Elsevier

Toxicology Letters

Volume 153, Issue 3, 28 November 2004, Pages 343-355
Toxicology Letters

Oltipraz, 3H-1,2-dithiole-3-thione, and sulforaphane induce overlapping and protective antioxidant responses in murine microglial cells

https://doi.org/10.1016/j.toxlet.2004.06.006Get rights and content

Abstract

Oltipraz (OPZ) is a known inducer of glutathione S-transferases and a mechanism-based inhibitor of cytochrome P450 1A2. Given the detoxification characteristics of this compound, the transcriptional effects of OPZ, along with the related naturally occurring compounds 3H-1,2-dithiole-3-thione (D3T) and sulforaphane (SF), were examined by gene expression profiling in murine BV-2 microglial cells, a neuronal macrophage cell type that mediates inflammatory responses in the brain. We show that the three compounds generate largely overlapping transcriptional changes in genes that are associated with detoxification and antioxidant responses. In addition, induction of an antioxidant/detoxification response in the microglial cells by OPZ, D3T, or SF was also able to protect cells from H2O2-induced toxicity and to attenuate the production of reactive oxygen species in response to lipopolysaccharide treatment of cells. These results show that OPZ, D3T, and SF activate overlapping changes in gene expression and that they can regulate detoxification/antioxidant responses in multiple cells types, including cell types known to have a role in the production of oxidative stress.

Introduction

Chemical inducers of antioxidant proteins and phase II enzymes are known to enhance the detoxification of carcinogens and thus protect against mutation and initiation of neoplasia (Kensler, 1997). However, the benefit of pharmacologically inducing a cellular antioxidant response is not limited to cancer chemoprevention. Two groups of chemical inducers identified from cruciferous vegetables are the isothiocyanates and dithiolethiones (Yates, 1998, Zhang et al., 1992). Sulforaphane (SF) and 3H-1,2-dithiole-3-thione (D3T) are representative compounds from each of these respective groups (Fig. 1) and both have been studied extensively as cancer chemoprotective agents. Oltipraz (OPZ), a substituted dithiolethione, has also been shown to induce cancer chemoprotective effects in animal models and is currently being evaluated in clinical studies as a chemopreventive agent for hepatocarcinogenesis (Kensler et al., 1999, Kensler et al., 1998, Zhang et al., 1997) (Fig. 1).

The cancer chemoprotective effects of OPZ have been attributed to both its ability to induce expression of GSTs and other phase II enzymes as well as its ability to inhibit the P450 catalyzed transformation of several procarcinogens (Clapper, 1998, Langouët et al., 2000, Langouët et al., 1997). In addition, recent gene expression profiling studies have demonstrated that D3T and SF can up-regulate a wide spectrum of antioxidative stress responsive genes in murine hepatocytes and primary intestinal epithelial cells, respectively (Kwak et al., 2003, Thimmulappa et al., 2002). Genes upregulated include NAD(P)H:quinone reductase, epoxide hydrolase, dihydrodiol dehydrogenase, γ-glutamylcysteine synthetase, heme-oxygenase-1 (HO-1), leukotriene B4 dehydrogenase, aflatoxin B1 dehydrogenase, and ferritin (Kwak et al., 2003, Thimmulappa et al., 2002). These drugs can also induce transcription of gene products that assist in clearing reactive oxidant species including SOD family members, catalase, glutathione peroxidase cycle members, glutathione (GSH), GSH reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), and HO-1.

Production of reactive electrophiles/oxidants in the brain is implicated in both neurodegeneration and cellular injury and has been linked to a variety of conditions including Alzheimer’s disease, stroke, multiple sclerosis, Parkinson’s disease, and others (Bo et al., 1994, Choi and Rothman, 1990, Eddleston and Mucke, 1993, Koutsilieri et al., 2002, Kreutzberg, 1996). Several studies have demonstrated that dietary compounds (examples include vitamin E and its derivatives) and other non-steroidal pharmacological agents (examples include thiazolidiniones, 2-methylaminochromans, pyrrolopyrimidines, etc.) that produce antioxidant effects may represent treatment avenues for chronic neurodegeneration (Bordet, 2002, McCulloch and Dewar, 2001, Moosmann and Behl, 2002). As many degenerative CNS disorders are associated with signs of CNS inflammation, brain specific immune effector cells, such as microglial cells, are thought to be important contributors to the disease process. Microglial cells are a subset of glial cells and produce a variety of cytokines and cytotoxic agents, including reactive oxygen and nitrogen species, when activated by cytokines, bacterial infection, injury, β-amyloid peptides, and other etiological agents (Choi, 1993, Eddleston and Mucke, 1993, Kreutzberg, 1996). As such, the generation of oxidative stress following microglial cell activation has been implicated in the pathogenesis of neuroinflammatory diseases.

In this study, gene expression profiling was used to examine the effects of SF, D3T, and OPZ on murine microglial cells. Recent studies have shown that tert-butylhydroquinone (tBHQ), an activator of the Nrf2 transcription factor pathway, and SF were able to protect microglial cells against oxidative stress by induction of antioxidant and detoxification pathways (Kraft et al., 2004, Lee et al., 2003, Shih et al., 2003). We demonstrate that the dithiolethiones, including the clinically relevant compound OPZ, are also able to induce a robust transcriptional antioxidant response in these cells. In addition, we provide evidence that treatment of microglial cells with OPZ, D3T, or SF protects against H2O2-induced toxicity and attenuates production of extracellular ROS following stimulation by LPS. Taken together, these data suggest that both isothiocyanates (e.g. SF) and dithiolethiones (e.g. OPZ and D3T) can attenuate oxidative stress and/or the damaging effect of chronic inflammation in neuronal model systems and macrophage systems.

Section snippets

Chemicals

OPZ and D3T were generously provided by Dr. Sophie Langouët (INSERM, Rennes, France). SF was purchased from LKT Laboratories, Inc. (St. Paul, Minnesota). Solutions of OPZ, D3T, and SF were prepared in DMSO and stored at 4 °C.

Cell culture

BV-2 cells, a murine microglial cell line, were kindly provided by Dr. Linda Van Eldik (Northwestern University). Cells were cultured at 37 °C and 5% CO2 in αMEM containing 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO/BRL, Grand Island, NY) as

Identification of genes induced by OPZ, D3T, and SF

Two-color cDNA microarray profiling was used to examine the transcriptional effects of OPZ, D3T, and SF on microglial cells. In each case, RNA from treated cells was compared to RNA isolated from mock-treated (DMSO) control cells. Cells were treated with OPZ, D3T, or SF for 12 h.

Following normalization of the gene expression data, genes that changed expression in each treatment group were identified (see Section 2). Genes that changed expression included 61 genes that were commonly up-regulated

Discussion

Inappropriate activation of glial cells has been linked to a variety of degenerative neurological diseases including Alzheimer’s disease, stroke, multiple sclerosis, Parkinson’s disease, and others (Bo et al., 1994, Choi and Rothman, 1990, Eddleston and Mucke, 1993, Koutsilieri et al., 2002, Kreutzberg, 1996). The current study shows that treatment of microglial cells with OPZ, D3T, and SF induces transcription of a number of genes involved in the antioxidant stress response (Table 1). Systems

Acknowledgments

In memory of Sarah Klenow. Thanks to Dr. K.A. Furge and the Bioinformatics Special Program at the Van Andel Research Institute for assistance in the microarray data analysis. This work was supported in part by the Camille and Henry Dreyfus Foundation (L.L.F.), the Michigan Economic Development Corporation (L.L.F., K.A.F., and B.B.H.), and the Kalamazoo College MacArthur and Hutchcroft funds (L.L.F.).

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    Present address: Functional Genomics Laboratory, Perinatology Research Branch of the National Institute of Child Health & Human Development (NICHD), Wayne State University School of Medicine, 3258 Scott Hall, 540 E. Canfield Ave., Detroit, MI 48201, USA

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