Biomedical subjects

S W Burchiel

Publications and source records attributed to S W Burchiel.

At least 19 recordsLinked to original sources

PubMed1997

Assessment of immunotoxicity by multiparameter flow cytometry.

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.

Allergens↗

PubMed1997

Depletion of glutathione by benzo(a)pyrene metabolites, ionomycin, thapsigargin, and phorbol myristate in human peripheral blood mononuclear cells.

Previous studies in this laboratory have shown that polycyclic aromatic hydrocarbons (PAHs) alter Ca2+ homeostasis and inhibit activation of both B and T lymphocytes obtained from rodents and humans. In the present studies, we demonstrate that alpha-naphthoflavone (ANF), an inhibitor of cytochrome P4501A activity, reduced the Ca2+ elevation produced by BaP in human peripheral blood mononuclear cell (HPBMC) lymphocytes. These results suggested that BaP metabolites may play a role in intracellular Ca2+ homeostasis in human lymphocytes. Reactive oxidative intermediates of BaP produced in HPMBC are known to be highly carcinogenic and have also been shown to be immunosuppressive. We examined the effects of benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), benzo(e)pyrene (BeP), and anthracene, as well as certain BaP metabolites, on the levels of intracellular Ca2+ and glutathione in HPBMC. While BaP, DMBA, BeP, and anthracene did not cause a statistically significant decrease in GSH in HPBMC at concentrations of 1 or 10 microM following a 6-, 48-, or 72-hr exposure, reactive BaP metabolites including 4,5-epoxide BaP and 7,8-diol-9,10-epoxide BaP consistently produced a 20-30% depletion of glutathione in HPBMC following a 6-hr treatment period. These BaP metabolites also elevated intracellular Ca2+ in HPBMC during a 6-hr incubation. Results of these experiments suggest that metabolism of BaP to certain epoxide metabolites may be responsible for sulfhydryl damage leading to transient GSH depletion and Ca2+ elevation. These results are consistent with the hypothesis that sulfhydryl damage by certain PAH metabolites may lead to altered Ca2+ homeostasis, leading to inhibition of cell activation and proliferation in HPBMC.

Benzo(a)pyrene↗

PubMed1997

Characterization of intracellular calcium responses produced by polycyclic aromatic hydrocarbons in surface marker-defined human peripheral blood mononuclear cells.

Previous studies have demonstrated that polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP) and 7,12-dimethybenz[a]anthracene (DMBA), and possibly 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), may exert their immunosuppressive effects by altering intracellular Ca2+ homeostasis in lymphocytes. In these studies, we examined the effects of two immunosuppressive PAHs (BaP and DMBA), two nonimmunosuppressive PAHs (benzo[e]pyrene (BeP) and anthracene (ANTH)), and TCDD on intracellular Ca2+ levels in surface marker-defined human peripheral blood mononuclear cells (HPBMC). BaP and DMBA, but not BeP and ANTH, were found to produce a time-dependent increase in intracellular Ca2+ with maximal effects achieved following 42- to 66-hr exposures. In a series of studies with HPBMC obtained from 10 donors exposed in vitro for 42 hr, BaP and DMBA were found to produce a significant increase in Ca2+ in CD3+ T cells, CD19+ B cells, and CD14+ monocytes. BeP and ANTH did not produce a statistically significant increase in Ca2+ in the group of donors, but occasionally produced an apparent nonspecific elevation of Ca2+ in HPBMC from individual donors. Interestingly, TCDD produced a small and statistically significant increase in Ca2+ only in B cells analyzed for the pooled 10 donors. Certain BaP metabolites, such as the 7,8-dihydrodiol and the 7,8-diol-9,10-epoxide, were more effective in elevating Ca2+ in HPBMC lymphocytes at 20 hr than was BaP. These results demonstrate in normal HPBMC that immunosuppressive PAHs alter intracellular Ca2+ homeostasis in B cells, T cells, and monocytes, and suggest that P450 metabolism may play an important role in the immunotoxicity of certain PAHs.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

Carcinogenic polycyclic aromatic hydrocarbons increase intracellular Ca2+ and cell proliferation in primary human mammary epithelial cells.

Previous studies have shown that polycyclic aromatic hydrocarbons (PAHs) mobilize intracellular Ca2+ in human T cells by inositol trisphosphate-dependent mechanisms resulting from activation of phospholipase C-gamma by SRC-related protein tyrosine kinases, thereby mimicking antigen-receptor activation. Ca2+ appears to play an important second messenger role in growth factor control of cell proliferation in human mammary epithelial cells (HMEC), such as the epidermal growth factor receptor pathway. The purpose of the present studies was to determine if PAHs are able to increase intracellular Ca2+ in primary cultures of HMEC and increase cell proliferation. Two carcinogenic and two non-carcinogenic PAHs were tested for their ability to increase intracellular Ca2+ in HMEC. The carcinogenic PAHs dimethylbenz[a]anthracene (DMBA) and benzo[a] pyrene (BaP) were able to cause Ca2+ elevation in HMEC at early time points (2 h) and caused sustained alterations in Ca2+ homeostasis (18 h). DMBA showed maximal effects at early time points (2 h), while BaP showed maximal effects on sustained Ca2+ (18 h). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent dioxin and tumor promoter, produced maximal Ca2+ elevation at 2 h, with a return to near baseline levels by 6 h. The non-carcinogenic PAHs benzo[e]pyrene and anthracene did not significantly alter intracellular Ca2+ at any time point. alpha-Naphthoflavone significantly reduced the Ca2+ response induced by BaP treatment, but not by DMBA or TCDD, suggesting that P450 1A or 1B metabolism of BaP may be important in the sustained Ca2+ elevating response. In evaluating the effects of BaP on HMEC proliferation, BaP was found to increase the number of cells recovered after 4 days in culture in the absence or presence of various concentrations of epidermal growth factor. These studies provide initial evidence that Ca2+ signaling may be associated with mitogenesis in HMEC, which may play a role in tumor promotion and progression produced by PAHs.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1996

Human T cells are highly sensitive to suppression of mitogenesis by polycyclic aromatic hydrocarbons and this effect is differentially reversed by alpha-naphthoflavone.

The immunosuppressive effects of polycyclic aromatic hydrocarbons (PAHs) on immune responses in rodents, both in vivo and in vitro, have been widely documented. However, few studies have addressed the immunotoxicity of PAHs in the human system. In this report, we examined the toxic effects of nine different PAHs on human peripheral blood T cell mitogenesis. We found that benzo(a)pyrene (BaP), 3-methylcholanthrene (3-MC), and 7,12-dimethylbenz(a)anthracene (DMBA) were highly immunotoxic in the human system, while dibenz(a,c)anthracene (DAC) and dibenz(a,h)anthracene (DAH) were of intermediate toxicity, 9,10-dimethylanthracene (DMA), benzo(e)pyrene (BeP), and benz(a)anthracene (BA) were mildly immunotoxic, and anthracene (ANTH) had no measureable toxicity at the concentrations tested. Our results using human lymphocytes differed from previous studies in rodents, in that BaP and 3-MC were the most immunotoxic PAHs in the human mitogenesis assay, while DMBA has long been regarded as the PAH that is most potently toxic to rodent T cell responses. We also showed that alpha-naphthoflavone (ANF), which functions as both an Ah receptor antagonist and an inhibitor of cytochrome P450 activity, was able to block the suppressive effects of both BaP and DMBA, but not 3-MC. This suggests that the immunotoxicity of 3-MC may be mediated through a different mechanism than BaP or DMBA. Addition of four different BaP metabolites directly to cultures of human mononuclear cells showed that the 7,8-dihydrodiol metabolite was the most toxic, and that this toxicity could be completely blocked by equimolar and 10-fold greater concentrations of ANF. The 7,8-dihydrodiol metabolite was probably further metabolized to the 7,8-diol epoxide, the toxicity of which could not be effectively reversed by ANF. The 4,5-epoxide metabolite was apparently cytotoxic at high concentrations (10 microM), while the 7-hydroxy metabolite had no overtly negative effects on proliferation.

Adult↗

PubMed1996

Inhibition of sarco-endoplasmic reticulum calcium ATPases (SERCA) by polycyclic aromatic hydrocarbons: lack of evidence for direct effects on cloned rat enzymes.

Previous studies have demonstrated that immunosuppressive polycyclic aromatic hydrocarbons (PAHs) disrupt Ca2+ homeostasis leading to inhibition of the Ca(2+)-dependent pathways of T cell and B cell activation. The sustained Ca(2+)-elevation produced by immunosuppressive PAHs may result from the inhibition of Ca(2+)-ATPases in the endoplasmic reticulum (SERCA). The purpose of the present study was to determine whether PAHs directly inhibit cloned SERCA enzymes, and whether there is any selectivity for certain isoforms. PAHs were examined for their effects on purified cloned rat SERCA enzymes, including SERCA1, SERCA2a and SERCA3, transiently expressed in human embryonic kidney (HEK) cells. Results showed that known SERCA inhibitors, thapsigargin (100 nM) and 2,5-di(t-butyl)-1,4-benzohydroquinone (10 mumol), completely inhibited all rat SERCA isoforms, whereas 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, benzo(e)pyrene, anthracene, 3-methyl-cholanthrene, 9,10-dimethylanthracene and benz(a)anthracene at concentrations as high as 10 mumol appeared to have little inhibitory effect on any of the SERCA. The results demonstrating that PAHs do not inhibit cloned SERCA enzymes suggest that metabolism may be required for PAH-induced inhibition, or that other cellular elements, not present in the HEK transfection model, may be required for activity.

Adenosine Triphosphate↗

PubMed1996

Molecular mechanisms of toxicant-induced immunosuppression: role of second messengers.

Changes in immunocompetence following chemical exposure have been established for a wide variety of unrelated agents. For the vast majority of immunotoxic compounds thus far identified, disruption of normal immune function is clearly mediated through direct interactions between the agent, or its metabolite, and immunocompetent cells. Regardless of whether this interaction occurs at the level of the cell membrane or at intracellular sites, basic regulatory processes mediated by second messengers are often altered. These alterations can ultimately result in immunologic dysfunction, which is most often manifested as immunosuppression. The specific disruptions in intracellular signaling produced by a number of immunotoxic compounds have now been identified, leading to a basic understanding of their molecular mechanism of action. Equally important, through the application of these agents as biological probes, new insights have been gained pertaining to which intracellular processes control which cellular functions within various populations of immunocompetent cells.

Adenylyl Cyclase Inhibitors↗

PubMed1995

Inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCA) by polycyclic aromatic hydrocarbons in HPB-ALL human T cells and other tissues.

Polycyclic aromatic hydrocarbons (PAHs) are immunosuppressive chemicals found in the environment that have been shown to disrupt intracellular Ca2+ homeostasis and Ca(2+)-dependent signaling in human and murine lymphocytes. Many PAHs produce a rapid and sustained increase in intracellular free Ca2+ in lymphocytes. The mechanism of persistent Ca2+ perturbation remains undefined. In the present studies, ATP-dependent 44Ca2+ uptake into vesicles prepared from a 15,000g supernatant of HPB-ALL human T cell lysates was significantly inhibited by 0.1, 1, and 10 microM concentrations of the immunotoxic PAHs 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BAP), benz[a]anthracene, and 9,10-dimethylanthracene, but not by the less immunotoxic compounds anthracene (ANT) and benzo[e]pyrene (BEP). Ca(2+)-ATPase catalytic activity was determined by quantitating hydrolysis of ATP in the presence or absence of PAHs, with known ATPase inhibitors included as controls. Formation of inorganic phosphate was significantly decreased (> 65% of control at 10 microM) by DMBA and BAP, whereas ANT and BEP caused only a slight reduction in activity (10% of control at 10 microM). Anthracene partially reversed the inhibitory effect of DMBA and BAP on ATP hydrolysis when agents were coincubated. Both DMBA and BAP, but not ANT and BEP, inhibited the activity of all known SERCA-type Ca(2+)-ATPases, while not affecting either Na+, K(+)-ATPase activity or plasma membrane Ca(2+)-ATPase activities. These results demonstrate that immunotoxic and carcinogenic polycyclic aromatic hydrocarbons have a thapsigargin-like effect in human lymphocytes and SERCA-containing tissues from various species. Inhibition of SERCA activity may play an important role in altered Ca2+ homeostasis in lymphocytes and other tissues.

Adenosine Triphosphate↗

PubMed1995

Role of alterations in Ca(2+)-associated signaling pathways in the immunotoxicity of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. The effects of PAHs on the immune system of various animals and models have been studied for at least 30 yr. Despite these efforts, the mechanism or mechanisms by which PAHs exert their effects on the immune system are still largely unknown. During recent years, the molecular events associated with lymphocyte activation and receptor-mediated signaling have become increasingly clear. Substantial progress has been made in understanding the molecular and cellular bases for toxicant-induced immune cell injury. Understanding mechanisms of drug or chemical effects on the immune system is an important area of research in the field of immunotoxicology, and indeed in all fields of toxicology. Mechanistic toxicology plays an important role in risk assessment and extrapolation of potential human health effects. In this review, we have summarized recent evidence that has examined the effects of PAHs on the immune system of animals and humans. In particular, we have focused on the effects of PAHs on cell signaling in lymphoid cells and have examined the hypothesis that PAHs alter lymphocyte activation via calcium-dependent mechanisms. Previously published reports are discussed, and new data obtained with murine B cells and cell lines are presented demonstrating the relationship between alterations in intracellular calcium and immune dysregulation. These data demonstrate a strong association between PAH-induced alterations in B- and T-lymphocyte activation and changes in calcium homeostasis.

Animals↗

PubMed1994

Inhibition of humoral immunity and mitogen responsiveness of lymphoid cells following oral administration of the heterocyclic food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to B6C3F1 mice.

In these studies, the food promutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was evaluated for its immunotoxicity in B6C3F1 mice following oral 5-day dosing at total doses of 50 and 150 mg/kg. Results indicated that PhIP produced a dose-dependent suppression of the humoral immune response of spleen cells to sheep erythrocytes, with a 50% decrease in the number of PFC detected at the 150 mg/kg dose of PhIP. A 40-90% inhibition of the phytohemagglutinin (PHA) response of spleen cells, mesenteric lymph nodes (MLNs), and Peyer's patch (PP) lymphocytes was seen in the treatment groups. The lipopolysaccharide (LPS) response was somewhat more variable and less affected with 20-30% inhibition observed in the spleen and PPs, whereas PhIP increased the LPS response in the MLNs. There was no effect of PhIP on cell recovery or viability in any of the treatment groups. Flow cytometry analysis revealed a depletion of T cells (Thy 1.2+ cells) and a slight increase in B cells (Ly5+ cells) in the PPs. The percentage of B and T cells present in the spleen and MLNs was unaffected by PhIP. These results demonstrate that the oral administration of PhIP produces immunotoxicity to mice, especially to lymphoid tissues present in the GI tract (i.e., PPs), and demonstrates that T cell mitogen (PHA) responses in PPs are the most sensitive indicator of PhIP-induced immunotoxicity.

Administration, Oral↗

PubMed1994

Polycyclic aromatic hydrocarbons decrease intracellular glutathione levels in the A20.1 murine B cell lymphoma.

Previous studies in this laboratory have shown that polycyclic aromatic hydrocarbons (PAHs) inhibit lymphocyte activation and alter intracellular Ca2+ homeostasis. Other investigators have demonstrated that intracellular Ca2+ may increase in lymphocytes following exposure to chemical oxidants or ionizing radiation. Cellular oxidants produce both a rise in intracellular Ca2+ and a decrease in intracellular levels of glutathione (GSH) in numerous cells and tissues. Therefore, the purpose of the present study was to determine whether PAHs alter intracellular levels of glutathione in lymphocytes. Using different, well-established glutathione assays, it was demonstrated in the A20.1 murine B lymphoma that PAHs induce a transient decrease in intracellular glutathione. A 10-25% decrease in reduced GSH was produced by benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, benz(a)anthracene, and anthracene within 2-4 hr of exposure. Benzo(e)pyrene did not alter intracellular levels of glutathione in A20.1 cells. We conclude that glutathione depletion may contribute to cell injury in lymphocytes exposed to PAHs.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1994

Persistence of calcium elevation in the HPB-ALL human T cell line correlates with immunosuppressive properties of polycyclic aromatic hydrocarbons.

The immunosuppressive synthetic methylated polycyclic aromatic hydrocarbon (PAH), 7,12-dimethylbenz[a]anthracene (DMBA), has been shown to cause both an immediate and a sustained elevation of free intracellular calcium (Ca2+) in human T cells. In the present studies, a series of anthracene- and pyrene-based PAHs were tested for rapid (3 min) and sustained (4 hr) Ca2+ mobilization in the HPB-ALL human T cell line measured by flow cytometry using Fluo-3 as a Ca2+ indicator. Immunosuppressive PAHs produced a sustained Ca2+ elevation for at least 4 hr, while weakly immunosuppressive PAHs caused only a transient increase in Ca2+. The immunosuppressive PAHs, DMBA, benzo[a]pyrene, dibenz[a,h]anthracene, and 9,10-dimethylanthracene, produced a sustained increase in intracellular Ca2+ in HPB-ALL cells. Those PAHs with moderate to minimal immunosuppressive properties (i.e., dibenz[a,c]anthracene, benz[a]anthracene, benzo[e]pyrene, and anthracene) produced small and transient Ca2+ mobilization responses in HPB-ALL cells. It appeared that methylation of anthracene at the 9,10-positions increased the duration of Ca2+ mobilization, whereas the addition of a benzene group in the "a" position was associated with a transient increase in Ca2+ levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, partially inhibited the rapid and sustained PAH-induced Ca2+ mobilization responses, while the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, had essentially no effect on PAH-induced Ca2+ elevation. It appears that the action of PAHs on PTKs is important in the rapid Ca2+ response of human T cells. However, additional biochemical mechanisms appear to be responsible for the sustained elevation of Ca2+ produced by PAHs in T cells. The results of these studies demonstrate that persistent elevation of intracellular Ca2+ by PAHs correlates with their known immunosuppressive properties.

Benzo(a)pyrene↗

PubMed1994

Analysis of radiolabeled CHO cell-derived rHuGM-CSF pharmacokinetics and biodistribution in rhesus monkeys following intravenous and subcutaneous injection.

The purpose of these studies was to examine the biodistribution and pharmacokinetics of radiolabeled human CHO cell-derived rHuGM-CSF in normal Rhesus monkeys (Macaca mulatta) following intravenous (i.v.) and subcutaneous (s.c.) injection. A dual radioisotope tracer technique was utilized to monitor the behavior of rHuGM-CSF in vivo. Recombinant HuGM-CSF was radiolabeled with I-123 (a 13.2 h half-life, 140 KeV pure gamma emitting radionuclide detected using gamma scintigraphic imaging) using a mild chloramine T reaction. A separate preparation of rHuGM-CSF radiolabeled with S-35 methionine by bioincorporation in tissue culture was mixed with the I-123-labeled protein, permitting comparison of data obtained from the two radiolabels. Two dose levels of rHuGM-CSF were used for i.v. bolus (15 and 300 micrograms/kg) and s.c. (10 and 100 micrograms/kg) studies. The results of these studies demonstrated that the co-administered I-123 rHuGM-CSF and S-35 rHuGM-CSF followed similar blood elimination kinetics after i.v. or s.c. injection. Following i.v. bolus injection, rHuGM-CSF was found to rapidly distribute to all central body cavity high blood flow organs, followed by rapid uptake in the kidneys and elimination in the urine. There were no differences in the pharmacokinetic values obtained for I-123- and S-35-labeled rHuGM-CSF nor for the two dose levels examined. Following, s.c. injection, I-123- and S-35-labeled rHuGM-CSF were found to reach maximal plasma levels after approximately 16 h. The primary route of elimination was the urine. Monkeys previously exposed to rHuGM-CSF were found to have circulating antibodies to rHuGM-CSF. Studies in these animals revealed a significantly altered distribution and clearance of radiolabeled rHuGM-CSF, with the majority of the injected activity being cleared by the liver.

Animals↗

PubMed1993

7,12-Dimethylbenz[a]anthracene activates protein-tyrosine kinases Fyn and Lck in the HPB-ALL human T-cell line and increases tyrosine phosphorylation of phospholipase C-gamma 1, formation of inositol 1,4,5-trisphosphate, and mobilization of intracellular calcium.

Previous studies have shown that the immunosuppressive and carcinogenic polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) impairs Ca(2+)-dependent transmembrane signaling in human and murine lymphocytes. The purpose of the present studies was to analyze potential mechanisms of immunosuppression by DMBA and to examine effects on Ca2+ homeostasis and antigen-receptor signaling in human T cells. DMBA produced a rapid and sustained increase in Ca2+ levels in HPB-ALL cells by release of cytoplasmic Ca2+. DMBA also inhibited anti-CD3/CD4 mobilization of Ca2+ in HPB-ALL cells, with half-maximal inhibition occurring at approximately 4 hr. Thus, the kinetics for initial Ca2+ mobilization and inhibition of the anti-CD3/CD4 response differed. The rapid rise in intracellular Ca2+ induced by DMBA alone was accompanied by a rapid but transient increase in inositol 1,4,5-trisphosphate and tyrosine phosphorylation of phospholipase C-gamma 1. The pattern of tyrosine phosphorylation induced by DMBA in HPB-ALL cells was remarkably similar to that induced by anti-CD3/CD4 activation. Thus, DMBA-induced phosphorylation may mimic antigen-receptor activation in T cells, which may lead to alterations in antigen responsiveness. The mechanism of DMBA-induced tyrosine phosphorylation of phospholipase C-gamma 1 may have been due to an increase in protein-tyrosine kinase activity, since it was found that DMBA produced a > 2-fold increase in the activity of the T-cell receptor-associated Src-family kinases Fyn and Lck. The kinetics of activation of protein-tyrosine kinases demonstrated that Fyn activity was increased within 10 min of exposure to DMBA, whereas maximal Lck activation required 30 min. Thus, it is likely that the Fyn kinase or other protein-tyrosine kinases may be responsible for the early tyrosine phosphorylation of phospholipase C-gamma 1, which results in inositol 1,4,5-trisphosphate release and mobilization of intracellular Ca2+.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1993

DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma.

The mechanism by which 7,12-dimethylbenz[a]anthracene (DMBA) produces cytotoxicity in lymphocytes was investigated in these studies using the murine A20.1 B cell lymphoma. Results show that in vitro exposure of these cells to 10-30 microM DMBA for 4 hr produced an increase in intracellular Ca2+, DNA fragmentation, and subsequent cell death. Elevation of Ca2+ and DNA fragmentation induced by DMBA were greatly pronounced when the A20.1 cells were exposed at high cell density (10(7) cells/ml). DMBA-induced DNA fragmentation and cell death were inhibited by coexposure of A20.1 cells to a calcium chelator (EDTA), a general nuclease and polymerase inhibitor (aurintricarboxylic acid), and a protein synthesis inhibitor (cycloheximide). These agents have been previously shown to inhibit apoptosis in lymphocytes and other cells exposed to chemical agents. We also found that cyclosporin A, an inhibitor of Ca(2+)-dependent pathways of T and B cell activation, prevented apoptosis in the A20.1 cell line. These results demonstrate that DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma by Ca(2+)-dependent pathways. The increased sensitivity of A20.1 at high cell density to Ca2+ elevation and DNA fragmentation suggests that cell to cell interactions may also be important in this process.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1992

DMBA-induced cytotoxicity in lymphoid and nonlymphoid organs of B6C3F1 mice: relation of cell death to target cell intracellular calcium and DNA damage.

The purpose of these studies was to evaluate the effects of 7,12-dimethylbenz[a]anthracene (DMBA) on intracellular free Ca2+ and DNA fragmentation in lymphoid cells obtained from the spleen, thymus, and Peyer's patches (PPs) of female B6C3F1 mice. Previous studies in our laboratories have shown that DMBA is cytotoxic to these lymphoid organs and that calcium homeostasis may be impaired following DMBA treatment. The results of the present studies show that a daily oral 14-day exposure of mice to DMBA produced a dose-dependent decrease in the number of viable cells recovered from the spleen, PPs, and thymus. Intracellular levels of Ca2+ were elevated in the spleen and PPs of mice receiving 140 mg/kg of DMBA. Extensive DNA fragmentation was detected in cells obtained from the spleen and PPs, as well as from the thymus. The thymus and PPs demonstrated DNA fragmentation at significantly lower doses of DMBA (42 mg/kg) than did the spleen (140 mg/kg). While cells obtained from the thymus did not demonstrate an elevation in Ca2+ produced by DMBA, in vitro exposure of isolated thymocytes to 3-30 microM DMBA for 4 hr produced significant elevation of intracellular Ca2+. A "ladder-like" pattern of DNA fragmentation was seen by agarose gel electrophoresis of DNA obtained from thymus cells treated with DMBA in vitro, suggesting DNA degradation by endonucleases. Collectively, these studies suggest that DMBA produces lymphotoxicity through an apoptosis-like mechanism involving fragmentation of genomic DNA by Ca(2+)-activated enzymes.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1992

Covalent binding of 7,12-dimethylbenz[a]anthracene to lymphoid and nonlymphoid tissues following oral administration to B6C3F1 mice.

Previous studies have shown that 7,12-dimethylbenz[a]anthracene (DMBA) is cytotoxic to various murine lymphoid tissues, including the spleen, thymus, mesenteric lymph nodes (MLNs), and Peyer's patches (PPs). In the present studies, we measured the amount of covalent binding of [3H]DMBA to lymphoid and nonlymphoid tissues and correlated these findings with the overall levels of [3H]DMBA (and derived substances) present in various tissues following a single oral administration to mice. Results show that [3H]DMBA was taken up relatively rapidly from the GI tract and that it was nearly completely eliminated within 24 hr via the feces. Peak plasma levels were obtained approximately 6 hr after gavage, and most organs (including brain, heart, liver, lung, kidney, spleen, and thymus) achieved their peak level of DMBA at this time. Maximal concentrations of DMBA were detected in gut-associated lymphoid tissues (i.e., PPs and MLNs) at 4 hr, during which time covalent binding of [3H]DMBA was also maximal. The time course for covalent binding was different in the liver, lung, thymus, and spleen, peaking at 6-12 hr. The amount of covalent binding of [3H]DMBA and derived metabolites in the spleen was more than twice that seen in the other tissues examined. Since the spleen has previously been found to be less sensitive to DNA fragmentation induced by DMBA than the PPs, these results suggest that covalent binding may not be the primary determinant of lymphotoxicity in these organs.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1992

Inhibition of calcium-dependent pathways of B-cell activation by DMBA.

The purpose of the experiments described in these studies was to determine the effects of 7,12-dimethylbenz[a]anthracene (DMBA) on B-cell activation produced by anti-IgD antibodies and interleukin-4 (IL-4). B and T cells are known to share many of the same biochemical pathways for cell activation by mitogen and antigen receptors. Previous studies in this laboratory have shown that DMBA inhibits mitogen-induced Ca2+ mobilization in murine and human T cells and produces an increase in intracellular Ca2+ in resting cells. The results of the present studies demonstrate that DMBA increases Ca2+ in resting B cells and inhibits B cell activation produced by anti-IgD antibodies, as measured by mobilization of free intracellular Ca2+ and [3H]thymidine incorporation. The proliferative response of B cells to insolubilized anti-IgD was suppressed only when cells were preexposed to DMBA. In contrast, IL-4 pathways of B-cell activation were insensitive to inhibition by DMBA, even when cells were preexposed. The induction of Class II MHC antigen (Ia) antigens on B cells by IL-4 was also found to be insensitive to DMBA treatment. These results suggest that DMBA suppresses only Ca(2+)-dependent pathways of B cell activation and indicate that altered Ca2+ homeostasis may be responsible for immunosuppression induced by this agent.

9,10-Dimethyl-1,2-benzanthracene↗