Germ cell mutagenicity of three metabolites of 1,3-butadiene in the rat: Induction of spermatid micronuclei by butadiene mono-, di-, and diolepoxides in vivo

Three metabolites of the industrial chemical 1,3-butadiene (BD), namely butadiene monoepoxide (BMO, 3,4-epoxy-1-butene), diepoxide (DEB, 1,2;3,4-diepoxybutane), and diolepoxide (DE, 3,4-epoxybutane-1,2-diol) were studied for germ cell mutagenicity using the rat spermatid micronucleus (MN) test. All three epoxides increased slightly, but significantly, the frequency of spermatid MN. The most sensitive stage to the action of BMO and DEB was preleptotene (meiotic S phase) harvested at 18-day time intervals after treatment. The dose-response for BMO followed a second order curve at this time interval, with maximum MN induction at the dose of 186 μmol/kg and lower induction at higher doses. Late stages of the meiotic prophase (late pachytene-diplotene-diakinesis) also showed some sensitivity to the three epoxides. Stem cell spermatogonia were affected by DEB as observed by a slight induction of spermatid micronuclei 50 days aftertreatment. No clear cytotoxic effects were observed by measuring testicular weight or cell numbers of seminiferous epithelial stage I 18 days after the treatments. DEB at the dose 387 μmol/kg caused a slight inhibition of spermatogonial DNA synthesis in stage I and a delay of meiotic DNA replication observed in stage XII 72 hr after treatment. Since BMO is able to induce spermatid MN in the rat, the present results, together with previous data, indicate that rat bone marrow MN results that are negative for both BD and BMO cannot directly predict mutagenicity in male germ cells. The results also emphasize that tissue-, species-, and strain-specific differences in metabolism have to be taken into account when the genetic risks of human butadiene exposure are evaluated. The results support the conclusion that 1,3-butadiene is a germ cell mutagen--possibly also in humans. Environ. Mol. Mutagen. 29:230–239, 1997. © 1997 Wiley-Liss, Inc.     

直接低渗法制备男性不育症精液生精细胞染色体观察

目的：研究男性不育症生殖细胞减数分裂过程、生精细胞染色体畸变与不育的关系。方法：选择不育门诊中１９例男性原发不肓患者精液和４例禁欲期正常男性志愿者精液，应用直接低渗法，获得各级生精细胞（精原细胞、初级／次级精母细胞、精细胞）分裂相。结果：不同样本精液中各级生精细胞分裂相分布有极显著性差异（Ｐ〈０．００５）；弱精症患者精液标本中主要为ＭＩ单价体、染色单体畸变，少精症和无精症主要为联会消失、减数分裂阻断。结论：直接低渗法能为诊断男性不育提供有价值的细胞遗传学信息。生精细胞染色体畸变、减数分裂过程异常是导致男性不肓的原因之一。     

Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes

Basal and follicle-stimulating hormone (FSH)-stimulated cyclic AMP (cAMP) productions by seminiferous tubular segments from irradiated adult rats were investigated at defined stages of the epithelial cycle when specific spermatogenic cells were low in number. Seven days post-irradiation, depletion of spermatogonia did not influence the basal cAMP production, but FSH response increased in stages II-VIII. Seventeen days post-irradiation when spermatocytes were low in number, there was a small increase in basal cAMP level in stages VII-VIII and FSH-stimulated cAMP production increased in stages VII-XII and XIII-I. At 38 days when pachytene spermatocytes and round spermatids (steps 1-6) were low in number, a decreased basal cAMP production was measured in stages II-VI and IX-XII. FSH-stimulated cAMP output increased in stages VII-XII but decreased in stages II-VI. At 52 days when all spermatids were low in number, basal cAMP levels decreased in all stages of the cycle, whereas FSH response was elevated only in stages VII-XII. All spermatogenic cell types seem to have an effect on cAMP production by the seminiferous tubule in a stage-specific fashion. Germ cells appear to regulate Sertoli cell FSH response in a paracrine way, and a part of cAMP may originate from spermatids stimulated by an unknown FSH-dependent Sertoli cell factor. The FSH-dependent functions may control such phenomena as spermatogonial proliferation, final maturation of spermatids, and onset of meiosis.     

Micronucleus assay in aquatic animals

Aquatic pollutants produce multiple consequences at organism, population, community and ecosystem level, affecting organ function, reproductive status, population size, species survival and thus biodiversity. Among these, carcinogenic and mutagenic compounds are the most dangerous as their effects may exert a damage beyond that of individual and may be active through several generations. The application of genotoxicity biomarkers in sentinel organisms allows for the assessment of mutagenic hazards and/or for the identification of the sources and fate of the contaminants. Micronucleus (MN) test as an index of accumulated genetic damage during the lifespan of the cells is one of the most suitable techniques to identify integrated response to the complex mixture of contaminants. MN assay is today widely applied in a large number of wild and transplanted aquatic species. The large majority of studies or programmes on the genotoxic effect of the polluted water environment have been carried out with the use of bivalves and fish. Haemocytes and gill cells are the target tissues most frequently considered for the MN determination in bivalves. The MN test was widely validated and was successfully applied in a large number of field studies using bivalves from the genera Mytilus. MN in fish can be visualised in different cell types: erythrocytes and gill, kidney, hepatic and fin cells. The use of peripheral erythrocytes is more widely used because it avoids the complex cell preparation and the killing of the animals. The MN test in fish erythrocytes was validated in laboratory with different species after exposure to a large number of genotoxic agents. The erythrocyte MN test in fish was also widely and frequently applied for genotoxicity assessment of freshwater and marine environment in situ using native or caged animals following different periods of exposure. Large interspecies differences in sensitivity for MN induction were observed. Further validation studies are needed in order to better characterise the different types of nuclear alterations and to clarify the role of biotic and abiotic factors in interspecies and inter-individual variability.     

Biocompatibility of magnesium particles evaluated by in vitro cytotoxicity and genotoxicity assays

Mg is a biodegradable biomaterial which may release particles (MP) to the environment. The possible cyto- and genotoxic effects of MP derived from magnesium powder (mesh 325) were analyzed on rat osteosarcoma UMR106 cells in order simulate the effect of Mg debris. Neutral red (NR) incorporation and acridine orange/ethidium bromide (AO/EB) staining techniques were used as endpoints to analyze the cytotoxic effects at 25-1000 μg/mL concentration range. Genotoxicity was estimated according to micronucleus (MN) formation and the Comet assay (CA). Results showed that MP size changes with time due to corrosion. Changes in lysosomal activity were observed after 24 h only at 1000 μg/mL. Accordingly, AO/EB staining showed a significant decrease in the number of living cells at 500 μg/mL. Transmission electronic microscopy showed MP internalization (60 and 200 nm diameter) in cells after 2-h treatment, whereas no MP was detected after 24 h. A significant dose-dependent increase in MN frequencies was observed at 25-100 μg/mL range (nontoxic range). DNA damage induction was assessed by CA only at 500 μg/mL. Results showed dose-dependent cytotoxic and genotoxic effects of MP on UMR106 cells with different threshold values of MP concentration.     

Histological changes of acrylamide-induced testicular lesions in mice

The effects of a single oral dose of 150 or 100 mg/kg acrylamide on the testis were studied histopathologically in prepubertal and adult mice over a period of 10 days following treatment. In the prepubertal mice, severe testicular damage such as vacuolation and swelling of the round spermatids, necrosis of the late elongated spermatids in stages I to VIII, abnormal meiosis in stage XIX, and a marked cellular exfoliation into the lumen were detected 1 day after administration of the 150 mg/kg dose. On Day 2, the damage was more pronounced. However, the testicular damage was repaired 7 to 10 days after treatment. In the adults, many tubules from stages I to VIII were affected, showing nuclear vacuolation in most of the round spermatids 1 day after administration of the 150 mg/kg dose. Meiosis in stage XIV was normal. In the prepubertal mice, nuclei of the round spermatids in stage I-III, especially step 1 spermatids had degenerated 1 day after administration of the 100 mg/kg dose. In the adults, the round spermatids had degenerated in stages I-II and IV-VIII 1 day after administration of the 100 mg/kg dose. These results suggested that the most vulnerable cell type among the spermatogenic cells was the round spermatid, especially that in the Golgi phase (stage I-III). Other stages and spermatogenic cells were relatively resistant to acrylamide testicular toxicity.     

Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide,adriamycin, and diepoxybutane

In testis, apoptosis is a way to eliminate damaged germ cells during their development. In this study, we evaluated the ability of three germ cell mutagens to induce apoptosis (or programmed cell death) at specific stages of rat seminiferous epithelial cycle. These chemicals include the cancer chemotherapy drugs etoposide and adriamycin and the butadiene metabolite diepoxybutane. According to our results, etoposide is a very potent inducer of apoptosis in male rat germ cells and the cell types most sensitive to it include all types of spermatogonia, zygotene, and early pachytene spermatocytes and meiotically dividing spermatocytes. Also, adriamycin causes an increase in apoptosis at specific stages of seminiferous epithelial cycle and the most sensitive cell types are type A_3–4 spermatogonia, preleptotene, zygotene, and early pachytene spermatocytes. Die poxybutane does not cause any significant increase in the frequency of apoptosis in rat testis. In addition, we studied whether p53 is taking part in the apoptotic response of spermatogenic cells by studying the levels of p53 protein in testis before and after chemical treatment. No accumulation of p53 in testis was seen after treatment with these three chemicals. The expression of two p53-regulated genes, p21^WAF1 and mdm2, was also studied but no increase in the levels of mRNA of these genes was observed after treatment. The results indicate that apoptosis should be taken into consideration when the genotoxic effects of chemicals are evaluated in germ cells. Environ. Mol. Mutagen. 31:133–148, 1998 © 1998 Wiley-Liss, Inc.     

The effect of combined therapy with ritodrine,erythromycin and verapamil on the frequency of micronuclei in peripheral blood lymphocytes of pregnant women

Abstract The main aim of this study was to investigate the genotoxic effect of combined pharmacotherapy applied in post-operative treatment of cervical cerclage in pregnant women over six days. This study included 19 phenotypically healthy pregnant women in mid-trimester with a diagnosis of cervical insufficiency, mean age 28±5.33. The frequency of micronuclei (MN) was estimated in peripheral blood lymphocytes of patients before surgical intervention and after the end of applied pharmacotherapy by application of cytokinesis block micronucleus (CBMN) test. Mean value of baseline MN frequency was 6.84±2.91 MN/1000 binucleated cells, and after the end of the applied therapy, 10.32±4.27 MN/1000 binucleated cells (P<0.001) were found. The data of cell proliferation index showed that the combined therapy did not induce significant difference in cell kinetics (P>0.05). Our results showed that combined pharmacotherapy treatment over six days significantly increased the frequency of MN in peripheral blood lymphocytes of pregnant women.     

Micronucleus assay for mouse alveolar Type II and Clara cells

The objective of our study was to develop a micronucleus (MN) assay for detecting genotoxic damage after inhalation exposure in mouse alveolar Type II and Clara cells, potential target cells for lung carcinogens. Ten male C57BL/6J mice were exposed to ethylene oxide (630 mg/m³) for 4 hr via inhalation; 10 unexposed mice serving as controls. 72 hr after the exposure, Clara cells and alveolar Type II cells were isolated using two different methods. Method 1 included a 15‐min trypsin lavage and a 2‐hr incubation of cell suspension. Method 2 involved a 30‐min trypsin lavage, Percoll gradient centrifugation, and a 48‐hr incubation for cell attachment. Nitro blue tetrazolium (NBT) ‐staining was applied to distinguish Clara cells. The frequency of micronuclei (MNi) was scored in NBT‐negative cells (defined as Type II cells in Method 2) and NBT‐positive cells (Clara cells). To detect possible differences between the techniques, MNi in Clara cells were analyzed from samples prepared by both methods. With Method 2, a clear increase in the mean frequency of micronucleated cells was seen in the exposed mice as compared with the controls, for both alveolar Type II and Clara cells. However, no significant increase in MN frequency was seen in Clara cells analyzed from samples prepared by Method 1. Based on our findings, mouse alveolar Type II and Clara cells seem to be suitable for MN analysis in studies aimed at identifying genotoxic lung carcinogens. Both alveolar Type II and Clara cells can be isolated using Method 2. Environ. Mol. Mutagen. 2010. © 2009 Wiley‐Liss, Inc.     

Catalytic inhibitors of topoisomerase II are DNA-damaging agents: induction of chromosomal damage by merbarone and ICRF-187

Merbarone is a catalytic inhibitor of topoisomerase II (topo II) that has been proposed to act primarily by blocking topo II-mediated DNA cleavage without stabilizing DNA-topo II-cleavable complexes. In this study merbarone was used as a model compound to investigate the genotoxic effects of catalytic inhibitors of topo II. The clastogenic properties of merbarone were evaluated using in vitro and in vivo micronucleus (MN) assays combined with CREST staining. For the in vitro MN assay, ICRF-187, a different type of catalytic inhibitor, and etoposide, a topo II poison, were used for comparison. Treatment of TK6 cells with all three of these drugs resulted in highly significant dose-related increases in kinetochore-lacking MN and, to a lesser extent, kinetochore-containing MN. In addition, a good correlation between p53 accumulation and MN formation was seen in the drug-treated cells. A mouse MN assay was performed to confirm that similar DNA-damaging effects would occur in vivo. Bone marrow smears from merbarone-treated B6C3F1 mice showed a dose-related increase in micronucleated polychromatic erythrocytes with a mean of 26 MN per 1000 cells being seen at the 60 mg/kg dose. Almost all MN lacked a kinetochore signal, indicating that merbarone was predominantly clastogenic under these conditions in vivo. The present study clearly shows that merbarone is genotoxic both in vitro and in vivo, and demonstrates the inaccuracy of earlier statements that merbarone and other catalytic inhibitors block the enzymatic activity of topo II without damaging DNA.     

cytogenetic and germ cell effects of phosphine inhalation by rodents: II. subacute exposures to rats and mice

Phosphine (PH₃) is a highly toxic grain fumigant to which there is significant human workplace exposure. To determine the in vivo cytogenetic effects of inhalation of PH₃, male F344/N rats and B6C3F1 mice were exposed to target concentrations of 0, 1.25, 2.5, or 5 ppm PH₃ for 6 hr/day for 9 days over an 11 -day period. Approximately 20 hr after the termination of exposures, blood was removed from the mice and rats by cardiac puncture and the lymphocytes cultured for analyses of sister chromatid exchanges and chromosome aberrations in rats and mice, and micronuclei (MN) in cytochalasin B-induced binucleated lymphocytes from mice. In addition, bone marrow (rats) and peripheral blood (mice) smears were made for the analysis of MN in polychromatic and normochromatic erythrocytes. No significant increase in any of the cytogenetic endpoints was found at any of the concentrations examined. These results indicate that concentrations of PH₃ up to 5 ppm are not genotoxic to rodents when administered by inhalation for 9 days during an 11 -day period as measured by several cytogenetic assays. To evaluate the effects of PH₃ on male germ cells, a dominant lethal test was conducted in male mice exposed to 5 ppm PH₃ for 10 days over a 12-day period and mated to groups of untreated females (2 females/male) on each of 6 consecutive 4-day mating intervals. None of the 6 groups of females exhibited a significant increase in percent resorptions. These results indicate that exposure to 5 ppm PH₃ by inhalation does not induce dominant lethality in male mouse germ cells at steps in spermatogenesis ranging from late differentiating spermatogonia/early primary spermatocytes through mature sperm. © 1994 Wiley-Liss, Inc.     

Protective effects of antioxidants on micronuclei induced by camphorquinone/N,N-dimethyl-p-toluidine employing in vitro mammalian test system

Camphorquinone (CQ) is widely used as an initiator in modern visible-light (VL) cured resin systems. CQ is also characterized as a potential allergenic compound. To date, there is growing concern that CQ may produce genetic damage by inducing mutation. In this study, CQ in the presence of reducing agent N,N-dimethyl-p-toluidine (DMT) with or without VL irradiation was analyzed for the induction of chromosomal aberrations indicated by micronuclei (MN) induced in CHO cells. Our data demonstrated that an increase in the numbers of MN was observed with CQ/DMT with or without VL irradiation (p < 0.05). Significant prolongation of cell cycles was observed by the treatment with CQ/DMT with or without VL irradiation (p < 0.05). In addition, VL irradiated CQ/DMT was found to exhibit significantly genotoxic and cytotoxic effects as compared with CQ/DMT alone (p < 0.05). Furthermore, to determine whether oxidative stress could modulate the MN induced by CQ/DMT with or without VL irradiation in CHO cells, cells were pre-treated with various antioxidants 10 mM N-acetyl-L-cysteine (NAC), 2 mM ascorbic acid, and 2 mM alpha-tocopherol. The pre-treatment with antioxidants could antagonize not only the increased MN cells but also the prolonged cell cycle induced by CQ/DMT with or without VL irradiation in CHO cells (p < 0.05). Our findings provide the evidences for the induction of MN by CQ/DMT employing mammalian test system, indicating clastogenic activity of CQ/DMT with or without VL irradiation in vitro. In addition, VL irradiated CQ/DMT exhibits higher genotoxic and cytotoxic effects than CQ/DMT alone. Moreover, NAC, ascorbic acid, and alpha-tocopherol act as the antagonists against the genotoxicity and cytotoxicity of CQ/DMT with or without VL irradiation.     

Dose-related clastogenic effects induced by benzene in bone marrow cells and in differentiating spermatogonia of Swiss CD1 mice.

Cancer hazard is due to genotoxic events in somatic cells and genetic hazard in the strict sense is due to mutagenic events in germ cells. The investigation of sensitivity differences between somatic and germinal cells is pertinent to the question whether a genotoxic carcinogen is also a germ cell mutagen. Cytogenetic damage induced by benzene in mice was evaluated by determining the frequencies of chromosomal aberrations in bone marrow and spermatogonial cells of male Swiss CD1 mice. First, the analysis was performed by administering 1 ml/kg (880 mg/kg) of benzene as a single oral dose and sampling either cell type after a wide range of times (6, 12, 18, 24, 30, 36, 42 and 48 h) to determine the time of maximum response. At this dose benzene showed high clastogenic activity in bone marrow cells with a peak between 24 and 30 h. In differentiating spermatogonia the frequency of aberrant cells was highest 24 h after treatment. The overall effect in spermatogonia was lower than in bone marrow cells. Second, the dose response was determined 24 h after treatment with two additional doses of benzene: 0.1 ml/kg (88 mg/kg) and 0.5 ml/kg (440 mg/kg) for bone marrow cells; 0.25 ml/kg (220 mg/kg) and 0.5 ml/kg (440 mg/kg) for differentiating spermatogonia. The clastogenic effect was dose dependent in both cell types. The frequencies of aberrant cells increased in a linear-quadratic manner in bone marrow and linearly in differentiating spermatogonia. Furthermore, the per cell damage was higher in bone marrow than in spermatogonial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Micronucleus monitoring of a floriculturist population from western Liguria,Italy

A biomonitoring study was carried out to investigate whether exposure to complex pesticide mixtures in ornamental crop production represents a potential genotoxic risk. Exposed and control subjects were selected in western Liguria (Italy). The area was chosen for its intensive use of pesticides. The main crops produced were roses, mimosas, carnations and chrysanthemums, as ornamental non-edible plants, and tomato, lettuce and basil, as edible ones. The levels of micronuclei (MN) were analysed in peripheral blood lymphocytes of 107 floriculturists (92 men and 15 women) and 61 control subjects (42 men and 19 women). A statistically significant increase in binucleated cells with micronuclei (BNMN) was detected in floriculturists with respect to the control population (4.41 +/- 2.14 MN/1000 cells versus 3.04 +/- 2.14, P < 0.001). The mean number of BNMN varied as a function of sex and age. Smoking habit had no effect on MN frequency. A positive correlation between years of farming and MN frequency in peripheral blood lymphocytes was observed (r = 0.30, P = 0.02). The conditions of exposure were also associated with an increase in cytogenetic damage, with a 28% higher MN frequency in greenhouse workers compared with subjects working only outdoors in fields. Workers not using protective measures during high exposure activities showed an increase in MN frequency. Our findings suggest a potential genotoxic risk due to pesticide exposure.     

Effect of chemicals on the duration of male meiosis in mice detected with laser scanning cytometry.

Aneuploidy studies in sperm such as the sperm-FISH assay require a precise knowledge of the duration of spermatogenesis, especially of the meiotic stages. This is important in order to sample sperm from the epididymis at appropriate intervals after animal treatment. However, aneugens may delay the cell cycle. The progression from meiotic divisions to epididymal sperm was determined by labelling the last S-phase before meiosis with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. In a time frame of 20--24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). We studied the effects of the chemicals acrylamide, colchicine, diazepam, griseofulvin, taxol, thiobendazole, trichlorfon and vinblastine on the duration of meiotic divisions in male mice. Colchicine treatment prolonged the duration of meiotic divisions by about 48 h. On days 21 and 22, the frequencies of BrdU-labelled sperm in the colchicine group were 11.7 and 9.4%, respectively, while they were 28.4 and 30.6%, respectively, in the concurrent controls (P > 0.01). On day 24 after treatment, the frequency of labelled sperm in the colchicine group reached the control level. Etoposide treatment resulted in an elevation of BrdU-labelled sperm at 23 rather than 22 days. The other chemicals showed no significant effect of prolonging meiotic cell cycle progression. On the basis of the colchicine and etoposide data, it is suggested that the effect of a chemical on the meiotic cell cycle progression is determined first in order to chose the appropriate sperm sampling time to detect aneuploidy induction.     

The micronucleus assay in human exfoliated urothelial cells: application in a genotoxicity study of workers exposed to a mineral jelly containing sodium nitrite and N-phenyl-1-naphthylamine

Exposure to certain chemical agents in occupational settings has been identified as carcinogenic to the human bladder. Micronucleus (MN) analysis in exfoliated urothelial cells is an interesting method for biomonitoring genetic damage in human populations. However, few studies have been performed in an occupational context. The aim of this study was to examine whether the occupational use of a mineral jelly induced a genotoxic risk for workers employed at a single factory producing bearings using the MN test on exfoliated urothelial cells. The prevalence of micronucleated exfoliated urothelial cells (MNC) was determined in 35 female workers with dermal exposure to the jelly and 41 female controls. The mean percentage of MNC (expressed as percent cells with MN per 1000 cells scored) observed in the exposed worker group was 0.46 +/- 0.11% (range 0-2.8) and in the control group 0.14 +/- 0.03% (range 0-0.8). There is a significant job effect (P = 0.0018, MANCOVA) on the prevalence of MNC, whereas age and smoking habit had no significant effect (P = 0.90 and 0.91, respectively). There is no interaction between job and smoking habit (P = 0.4421). Exposure to the mineral jelly appeared to be the main factor inducing the increased prevalence of MNC. This may be due to the presence of mutagens/carcinogens in the jelly: an aromatic amine, N-phenyl-1-naphthylamine (CAS no. 90-30-2), which is carcinogenic in mice, or sodium nitrite (CAS no. 7632-00-0), which is genotoxic in human cell systems. In conclusion, these results suggest that use of the mineral jelly could present a genotoxic risk for workers. We think that the MN assay on exfoliated cells could be valuable for biological monitoring purposes in occupational contexts as a marker of significant exposure to bladder mutagenic/carcinogenic agents.     

Persistence of micronuclei in peripheral blood normochromatic erythrocytes of subchronically benzene-treated male mice

The kinetics of micronucleus (MN) induction and decline in blood normochromatic erythrocytes (NCE) of mice subchronically exposed to benzene was investigated during and after exposure. Swiss (ICR) male mice (10/group) were given 0.0, 36.6, 73.2, and 146.4 mg/kg body weight benzene by gavage daily for 14 days, except for days 5 and 10. The frequency of MN increased significantly (P <.001) during benzene treatment as a function of both concentration and time. Eleven days after exposure the levels of MN were higher than those observed at the end of exposure. After an initial rapid decline in the frequency of MN from 11 to 18 days postexposure, the decline became linear with time through 60 days postexposure. Using linear regression analysis, the MN level in each treatment group was predicted to reach control levels by approximately 85 days post-treatment. Dose-dependent suppression and recovery of erythropoiesis, estimated by polychromatic erythrocyte frequency, were observed in the 1st and 2nd weeks of exposure, respectively. Red blood cell (RBC) production was markedly increased in the first 3 weeks after benzene treatment. At later times the rate of production of the RBC returned to normal and may account for the linear decline observed in MN frequency. This research indicates that the frequency of MN is dose and duration dependent, while the decline in MN frequency after the end of benzene exposure can be related to changes in the kinetics of erythropoiesis.     

A factor present in human milk, but not colostrum, which is cytotoxic for human lymphocytes

This study reports the presence of a factor in human milk which is cytotoxic to both autologous and heterologous human peripheral blood mononuclear cells (PBMC). The cytotoxic effect was measured by the release of 51Cr from labelled cells or by the inability of PBMC to exclude trypan blue following exposure to milk. The cytotoxic factor was not dialysable or sensitive to heating at 56 degrees C for 30 min, and could not be adsorbed from milk by PBMC. It lysed lymphocytes harvested from colostrum and from autologous or heterologous milk. The cytotoxic factor in the milk was different from the factor found in colostrum which inhibits the proliferation of mitogen stimulated PBMC, but which is not cytotoxic. Testing of serial samples of milk from the same mother indicated that the factor could be detected in the milk after 3-4 days post partum. We conclude that factors cytotoxic for human PBMC appear in human milk early in lactation.