Biomedical subjects
R D Irons
Publications and source records attributed to R D Irons.
An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure.
Benzene-induced myelotoxicity can be reproduced by the coadministration of two principal metabolites, phenol and hydroquinone. Coadministration of phenol (75 mg/kg) and hydroquinone (25-75 mg/kg) twice daily to B6C3F1 mice for 12 days resulted in a significant loss in bone marrow cellularity in a manner exhibiting a dose-response. One explanation for this potentiation is that phenol stimulates the peroxidase-dependent metabolism of hydroquinone. Addition of phenol to incubations containing horseradish peroxidase, H2O2, and hydroquinone resulted in a stimulation of both hydroquinone removal and benzoquinone formation. Stimulation occurred with phenol as low as 100 microM and with very low concentrations of horseradish peroxidase. When boiled rat liver protein was added to identical incubations containing [14C]hydroquinone, the level of radioactivity recovered as protein bound increased by 37% when phenol was added. Similar results were observed when [14C]hydroquinone was incubated in the presence of activated human leukocytes. Hydroquinone binding was increased by approximately 70% in the presence of phenol. Phenol-induced stimulation of hydroquinone metabolism and benzoquinone formation represents a likely explanation for the bone marrow suppression associated with benzene toxicity.
Selective activation of endogenous ecotropic retrovirus in hematopoietic tissues of B6C3F1 mice during the preleukemic phase of 1,3-butadiene exposure.
1,3-Butadiene (BD), a comonomer used in the production of synthetic rubber, is a rodent carcinogen. We have observed a marked increase in the incidence of thymic lymphoma in male B6C3F1 relative to NIH Swiss mice chronically exposed to BD in the absence of demonstrable differences in bone marrow (target organ) toxicity. Increased expression of murine leukemia virus (MuLV) antigens was also observed on lymphomas from BD-exposed B6C3F1 mice. Because NIH Swiss mice do not usually express endogenous retroviruses and their ecotropic proviral sequences are not intact, these findings provide presumptive evidence of a role for endogenous retrovirus sequences in BD-induced lymphoma in the B6C3F1 mouse. The present study was conducted to examine the expression and behavior of endogenous retroviruses in these strains during the preleukemic phase of BD exposure. Chronic exposure to BD (1250 ppm) 6 hr/day, 5 days/wk for 3 to 21 weeks increased markedly the quantity of ecotropic retrovirus recoverable from bone marrow, thymus, and spleen of B6C3F1 mice. However, expression of other endogenous retroviruses (xenotropic, MCF-ERV) was not enhanced. No viruses of any type were found in similarly treated NIH Swiss mice. The mechanism of this increase in ecotropic retrovirus in B6C3F1 mice is believed to be de novo activation in greater numbers of cells because changes in the Fv-1 tropism of the replicating viruses or changes in Fv-1 host restriction were not found. Endogenous retroviruses are thus implicated in BD-induced leukemogenesis in B6C3F1 mice. Further studies will examine the role of retrovirus in BD-induced leukemogenesis and the mechanisms of activation of ecotropic proviral sequences in murine cells.
Chromosome aberrations in mouse bone marrow cells following in vivo exposure to 1,3-butadiene.
Chronic exposure to 1,3-butadiene (BD) results in a marked increase in the incidence of thymic lymphoma in male B6C3F1 relative to NIH Swiss mice whereas no demonstrable differences in bone marrow (target organ) toxicity exist. Repeated exposure to BD is known to produce a macrocytic anemia and an increase in the frequency of micronuclei in circulating erythrocytes in both strains. The present study was undertaken to determine if chromosomal breakage, aneuploidy or both reflect differences in BD leukemogenicity observed between B6C3F1 and NIH Swiss mice. Mice were exposed to a single concentration of BD (1250 p.p.m.) for 6 h. Bone marrow cell preparations were made at 24, 48, 72 and 96 h after cessation of exposure. In both strains comparable increases in the frequency of chromosomal aberrations (of the chromatid type) were observed following exposure to BD. Significant differences in the number of chromosomes were not observed, although a pattern of chromosomal loss in cells from treated animals was observed. These results indicate that BD-treatment in vivo produces significant increases in chromatid aberrations but not aneuploidy in both strains. Therefore it is concluded that bone marrow toxicity, including cytogenetic abnormalities, is not predictive of leukemogenicity in these mice.
Macrocytic-megaloblastic anemia in male NIH Swiss mice following repeated exposure to 1,3-butadiene.
Thymic lymphoma/leukemia is the major cause of death in B6C3F1 mice chronically exposed to 1,3-butadiene (BD). Similar to radiation-induced murine thymic lymphoma, the bone marrow is also a major target organ. Because of the association of murine thymic lymphoma with endogenous type-C murine leukemia retroviruses (MuLV) present in the germ line of most strains of laboratory mice, including B6C3F1 and its parent strains, we examined the effects of BD exposure on NIH Swiss mice which do not possess intact endogenous ecotropic MuLV. Male NIH Swiss mice exhibited a macrocytic-megaloblastic anemia following inhalation of 1250 ppm BD for 6 weeks. Treatment-related changes included decreases in circulating erythrocytes, total hemoglobin, and hematocrit and an increase in mean corpuscular volume. An eightfold increase in circulating micronuclei was also observed. The anemia was not accompanied by a significant alteration in mean corpuscular hemoglobin concentration, an increase in circulating reticulocytes, or an increase in circulating nucleated erythrocytes. These findings are consistent with a treatment-related macrocytic-megaloblastic anemia and indicate that the bone marrow is an important target for BD toxicity in mice independent of MuLV background and expression.
Macrocytic-megaloblastic anemia in male B6C3F1 mice following chronic exposure to 1,3-butadiene.
In the present study exposure to 1,3-butadiene (BD) resulted in a macrocytic-megaloblastic anemia in male B6C3F1 mice following chronic inhalation of 1250 ppm for 6 to 24 weeks. Treatment-related changes evident after 6 weeks of exposure included a decrease in circulating erythrocytes, total hemoglobin, and hematocrit and an increase in mean corpuscular volume. A leukopenia, due primarily to a decrease in segmented neutrophils, and a five- to sixfold increase in circulating micronuclei were observed after 6 and 24 weeks of exposure. These changes were not accompanied by a significant alteration in mean corpuscular hemoglobin concentration, an increase in circulating reticulocytes, or circulating nucleated erythrocytes. A consistent treatment-related alteration in bone marrow cellularity was not found. However, flow cytofluorometric analysis of bone marrow DNA cell cycle kinetics revealed a 44% increase in proliferative index relative to controls, due primarily to an increase in the proportion of cells in S phase. These findings are consistent with a treatment-related macrocytic-megaloblastic anemia and indicate the bone marrow to be an important target organ for BD toxicity.
The role of reactive intermediates in sulfhydryl-dependent immunotoxicity: interference with microtubule assembly and microtubule-dependent cell function.
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Altered hematopoietic stem cell development in male B6C3F1 mice following exposure to 1,3-butadiene.
The effects of the murine lymphomagen, 1,3-butadiene (BD), on the proliferation and differentiation of hematopoietic stem cells were examined in male B6C3F1 mice. Exposure to 1250 ppm BD for 6 weeks resulted in no demonstrable alteration in the frequency of spleen colony-forming units (CFU-S); however, colonies derived from treated animals were smaller than those from controls. The absence of any difference in the frequency of CFU-GM after 6 weeks exposure suggests that BD produces an alteration in the relative proportion of immature to mature pluripotent stem cells in BD-exposed animals. This was confirmed by the examination of the effects of BD on stem cell development in long-term bone marrow culture. After 14 days, the number of CFU-GM derived from cultures of animals exposed for 6 weeks was reduced compared to controls. However, at 28 days an increase relative to controls was observed. This shift in the course of differentiation of the granulocyte/macrophage precursor cell, as assessed by the CFU-GM, provides further evidence that there is an increase in the relative frequency of primitive or immature stem cells in BD-treated mice. After a 30-31 week exposure to BD, a decrease in the numbers of both CFU-S and CFU-GM was observed. These findings indicate that BD causes alterations in stem cell development and suggest that alterations in bone marrow stem cells may play an essential role in the pathogenesis of BD-induced thymic lymphoma.
Effect of short-term inhalation exposure to 1,3-butadiene on murine immune functions.
Interest in 1,3-butadiene (BD) as a potential immunomodulator was prompted by reports of an increased incidence of neoplasia in humans exposed to BD during the manufacture of styrene-butadiene synthetic rubber, and by a recent study which demonstrated a high incidence of thymic lymphomas in B6C3F1 mice. B6C3F1 mice were exposed to 1250 ppm BD by inhalation 6 hr per day, 5 days per week, for 6 or 12 weeks. Immune function assays were selected to evaluate specific humoral and cell-mediated immunity and spontaneous cytotoxicity; lymphoid organ histopathology was also evaluated. A slight decrease in antibody plaque-forming cells (PFC) per spleen was observed in exposed mice, although PFC per 10(6) splenic lymphocytes was normal. Significant extramedullary hematopoiesis and erythroid hyperplasia was observed in spleens from exposed mice, and correlated with a twofold increase in thymidine incorporation in spontaneously proliferating splenocytes. No differences in proliferation to alloantigens were demonstrable between control and BD-exposed splenocytes. Mitogenesis by phytohemagglutinin, Concanavalin A, and lipo polysaccharide was suppressed in splenocytes from exposed mice, but may have been due to the cellular dilution effect of hematopoietic activity. Cytotoxic T-lymphocyte generation was suppressed after a 6-week exposure to BD, but was comparable to controls after 12 weeks of exposure. No differences in spontaneous cytotoxicity were observed between control and exposed mice. Overall, no persistent immunological defects were detectable after inhalation exposure to this tumorigenic agent.
Effect of sampling site and collection method on variations in baseline clinical pathology parameters in Fischer-344 rats. II. Clinical hematology.
An analysis of the influence of blood-sampling site and collection method on peripheral hematology parameters was conducted in age-matched male Fischer-344 rats. Sites examined for blood collection were the right ventricle, abdominal aorta, abdominal vena cava, retroorbital plexus, and tail. Collection methods used included syringe (10 ml), Vacutainer(s) (3 ml or 3 ml X 2), and capillary tube. Blood collected from the tail exhibited leukocyte counts approximately twice those of samples samples from other sites. Blood collected from the retroorbital plexus and tail exhibited significant variations in white blood cell count, red blood cell count, hemoglobin, and hematocrit, and differences in leukocyte differential counts of lymphocytes and neutrophils when compared with other sites. Blood collected from the abdominal aorta and in a second Vacutainer from the right ventricle exhibited lower erythrocyte, leukocyte, and platelet counts than that collected from other sites with the exception of the platelet count from tail blood which was lower than that from all other sites. Although parameter values vary with sample site selection, those obtained from right ventricle blood were the least variable and the most consistent when compared with all other methods.
The effects of diet on the toxicity of nitroaromatic chemicals in rodents.
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Evidence for direct action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic epithelium.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) acts on selected targets within the immune system to produce a characteristic profile of pathologic responses typified by thymic atrophy, suppressed cellular immunity, and inhibition of antibody production to T-lymphocyte-dependent antigens. Studies in inbred mice differing in sensitivity to TCDD indicate that TCDD-induced thymic atrophy is mediated by a receptor protein (designated the Ah receptor). To study the cellular and molecular basis for TCDD-induced thymic atrophy, primary cultures of thymic epithelial (TE) cells were established from C57BL/6 mice, a strain sensitive to TCDD. Treatment of TE monolayers with TCDD (0.1 to 10 nM) resulted in the altered maturation of cocultured syngeneic thymocytes as judged by suppression (40% of control at 10 nM TCDD) of TE-dependent responsiveness of thymocytes to the mitogens concanavalin A and phytohemagglutinin. TE-conditioned medium enhanced the mitogen responsiveness of thymocytes three- to four-fold; however, the enhanced mitogen response mediated by the TE-conditioned medium was not suppressed in thymocytes incubated in medium collected from TCDD-treated cultures or in TE-conditioned medium to which TCDD (10 nM) had been added directly. The suppression of TE-dependent maturation of thymocytes was concentration dependent (EC50 approximately 1 nM) and stereospecific, suggesting involvement of the Ah receptor. The Ah receptor in cytosol fractions from cultured TE cells was measured directly and was found to be present at a concentration 3 and 3.5 times greater than that measured in whole thymus and thymocytes, respectively. The results of this study indicate that TCDD can act directly on epithelial target cells in the thymus: one consequence of this action appears to be the altered thymus-dependent maturation of T-lymphocyte precursors, mediated through direct cell-cell contact between thymocytes and TE cells.
Effect of sampling site and collection method on variations in baseline clinical pathology parameters in Fischer-344 rats. 1. Clinical chemistry.
An analysis of the influence of blood sampling site and collection method on clinical chemistry parameters was conducted in male Fischer-344 rats. Sampling sites compared included the right ventricle, aorta, vena cava, retroorbital sinus, and tail. Methods of collection included Vacutainer (3 ml sample), syringe (exsanguination), and capillary tube. Nineteen frequently measured clinical chemistry procedures were determined, including serum enzymes, cholesterol, and triglycerides. Significant differences were noted for almost every parameter as a function of sampling site. Samples collected from the right ventricle with a Vacutainer produced the most representative results for most but not all procedures when compared to the overall mean (for all methods). The largest differences were encountered in samples obtained from tail and retroorbital plexus. Exanguination also resulted in significant differences for some parameters when compared to smaller sample volumes obtained from the same site. It was concluded that sampling site and collection method can be a major source of variation in clinical chemistry measurements, and in selection of an appropriate method, one should consider which parameters are likely to be of major interest.
Quinones as toxic metabolites of benzene.
Occupational exposure to benzene has long been associated with toxicity to the blood and bone marrow, including lymphocytopenia, pancytopenia, aplastic anemia, acute myelogenous leukemia, and possibly lymphoma. A variety of studies have established that benzene itself is not the toxic species but requires metabolism to reactive intermediates. The bioactivation of benzene is complex. Both primary and secondary oxidation of benzene and its metabolites are mediated via cytochrome P-450 in the liver, although the role of secondary metabolism in the bone marrow is not clear. Toxicity is associated with the dihydroxy metabolites, hydroquinone and catechol, which concentrate in bone marrow. Hydroquinone and its terminal oxidation product, p-benzoquinone, have been demonstrated to be potent suppressors of cell growth in culture. Suppression of lymphocyte blastogenesis by these compounds is a sulfhydryl-dependent process and occurs at concentrations that do not result in cell death, or in detectable alterations in energy metabolism, intracellular glutathione concentration, or protein synthesis. Recent studies suggest that these compounds and other membrane-penetrating sulfhydryl alkylating agents, such as N-ethylmaleimide and cytochalasin A, and endogenous regulatory molecules, such as soluble immune response suppressor (SIRS), interfere with microtubule assembly in vitro and selectively interfere with microtubule-dependent cell functions at identical concentrations. These agents appear to react with nucleophilic sulfhydryl groups essential for guanosine triphosphate binding to tubulin that are particularly sensitive to sulfhydryl-alkylating agents.
Studies of immune function and host resistance in B6C3F1 mice exposed to formaldehyde.
A series of immune function and host resistance parameters were examined in female B6C3F1 mice following a 21-day (6 hr/day) inhalation exposure to 15 ppm of formaldehyde (HCHO). Immune parameters examined included delayed hypersensitivity to keyhole limpet hemocyanin, antibody plaque-forming cell response to sheep erythrocytes (T-lymphocyte-dependent antigen) and TNP-Ficoll (T-lymphocyte-independent antigen), lymphoid organ weights and histopathology, routine hematology, bone marrow cellularity and CFU progenitor cell enumeration, lymphocyte subpopulation quantitation by cell surface markers, mitogen-induced lymphocyte blastogenesis, macrophage function parameters, and host resistance to challenge with the bacterium Listeria monocytogenes and transplantable tumor cells. Lymphoid organ weight, bone marrow cellularity, and hematology parameters were unchanged in HCHO exposed mice. Similarly, the percentage of T and B lymphocytes and their proliferative responses to mitogens were not significantly altered. Antibody (IgM) plaque-forming cell response following antigen challenge was unchanged. Macrophage function was normal although some evidence of enhanced H2O2 production associated with elevated bactericidal activity was observed in resident macrophages. Resistance to challenge with the bacteria Listeria monocytogenes was significantly enhanced, while resistance to tumor challenge remained unchanged. No evidence of immunosuppression following short-term exposure to HCHO was observed.
Soluble immune response suppressor (SIRS) inhibits microtubule function in vivo and microtubule assembly in vitro.
Soluble immune response suppressor (SIRS) is a product of concanavalin A-stimulated murine T cells that, when activated or oxidized by macrophages or H2O2 (SIRSox), suppresses in vitro immune responses and inhibits cell division by normal and neoplastic cells. SIRSox is inactivated by a variety of electron donors, which suggests that SIRSox may be an oxidizing agent. Incubation of lymphocytes with SIRSox, but not with SIRS, partially reversed concanavalin A-mediated inhibition of capping of membrane immunoglobulin on B cells, and disrupted the cytoplasmic array of microtubules visualized by fluorescence microscopy. SIRSox also inhibited microtubule assembly in vitro in a concentration-dependent manner. Inactivation of SIRSox by dithiothreitol prevented SIRSox-mediated reversal of inhibition of capping and inhibition of microtubule assembly. These results reveal a pattern of SIRSox activity similar to sulfhydryl-dependent cytoskeletal disrupting agents (e.g., N-ethylmaleimide, cytochalasin A, p-benzoquinone), and suggest that SIRSox-mediated suppression of proliferation may involve interference with sulfhydryl-dependent cytoskeletal events critical for cell division.
Phytohemagglutinin-induced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment.
Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25- and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25- or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunofluorescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.
Alteration of lymphocyte function by quinones through a sulfhydryl-dependent disruption of microtubule assembly.
The cytoskeleton plays a pivotal role in lectin-induced lymphocyte blastogenesis. Microtubule disrupting agents, many of which are sulfhydryl (SH) reagents, interfere with cytoskeletal-dependent cell functions including lymphocyte blastogenesis and agglutination. For example, hydroquinone (HQ) and N-ethylmaleimide (NEM) inhibit lectin-stimulated lymphocyte blastogenesis and agglutination at concentrations (10(-5)M) that do not reduce cell viability or ATP production. Indicative of the SH-specificity of these effects, only L-cysteine protects against HQ or NEM inhibition of blastogenesis and agglutination. Other compounds, including L-serine, DL-lysine and imidazole, have no protective effect. These and other findings previously reported suggest a selective interaction of HQ, or its oxidation product, p-benzoquinone (p-BQ) with SH groups critical to early G1 events associated with lymphocyte activation. These compounds show similar SH specificity in inhibiting microtubule assembly in vitro. The subcellular target specificity (cytoskeleton) exhibited by these compounds was compared to that of Adriamycin (ADR), a complex polycyclic quinone with known immunotoxic activity. ADR inhibited microtubule assembly in vitro and inhibited lymphocyte blastogenesis, however, these effects were not correlated with a loss of agglutination nor was toxicity protected against by the addition of SH compounds. The combination of cell culture methods together with application of techniques to measure microtubule assembly in vitro provides an effective means to discriminate between agents that selectively interfere with cytoskeletal-dependent function and those producing non-specific effects associated with cell death, such as decreased energy production or increased membrane permeability.