Daniel Wierda

Suppression of bone marrow stromal cell function by benzene and hydroquinone is ameliorated by indomethacin.

Administration of benzene to mice will inhibit bone marrow stromal cell-supported hemopoiesis in culture. Hydroquinone, a major metabolite of benzene, will cause a similar inhibition of stromal cell function in vitro. Stromal cells produce both an inducer (colony-stimulating factor) and an inhibitor (prostaglandin E2; PGE2) of hemopoiesis. This research was conducted to determine if prostaglandin synthesis is involved in the suppression of stromal cell function by benzene and hydroquinone. Male B6C3F1 mice were administered benzene (100 mg/kg), indomethacin (1 mg/kg), or benzene plus indomethacin twice a day for 4 consecutive days. On Day 5 bone marrow cells were removed to determine the effect of treatment. In a second series of experiments mouse bone marrow stromal cells in culture were treated with hydroquinone (10(-7) to 10(-4) M), indomethacin (10(-6) M), or a combination of hydroquinone plus indomethacin. Stromal cell function was based on the ability of the treated stromal cells to support granulocyte/monocyte colony development in coculture. The results demonstrated that preadministration of indomethacin in vivo ameliorated benzene-induced inhibition of bone marrow stromal cell function. In vitro, indomethacin ameliorated hydroquinone toxicity to stromal cell function. Benzene administration in vivo induced elevated PGE2 in bone marrow samples which were prevented by preadministration of indomethacin. However, hydroquinone in vitro did not induce a consistent increase in PGE2 levels. These results suggested that toxicity to stromal cells was not due solely to increased prostaglandin synthetase activity.

In vitro effects of benzene metabolites on mouse bone marrow stromal cells

Benzene exposure can result in bone marrow myelotoxicity. We examined the effects of benzene metabolites on bone marrow stromal cells of the hemopoietic microenvironment. Male B6C3F1 mouse bone marrow adherent stromal cells were plated at 4 X 10(6) cells per 2 ml of DMEM medium in 35-mm tissue culture dishes. The growing stromal cell cultures were exposed to log 2 doses of five benzene metabolites: hydroquinone, benzoquinone, phenol, catechol, or benzenetriol for 7 days. The dose which caused a 50% decrease in colony formation (TD50) was 2.5 X 10(-6) M for hydroquinone, 17.8 X 10(-6) M for benzoquinone, 60 X 10(-6) M for benzenetriol, 125 X 10(-6) M for catechol, and 190 X 10(-6) M for phenol. We next examined the effect of benzene metabolites on the ability of stromal cells to influence granulocyte/monocyte colony growth (G/M-CFU-C) in a coculture system. Adherent stromal cells were plated and incubated for 14 days and then exposed to a benzene metabolite. After 3 days the medium and metabolite were removed and an agar:RPMI layer containing 10(6) fresh bone marrow cells was placed over the stromal layer. After incubation for 7 days the cultures were scored for G/M colony formation. Hydroquinone and benzoquinone were most toxic, while catechol and benzenetriol inhibited colony growth only at high doses. These results indicate that injured bone marrow stromal cells may be a significant factor in benzene-induced hemotoxicity.

Macrophage regulation of myelopoiesis is altered by exposure to the benzene metabolite hydroquinone

Hydroquinone, a myelotoxic metabolite of benzene, decreases the ability of murine bone marrow stromal cells to support myelopoiesis in vitro. Bone marrow stroma consists of macrophages and fibroblastoid stromal cells that participate coordinately in regulating myelopoiesis. The goal of this study was to determine if macrophage or fibroblastoid cell function is more sensitive to the myelotoxic actions of hydroquinone. To address this question, we developed purified populations of macrophages and fibroblastoid stromal cells and treated each population with hydroquinone. These cells were reconstituted together with nontreated cells of the opposite type and assayed for their ability to support the formation of granulocyte and macrophage colonies in an agar overlay. Reconstituted cultures containing hydroquinone-treated macrophages supported fewer colonies than did corresponding cultures containing untreated macrophages. Reconstituted cultures containing hydroquinone-treated fibroblastoid stromal cells were not affected. Moreover, hydroquinone reduced detectable interleukin-1 activity in purified macrophage cultures stimulated with lipopolysaccharide. These results indicate that hydroquinone selectively interferes with macrophage function possibly, in part, via alteration of macrophage interleukin-1 secretion.

Modulation of stromal cell function in DBA/2J and B6C3F1 mice exposed to benzene or phenol.

Inbred B6C3F1 (B6) mice are more resistant to benzene myelotoxicity than are inbred DBA/2J (D2) mice. This difference may be due to increased sensitivity of the hemopoietic microenvironment in D2 mice to benzene or benzene metabolites relative to B6 mice. The objectives of this investigation were to determine whether stromal cells which support hemopoiesis in the marrow were more sensitive to benzene in D2 mice than in B6 mice and to determine whether these strains would continue to express differences in susceptibility to phenol, an oxidative metabolite of benzene. Mice were given benzene (100 mg/kg) or phenol (100 mg/kg) intraperitoneally, twice a day for four consecutive days. On Day 5 marrow cell suspensions were removed from mice given benzene or phenol and from controls, plated in culture and assayed for (1) the relative number of adherent stromal cell (ASC) colonies present, (2) the number of granulocyte/monocyte precursors from benzene or phenol treated mice that could be supported by ASC from normal mice in coculture, and (3) the number of granulocyte/monocyte precursors from normal mice which could be supported by ASC from benzene or phenol treated mice. After benzene administration, only reductions in body weight and marrow cellularity followed the expected pattern and were reduced to a greater extent among D2 mice than B6 mice. Benzene had no significant effect on ASC colonies or on the number of granulocyte/monocyte precursors present. In contrast, the ability of ASC to support hemopoiesis of granulocyte/monocytes from normal donors was reduced to a greater degree among B6 mice than among D2 mice which paradoxically showed an increase in the ability to support hemopoiesis in coculture. Phenol significantly reduced the ability of ASC to support hemopoiesis of granulocyte/monocyte precursors but no preferential effect between strains was evident. These results suggest that benzene, but not phenol, is metabolized differently between the two strains and that bone marrow stromal cells, components of the hemopoietic microenvironment, are sensitive targets for benzene or its oxidative metabolites.

Chlorphentermine Suppresses the Phosphatidylinositol Pathway in Concanavalin A-Activated Mouse Splenic Lymphocytes

We have previously demonstrated that the chlorphentermine (CP)1-induced impairment in lymphocyte blastogenesis involves drug-induced inhibition of an event which occurs very early during lymphocyte activation. An early event, which is associated with mitogen-induced lymphocyte activation, involves the hydrolysis of phosphatidylinositol by phospholipase C to yield inositol phosphates and diacylglycerol as products. Inositol phosphates and diacylglycerol then function as mediators of a trans-membrane signal for the continuation of the cellular response. It was the purpose of the present study to determine the effects of CP on this phosphatidylinositol pathway. We demonstrated that formation of inositol phosphates in lymphocytes increases progressively above control over a 2 hour period following concanavalin A (Con A)-stimulation. In contrast, lymphocytes pre-incubated with 10(-5)M CP for 60 min, then stimulated with Con A for 2 hours in the presence of 10(-5)M CP, exhibit a significantly depressed inositol phosphate formation. In addition, CP also inhibited the activity of phospholipase C (IC50 = 0.58 mM), the enzyme responsible for the formation of inositol phosphates during lymphocyte activation. Further, lymphocytes activated in a manner that bypasses the phosphatidylinositol pathway are not inhibited by 10(-7)M or 10(-9)M CP as are cells activated with Con A. These results suggest that the suppression of the phosphatidylinositol pathway may be involved in the inhibition by CP of lymphocyte blastogenesis induced by Con A.

Morphological and functional changes in mouse splenic lymphocytes following in vivo and in vitro exposure to chlorphentermine

With repeated administration to animals, the cationic, amphiphilic drug, chlorphentermine (CP), has been shown by others to induce a phospholipidosis in lymphocytes. In the present study mouse splenic lymphocytes, exposed to CP, either in vivo or in vitro, developed morphological changes consistant with the induction of phospholipidosis. In addition, CP induced functional changes in lymphocytes. Mice, treated with CP in vivo, demonstrated a significantly depressed ability to generate a delayed hypersensitivity response or to produce antibody-secreting cells against de novo antigens. Mouse splenic lymphocytes, exposed to 10(-7) M CP for 3 days in vitro, demonstrated a significantly depressed blastogenic response to the mitogens phytohemagglutinin, concanavalin A and lipopolysaccharide. CP inhibited an event that occurred early during lymphocyte activation, but was subsequent to mitogen/receptor coupling. In addition, CP significantly depressed the increased uptake of choline that occurs in lymphocytes following cellular activation. Since the presence of phospholipidosis is indicative of an impairment in phospholipid metabolism, these results taken together provide evidence for a relationship between this phenomenon and altered immune function.

Partial characterization of bone marrow hemopoiesis in mice after cisplatin administration

After cessation of cisplatin (cis-dichlorodiammineplatinum) chemotherapy, selective recovery of certain cell lineages occurs during bone marrow hemopoiesis. To further investigate the process of selective hemopoiesis, we combined the use of buoyant density gradient separation, morphology, and lymphocyte function assays to characterize changes in the hemopoiesis of immature and mature marrow cells after exposure to cisplatin. A single, cytotoxic dose of cisplatin was administered to B6D2F1 male mice, and marrow cell suspensions were taken from these mice 3 and 7 days later to characterize hemopoietic recovery. Morphology and buoyant density separation of marrow cells revealed that the most significant changes in cellular composition occurred at Day 3. At this time, there was an increase in immature white blood cells (WBCs) and immature polymorphoneutrophils (PMNs) with a concomitant reduction in immature red blood cells (RBCs). By Day 7, the normal proportion of immature RBCs, immature WBCs, and PMNs was restored; however, the buoyant distribution patterns for PMNs indicated that a greater proportion of immature PMNs was still present relative to marrow suspensions from normal mice. Fewer lymphocytes were also observed in marrows from the Day 7 group when compared with controls. Lymphocyte function tests indicated reduced mitogen responsiveness of lymphocytes from both Day 3 and Day 7; however, more immature lymphocytes were present after 7 days than were seen with either normal or Day 3 marrow suspensions. Overall, the results indicated that hemopoiesis proceeded through a specific hierarchy which began with the restoration of the erythrocyte line followed by the leukocyte cell lines. Lymphocyte recovery lagged behind the restoration of all the cell lineages examined.

The measurement of chemiluminescence, aggregation, and 5-hydroxy-6,8,11,14-eicosatetraenoic acid production of n-formyl-methioninyl-leucyl-phenylalanine-stimulated human polymorphonuclear leukocytes

Abstract Polymorphonuclear leukocytes (PMNs) which had been treated with nordihydroguaiaretic acid (NDGA) or indomethacin were stimulated with varying doses of n -formyl-methioninyl-leucyl-phenylalanine (FMLP). Aggregation and chemiluminescence (CL) were simulta-neously measured. Supernatant solutions were obtained and saved from cells stimulated with 10 −6 M FMLP. These solutions were assayed for 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) using radioimmunoassay (RIA) techniques and used as a measure of 5-lipoxygenase (LO) activity. It was found that 10 −5 M NDGA inhibited CL from PMNs stimulated with FMLP. However, NDGA had much less effect on aggregation of these cells. Indomethacin had little effect on PMN aggregation and less effect on CL than NDGA following FMLP stimulation. Indomethacin (10 −5 M ) did inhibit CL stimulated with FMLP at FMLP doses of 10 −7 and 10 −6 M but had no effect of CL at FMLP doses of 10 −5 M . Finally, RIA for 5-HETE showed that the effects of NDGA and indomethacin on 5-HETE production could be correlated with their effects on CL responses of cells stimulated with 10 −6 M FMLP. These results support two conclusions. First, PMN-CL and LO activity may be correlated. Second, PMN-CL is not necessarily associated with PMN aggregation. These observations may indicate that LO products are not necessary for PMN aggregation to occur.

Studies of luminol-dependent whole-blood chemiluminescence induced by platelet-activating factor (PAF)

Abstract A whole-blood luminol-dependent chemiluminescence (CL) assay stimulated by platelet-activating factor (PAF) is reported. Factors investigated using this assay were dilution of blood, dose responsiveness of PAF, and inactivation of PAF. The final conditions chosen for the assay were a 1:10 dilution of whole blood and 2 × 10−4 M luminol. Nordihydroguaiaretic acid (NDGA) and indomethacin (INDO), which inhibit lipoxygenase and cyclooxygenase, decreased luminol-dependent whole-blood chemiluminescence by 59 and 17%, respectively. Superoxide dismutase and catalase were relatively ineffective inhibitors of CL (28 and 22%, respectively) while sodium azide was most inhibitory (84%). Although these studies were not entirely conclusive, lipoxygenase and myeloperoxidase appear to be important in CL elicited by PAF in this whole-blood CL assay. This system may serve as a useful screening system for measuring the effect of PAF in the blood of different individuals without extensive separation of cell types. It has the distinct advantage of being closer to in situ Conditions.

Perinatal Immunotoxicity of Benzene Toward Mouse B Cell Development

Benzene is widely used by chemical industries and exposure to benzene has been shown experimentally to be immunotoxic in adult animals. This study addressed whether exposure of fetuses in utero to benzene compromises the development of fetal B lymphopoiesis and whether B-lymphocyte development recovers postnatally. Pregnant BALB/C dams were given intraperitoneal injections of benzene (100 mg/kg, twice daily) from day 12.5 of gestation through day 19.5 of gestation. Phenotypic analysis revealed that fetal liver cell suspensions from embryos exposed in utero contained fewer pre-B cells and B cells than corresponding controls. Fetal liver cell cultures established from these embryos also produced fewer B cells. In contrast, levels of pre-B cells were elevated in the livers of 8-day-old neonates that had been exposed to benzene in utero. Moreover, responsiveness to the B-cell mitogen, LPS, was significantly decreased in spleen cell cultures derived from these neonates. These results indicate that in utero exposure to high concentrations of benzene alters fetal B lymphopoiesis and may compromise immune responsiveness postnatally.