Storage in methanol of smears intended for acridine orange staining.
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Acridine Orange↗
Biomedical subjects
H Norppa
Publications and source records attributed to H Norppa.
Induction of micronuclei in cultured human lymphocytes treated with vinblastine before and after mitogen stimulation.
The analysis of micronuclei (MN) in cultured human lymphocytes can, in principle, detect exposure to clastogens and aneuploidogens alike. As aneuploidogens such as spindle poisons usually act on dividing cells, it is not clear how an in vivo exposure of resting peripheral lymphocytes to an aneuploidogen could be transmitted and expressed as MN in cultured lymphocytes. This question is fundamental in judging if cultured lymphocytes can be used to detect in vivo exposure to aneuploidogens. In the present study, in vivo exposure of resting lymphocytes to an aneuploidogen was simulated in vitro by a 24-h pulse treatment of human lymphocytes with vinblastine sulfate (VBL) before mitogen stimulation, followed by two washes and culture in the presence of phytohemagglutinin (PHA) for 72 h. This treatment protocol did result in an increased MN frequency, but only at the highest concentration of VBL available for analysis (100 ng/ml). A more effective response, with a significant effect already at 40 ng/ml, was obtained when the 24-h pulse treatment was started at 24 h of PHA-stimulated 72-h cultures. Still much lower concentrations of VBL (1 or 2.5 ng/ml) were effective, when the treatment, started 24 h after culture initiation, was continued for 48 or 72 h (respectively) until cell harvest. These results demonstrate that MN induction by VBL depends, as expected, on the duration and timing of exposure, reflecting the availability of dividing cells during the treatment. The positive MN response obtained in the pulse-treated unstimulated lymphocytes may reflect an effect initiated in the resting stage and retained until mitosis or residual VBL left in the cells or in the cell suspension, despite the washes.
Adult↗
Analysis of chromosomal aberrations, sister-chromatid exchanges and micronuclei in peripheral lymphocytes of pharmacists before and after working with cytostatic drugs.
The frequencies of chromosome aberrations, SCEs and micronuclei (cytokinesis-block method) in blood lymphocytes were compared among six nonsmoking female pharmacists before and after 1 year of working with cytostatic drugs. All possible precautions were taken to avoid exposure to cytostatics, including proper protective clothing and a monitored, negative-pressured working environment with vertical laminar flow cabinet. As referents, an age-matched group of six nonsmoking female hospital workers not dealing with cytostatics was simultaneously sampled twice with the same time interval. The pharmacists showed a marginally higher mean frequency of SCEs/cell (6.3; P = 0.049) after the working period than 1 year earlier (5.8). On the other hand, the referents, with no obvious exposure, had a higher mean number of cells with chromatid-type aberrations, gaps excluded, in the second sampling (2.0%; P = 0.048) than in the first one (0.5%). In addition, a slight (P = 0.055) trend towards a higher frequency of micronucleated binucleate cells was observed in the second sampling for both the exposed and control subjects. As such findings suggest technical variation in the cytogenetic parameters, the small difference observed in SCEs for the pharmacists between the two samplings was probably not related to the cytostatics exposure. No statistically significant differences were observed for any of the cytogenetic parameters in comparisons between the pharmacists and the referents. The findings suggest that caution should be exercised in comparing results obtained from two different samplings in prospective cytogenetic studies.
Adult↗
Analysis of chromosomal aberrations, sister chromatid exchanges and micronuclei among power linesmen with long-term exposure to 50-Hz electromagnetic fields.
Chromosomal aberrations, sister chromatid exchanges (SCEs), replication indices and micronuclei in peripheral blood lymphocytes were analysed among 27 nonsmoking power linesmen with considerable long-term exposure to 50-Hz electromagnetic (EM) fields, and among 27 nonsmoking telephone linesmen serving as a reference group, pairwise matched with the exposed workers for age and geographical region. Blood samples from the two groups were collected, cultured and analysed in parallel. No differences between the groups were observed on analysis of SCEs, replication indices or micronuclei. However, the mean rate of lymphocytes with chromatid-type breaks was higher among the power linesmen (0.96% gaps excluded, 1.41% gaps included) than among the reference group (0.44% and 0.70%, respectively). The excess of aberrant cells was concentrated among those power linesmen who had worked earlier in their life. Although the interpretation is somewhat complicated by the confounding effect of previous smoking, these results suggest that exposure to 50-Hz EM fields is associated with a slight increase in chromatid breaks.
Adult↗
Detection of whole chromosomes in micronuclei of cytokinesis-blocked human lymphocytes by antikinetochore staining and in situ hybridization.
The effect of cytochalasin B (Cyt-B; 3 and 6 micrograms/ml; for the last 28 h) on micronuclei (MN) was studied in 72-h purified lymphocyte cultures of three male donors. The frequency of MN was much higher in multinucleate cells (mean 100-204 MN per 1000 cells) than in binucleate cells (mean 8.2-21.0 MN per 1000 cells), tetranucleate cells containing more MN than trinucleate cells. The presence of whole chromosomes in the MN was studied in two separate experiments by immunofluorescence using antikinetochore (CREST) serum and by a centromeric alphoid DNA oligomer probe (in situ hybridization, ISH). In the tri- and tetra-nucleate cells produced by Cyt-B, MN were clearly more often kinetochore-positive (K+) (mean 82-86%) and centromere-positive (C+) (mean 73-83%) than in mononucleate cells of cultures containing no Cyt-B (mean 63% for CREST and 50% for ISH), indicating that most of the excess MN in the multinucleate cells were due to whole chromosomes. The binucleate lymphocytes had about as high prevalence of K+MN (mean 79-84%) as the tri- and tetra-nucleate cells, despite their low MN count. Also in the ISH analysis, the majority of MN in binucleate cells were positively stained (mean 58-62%). If it is assumed that the extra labelled MN are due to Cyt-B, the present findings suggest that Cyt-B could be responsible for approximately 45-57% (CREST data) or approximately 17-23% (ISH data) of MN in binucleate cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Micronucleus assay in lymphocytes as a tool to biomonitor human exposure to aneuploidogens and clastogens.
The analysis of micronuclei (MN) in cultured human lymphocytes is, in principle, able to detect exposure to clastogens and aneuploidogens alike. There is, however, no clear evidence from human biomonitoring studies or animal experiments showing that in vivo exposure of resting lymphocytes to an aneuploidogen could actually be expressed as MN in cultured lymphocytes. In vitro, a pulse treatment of human lymphocytes with vinblastine, an aneuploidogen, did result in MN induction even if performed before mitogen stimulation, although a much more pronounced effect was obtained in actively dividing lymphocyte cultures. On the other hand, it is probable that a considerable portion of "spontaneous" MN contain whole chromosomes, their contribution increasing with age. It also seems that cytochalasin B, used for the identification of second cell cycle interphase cells in the MN assay, is able to slightly increase the level of MN with whole chromosomes. If MN harboring chromosome fragments represent a minority of the total MN frequency, there may be difficulties in detecting a weak effect in this fraction of MN against the background of MN with whole chromosomes. This would reduce the sensitivity of the assay in detecting clastogens, unless MN with whole chromosomes and chromosome fragments are distinguished from each other. That a problem may exist in sensitivity is suggested by the difficulty in demonstrating MN induction by smoking, an exposure capable of inducing chromosome aberrations. The sensitivity of the lymphocyte MN assay could be increased by detecting kinetochore or centromere in MN, or by automation, allowing more cells to be analyzed.
Aneuploidy↗
Monitoring occupational exposure to styrene from hemoglobin adducts and metabolites in blood.
Monitoring occupational exposure to styrene was achieved through quantification of adducts of styrene 7,8-oxide to N-terminal valine in hemoglobin (Hb) on the basis of the enrichment of adducted globin chains by ion-exchange chromatography and gas chromatographic-mass spectrometric analysis by the use of the N-alkyl Edman method. Application to blood samples from reinforced plastics workers exposed to styrene and from referents showed Hb adduct levels correlating with the blood styrene glycol and urinary mandelic acid concentrations. The blood styrene glycol and styrene 7,8-oxide levels of the exposed workers averaged 2.5 mumol.l-1 (17 subjects) and 0.09 mumol.l-1 (7 subjects), respectively. The blood styrene glycol and urinary mandelic acid content (mean 9.5 mmol.l-1, 17 subjects) suggested a styrene concentration of about 300 mg.m-3 (75 ppm) in the workplace air. The Hb adduct levels were low (mean 28 pmol.g-1), indicating rapid detoxification of styrene 7,8-oxide in humans.
Adult↗
Chromosome aberrations in peripheral lymphocytes of workers employed in the plywood industry.
Workers employed in sawmills and in the manufacture of plywood are exposed to potentially mutagenic chemical aromatic emissions from wood. However, very little is known about the exposure to these natural wood components. In an attempt to determine whether such exposure could have clastogenic effects, a group of 13 male nonsmoking employees mainly from the beginning of the wood-processing line of three plywood mills and 15 matched nonsmoking referents were studied for chromosome aberrations in blood lymphocytes. A statistically significant elevation of the frequency of cells with chromatid-type breaks (mean 2.1%), as compared with the corresponding frequency of the referents (mean 1.0%), was observed for the lymphocytes of the wood workers. These results lend support to previous studies which suggested that wood-drying fumes may be carcinogenic.
Adult↗
Genetic toxicity of 1,3-butadiene and styrene.
1,3-Butadiene and styrene (vinyl benzene) are indirect genotoxins, which require metabolic activation to an epoxide form in order to bind covalently to DNA. Styrene 7,8-oxide, the active metabolite of styrene, is a carcinogen in rodents and has been shown to be genotoxic in most in-vitro test systems and at various genetic endpoints. The few studies available on the genotoxicity of styrene 7,8-oxide in vivo have yielded negative or (in mice) weakly positive results. Styrene is not usually genotoxic in vitro in assays employing a microsomal preparation from rat liver for metabolic activation, but positive effects have been obtained when other sources of metabolic activation, such as human erythrocytes, were provided. In vivo, styrene has been found repeatedly to be weakly genotoxic in the assay for sister chromatid exchange, especially in mice. Cytogenetic damage (usually chromosomal aberrations) has been reported in many studies of workers, mainly from the reinforced plastics industry where ambient concentrations of styrene may be high (50-100 ppm), while most negative findings are associated with exposure to lower levels. Butadiene is metabolized to two reactive forms, 1,2-epoxy-3-butene and further to 1,2:3,4-diepoxybutane, both of which are genotoxic in various test systems in vitro. The lowest effective dose of the latter is 1-2 orders of magnitude higher than that of the respective monoepoxide. Butadiene itself has not been tested extensively for genotoxicity in vitro. A species-specific difference in the responses of mice and rats at various cytogenetic end-points is seen in vivo, the lowest effective concentrations in rats being clearly higher than those in mice.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Sister-chromatid exchanges induced by vinyl esters and respective carboxylic acids in cultured human lymphocytes.
Vinyl acetate--an efficient inducer of sister-chromatid exchanges (SCEs)--is known to be hydrolyzed in mammalian cells into acetic acid and acetaldehyde, the latter being the likely metabolite responsible for the SCE induction. As similar hydrolysis to acetaldehyde and to a carboxylic acid is also expected for other vinyl esters, five such compounds--vinyl formate, vinyl chloroformate, vinyl propionate, vinyl crotonate and vinyl-2-ethylhexanoate--and five carboxylic acids--formic acid, acetic acid, propionic acid, crotonic acid and 2-ethylhexanoic acid--were tested for their ability to induce SCEs in cultured (72 h) human lymphocytes with a 48-h treatment, starting at 24 h after culture initiation. Vinyl formate, vinyl propionate and vinyl crotonate induced a clear dose-dependent increase in the number of SCEs/cell at concentrations of 0.125-0.5 mM and vinyl chloroformate at 0.063-1 mM, i.e., at roughly the same concentration range as vinyl acetate and acetaldehyde. Vinyl-2-ethylhexanoate required slightly higher concentrations (0.25-4 mM) for SCE induction. All of the carboxylic acids tested also elevated SCEs, but only slightly. Formic acid and crotonic acid produced some SCE increase at a concentration of 10 mM, acetic acid at 5 and 10 mM and propionic acid at 2.5 mM. 2-Ethylhexanoic acid induced SCEs at a lower concentration range (0.63-2.5 mM) than the other acids. The positive concentrations of the first three carboxylic acids lowered the pH of the culture medium immediately after the treatment by 0.5-1.0 pH unit (lowest observed pH 6.53). The pH differences from the control cultures became smaller in measurements done 24 h and 48 h after the beginning of treatment. Propionic acid and 2-ethylhexanoic acid affected medium pH only slightly (maximum drop 0.2 pH units) at the concentrations that induced SCEs. The results lend support to the idea that the efficient SCE induction observed with the vinyl esters results from the formation of acetaldehyde, with carboxylic acids--with the possible exception of 2-ethylhexanoic acid--playing no significant role. The slight SCE induction obtained with the carboxylic acids cannot be explained by lowered pH alone.
Carboxylic Acids↗
Induction of sister-chromatid exchanges by 2-aminofluorene in cultured human lymphocytes with and without erythrocytes.
2-Aminofluorene (2-AF), an indirect mutagen reported to be metabolically activated by erythrocytes in the Salmonella mutagenicity test, was studied for the induction of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro with (whole-blood cultures) and without erythrocytes (isolated lymphocyte cultures). 2-AF (0.025-0.8 mM) was present in the cultures for the last 48 h of 72-h cultures. In both types of culture, SCEs increased in a dose-dependent manner, with a statistically significant elevation already at the lowest concentration of 2-AF tested and maximum responses of 2.4-fold (whole blood) and 2.1-fold (isolated lymphocytes), in comparison with mean SCEs/cell in control cultures, at 0.4 and 0.2 mM concentrations (respectively). Thus, the induction of SCEs by 2-AF was not dependent on the presence of erythrocytes. Styrene (2 mM), a positive control chemical known to require erythrocytes for efficient SCE induction in vitro, was shown to produce a 4.9-fold increase in SCEs in whole-blood cultures, but only a slight (1.3-fold) effect in isolated lymphocyte cultures. The results suggest that leukocytes, but not erythrocytes, are important in the metabolic activation of 2-AF in the human lymphocyte SCE assay.
Adult↗
Mouse bone marrow micronucleus test using flow cytometry.
At present, the micronucleus test is the most popular short-term assay for the in vivo detection of clastogens or spindle poisons. As micronuclei are rare events, it has been argued that the conventional microscopical analysis based on 500-2000 cells per animal does not provide enough sensitivity. In addition, the manual scoring of micronuclei is time-consuming and tedious. As an attempt to solve these problems, an automated method for the analysis of micronuclei in mouse bone marrow erythrocytes was developed using flow cytometry. Femoral bone marrow cells, from mice treated with known in vivo clastogens [mitomycin C (MMC): 0.5, 1 or 2 mg/kg body weight intraperitoneally; benzene: 1000, 2000 or 4000 mg/kg body weight orally] or vehicles for the clastogens (physiological saline, olive oil; for control animals), were fixed with 1% glutaraldehyde, suspended in 70% ethanol for storing, stained with 4',6-diamidino-2-phenylindole (0.5 microgram/ml) and analyzed (50,000 cells per sample) in an EPICS V flow cytometer using a Coherent 5W UV laser at 150-200 mV. The frequency of micronucleated erythrocytes (MNEs) was calculated from the cytometer, by a computer program involving model fitting to normal Gaussian distribution. Correlations between MNE frequencies obtained by manual microscopic observations (1000-2000 cells per animal) and by the flow cytometric measurements were good for both group results (means of 4-5 mice; linear correlation coefficient r = 0.89-0.998) and individual data (r = 0.82-0.96). Repeated experiments with MMC showed good reproducibility for the flow cytometric scoring of micronuclei.
Algorithms↗
Flow cytometric micronucleus test with mouse peripheral erythrocytes.
Flow cytometric (FC) analysis was applied to micronucleus test with mouse peripheral blood erythrocytes. The method is based on the measurement of peak fluorescence (PFL) of sphered glutaraldehyde-fixed erythrocytes before and after staining with 4',6-diamidino-2-phenylindole (DAPI), in an EPICS V flow cytometer. The frequency of micronucleated erythrocytes (MNEs) is calculated by a computer program comparing PFL data obtained with and without DAPI. To evaluate the method, male ddY mice were treated with 6-mercaptopurine and benzene and blood was collected from tail vein at intervals of 4-7 days. Both microscopic and FC analysis showed a steady increase in the incidence of MNEs, reaching a plateau when about a month had passed from the start of the treatments. The effects of benzo[a]pyrene, mitomycin C,N-ethyl-N-nitrosourea, bromodichloromethane and potassium chromate (VI) were also studied with both the manual and FC assay in samples collected a week after five weekly treatments. The percentages of MNEs obtained manually and by the FC measurements showed good correlation, the former three chemicals being positive and the latter two negative or, in the FC analysis, difficult to classify. Because of the high number of cells examined (50,000/animal), the FC analysis was probably more sensitive than the manual method where only 2000 cells were scored per animal. This was further suggested by (i) steady time responses, also for individual animals, in the FC results on 6-mercaptopurine and benzene, (ii) overall reduced inter-individual variation in the FC measurements, and (iii) detection of MNE induction by mitomycin C at a lower dose level with the FC than the manual analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Algorithms↗
Single-strand breaks, chromosome aberrations, sister-chromatid exchanges, and micronuclei in blood lymphocytes of workers exposed to styrene during the production of reinforced plastics.
Chromosome aberrations (CA), micronuclei (MN, cytokinesis-block [CB] method), and sister-chromatid exchanges (SCE) were analysed in blood lymphocytes of 17 workers and 17 control subjects. The mean urinary mandelic acid level (average 9.4 mmol/l) and styrene glycol in blood (average 2.5 mumol/l) implied exposure to about 300 mg/m3 of styrene in the plant. The number of CA was significantly higher in non-smoking workers compared with nonsmoking controls. A significant correlation was observed between duration of exposure and individual CA level of all workers. No significant effects were observed in MN or SCE. Single-strand breaks (SSB) in DNA of isolated lymphocytes were studied in nine of the workers and eight of the controls by the DNA-unwinding technique. The results showed an increase in SSB among the exposed workers. The present findings support earlier reports on the increase of structural CA in blood lymphocytes of workers in the reinforced plastic industry, and also show that SSBs are elevated in such workers.
Chromosome Aberrations↗
Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures.
The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5-6 micrograms/ml for the last 28 h) was 9-21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 micrograms/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5-12 micrograms/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (greater than or equal to 3 poles) divisions which had a high frequency of anaphase aberrations (39-53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided greater than or equal to 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.
Adult↗
1,3-Butadiene and its epoxides induce sister-chromatid exchanges in human lymphocytes in vitro.
Sister-chromatid exchanges (SCEs) were induced in human lymphocytes by 1,3-butadiene and its epoxides 3,4-epoxy-1-butene and 1,2:3,4-diepoxybutane. After a pulse treatment of 2 h, 1,3-butadiene produced a weak but reproducible increase in SCEs both with and without S9 mix. The response was similar in cultures of whole blood and of isolated lymphocytes. The 2 epoxide metabolites of butadiene, studied in whole-blood lymphocyte cultures without exogenous metabolic activation, were highly active SCE inducers. The lowest effective concentrations of butadiene, monoepoxybutene, and diepoxybutane were 2000 microM, 25 microM and 0.5 microM, respectively. A slight but dose-dependent increase in SCEs was also observed without an exogenous metabolic system after a 48-h treatment with 1,3-butadiene. Already the lowest concentration tested (500 microM) was effective. Again, the response was similar in cultures of whole blood and isolated lymphocytes, suggesting that the lymphocytes are capable of metabolically activating 1,3-butadiene.
Butadienes↗
Determination of 6-thioguanine resistant lymphocytes in human blood by immunohistochemical antibromodeoxyuridine staining.
An immunohistochemical method for the determination of 6-thioguanine resistant (TGr) lymphocytes in human blood samples has been developed. The new technique is a modification of the autoradiographic assay and uses labelling of DNA synthesizing cells by bromodeoxyuridine (BrdU) instead of tritiated thymidine. The label is detected immunohistochemically by using a monoclonal antibody against BrdU in single-stranded DNA. Thawed cryopreserved isolated mononuclear cells are cultured with (TG cultures) and without (control cultures) TG for 40 h, the last 16 h with BrdU. The cells are harvested, fixed and put on microscopic slides; for the TG cultures, all material is used, while slides from the control culture are prepared at a dilution of 1:300. The immunohistochemical staining of the slides involves denaturation of DNA in formamide, incubation with anti-BrdU-DNA antibody, followed by a secondary antibody conjugated with peroxidase and staining with diaminobenzidine. Nuclei that have incorporated BrdU appear brown while all other material remains unstained. The number of all labelled nuclei is scored similarly in control and TG slides and the frequency of TGr lymphocytes is obtained by dividing the number of labelled nuclei in TG slides with that (multiplied by the dilution factor) in control slides. The anti-BrdU method will much simplify and speed up the microscopical evaluation of the TG variants and is very suitable for automation.
Bromodeoxyuridine↗
Styrene revisited--exposure assessment and risk estimation in reinforced plastics industry.
A survey performed in 32 workshops in reinforced plastics industry showed the mean TWA 8 h concentration of styrene in personal air to be 43 ppm (range 5-182 ppm) among laminators and 11 ppm (range 1-133 ppm) among other workers. The biological measurement of urinary mandelic acid + phenylglyoxylic acid showed mean values of 2.4 mmol/l among laminators without respirators and 1.3 mmol/l when respirators were used. No effects of work related exposure were detected in the cytogenetic parameters, chromosome aberrations, sister chromatid exchanges or micronuclei analyzed in peripheral blood lymphocytes. Grading of the exposure on the basis of lamination method, years of exposure, daily laminating time, air styrene concentration and urinary mandelic acid among laminators, neither revealed any dose dependency.
Air Pollutants, Occupational↗