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

doi:10.1016/0041-008X(92)90016-L

Toxicology and Applied Pharmacology

Volume 113, Issue 1, March 1992, Pages 126-132

https://doi.org/10.1016/0041-008X(92)90016-L Get rights and content

Abstract

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 μm 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 Ca²⁺-activated enzymes.

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Induction of intracellular calcium concentration by environmental benzo(a)pyrene involves a β2-adrenergic receptor/adenylyl cyclase/Epac-1/inositol 1,4,5-trisphosphate pathway in endothelial cells
2012, Journal of Biological Chemistry
Citation Excerpt :
This binding mode most likely allows extensive interactions of B(a)P with Phe-193, Tyr-199, Phe-289, and Phe-290 on β2ADR and partially overlaps the carazolol binding site (Fig. 7C). Exposure to PAHs is well known to trigger an early increase of [Ca2+]i, which is thought to participate to the up-regulation of various genes targeted by the PAHs-activated transcription factor AhR (5, 50). The aim of the present study was to characterize the initial events implicated in this calcium mobilization by PAHs.
Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (B(a)P) are widely distributed environmental contaminants, known as potent ligands of the aryl hydrocarbon receptor (AhR). These chemicals trigger an early and transient increase of intracellular calcium concentration ([Ca²⁺]_i), required for AhR-related effects of PAHs. The mechanisms involved in this calcium mobilization were investigated in the present study. We demonstrated that B(a)P-mediated [Ca²⁺]_i induction was prevented in endothelial HMEC-1 cells by counteracting β2-adrenoreceptor (β2ADR) activity using pharmacological antagonists, anti-β2ADR antibodies, or siRNA-mediated knockdown of β2ADR expression; by contrast, it was strongly potentiated by β2ADR overexpression in human kidney HEK293 cells. B(a)P was shown, moreover, to directly bind to β2ADR, as assessed by in vitro binding assays and molecular modeling. Pharmacological inhibition and/or siRNA-mediated silencing of various signaling actors acting downstream of β2ADR in a sequential manner, such as G protein, adenylyl cyclase, Epac-1 protein, and inositol 1,4,5-trisphosphate (IP₃)/IP₃ receptor, were next demonstrated to prevent B(a)P-induced calcium signal. Inhibition or knockdown of these signaling elements, as well as the use of chemical β-blockers, were finally shown to counteract B(a)P-mediated induction of cytochrome P-450 1B1, a prototypical AhR target gene. Taken together, our results show that B(a)P binds directly to β2ADR and consequently utilizes β2ADR machinery to mobilize [Ca²⁺]_i, through activation of a G protein/adenylyl cyclase/cAMP/Epac-1/IP₃ pathway. This β2ADR-dependent signaling pathway activated by PAHs may likely be crucial for PAH-mediated up-regulation of AhR target genes, thus suggesting a contribution of β2ADR to the health-threatening effects of these environmental pollutants.
Low-dose synergistic immunosuppression of T-dependent antibody responses by polycyclic aromatic hydrocarbons and arsenic in C57BL/6J murine spleen cells
2010, Toxicology and Applied Pharmacology
Polycyclic aromatic hydrocarbons (PAHs) and arsenic are both environmental agents that are known to have significant immunotoxicity. Previous studies have shown that PAH exposure of spleen cells in vitro produces significant immune suppression of humoral immunity, especially when P450 activation products are examined. Exposure to arsenic, particularly sodium arsenite, has also been found to be suppressive to antibody responses in vitro and in vivo. The purpose of the present studies was to examine the immunotoxicity of PAHs and arsenite following coexposures with the theory being that the agents may exert synergistic actions, which might be based on their different mechanisms of action. Spleen cells were isolated from male C57BL/6J wild-type mice and treated with PAHs and/or arsenic (arsenite or arsenate). Immunotoxicity assays were used to assess the T-dependent antibody response (TDAR) to sheep red blood cells (SRBCs), measured by a direct plaque-forming cell (PFC) assay. Cell viability was measured by trypan blue staining. Spleen cell viability was not altered following 4 days of PAH and/or arsenic treatment. However, the TDAR demonstrated suppression by both PAHs and arsenic in a concentration-dependent manner. p53 was also induced by NaAsO₂ (As³⁺) and PAHs alone or in combination. The PAHs and their metabolites investigated included benzo[a]pyrene (BaP), BaP-7,8-diol, BaP-7,8-diol-9,10-epoxide (BPDE), 7,12-dimethylbenz[a]anthracene (DMBA), DMBA-3,4-diol, dibenzo[a,l]pyrene (DB[a,l]P). PAH metabolites were found to be more potent than parent compounds in producing immunosuppression and inducing p53 expression. Interestingly, DB[a,l]P, a potent carcinogenic PAH not previously characterized for immunotoxicity, was also found to be strongly immunosuppressive. Arsenite (NaAsO₂, As³⁺) was found to produce immunosuppression at concentrations as low as 0.5 μM and was immunosuppressive at a 10-fold lower concentration than sodium arsenate (Na₂HAsO₄, As⁵⁺). Coexposure of spleen cell cultures to PAHs and As³⁺, both at individual low-effect concentrations, was found to produce profound suppression of the TDAR demonstrating synergy between these two chemical classes of agents.
Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice
2007, Toxicology Letters
Cadmium immunotoxicity in rodents is primarily characterized by marked thymic damage and splenomegaly. To understand the toxicity of Cd on lymphoid cells in vivo, a single dose of Cd as CdCl₂ (1.8 mg/kg, i.p.) was administered to male BALB/c mice and cytotoxicity (MTT assay), oxidative stress indicators (glutathione, reactive oxygen species) and apoptotic markers (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) were assessed in thymic and splenic single cell suspensions, at various time intervals. Lowering of body weight gain and cellularity and a loss in cell viability was seen in the Cd treated mice. The earliest significant increase in ROS at 18 h, followed by mitochondrial membrane depolarization, caspase-3 activation and GSH depletion at 24 h in spleen and later at 48 h in thymus, strongly implicate the possible involvement of ROS. A pronounced inhibition of cell proliferative response at 48 h and 72 h may also be linked to Cd induced apoptosis. The morphological alterations including thymic cortical cell depletion and an increase in red pulp with diminished white pulp in spleen were observed at 48 h and beyond.
The splenic cells appeared more susceptible than thymus cells to the adverse effects of Cd. The present study, therefore, demonstrates potentiation of oxidative stress followed by mitochondrial-caspase dependent apoptotic pathway. This may, in part, be responsible for causing suppression of cell proliferative response, thymic atrophy and splenomegaly.
The effects of polycyclic aromatic hydrocarbons on the immune system of fish: A review
2006, Aquatic Toxicology
Polycyclic aromatic hydrocarbons are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. This review summarizes the diverse literature on the effects of these pollutants on innate and acquired immunity in fish and the mechanism of PAH-induced immunotoxicity. Among innate immune parameters, many authors have focused on macrophage activities in fish exposed to polycyclic aromatic hydrocarbons. Macrophage respiratory burst appears especially sensitive to polycyclic aromatic hydrocarbons. Among acquired immune parameters, lymphocyte proliferation appears highly sensitive to polycyclic aromatic hydrocarbon exposure. However, the effects of polycyclic aromatic hydrocarbons on both specific and non-specific immunity are contradictory and depend on the mode of exposure, the dose used or the species studied. In contrast to mammals, fewer studies have been done in fish to determine the mechanism of polycyclic aromatic hydrocarbon-induced toxicity. This phenomenon seems to implicate different intracellular mechanisms such as metabolism by cytochrome P4501A, binding to the Ah-receptor, or increased intracellular calcium. Advances in basic knowledge of fish immunity should lead to improvements in monitoring fish health and predicting the impact of polycyclic aromatic hydrocarbons on fish populations, which is a fundamental ecotoxicological goal.
Dibenzo[a,l]pyrene induced DNA adduct formation in lung tissue in vivo
2005, Cancer Letters
Polycyclic aromatic hydrocarbons (PAHs) are environmental carcinogens present in the atmosphere from combustion sources such as cigarette smoke, diesel exhaust, residential heating processes, and industrial coke production. To date, dibenzo[a,l]pyrene (DBP) has been found to be the strongest tumor-initiating PAH ever tested in rodent skin and mammary tumor models. Here we show for the first time that systemic exposure to DBP causes DNA damage in mouse lung tissue. C57BL/6 mice were gavaged with 1, 5 or 20 mg DBP/kg body weight, daily for 10 days. Toxicity of DBP was revealed by a decrease in body and organ weight of mice while no apparent cell death was observed on P815 mastocytoma cells (allograft model) in vitro. However, treatment of P815 cells in vitro with the ultimate carcinogenic metabolite of DBP, the fjord region (−)-anti-11,12-diol 13,14-epoxide [(−)-anti-DBPDE], resulted in the total loss of cell viability. Lungs from the animals were removed and subjected to DBP-DNA adduct analysis. A dose dependent adduct formation was revealed by ³³P-postlabeling analysis of DNA from lung tissue. The majority of DNA adducts formed in lungs of mice after systemic exposure to DBP were contributed by (−)-anti-DBPDE. The data from this in vivo model are consistent with previous metabolic activation results obtained with DBP in human cells in culture.
3-Methylcholanthrene induces lymphocyte and phagocyte apoptosis in common carp (Cyprinus carpio L) in vitro
2004, Aquatic Toxicology
Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental pollutant that are known to be carcinogenic and immunotoxic. In mammals it was suggested that PAH compromise the immune system in part through the induction of programed cell death (apoptosis). In fish, no study has reported the importance of this physiological process in PAH-induced immunotoxicity. We have therefore investigated the capacity of 3-methylcholanthrene to induce lymphocyte and phagocyte apoptosis in carp. By three criteria (exposition of phosphatidylserine at the outer cell membrane, chromatin condensation and fragmentation, and decreased cell size) the data indicate that 3-methylcholanthrene (3-MC) treatment (from 20 to 200 μM) during 24 h produces apoptosis in both lymphocytes and phagocytes. In order to evaluate whether 3-MC induced apoptosis is related to the metabolic activation of 3-MC or 3-MC Ah-R binding, co-exposure experiments with 3-MC and alpha-naphtoflavone (a-NF), a compound that inhibits metabolic activation of 3-MC and 3-MC Ah-R binding were performed. While a-NF did not prevent 3-MC-induced apoptosis, the compound itself was found to be a strong inducer of apoptosis. There results might indicate that metabolic activation of 3-MC or 3-MC Ah-R binding is not causally linked to apoptosis. However, since 3-MC, a-NF and 3-MC + a-NF treatments produce the same sustained increase (3 h minimum) in intracellular calcium level, it is possible that this phenomenon is implicated in the induction of programmed cell death by these hydrocarbons.

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²: Supported in part by NIH Grant GM-41564.

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Toxicology and Applied Pharmacology

Abstract

References (24)

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

Toxicol. Appl. Pharmacol.

Inhibition of lymphocyte activation in splenic and gut-associated lymphoid tissues following oral exposure of mice to 7,12-dimethylbenz[a]anthracene

Toxicol. Appl. Pharmacol.

Persistent immunosuppression of humoral immunity produced by 7,12-dimethylbenz[a]anthracene (DMBA) in B6C3F1 mice: Correlation with changes in spleen cell surface markers detected by flow cytometry

Int. J. Immunopharmacol.

Alterations in mitogen-induced calcium mobilization and intracellular free calcium produced by 7,12-dimethylbenz[a]anthracene (DMBA) in the Jurkat human T cell line

Int. J. Immunopharmacol.

Suppression of local gut-associated immunity compared to splenic immunity following oral 7,12-dimethylbenz[a]anthracene ingestion in B6C3F1 mice

Fundam. Appl. Toxicol.

Chemical separation of helper cell function and delayed hypersensitivity responses

Cell. Immunol.

Examination of bone marrow, immunologic parameters and host susceptibility following pre- and postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Int. J. Immunopharmacol.

Early loss of large genomic DNA in vivo with accumulation of Ca²⁺ in the nucleus during acetaminophen-induced liver injury

Toxicol. Appl. Pharmacol.

Immunosuppression following exposure to 7,12-dimethylbenz[a]anthracene (DMBA) in Ah-responsive and Ah-nonresponsive mice

Toxicol. Appl. Pharmacol.

Immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in strains of mice with different susceptibility to induction of aryl hydrocarbon hydroxylase

Toxicol. Appl. Pharmacol.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in $C57B1 6$ mice

Toxicol. Appl. Pharmacol.

Immunosuppression following 7,12-dimethylbenz[a]anthracene exposure in B6C3F1 mice. Effects on humoral immunity and host resistance

Toxicol. Appl. Pharmacol.

Cited by (44)

Induction of intracellular calcium concentration by environmental benzo(a)pyrene involves a β2-adrenergic receptor/adenylyl cyclase/Epac-1/inositol 1,4,5-trisphosphate pathway in endothelial cells

Low-dose synergistic immunosuppression of T-dependent antibody responses by polycyclic aromatic hydrocarbons and arsenic in C57BL/6J murine spleen cells

Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice

The effects of polycyclic aromatic hydrocarbons on the immune system of fish: A review

Dibenzo[a,l]pyrene induced DNA adduct formation in lung tissue in vivo

3-Methylcholanthrene induces lymphocyte and phagocyte apoptosis in common carp (Cyprinus carpio L) in vitro

Regular articleDMBA-induced cytotoxicity in lymphoid and nonlymphoid organs of B6C3F1 mice: Relation of cell death to target cell intracellular calcium and DNA damage

Abstract

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Int. J. Immunopharmacol.

Int. J. Immunopharmacol.

Fundam. Appl. Toxicol.

Cell. Immunol.

Int. J. Immunopharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

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