Increased expression of adhesion proteins (MAC-1) and altered metabolic response in human leukocytes exposed to surfactant in vitro.

In this study the modulatory effects of a well-defined surfactant preparation on blood leukocytes were investigated. The expression of the cell surface receptor MAC-1 was analyzed by flow cytofluorometry, and the metabolic response was measured by a chemiluminescence technique. An increase (p less than 0.05) in the MAC-1 receptor expression was observed in the granulocytes but not in the monocytes. There was a decrease in the metabolic response of the leukocytes after stimulation with phorbol myristate acetate (PMA) and a delay (p less than 0.01 for both) in the peak activity. Formyl-methionyl-leucyl-phenylalanine (fMLP) caused an increased peak (p less than 0.01). Thus, the surfactant preparation had a modulatory effect on blood leukocytes with regard to the expression of the cell surface receptor MAC-1 and the metabolic response.

Human cytogenetic biomonitoring using flow-cytometric analysis of micronuclei in transferrin-positive immature peripheral blood reticulocytes.

We have developed a method to isolate and analyze nascent human reticulocytes in peripheral blood for the presence of micronuclei (MN). For a very short time peripheral reticulocytes show residual expression of the transferrin receptor. Using immunomagnetic separation of cells expressing the transferrin receptor, a population of immature reticulocytes (Trf-Ret) was isolated from peripheral blood. In humans, the spleen actively removes micronucleated erythrocytes but during the short lifetime of the isolated Trf-Ret only a fraction (less than about 20%) of the MN-containing reticulocytes will have been eliminated. Cells were stained with the fluorescent dyes Thiazole Orange for RNA and Hoechst 33342 for DNA and analyzed by flow cytometry and fluorescence microscopy. Baseline frequencies of MN-Trf-Ret on a group of healthy donors were found to be 1.1% for males and 1.4% for females; however, the gender difference was not significant. The frequency of MN-Trf-Ret in the studied group increased with age, and was dependent on blood group. In three donors studied over 4 months, the baseline level remained stable. In cancer patients treated with radiation or chemotherapy, the frequency of MN-Trf-Ret increased 10- to 20-fold after 1-4 days, depending on the treatment. A high correlation between flow and manual analysis of MN-Trf-Ret was seen. We believe the method has a high potential as a sensitive and rapid method for biological monitoring in presumed exposed groups and individuals.

Flow cytometric analysis of micronucleus induction in mouse erythrocytes by gamma-irradiation at very low dose-rates.

Male CBA-S mice were subjected to protracted gamma-irradiation. Two groups of five animals were each exposed to dose rates of 6 and 30 mGy/day for 56 days, respectively, upon which irradiation was terminated and the groups were followed for an additional 49 days. Frequencies of micronucleated poly- and normochromatic erythrocytes in peripheral blood samples were determined before (day 0), during (day 14, 28 and 56), and after (day 70 and 105) irradiation using flow cytometry. A second experiment was performed as above, but with exposure limited to 7 days. Frequencies of micronucleated polychromatic erythrocytes in bone marrow and peripheral blood were determined. Significantly elevated frequencies of micronuclei in peripheral blood polychromatic erythrocytes were found for the 30-mGy/day dose group on day 14, 28 and 56 and for the 6-mGy/day dose group on day 28 and 56. In normochromatic erythrocytes from peripheral blood significantly elevated frequencies were found on all sampling occasions with mouse given 30 mGy/day, while those given 6 mGy/day showed significantly elevated frequencies on day 28, 56 and 70. The frequencies of micronucleated polychromatic erythrocytes were found to be similar in bone marrow and peripheral blood, while the frequencies of micronucleated normochromatic erythrocytes were consistently lower than for micronucleated polychromatic erythrocytes at all samplings for all groups. On day 28 the frequencies (mean +/- SE) of micronucleated polychromatic erythrocytes in peripheral blood were 0.0016 +/- 0.0001 for the control group, 0.0019 +/- 0.0001 for the 6-mGy/day group and 0.0028 +/- 0.0003 for the 30-mGy/day group. The results show an elevated induction of micronuclei in erythroblasts at a dose rate of approximately 3 mGy per cell cycle.

Effects of extended low-dose-rate exposure to 137Cs detected by flow-cytometric enumeration of micronucleated erythrocytes in mouse peripheral blood.

An automated variant the of micronucleus assay for erythrocytes in mouse peripheral blood has recently been developed. The flow-cytometric technique used allows very large numbers of erythrocytes to be analysed with a relatively small effort. Here we report the potential of this method to detect a response to extended low-dose-rate exposure to gamma-irradiation. The mice were irradiated with a 137Cs source at a dose rate of 4.8 cGy/day for 26 days. Sampling was continued for another 39 days after irradiation. Elevated frequencies compared with the control group were found at days 2, 9 and 20 after the start of the irradiation for micronucleated polychromatic erythrocytes, and at days 9, 20, 29, 42, 51 and 65 for micronucleated normochromatic erythrocytes.

Flow cytometric analysis of micronucleus induction in mice by internal exposure to 137Cs at very low dose rates.

Internal radiation from 137Cs, intraperitoneally injected into mice, induced chromosome damage seen as micronuclei in erythrocytes of peripheral blood harvested 72 h after injection and analysed with flow cytometry. The retention of injected 137Cs activity was determined and the absorbed doses obtained from the beta-radiation of 137Cs were calculated for the whole bodies and bone marrow of the treated mice. The absorbed doses during the most relevant period for micronucleus induction were 2.7-18.3 mGy per day. The dose to the bone marrow during the same period was calculated to be 6-44 mGy per day. A linear dose response relationship was found.

Absence of genomic instability in mice following prenatal low dose-rate gamma-irradiation.

PURPOSE: To determine whether mice exposed to an extended low dose of gamma-irradiation during most of their prenatal period express increased frequencies of micronucleated polychromatic erythrocytes (fMPCE) and/or micronucleated normochromatic erythrocytes (fMNCE) several weeks after the end of irradiation. METHODS: Female CBA/Ca mice were gamma-irradiated for an average of 16 days during their pregnancy. The mice were exposed to dose rates of 0, 44, 99 and 265 mGy/day. At 1-2 days prior to parturition the mice were removed from exposure. Then, 36 days after birth, peripheral blood was drawn from all offspring (74 mice). Using flow-cytometer-based analysis, the frequencies of MPCE and MNCE were determined. From each animal about 170,000 PCE were analysed. RESULTS: No delayed effects in terms of higher fMPCE or fMNCE were observed among the in utero exposed mice of either gender. On the contrary, a significant (p<0.001) reduction of fMPCE was found among the male offspring exposed at the highest dose rate. CONCLUSION: Gamma-irradiation of mice during their prenatal stage did not induce damage in erythroid stem cells that can be detected as persistent or delayed chromosome aberrations (i.e. micronucleated erythrocytes) at 35 days after the end of exposure.

Improved lung function and quality of life following increased elastic recoil after lung volume reduction surgery in emphysema.

Lung volume reduction surgery for severe emphysema with removal of 20-30% of the most destroyed parts of the lung parenchyma has been reported to improve lung function substantially. Increased elastic recoil has been suggested as one underlying mechanism for the improvement. Fourteen patients, seven men and seven women with a mean age of 62 years, who underwent bilateral lung volume reduction surgery have been followed up for 3 months. We here report the data on quality of life, lung function and elastic recoil. FEV1.0 increased by a mean of 26% from 0.581 to 0.731 (P < 0.01). The mean TLC was reduced by 16% from 8.91 to 7.51 (P < 0.001). The level of hyperinflation decreased as implied by a reduction in the ratio of RV to TLC from 0.70 to 0.60 (P < 0.001). The pulmonary elastic recoil improved, with an increase in the transpulmonary pressure at maximal inspiration (PelTLC) from 0.95 kPa to 1.35 kPa (P < 0.05) and an average increase in the coefficient of retraction PelTLC/TLC) from 0.12 kPa l-1 to 0.19 kPa l-1 (P < 0.01). The resting PaO2 increased from a mean of 8.7 kPa to 9.8 kPa (P < 0.01). The patients reported a high degree of subjective improvement according to the St. George's Respiratory Questionnaire and the working capacity on a bicycle increased by 26% from a mean of 38 W to 48 W (P < 0.01). The promising short-term results of lung volume reduction surgery for severe emphysema appear to be related to improved pulmonary elastic recoil.

Mechanism of the lethal and mutagenic effects of phenoxyacetic acids in Saccharomyces cerevisiae.

MCPA and salicylic acid, two compounds with similar structures and almost the same dissociation pattern, were tested for lethal and mutagenic effects on, and uptake by, cells of Saccharomyces cerevisiae strain rad18. The results obtained with the two compounds were similar, suggesting a common mechanism of action. It is proposed that they act by increasing the concentration of hydrogen ions within the cell, so that killing and mutation occur. Mutations were induced only when killing reached 95--99%. The compounds are considered weak mutagens for yeast cells. The methyl ester of MCPA also induced killing and reverse mutation, but only at concentrations about 100 times higher than for the undissociated acid. MCPA methyl ester did not increase the number of revertants in the Salmonella/liver microsome test. It is suggested that the effects of the methyl ester of MCPA depends on the ester being hydrolysed to the acid by yeast cells and the liver microsome preparation.

Induction of mitotic recombination with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Saccharomyces cerevisiae. A comparison between treatment in vitro and in the host-mediated assay.

Two methods, treatment in vitro and the host-mediated assay method, were compared in their ability to demonstrate the induction of MNNG of nitotic recombination in a diploid strain of Saccharomyces cerevisiae. MNNG had a strong activity in vitro but not in the host-mediated assay at the concentrations tested. When the genetic effects of MNNG have been tested in different test systems, sometimes negative, sometimes positive results have been obtained. The relevance of different tests for risk evaluation is discussed, and it is concluded from the data on MNNG that tests on whole mammals may sometimes give false negative results because the cells tested are, in parts of the body, less accessible to the mutagen. Increasing doses of MNNG by treatment in vitro gave decreasing frequencies of mitotic recombination, indicating damage to the recombinational system in the cells. Dose-response relationships for recombination and mutation are discussed.

Mitotic gene conversion induced in yeast by isoniazid. influence of a transition metal and of the physiological conditions of the cells.

Isoniazid (INH) induced mitotic gene conversion at the trp5 locus of strain D7 of Saccharomyces cerevisiae. A suggested mechanism involving autoxidation of INH and production of hydrogen peroxide is supported by the facts that (i) the presence of a transition metal, Mn(II), greatly enhanced the effect of INH, (ii) cells stored for a long time in the refrigerator are much more sensitive to INH + Mn(II) than fresh cells probably due to loss of catalase and/or peroxidase activity, (iii) presence of S9 mix during the treatment eliminated the effect of INH + Mn(II).

Human lymphocytes resistant to 6-thioguanine: restrictions in the use of a test for somatic mutations arising in vivo studied by flow-cytometric enrichment of resistant cell nuclei.

A method is presented that facilitates the enumeration of 6-thioguanine(TG)-resistant human lymphocytes from peripheral blood. By the use of flow cytometry, nuclei from cells stimulated to divide in vitro and having reached their late S and G2 phases of growth, were sorted out from cultures incubated for 48 h, the last 6 h in medium containing [3H]thymidine. The out-sorted nuclei were subjected to autoradiography, and the labelling index was determined. The frequency of TG-resistant cells was calculated from the quotient (labelling index in the presence of TG/labelling index in the absence of TG). Over a period of several months, two blood donors serving as internal standards showed a variation of 20% in their frequencies of resistant lymphocytes. In a referent group of 48 blood donors, inter-individual differences in the frequency of resistant cells (range 10(-5)-10(-4) ) were established. There was a positive correlation between an increased frequency of resistant cells and the age of the blood donors. Patients treated with cytostatics or psoralen and ultraviolet radiation (PUVA) showed elevated frequencies of resistant cells (about 10(-3)-10(-2) ). We suggest that at least a part of the resistant cells scored are phenocopies and thus have not originated from somatic mutation. The high number of phenocopies after treatments with cytostatics or PUVA may depend on a temporary depletion from the cells of functioning HGPRT enzyme owing to a transcriptional block caused by the agents combined with a rather fast turnover of the enzyme. We conclude that there are restrictions in the use of this system for determining genotoxic effects in vivo.

Resistance to 6-thioguanine in spontaneously cycling and in mitogen-stimulated human peripheral lymphocytes.

Spontaneously cycling lymphocytes (in cell division in cultures without addition of phytohemagglutinin, PHA) go through the various phases of the first division with the same kinetics as PHA-stimulated cells. In samples from 10 referents, the frequency of spontaneously cycling lymphocytes varied from 8.9 X 10(-5)-9.5 X 10(-3) as indicated with autoradiography on cells in (S + G2) phase determined by flow sorting. In PHA-stimulated samples from the same persons the frequency of 6-thioguanine (TG)-resistant variants was between 4 X 10(-7) and 2.6 X 10(-6), which indicates that most of the spontaneously cycling cells were TG-sensitive. With lymphocytes from one of the referents it was found that: (i) in the presence of 2 X 10(-4) M TG, more than 97% of the spontaneously cycling cells were inhibited before or in early S phase, and (ii) when a flow cytometer was set to sort out TG-resistant cells in late S + G2 phase after 48 h incubation in medium with PHA, the contribution of TG-resistant cells from the spontaneously cycling fraction amounted to less than 2% of the total number of resistant cells.

Erythropoiesis and the induction of micronuclei in mouse spleen determined by flow cytometry.

Erythrocytes from the spleen of CBA mice have been prepared for analyses by flow cytometry. About 80% of the polychromatic erythrocytes (PCE) in the spleen originate from erythropoiesis in the spleen, while the remaining 20% come from the peripheral blood. Analyses of the RNA content of PCE revealed that splenic PCE do not mature into normochromatic erythrocytes (NCE) in the spleen but leave the organ at a more immature stage. A considerable part of the PCE from bone marrow also mature into NCE in the bone marrow. The rate of RNA breakdown in PCE follows an exponential function. Time-courses for the appearance of micronucleated PCE (MPCE) from spleen and from bone marrow were determined by analysis of samples taken with short intervals after an acute dose of 0.1 Gy X-rays. The time-courses were identical for MPCE from the spleen and the bone marrow. The frequency of MPCE (fMPCE) starts to increase at about 10 h after irradiation and reaches its maximum after about another 20 h upon which fMPCE returns to control level. The first induced MPCE in peripheral blood appear at about 20 h after irradiation. The effects of the carcinogen DMBA, 9,10-dimethyl-1,2-benzanthracene, at low doses were determined in PCE from spleen and bone marrow. The sensitivity was found to be about the same for erythroblasts in the spleen and the bone marrow. Protracted exposure to gamma-irradiation at a very low dose rate (44 mGy/day) gave a similar increase of fMPCE in bone marrow and spleen. The suitability of using splenic erythrocytes in the micronucleus test is discussed.

Spontaneous and radiation-induced micronuclei in erythrocytes from four species of wild rodents: a comparison with CBA mice.

Almost 100 animals of 4 different species of small wild rodents (bank vole, Clethrionomys glareolus; field vole, Microtus agrestis; yellow-necked mouse, Apodemus flavicollis; and wood mouse, Apodemus sylvaticus) were trapped in central Sweden and used in experiments to determine the spontaneous and radiation-induced frequencies of polychromatic (fMPCE) and normochromatic erythrocytes (fMNCE) from bone marrow (bm) and peripheral blood (pb) using flow cytometric analysis. The results were compared with those from similar experiments with CBA mice. The saving of time and labour by the use of the flow cytometer-based analysis was a prerequisite for this study in which about 135 million PCE were analysed. The two species of voles had a mean background fMPCE (bm) of about the same value as CBA mice, while the yellow-necked mice had about five times higher fMPCE (bm). Wood mice had more than twice the fMPCE (bm) compared to CBA mice. Between individual animals in each of the 4 species, the background fMPCE (bm) varied more than between individual CBA mice, and the elimination of micronucleated erythrocytes was considerable. When exposed to ionizing radiation, the voles did not show a significant response. The response of the two Apodemus species was similar to that of the CBA mice, although it varied between individual animals and was not correlated to their background fMPCE. This study indicates that bank voles and field voles are unsuitable testing objects in the in vivo micronucleus assay. On the other hand, yellow-necked mice and wood mice seem to be useful in this test. Since the variation between individuals is considerable in wild Apodemus mice, large groups will be needed for obtaining statistically significant results when exposure to a genotoxic agent is low. Alternatively, repeated samples can be taken from individual wild mice to study the effect of a decreased exposure after keeping the animals for a period of time in an uncontaminated environment.

The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood.

The time-course of micronucleated polychromatic erythrocytes (MPCE) in mouse bone marrow and peripheral blood, induced by an acute 0.1 Gy dose of X-rays, was determined using flow cytometric analysis, which made frequent sampling possible and allowed use of a dose low enough not to affect erythroid cell proliferation. The frequency of MPCE (fMPCE) began to increase in the bone marrow at 10 h after irradiation and reached a maximum at 28 h after irradiation. In the peripheral blood fMPCE began to increase at 20 h after irradiation and peaked at about 40 h after irradiation. The time-course found is discussed on the basis of data on the differentiation of erythroid cells. The results indicate that the micronuclei registered in polychromatic erythrocytes may originate from lesions induced not only during the last cell cycle but also during earlier ones. After an acute dose of 1.0 Gy of X-rays the maximum fMPCE was delayed both in bone marrow and peripheral blood reflecting an effect on the cell cycle progression of erythroblasts.

The micronucleus test in rat erythrocytes from bone marrow, spleen and peripheral blood: the response to low doses of ionizing radiation, cyclophosphamide and vincristine determined by flow cytometry.

The frequency of micronucleated polychromatic erythrocytes (fMPCE) was determined in samples from bone marrow, spleen and peripheral blood of rats exposed to low doses of X-rays, cyclophosphamide or vincristine. The fMPCE values were lower in the peripheral blood than in bone marrow or spleen. This is due to the elimination of MPCE from the circulating blood, which was confirmed by the results from prolonged exposure of rats to gamma-radiation. When the analysis was restricted to the youngest PCE in peripheral blood, the sensitivity of the assay was considerably improved. This can be reproducibly achieved with the flow cytometric analysis.

Low dose effects of chemicals as assessed by the flow cytometric in vivo micronucleus assay.

Using flow cytometric automation of the mouse in vivo, micronucleus assay increases the sensitivity of the test. This is achieved through a very large increase in the number of cells scored, by a factor of 100x, which in turn greatly reduces the sampling error. With this method, dose-response relationships of in vivo micronucleus induction for four model agents mitomycin C (MMC), diepoxybutane (DEB), cyclophosphamide (CPA), and colchicine (COL) were studied at low dose levels. For the three clastogens MMC, DEB and CPA, linear dose-response relationships were found over the dose ranges studied, even in the very low dose region (defined as the dose region where the frequency of micronucleated erythrocytes is less than twice the baseline frequency). This is consistent with the view that no threshold should exist for genotoxic agents which target DNA. For COL a dose range was found, in which the frequency of micronucleated erythrocytes did not increase with dose, possibly indicating an in vivo threshold. The flow cytometric in vivo micronucleus assay represents one possibility for in vivo low dose-response studies.

The influence of pH on the effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) on Saccharomyces cerevisiae and Salmonella typhimurium.

The genetic effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) have been investigated in cells of the yeast Saccharomyces cerevisiae and of the bacterium Salmonella typhimurium in experiments in vitro and in vivo. Experiments in vitro showed that the killing of both yeast and bacteria is dependent on the pH in the treatment solution of 2,4-D. A dose-dependent increase of the frequency of mitotic gene conversion and mitotic recombination in yeast was observed at pH 4.50 and 4.30. In experiments in vitro with two strains of Salmonella no significant increase of the number of revertants to prototrophy was obtained. The positive correlation between survival of cells and dissociation of 2,4-D in the pH region 2.8-5.0 indicates that the cells are unable to take up dissociated 2,4-D. Therefore the survival is high at a high pH when most 2,4-D is in dissociated form, and the survival is low at a relatively low pH when more of the 2,4-D is in its undissociated form. No genetic effects were induced by oral administration of tolerable doses of 2,4-D in host-mediated assays using mice as hosts and yeast or Salmonella as indicator cells.

Autoradiographic detection of HPRT variants of human lymphocytes resistant to RNA synthesis inhibition.

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.