Toxicant-Induced Oxidative Stress in Cancer

Brad L. Upham*,{dagger},1 and James G. Wagner*,{ddagger}

* National Food Safety and Toxicology Center, {dagger} Department of Pediatrics and Human Development, and {ddagger} Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824-1317

ABSTRACT

The article highlighted in this issue is "The Role of Oxidative Stress in Indium Phosphide-Induced Lung Carcinogenesis in Rats" by Barbara C. Gottschling, Robert R. Maronpot, James R. Hailey, Shyamal Peddada, Cindy R. Moomaw, James E. Klaunig, and Abraham Nyska (pp. 28–40). The article integrates a traditional pathologic study of toxicant-induced pulmonary carcinogenesis with an immunohistologic assessment of oxidative stress, thereby determining a potential mechanism of action of a toxicant, specifically indium phosphide.

Otto Warburg was the first scientist to implicate oxygen in cancer (Warburg, 1956Go) as far back as the 1920s. However, the underlying mechanism by which oxygen might contribute to the carcinogenic process was undetermined for many years. The discovery of superoxide dismutase in 1968 by McCord and Fridovich (1968) led to an explosion of research on the role of reactive oxygen in the pathologies of biological organisms. Reactive oxygen has been specifically connected with not only cancer but also many other human diseases (Allen and Tresini, 2000Go; Hippeli et al., 1999Go). For many years, research in oxidative stress focused primarily on determining how reactive oxygen species (ROS) damage cells by indiscriminate reactions with the macromolecular machinery of a cell, particularly lipids, proteins and DNA. We know in great detail how ROS react with lipids leading to the peroxidation of biological membranes and resulting in necrotic lesions (Gille and Sigler, 1995Go) and how ROS react with the nucleotides of DNA leading to potential mutations (Cadet et al., 1997Go; Gille and Sigler, 1995Go). However, many chronic diseases associated with oxidative stress, such as cancer, are not always a consequence of tissue necrosis, DNA mutations, or protein damage but rather to altered gene expression through epigenetic mechanisms (Trosko and Ruch, 1998Go; Upham et al., 1998Go). For example, organic and hydrogen peroxides have been shown to act as tumor promoters and not as initiators (Slaga et al., 1981Go; Klein-Szanto and Slaga, 1982Go; Gimenez-Conti, et al., 1998Go; Mitchel et al., 1987Go), indicating that these oxidants are not mutagens but rather epigenetic toxicants. In the past decade, much research has shifted to the understanding of how ROS can reversibly control the expression of genes at noncytotoxic doses (Allen and Tresini, 2000Go). In this regard, at least 127 genes and signal transducing proteins have been reported to be sensitive to reductive and oxidative (redox) states in the cell (Allen and Tresini, 2000Go).

Although many intracellular signaling pathways are known to be redox-sensitive, the mitogen activated protein kinase (MAPK) and nuclear factor-kB (NF{kappa}B) signal transduction pathways have been examined far more than any other pathways (Allen and Tresini, 2000Go; Gabbita et al., 2000Go; Hensley et al., 2000Go). These two pathways either directly or indirectly transduce most redox responses (Allen and Tresini, 2000Go). MAPK is not only activated by ROS (Guyton et al., 1996Go) but actually requires the presence of H2O2 (Sundaresan et al., 1995Go). These authors demonstrated that the extracellular ligand, platelet derived growth factor (PDGF), induces a burst of H2O2 in vascular smooth muscle cells, which in turn activates MAPK. The activation of MAPK by PDGF is prevented by the transfection of catalase, which eliminates H2O2, into these cells. Downstream events of MAPK activation, such as proliferation and increased cell motility, are also inhibited by catalase. This is one of several studies demonstrating that endogenous growth factors (extracellular ligands) generate ROS, which are then required downstream in intracellular signaling to successfully transmit their signals to the nucleus (Lander, 1997Go).

The successful transmission of an extracellular signal from the membrane to the nucleus via intracellular signaling pathways in solid tissue cell types is also dependent upon intercellular signals through gap junctions (Trosko et al., 1998Go; Upham et al., 1998Go). Not surprisingly, ROS have also been demonstrated to reversibly inhibit gap junctional intercellular communication (GJIC) at noncytotoxic levels (Upham et al., 1997Go). If gap junctions were not closed, then the H2O2 generated by extracellular ligands could escape through gap junctions into neighboring cells, thereby potentially diluting to a level that would be insufficient for MAPK-dependent activation of transcription factors. These examples demonstrate how extra-, intra-, and intercellular signaling pathways might interact to coordinate the epigenetic expression of genes in response to ROS.

In addition to oxidants serving normal roles as subcellular messengers in gene regulatory and signal transduction pathways, antioxidants have also been demonstrated to serve as subcellular messengers for normal cell function (Allen and Tresini, 2000Go). For example, a major H2O2-scavenging pathway is the two-electron reduction of H2O2 catalyzed by glutathione peroxidase, which clearly serves a protective role against peroxide-dependent oxidative injury. However, depletion of intracellular pools of glutathione (GSH), by inhibiting the rate-limiting step of its biosynthesis, paradoxically reverses the biological effect of H2O2 in several systems. For example, inhibition of GJIC (Upham et al., 1997Go), induction of c-jun (Kuo et al., 1996Go), and activation of NF{kappa}B (Ginn-Pease and Whisler, 1996Go) by H2O2 were completely reversed when the cellular systems were depleted of GSH. This indicates that these signaling pathways required not only H2O2 but also GSH. Inhibition of GJIC and the induction of early-response genes are hallmarks of tumor promotion, and in the results just described, a reduction in the natural antioxidant GSH could also potentially protect a cell from proliferative responses to extracellular ligands.

In the highlighted article, Gottschling and coworkers demonstrate that indium phosphide-induced oxidative stress strongly correlates with the progression of pulmonary lesions from hyperplasia to the formation of squamous cysts. Although a causal link between oxidative stress and cancer was not established and no determination was made as to whether altered cell signaling or cellular damage was the critical event of hyperplasia, the results of their study demonstrate how immunohistochemical techniques can be used to identify a potential mechanism of toxic insult from the results of a standard-type chronic NTP study.

The four markers used to assess oxidative stress in this study were 8-hydroxydeoxyguanosine (8-OHdG), glutathione-S-transferase Pi (GST-Pi), inducible nitric oxide synthase (i-NOS), and cyclooxygenase-2 (COX-2). A summary of their findings in animals exposed to chronic levels of indium phosphide is presented in Table 1Go.


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TABLE 1 Levels of Oxidative Stress Markers in Animals after Chronic Exposure to Indium Phosphide
 
Indium phosphide inhalation resulted in severe pulmonary inflammation that correlated with the infiltration of reactive oxygen generating immune cells. Macrophages exhibited high levels of i-NOS and COX-2, the inducible forms of nitric oxide synthase and cyclooxygenase. Similar results were also observed in a study of human patients with primary lung cancer who exhibited an upregulation of i-NOS and subsequent increase in exhaled NO (Moilanen et al., 1998Go). Nitric oxide (NO) is a reactive oxygen intermediate implicated in many cell signaling pathways. Although the role of NO in tumor biology remains controversial, most data indicates that NO promotes tumor progression (Orucevic et al., 1999Go). Similarly, COX-2 expression in lung macrophages has been correlated with a procarcinogenic state of the lung (Yao et al., 2000Go; Hosomi et al., 2000Go; Fosslien, 2000Go). Macrophage-derived prostanoids might contribute to the progression of hyperplasia in nearby epithelial cells. More important in this study is the high expression of COX-2 in epithelial cells of adenomas and squamous cysts. These findings are consistent with results from human studies, where COX-2 expression was elevated in well-defined carcinomas (Wolff et al., 1998Go).

Gottschling and coworkers found increased levels of 8-OHdG and GST-Pi in lung epithelial cells. Although 8-OHdG levels have been used as a marker for oxidative stress for many years, only recently has immunohistochemical labeling of 8-OHdG in paraffin embedded pulmonary tissue been demonstrated (Kawashima et al., 2000Go). While implicated in G-C to T-A transversions, this marker does not always correlate with genotoxicity. For example, increased 8-OHdG induced by peroxisome proliferators, which are classified as nongenotoxic carcinogens, failed to correlate with tumor multiplicity (Cattley and Preston, 1995Go). Similarly, induction of 8-OHdG by menadione did not correlate with the initiation (genotoxic) stage of cancer but rather with promotion, a nongenotoxic stage. Despite the weak link to tumorigenicity, 8-OHdG is a good indicator of oxidative stress. In contrast, GST-Pi is an excellent marker that correlates well with tumorigenicity and oxidative stress. The GST genes possess AP-1 response elements that bind to AP-1 composed of heterodimers of c-jun and c-fos (Daniel, 1993Go). Reactive oxygen species are transduction signals that activate c-fos and c-jun genes through MAPK signal transduction pathways (Allen and Tresini, 2000Go), thereby linking GST gene expression with ROS. An important function of GST in response to oxidative stress is its ability to conjugate GSH with lipid peroxidation products (Rao and Shaha, 2000Go), which have also been linked to signal transduction (Leonarduzzi et al., 2000Go).

In summary, oxidative stress is a known mediator of cancer. Although the oxidative mechanisms of cancer remain uncertain, we do know that reactive oxygen can play a role at two levels. One is oxidative damage to DNA that could lead to the mutational events of initiation and progression. The other is interruption of reactive oxygen-dependent cell signaling pathways controlling gene expression that contribute to all stages of cancer, in particular tumor promotion. Although oxidative stress was not causally linked to the carcinogenicity of indium phosphide, the research presented by Gottschling et al. in this issue of Toxicological Sciences indicates a strong correlation of indium phosphide-induced oxidative stress and cancer. More importantly, perhaps, this manuscript represents an extension of the investigative tools in the pathologist's armamentarium. Due to methodological constraints, pathologists have previously been limited in their ability to provide mechanistic descriptions of toxicity. Recent developments in immunohistochemical, molecular, and genomic technologies provide new and powerful techniques for the toxicologic pathologist. In this regard, a reexamination of past NTP studies and early work on pulmonary tumors induced by cigarette smoke and fibers may reveal insights into the oxidative mechanism of carcinogenesis. Further methodological development will provide more accurate and predictive markers to determine how toxicants can oxidatively induce cancer.

NOTES

1 To whom correspondence should be addressed at Department of Pediatrics and Human Development, 243 Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824-1317. Fax: (517) 432-6340. E-mail: upham{at}pilot.msu.edu. Back

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