Intercorrelations and sources of variability in three mutagenicity assays: a population-based study

The purpose of this study was to evaluate the intercorrelation between three genetic assays in 112 subjects. The group was pooled from two originally separate but homogeneous subgroups of 56 persons each. Procedures included assays for hprt mutant frequencies, micronuclei in human lymphocytes, and mutations at the glycophorin A (gpa) loci. We found no statistically significant or biologically important intercorrelations among the three biomarkers. We did, however, observe significant correlations between log_e hprt mutant frequency and cloning efficiency (inverse correlation for these 2 variables), age and log_e hprt mutant frequency, an inverse relationship between cloning efficiency and age, and an important differential sex effect favoring a greater micronuclei frequency in females than males. No significant correlations between the covariates of interest and glycophorin A variant frequencies NN or NO were observed. Using multivariable linear regression, age was found to account for the majority of the variability in hprt mutant frequency (greater than sex and/or smoking); for micronuclei data, only sex contributed a statistically significant and biologically important proportion to the total variation. We conclude that despite observing no significant intercorrelations between the three assays performed simultaneously from the same individuals in a large population database, a significant correlation between age and hprt mutant frequency and an inverse association between cloning efficiency and hprt do exist; furthermore, we verified the strong differential sex-specific effect on micronucleus frequencies.     

Multiparametric flow cytometric analysis of radiation-induced micronuclei in mammalian cell cultures.

A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences. The frequencies of radiation-induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei. Dose effect curves and the time-dependent induction of micronuclei were measured for two different mouse cell lines.     

Establishment of dose-response relationships between doses of Cs-137 γ-rays and frequencies of micronuclei in human peripheral blood lymphocytes

It has been suggested that the yield of micronuclei in human peripheral blood lymphocytes could be used as a biological dosimeter in cases of radiation exposure. In the present study micronuclei were induced in lymphocytes by exposing human blood samples in vitro to various doses of Cs-137 γ-rays. The blood samples were then cultivated using the cytokinesis block method. Coded programs were employed to establish the relationships between the frequencies of micronuclei and various doses of γ-rays. The best fit was obtained by the linear-quadratic model, Y = c + aD + bd², where Y is the yield of micronuclei, D is the dose in Gy and c, a, b, are constants. It seems there is a correlation between the yields of MN in mononuclear cells and the corresponding doses of radiation. Therefore an attempt was made to include these MN in the calculation of the dose-response relationship.     

Increased frequencies of micronuclei in T8 lymphocytes of smokers

Micronucleus frequencies and mitotic indices were analyzed in B, T4, and T8 lymphocytes from 40 smokers and 42 non-smoking referents. The highest level of micronuclei was found in T4 cells followed by T8 and B cells. These differences were statistically significant. There were statistically significant linear correlations between the micronucleus frequencies of all three subsets. There was a statistically significant effect of smoking only in the T8 cells. Smoking also increased the number of neutrophilic granulocytes and lymphocytes in the peripheral blood. There was a statistically significant effect of age on the micronucleus frequencies in T4 and T8 lymphocytes. The mitotic indices did not have any effect on the micronucleus frequencies and they were not influenced by smoking, age or sex.     

The micronuclei frequencies in the buccal cell epithelium of young people of different age and gender in Ukraine

The results of study of micronuclei (MN) frequencies among the participants of Ukrainian school biological olympiads are presented. Totally 266 persons have been inspected. The distribution of MN frequencies correspond to the Poisson's distribution with lambda = 2.5. The average frequency of micronuclei in males was 2.4 +/- 0.15%, in females it was 2.7 +/- 0.14%. The difference of the average MN frequencies for these two groups was statistically insignificant. The individual micronuclei frequencies varied from 0 to 8.3%, the average MN frequency in the general group was 2.5 +/- 0.11%, (limits 2-5%). The micronuclei frequencies in different age groups of males and females were compared. Significantly higher MN frequencies in females than in males at the age of sixteen were detected. The age-related changes of micronuclei frequencies (14-18 age) were different for females compared to males.     

Chromosomal aberrations and sister-chromatid exchanges in workers exposed to styrene

Few studies exist about chromosomal damage in workers occupationally exposed to styrene. In the present study, chromosomal aberrations and SCEs were analyzed from cultures of peripheral lymphocytes of workers employed in 6 different reinforced-plastics industries with styrene air exposure levels ranging from 30 to 400 mg/mc. A control group was selected on the base of sex, age and smoking habit. We examined 50-h cultures (for chromosomal-aberrations) and 72-h cultures (for SCEs) for each individual. All workers exposed to styrene, as compared with controls, showed significantly increased frequencies of chromosomal aberrations, while SCEs were significantly increased at 4 of the 6 plants. High SCE values appeared with styrene air concentrations higher than 200 mg/mc. Apart from the possible presence and role of other interfering chemicals in the various plants, chromosomal aberrations seem to be more sensitive than SCEs for the detection of chromosomal damage caused by exposure to low doses of styrene.     

Chromosomal aberrations and sister-chromatid exchanges in workers exposed to styrene

Few studies exist about chromosomal damage in workers occupationally exposed to styrene. In the present study, chromosomal aberrations and SCEs were analyzed from cultures of peripheral lymphocytes of workers employed in 6 different reinforced-plastics industries with styrene air exposure levels ranging from 30 to 400 mg/mc. A control group was selected on the base of sex, age and smoking habit. We examined 50-h cultures (for chromosomal-aberrations) and 72-h cultures (for SCEs) for each individual.All workers exposed to styrene, as compared with controls, showed significantly increased frequencies of chromosomal aberrations, while SCEs were significantly increased at 4 of the 6 plants. High SCE values appeared with styrene air concentrations higher than 200 mg/mc.Apart from the possible presence and role of other interfering chemicals in the various plants, chromosomal aberrations seem to be more sensitive than SCEs for the detection of chromosomal damage caused by exposure to low doses of styrene.     

Nuclear anomalies in the buccal cells of calcite factory workers

The micronucleus (MN) assay on exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans. To determine the genotoxic effects of calcite dust that forms during processing, MN assay was carried out in exfoliated buccal cells of 50 (25 smokers and 25 non-smokers) calcite factory workers and 50 (25 smokers and 25 non-smokers) age- and sex-matched control subjects. Frequencies of nuclear abnormalities (NA) other than micronuclei, such as binucleates, karyorrhexis, karyolysis and 'broken eggs', were also evaluated. Micronuclei and the other aforementioned anomalies were analysed by two way analysis of covariance. The linear correlations between the types of micronucleus and nuclear abnormalities were determined by Spearman's Rho. There was a positive correlation between micronuclei and other types of nuclear abnormalities in accordance with the Spearman's Rho test. Results showed statistically significant difference between calcite fabric workers and control groups. MN and NA frequencies in calcite fabric workers were significantly higher than those in control groups (p < 0.05). The results of this study indicate that calcite fabric workers are under risk of significant cytogenetic damage.     

DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabeling technique. In lungs after 21 days of exposure to 1500 mg/m3 the level of 7-SO-guanine was 23.0+/-11.9 adducts/10(8) normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6+/-0.2 adducts/10(8) normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood (r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4+/-2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural elimination of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei (r=0.721, P=0.028).     

Cytogenetic monitoring of fishermen with environmental mercury exposure

Due to high mercury levels in many Mediterranean aquatic organisms, people who live in this area and consume large amounts of seafood are exposed to a toxicological hazard. A group of 51 fishermen exposed to mercury through eating contaminated seafood from the northern Tyrrhenian Sea underwent cytogenetic monitoring. This work is part of a research project consisting of the evaluation of micronuclei (MN), chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in peripheral blood lymphocytes. Here we present data on mercury levels in blood and on micronucleus frequencies in peripheral blood lymphocytes of fishermen. The range of mercury concentrations in blood was 10.08–304.11 ng/g fresh weight, the average was 88.97±54.09 ng/g. Micronucleus frequency was defined with at least 2000 binucleated cells scored for each person; the average was 8.74 ± 2.56 expressed on 1000 binucleated cells. A statistical correlation was found between MN frequency and total mercury concentration in blood (p = 0.00041, r = 0.674), as well as between MN frequency and age (p = 0.017). No other parameters taken into account correlated with MN frequency.     

Monitoring of carbamate and organophosphate resistance levels in Nilaparvata lugens based on bioassay and quantitative sequencing

The resistance levels to carbamate (CB) and organophosphate (OP) insecticides were determined by topical application in 14 field strains of Nilaparvata lugens. The resistance levels of N. lugens to CB and OP were 1.3–47.5-fold and 1.4–14.4-fold higher than a susceptible strain, respectively. A quantitative sequencing (QS) protocol was established to determine the allele frequencies of four acetylcholinesterase point mutations putatively associated with CB and OP resistance. The allele frequencies of the four mutations (G119A, F/Y330S, F331H and I332L) in field strains' ranges are ca. 0.0–51.7%, 0.0–88.9%, 5.1–56.0% and 6.7–57.3%, respectively. The F331H and I332L were tightly linked to each other, suggesting that these mutations may occur simultaneously. In correlation analysis, G119A was not well correlated with actual resistance levels (r² = < 0.232), whereas F331H and I332L showed a better correlation with the resistance levels of benzofuranyl methylcarbamates (r² = 0.595). This finding indicates that F331H and I332L mutation frequencies may be used as molecular markers for detecting carbamate resistance in N. lugens. A QS protocol detecting the F331H and I332L mutation frequencies could therefore be employed as a supportive tool for the rapid monitoring of CB insecticide resistance levels in N. lugens.     

Chromosome damage and micronucleus formation in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cellular telephone frequency (847.74 MHz, CDMA)

Peripheral blood samples collected from four healthy nonsmoking human volunteers were diluted with tissue culture medium and exposed in vitro for 24 h to 847.74 MHz radiofrequency (RF) radiation (continuous wave), a frequency employed for cellular telephone communications. A code division multiple access (CDMA) technology was used with a nominal net forward power of 75 W and a nominal power density of 950 W/m(2) (95 mW/cm(2)). The mean specific absorption rate (SAR) was 4.9 or 5.5 W/kg. Blood aliquots that were sham-exposed or exposed in vitro to an acute dose of 1.5 Gy of gamma radiation were included in the study as controls. The temperatures of the medium during RF-radiation and sham exposures in the Radial Transmission Line facility were controlled at 37 +/- 0.3 degrees C. Immediately after the exposures, lymphocytes were cultured at 37 +/- 1 degrees C for 48 or 72 h. The extent of genetic damage was assessed from the incidence of chromosome aberrations and micronuclei. The kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation-exposed and sham-exposed lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, excess fragments, binucleate cells, and micronuclei. The response of gamma-irradiated lymphocytes was significantly different from that of both RF-radiation-exposed and sham-exposed cells for all of these indices. Thus there was no evidence for induction of chromosome aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 847.74 MHz RF radiation (CDMA) at SARs of 4.9 or 5.5 W/kg.     

Comparative evaluation of the DNA damage response in two Peruvian marine bivalves exposed to changes in temperature

The comparison of temperature responses of two mytilids from the high (Brachidontes purpuratus) and low (Semimytilus algosus) intertidal zone of the Peruvian coast was carried out focusing on the production of micronucleus and nuclear abnormalities in gill tissue. Two temperatures (23 and 11°C) were evaluated, in presence of three mitomycin C concentrations as a stressor (0.02, 0.04 and 0.06 × 10^?6), simulating hypothetical El Niño and La Niña conditions. Responses to extreme temperatures between both species were significantly different (P = 0.008). Frequency of micronuclei and nuclear abnormalities in S. algosus did not differ statistically among the temperature treatments, whereas in B. purpuratus there was an observed significant decrease in nuclear abnormalities (P = 0.012) and micronuclei frequencies (P = 0.002) between both temperature treatments. The low frequency of micronuclei observed at high temperature and mitomycin C suggests a better efficiency to stress resistance, as occurs during El Niño events, of the species from the higher intertidal zone of B. purpuratus compared to S. algosus from the lower intertidal zone.     

Radionuclide determination of absolute LV volumes: Interstudy,interobserver and intraobserver variances

The results of 22 absolute left ventricular volume (LVV) determinations by a radionuclide (RN) method are compared to the results obtained by contrast ventriculography (CV). Another 10 patients were analysed in order to evaluate the interstudy, interobserver and intraobserver variances. Good correlation was shown between the RN and CV measurements of the end diastolic volume (EDV), end systolic volume (ESV), stroke volume (SV) and ejection fraction (EF), but the RN method overestimates the EDV and ESV. The EF was underestimated, but no difference could be shown for the SV. On the inter- and intraobserver levels, regression analysis yielded excellent correlation (r > 0.99 in all cases) with no statistically significant difference (P < 0.05). The interstudy variance was minimal as indicated by regression analysis (r > 0.87) and no statistically significant difference (P < 0.05) could be shown between studies. The results indicate that the RN method of LVV determination can be used in intervention studies over a limited period.     

Measurement of plasma testosterone by gas chromatography-negative-ion mass spectrometry using pentafluoropropionic derivatives

Plasma testosterone was measured by gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-MS). The testosterone was extracted from plasma using home-made Extrelut columns and diethyl ether elution. It was quantified as the pentafluoropropionate (PFP) derivative by selected-ion monitoring at m/z 560 (testosterone) and 563 (d₃-testosterone), accounting for about 34% of the total ion. The characteristics of the method were: extraction recovery about 95%; linearity over the range 1.7–71.5 nmol l⁻¹ with linear regression equation y = 1.41x + 0.0217, r = 0.999; detection limit 3.5 fmol injected with a signal-to-noise ratio of 7.4; within-day variation, 3% for GC-MS, and 5.8% for the whole process; day-to-day coefficient of variation, 6.6–11%, depending on the concentrations. There was a good correlation between the results obtained by GC-MS and RIA (r = 0.994), but the GC-MS values were significantly lower (p < 0.05) than those obtained by RIA.     

Measurement of micronuclei by cytokinesis-block method in cultured Chinese hamster cells: comparison with types and rates of chromosome aberrations

The cytokinesis-block method of Fenech and Morley (1985) has been tested for the enumeration and characterization of micronuclei in exponentially growing Chinese hamster cells in culture. The consistent dose-response relations were obtained in cultures treated with mitomycin C, caffeine and colcemid. Comparison with the chromosome aberration frequencies indicated that approximately 30% of the acentric chromosomes are expressed as micronuclei in the mitomycin C and caffeine treated cells. The size distribution of the micronuclei suggested that the base-line frequency of micronuclei is mainly a reflection of mitotic dysfunctions rather than chromosome structural aberrations.     

Micronucleation by mitosis inhibitors in developing microspores of Spathiphyllum wallisii Regel

Key message

We developed an efficient protocol for chromosome scattering in Spathiphyllum microspores. The effects of plant material, developmental age, genotype and antimicrotubular toxin type, exposure and concentration were evaluated.

Abstract

Asymmetric hybridization through microprotoplast-mediated chromosome transfer (MMCT) is a known method for overcoming sexual breeding barriers between distantly related plant species. To obtain microprotoplasts, it is necessary to induce mass micronucleation either in somatic or gametic cells. We have tested the efficiency for micronuclei induction of five mitosis inhibitors, amiprophos-methyl (APM), butamiphos (BUT), chlorpropham (CIPC), oryzalin (ORY) and propyzamide (PRO), on developing microspores of diploid Spathiphyllum wallisii Regel. Besides the used toxins, also the effect of their concentrations and incubation period as well as plant genotypes and material was tested. We observed micronuclei (MNi) in pollen mother cells, dyads and tetrads as well as other abnormalities such as ball metaphases and chromosome bridges. The flower position on the spadix and the type of starting material (dissected anthers vs. complete spadices) did not significantly influence micronucleation frequencies. The highest micronucleation index of 86 % was obtained in microspores treated with 10 μM ORY during 72 h. All six genotypes tested formed micronuclei after this particular treatment, although the efficiency varied between cultivars. Next to ORY, CIPC was also a very efficient MNi inducer. The average number of MNi found in micronucleated cells varied between 1.67–6.44 for CIPC and 0.83–5.50 for ORY. The maximal number of MNi observed was 12 for CIPC and 9 for ORY. Our results demonstrate that CIPC and ORY can be applied for mass micronucleation on developing microspores of S. wallisii as a first step of MMCT in aroid interspecific or intergeneric breeding.     

Chromosomal aberrations and micronuclei in lymphocytes of breast cancer patients after an accident during radiotherapy with 8 MeV electrons

In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.     

Plasma progesterone levels in superovulated cattle in relation to hatched embryo sizes,ovulation rates,and premature regression of corpora lutea

Progesterone concentrations were measured in peripheral plasma of 62 cattle treated with PMSG. There was good correlation (r = 0.92) between a single day-10 value and the sum of daily values up to day 12 (19 animals) or day 13 (17 animals). The day-10 progesterone level was correlated strongly with ovulation rate (r = 0.76; 55 animals) but to a negligible extent with the sizes of 306 day-13 embryos from 47 donors (r = 0.17). Premature regression of corpora lutea, encountered in 14 (7.3%) of 191 flushed donors, began between days 5 and 8 and was reflected in the progesterone profiles of seven animals that were serially sampled.