Klaunig J.E., Shi Y. (2009). Assessment of gap junctional intercellular communication. Curr Protoc Toxicol. 2009 Aug;Chapter 2:Unit2.17. DOI: 10.1002/0471140856.tx0217s41. PMID: 20941698.
Abstract. Gap junctions are important plasma membrane organelles through which most cells in normal tissues communicate with each other. They exist in two neighboring cells and each cell contributes half of the structure. One gap junction consists of two hexameric connexons that dock with each other to create a channel. Six of the basic subunits referred to as connexins form a connexon. Less than one hundred to several thousand gap junction channels cluster together in the plane of the membrane. The gap junction channels serve as a regulated conduit for the intercellular exchange of small molecules. Maintenance of the integrity of gap junctional intercellular communication (GJIC) is important and required for normal electrical coupling, homeostasis, and embryogenesis. Aberrations of gap junctions have been related to human diseases such as cancer, cardiac arrhythmia, Charcot-Marie-tooth disease, and visceroatrial heterotaxia syndrome. This unit describes methods for measuring gap junctional intercellular communication using primary mouse hepatocytes as a model. Focus is only on functional evaluation based on dye coupling. Other methods, such as intracellular calcium waves and dual patch clamp, have been used to measure gap junctional communication, but these are not described in this unit.
180. Cohen S.M., Storer R.D., Criswell K.A., Doerrer N.G., Dellarco V.L., Pegg D.G., Wojcinski Z.W., Malarkey D.E., Jacobs A.C., Klaunig J.E., Swenberg J.A., Cook J.C. (2009). Hemangiosarcoma in rodents: mode-of-action evaluation and human relevance. Toxicol Sci. 2009 Sep;111(1):4-18. DOI: 10.1093/toxsci/kfp131. PMID: 19525443.
Abstract. Although rarely occurring in humans, hemangiosarcomas (HS) have become important in evaluating the potential human risk of several chemicals, including industrial, agricultural, and pharmaceutical agents. Spontaneous HS arise frequently in mice, less commonly in rats, and frequently in numerous breeds of dogs. This review explores knowledge gaps and uncertainties related to the mode of action (MOA) for the induction of HS in rodents, and evaluates the potential relevance for human risk. For genotoxic chemicals (vinyl chloride and thorotrast), significant information is available concerning the MOA. In contrast, numerous chemicals produce HS in rodents by nongenotoxic, proliferative mechanisms. An overall framework is presented, including direct and indirect actions on endothelial cells, paracrine effects in local tissues, activation of bone marrow endothelial precursor cells, and tissue hypoxia. Numerous obstacles are identified in investigations into the MOA for mouse HS and the relevance of the mouse tumors to humans, including lack of identifiable precursor lesions, usually late occurrence of the tumors, and complexities of endothelial biology. This review proposes a working MOA for HS induced by nongenotoxic compounds that can guide future research in this area. Importantly, a common MOA appears to exist for the nongenotoxic induction of HS, where there appears to be a convergence of multiple initiating events (e.g., hemolysis, decreased respiration, adipocyte growth) leading to either dysregulated angiogenesis and/or erythropoiesis that results from hypoxia and macrophage activation. These later events lead to the release of angiogenic growth factors and cytokines that stimulate endothelial cell proliferation, which, if sustained, provide the milieu that can lead to HS formation.
179. Lee L., Alloosh M., Saxena R., Van Alstine W., Watkins B.A., Klaunig J.E., Sturek M., Chalasani N. (2009) Nutritional model of steatohepatitis and metabolic syndrome in the Ossabaw miniature swine. Hepatology. 2009 Jul;50(1):56-67. DOI: 10.1002/hep.22904. PMID: 19434740.
Abstract. Miniature pigs residing in the Ossabaw Island (Ossabaw pigs) exhibit a thrifty genotype, and when fed a high-calorie diet they consistently develop metabolic syndrome defined by obesity, insulin resistance, hypertension, and dyslipidemia. We conducted a study to induce steatohepatitis in Ossabaw pigs by dietary manipulation. Pigs were fed standard chow (controls, n = 15), high-fructose diet (20% kcal from fructose and 10.5% kcal from fat) (fructose group, n = 9), atherogenic diet (20% kcal from fructose and 46% kcal from fat and 2% cholesterol and 0.7% cholate by weight) (atherogenic diet group, n = 13), and modified atherogenic diet (different source of fat and higher protein but lower choline content) (M-Ath diet group, n = 7). All animals were sacrificed at 24 weeks after dietary intervention. The high-fructose group had significant weight gain, hypertension, and insulin resistance but showed normal liver histology. The atherogenic diet group had metabolic syndrome and abnormal liver histology consisting of significant microvesicular steatosis and fatty Kupffer cells but no ballooning or fibrosis. The M-Ath diet group developed severe metabolic syndrome and markedly abnormal liver histology with macrovesicular and microvesicular steatosis, fatty Kupffer cells, extensive hepatocyte ballooning, and pericellular/perisinusoidal fibrosis. Compared with controls, the M-Ath diet group had significantly lower serum adiponectin but higher serum leptin and tumor necrosis factor (TNF) levels and higher hepatic triglyceride and malondialdehyde levels. Conclusion: Ossabaw pigs fed a modified atherogenic diet develop severe metabolic syndrome and abnormal liver histology with close resemblance to human nonalcoholic steatohepatitis (NASH).
178. Pu X., Kamendulis L.M., Klaunig J.E. (2009). Acrylonitrile-induced oxidative stress and oxidative DNA damage in male Sprague-Dawley rats. Toxicol Sci. 2009 Sep;111(1):64-71. DOI: 10.1093/toxsci/kfp133. PMID: 19546159.
Abstract. Studies have demonstrated that the induction of oxidative stress may be involved in brain tumor induction in rats by acrylonitrile. The present study examined whether acrylonitrile induces oxidative stress and DNA damage in rats and whether blood can serve as a valid surrogate for the biomonitoring of oxidative stress induced by acrylonitrile in the exposed population. Male Sprague-Dawley rats were treated with 0, 3, 30, 100, and 200 ppm acrylonitrile in drinking water for 28 days. One group of rats were also coadministered N-acetyl cysteine (NAC) (0.3% in diet) with acrylonitrile (200 ppm in drinking water) to examine whether antioxidant supplementation was protective against acrylonitrile-induced oxidative stress. Direct DNA strand breakage in white blood cells (WBC) and brain was measured using the alkaline comet assay. Oxidative DNA damage in WBC and brain was evaluated using formamidopyrimidine DNA glycosylase (fpg)-modified comet assay and with high-performance liquid chromatography-electrochemical detection. No significant increase in direct DNA strand breaks was observed in brain and WBC from acrylonitrile-treated rats. However, oxidative DNA damage (fpg comet and 8’hydroxyl-2-deoxyguanosine) in brain and WBC was increased in a dose-dependent manner. In addition, plasma levels of reactive oxygen species (ROS) increased in rats administered acrylonitrile. Dietary supplementation with NAC prevented acrylonitrile-induced oxidative DNA damage in brain and WBC. A slight, but significant, decrease in the GSH:GSSG ratio was seen in brain at acrylonitrile doses > 30 ppm. These results provide additional support that the mode of action for acrylonitrile-induced astrocytomas involves the induction of oxidative stress and damage. Significant associations were seen between oxidative DNA damage in WBC and brain, ROS formation in plasma, and the reported tumor incidences. Since oxidative DNA damage in brain correlated with oxidative damage in WBC, these results suggest that monitoring WBC DNA damage maybe a useful tool to assess acrylonitrile-induced oxidative stress in humans.
177. Harvilchuck J.A., Pu X., Klaunig J.E., Carlson G.P. (2009). Indicators of oxidative stress and apoptosis in mouse whole lung and Clara cells following exposure to styrene and its metabolites. Toxicology. 2009 Oct 29;264(3):171-8. DOI: 10.1016/j.tox.2009.08.001. PMID: 19666080.
Abstract. In mice, styrene is hepatotoxic, pneumotoxic, and causes lung tumors. One explanation for the mechanism of toxicity is oxidative stress/damage. Previous studies have shown decreased glutathione levels, linked to increased apoptosis, in lung homogenates and isolated Clara cells 3 h following styrene or styrene oxide (SO) administration or in vitro exposure. The objective of the current studies was to determine what effects styrene and its active metabolites, primarily styrene oxide, had on indicators of oxidative stress and attendant apoptosis in order to understand better the mechanism of styrene-induced toxicity. Three hours following in vitro exposure of Clara cells to styrene or SO there were increases in reactive oxygen species (ROS). Following administration of styrene or styrene oxide ip, increases in ROS, superoxide dismutase (SOD), and 8-hydroxydeoxyguanosine (8-OHdG) formation were observed. Since increases in ROS have been linked to increases in apoptosis ratios of bax/bcl-2, mRNA and protein expression were determined 3-240 h following the administration of styrene and R-styrene oxide (RSO). The bax/bcl-2 mRNA ratio increased 12 and 24 h following R-SO and 120 h following styrene administration. However, the bax/bcl-2 protein ratio was not increased until 240 h following R-SO, and 24 and 240 h following styrene administration. However, only a slight increase in caspase 3 was observed. These results indicated that oxidative stress occurred 3h following styrene or styrene oxide as evidenced by increased ROS and SOD. This increased ROS may be responsible for the increased 8-OHdG formation. Our findings of limited apoptosis in Clara cells following acute exposure to styrene or SO are in agreement with others and may reflect the minimal extent to which apoptosis plays a role in acute styrene toxicity. It is clear, however, that oxidative stress and oxidative effects on DNA are increased following exposure to styrene or styrene oxide, and these may play a role in the lung tumorigenesis in mice.
176. de Peyster A., Rodriguez Y., Shuto R., Goldberg B., Gonzales F., Pu X., Klaunig J.E. (2008). Effect of oral methyl-t-butyl ether (MTBE) on the male mouse reproductive tract and oxidative stress in liver. Reprod Toxicol. 2008 Nov-Dec;26(3-4):246-53. DOI: 10.1016/j.reprotox.2008.08.009. PMID: 18824092.
Abstract. MTBE is found in water supplies used for drinking and other purposes. These experiments follow up on earlier reports of reproductive tract alterations in male mice exposed orally to MTBE and explored oxidative stress as a mode of action. CD-1 mice were gavaged with 400-2000 mg/kg MTBE on days 1, 3, and 5, injected i.p. with hCG (2.5 IU/g) on day 6, and necropsied on day 7. No effect was seen in testis histology or testosterone levels. Using a similar dosing protocol, others had initially reported disruption of seminiferous tubules in MTBE-gavaged mice, although later conclusions published were consistent with our findings. Another group had also reported testicular and other reproductive system abnormalities in male BALB/c mice exposed for 28 days to 80-8000 microg/ml MTBE in drinking water. We gave these MTBE concentrations to adult mice for 28 days and juvenile mice for 51 days through PND 77. Evidence of oxidative stress was examined in liver homogenates from the juvenile study using MDA, TEAC and 8OH2hG as endpoints. MTBE exposures at the levels examined indicated no significant changes in the male mouse reproductive tract and no signs of hepatic oxidative stress. This appears to be the first oral MTBE exposure of juvenile animals, and also the first to examine potential for MTBE to cause oxidative stress in vivo using a typical route of human exposure.
175. Klaunig J.E. (2008) Acrylamide carcinogenicity. J Agric Food Chem. 2008 Aug 13;56(15):5984-8. DOI: 10.1021/jf8004492. PMID: 18624430.
Abstract. The induction of cancer by chemicals is a multiple-stage process. Acrylamide is carcinogenic to experimental mice and rats, causing tumors at multiple organ sites in both species when given in drinking water or by other means. In mice, acrylamide increased the incidence and multiplicity of lung tumors and skin tumors. In two bioassays in rats, acrylamide administered in drinking water consistently induced mesotheliomas of the testes, thyroid tumors, and mammary gland tumors. In addition, brain tumors appeared to be increased. In one of the rat bioassays, pituitary tumors, pheochromocytomas, uterine tumors, and pituitary tumors were noted. The conversion of acrylamide metabolically to the reactive, mutagenic, and genotoxic product, glycidamide, can occur in both rodent and humans. Glycidamide and frequently acrylamide have been positive for mutagenicity and DNA reactivity in a number of in vitro and in vivo assays. The effects of chronic exposure of glycidamide to rodents have not been reported. Epidemiologic studies of workers for possible health effects from exposures to acrylamide have not shown a consistent increase in cancer risk. Although an increase in the risk for pancreatic cancer (almost double) was seen in highly exposed workers, no exposure response relationship could be determined. The mode of action remains unclear for acrylamide-induced rodent carcinogenicity, but support for a genotoxic mechanism based on in vitro and in vivo DNA reactivity assays cannot be ruled out. In addition, the pattern of tumor formation in the rat following chronic exposure supports a genotoxic mode of action but also suggests a potential role of endocrine modification.
174. Novotny N.M., Grosfeld J.L., Turner K.E., Rescorla F.J., Pu X., Klaunig J.E., Hickey R.J., Malkas L.H., Sandoval J.A. (2008). Oxidative status in neuroblastoma: a source of stress? J Pediatr Surg. 2008 Feb;43(2):330-4. DOI: 10.1016/j.jpedsurg.2007.10.040. PMID: 18280284.
Abstract. Reactive oxygen species have been shown to be initiators/promotors of tumorigenesis. Because evidence supports the role of increased oxidative stress in solid tumors, we sought to establish this relationship in neuroblastoma (NB). The aim of the study was to investigate the extent of oxidative DNA damage and antioxidative status in a progressive animal model of human NB. Tumors were induced in the left kidneys of nude mice by the injection of cultured human NB cells (10(6)). Blood was collected from tumor-bearing mice and controls at 2, 4, and 6 weeks. Peripheral blood leukocyte oxidative DNA damage was determined using single-cell gel electrophoresis (comet assay), and plasma antioxidant capacity was assessed by the Trolox equivalent antioxidant capacity method. Levels of oxidative DNA damage in peripheral blood leukocytes of NB-bearing mice were increased by 166%, 110%, and 87% as compared with healthy controls at 2, 4, and 6 weeks, respectively. Plasma total antioxidant values for tumor-bearing mice were not significantly different from control mice. Our results indicate an increase of oxidative stress in an animal model of human NB, especially in the early stages of growth. Yet, we did not observe an appreciable response in plasma antioxidant activity. Because an altered redox status has been implicated in tumor maintenance and progression, these findings support the notion of a complex oxidant-antioxidant imbalance contributing to NB growth.
173. Hahn N.M., Kelley M.R., Klaunig J.E., Koch M.O., Li L., Sweeney C.J. (2007). Constitutional polymorphisms of prostate cancer: prognostic and diagnostic implications. Future Oncol. 2007 Dec;3(6):665-82. PMID: 18041919.
Abstract. Prostate cancer is the most common cancer diagnosis in men. While often perceived as a slow, indolent malignancy, prostate cancer trails only lung cancer among cancer-related mortality in men. Current diagnosis and treatment algorithms are plagued by overdiagnosis of non-lethal indolent prostate cancer with no proven means to predict, detect, and prevent aggressive lethal prostate cancer in men most at risk. These challenges are particularly concerning for African-American men who demonstrate increased rates of prostate cancer incidence and mortality when compared to other ethnic groups. With the completion of the human genome project, technology and techniques now exist to differentiate cancer from normal tissues based on the expression patterns of thousands of genes assessed simultaneously on a single microarray gene ‘chip’. This platform has greatly improved our understanding of genes that regulate tumor behavior once cancer is established. Microarrays can also be utilized in patients without cancer to determine which patients are at high risk for tumor development and in need of rational prevention strategies. Constitutional single nucleotide polymorphisms (SNPs) are one source of genetic variation and may serve as a means to identify these high-risk individuals. SNPs are single nucleotide base pair changes within a gene which occur in one percent or more of the population. SNPs can contribute to a disease state by altering the function of a protein encoded by a gene without affecting gene expression. This review will examine the current understanding of constitutional SNPs associated with prostate cancer carcinogenesis, highlight two current diagnostic array platforms and discuss implications for future prevention and screening programs.
172. Calabrese E.J., Bachmann K.A., Bailer A.J., Bolger P.M., Borak J., Cai L., Cedergreen N., Cherian M.G., Chiueh C.C., Clarkson T.W., Cook R.R., Diamond D.M., Doolittle D.J., Dorato M.A., Duke S.O., Feinendegen L., Gardner D.E., Hart R.W., Hastings K.L., Hayes A.W., Hoffmann G.R., Ives J.A., Jaworowski Z., Johnson T.E., Jonas W.B., Kaminski N.E., Keller J.G., Klaunig J.E. et al. (2007). Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hormetic dose-response framework. Toxicol Appl Pharmacol. 2007 Jul 1;222(1):122-8. PMID: 17459441.
Abstract. Many biological subdisciplines that regularly assess dose-response relationships have identified an evolutionarily conserved process in which a low dose of a stressful stimulus activates an adaptive response that increases the resistance of the cell or organism to a moderate to severe level of stress. Due to a lack of frequent interaction among scientists in these many areas, there has emerged a broad range of terms that describe such dose-response relationships. This situation has become problematic because the different terms describe a family of similar biological responses (e.g., adaptive response, preconditioning, hormesis), adversely affecting interdisciplinary communication, and possibly even obscuring generalizable features and central biological concepts. With support from scientists in a broad range of disciplines, this article offers a set of recommendations we believe can achieve greater conceptual harmony in dose-response terminology, as well as better understanding and communication across the broad spectrum of biological disciplines.
171. Klaunig J.E., Babich M.A., Cook J.C., David R.M., DeLuca J.G., McKee R.H., Peters J.M., Roberts R.A., Fenner-Crisp P.A. (2007). PPARalpha and effects of TCE. Environ Health Perspect. 2007 Jan;115(1):A14-5. PMID: 17366801.
Abstract. Key issues in the role of peroxisome proliferator-activated receptor agonism and cell signaling in trichloroethylene toxicity.
170. Roberts R.A., Ganey P.E., Ju C., Kamendulis L.M., Rusyn I., Klaunig J.E. (2007) Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis. Toxicol Sci. 2007 Mar;96(1):2-15. Review. PMID: 17122412.
Abstract. Kupffer cells are resident macrophages of the liver and play an important role in its normal physiology and homeostasis as well as participating in the acute and chronic responses of the liver to toxic compounds. Activation of Kupffer cells directly or indirectly by toxic agents results in the release of an array of inflammatory mediators, growth factors, and reactive oxygen species. This activation appears to modulate acute hepatocyte injury as well as chronic liver responses including hepatic cancer. Understanding the role Kupffer cells play in these diverse responses is key to understanding mechanisms of liver injury. Idiosyncratic drug-induced liver disease results in morbidity and mortality, impacting severely on the development of new pharmacological agents. Modulation of the response of Kupffer cells by drugs has been suggested as a cause for the idiosyncratic response. Similarly, liver damage seen in chronic ethanol consumption appears to be modulated by Kupffer cell activation. More recent evidence has noted a contributory role of Kupffer cell activation in the process of hepatic carcinogenesis. Several nongenotoxic carcinogens, for example, activate Kupffer cells resulting in the release of cytokines and/or reactive oxygen species that induce hepatocyte cell proliferation and may enhance clonal expansion of preneoplastic cells leading to neoplasia. Kupffer cells therefore appear to play a central role in the hepatic response to toxic and carcinogenic agents. Taken together, the data presented in this symposium illustrate to the toxicologist the central role played by Kupffer cells in mediating hepatotoxicity.
169. Pu X., Kamendulis L.M., Klaunig J.E. (2006). Acrylonitrile-induced oxidative DNA damage in rat astrocytes. Environ Mol Mutagen. 2006 Oct;47(8):631-8. PMID: 16917936.
Abstract. Chronic administration of acrylonitrile results in a dose-related increase in astrocytomas in rat brain, but the mechanism of acrylonitrile carcinogenicity is not fully understood. The potential of acrylonitrile or its metabolites to induce direct DNA damage as a mechanism for acrylonitrile carcinogenicity has been questioned, and recent studies indicate that the mechanism involves the induction of oxidative stress in rat brain. The present study examined the ability of acrylonitrile to induce DNA damage in the DI TNC1 rat astrocyte cell line using the alkaline Comet assay. Oxidized DNA damage also was evaluated using formamidopyrimidine DNA glycosylase treatment in the modified Comet assay. No increase in direct DNA damage was seen in astrocytes exposed to sublethal concentrations of acrylonitrile (0-1.0 mM) for 24 hr. However, acrylonitrile treatment resulted in a concentration-related increase in oxidative DNA damage after 24 hr. Antioxidant supplementation in the culture media (alpha-tocopherol, (-)-epigallocathechin-3 gallate, or trolox) reduced acrylonitrile-induced oxidative DNA damage. Depletion of glutathione using 0.1 mM DL-buthionine-[S,R]-sulfoximine increased acrylonitrile-induced oxidative DNA damage (22-46%), while cotreatment of acrylonitrile with 2.5 mM L-2-oxothiazolidine-4-carboxylic acid, a precursor for glutathione biosynthesis, significantly reduced acrylonitrile-induced oxidative DNA damage (7-47%). Cotreatment of acrylonitrile with 0.5 mM 1-aminobenzotriazole, a suicidal inhibitor of cytochrome P450, prevented the oxidative DNA damage produced by acrylonitrile. Cyanide (0.1-0.5 mM) increased oxidative DNA damage (44-160%) in astrocytes. These studies demonstrate that while acrylonitrile does not directly damage astrocyte DNA, it does increase oxidative DNA damage. The oxidative DNA damage following acrylonitrile exposure appears to arise mainly through the P450 metabolic pathway; moreover, glutathione depletion may contribute to the induction of oxidative DNA damage by acrylonitrile.
168. Corthals S.M., Kamendulis L.M., Klaunig J.E. (2006) Mechanisms of 2-butoxyethanol-induced hemangiosarcomas. Toxicol Sci. 2006 Aug;92(2):378-86. PMID: 16675516.
Abstract. Chronic exposure to 2-butoxyethanol increased liver hemangiosarcomas in male mice. The mechanism for the selective induction of hemangiosarcomas by 2-butoxyethanol is unknown but has been suggested to occur through non-DNA-reactive mechanisms. The occurrence of liver hemangiosarcomas in male mice has been linked to oxidative damage subsequent to RBC hemolysis and iron deposition and activation of macrophages (Kupffer cells) in the liver, events that exhibit a threshold in both animals and humans. 2-Butoxyethanol is metabolized to 2-butoxyacetaldehyde and 2-butoxyacetic acid, and although the aldehyde metabolite is short lived, the potential exists for this metabolite to cause DNA damage. The present study examined whether 2-butoxyethanol and its metabolites, 2-butoxyacetaldehyde and 2-butoxyacetic acid, damaged mouse endothelial cell DNA using the comet assay. No increase in DNA damage was observed following 2-butoxyethanol (1-10mM), 2-butoxyacetaldehyde (0.1-1.0mM), or 2-butoxyacetic acid (1-10mM) in endothelial cells after 2, 4, or 24 h of exposure. Additional studies examined the involvement of hemolysis and macrophage activation in 2-butoxyethanol carcinogenesis. DNA damage was produced by hemolyzed RBCs (10 x 10(6), 4 h), ferrous sulfate (0.1-1.0 microM; 2-24 h), and hydrogen peroxide (50-100 microM; 1-4 h) in endothelial cells. Hemolyzed RBCs also activated macrophages, as evidenced by increased tumor necrosis factor (TNF) alpha, while neither 2-butoxyethanol nor butoxyacetic acid increased TNF-alpha from macrophages. The effect of activated macrophages on endothelial cell DNA damage and DNA synthesis was also studied. Coculture of endothelial cells with activated macrophages increased endothelial cell DNA damage after 4 or 24 h and increased endothelial cell DNA synthesis after 24 h. These data demonstrate that 2-butoxyethanol and related metabolites do not directly cause DNA damage. Supportive evidence also demonstrated that damaged RBCs, iron, and/or products from macrophage activation (possibly reactive oxygen species) produce DNA damage in endothelial cells and that activated macrophages stimulate endothelial cell proliferation. These events coupled together provide the events necessary for the induction of hemangiosarcomas by 2-butoxyethanol.
167. Holsapple M.P., Pitot H.C., Cohen S.M., Boobis A.R., Klaunig J.E., Pastoor T., Dellarco V.L., Dragan Y.P. (2006). Mode of action in relevance of rodent liver tumors to human cancer risk. Toxicol Sci. 2006 Jan;89(1):51-6. PMID: 16221960.
Abstract. Hazard identification and risk assessment paradigms depend on the presumption of the similarity of rodents to humans, yet species specific responses, and the extrapolation of high-dose effects to low-dose exposures can affect the estimation of human risk from rodent data. As a consequence, a human relevance framework concept was developed by the International Programme on Chemical Safety (IPCS) and International Life Sciences Institute (ILSI) Risk Science Institute (RSI) with the central tenet being the identification of a mode of action (MOA). To perform a MOA analysis, the key biochemical, cellular, and molecular events need to first be established, and the temporal and dose-dependent concordance of each of the key events in the MOA can then be determined. The key events can be used to bridge species and dose for a given MOA. The next step in the MOA analysis is the assessment of biological plausibility for determining the relevance of the specified MOA in an animal model for human cancer risk based on kinetic and dynamic parameters. Using the framework approach, a MOA in animals could not be defined for metal overload. The MOA for phenobarbital (PB)-like P450 inducers was determined to be unlikely in humans after kinetic and dynamic factors were considered. In contrast, after these factors were considered with reference to estrogen, the conclusion was drawn that estrogen-induced tumors were plausible in humans. Finally, it was concluded that the induction of rodent liver tumors by porphyrogenic compounds followed a cytotoxic MOA, and that liver tumors formed as a result of sustained cytotoxicity and regenerative proliferation are considered relevant for evaluating human cancer risk if appropriate metabolism occurs in the animal models and in humans.
Conroy S.K., McDonald B.C., Smith D.J., Moser L.R., West J.D., Kamendulis L.M., Klaunig J.E., Champion V.L., Unverzagt F.W., Saykin A.J. (2005). Alterations in brain structure and function in breast cancer survivors: effect of post-chemotherapy interval and relation to oxidative DNA damage. Toxicol Sci. 2005 Oct;87(2):328-36. PMID: 23263697
Abstract. Neuroimaging studies have begun to uncover the neural substrates of cancer and treatment-related cognitive dysfunction, but the time course of these changes in the years following chemotherapy is unclear. This study analyzed multimodality 3T MRI scans to examine the structural and functional effects of chemotherapy and post-chemotherapy interval (PCI) in a cohort of breast cancer survivors (BCS; n = 24; PCI mean 6, range 3-10 y) relative to age- and education-matched healthy controls (HC; n = 23). Assessments included voxel-based morphometry for gray matter density (GMD) and fMRI for activation profile during a 3-back working memory task…
166. Seed J., Carney E.W., Corley R.A., Crofton K.M., DeSesso J.M., Foster P.M., Kavlock R., Kimmel G., Klaunig J. et al. (2005). Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 Oct-Nov;35(8-9):664-72. PMID: 16417033
Abstract. A complete mode of action human relevance analysis–as distinct from mode of action (MOA) analysis alone–depends on robust information on the animal MOA, as well as systematic comparison of the animal data with corresponding information from humans. In November 2003, the International Life Sciences Institute’s Risk Science Institute (ILSI RSI) published a 2-year study using animal and human MOA information to generate a four-part Human Relevance Framework (HRF) for systematic and transparent analysis of MOA data and information. Based mainly on non-DNA-reactive carcinogens, the HRF features a “concordance” analysis of MOA information from both animal and human sources, with a focus on determining the appropriate role for each MOA data set in human risk assessment. With MOA information increasingly available for risk assessment purposes, this article illustrates the further applicability of the HRF for reproductive, developmental, neurologic, and renal endpoints, as well as cancer. Based on qualitative and quantitative MOA considerations, the MOA/human relevance analysis also contributes to identifying data needs and issues essential for the dose-response and exposure assessment steps in the overall risk assessment.
165. Klaunig J.E. (2005). Cancer biology and hormesis: commentary. Crit Rev Toxicol. 2005 Jul;35(6):593-4. PMID: 16422395.
Abstract. The observation of biphasic dose-response relationships following exposure to pharmacological and toxicological agents has been well documented. In this review Dr. Calabrese, using published data on human tumor cell lines treated with a variety of agents has provided additional support for the demonstration of hormesis in the cancer process. While this review has restricted the examination to human tumor cell lines, this limitation dose not take away from the value of the treatise and helps to point out the need for further analysis of the biphasic does response in other cancer models including in vivo carcinogenesis studies and human cancer epidemiology. This issue is further enhanced when the potential mechanisms for hormetic responses in the cancer cells are discussed, since the same mechanisms participate in the carcinogenesis process. Overall, this review provides an excellent opening examination into the definition of biphasic dose-response effects of toxic and pharmacological agents in cancer cells.
164. Shankar S.S., Dubé M.P., Gorski J.C., Klaunig J.E., Steinberg H.O. (2005). Indinavir impairs endothelial function in healthy HIV-negative men. Am Heart J. 2005 Nov;150(5):933. PMID: 16290967.
Abstract. Potent antiretroviral treatment has drastically reduced mortality in HIV-infected patients but may accelerate atherosclerotic disease, which could be partially mediated via endothelial dysfunction. In 8 HIV-negative healthy males, leg blood flow responses to intraartery infusions of methacholine chloride (Mch), sodium nitroprusside, and NG-mono-methyl-L-arginine (L-NMMA) were measured before and after 4 weeks of daily oral indinavir. In the same subjects, we also assessed the effect of indinavir on lipids, insulin sensitivity, markers of inflammation, as well as oxidative stress. After 4 weeks of indinavir, the endothelium-dependent response to methacholine chloride was impaired (195% +/- 38% vs 83% +/- 13%, P < .05), the response to NG-mono-methyl-L-arginine (nitric oxide-dependent tone) was nearly abrogated (-30% +/- 4% vs -1% +/- 11%, P < .05), whereas the endothelium-independent response to sodium nitroprusside remained unchanged. Fasting insulin levels increased from 5.8 +/- 1.2 to 7.0 +/- 1.4 microU/mL (P < .05), and HOMA-IR scores increased from 1.3 +/- 0.3 to 1.6 +/- 0.3 U (P < .05). There were no changes in blood pressure, lipids, markers of inflammation, or oxidative stress. Four weeks of the HIV-1 protease inhibitor indinavir, in the absence of HIV-1 infection, causes vascular dysfunction most likely at the level of endothelial nitric oxide production. The vascular dysfunction may be mediated partially by the concomitant induction of insulin resistance but other mechanisms cannot be ruled out.
163. Kirman C.R., Gargas M.L., Marsh G.M., Strother D.E., Klaunig J.E., Collins J.J., Deskin R. (2005). Cancer dose–response assessment for acrylonitrile based upon rodent brain tumor incidence: use of epidemiologic, mechanistic, and pharmacokinetic support for nonlinearity. Regul Toxicol Pharmacol. 2005 Oct;43(1):85-103. PMID: 16099568.
Abstract. A cancer dose-response assessment was conducted for acrylonitrile (AN) using updated information on mechanism of action, epidemiology, toxicity, and pharmacokinetics. Although more than 10 chronic bioassays indicate that AN produces multiple tumors in rats and mice, a number of large, well-conducted epidemiology studies provide no evidence of a causal association between AN exposure and cancer mortality of any type. The epidemiological data include early industry exposures that are far higher than occur today and that approach or exceed levels found to be tumorigenic in animals. Despite the absence of positive findings in the epidemiology data, a dose-response assessment was conducted for AN based on brain tumors in rats. Mechanistic studies implicate the involvement of oxidative stress in rat brain due to a metabolite (2-cyanoethylene oxide or CEO, cyanide), but do not conclusively rule out a potential role for the direct genotoxicity of CEO. A PBPK model was used to predict internal doses (peak CEO in brain) for 12 data sets, which were pooled together to provide a consistent characterization of the dose-response relationship for brain tumor incidence in the rat. The internal dose corresponding to a 5% increase in extra risk (ED 05=0.017 mg/L brain) and its lower confidence limit (LED 05=0.014 mg/L brain) was used as the point of departure. The weight-of-evidence supports the use of a nonlinear extrapolation for the cancer dose-response assessment. A quantitative comparison of the epidemiology exposure-response data (lung and brain cancer mortality) to the rat brain tumor data in terms of internal dose adds to the confidence in the nonlinear extrapolation. Uncertainty factors of 200 and 220 (for the oral and inhalation routes, respectively) were applied to the LED 05 to account for interspecies variation, intraspecies variation, and the severity of the response measure. Accordingly, oral doses below 0.009 mg/kg-day and air concentrations below 0.1mg/m(3) are not expected to pose an appreciable risk to human populations exposed to AN.
162. Nie Y., Schoepp D.D., Klaunig J.E., Yard M., Lahiri D.K., Kubek M.J. (2005). Thyrotropin-releasing hormone (protirelin) inhibits potassium-stimulated glutamate and aspartate release from hippocampal slices in vitro. Brain Res. 2005 Aug 23;1054(1):45-54. PMID: 16055093.
Abstract. Excess excitatory amino acid release is involved in pathways associated with seizures and neurodegeneration. Thyrotropin-releasing hormone (TRH; protirelin), a brain-derived tripeptide, has shown efficacy in the treatment of such disorders, yet its mechanism of neuroprotection is poorly understood. Using superfused hippocampal slices, we tested the hypothesis that TRH could inhibit evoked glutamate/aspartate release in vitro. Rat hippocampal slices were first equilibrated in oxygenated Krebs buffer (KRB) (120 min) then superfused for 10 min with KRB (control), or KRB containing 0.1, 1, or 10 microM TRH respectively, prior to and during 5 min depolarization with high potassium KRB (50 mM [K(+)] +/- TRH). Fractions (1 min) were collected during the 5 min stimulation and for an additional 10 min thereafter and analyzed for glutamate and aspartate by HPLC. TRH had no effect on baseline glutamate/aspartate release, while all three TRH doses significantly (P < 0.05) inhibited peak 50 mM [K(+)]-stimulated glutamate/aspartate release, and glutamate remained below control (P < 0.05) at 15 min post stimulation. A 5 min pulse of TRH (10 microM) had no affect on basal glutamate/aspartate release, whereas the TRH pre-pulsed slices failed to release glutamate/aspartate by [K(+)]-stimulation given 15 min later. These results are the first to show a potent and prolonged inhibitory effect of TRH on evoked glutamate/aspartate release in vitro. These initial studies suggest that exogenous and/or endogenous TRH may function, in part, to modulate excess glutamate release in specific CNS loci. Additional studies are in progress to fully understand the mechanism of this potent effect of TRH and its implication in various CNS disorders.
161. Kamendulis L.M., Klaunig JE. (2005). Species differences in the induction of hepatocellular DNA synthesis by diethanolamine. Toxicol Sci. 2005 Oct;87(2):328-36. PMID: 16014740.
Abstract. Diethanolamine increased the incidence and multiplicity of liver tumors in the mouse following chronic exposure. Diethanolamine is known to inhibit cellular choline uptake. Since choline deficiency produces tumors in rodents, diethanolamine, through choline depletion, may result in tumor development in rodents. The potential for diethanolamine to function through this mode of action in humans is not known. The present studies examined the effect of diethanolamine (0-500 mug/ml) and choline depletion on DNA synthesis and changes in expression of genes involved in cell growth pathways in primary cultures of mouse, rat, and human hepatocytes. In mouse and rat hepatocytes DNA synthesis was increased following treatment with 10 mug/ml diethanolamine and higher (3- to 4-fold over control). In contrast, diethanolamine failed to increase DNA synthesis in human hepatocytes. Incubation of hepatocytes in medium containing reduced choline (1/10 to 1/100 of normal medium; 0.898 to 0.0898 mg/l vs. 8.98 mg/l) increased DNA synthesis (1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively); however, choline depletion did not induce DNA synthesis in human hepatocytes. Mouse and rat hepatocytes incubated in medium supplemented with 2- to 50-fold excess choline reduced diethanolamine-induced DNA synthesis to control levels or below. Gene expression analysis of mouse and rat hepatocytes following diethanolamine treatment showed increases in genes associated with cell growth and decreases in expression of genes involved in apoptotic pathways. These results support the hypothesis that choline depletion is central to the mode of action for the induction of rodent hepatic neoplasia by diethanolamine. Furthermore, since diethanolamine treatment or choline depletion failed to induce DNA synthesis in human hepatocytes, these results suggest that humans may not be at risk from the carcinogenic effects of diethanolamine.
160. Klaunig J.E., Kamendulis L.M. (2005). Mechanisms of acrylamide induced rodent carcinogenesis. Adv Exp Med Biol. 2005;561:49-62. PMID: 16438288.
Abstract. Acrylamide is a monomer of polyacrylamide, used in biochemistry, in paper manufacture, in water treatment, and as a soil stabilizer. The monomer can cause several toxic effects and has the potential for human exposure either through the environment or from occupational exposure. Recently, additional concern for the potential toxicity of acrylamide in humans has arisen with the finding of acrylamide formation in some processed foods. It has been established that following chronic exposure, rats exhibited an increase in the incidence of adrenal pheochromocytomas, testicular mesotheliomas, thyroid adenomas and mammary neoplasms in F344 rats. This has raised increased concerns regarding the carcinogenic risk to humans from acrylamide exposure. Studies examining the DNA reactivity of acrylamide have been performed and have had differing results. The tissue and organ pattern of neoplastic development seen in the rat following acrylamide exposure is not consistent with that seen with other strictly DNA reactive carcinogens. Based on the pattern of neoplastic development, it appears that acrylamide is targeting endocrine sensitive tissues. In the current monograph, studies on the effect of acrylamide on DNA reactivity and on altered cell growth in the target tissues in the rat are reported. DNA synthesis was examined in F344 rats treated with acrylamide (0, 2, or 15 mg/kg/day) for 7, 14, or 28 days. Acrylamide increased DNA synthesis in the target tissues (thyroid, testicular mesothelium, adrenal medulla) at all doses and time points examined. In contrast, in a non-target tissue (liver), no increase in DNA synthesis was seen. Examination of DNA damage using single cell gel electrophoresis (the Comet assay) showed an increase in DNA damage in the target tissues, but not in non-target tissue (liver). In addition, a cellular transformation model, (the Syrian Hamster Embryo (SHE) cell morphological transformation model), was used to examine potential mechanisms for the observed carcinogenicity of acrylamide. SHE cell studies showed that glutathione (GSH) modulation by acrylamide was important in the cell transformation process. Treatment with a sulfhydryl donor compound (NAC) reduced acrylamide transformation while depletion of GSH (BSO) resulted in an enhancement of transformation. In summary, acrylamide caused both an increase in DNA synthesis and DNA damage in mammalian tissues and cells suggesting that DNA reactivity and cell proliferation, in concert, may contribute to the observed acrylamide-induced carcinogenicity in the rat and has implication on the possible risk for human neoplasm development.
159. Klaunig J.E., Kamendulis L.M. (2005). Mode of action of butoxyethanol-induced mouse liver hemangiosarcomas and hepatocellular carcinomas. Toxicol Lett. 2005 Mar 28;156(1):107-15. PMID: 15705491.
Abstract. Chronic exposure to 2-butoxyethanol resulted in an increase in liver hemangiosarcomas and hepatic carcinomas in male mouse liver. No increase in liver neoplasia was observed in similarly exposed male and female rats or female mice. We have proposed that the production of liver neoplasia in the male mouse is the result of oxidative damage secondary to the hemolytic deposition of iron in the liver. Our working hypothesis is that the mode of action of butoxyethanol-induced mouse liver hemangiosarcomas and hepatic neoplasia involves the metabolism of 2-butoxyethanol to butoxyacetic acid which results in the induction of RBC hemolysis. This hemolytic response is translated into the accumulation of iron in both liver hepatocytes and Kupffer cells. The Kupffer cell response to this insult is two-fold: (1) the production of oxidative species-through both Kupffer cell activation and through the Fenton reaction involving iron and (2) the production of cytokines (for example TNF alpha). The induction of reactive oxygen species can, if not scavenged, produce oxidative DNA damage (the formation of OH8dG), as well as increase cell growth through modulation of gene expression. While the reactive oxygen species generation would occur in the both rats and mice, the ability of the rat to detoxify the reactive oxygen species would preclude the remaining steps from occurring. In contrast, in the mouse, the reactive oxygen species would override antioxidant defense mechanisms and allow the proposed mode of action to move forward. Our results to date in male B6C3F1 mice and male F344 rats treated with 2-butoxyethanol (via daily gavage; five times per week) at doses of 0, 225, 450, and 900 mg/kg/day (mice) and 0, 225, 450 mg/kg/day (rats), respectively, showed: an increase in hemolysis in 2-butoxyethanol treated rats and mice in a dose-dependent manner, in addition, an increase in the percent of iron stained Kupffer cells in the liver was observed following treatment with 450 and 900 mg/kg of 2-butoxyethanol in mice and 225 and 450 mg/kg of 2-butoxyethanol in rat. With the iron deposition, a biphasic increase in oxidative damage (OH8dG and malondialdehyde) was seen in mouse liver after treatment with 2-butoxyethanol. In contrast, no increase in oxidative damage was observed in the rat liver at any of the doses examined. Concomitant with the increase in oxidative damage, Vitamin E levels were similarly reduced by 2-butoxyethanol in both mice and rat liver. However, the basal level of Vitamin E in rat liver was 2.5-fold greater than in mouse liver. A biphasic induction of DNA synthesis was seen following 2-butoxyethanol in the mouse. In mouse liver, increased DNA synthesis was observed in hepatocytes at 90 days and in endothelial cells at 7 and 14 days at all doses. No change in DNA synthesis was seen in 2-butoxyethanol treated rat liver. No apparent differences in apoptosis and mitosis in the liver were observed in mouse and rat liver between 2-butoxyethanol treatment groups and untreated controls. These results suggest that the induction of DNA synthesis, possibly from oxidative stress and/or Kupffer cell activation, occurs selectively in the mouse liver, in endothelial cells and in hepatocytes following exposure to 2-butoxyethanol, and support the hypothesis proposed above.