National Toxicology Program and the National Center for Toxicogenomics, National Institute of Environmental Health Sciences, 111 Alexander Drive, Research Triangle Park, North Carolina 27709
ABSTRACT
The article highlighted in this issue is "Hepatic Effects of 2-Butoxyethanol in Rodents" by A. M. Siesky, L. M. Kamendulis, and J. E. Klaunig (pp. 252260).
The observation that similar treatments produce unique outcomes in different sexes or different species often provides a model for mechanistic studies. As an example unrelated to the present paper in this issue, the observation was made that male and female rats exhibited highly different responses to chronic chemical exposure in the kidney, which allowed scientists to characterize the sex-specific responses to chemicals that induce 2u-globulin nephropathy. This syndrome is produced by a variety of chemicals and is manifested by accumulation of
2u-globulin in the kidney, resulting in compensatory cell proliferation and renal tubular tumors.
2u-globulin is a low molecular weight protein (18,700 daltons) synthesized in the liver of male but not female rats. Following secretion from the liver,
2u-globulin is filtered in the glomerulus and slowly hydrolyzed in the proximal tubule. There is a strict requirement that a chemical or a metabolite physically bind to the
2u-globulin to produce nephropathy, although covalent, irreversible binding is not required. When the chemical-
2u-globulin complex is filtered by the kidney, it accumulates in phagolysosomes in the proximal tubule region and produces cytotoxicity, which results in subsequent regenerative hyperplasia (Lehman-McKeeman, 1997
). Several nongenotoxic chemicals, including d-limonene, unleaded gasoline, jet fuels, and 1,4-dichlorobenzene produce sustained nephrotoxicity, which appears to be the causative factor in their induction of renal cancer (Borghoff et al., 1990
; Borghoff and Lagarde, 1993
; Swenberg et al., 1992
). Female rats, mice, and NBR rats do not accumulate
2u-globulin in their kidneys, do not produce nephrotoxicity and regenerative hyperplasia, and do not develop renal tumors following chemical exposure. These data are now accepted by regulatory agencies to discount the risk to humans by chemicals that are shown to induce
2u-globulin nephropathy (Baetcke, 1991
IARC, 1998
).
An observation that two species of rodents differ dramatically in their responses to chemical treatment also offers a unique opportunity to study mechanisms of chemical toxicity across species and allows insight into the use of such data for human risk assessment. The manuscript highlighted in this issue exploits the differential response of rats and mice to chronic 2-butoxyethanol (ethylene glycol monobutyl ether) exposure (Siesky et al., 2002). Male mice were found to be highly sensitive to hepatocarcinogenesis following chronic exposure to 2-butoxyethanol. Male mice exposed to 2-butoxyethanol by inhalation for 2 years had significantly increased incidence of hemangiosarcoma of the liver relative to controls and exceeded the range in historical controls. In addition, there were possible exposure-related increases in the incidence of hepatocellular carcinoma. Incidences of hemosiderin (iron deposition) pigmentation in the Kupffer cells were significantly increased in mice. The incidences of splenic hematopoietic cell proliferation and hemosiderin pigmentation were generally increased, and the incidences of bone marrow hyperplasia were also increased in mice (NTP, 2000
). These data lead the authors to hypothesize that the increased incidence of liver tumors in mice was a result of increased oxidative stress and Kupffer cell activation arising from iron accumulation in the liver secondary to red blood cell hemolysis. This is a testable hypothesis since rats were found to be resistant to the hepatocarcinogenicity of 2-butoxyethanol and should, therefore, respond less to the possible prooxidant effects of 2-butoxyethanol than the more sensitive mice species.
The hypothesis tested by these researchers is that 2-butoxyethanol, via the action of its primary metabolite 2-butoxyacetic acid, induces hemolysis with concomitant accumulation of iron in the liver. Iron is a prooxidant, and high levels of iron produce oxidant stress either directly by producing reactive oxygen species by the Fenton reaction or by activation of Kupffer cells, which in turn produce reactive oxygen species and release cytokines that may suppress apoptosis and increase cell proliferation, both of which are shown to be associated with increased susceptibility for cancer development. This is a testable hypothesis due to the species specificity of the carcinogenicity assay for the hypothesis to be confirmed, the mouse must be shown to produce greater responses to 2-butoxyethanol exposure than rats.
Interestingly, mice and rats both were sensitive to the hemolytic action of 2-butoxyethanol, discounting any theories of differential metabolism between the two species (Siesky et al., 2002, Fig. 2). Hematocrit decreased, spleen weight increased, and Kupffer cells were activated in both rats and mice. Thus, one would question if the authors' theories are correct.
The solution to the differential response to 2-butoxyethanol was shown to be based on the species-specific response to reactive oxygen species. Mice accumulate oxidized DNA bases, especially 8-hydroxydeoxyguanosine, and experience lipid peroxidation and increased rates of cell proliferation, whereas rats had none of these outcomes. Oxidized DNA bases are precursors to mutations which in turn lead to cancer formation. Lipid peroxidation makes cells leaky and causes them to die, resulting in compensatory cell proliferation, another risk factor for tumor development. To further clarify cell type undergoing the increase in cell proliferation, the authors demonstrated that the cell proliferation occurs both in hepatocytes and endothelial cells in mice, both target cell types for 2-butoxyethanol-induced neoplasia. This is an elegant approach.
These data certainly demonstrate many possible explanations for the differential tumorigenic response of mice and rats to 2-butoxyethanol exposure, but the authors did not stop before asking the more intriguing question: What is the basis for these species differences with regard to responses to reactive oxygen species? The authors measured the amount of the antioxidant vitamin E and the rate of depletion following oxidant stress by 2-butoxyethanol in rats and mice. Their results were fascinating; while 2-butoxyethanol depleted the vitamin E content in both species, the basal level of vitamin E is much higher in the rat than the mouse. Indeed, even after vitamin E depletion by 2-butoxyethanol in the rat, the reduced levels were still higher than in the untreated mouse. Therefore, the progression of toxicity observed in the mouse and the resistance to toxicity in the rat appear to result from different antioxidant levels in the different species!
So what does this mean for humans? Are we sensitive like mice or resistant like rats? The authors conclude humans should be highly resistant to accumulation of iron in the liver and are not at risk for hepatic toxicity by the reactive oxygen species mechanism following exposure to 2-butoxyethanol. Recent studies have demonstrated the resistance to 2-butoxyacetic acid hemolysis by human red blood cells (Udden, 2000), indicating the initial hemolysis after exposure to 2-butoxyethanol is unlikely to cause iron accumulation in the liver. Additionally, levels of vitamin E in human liver is high, approximately 100-fold higher than in the mouse (Rocci et al., 1997
). Therefore, the oxidative stress mechanism that results in the tumorigenic response in the mouse but not the rat is unlikely to occur in humans. A mouse is not a rat is not a human.
NOTES
1 To whom correspondence should be addressed. Fax: (919) 541-4632. E-mail: cunning1{at}niehs.nih.gov.
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