2000-2004

 

2004

158. Lafferty J.S., Kamendulis L.M., Kaster J., Jiang J., Klaunig J.E. (2004). Subchronic acrylamide treatment induces a tissue-specific increase in DNA synthesis in the rat. Toxicol Lett. 2004 Dec 1;154(1-2):95-103. PMID: 15475183.

Abstract. Chronic treatment with acrylamide results in increased incidence of adrenal (pheochromocytoma), testicular (mesotheliomas) and thyroid (adenoma) neoplasia in male rats. While acrylamide has been demonstrated to be DNA reactive, the tissue pattern of neoplasm induction by acrylamide suggests other mechanisms in addition to DNA reactivity may be involved in the carcinogenesis of this compound. The present studies were performed to determine whether acrylamide or an acrylamide metabolite altered cell growth in the neoplastic target tissues in the rat. DNA synthesis, mitosis and apoptosis were examined in F344 and Sprague-Dawley male 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 for tumor development (thyroid, testicular mesothelium, adrenal medulla) in both rat species. In contrast, cell growth was not altered in the liver and adrenal cortex (non-target tissues for acrylamide carcinogenesis). No changes in apoptosis or mitosis were observed in any of the tissues examined. Inhibition of oxidative metabolism of acrylamide using 1-aminobenzotriazole reduced acrylamide-induced DNA synthesis only in the adrenal medulla, having no apparent effect in the testicular mesolthelium or thyroid. In summary, acrylamide produced a selective increase in DNA synthesis that correlates with the previously reported tumor target tissues.

157. Boatman R., Corley R., Green T., Klaunig J., Udden M. (2004). Review of studies concerning the tumorigenicity of 2-butoxyethanol in B6C3F1 mice and its relevance for human risk assessmentJ Toxicol Environ Health B Crit Rev. 2004 Sep-Oct;7(5):385-98. PMID: 15371241

Abstract. The U.S. National Toxicology Program (NTP) has completed 2-yr inhalation exposures in rats and mice with 2-butoxyethanol (BE). This review concerns the most significant findings from those studies and describes recent research into the mechanistic aspects of BE-mediated tumorigenesis in the mouse and the relevance of such effects to humans. Two tumor types were increased in B6C3F1 mice leading to the classification of “some evidence” of carcinogenicity: liver hemangiosarcomas in male mice and forestomach tumors in female mice (primarily benign papillomas). The results of research collected to date indicate that the tumorigenesis noted for BE was produced by indirect mechanisms. In particular, the occurrence of liver hemangiosarcomas in male mice has been linked to oxidative damage subsequent to red blood cell hemolysis and iron deposition in this organ. Oral administration of BE in mice up to 600 mg/kg/d for up to 90 d produces a dose-related increase in iron (Perl’s staining) in Kupffer cells and hepatocytes, increased DNA synthesis in endothelial cells, and enhanced oxidative damage. Further, iron alone, and not BE or BAA, is responsible for producing oxidative damage in cultured hepatocytes from rats or mice. Forestomach neoplasms in female mice were most likely a result of prolonged exposure-induced irritation with compensatory hyperplasia and subsequent tumor promotion. This mechanism is supported by studies indicating elevated levels of BE and BAA in the mouse forestomach tissues and stomach contents following multiple routes of exposure, forestomach epithelial cell cytotoxicity and cell proliferation following administration of BE and BAA, and the increased capacity of forestomach tissues from female mice to metabolize BE to the more irritating metabolite, BAA. The current article summarizes the results of a number of in vivo and in vitro studies designed to elucidate the underlying mechanisms of tumorigenesis by BE in the mouse and discusses the relevance of these for human risk.

156. Rusyniak D.E., Tandy S.L., Kamendulis L.M., Sprague J.E., Klaunig J.E. (2004). The effects of ecstasy (MDMA) on rat liver bioenergetics. Acad Emerg Med. 2004 Jul;11(7):723-9. PMID: 15231458.

Abstract. Use of the drug ecstasy (3,4-methylenedioxymethamphetamine [MDMA]) can result in life-threatening hyperthermia. Agents that uncouple mitochondrial oxidative phosphorylation are known to cause severe hyperthermia. In the present study, the authors tested the hypothesis that MDMA directly uncouples oxidative phosphorylation in rat liver mitochondria. Effects on mitochondrial bioenergetics were assessed both in vitro and ex vivo. In vitro studies consisted of measuring the effects of MDMA (0.1-5.0 mmol/L) on states of respiration in isolated rat liver mitochondria and on mitochondrial membrane potential in a rat liver cell line. In ex vivo studies, mitochondrial rates of respiration were measured in the livers of rats one hour after treatment with MDMA (40 mg/kg subcutaneously). With the in vitro mitochondrial preparations, only concentrations of 5 mmol/L MDMA showed evidence of uncoupling with a slight increase in state 4 respiration and a corresponding decrease in the respiratory control index. MDMA (0.1-5.0 mmol/L) failed to decrease the mitochondrial membrane potential in 3,3-dihexyloxacarbocyanide iodide-stained WB-344 cells after either one or 24 hours of incubation. Ex vivo rates of respiration obtained from the livers of rats one hour after treatment with MDMA (40 mg/kg subcutaneously) showed no evidence of mitochondrial uncoupling. These data suggest that while high concentrations of MDMA have some mild uncoupling effects in isolated mitochondria, these effects do not translate to cell culture or ex vivo studies in treated animals. These data do not support the view that the hyperthermia induced by MDMA is from a direct effect on mitochondrial oxidative phosphorylation.

155. Chirase N.K., Greene L.W., Purdy C.W., Loan R.W., Auvermann B.W., Parker D.B., Walborg E.F. Jr, Stevenson D.E., Xu Y., Klaunig J.E. (2004). Effect of transport stress on respiratory disease, serum antioxidant status, and serum concentrations of lipid peroxidation biomarkers in beef cattle. Am J Vet Res. 2004 Jun;65(6):860-4. PMID: 15198229.

Abstract. To determine the effect of transportation stress on serum concentrations of oxidative stress biomarkers of calves. 105 crossbred beef steer calves (mean [+/-SD] body weight, 207 +/- 21.2 kg). Calves were assembled at 1 location in Tennessee, and pretransit (day -3) blood samples were collected. Calves were allotted randomly by body weight into 2 groups. Calves were transported 1,930 miles to a feedlot in Texas, and 1 group received tilmicosin phosphate (33 microg/kg, s.c.) upon arrival. Calves were weighed and blood samples collected on the day of arrival (day 1) and on days 15, 22, and 28. Calves were scored daily for signs of bovine respiratory disease (BRD). Serum total antioxidant capacity (TACA) and serum malondialdehyde (MDA) concentrations were determined. Transportation stress significantly decreased mean serum TACA concentrations (from 147 +/- 31.2 U/mL to 133 +/- 20.1 U/mL) and significantly increased serum MDA concentrations (from 10.9 +/- 18.3 microg/mL to 30.2 +/- 50.5 microg/mL). Calves that died had a 43% increase in serum MDA concentration on day 1, compared with calves that lived (42.2 +/- 67.0 microg/mL vs 29.4 +/- 49.4 microg/mL, respectively). Calves that had > or =3 episodes of BRD had 2-fold higher serum MDA concentrations on day 1 than healthy calves. Tilmicosin-treated calves had a 20.8% significantly greater average daily gain and significantly greater serum TACA concentration than nontreated calves on day 28. Transportation stress increases serum concentrations of oxidative stress biomarkers that are related to episodes of BRD and mortality in calves.

154. Cohen S.M., Klaunig J., Meek M.E., Hill R.N., Pastoor T., Lehman-McKeeman L., Bucher J., Longfellow D.G., Seed J., Dellarco V., Fenner-Crisp P., Patton D. (2004). Evaluating the human relevance of chemically induced animal tumors. Toxicol Sci. 2004 Apr;78(2):181-6. PMID: 14737005.

Abstract. Defining the mode(s) of action by which chemicals induce tumors in laboratory animals has become a key to judgments about the relevance of such tumor data for human risk assessment. Frameworks for analyzing mode of action information appear in recent U.S. EPA and IPCS publications relating to cancer risk assessment. This FORUM paper emphasizes that mode of action analytical frameworks depend on both qualitative and quantitative evaluations of relevant data and information: (1) presenting key events in the animal mode of action, (2) developing a “concordance” table for side-by-side comparison of key events as defined in animal studies with comparable information from human systems, and (3) using data and information from mode of action analyses, as well as information on relative sensitivity and exposure, to make weight-of-evidence judgments about the relevance of animal tumors for human cancer assessments. The paper features a systematic analysis for using mode of action information from animal and human studies, based in part on case examples involving environmental chemicals and pharmaceuticals

153. Klaunig J.E., Kamendulis L.M. (2004). The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol. 2004;44:239-67. PMID: 14744246.

Abstract. Chemical carcinogenesis follows a multistep process involving both mutation and increased cell proliferation. Oxidative stress can occur through overproduction of reactive oxygen and nitrogen species through either endogenous or exogenous insults. Important to carcinogenesis, the unregulated or prolonged production of cellular oxidants has been linked to mutation (induced by oxidant-induced DNA damage), as well as modification of gene expression. In particular, signal transduction pathways, including AP-1 and NFkappaB, are known to be activated by reactive oxygen species, and they lead to the transcription of genes involved in cell growth regulatory pathways. This review examines the evidence of cellular oxidants’ involvement in the carcinogenesis process, and focuses on the mechanisms for production, cellular damage produced, and the role of signaling cascades by reactive oxygen species.

2003

152. Klaunig J.E., Babich M.A., Baetcke K.P., Cook J.C., Corton J.C., David R.M., DeLuca J.G., Lai D.Y., McKee R.H., Peters J.M., Roberts R.A., Fenner-Crisp P.A. (2003). PPARalpha agonist-induced rodent tumors: modes of action and human relevance. Crit Rev Toxicol. 2003;33(6):655-780. PMID: 14727734.

Abstract. Widely varied chemicals–including certain herbicides, plasticizers, drugs, and natural products–induce peroxisome proliferation in rodent liver and other tissues. This phenomenon is characterized by increases in the volume density and fatty acid oxidation of these organelles, which contain hydrogen peroxide and fatty acid oxidation systems important in lipid metabolism. Research showing that some peroxisome proliferating chemicals are nongenotoxic animal carcinogens stimulated interest in developing mode of action (MOA) information to understand and explain the human relevance of animal tumors associated with these chemicals. Studies have demonstrated that a nuclear hormone receptor implicated in energy homeostasis, designated peroxisome proliferator-activated receptor alpha (PPARalpha), is an obligatory factor in peroxisome proliferation in rodent hepatocytes. This report provides an in-depth analysis of the state of the science on several topics critical to evaluating the relationship between the MOA for PPARalpha agonists and the human relevance of related animal tumors. Topics include a review of existing tumor bioassay data, data from animal and human sources relating to the MOA for PPARalpha agonists in several different tissues, and case studies on the potential human relevance of the animal MOA data. The summary of existing bioassay data discloses substantial species differences in response to peroxisome proliferators in vivo, with rodents more responsive than primates. Among the rat and mouse strains tested, both males and females develop tumors in response to exposure to a wide range of chemicals including DEHP and other phthalates, chlorinated paraffins, chlorinated solvents such as trichloroethylene and perchloroethylene, and certain pesticides and hypolipidemic pharmaceuticals. MOA data from three different rodent tissues–rat and mouse liver, rat pancreas, and rat testis–lead to several different postulated MOAs, some beginning with PPARalpha activation as a causal first step. For example, studies in rodent liver identified seven “key events,” including three “causal events”–activation of PPARalpha, perturbation of cell proliferation and apoptosis, and selective clonal expansion–and a series of associative events involving peroxisome proliferation, hepatocyte oxidative stress, and Kupffer-cell-mediated events. Similar in-depth analysis for rat Leydig-cell tumors (LCTs) posits one MOA that begins with PPARalpha activation in the liver, but two possible pathways, one secondary to liver induction and the other direct inhibition of testicular testosterone biosynthesis. For this tumor, both proposed pathways involve changes in the metabolism and quantity of related hormones and hormone precursors. Key events in the postulated MOA for the third tumor type, pancreatic acinar-cell tumors (PACTs) in rats, also begin with PPARalpha activation in the liver, followed by changes in bile synthesis and composition. Using the new human relevance framework (HRF) (see companion article), case studies involving PPARalpha-related tumors in each of these three tissues produced a range of outcomes, depending partly on the quality and quantity of MOA data available from laboratory animals and related information from human data sources.

151. Cohen S.M., Meek M.E., Klaunig J.E., Patton D.E., Fenner-Crisp P.A. (2003). The human relevance of information on carcinogenic modes of action: overview. Crit Rev Toxicol. 2003;33(6):581-9. PMID: 14727732.

Abstract. Risk assessment policies and practice place increasing reliance on mode of action (MOA) data to inform conclusions about the human relevance of animal tumors. In June 2001, the Risk Science Institute of the International Life Sciences Institute formed a workgroup to study this issue. The workgroup divided into two subgroups, one developing and testing a “framework” for MOA relevance analysis and the other conducting an in-depth analysis of peroxisome proliferation-activated receptor (PPAR)alpha activation as the MOA for some animal carcinogens. This special issue of Critical Reviews in Toxicology presents the scientific reports emerging from this activity. These reports serve several purposes. For risk assessors in and out of government, they offer a new human relevance framework (HRF) that complements and extends existing guidance from other organizations. Regarding the specific MOA for peroxisome proliferating chemicals, these reports offer a state-of-the-science review of this important MOA and its role in tumorigenesis in three different tissues (liver, testis, and pancreas). The case studies in these reports present models for using MOA information to evaluate the hazard potential for humans. The cases also illustrate the substantial impact of a complete human relevance analysis, as distinct from an animal MOA analysis alone, on the nature and scope of risk assessment.

150. Lin S., Wei X., Xu Y., Yan C., Dodel R., Zhang Y., Liu J., Klaunig J.E., Farlow M., Du Y. (2003). Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons. Life Sci. 2003 Feb 21;72(14):1635-41. PMID: 12551752.

Abstract. Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS). Anti-oxidants by inhibiting free radical generation, protect neurons against 6-OHDA-induced neurotoxicity. In this study, we investigated whether or not minocycline, a neuroprotective compound, could directly protect neurons against 6-OHDA-induced neurotoxicity and inhibit 6-OHDA-induced free radical production in cultured rat cerebellar granule neurons (CGN). We now report that exposure of CGN to 6-OHDA (100 microM) resulted in a significant increase in free radical production with death of 86% of CGN. Pretreatment with minocycline (10 microM) for 2 h prevented 6-OHDA-induced free radical generation and neurotoxicity. Furthermore, minocycline also attenuated H(2)O(2)-induced neurotoxicity. Our results suggest that minocycline blocks 6-OHDA-induced neuronal death possibly by inhibiting 6-OHDA-induced free radical generation in CGN. Both the antioxidative and neuroprotective effects of minocycline may be beneficial in the therapy of Parkinson’s disease and other neurodegenerative diseases.

2002

149. Teuschler L., Klaunig J., Carney E., Chambers J., Conolly R., Gennings C., Giesy J., Hertzberg R., Klaassen C., Kodell R., Paustenbach D., Yang R. (2002). Support of science-based decisions concerning the evaluation of the toxicology of mixtures: a new beginning. Toxicol Pharmacol. 2002 Aug;36(1):34-9. PMID: 12383716.

Abstract.

148. Kamendulis L.M., Isenberg J.S., Smith J.H., Pugh G. Jr, Lington A.W., Klaunig J.E. (2002). Comparative effects of phthalate monoesters on gap junctional intercellular communication and peroxisome proliferation in rodent and primate hepatocytes. J Toxicol Environ Health A. 2002 Apr 26;65(8):569-88. PMID: 11995694.  

Abstract. Several phthalate esters, compounds used as plasticizers in a variety of commercial products, have been shown to induce hepatic tumors in rodents. In this study, the comparative effects of phthalate monoesters on inhibition of gap junctional intercellular communication and induction of peroxisomal beta-oxidation were assessed in primary cultured hepatocytes from rats, mice, hamsters, cynomolgus monkeys, and humans. A human liver cell line was also utilized. Eight monoesters examined included mono-2-ethylhexyl phthalate (MEHP), mono-n-octyl phthalate (MNOP), mono-isononyl phthalate (MINP, 3 types, -1, -2, and -3), mono-isoheptyl phthalate (MIHP), mono-isodecyl phthalate (MIDP), and mono-(heptyl, nonyl, undecyl) phthalate (M711P). Gap junctional intercellular communication was measured 4 and 24 h after treatment by lucifer yellow dye coupling. Gap junctional intercellular communication was inhibited in rat and mouse hepatocytes by all eight monoesters in a concentration-dependent manner. In most cases, gap junctional intercellular communication was significantly reduced at the lowest concentrations tested (50 pM). Inhibition of gap junctional intercellular communication in rodent cells was substantially reversed within 24 h of monoester removal. In contrast, cell-to-cell communication was not inhibited in hamster, cynomolgus, or human hepatocytes or in a human liver cell line at any concentration examined. In rat hepatocytes, peroxisomal beta-oxidation was elevated after treatment with MEHP, MINP, MIHP, and MIDP but not MNOP or M711P, and with all but MIHP in mouse hepatocytes. The eight phthalates produced no marked change on peroxisomal beta-oxidation in hepatocytes from other species. These data provide additional evidence that the toxicological effects of phthalate esters are species specific.

147. Siesky A.M., Kamendulis L.M., Klaunig J.E. (2002). Hepatic effects of 2-butoxyethanol in rodents. Toxicol Sci. 2002 Dec;70(2):252-60. PMID: 12441370.

Abstract. Chronic inhalation of 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 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. This occurs selectively in the male mouse and leads either directly or indirectly to liver neoplasia. To address this proposal, male B6C3F1 mice and male F344 rats were 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, and 450 mg/kg/day (rats) respectively. Following treatment for 7, 14, 28, and 90 days, DNA synthesis, oxidative damage, hematocrit, and iron deposition were measured in the livers. An increase in hemolysis (measured by a decrease in hematocrit and increase in relative spleen weight) was observed in 2-butoxyethanol-treated rats and mice in a dose-dependent manner. An increase in the percentage of iron-stained Kupffer cells 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 rats. A biphasic increase in oxidative damage (8-hydroxydeoxyguanosine and malondialdehyde) was seen in mouse liver after 7 and 90 days of treatment with 2-butoxyethanol, whereas no increases were observed in treated rat liver. Vitamin E levels were reduced by 2-butoxyethanol treatment in both mice and rat liver; however, the basal level of vitamin E was approximately 2.5-fold higher in rat than in mouse liver. A similar biphasic induction of DNA synthesis was seen following 2-butoxyethanol treatment in the mouse. In the 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 DNA synthesis, possibly from oxidative stress or Kupffer cell activation, occurs selectively in the mouse liver, primarily in endothelial cells (a target of 2-butoxyethanol neoplasia), following exposure to 2-butoxyethanol.

146. Kamendulis L.M., Zhang H., Wang Y., Klaunig J.E. (2002). Morphological transformation and oxidative stress induced by cyanide in Syrian hamster embryo (SHE) cells. Toxicol Sci. 2002 Aug;68(2):437-43. PMID: 12151639.

Abstract. Cyanide is a well-established poison known for its rapid lethal action and toxicity. Although long-term mammalian studies examining the carcinogenic potential of cyanide have not been previously reported, cyanide was reported to be positive in Salmonella typhimurium mutagenesis assay and induced aneuploidy in Drosophila. To further evaluate the carcinogenic potential of cyanide, the ability of cyanide to induce morphological transformation in Syrian hamster embryo (SHE) cells was studied. Cyanide induced a dose-dependent increase in morphological transformation in SHE cells following a 7-day continuous treatment. A significant increase in transformation was observed at potassium cyanide doses of 200 microM and greater. Transformation induced by cyanide was inhibited in a dose-related manner by vitamin E, suggesting a role of oxidative stress in the induction of morphological transformation by cyanide. Further, it was shown that 500 microM cyanide induced oxidative DNA damage in SHE cells, evidenced by the formation of 8-hydroxy-2′-deoxyguanosine (50-66% increase over control). The induction of oxidative stress by cyanide involved an early and temporal inhibition of antioxidant enzymes (catalase and superoxide dismutase) as well as an increased production of reactive oxygen species (1.5- to 2.0-fold over control).

145. Park J., Kamendulis L.M., Klaunig J.E. (2002). Mechanisms of 2-butoxyethanol carcinogenicity: studies on Syrian Hamster Embryo (SHE) cell transformation. Toxicol Sci. 2002 Jul;68(1):43-50. PMID: 12075109.  

Abstract. Previous studies showed that 2-butoxyethanol increased liver tumors in B6C3F1 mice following chronic exposure. While the mechanism of 2-butoxyethanol-induced liver carcinogenicity has not been defined, 2-butoxyethanol has been shown to induce hemolysis in rodents via 2-butoxyacetic acid, the major metabolite of 2-butoxyethanol. This toxic effect, coupled with the observation that continued treatment with 2-butoxyethanol results in hemosiderin deposition in the liver, has led to our hypothesis that liver carcinogenicity by 2-butoxyethnaol is mediated via oxidative stress (iron catalyzed) and Kupffer cell activation. The present study used Syrian Hamster Embryo (SHE) cell transformation, a surrogate in vitro model for carcinogenesis in vivo, to examine whether 2-butoxyethanol, 2-butoxyacetic acid, or iron (ferrous sulfate) produced cell transformation. SHE cells were treated with either 2-butoxyethanol (0.5-20 mM), 2-butoxyacetic acid (0.5-20 mM), or ferrous sulfate (0.5-75 microg/ml) for 7 days. 2-Butoxyethanol and 2-butoxyacetic acid did not induce cellular transformation. In contrast, treatment with ferrous sulfate (2.5 and 5.0 microg/ml) increased morphological transformation. Cotreatment of ferrous sulfate with the antioxidants alpha-tocopherol (vitamin E) or (-)-epigallocatechin-3-gallate (EGCG) prevented ferrous sulfate-induced transformation, suggesting the involvement of oxidative stress in SHE cell transformation. The level of oxidative DNA damage (OH8dG) increased following ferrous sulfate treatment in SHE cells; additionally, using single cell gel electrophoresis (comet assay), ferrous sulfate treatment produced an increase in DNA damage. Both DNA lesions were decreased by cotreatment of ferrous sulfate with antioxidants. These data support our proposal that iron, produced indirectly through hemolysis, and not 2-butoxyethanol or its metabolite 2-butoxyacetic acid, is responsible for the observed carcinogenicity of 2-butoxyethanol.

144. Zhang H., Kamendulis L.M., Klaunig J.E. (2002). Mechanisms for the induction of oxidative stress in Syrian hamster embryo cells by acrylonitrile. Toxicol Sci. 2002 Jun;67(2):247-55. PMID: 12011484.

Abstract. Chronic administration of acrylonitrile to rats resulted in an increase in the incidence of glial neoplasms of the brain. Recent studies have shown that acrylonitrile induces oxidative stress in rat brain and cultured rat glial cells. Acrylonitrile also induces morphological transformation concomitant with an increase in the formation of oxidized DNA in Syrian Hamster Embryo (SHE) cells in a dose-dependent manner. The mechanism for the induction of oxidative stress in SHE cells remains unresolved. The present study examined the effects of acrylonitrile on enzymatic and nonenzymatic antioxidants in SHE cells. SHE cells were treated with subcytolethal doses of acrylonitrile (0, 25, 50, and 75 microg/ml) for 4, 24, and 48 h. Acrylonitrile (50 microg/ml and 75 microg/ml) increased the amount of reactive oxygen species in SHE cells at all time points. Glutathione (GSH) was depleted and catalase and superoxide dismutase activities were significantly decreased in SHE cells after 4 h of treatment. The inhibition of these antioxidants was temporal, returning to control values or higher after 24 and 48 h. Xanthine oxidase activity was increased following 24 and 48 h treatment with acrylonitrile. 1-aminobenzotriazole, a suicidal P450 enzyme inhibitor, attenuated the effects of acrylonitrile on catalase and xanthine oxidase in SHE cells, suggesting that P450 metabolism is required for acrylonitrile to produce its effects on these enzymes. Additional studies showed that in the absence of metabolic sources acrylonitrile had no effect on either catalase or superoxide dismutase activity. These results suggest that the induction of oxidative stress by acrylonitrile involves a temporal decrease in antioxidants and increase in xanthine oxidase activity that is mediated by oxidative metabolism of acrylonitrile.

143. Park J., Kamendulis L.M., Klaunig J.E. (2002). Effects of 2-butoxyethanol on hepatic oxidative damage. Toxicol Lett. 2002 Jan 5;126(1):19-29. PMID: 11738267.

Abstract. 2-Butoxyethanol has been reported to induce an increase in liver tumors in male B6C3F1 mice following chronic inhalation while rats, similarly treated, showed no increase in liver tumors. The mechanism for the selective induction of cancer in mouse liver is unknown, however, 2-butoxyethanol has been shown to induce hemolysis in mice, resulting in an accumulation of hemosiderin (iron) in the liver. Previous studies by our group and others have shown that mouse liver compared to other rodent species has a lower antioxidant capacity and appears to be more susceptible to chemically-induced oxidative damage. Since iron is known to produce hydroxyl radicals (through the Fenton reaction), we have proposed that the 2-butoxyethanol-induced iron overload (through hemolysis) may contribute to the induction of liver neoplasia in the mouse. In the present studies, 2-butoxyethanol induced oxidative stress in the liver of mice following 7-day treatment by gavage. These studies also examined whether 2-butoxyethanol, 2-butoxy acetic acid (a major metabolite of 2-butoxyethanol) or iron (FeSO(4)) produced oxidative stress in mouse and rat hepatocytes. Oxidative stress was examined by measuring oxidative DNA damage (OH8dG), lipid peroxidation (MDA formation) and cellular vitamin E concentrations. Neither 2-butoxyethanol or 2-butoxyacetic acid induced changes in the oxidative stress parameters examined in either rat or mouse hepatocytes. In contrast, FeSO(4) produced a dose-related increase in OH8dG and MDA and a decrease in vitamin E levels following 24 h treatment. Mouse hepatocytes were more sensitive than rat hepatocytes to the oxidative damage induced by the FeSO(4). FeSO(4)-induced oxidative stress was not increased by co-treatment of FeSO(4) with either 2-butoxyethanol or 2-butoxy acetic acid. These results support the proposal that the induction of hepatic oxidative stress by 2-butoxyethanol in vivo occurs secondary to induction of hemolysis and iron deposition in the liver rather than as a direct action of 2-butoxyethanol or its main metabolite, 2-butoxy acetic acid.

142. Park J., Kamendulis L.M., Friedman M.A., Klaunig J.E. (2002). Acrylamide-induced cellular transformation. Toxicol Sci. 2002 Feb;65(2):177-83. PMID: 11812921.

Abstract. Acrylamide is a monomer of polyacrylamide, whose products are used in biochemistry, the manufacture of paper, water treatment, and as a soil stabilizer. While polymeric acrylamide is nontoxic, the monomer can cause several toxic effects and has the potential for human occupational exposure. While acrylamide is not mutagenic in prokaryotic mutagenesis assays, chronic acrylamide treatment in rodents has been shown to produce tumors in both rats and mice. The mechanism for the induction of tumors by acrylamide is not known. In the present study, we examined the possibility that acrylamide might induce cellular transformation, using Syrian hamster embryo (SHE) cell morphological transformation as well as potential mechanisms for the cellular transformation. Results showed that treatment with 0.5 mM and higher concentrations of acrylamide continuously for 7 days induced morphological transformation. Cotreatment with acrylamide and N-acetyl-L-cysteine (NAC), a sulfhydryl group donor, resulted in the reduction of acrylamide-induced morphological transformation in SHE cells. Cotreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, and acrylamide produced no change in morphological transformation when compared to acrylamide treatment only. Cotreatment with acrylamide and DL-buthionone-[S,R]-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, increased the percent of morphologically transformed colonies compared to acrylamide treatment alone. Acrylamide reduced GSH levels in SHE cells, and cotreatment with acrylamide and NAC prevented the acrylamide-induced reduction of GSH. BSO treatment with acrylamide enhanced the depletion of GSH. These results suggest that acrylamide itself, but not oxidative P450 metabolites of acrylamide appear to be involved in acrylamide-induced cellular transformation and that cellular thiol status (possibly GSH) is involved in acrylamide-induced morphological transformation.

2001

141. Isenberg J.S., Kamendulis L.M., Ackley D.C., Smith J.H., Pugh G. Jr, Lington A.W., McKee R.H., Klaunig J.E. (2001). Reversibility and persistence of di-2-ethylhexyl phthalate (DEHP)- and phenobarbital-induced hepatocellular changes in rodents. Toxicol Sci. 2001 Dec;64(2):192-9. PMID: 11719701.

Abstract. The tumor promotion stage of chemical carcinogenesis has been shown to exhibit a persistence of cellular effects during treatment and the reversibility of these changes upon cessation of treatment. Inhibition of gap-junctional intercellular communication and increased replicative DNA synthesis appear to be important in this process. The present study assessed the persistence and reversibility of gap-junctional intercellular communication inhibition, peroxisomal proliferation, and replicative DNA synthesis in livers from male F344 rats and B6C3F1 mice. Dietary administration of 20,000 mg/kg DEHP to male rats for 2 weeks decreased intercellular communication (67% of control) and enhanced replicative DNA synthesis (4.8-fold over control). Elevation of the relative liver weight and the induction of peroxisomal beta oxidation were also observed following treatment with 20,000 mg/Kg DEHP for 2 weeks. Following DEHP administration at a dose of 6000 mg/kg for 18 months, inhibition of gap-junctional intercellular communication persisted, and the relative liver weight and induction of peroxisomal beta oxidation remained elevated in both rats and male B6C3F1 mice. Treatment of rats and mice with phenobarbital for 18 months (500-mg/kg diet) also produced an increase in relative liver weight and a decrease in cell-to-cell communication. In recovery studies in which DEHP was administered to male F344 rats for 2 weeks and then withdrawn, the relative liver weight, rate of peroxisomal beta oxidation, increase in replicative DNA synthesis, and inhibition of gap-junctional intercellular communication returned to control values within 2 to 4 weeks after DEHP treatment ceased. Recovery studies with phenobarbital produced similar results. The primary active metabolite of DEHP, mono-2-ethylhexyl phthalate (MEHP), was detected in the livers of animals treated with DEHP for greater than 2 weeks. However, it could not be detected after removal of DEHP from the diet for 2 weeks. This study demonstrated that inhibition of gap-junctional intercellular communication, along with indicators of peroxisomal proliferation, including increased relative liver weight and enhanced peroxisomal beta oxidation, persist while DEHP treatment continues but reverses when treatment is stopped. Studies with phenobarbital produced a similar pattern of response.

140. Gottschling B.C., Maronpot R.R., Hailey J.R., Peddada S., Moomaw C.R., Klaunig J.E., Nyska A. (2001). The role of oxidative stress in indium phosphide-induced lung carcinogenesis in rats. Toxicol Sci. 2001 Nov;64(1):28-40. PMID: 11606799.

Abstract. Indium phosphide (IP), widely used in the microelectronics industry, was tested for potential carcinogenicity. Sixty male and 60 female Fischer 344 rats were exposed by aerosol for 6 h/day, 5 days/week, for 21 weeks (0.1 or 0.3 mg/m(3); stop exposure groups) or 105 weeks (0 or 0.03 mg/m(3) groups) with interim groups (10 animals/group/sex) evaluated at 3 months. After 3-month exposure, severe pulmonary inflammation with numerous infiltrating macrophages and alveolar proteinosis appeared. After 2 years, dose-dependent high incidences of alveolar/bronchiolar adenomas and carcinomas occurred in both sexes; four cases of squamous cell carcinomas appeared in males (0.3 mg/m(3)), and a variety of non-neoplastic lung lesions, including simple and atypical hyperplasia, chronic active inflammation, and squamous cyst, occurred in both sexes. To investigate whether inflammation-related oxidative stress functioned in the pathogenesis of IP-related pulmonary lesions, we stained lungs of control and high-dose animals immunohistochemically for four markers indicative of oxidative stress: inducible nitric oxide synthase (i-NOS), cyclooxygenase-2 (COX-2), glutathione-S-transferase Pi (GST-Pi), and 8-hydroxydeoxyguanosine (8-OHdG). Paraffin-embedded samples from the 3-month and 2-year control and treated females were used. i-NOS and COX-2 were highly expressed in inflammatory foci after 3 months; at 2 years, all four markers were expressed in non-neoplastic and neoplastic lesions. Most i-NOS staining, mainly in macrophages, occurred in chronic inflammatory and atypical hyperplastic lesions. GST-Pi and 8-OHdG expression occurred in cells of carcinoma epithelium, atypical hyperplasia, and squamous cysts. These findings suggest that IP inhalation causes pulmonary inflammation associated with oxidative stress, resulting in progression to atypical hyperplasia and neoplasia.

139. Xue H., Aziz R.M., Sun N., Cassady J.M., Kamendulis L.M., Xu Y., Stoner G.D., Klaunig J.E. (2001). Inhibition of cellular transformation by berry extracts. Carcinogenesis. 2001 Feb;22(2):351-6. Erratum in: Carcinogenesis 2001 May;22(5):831-3. PMID: 11181460.

Abstract. Recent studies have examined and demonstrated the potential cancer chemopreventive activity of freeze-dried berries including strawberries and black raspberries. Although ellagic acid, an abundant component in these berries, has been shown to inhibit carcinogenesis both in vivo and in vitro, several studies have reported that other compounds in the berries may also contribute to the observed inhibitory effect. In the present study, freeze-dried strawberries (Fragara ananassa, FA) or black raspberries (Rubus ursinus, RU) were extracted, partitioned and chromatographed into several fractions (FA-F001, FA-F003, FA-F004, FA-F005, FA-DM, FA-ME from strawberries and RU-F001, RU-F003, RU-F004, RU-F005, RU-DM, RU-ME from black raspberries). These extracts, along with ellagic acid, were analyzed for anti-transformation activity in the Syrian hamster embryo (SHE) cell transformation model. None of the extracts nor ellagic acid by themselves produced an increase in morphological transformation. For assessment of chemopreventive activity, SHE cells were treated with each agent and benzo[a]pyrene (B[a]P) for 7 days. Ellagic acid, FA-ME and RU-ME fractions produced a dose-dependent decrease in transformation compared with B[a]P treatment only, while other fractions failed to induce a significant decrease. Ellagic acid, FA-ME and RU-ME were further examined using a 24 h co-treatment with B[a]P or a 6 day treatment following 24 h with B[a]P. Ellagic acid showed inhibitory ability in both protocols. FA-ME and RU-ME significantly reduced B[a]P-induced transformation only when co-treated with B[a]P for 24 h. These results suggest that a methanol extract from strawberries and black raspberries may display chemopreventive activity. The possible mechanism by which these methanol fractions (FA-ME, RU-ME) inhibited cell transformation appear to involve interference of uptake, activation, detoxification of B[a]P and/or intervention of DNA binding and DNA repair.

138. Kamendulis L.M., Kolaja K.L., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (2001). Comparative effects of dieldrin on hepatic ploidy, cell proliferation, and apoptosis in rodent liver. J Toxicol Environ Health A. 2001 Jan 26;62(2):127-41. PMID: 11209821.

Abstract. Dieldrin-induced hepatocarcinogenesis, which is seen only in the mouse, apparently occurs through a nongenotoxic mechanism. Previous studies have demonstrated that dieldrin induces hepatic DNA synthesis in mouse, but not rat liver. A number of nongenotoxic hepatocarcinogens have been shown to increase hepatocyte nuclear ploidy following acute and subchronic treatment in rodents, suggesting that an induction of hepatocyte DNA synthesis may occur without a concomitant increase in cell division. The current study examined the effects of dieldrin on changes in hepatocyte DNA synthesis, mitosis, apoptosis, and ploidy in mouse liver (the sensitive strain and target tissue for dieldrin-induced carcinogenicity) and the rat liver (an insensitive species). Male F344 rats and B6C3F1 mice were treated with 0, 1, 3, or 10 mg dieldrin/kg diet and were sampled after 7, 14, 28, or 90 d on diet. Liver from mice fed 10 mg dieldrin/kg diet exhibited significantly increased DNA synthesis and mitosis at 14, 28, or 90 d on diet. In rats, no increase in DNA synthesis or mitotic index was observed. The apoptotic index in liver of mice and rats did not change over the 90-d study period. Exposure of mice to only the highest dose of dieldrin produced a significant increase in octaploid (8N) hepatocytes and a decrease in diploid (2N) hepatocytes, which were restricted primarily to centrilobular hepatocytes, with the periportal region showing little or no change from control. No changes in hepatocyte nuclear ploidy were observed in the rat. This study demonstrates that exposure to high concentrations of dieldrin is accompanied by increased nuclear ploidy and mitosis in mouse, but not rat, liver. It is proposed that the observed increase in nuclear ploidy in the mouse may reflect an adaptive response to dieldrin exposure.

2000

137. Kingery J.R., Sowinski K.M., Kraus M.A., Klaunig J.E., Mueller B.A. (2000). Vancomycin assay performance in patients with end-stage renal disease receiving hemodialysis. Pharmacotherapy. 2000 Jun;20(6):653-6. PMID: 10853620.

Abstract. To compare the performance of polyclonal fluorescence polarization immunoassay (pFPIA) with that of enzyme-multiplied immunoassay technique (EMIT) in patients receiving vancomycin and hemodialysis. Outpatient hemodialysis center. Seven subjects with end-stage renal disease treated with hemodialysis 3 times/week with a cellulose triacetate hemodialyzer. Subjects received vancomycin 1000 mg intradialytically during the first study session and 750 mg every other hemodialysis session thereafter for 4 weeks. Blood samples were obtained throughout the study, and vancomycin serum concentrations were determined by pFPIA and EMIT. The mean +/- SD difference (estimate of bias) between assays was -1.10 +/- 1.35 mg/L. The limits of agreement (mean difference +/- 1.96 x SD) between them were -3.80-1.60 mg/L. Our data suggest that the manufacturer’s changes in the vancomycin pFPIA eliminated overestimation of drug concentrations in patients undergoing high-permeability hemodialysis

136. Klaunig J.E., Kamendulis L.M., Xu Y. (2000). Epigenetic mechanisms of chemical carcinogenesis. Hum Exp Toxicol. 2000 Oct;19(10):543-55. PMID: 11211991.

Abstract. Chemically induced cancer is a multi-step process involving damage to the genome initially followed by clonal expansion of the DNA damaged cell eventually leading to a neoplasm. Chemical carcinogens have been shown to impact at all of the stages of the tumorigenesis process. It has become apparent that chemical and physical agents that induce cancer may do so through several different cellular and molecular mechanisms. Epigenetic (nongenotoxic) chemical carcinogens are those agents that function to induce tumor formation by mechanisms exclusive of direct modification or damage to DNA. These agents appear to modulate cell growth and cell death and exhibit dose response relationships between exposure and tumor formation. The exact and/or exclusive mechanisms by which these agents function have not been established, however, changes in cell growth regulation and gene expression are important to tumor formation. This review focuses on several potential mechanisms and cellular processes that may be involved in nongenotoxic chemical carcinogenesis.

135. Pugh G. Jr, Isenberg J.S., Kamendulis L.M., Ackley D.C., Clare L.J., Brown R., Lington A.W., Smith J.H., Klaunig J.E. (2000). Effects of di-isononyl phthalate, di-2-ethylhexyl phthalate, and clofibrate in cynomolgus monkeys. Toxicol Sci. 2000 Jul;56(1):181-8. PMID: 10869467.

Abstract. The effects of the peroxisome proliferators di-isononyl phthalate (DINP) and di-2-ethylhexyl phthalate (DEHP) were evaluated in young adult male cynomolgus monkeys after 14 days of treatment, with emphasis on detecting hepatic and other effects seen in rats and mice after treatment with high doses of phthalates. Groups of 4 monkeys received DINP (500 mg/kg/day), DEHP (500 mg/kg/day), or vehicle (0.5% methyl cellulose, 10 ml/kg) by intragastric intubation for 14 consecutive days. Clofibrate (250 mg/kg/day), a hypolipidemic drug used for cholesterol reduction in human patients was used as a reference substance. None of the test substances had any effect on body weight or liver weights. Histopathological examination of tissues from these animals revealed no distinctive treatment-related effects in the liver, kidney, or testes. There were also no changes in any of the hepatic markers for peroxisomal proliferation, including peroxisomal beta-oxidation (PBOX) or replicative DNA synthesis. Additionally, in situ dye transfer studies using fresh liver slices revealed that DINP, DEHP, and clofibrate had no effect on gap junctional intercellular communication (GJIC). None of the test substances produced any toxicologically important changes in urinalysis, hematology, or clinical chemistry; however, clofibrate produced some emesis, small increases in serum triglyceride, decreased calcium, and decreased weights of testes/epididymides and thyroid/parathyroid. The toxicological significance of these small changes is questionable. The absence of observable hepatic effects in monkeys at doses that produce hepatic effects in rodents suggests that DINP, DEHP, and clofibrate would also not elicit in primates other effects such as liver cancer. These data, along with results from in vitro hepatocyte studies, indicate that rodents are not good animal models for predicting the hepatic effects of phthalates in primates, including humans.

134. Smith J.H., Isenberg J.S., Pugh G. Jr, Kamendulis L.M., Ackley D., Lington A.W., Klaunig J.E. (2000). Comparative in vivo hepatic effects of Di-isononyl phthalate (DINP) and related C7-C11 dialkyl phthalates on gap junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX), and DNA synthesis in rat and mouse liver. Toxicol Sci. 2000 Apr;54(2):312-21. PMID: 10774813.

Abstract. The short-term hepatic effects of DINP (CAS 68515-48-0, designated DINP-1) in rats and mice were evaluated at tumorigenic and nontumorigenic doses from previous chronic studies. Groups of male F344 rats were fed diets with DINP-1 at concentrations of 0, 1000, or 12,000 ppm and male B6C3F1 mice at 0, 500, or 6000 ppm DINP-1. After 2 or 4 weeks of treatment, changes in liver weight, gap junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX), and replicative DNA synthesis were examined. In addition, hepatic and serum concentrations of the parent compound and major metabolites were determined. Relative to controls in both species, increased liver weight and PBOX at the high dose of DINP-1 were consistent with peroxisomal proliferation. Hepatic GJIC was inhibited and DNA synthesis was increased at the high dose of DINP-1, which is also consistent with the tumorigenic response in rats and mice reported in other chronic studies at these doses. These hepatic effects were not observed at the low doses of DINP-1. At comparable low doses of DINP-1 in other chronic studies, no liver tumors were observed in rats and mice. The monoester metabolite (MINP-1) was detected in the liver at greater concentrations in mice than rats. This result is also consistent with the dose-response observations in rat and mouse chronic studies. Additionally, other structurally similar dialkyl phthalate esters ranging from C7 to C11 were evaluated using a similar protocol for comparison to DINP-1; these included an alternative isomeric form of DINP (DINP-A), di-isodecyl phthalate (DIDP), di-isoheptyl phthalate (DIHP), di-heptyl, nonyl undecyl phthalate (D711P), and di-n-octyl phthalate (DNOP). Collectively, these data indicate that in rats and mice, DINP-1 and other C7-C11 phthalates exhibit a threshold for inducing hepatic cellular events. Further, where previous chronic data were available for these compounds, these phthalates elicited hepatic effects at doses that correlated with the tumorigenic response. Overall, these studies suggest a good correlation between the inhibition of GJIC when compared with the data on production of liver tumors in chronic studies

133. Zhang H., Xu Y., Kamendulis L.M., Klaunig J.E. (2000). Morphological transformation by 8-hydroxy-2′-deoxyguanosine in Syrian hamster embryo (SHE) cells. Toxicol Sci. 2000 Aug;56(2):303-12. PubMed PMID: 10910988.

Abstract. 8-Hydroxy-2′-deoxyguanosine (OH8dG) is one of the most prevalent oxidative DNA modifications found in eukaryotic cells. Previous studies have suggested an association between OH8dG formation and carcinogenesis. However, it is unclear whether OH8dG formation results in the necessary genotoxic events for cancer development. In the present study, the formation of OH8dG and its ability to transform Syrian hamster embryo (SHE) cells was examined. Methylene blue, a photosensitizer that in the presence of light can generate singlet oxygen by a type II mechanism, was used to produce oxidative DNA damage (predominantly OH8dG) in SHE cells. Photoactivated methylene blue produced a dose-dependent increase in OH8dG as well as a dose-dependent increase in morphological transformation in SHE cells. SHE cells transfected with DNA that contained increasing concentrations of OH8dG displayed a dose-dependent increase in morphological transformation. Treatment with beta-carotene (a singlet oxygen quencher) inhibited both the formation of OH8dG and the induction of morphological transformation in photoactivated methylene blue-treated SHE cells. These results suggest that formation of OH8dG can induce morphological transformation and provide further support for a role of OH8dG formation in the carcinogenesis process.

132. Zhang H., Kamendulis L.M., Jiang J., Xu Y., Klaunig J.E. (2000). Acrylonitrile-induced morphological transformation in Syrian hamster embryo cells. Carcinogenesis. 2000 Apr;21(4):727-33. PMID: 10753209.

Abstract. Acrylonitrile (ACN) is a monomer used in the synthesis of rubber, fibers and plastics. Previous studies demonstrated that ACN induces brain neoplasms (predominately astrocytomas) in rats following chronic treatment. While the mechanisms of ACN-induced glial cell carcinogenicity have not been completely elucidated, investigations by our group and others have suggested a role for the induction of oxidative stress and the resultant oxidative damage in this process. In vitro cell transformation models are useful for detecting and studying the mechanisms of chemical carcinogenesis. Cell transformation by chemical carcinogens in Syrian hamster embryo (SHE) cells exhibits a multistage process similar to that observed in vivo, for both non-genotoxic and genotoxic carcinogens. In the present study, the ability of ACN to induce morphological transformation and oxidative damage was examined in SHE cells. ACN induced an increase in morphological transformation at doses of 50, 62.5 and 75 microg/ml (maximum sub-toxic dose tested) following 7 days of continuous treatment. SHE cells exposed to ACN for 24 h failed to increase morphological transformation. Morphological transformation by ACN was inhibited by co-treatment with the antioxidants alpha-tocopherol and (-)-epigallocathechin-3 gallate (EGCG) for 7 days. Treatment of SHE cells with 75 microg/ml ACN produced a significant increase in 8-hydroxy-2′-deoxyguanosine that was also inhibited by co-treatment with alpha-tocopherol or EGCG. These results support the proposal that oxidative stress and the resulting oxidative damage is involved in ACN-induced carcinogenicity

131. Isenberg J.S., Kamendulis L.M., Smith J.H., Ackley D.C., Pugh G. Jr, Lington A.W., Klaunig J.E. (2000). Effects of Di-2-ethylhexyl phthalate (DEHP) on gap-junctional intercellular communication (GJIC), DNA synthesis, and peroxisomal beta oxidation (PBOX) in rat, mouse, and hamster liver. Toxicol Sci. 2000 Jul;56(1):73-85. PMID: 10869455.

Abstract. The present study evaluated the effect of di-2-ethylhexyl phthalate (DEHP) on gap-junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX) activity, and replicative DNA synthesis in several rodent species with differing susceptibilities to peroxisome proliferator-induced hepatic tumorigenesis. A low (non-tumorigenic) and high (tumorigenic) dietary concentration of DEHP was administered to male F344 rats for 1, 2, 4, and 6 weeks. Additionally, a previously non-tumorigenic dose (1000 ppm) and tumorigenic dose of DEHP (12,000 ppm), as determined by chronic bioassay data, were examined following 2 weeks dietary administration. Male B6C3F1 mice were fed the non-tumorigenic concentration, 500 ppm, and the tumorigenic concentration, 6000 ppm, of DEHP for two and four weeks. The hepatic effects of low and high concentrations of DEHP, 1000 and 6000 ppm, were also examined in male Syrian Golden hamsters (refractory to peroxisome proliferator-induced tumorigenicity). In rat and mouse liver, a concentration-dependent increase in the relative liver weight, PBOX activity, and replicative DNA synthesis was observed at the earliest time point examined. Concurrent to these observations was an inhibition of GJIC. In hamster liver, a slight increase in the relative liver weight, PBOX activity, and replicative DNA synthesis was observed. However, these effects were not of the same magnitude or consistency as those observed in rats or mice. Furthermore, DEHP had no effect on GJIC in hamster liver at any of the time points examined (2 and 4 weeks). HPLC analysis of DEHP and its primary metabolites, mono-2-ethylhexyl phthalate (MEHP), and phthalate acid (PA), indicated a time- and concentration-dependent increase in the hepatic concentration of MEHP. At equivalent dietary concentrations and time points, the presence of MEHP, the primary metabolite responsible for the hepatic effects of DEHP, demonstrated a species-specific response. The largest increase in the hepatic concentration of MEHP was observed in mice, which was greater than the concentration observed in rats. The hepatic concentration of MEHP was lowest in hamsters. Hepatic concentrations of DEHP and phthalic acid were minimal and did not correlate with concentration and time. Collectively, these data demonstrate the inhibition of hepatic GJIC and increased replicative DNA synthesis correlated with the observed dose- and species-specific tumorigenicity of DEHP and may be predictive indicators of the nongenotoxic carcinogenic potential of phthalate esters.

130. Isenberg J.S., Klaunig J.E. (2000). Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in liver cells. Toxicol Sci. 2000  Feb;53(2):340-51. PMID: 10696782.

Abstract. Rotenone inhibits spontaneously and chemically induced hepatic tumorigenesis in rodents through the induction of apoptosis. However, the mechanism for the induction of apoptosis by rotenone has not been defined. Mitochondrial dysfunction, in particular the induction of the mitochondrial membrane permeability transition (MPT), has been implicated in the cascade of events involved in the induction of apoptosis. Inhibition of the mitochondrial electron-transport chain reduces the mitochondrial transmembrane potential (delta(psi)m), which may induce the formation of the mitochondrial permeability transition pore and the subsequent MPT. Fluorescent microscopy of Hoechst 33258-stained WB-F344 cells, a rat-liver cell line, was utilized to examine the effect of the mitochondrial respiratory chain inhibitor, rotenone (0.5-5 microM), atractyloside (5-10 microM), and cyclosporin A (2.5-10 microM) on apoptosis. A time- and concentration-dependent increase in liver cell apoptosis was observed following treatment with rotenone and atractyloside (11.7- and 7.7-fold, respectively, over solvent control). Cotreatment with 7.5- and 10 microM-cyclosporin A for 12 h inhibited the apoptogenicity of 5-microM rotenone treatment. A similar effect was observed following cyclosporin A cotreatment with atractyloside. Rotenone induced a rapid increase in apoptosis (within 20 min of treatment). By 2 h of treatment, the morphological appearance of apoptosis was similar to that observed in cultures treated continuously with rotenone for 12 h. Inhibition studies demonstrated that cyclosporin A prevented apoptosis if the exposure to it occurred prior to the 20-min threshold necessary to induce apoptosis by rotenone. Mitochondrial function was examined by staining with the mitochondrial membrane potential (delta(psi)m)-sensitive fluorochrome, MitoTracker Red (CMXRos) and confirmed utilizing cytofluorometric analysis of DiOC6(3)-stained cells. Rotenone (5.0-microM) and atractyloside (5.0-microM) reduced the percent of CMXRos or DiOC6(3)-positive (delta(psi)m-positive) liver cells within 15 min and throughout the duration of the study (6 h) to approximately 65-80% and 50-80% of control. However, cotreatment with concentrations of cyclosporin A that inhibited the apoptogenicity of rotenone and atractyloside prevented the rotenone- and atractyloside-induced reduction of the delta(psi)m. Therefore, the apoptogenic effect of rotenone and atractyloside appears to occur rapidly (within 20 min) and is irreversible once mitochondrial damage occurs. The inhibition of the rotenone- and atractyloside-induced apoptosis and mitochondrial dysfunction by cyclosporin A suggests the MPT may be involved in the induction of apoptosis by rotenone.

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