previous studies. The analysis of statistical parameters such as the Weighted Chi Squared (χ2) and p-value suggests that using distinct curves for each sex, rather than a unified biodosimetry formula for pooled data, ensures more accurate radiation dose estimates. Consequently, the findings of this study provide us with the assurance to utilize the derived calibration curve of MN for upcoming biological dosimetry needs in Iran. However, to enhance the reliability of results, it's essential to use biodosimetry with diverse assays and consider all clinical and physical parameters, given the limitations of cytogenetic assays.

Telomere Length, Mitochondrial DNA, and Micronucleus Yield in Response to Oxidative Stress in Peripheral Blood Mononuclear Cells.

Telomere shortening, chromosomal damage, and mitochondrial dysfunction are major initiators of cell aging and biomarkers of many diseases. However, the underlying correlations between nuclear and mitochondrial DNA alterations remain unclear. We investigated the relationship between telomere length (TL) and micronucleus (MN) and their association with mitochondrial DNA copy number (mtDNAcn) in peripheral blood mononuclear cells (PBMCs) in response to 100 μM and 200 μM of hydrogen peroxide (H2O2) at 44, 72, and 96 h. Significant TL shortening was observed after both doses of H2O2 and at all times (all p < 0.05). A concomitant increase in MN was found at 72 h (p < 0.01) and persisted at 96 h (p < 0.01). An increase in mtDNAcn (p = 0.04) at 200 µM of H2O2 was also found. In PBMCs treated with 200 µM H2O2, a significant inverse correlation was found between TL and MN (r = -0.76, p = 0.03), and mtDNA content was directly correlated with TL (r = 0.6, p = 0.04) and inversely related to MN (r = -0.78, p = 0.02). Telomere shortening is the main triggering mechanism of chromosomal damage in stimulated T lymphocytes under oxidative stress. The significant correlations between nuclear DNA damage and mtDNAcn support the notion of a telomere-mitochondria axis that might influence age-associated pathologies and be a target for the development of relevant anti-aging drugs.

Sex and dose rate effects in automated cytogenetics.

Testing and validation of biodosimetry assays is routinely performed using conventional dose rate irradiation platforms, at a dose rate of approximately 1Gy/min. In contrast, the exposures from an improvised nuclear device will be delivered over a large range of dose rates with a prompt irradiation component, delivered in less than 1μs, and a protracted component delivered over hours and days. We present preliminary data from a large demographic study we have undertaken for investigation of age, sex and dose rate effects on dicentric and micronucleus yields. Our data demonstrate reduced dicentric and micronucleus yields at very high dose rates. Additionally, we have seen small differences between males and females, with males having slightly fewer micronuclei and slightly more dicentrics than females, at high doses.

Molecular and Cellular Responses to Ionization Radiation in Untransformed Fibroblasts from the Rothmund–Thomson Syndrome: Influence of the Nucleo-Shuttling of the ATM Protein Kinase

The Rothmund–Thomson syndrome (RTS) is a rare autosomal recessive disease associated with poikiloderma, telangiectasias, sun-sensitive rash, hair growth problems, juvenile cataracts and, for a subset of some RTS patients, a high risk of cancer, especially osteosarcoma. Most of the RTS cases are caused by biallelic mutations of the RECQL4 gene, coding for the RECQL4 DNA helicase that belongs to the RecQ family. Cellular and post-radiotherapy radiosensitivity was reported in RTS cells and patients since the 1980s. However, the molecular basis of this particular phenotype has not been documented to reliably link the biological and clinical responses to the ionizing radiation (IR) of cells from RTS patients. The aim of this study was therefore to document the specificities of the radiosensitivity associated with RTS by examining the radiation-induced nucleo-shuttling of ATM (RIANS) and the recognition and repair of the DNA double-strand breaks (DSB) in three skin fibroblasts cell lines derived from RTS patients and two derived from RTS patients’ parents. The results showed that the RTS fibroblasts tested were associated with moderate but significant radiosensitivity, a high yield of micronuclei, and impaired DSB recognition but normal DSB repair at 24 h likely caused by a delayed RIANS, supported by the sequestration of ATM by some RTS proteins overexpressed in the cytoplasm. To our knowledge, this report is the first radiobiological characterization of cells from RTS patients at both molecular and cellular scales.

Replication stress drives chromosomal instability in fibroblasts of childhood cancer survivors with second primary neoplasms

New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary neoplasms (SPNs) as a late consequence of genotoxic cancer therapies with the highest risk among former childhood cancer patients. Since risk factors and predictive biomarkers for therapy-associated SPN remain unknown, we examined the sensitivity to mild replication stress as a driver of genomic instability and carcinogenesis in fibroblasts from 23 long-term survivors of a pediatric first primary neoplasm (FPN), 22 patients with the same FPN and a subsequent SPN, and 22 controls with no neoplasm (NN) using the cytokinesis-block micronucleus (CBMN) assay. Mild replication stress was induced with the DNA-polymerase inhibitor aphidicolin (APH). Fibroblasts from patients with the DNA repair deficiency syndromes Bloom, Seckel, and Fanconi anemia served as positive controls and for validation of the CBMN assay supplemented by analysis of chromosomal aberrations, DNA repair foci (γH2AX/53BP1), and cell cycle regulation. APH treatment resulted in G2/M arrest and underestimation of cytogenetic damage beyond G2, which could be overcome by inhibition of Chk1. Basal micronuclei were significantly increased in DNA repair deficiency syndromes but comparable between NN, FPN, and SPN donors. After APH-induced replication stress, the average yield of micronuclei was significantly elevated in SPN donors compared to FPN (p = 0.013) as well as NN (p = 0.03) donors but substantially lower than for DNA repair deficiency syndromes. Our findings suggest that mild impairment of the response to replication stress induced by genotoxic impacts of DNA-damaging cancer therapies promotes genomic instability in a subset of long-term cancer survivors and may drive the development of an SPN. Our study provides a basis for detailed mechanistic studies as well as predictive bioassays for clinical surveillance, to identify cancer patients at high risk for SPNs at first diagnosis.

Quantitative Correlations between Radiosensitivity Biomarkers Show That the ATM Protein Kinase Is Strongly Involved in the Radiotoxicities Observed after Radiotherapy.

Tissue overreactions (OR), whether called adverse effects, radiotoxicity, or radiosensitivity reactions, may occur during or after anti-cancer radiotherapy (RT). They represent a medical, economic, and societal issue and raise the question of individual response to radiation. To predict and prevent them are among the major tasks of radiobiologists. To this aim, radiobiologists have developed a number of predictive assays involving different cellular models and endpoints. To date, while no consensus has been reached to consider one assay as the best predictor of the OR occurrence and severity, radiation oncologists have proposed consensual scales to quantify OR in six different grades of severity, whatever the organ/tissue concerned and their early/late features. This is notably the case with the Common Terminology Criteria for Adverse Events (CTCAE). Few radiobiological studies have used the CTCAE scale as a clinical endpoint to evaluate the statistical robustness of the molecular and cellular predictive assays in the largest range of human radiosensitivity. Here, by using 200 untransformed skin fibroblast cell lines derived from RT-treated cancer patients eliciting OR in the six CTCAE grades range, correlations between CTCAE grades and the major molecular and cellular endpoints proposed to predict OR (namely, cell survival at 2 Gy (SF2), yields of micronuclei, recognized and unrepaired DSBs assessed by immunofluorescence with γH2AX and pATM markers) were examined. To our knowledge, this was the first time that the major radiosensitivity endpoints were compared together with the same cohort and irradiation conditions. Both SF2 and the maximal number of pATM foci reached after 2 Gy appear to be the best predictors of the OR, whatever the CTCAE grades range. All these major radiosensitivity endpoints are mathematically linked in a single mechanistic model of individual response to radiation in which the ATM kinase plays a major role.

Usher Syndrome Belongs to the Genetic Diseases Associated with Radiosensitivity: Influence of the ATM Protein Kinase.

Usher syndrome (USH) is a rare autosomal recessive disease characterized by the combination of hearing loss, visual impairment due to retinitis pigmentosa, and in some cases vestibular dysfunctions. Studies published in the 1980s reported that USH is associated with cellular radiosensitivity. However, the molecular basis of this particular phenotype has not yet been documented. The aim of this study was therefore to document the radiosensitivity of USH1—a subset of USH—by examining the radiation-induced nucleo-shuttling of ATM (RIANS), as well as the functionality of the repair and signaling pathways of the DNA double-strand breaks (DSBs) in three skin fibroblasts derived from USH1 patients. The clonogenic cell survival, the micronuclei, the nuclear foci formed by the phosphorylated forms of the X variant of the H2A histone (ɣH2AX), the phosphorylated forms of the ATM protein (pATM), and the meiotic recombination 11 nuclease (MRE11) were used as cellular and molecular endpoints. The interaction between the ATM and USH1 proteins was also examined by proximity ligation assay. The results showed that USH1 fibroblasts were associated with moderate but significant radiosensitivity, high yield of micronuclei, and impaired DSB recognition but normal DSB repair, likely caused by a delayed RIANS, suggesting a possible sequestration of ATM by some USH1 proteins overexpressed in the cytoplasm. To our knowledge, this report is the first radiobiological characterization of cells from USH1 patients at both molecular and cellular scales.

The cytokinesis-block micronucleus assay for cryopreserved whole blood

Purpose The cytokinesis-block micronucleus (MN) assay is a widely used technique in basic radiobiology research, human biomonitoring studies and in vitro radiosensitivity testing. Fresh whole blood cultures are commonly used for these purposes, but immediate processing of fresh samples can be logistically challenging. Therefore, we aimed at establishing a protocol for the MN assay on cryopreserved whole blood, followed by a thorough evaluation of the reliability of this assay for use in radiosensitivity assessment in patients. Materials and methods Whole blood samples of 20 healthy donors and 4 patients with a primary immunodeficiency disease (PID) were collected to compare the results obtained with the MN assay performed on fresh versus cryopreserved whole blood samples. MN yields were scored after irradiation with 220 kV X-rays (dose rate 3 Gy/min), with doses ranging from 0.5–2 Gy. Results The application of the MN assay on cryopreserved blood samples was successful in all analyzed samples. The radiation-induced MN and NDI scores in fresh and cryopreserved blood cultures were found to be similar. Acceptable inter-individual and intra-individual variabilities in MN yields were observed. Repeated analysis of cryopreserved blood cultures originating from the same blood sample, thawed at different time points, revealed that MN values remain stable for cryopreservation periods up to one year. Finally, radiosensitive patients were successfully identified using the MN assay on cryopreserved samples. Conclusions To our knowledge, this study is the first report of the successful use of cryopreserved whole blood samples for application of the MN assay. The data presented here demonstrate that the MN assay performed on cryopreserved whole blood is reliable for radiosensitivity testing. Our results also support its wider use in epidemiological, biomonitoring and genotoxicity studies. The presented method of cryopreservation of blood samples might also benefit other assays.

Cytogenetic monitoring of peripheral blood lymphocytes from medical radiation professionals occupationally exposed to low-dose ionizing radiation

In order to assess the health risk of low-dose radiation to radiation professionals, monitoring is performed through chromosomal aberration analysis and micronuclei (MN) analysis. MN formation has drawbacks for monitoring in the low-dose range. Nucleoplasmic bridge (NPB) analysis, with a lower background level, has good dose-response relationships at both high and relatively low dose ranges. Dicentric and ring chromosomes were analyzed in 199 medical radiation professionals, and NPB/MN yields were analyzed in 205 radiation professionals. The effects of sex, age of donor, types of work, and length of service on these cytogenetic endpoints were also analyzed. The yields of the three cytogenetic endpoints were significantly higher in radiation professionals versus controls. Frequencies of dicentric plus ring chromosomes were affected by length of service. NPB frequencies were influenced by type of work and length of service. MN yields were affected not only by types of work and length of service but also by donor sex and age. In conclusion, dicentric plus ring chromosomes, NPB, and MN can be induced by low-dose radiation in radiation professionals. NPB is a potential biomarker to assess the health risk of occupational low-dose radiation exposure.

A scanning and image processing system with integrated design for automated micronucleus scoring

Purpose: Our aim was to design a compact and cost-effective optical microscopic system for automated non-fluorescent micronucleus (MN) scoring whose performance can reach the accuracy of visual scoring with the help of minimal user interaction and also gives an option for fully automatic scoring with an accuracy suitable for triage purposes.Materials and methods: The concept of Radometer MN-Series (RS-MN) microscopic system designed by Radosys was to develop hardware and software layers in parallel in order to optimize the performance in automated MN scoring. A MN assay slide is automatically scanned by the RS-MN then the binucleated cells and micronuclei are automatically identified. Processing 1000 cells takes 10–60 minutes by automatic scoring (scanning plus image processing) depending on sample quality and required accuracy. The manual revision of the cell gallery takes an extra 10 minutes per sample. Dose response curves are determined for manual, automatic and semi-automatic scoring methods.Results: The combination of object-sensitive autofocusing method and the multi-layer image acquisition is able to reduce the minimum resolvable dose by 14%. The MN yields obtained from the manual, semi-automatic and automatic scoring methods are well correlating (Pearson’s correlation coefficients are between 0.977 and 0.998). In order to compare the reliability of the results of visual and automatic scoring, an extended analysis on uncertainty contributors was performed. For a dose of 1 Gy, the estimated relative uncertainty from the Poisson characteristics of MN yield is 17–19% for the manual and 20–38% for the automated scoring. Other uncertainty factors (differences in donor radiosensitivity, scorer performance, and sample preparations) can contribute to this error fall within a similar range: 3–16%. Taking into account all of the possible uncertainties, the minimum resolvable dose for the manual (0.48 Gy) is the two-thirds of that of the automatic scoring (0.61 Gy).Conclusions: The results verify that the fully automatic mode of RS-MN is suitable for triage purposes. The performance of the user interacted semi-automatic mode is comparable with the reference manual scoring. Its performance reaches up to other non-fluorescent automatic systems and offers a compact and cost-effective alternative.

Radiosensitization by Radiofrequency Fields is Correlated with Micronucleus Yield and Proliferative Index

Radiosensitization by Radiofrequency Fields is Correlated with Micronucleus Yield and Proliferative Index

Radioprotective and Antimutagenic Effects of Pycnanthus angolensis Warb Seed Extract against Damage Induced by X Rays.

Although different studies have demonstrated different applications of Pycnanthus angolensis extracts in traditional African and Asian medicine, its possible antimutagenic or genoprotective capacities have never been explored. We studied these capabilities of Pycnanthus angolensis seed extract (PASE) by means of the two micronucleus assays, determining the frequency of micronucleus (MN) yield in mouse bone marrow (in vivo) and in human lymphocytes blocked by cytochalasin B (in vitro). PASE exhibited a significant genoprotective capacity (p < 0.001) against X-rays with a protection factor of 35% in both in vivo and in vitro assays. Further, its radioprotective effects were determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) cell viability test in two cell lines: one being radiosensitive (i.e., human prostate epithelium (PNT2) cells) and the other being radioresistant (i.e., B16F10 melanoma cells). In the radiosensitive cells, PASE showed a protection factor of 35.5%, thus eliminating 43.8% of X-ray-induced cell death (p < 0.001) and a dose reduction factor of 2.5. In the radioresistant cells, a protection factor of 29% (p < 0.001) with a dose reduction factor of 4 was realized. PASE elicited a greater radioprotective capacity than the substances currently used in radiation oncology and, thus, could be developed as a nutraceutical radioprotectant for workers and patients exposed to ionizing radiation.

Some mutations in the xeroderma pigmentosum D gene may lead to moderate but significant radiosensitivity associated with a delayed radiation-induced ATM nuclear localization

Purpose: Xeroderma Pigmentosum (XP) is a rare, recessive genetic disease associated with photosensitivity, skin cancer proneness, neurological abnormalities and impaired nucleotide excision repair of the UV-induced DNA damage. Less frequently, XP can be associated with sensitivity to ionizing radiation (IR). Here, a complete radiobiological characterization was performed on a panel of fibroblasts derived from XP-group D patients (XPD).Materials and methods: Cellular radiosensitivity and the functionality of the recognition and repair of chromosome breaks and DNA double-strand breaks (DSB) was evaluated by different techniques including clonogenic cell survival, micronuclei, premature chromosome condensation, pulsed-field gel electrophoresis, chromatin decondensation and immunofluorescence assays. Quantitative correlations between each endpoint were analyzed systematically.Results: Among the seven fibroblast cell lines tested, those derived from three non-relative patients holding the p.[Arg683Trp];[Arg616Pro] XPD mutations showed significant cellular radiosensitivity, high yield of residual micronuclei, incomplete DSB recognition, DSB and chromosome repair defects, impaired ATM, MRE11 relocalization, significant chromatin decondensation. Interestingly, XPD transduction and treatment with statins and bisphosphonates known to accelerate the radiation-induced ATM nucleoshuttling led to significant complementation of these impairments.Conclusions: Our findings suggest that some subsets of XPD patients may be at risk of radiosensitivity reactions and treatment with statins and bisphosphonates may be an interesting approach of radioprotection countermeasure. Different mechanistic models were discussed to better understand the potential specificity of the p.[Arg683Trp];[Arg616Pro] XPD mutations.

Role of Endoplasmic Reticulum and Mitochondrion in Proton Microbeam Radiation-Induced Bystander Effect.

It is well known that mitochondria and the endoplasmic reticulum (ER) play important roles in radiation response, but their functions in radiation-induced bystander effect (RIBE) are largely unclear. In this study, we found that when a small portion of cells in a population of human lung fibroblast MRC-5 cells were precisely irradiated through either the nuclei or cytoplasm with counted microbeam protons, the yield of micronuclei (MN) and the levels of intracellular reactive oxygen species (ROS) in nonirradiated cells neighboring irradiated cells were significantly increased. Mito/ER-tracker staining demonstrated that the mitochondria were clearly activated after nuclear irradiation and ER mass approached a higher level after cytoplasmic irradiation. Moreover, the radiation-induced ROS was diminished by rotenone, an inhibitor of mitochondria activation, but it was not influenced by siRNA interference of BiP, an ER regulation protein. While for nuclear irradiation, rotenone-enhanced radiation-induced ER expression, and BiP siRNA eliminated radiation-induced activation of mitochondria, these phenomena were not observed for cytoplasmic irradiation. Bystander MN was reduced by rotenone but enhanced by BiP siRNA. When the cells were treated with both rotenone and BiP siRNA, the MN yield was reduced for nuclear irradiation but was enhanced for cytoplasmic irradiation. Our results suggest that the organelles of mitochondria and ER have different roles in RIBE with respect to nuclear and cytoplasmic irradiation, and the function of ER is a prerequisite for mitochondrial activation.

Micronuclei as a marker for medical screening of subjects continuously occupationally exposed to low doses of ionizing radiation

Context: Genotoxicity assays are widely employed in human biomonitoring studies to assess genetic damage inflicted by genotoxic agents.Objective: Evaluation of micronuclei (MN) as a screening marker of occupational ionizing radiation (IR) exposure.Materials and methods: Using micronucleus test, peripheral blood lymphocytes (PBL) of 402 control and exposed subjects were screened for genetic damage.Results: The mean frequencies of micronucleus test parameters were significantly higher in exposed persons. Increase of micronucleus yield with duration of exposure (DOE) by 0.303MN/year was revealed.Discussion and conclusion: The obtained data encourage us to consider MN as valuable markers for preventive medical screening of occupationally exposed groups.

Cytogenetic damage of radiotherapy in long-term head and neck cancer survivors

Purpose: To evaluate cytogenetic damage of radiotherapy (RT) and chemoradiotherapy (CRT) in long-term head and neck cancer survivors.Materials and methods: This study included 20 patients treated with RT (10 patients) or CRT (10 patients) for head and neck cancer. Nine healthy volunteers were included as control subjects. Cytochalasin B-blocked micronucleus (CBMN) assay was used to evaluate cytogenetic damage. To evaluate micronucleus (MN) by CBMN, the venous blood samples were drawn median 68 months (range 60–239 months) after the completion of treatment (RT or CRT) for head and neck cancer.Results: Nuclear division index (NDI) and number of MN in mononuclear and binuclear lymphocytes were significantly higher in patients with head and neck cancer than in control subjects [1.19 (1.08–1.47) vs. 1.07 (1.04–1.14), p < 0.001; 11.0 (2.0–22.0) vs. 1.0 (0–3.0), p < 0.001 and 15.0 (5.0–45.0) vs. 9.0 (2.0–15.0), p = 0.020, respectively]. NDI and number of MN in mononuclear lymphocytes were significantly lower in control subjects compared patients received CRT and those received only RT, but there was no significant difference between patients received CRT and those received only RT. Number of MN in binuclear lymphocytes was significantly lower in control subjects compared to patients received CRT, but there was no significant difference between control subjects and those received only RT. Also there was no significant difference between patients received CRT and those received only RT in terms of number of MN in binuclear lymphocytes.Conclusions: MN frequency of mononuclear and binuclear lymphocytes in medical follow-up of patients with head and neck cancer after RT could be important in evaluating cytogenetic damage of RT. However, further investigations are needed to provide quantitative correlations between MN yields and the clinical features in post-radiotherapy period.

In vitro RABiT measurement of dose rate effects on radiation induction of micronuclei in human peripheral blood lymphocytes.

Developing new methods for radiation biodosimetry has been identified as a high-priority need in case of a radiological accident or nuclear terrorist attacks. A large-scale radiological incident would result in an immediate critical need to assess the radiation doses received by thousands of individuals. Casualties will be exposed to different doses and dose rates due to their geographical position and sheltering conditions, and dose rate is one of the principal factors that determine the biological consequences of a given absorbed dose. In these scenarios, high-throughput platforms are required to identify the biological dose in a large number of exposed individuals for clinical monitoring and medical treatment. The Rapid Automated Biodosimetry Tool (RABiT) is designed to be completely automated from the input of blood sample into the machine to the output of a dose estimate. The primary goal of this paper was to quantify the dose rate effects for RABiT-measured micronuclei in vitro in human lymphocytes. Blood samples from healthy volunteers were exposed in vitro to different doses of X-rays to acute and protracted doses over a period up to 24 h. The acute dose was delivered at ~1.03 Gy/min and the low dose rate exposure at ~0.31 Gy/min. The results showed that the yield of micronuclei decreases with decreasing dose rate starting at 2 Gy, whereas response was indistinguishable from that of acute exposure in the low dose region, up to 0.5 Gy. The results showed a linear-quadratic dose-response relationship for the occurrence of micronuclei for the acute exposure and a linear dose-response relationship for the low dose rate exposure.

The cytokinesis-blocked micronucleus assay: dose-response calibration curve, background frequency in the population and dose estimation.

An in vitro study of the dose responses of human peripheral blood lymphocytes was conducted with the aim of creating calibrated dose-response curves for biodosimetry measuring up to 4 Gy (0.25-4 Gy) of gamma radiation. The cytokinesis-blocked micronucleus (CBMN) assay was employed to obtain the frequencies of micronuclei (MN) per binucleated cell in blood samples from 16 healthy donors (eight males and eight females) in two age ranges of 20-34 and 35-50 years. The data were used to construct the calibration curves for men and women in two age groups, separately. An increase in micronuclei yield with the dose in a linear-quadratic way was observed in all groups. To verify the applicability of the constructed calibration curve, MN yields were measured in peripheral blood lymphocytes of two real overexposed subjects and three irradiated samples with unknown dose, and the results were compared with dose values obtained from measuring dicentric chromosomes. The comparison of the results obtained by the two techniques indicated a good agreement between dose estimates. The average baseline frequency of MN for the 130 healthy non-exposed donors (77 men and 55 women, 20-60 years old divided into four age groups) ranged from 6 to 21 micronuclei per 1000 binucleated cells. Baseline MN frequencies were higher for women and for the older age group. The results presented in this study point out that the CBMN assay is a reliable, easier and valuable alternative method for biological dosimetry.

The influence of blood storage time and general anaesthesia on chromosomal radiosensitivity assessment.

The micronucleus assay (MN assay) is a well-established assay in genetic toxicology, biomonitoring of mutagen-exposed populations and chromosomal radiosensitivity testing. To evaluate the effect of storage time on the chromosomal radiosensitivity assessment in lymphocytes, micronuclei (MN) yields in blood samples received and processed on the same day were compared with MN yields obtained when blood cultures were set up 24 and 48h after blood sampling. Furthermore, the influence of general anaesthesia on MN and binucleated cells (BN) yields in the MN assay was considered. Blood samples of 10 healthy donors were irradiated and blood cultures were set up during the same day of blood sampling or with a delay of 24 or 48h. The MN assay was also performed on two blood samples from 60 women undergoing breast surgery. The first blood sample was taken before general anaesthesia and the second sample, 2h after anaesthesia induction. Fifty percent of the blood samples were transported to the cytogenetics lab within 2h while the other 50% reached the lab after 24h. The results of this study show a decrease in BN and an increase in MN yields with increasing storage time before irradiation and setting up of the MN assay for both healthy controls and patients. The administration of general anaesthesia in patients resulted in lower BN yields, higher spontaneous MN yields but no differences in radiation-induced MN yields. In conclusion, this study indicates that the time between blood sampling and the in vitro irradiation of the samples for the MN assay influences the MN yields. Delays of more than 24h should be avoided. To assess chromosomal radiosensitivity in patients, blood samples should be taken before induction of general anaesthesia as anaesthesia can have an impact on the reliability of the MN results.

The micronucleus assay in mammalian cells in vitro to assess health benefits of various phytochemicals

We evaluated the protective effects of Gentiana lutea extracts (GLEx) and 6-Gingerol (6-G) on clastogenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz(α) anthracene (DMBA) in vitro on HepG2 cells using the frequencies of induced micronuclei (MN) as the end point. Pre-, post- and simultaneous treatments with GLEx or 6-G and the carcinogens were carried out. Both GLEx post- and simultaneous treatments reduced the frequencies of MN induced by MNNG and DMBA. Probably this effect is due to an increase of cytostasis and a physico-chemical interaction between GLEx and DMBA under simultaneous treatment. Pre- and simultaneous treatments with 6-G significantly reduced the yield of MNNG-induced micronuclei without affecting % of cytostasis. Simultaneous treatment with 6-G plus DMBA resulted in reduction in the frequency of MN and an increase in cytotoxicity compared to sample treated alone with DMBA, whereas a post-treatment, caused a significant decrease in the yield of MN compared with DMBA alone without any cytotoxic effect. These results are compared with our earlier data obtained in the same system with other phytochemicals. It is concluded that for a critical evaluation of the protective effects of phytochemicals, both the influence on the induced MN and induced cytostasis have to be considered.