Metabolism of benzene and phenol in macrophages in vitro and the inhibition of RNA synthesis by benzene metabolites

Benzene may affect hemopoiesis by damaging the bone marrow stroma that provides the microenvironment for hemopoiesis. A possible target of benzene toxicity in the stroma is the macrophage, which is a major source of protein factors required for the proliferation and differentiation of progenitor cells. As an initial approach towards understanding whether benzene inhibits hemopoietic factor production in bone marrow stroma, the metabolism of benzene and phenol has been studied and the effect of benzene and its metabolites on macrophage RNA synthesis has been examined. Benzene is not metabolized in macrophages but phenol, the major metabolite of benzene in bone marrow, is converted by peroxidase in the macrophage to both free metabolites and species which covalently bind to cellular macromolecules. Benzene and its metabolites inhibited RNA synthesis in a dose-dependent manner, with 50% inhibitory concentrations of 5 × 10^–3M for benzene, 2.5 × 10^–3 M for phenol, 2.5 × 10^–5 M for hydroquinone, and 6 × 10^–6 M for p-benzoquinone; this inhibition was not attributable to loss of cell viability. Benzene, possibly by an inhibition of uridine transport into macrophages, and phenol, by its conversion to covalently binding species, inhibit RNA synthesis in macrophages and thus may inhibit the synthesis of colony stimulating factors required for hemopoiesis.Abbreviations CFU-G / M colony forming unit-granulocyte / macrophage - FCS fetal calf serum - IC₅₀ molar concentration causing 50% inhibition - PBS phosphate buffered saline     

Estradiol metabolites as isoform-specific inhibitors of human glutathione S-transferases

Numerous studies have suggested that the lifetime dose of unopposed estrogen is a significant risk factor for breast and uterine cancer. Estradiol (E2) plays a putative role as a tumor promoter through interaction with estrogen receptors but can also be metabolized to redox active and/or mutagenic semiquinones and quinones. Similarly, equine estrogens (components of certain hormone replacement therapy preparations) are converted to quinone metabolites. The use of hormone replacement therapy has also been associated with increased breast and endometrial cancer risk. Recently, metabolites of certain equine estrogens have been shown to inhibit human glutathione S-transferases (hGSTs). Since E2 and equine estrogens share similarities in other biological interactions, we have investigated the inhibitory capacity of endogenously formed E2 metabolites toward various hGSTs. The quinone metabolite of 2-hydroxy-17-beta-estradiol (2-OH-E2) was synthesized, and inhibition of hGST-mediated biotransformation of model substrates was assessed. Inhibition of purified recombinant hGSTM1-1 and hGSTA1-1 occurred in a concentration-dependent manner with IC50-values of approximately 250 and 350 nM, respectively. hGSTs M2-2, P1-1 and T1-1 were significantly less sensitive to inhibition. Specific glutathione-conjugates of the estrogen quinone also potently inhibited hGSTM1-1 and hGSTA1-1. Mass spectrometry data indicate that the inhibition was not mediated via covalent adduction. Although we have demonstrated hGST inhibition via E2 metabolites, our findings indicate that the isoform specificity and potency of GST inhibition by endogenous E2 metabolites is different than that of equine estrogen metabolites.     

The inhibition of mitochondrial DNA replication in vitro by the metabolites of benzene, hydroquinone and p-benzoquinone

Rat liver mitochondria incubated with the metabolites of benzene, p-benzoquinone or 1,2,4-benzenetriol, showed a dose-dependent inhibition of [3H]dTTP incorporation into mtDNA with median inhibitory concentrations of 1 mM for each compound. Benzene and the metabolites phenol, catechol and hydroquinone did not inhibit at concentrations up to 10 mM. Similarly, incubation of p-benzoquinone or hydroquinone with rabbit bone marrow mitochondria showed a dose-dependent inhibition of mtDNA synthesis with 50% inhibition at 1 mM and 10 mM, respectively. That these metabolites inhibit mitochondrial replication was evidenced by the fact that [3H]dTTP incorporation into characteristic 38S, 27S and 7S mitochondrial replication intermediates was decreased by the quinones, as analyzed on 5-20% neutral sucrose velocity gradients. p-Benzoquinone, hydroquinone and 1,2,4-benzenetriol inhibited the activity of partially purified rat liver mtDNA polymerase gamma using either activated calf thymus DNA or poly(rA) X p(dT)12-18 as primer/template, with 50% inhibitory concentrations of 25 microM, 25 microM and 180 microM, respectively. Preincubation of the metabolites with polymerase gamma or primer/template, followed by removal of the unreacted metabolite by gel filtration, indicated that inhibition resulted from interaction of the metabolites with the enzyme, rather than with the template. Binding appeared to involve a sulfhydryl residue on the enzyme since the binding of [14C]hydroquinone was prevented by N-ethylmaleimide. The ability of hydroquinone or p-benzoquinone to inhibit binding of [14C]hydroquinone to the enzyme suggests that the compounds bind to a common site or are converted to a common intermediate. Inhibition of, or changes in, replication in mitochondria of bone marrow cells by hydroquinone and p-benzoquinone may explain the changes in the mitochondrial genome observed in marrow stem cells in acute myelogenous leukemia and may suggest a mechanism for benzene leukemogenesis.     

Inhibition of heme synthesis in bone marrow cells by succinylacetone: effect on globin synthesis

The effects of 4,6-dioxoheptanoic acid (succinylacetone, SA), an inhibitor of delta-aminolevulinic acid dehydratase, on total iron uptake, heme synthesis, and globin synthesis were studied in rat marrow cells in culture in order to examine the coordination of heme and globin synthesis. SA inhibited heme synthesis in both control and erythropoietin-stimulated cells in a dose-dependent fashion; at 10(-3) M, inhibition was complete, whereas at 10(-7) M, there was no significant effect. Inhibition of total iron uptake was also dose-dependent although, at 10(-3) M, it was not complete. The inhibition of heme synthesis by SA was partially overcome by addition of 10(-4) M porphobilinogen or protoporphyrin IX. SA caused an almost complete suppression of globin formation in both erythropoietin-stimulated and unstimulated cells as early as five hours after the addition of the inhibitor. When inhibition of heme synthesis was incomplete, globin synthesis was partially inhibited. These results indicate that heme synthesis is required for erythropoietin-mediated induction of globin synthesis in cultured bone marrow cells.     

Effects of genistein on expression of bone markers during MC3T3-E1 osteoblastic cell differentiation

In this in vitro study, the hypothesis that the beneficial effects of dietary genistein on bone are through the modulation of the bone marker synthesis by osteoblastic MC3T3-E1 cells was tested, and the possible roles of estrogen receptors in the actions of genistein on osteoblastic cells were also examined. Interleukin-6 production was decreased 40% to 60% in osteoblastic cells treated with genistein from either day 8-16 or day 12-16, at dietarily achievable concentrations (10(-10) to 10(-8) M) (P<0.05). The mRNA expression of osteoprotegerin increased about 140% in cells treated from with genistein day 4-8 at a concentration of 10(-8) M (P<0.05). The ratio of estrogen receptor-alpha to beta expression increased 10-fold from day 0 to 12 of culture (P<0.05). Correlating with this time-dependent variation in estrogen receptor expression, treatments of 17beta-estradiol and genistein had opposite dose patterns on the ratio of estrogen receptor-alpha to beta expression following treatment from day 4 to 6 compared to from day 0 to 2. The addition of ICI-182,780, an estrogen receptor blocker, reduced the inhibitory effect of genistein on IL-6 production by 30-50%. In summary, these findings suggest that the beneficial skeletal effects of genistein, at dietarily achievable levels, appear to be mediated, at least in part, by interleukin-6 and osteoprotegerin, and estrogen receptors play important roles in the inhibition of interleukin-6 synthesis by genistein in osteoblastic MC3T3-E1 cells.     

The effect of alkylresorcinol on the respiration and synthesis of nucleic acids and proteins in isolated thymocytes

The effect of the membranotropic agent alkylresorcinol 5C10 on the respiration, nucleic acid and protein synthesis in isolated thymocytes was studied. Within the 5C10 concentration range of 10(-7)-10(-5) M, the inhibition of respiration and incorporation of labelled precursors into thymocyte proteins and DNA was observed. In case of respiration and protein synthesis, a 50% inhibition was observed at alkylresorcinol concentrations of 10(-8) and 0.5.10(-5) M, respectively. The rate of 3H-thymidine incorporation into DNA progressively decreased already at 5C10 concentration of 10(-7) M. At 10(-6) M alkylresorcinol its inhibiting effect on DNA synthesis was about 30% and it did not change with a further rise in the inhibitor concentration up to 10(-5) M. In contrast, the rate of RNA synthesis significantly increased (ca. by 20%) within the alkylresorcinol concentration range of 10(-6)-10(-5) M. At 5C10 concentrations above 10(-5) M, the state of thymocytes in the preagglutination period appeared to be critical and was characterized by a dramatic inhibition of all the parameters under study. The experimental results suggest that alkylresorcinol 5C10 causes the inhibition of processes that are functionally coupled with biological membranes.     

重组白介素6-假单胞菌外毒素融合蛋白对正常BN大鼠骨髓粒系造血的作用

本文报道了重组白介素6-假单胞菌外毒素融合蛋白(IL-6-PE40)对正常BN大鼠骨髓粒系造血的体外效应。10ng/ml的IL-6-PE40对高表达IL-6受体的U266骨髓瘤细胞的蛋白质合成抑制率为50％,1000ng/ml则为100％。1～1000ng/mlIL-6-PE40对正常骨髓未纯化细胞的CFU-GM集落形成和DNA合成无明显抑制,但IL-6却具有一定的刺激效应。提示正常骨髓粒系祖细胞和骨髓细胞可能不表达IL-6受体,IL-6-PE40对粒系造血仍具有某些细胞毒作用,但被IL-6-PE40中的IL-6极大地削弱了。     

Investigation of benzene oxide in bone marrow and other tissues of F344 rats following metabolism of benzene in vitro and in vivo.

This study examines the initial activation of benzene, exploring key aspects of its metabolism by measurement of benzene oxide (BO) and BO-protein adducts in vitro and in vivo. To assess the potential influence of various factors on the production of BO, microsomes were prepared from tissues that were either targets of benzene toxicity, i.e. the bone marrow and Zymbal glands, or not targets, i.e. liver and kidneys, of control and acetone-treated F344 rats. No BO or phenol was detected in microsomal preparations of bone marrow or Zymbal glands (less than 0.007 nmol BO/mg protein and 0.7 nmol phenol/mg protein). On the other hand, BO and phenol were readily detected in preparations of liver and kidney microsomes and acetone pretreatment resulted in a 2-fold (kidney) increase or 3.7-fold (liver) increase in production of these metabolites. Initial rates of BO production in the liver isolates were 30 (control) to 50 (acetone-treated) times higher than in the corresponding kidney tissues. The estimated half-life of BO in bone marrow homogenates was 6.0 min and the second-order reaction rate constant was estimated to be 1.35 x 10(-3) l (g bone marrow)(-1) (h)(-1). These kinetic constants were used with measurements of BO-bone marrow adducts in F344 rats, receiving a single gavage dosage of 50-400 mg benzene (kg body weight)(-1) (McDonald, T.M., et al. (1994), Cancer Res. 54, 4907-4914), to predict the bone marrow dose of BO. Among the rats receiving 400 mg (kg body weight) (-1), a BO dose of 1.13 x 10(3) nM BO-h was estimated for the bone marrow, or roughly 40% of the corresponding blood dose predicted from BO-albumin adducts. Together these data suggest that, although BO is not produced at detectable levels in the bone marrow or Zymbal glands of F344 rats, BO is rapidly distributed via the bloodstream to these tissues where it may play a role in toxicity.     

Captopril inhibits ouabain-sensitive Na+/K+-ATPase

Captopril has been reported to inhibit ouabain-sensitive Na+/K+-ATPase activity in erythrocyte membrane fragments. We investigated the effect of captopril on two physiological measures of Na+/K+ pump activity: 22Na+ efflux from human erythrocytes and K+-induced relaxation of rat tail artery segments. Captopril inhibited 22Na+ efflux from erythrocytes in a concentration-dependent fashion, with 50% inhibition of total 22Na+ efflux at a concentration of 4.8 X 10(-3) M. The inhibition produced by captopril (5 X 10(-3) M) and ouabain (10(-4) M) was not greater than that produced by ouabain alone (65.3 vs. 66.9%, respectively), and captopril inhibited 50% of ouabain-sensitive 22Na+ efflux at a concentration of 2.0 X 10(-3) M. Inhibition by captopril of ouabain-sensitive 22Na efflux was not explained by changes in intracellular sodium concentration, inhibition of angiotensin-converting enzyme or a sulfhydryl effect. Utilizing rat tail arteries pre-contracted with norepinephrine (NE) or serotonin (5HT) in K+-free solutions, we demonstrated dose-related inhibition of K+-induced relaxation by captopril (10(-6) to 10(-4) M). Concentrations above 10(-4) M did not significantly inhibit K+-induced relaxation but did decrease contractile responses to NE, although not to 5HT. Inhibition of K+-induced relaxation by captopril was not affected by saralasin, teprotide or indomethacin. We conclude that captopril can inhibit membrane Na+/K+-ATPase in intact red blood cells and vascular smooth muscle cells. The mechanism of pump suppression is uncertain, but inhibition of ATPase should be considered when high concentrations of captopril are employed in physiological studies.     

The mitogenic activity of endothelin-1 on megakaryocytopoiesis in vitro

Endothelin (ET)-1 induces proliferation of various cells including smooth muscle cells, fibroblasts, glomerular mesangial cells, endothelial cells and osteoblasts. ET-1 also stimulates synthesis of interleukin (IL)-6 in endothelial and bone marrow stromal cells of rat. It is well known that IL-6 modulates megakaryocytopoiesis. Some studies have indicated that megakaryocytes express both ET receptors and they are targets for ET. Therefore we planned to examine the effects of ET-1 on the growth of normal megakaryocytic cells in rat bone marrow primary cell culture. Bone marrow cells were cultured at 37 degrees C, in an incubator atmosphere of 5% CO2 in air and 95% relative humidity for nine days. ET-1 at 10(-7), 10(-8 ) and 10(-11) M, and control with saline were added at the beginning of the experiment protocol. At each day, plasma clots were stained using direct-coloring thiocholin method for acetylcholinesterase activity. Although 10(-7) M ET-1 did not change the proliferation of megakaryocytic cells, this could be due to the presence of over crowded fibroblasts in the same environment. 10(-8) M ET-1 stimulated megakaryocytic cell growth to 234% over the control on the fifth day. ET-1 at a concentration of 10(-11) M also rised the megakaryocytic cell number significantly reaching up to 86% at the sixth day. Our results indicate that ET-1 may modulate the growth of megakaryocytic cells by an autocrine and/or paracrine action.     

Colony-stimulating factor-1 modulates alpha 2-macroglobulin receptor expression in murine bone marrow macrophages.

Primary cultures of murine bone marrow macrophages (BMMs) were prepared from marrow cell suspensions. These cells expressed specific receptors that recognized the transformed conformation of human alpha 2-macroglobulin (alpha 2M) generated by reaction with CH3NH2. alpha 2M receptor expression was regulated by colony-stimulating factor-1 (CSF-1). The BMMs were deprived of CSF-1 for 6 h and then treated with different concentrations of the purified cytokine. After 18 h, binding of 125I-alpha 2M-CH3NH2 was examined at 4 degrees C. Analysis of the saturation isotherms and Scatchard transformations indicated that the KD was not affected by CSF-1 (1.9-2.4 nM), whereas the maximum specific radioligand binding capacity (Bmax) was increased from 5.6 x 10(4) receptors/cell in the absence of CSF-1 to 2.2 x 10(5) and 2.6 x 10(5) receptors/cell for BMMs treated with 1,000 and 10,000 units/ml CSF-1, respectively. The difference in total cellular protein after exposure to different levels of CSF-1 for 18 h was small (1.50-1.92 ng/cell) and not statistically significant. A 6-12-h lag phase was identified between the time of CSF-1 exposure and increased alpha 2M receptor expression. Cycloheximide completely blocked the increase in alpha 2M receptor expression when added simultaneously with the CSF-1; greater than 50% inhibition was observed when the cycloheximide was added up to 8 h later. The RNA synthesis inhibitors, actinomycin D and daunomycin, prevented increased alpha 2M receptor expression when added up to 4 h after the CSF-1, but had no effect at 8 h. At 37 degrees C, uptake and digestion of 125I-alpha 2M-CH3NH2 was increased in BMMs treated with 1,000 units/ml CSF-1 for 18 h compared with untreated cells. These studies demonstrate that CSF-1 increases the expression of alpha 2M receptors in BMMs through a pathway that requires new RNA and protein synthesis. We hypothesize that increased alpha 2M receptor expression may play an important role in cellular growth and differentiation.     

Estradiol and raloxifene decrease the formation of multinucleate cells in human bone marrow cultures

Estrogen (E2) deficiency is responsible for increased bone turnover in the postmenopausal period, and it can be prevented by estrogen replacement therapy. The way estrogen acts on bone cells is not fully understood. Human bone marrow cell cultures may be a reliable model for studying the action of steroids on osteoclastogenesis in vitro. We examine the effects of estradiol and Raloxifene, a selective estrogen receptor modulator, on human primary bone marrow cells cultured for 15 days. 17beta-estradiol and Raloxifene significantly decreased the number of tartrate-resistant acid phosphatase multinucleate cells from osteoclast precursors on day 15. Estrogen receptor alpha (ER-alpha) mRNA was present in bone marrow mononuclear cells cultured for 5 days, but there was no estrogen receptor beta (ER-beta) mRNA, suggesting that this effect was mediated by ER-alpha. 15-day cultures no longer contained ER-alpha mRNA, suggesting that estrogen acts on early events of osteoclast differentiation. Finally, 10-8 M 17beta-estradiol has no effect on the release of IL-6 and IL-6-sr into the medium of marrow mononuclear cells cultured for 5 or 15 days. Osteoclast apoptosis was not affected by estradiol or Raloxifene after 15 days of culture under our conditions. In conclusion, we have shown that both estradiol and Raloxifene inhibit osteoclast differentiation in human bone marrow mononuclear cultures. The biological effect that can mimic in vivo differentiation could be mediated through ER-alpha.     

Effect of an anti-murine transferrin receptor-ricin A conjugate on bone marrow stem and progenitor cells treated in vitro

A monoclonal antibody with specificity for murine transferrin receptor was conjugated with the toxic A subunit of ricin. The dose range, specificity, and kinetics of inhibition of protein synthesis of the conjugate were determined on the murine T-lymphoma cell line, BW5147. When toxin was present throughout the period of culture, in vitro myeloid (CFUc) and erythroid (CFUe and BFUe) bone marrow colonies were inhibited by doses of conjugate comparable to those that inhibit protein synthesis in murine cell lines (IC50 of 5 X 10(-11)M). Bone marrow exposed briefly (30 min to 6 h) to anti-transferrin receptor antibody-ricin A conjugate was assayed for myeloid (CFUc) and erythroid (CFUe and BFUe) progenitors in vitro and for in vivo spleen colony formation (CFUs). Only CFUe were depleted by this pulse exposure, consistent with the higher frequency of proliferating cells and transferrin receptor expression in the CFUe population relative to other progenitors.     

Effects of urinary proteins from certain leukemics upon macromolecular synthesis and enzyme levels in bone marrow cultures.

Urinary proteins from human leukemic patients have been found to alter quantitatively macromolecular synthesis in primary mouse bone marrow cultures. Urinary protein-stimulated incorporation of [3H]uridine into RNA was found after 1 day of culture. Increased levels of adenine phosphoribosyltransferase and lysozyme were demonstrable at 3 and 5 days, respectively, with urinary protein-supplemented cultures. The incorporation of 3H-labeled deoxynucleosides into DNA was higher in the presence of urinary proteins after 2 days of culture. The rate of incorporation of [3H]deoxyuridine into DNA was strongly inhibited by 10(-5) M Methotrexate and 10(-6) M 5-fluorodeoxyuridine, however, the effect of urinary proteins on incorporation of [3H]uridine into RNA and lysozyme accumulation were not inhibited. Urinary proteins also stimulated the formation of "colonies" (groups of at least 30 cells) in media containing methylcellulose. This latter phenomenon was also not inhibited by 10(-5) M Methotrexate or 10(-6) M 5-fluorodeoxyuridine. The results of these studies are consistent with the postulate that in the presence of human urinary proteins, mouse bone marrow cells in culture proceed to a phenotype characteristic of circulating peripheral white cells.     

Prostaglandin-related bone resorption in cultured neonatal mouse calvaria: evaluation of biopotency of nonsteroidal anti-inflammatory drugs

The objective of this study was the development of an assay based on suppression of endogenous prostaglandin synthesis in cultured neonatal mouse calvaria for evaluation of the biopotency of nonsteroidal anti-inflammatory drugs in bone. In preliminary trials, osteolytic activity due to spontaneous prostaglandin production over a 72 h culture period was found highly variable, and could not be stabilized by addition of the common precursor arachidonic acid to the culture medium. Eventually, continuous exposure of mouse calvaria to moderate concentrations of thrombin (greater than or equal to 14 U/ml medium) proved to be satisfactory to achieve stable rates of bone resorption through continuous stimulation of prostaglandin synthesis from endogenous sources. Notably, the extent of net calcium release into the medium was highly reproducible in different experiments. As an example for possible applications of the bioassay, the ability of acemetacin to interfere with prostaglandin synthesis in bone, which had not been assessed before, was evaluated in a comparative assay with indomethacin and acetylsalicylic acid. While 1 X 10(-8) M acemetacin appeared to augment thrombin-induced bone resorption, as did 5 X 10(-6) M acetylsalicylic acid, a dose-dependent inhibition of calcium release was observed between 10(-7)-10(-5) M acemetacin. In this respect, the biopotency of indomethacin was 50 times higher than that of acemetacin and exceeded that of acetylsalicylic acid by a factor of more than 2000. These data could be useful for the appraisal of multiple effects of the investigated drugs on prostaglandin-related bone turnover.     

Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1-84) at 10(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo protein synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.