Influences of gender, development, pregnancy and ethanol consumption on the hematotoxicity of inhaled 10 ppm benzene

The hematotoxic effects of benzene in both humans and animals are well documented. Current estimates concerning the risks associated with benzene exposure are usually based on adult, male cohort studies; however, there are indications that females may respond differently than males to benzene and that fetuses may respond differently than adults. Another factor to be considered in risk estimates is the impact of personal habits. In experimental animals, ethanol consumption is known to increase the hematotoxicity of benzene; therefore, alcohol consumption may also alter the potential risk of individuals exposed to benzene. To address some of the factors that may confound risk estimates for benzene exposure, a series of experiments were performed. Age-matched male as well as pregnant and virgin female Swiss Webster mice were exposed to 10 ppm benzene for 6 h a day over 10 consecutive days (days 6 through 15 of gestation for the pregnant females). Half of the animals also received 5% ethanol in the drinking water during this period. On day 11, bone marrow cells from the adults and liver cells from the fetuses were assayed for the numbers of erythroid colony-forming units (CFU-e). CFU-e assays were also performed on bone marrow cells isolated from 6-week postpartum dams exposed during gestation and from in utero-exposed 6-week old males and females. Gender differences were clearly observed in the responses to the various exposure protocols. Depressions in CFU-e numbers were only seen in male mice while elevations in CFU-e numbers were only seen in female mice. Male mice exposed as adults for 10 days to benzene (B), ethanol (E) or benzene+ethanol (B+E) exhibited depressed CFU-e levels as did male fetal mice exposed to B in utero. In addition, adult male mice which had been exposed in utero to either B or to E individually displayed depressed CFU-e levels. In contrast, none of the groups of female mice exhibited any depressions in CFU-e numbers after any of the exposures. Elevations in CFU-e numbers were observed among pregnant females exposed to E and among adult females exposed to B+E in utero. In summary, a majority (6/9) of the exposure protocols produced depressions in the CFU-e numbers of male mice, whereas a majority (7/9) of the exposure protocols produced no changes in the CFU-e numbers of female mice. Those changes that were observed in females consisted of elevations of CFU-e numbers. These results suggest that the male erythron is more susceptible than the female erythron to the hematotoxicants benzene and ethanol, regardless of whether exposures occur in utero or during adulthood.     

Synthesis of arylidenehydrazono- and glycopyranosylhydrazino-sulfonylbenzylidene-2,4-imidazolidinediones as potential antiviral and antitumoral agents

Low-intensity pulsed ultrasound recently has been shown to accelerate long bone fracture healing, but its effect on bone growth and development is unknown. The longitudinal growth and bone density of the femur and tibia in young rats was measured after application of an ultrasound transducer emitting 1.5-MHz pulsed ultrasound (30 mW/cm2, SATA) for 20 min/day. After 28 days, no length difference was detected (< or = 2%) compared to the sham-treated leg or to unexposed controls. Also, no significant difference in bone mineral density (BMD) of the femur or tibia was found (< or = 6%). In a repeated experiment in which a periosteal trauma stimulus was created in the femoral diaphysis, the ultrasound also had no effect on growth or BMD. This results suggests that physeal bone growth is far less sensitive to this level of ultrasound application than is fracture repair. This may be related to the cascade of cellular events and regulatory factors that are present after a fracture.     

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.     

Erythroid colony formation (CFUe) in fetal liver and adult bone marrow and spleen from the mouse

The methyl cellulose modification of the CFUe technique has been applied to 14 day fetal liver and adult bone marrow and spleen from CBA/CA mice. Optimized doses of fetal calf serum, alpha-thioglycerol, erythropoietin and cell suspensions have been obtained from dose response curves in order to standardize the technique. The slopes of the erythropoietin and cell dose response curves indicate a greater sensitivity by fetal liver to the hormone than bone marrow or spleen. The proportion of cells in the DNA synthesis phase of the cell cycle, as measured by the CFUe technique, has been estimated by administering hydroxyurea. Two hours after the drug was injected, 89% of fetal liver cells, 71% of bone marrow cells and 81% of spleen cells were found to be in the S-phase.     

A test of the hypothesis that diagnostic ultrasound disrupts myelination in neonatal rats

Neonatal rats were exposed or sham exposed for 30 min to pulsed ultrasound [2.25 MHz carrier frequency, 1 microsecond pulse length, 50 Hz pulse repetition frequency (PRF), 50 W/cm2 Imax, 2 mW/cm2 ITA], euthanised and prepared for electron microscopic analysis of the nodes of Ranvier of the dorsal and ventral roots of the spinal cord. There was also a cage control. All materials were processed and scored blindly, evaluating whether perinodal myelin was normal. Rats from all regimens had areas of disrupted myelination. There was no statistically significant difference among the regimens for absence of myelination. The results did not confirm an earlier report that diagnostic ultrasound disrupts myelination in neonatal rats.     

A stable RAG1-RAG2-DNA complex that is active in V(D)J cleavage

A well-known characteristic of gamma delta T cells is that they are produced in waves during ontogeny, with cells expressing T-cell receptor V gamma 5 appearing early in fetal thymic ontogeny, followed by V gamma 6, then by other gamma delta T-cell types. In addition, evidence exists to suggest that the potential of haemopoietic precursors to generate different types of gamma delta T cells changes in ontogeny. We have used these observations as the basis for an extensive study of the potential for haemopoietic precursors isolated from fetal liver, neonatal spleen and adult bone marrow to reconstitute severe combined immunodeficient (SCID) mice. Mice that were reconstituted as newborns with fetal liver cells most closely resembled normal C.B-17 mice with respect to both lymphocyte numbers and subsets, while mice reconstituted with adult bone marrow had fewer cells than normal mice. This deficit spanned both T and B cells in all organs examined. Among the gamma delta T-cell subsets examined, the ability to reconstitute V gamma 4+ cells was particularly dependent on the ontogenic age of the reconstituting presursors, with fetal liver cells having the greatest capacity to generate V gamma 4+ cells, and adult bone marrow cells the least. The vast majority of the T cells produced in the reconstituted mice were of donor origin, and the level of reconstitution was found to be dependent upon some factor other than the presursor frequency.     

Frequency analysis of the T cell precursors in the thymus

A frequency determination of the T cell precursors in murine adult and fetal thymuses as well as in the bone marrow and fetal liver was made. Cells were serially diluted and injected into deoxyguanosine-treated fetal thymus lobes with a microinjector, and the lobes were cultured for 12 to 21 days. The lobes in which T cell development did not occur were discriminated from those in which T cells developed, and the precursor frequency was determined by Poisson probability distribution analysis. The precursor cell frequencies in adult bone marrow cells (2.4 x 10(-5)) and fetal liver cells (1.7 x 10(-4)) were comparable to those determined previously in in vivo intrathymus transfer experiments. The present study further shows that only a small fraction of fetal thymus cells (0.9-5.0 x 10(-2)), CD4-8- adult thymocytes (1.6 x 10(-2)), and Thy-1 low positive adult thymocytes (3.3 x 10-4)) retain the precursor activity.     

Is myringoplasty worth while?

1. We have used fetal rats to study the following aspects of the development of hemopoiesis: (a) content of hemopoietic stem cells in fetal bone marrow, liver, and peripheral blood and (b) origin of hemopietic cells in the developing mammalian bone marrow. 2. In the studies we utilized the diffusion chamber technique to study the content of stem cells committed to granulopoiesis. The number of myelopoietic stem cells in liver peripheral blood and in "bone marrow" of 18-day-old rats is nearly identical. Since in "bone marrow" a considerable number of peripheral blood cells are present in the vessels at that time, whereas extravascular cells consist only of mesenchymal cells, one might assume that these peripheral blood cells give rise to granulocytic precursors in the cultures. Morphologically these cells are "blast" cells and lymphocytes. 3. Based on cell labeling indices of radioautograms, derived from continuous infusion of pregnant rats with [3H]thymidine, it could be shown that in the perichondral area mesenchymal cells of the fetus and newborn have a slow rate of DNA turnover whereas "bone marrow" cells are in an active state of profileration. 4. In further support of this it was also shown that injections of hydroxyurea (an agent which destroys cells in DNA synthesis) has vitrually no effect on perichondral mesenchymal cells whereas "bone marrow" cells were completely blocked in their ability to support myelopoietic differentiation in the diffusion chamber implants. 5. The conclusions would therefore be that (a) local perichondral cells, i.e., mesenchymal cells, do not contribute to marrow hemopoiesis, (b) matrix cells of the developing bone marrow cannot reconstitute hemopoiesis, and (c) hemopoietic bone marrow cells develop from migrating peripheral "stem cells", one of the sources being the liver.     

Avoidance by rats of illumination with low power nonionizing electromagnetic energy.

Results of 2 experiments with a total of 18 female and 8 male Sprague-Dawley rats indicate that Ss spent more time in the halves of shuttle boxes that were shielded from illumination by 1.2-GHz microwave energy than in the unshielded halves. In Exp I, Ss avoided the energy when it was presented as 30-msec pulses with a pulse repetition rate of 100 pulses/sec (pps). The average power density was about .6 mW/cm2, and the peak power density was about 200 mW/cm2. In Exp II, the energy was presented both continuously and in pulse-modulated form (i.e., .5-msec exponentially decaying pulses at a rate of 1,000 pps). The average power density of the continuous energy was 2.4 mW/cm2, and the average power density of the pulse-modulated energy was .2 mW/cm2. The peak power density of the modulated energy was 2.1 mW/cm2. Results show that Ss avoided the pulsed energy, but not the continuous energy. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Comparative studies of the mitogenic effects of epidermal growth factor and transforming growth factor-alpha and the expression of various growth factors in neoplastic and non-neoplastic prostatic cell lines

Murine acute myeloid leukemia is characterized by chromosome 2 aberrations, and genesis of the marker chromosome 2 by radiation is suspected to be an initiating event of radiation leukemogenesis. A detailed analysis of the type and frequency of chromosome 2 aberrations in murine bone marrow cells at an early stage after irradiation is provided here. A total of 40 male C3H/He mice was exposed to 137Cs gamma-ray at a dose of 1, 2 or 3 Gy, and sacrificed 24 hours after irradiation. Metaphase samples prepared from bone marrow cells were Q-banded for karyotyping or painted with DNA probes specific to chromosome 2. In 5 mice analyzed by karyotyping, one mouse showed high frequency of the marker aberrations as well as other chromosome 2 aberrations. Chromosome painting analysis for the rest of the mice also detected 3 animals showing significantly high frequencies of chromosome 2 aberrations. Dose-dependence of the frequencies was observed even among those mice that tended to be sensitive. The results indicated that there was a subgroup of mice carrying hypersensitive chromosome 2. The subgroup could be leukemia-sensitive if radiation-induced chromosome aberrations are responsible for an early change in myeloid leukemogenesis.     

Transplantation of stromal cells transduced with the human IL3 gene to stimulate hematopoiesis in human fetal bone grafts in non-obese, diabetic-severe combined immunodeficiency mice

The non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mouse is a convenient host for human hematopoietic tissues and cells. Human fetal bone fragments engrafted subcutaneously in NOD-SCID mice sustain human hematopoiesis for several months. MS5 murine bone marrow stromal cells were transfected by electroporation with a plasmid containing the human interleukin-3 gene. As expected, stably transfected hu-IL3-MS5 cells supported human hematopoiesis in vitro more efficiently than MS5 cells. hu-IL3-MS5 cells were then injected intravenously into hu-NOD-SCID mice to test their ability to home to the mouse and/or human bone marrow, and to evaluate the role of hu-IL3 secretion on human hematopoiesis in vivo. hu-IL3 was detected in the mouse serum for up to an observation time of 8 weeks. hu-IL3-MS5 cells engrafted the bone marrow, spleen, liver and lungs of the mice but also the human bone graft. The presence of hu-IL3-MS5 cells in the human bone significantly stimulated local human hematopoiesis. This setting could be used to model the bone marrow homing of intravenously injected stromal cells or stromal cell precursors. The same experimental principle could also be applied in a therapeutic perspective to malignant human bone marrow hematopoiesis.     

Granulocyte colony-stimulating factor crosses the placenta and stimulates fetal rat granulopoiesis

We studied the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration to pregnant rats upon fetal and neonatal myelopoiesis. Pregnant rats were treated with rhG-CSF twice daily for 2, 4, and 6 days before parturition. rhG-CSF crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. Peak fetal serum levels were 1,000-fold lower than levels detected in the dam. Hematopoietic effects of rhG-CSF were assessed by cytologic analysis of the newborn blood, spleen, bone marrow, thymus, and liver. White blood cell counts were increased twofold to fourfold in newborns. This increase was due to circulating numbers of polymorphonuclear cells (PMN). rhG-CSF induced a myeloid hyperplasia in the newborn marrow consisting of immature and mature myeloid cells in the day-2 and day-4 treated pups. Bone marrow of pups treated for 6 days contained mostly hyper-segmented PMN with little or no increase in myeloid precursors. An increase in the number of postmitotic (PMN, bands, and metamyelocytes) and mitotic (promyeloblasts, myeloblasts, and metamyeloblasts) myeloid cells in the spleen of neonates was observed. No change was detected in splenic lymphocytes or monocytes. No effect of rhG-CSF was noted in the newborn liver or thymus. These results demonstrate that maternally administered rhG-CSF crosses the placenta and specifically induces bone marrow and spleen myelopoiesis in the fetus and neonate. The significant myelopoietic effects of rhG-CSF at low concentrations in the fetus suggest an exquisite degree of developmental sensitivity to this cytokine and may provide enhanced defense mechanisms to the neonate.     

Effects of low-intensity ultrasound on the central nervous system of primates

The brains of anesthesized squirrel monkeys were exposed to 2.25 to 5 MHz ultrasound at low intensities (average power from 3 mW/cm2 to 0.9 W/cm2). The exposure produced evoked potentials recorded by EEG electrodes chronically implanted in the midline parietal region. Computer analysis of the waveforms showed that ultrasound produced a transient upward shift in both the peak frequency and in its amplitude. Complete adaptation occurred with 3 min of continuous exposure to either CW or pulsed irradiation.     

Successful reconstitution of human hematopoiesis in the SCID-hu mouse by genetically modified, highly enriched progenitors isolated from fetal liver

Highly purified CD34++CD38-Lin- hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli beta-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the beta-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38-Lin- cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.     

Effects of gestational exposure to a video display terminal-like magnetic field (20-kHz) on CBA/S mice

Possible adverse effects of magnetic fields (MFs) on reproduction have been an open question. To verify the embryo-lethal effect of pulsed MF of the type emitted by video display terminals (VDTs) reported previously in CBA/S mice, a developmental toxicity study was conducted in animals of the same origin. Mated CBA/S mice (80-86 pregnant animals per group) were exposed to a 20-kHz MF with sawtooth waveform continuously from gestational day 0-18. The flux density of the vertical MF was 15 microT peak-to-peak (150 mG). This field was previously reported to increase the number of resorptions in CBA/S mice. On gestational day 18, the dams were killed and blood and bone marrow samples were taken for hematology and micronuclei analysis, respectively. The number of corpora lutea was counted and the content of the uterus examined. There were no statistically significant differences in maternal or fetal body weights, number of corpora lutea, implantations, resorptions, dead and live fetuses, or external and skeletal malformations. MF did not alter the number of blood cells or cause micronuclei in bone marrow erythrocytes in the dams. The mean number of resorptions was slightly but not statistically significantly, higher in the MF group than in controls. The results do not indicate marked developmental, hematological, or clastogenic effects of 20-kHz MFs.     

Characterization of the growth factor activity of amniotic fluid on cells from hematopoietic and lymphoid organs of different life stages

We investigated the proliferation-promoting effects of murine amniotic fluid (MAF) on in vitro cultured cells originally obtained from murine hematopoietic and lymphoid organs at different life stages. MAF promoted proliferation of the fetal liver cells (FLC), newborn spleen cells and adult bone marrow cells. The proliferation-promoting activity of MAF was extended to liver cells and spleen cells from mice younger than 2 weeks old. MAF did not, however, promote the proliferation of newborn or adult thymocytes, or of spleen cells, liver cells or peritoneal cells, liver cells or peritoneal cells from 2-week-old or older mice. Rather, it partially inhibited the proliferation of spleen cells, thymocytes and peritoneal cells from 1-year-old mice. These results suggest that MAF contains growth factors for hematopoietic stem cells but not for either mature or immature T lymphocytes. Supporting this view, the MAF activity was partially neutralized by a polyclonal anti-mouse stem cell factor (SCF) antibody. Moreover, the immunoblotting of MAF against anti-mouse SCF antibody revealed a band at 30-32 kDa corresponding to the previously reported SCF. Interestingly, MAF was able to maintain FLC and adult bone marrow cells alive in culture for a relatively long time (2 weeks). The MAF activity was further shown to be partially and cell type-dependently antagonized by TNF-alpha and TGF-beta. These results provided evidence that MAF contains potentially multiple growth factors preferentially affecting the early stage of hematopoiesis, one of which is SCF.     

Interactions between c-kit and stem cell factor are not required for B-cell development in vivo

The receptor-type tyrosine kinase, c-kit is expressed in hematopoietic stem cells (HSC), myeloid, and lymphoid precursors. In c-kit ligand-deficient mice, absolute numbers of HSC are mildly reduced suggesting that c-kit is not essential for HSC development. However, c-kit- HSC cannot form spleen colonies or reconstitute hematopoietic functions in lethally irradiated recipient mice. Based on in in vitro experiments, a critical role of c-kit in B-cell development was suggested. Here we have investigated the B-cell development of c-kit-null mutant (W/W) mice in vivo. Furthermore, day 13 fetal liver cells from wild type or W/W mice were transferred into immunodeficient RAG-2-/- mice. Surprisingly, transferred c-kit- cells gave rise to all stages of immature B cells in the bone marrow and subsequently to mature conventional B2, as well as B1, type B cells in the recipients to the same extent as transferred wild type cells. Hence, in contrast to important roles of c-kit in the expansion of HSC and the generation of erythroid and myeloid lineages and T-cell precursors, c-kit- HSC can colonize the recipient bone marrow and differentiate into B cells in the absence of c-kit.     

Toxicity of fumonisin B1 to B6C3F1 mice: a 14-day gavage study

Fumonisin B1 (FB1) is a fungal toxin produced by members of the genus Fusarium. Ingestion of FB1 causes species-specific neurotoxic, nephrotoxic, hepatotoxic and pulmonary effects. The clinical, haematological and pathological responses of adult male and female B6C3F1 mice to FB1 were assessed following 14 daily gavage doses ranging from 1 to 75 mg FB1/kg body weight/day. There were no consistent sex-related changes. Although all responses were modest, the most notable effects of FB1 were on the liver, bone marrow, adrenals and kidneys. In the liver, hepatocellular single cell necrosis, mitosis and anisokaryosis were observed, accompanied by elevated serum ALT. In the kidneys, minor histopathological changes were confined to female mice, while mild decreases in ion transport and increases in blood urea nitrogen were seen only in males. Small changes in glutathione levels were observed in the kidneys and livers of male mice. Adrenal cortical cell vacuolation was observed at 15 mg FB1/kg and higher in females and from 35 mg FB1/kg in males. Serum cholesterol was elevated in both male and female mice, possibly due to FB1-induced changes in lipid metabolism in the liver and adrenals. Although bone marrow cell numbers were unchanged, increases in vacuolated myeloid cells and lymphocytes were observed in female mice. In general, the degree of changes observed indicate that mice are not as sensitive a model of FB1 toxicity as rats.     

Ontogeny of the heavy chain immunoglobulin repertoire in fetal liver and bone marrow

We studied the kinetics of maturation of B cell progenitors in the mouse embryo, from day 15 of development to birth, both in liver and bone marrow. The analysis of Ig heavy chain rearrangements at different time points of late fetal development shows that oligoclonal patterns of V(H)-D-J(H) rearrangements are detected by day 15 in fetal liver. The pattern is polyclonal and diverse by day 17; however, 80% of the rearrangements are nonproductive. In bone marrow, the pattern of rearrangements is less diverse at birth, although the percentage of nonproductive rearrangements approaches adult bone marrow levels (35-40%). After day 17 in fetal liver, there is a sudden reversal in the percentage of nonproductive rearrangements that reaches 33% at day 19 (birth). Maturation of B cells, as measured by the fraction of surface Ig+ in total B220+ cells and the presence of N sequence additions in V(H)-D-J(H) joints, occurs in the marrow before fetal liver. These results demonstrate that the lymphopoietic environment in fetal liver and bone marrow of animals at the same stage of development is functionally distinct.