129. Brophy D.F., Sowinski K.M., Kraus M.A., Moe S.M., Klaunig J.E., Mueller B.A. (1999). Small and middle molecular weight solute clearance in nocturnal intermittent peritoneal dialysis. Perit Dial Int. 1999 Nov-Dec;19(6):534-9. PMID: 10641773.

Abstract.To determine the dialysate-to-plasma (D/P) concentration ratios and peritoneal dialytic clearance (CI(D)) of substances with a wide range of molecular weights in subjects receiving a simulated nocturnal intermittent peritoneal dialysis (NIPD) session. Open-label single-dose study. Six end-stage renal disease patients undergoing peritoneal dialysis (PD). Clinical research center of a university-affiliated hospital. Subjects received intravenous gentamicin and vancomycin on the first day of the study. Subjects received no PD until their return on the following day, when subjects underwent a simulated NIPD session utilizing four 2- to 2.5-L peritoneal dialysate dwells of 2 hours. Blood and dialysate samples were collected immediately before the session and after each dialysate dwell for determination of urea, creatinine, gentamicin, vancomycin, and beta2-microglobulin (beta2M) concentrations. Each solute’s D/P concentration ratio and peritoneal CI(D) were calculated. The (mean +/- SD) 2-hour D/P concentration ratios were 0.78 +/- 0.05 (urea), 0.49 +/- 0.11 (creatinine), 0.38 +/- 0.08 (gentamicin), 0.11 +/- 0.06 (vancomycin), and 0.07 +/- 0.03 (beta2M). Peritoneal CI(D) values (mL/min of dialysis) were 19.0 +/- 2.8 (urea), 12.1 +/- 3.5 (creatinine), 8.4 +/- 2.8 (gentamicin), 2.7 +/- 1.5 (vancomycin), and 1.7 +/- 0.8 (beta2M). The D/P concentration ratios and peritoneal CI(D) values for urea, creatinine, and gentamicin were significantly different from vancomycin and beta2M (repeated measures ANOVA, p < 0.05). Beta2-microglobulin peritoneal CI(D) was strongly related to gentamicin peritoneal CI(D) (r = 0.96, p < 0.05). Small molecular weight solutes have significantly greater D/P and peritoneal CI(D) than middle molecular weight solutes in NIPD. In NIPD, daily peritoneal CI(D) of beta2M is lower than that reported in continuous ambulatory PD. NIPD also results in lower drug CI(D) than that reported in continuous ambulatory PD studies.

128. Klaunig J.E., Kamendulis L.M. (1999). Mechanisms of cancer chemoprevention in hepatic carcinogenesis: modulation of focal lesion growth in mice. Toxicol Sci. 1999 Dec;52(2 Suppl):101-6. PMID: 10630597.

Abstract. Studies in our laboratory have concentrated on further understanding the mechanism by which chemicals induce cancer and the means to prevent or retard this process. Recent investigations have revolved around the role of oxidative stress and oxidative damage in the induction of cancer by nongenotoxic carcinogens. Hepatocarcinogenic compounds including selective chlorinated hydrocarbons appeared to induce oxidative stress in the liver. This oxidative stress and oxidative damage in turn may be responsible for the tumor-promoting activity of these compounds. Reduction of oxidative damage by antioxidants, or dietary-restriction, results in an ablation of the induction of selective cell growth by these agents. The oxidative stress induced by nongenotoxic agents may influence cell proliferation and/or apoptosis in the preneoplastic cells. Our studies with nongenotoxic hepatic carcinogens showed a dose-dependent increase in oxidative stress and an increase in hepatic focal lesion growth. Antioxidant dietary supplementation or caloric restriction prevented the lesion growth. This appeared to be through an increase in apoptosis in the hepatic lesions.

127. Stevenson D.E., Walborg E.F. Jr, North D.W., Sielken R.L. Jr, Ross C.E., Wright A.S., Xu Y., Kamendulis L.M., Klaunig J.E. (1999). Monograph: reassessment of human cancer risk of aldrin/dieldrin. Toxicol Lett. 1999 Oct 5;109(3):123-86. PMID: 10555138.

Abstract. In 1987, the US Environmental Protection Agency (EPA) classified aldrin and dieldrin as category B2 carcinogens, i.e. probable human carcinogens, based largely on the increase in liver tumors in mice fed either organochlorine insecticide. At that date, the relevant epidemiology was deemed inadequate to influence the cancer risk assessment. More time has now elapsed since early exposures of manufacturing workers to aldrin/dieldrin; therefore, updated epidemiological data possess more power to detect exposure-related differences in cancer risk and mortality. Also, recent experimental studies provide a plausible mode of action to explain the mouse specificity of dieldrin-induced hepatocarcinogenesis and call into question the relevance of this activity to human cancer risk. This monograph places this new information within the historic and current perspectives of human cancer risk assessment, including EPA’s 1996 Proposed Guidelines for Carcinogen Risk Assessment. Updated epidemiological studies of manufacturing workers in which lifetime exposures to aldrin/dieldrin have been quantified do not indicate increased mortality or cancer risk. In fact, at the middle range of exposures, there is evidence of a decrease in both mortality from all causes and cancer. Recent experimental studies indicate that dieldrin-induced hepatocarcinogenesis in mice occurs through a nongenotoxic mode of action, in which the slow oxidative metabolism of dieldrin is accompanied by an increased production of reactive oxygen species, depletion of hepatic antioxidant defenses (particularly alpha-tocopherol), and peroxidation of liver lipids. Dieldrin-induced oxidative stress or its sequelae apparently result in modulation of gene expression that favors expansion of initiated mouse, but not rat, liver cells; thus, dieldrin acts as a nongenotoxic promoter/accelerator of background liver tumorigenesis in the mouse. Within the framework of EPA’s Proposed Guidelines for Carcinogen Risk Assessment, it is proposed that the most appropriate cancer risk descriptor for aldrin/dieldrin, relating to the mouse liver tumor response, is ‘not likely a human carcinogen’, a descriptor consistent with the example of phenobarbital cited by EPA.

126. Lahiri, D.K., Xu, Y., Klaunig, J.E., Baiyewu, O., Ogunniyi, A., Hall, K., Hendrie, H., Sahota, A. (1999). Effect of Oxidative Stress on DNA Damage and Beta-Amyloid Precursor Proteins in Lymphoblastoid Cell Lines from a Nigerian Population. Annals of New York Academy of Sciences, 893, 331-336. PMID: 10672260.

Abstract. The epsilon 4 allele of apolipoprotein E (APOE) is strongly associated with late-onset Alzheimer’s disease (AD) in Caucasian populations, but our studies suggest that APOE epsilon 4 is not a risk factor for AD in Nigerian blacks and is a weak risk factor in African-Americans. The prevalence of AD is lower in Nigerians than in African-Americans. Increased oxidative damage to macromolecules in brain tissue by reactive oxygen species (ROS) has been reported in AD. Here we examined the effects of endogenous and induced oxidative stress on total (nuclear and mitochondrial) DNA damage in lymphoblastoid cell lines (5 probable AD and 3 controls) from Ibadan, Nigeria. Cells were exposed to 200 microM t-butyl peroxide (a generator of ROS) for 4 hours. Total DNA was isolated and digested with nuclease P1 and alkaline phosphatase. DNA fragments were separated by HPLC and the levels of 8-hydroxy-2′-deoxyguanosine (OH8dG, an indicator of DNA damage) and deoxyguanosine (dG) determined. We did not detect a significant difference in the OH8dG/dG ratio in untreated or treated cell lines in the two groups, and this was independent of APOE genotype. We also examined, by Western blotting, the level of beta-amyloid precursor protein (APP) which is involved in AD. The level of the heat shock protein (HSP-70) was examined as a control. There was a slight decrease in levels of APP and HSP-70 following treatment. Studies in cell lines from Caucasian subjects have shown an increase in mitochondrial DNA damage following oxidative challenge. Our preliminary results suggest that African populations are less vulnerable to chemical-induced oxidative DNA damage

125. Kamendulis L.M., Jiang J., Xu Y., Klaunig J.E. (1999). Induction of oxidative stress and oxidative damage in rat glial cells by acrylonitrile. Carcinogenesis. 1999 Aug;20(8):1555-60. PMID: 10426806.

Abstract. Chronic treatment of rats with acrylonitrile (ACN) resulted in a dose-related increase in glial cell tumors (astrocytomas). While the exact mechanism(s) for ACN-induced carcinogenicity remains unresolved, non-genotoxic and possibly tumor promotion modes of action appear to be involved in the induction of glial tumors. Recent studies have shown that ACN induced oxidative stress selectively in rat brain in a dose-responsive manner. The present study examined the ability of ACN to induce oxidative stress in a rat glial cell line, a target tissue, and in cultured rat hepatocytes, a non-target tissue of ACN carcinogenicity. Glial cells and hepatocytes were treated for 1, 4 and 24 h with sublethal concentrations of ACN. ACN induced an increase in oxidative DNA damage, as evidenced by increased production of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) in glial cells but not in rat hepatocytes. Hydroxyl radical formation following ACN treatment was also selectively increased in glial cells. Following 1 and 4 h of ACN exposure, the levels of the non-enzymatic antioxidant glutathione, as well as the activities of the enzymatic antioxidants catalase and superoxide dismutase were significantly decreased in the rat glial cells. Lipid peroxidation and the activity of glutathione peroxidase were not affected by ACN treatment in rat glial cells. No changes in any of these biomarkers of oxidative stress were observed in hepatocytes treated with ACN. These data indicate that ACN selectively induced oxidative stress in rat glial cells.

124. Kamendulis L.M., Jiang J., Zhang H., deFeijter-Rupp H., Trosko J.E., Klaunig J.E. (1999). The effect of acrylonitrile on gap junctional intercellular communication in rat astrocytes. Cell Biol Toxicol. 1999 Jun;15(3):173-83. PMID: 10580550.

Abstract. Rats chronically exposed to acrylonitrile (ACN) have shown a dose-dependent increase in the incidence of astrocytomas in the brain. The mechanism(s) by which ACN induces cancer in rodents has not been established. ACN does not appear to be directly genotoxic in the brain and thus a nongenotoxic mode of action has been proposed. Inhibition of gap junctional intercellular communication (GJIC) has been shown to be a property of many nongenotoxic carcinogens. The present study examined the effects of ACN on GJIC in a rat astrocyte transformed cell line, DI TNC1 cells (a target cell for ACN carcinogenicity) and primary cultured hepatocytes (a nontarget cell for ACN carcinogenicity). ACN inhibited GJIC in rat astrocytes in a dose-dependent manner. Inhibition of GJIC was observed following 2 h treatment with 0.10 mmol/L and 1.00 mmol/L ACN. However, in primary cultured hepatocytes, ACN exposed did not result in inhibition of GJIC even after 48 h of continued treatment. In the astrocytes, GJIC inhibition plateaued after 4 h of treatment and remained blocked throughout the entire experimental period examined. Inhibition of GJIC in DI TNC1 cells was reversed by removal of ACN from the culture medium after 4 or 24 h of treatment. Cotreatment of astrocytes with vitamin E reduced the effect of ACN-induced inhibition of GJIC. Similarly, inhibition of GJIC was prevented by treatment with 2-oxothiazolidine-4-carboxylic acid (OTC), a precursor of glutathione synthesis. Decreasing cellular glutathione by treatment with buthionine sulfoxamine alone (without ACN) did not affect GJIC in astrocytes. Collectively, these results demonstrate that treatment with ACN caused a selective inhibition of GJIC in rat DI TNC1 astrocytes (the target cell type), but not in rat hepatocytes (a nontarget tissue). Inhibition of GJIC in astrocytes was reversed by treatment with antioxidants and suggests a potential role for oxidative stress in ACN-induced carcinogenesis.

123. Herrman C.E., Sanders R.A., Klaunig J.E., Schwarz L.R., Watkins J.B. 3rd. (1999). Decreased apoptosis as a mechanism for hepatomegaly in streptozotocin-induced diabetic rats. Toxicol Sci. 1999 Jul;50(1):146-51. PMID: 10445763.

Abstract. Insulin-dependent diabetes mellitus in both humans and animals leads to structural and functional changes including hepatomegaly. This study examined hypertrophy, hyperplasia, and apoptosis, three basic aspects of tissue growth, in livers of Sprague-Dawley and Wistar rats made diabetic by iv injection of streptozotocin 8, 30, or 90 days previously. Immunohistochemical measurement of proliferating cell nuclear antigen revealed that hepatic DNA labeling indices were similar in normal control animals and diabetic rats 30 or 90 days post diabetic induction, but were reduced to 45 to 50% of control in insulin-treated diabetic animals, perhaps due to altered receptor activity or to partial insulin resistance, as reported previously. Flow cytometry indicated a 613% increase in diploid hepatocytes in the livers of diabetic rats 30 days after the onset of diabetes, compared to control. Diabetic livers contained 29% fewer tetraploid cells, 81% fewer octaploid cells, and 20% more binucleated hepatocytes than normal controls. At 90 days, the overall smaller size of hepatocytes in diabetic tissue was evidenced by more cells per area. Insulin treatment prevented some of these changes, but did not restore ploidy to a normal distribution. Mitosis, while 300% of normal at 8 days after streptozotocin injection, was reduced to 25% of normal after 90 days of diabetes. The morphological evidence of apoptosis was decreased by 23% to 76% in the diabetic liver, and was reversed but not normalized by insulin treatment. This study indicates that the hepatomegaly observed in streptozotocin-induced experimental diabetes may be due primarily to early hyperplasia, and later decreased apoptosis.

122. Klaunig J.E., Xu Y., Han C., Kamendulis L.M., Chen J., Heiser C., Gordon M.S., Mohler E.R. 3rd. (1999). The effect of tea consumption on oxidative stress in smokers and nonsmokers. Proc Soc Exp Biol Med. 1999 Apr;220(4):249-54. PMID: 10202398.

Abstract. While the anticarcinogenic effects of tea in animal models have been reported by several groups, human epidemiological studies examining tea consumption and cancer prevention have produced equivocal results. The beneficial properties of tea to human health may be related to the antioxidant properties of tea components. However, little evidence has been provided that tea consumption can either increase the antioxidant capacity or decrease oxidative stress in humans. In the present study, the effects of tea treatment (green tea) on biomarkers of oxidative stress were investigated in smokers and nonsmokers in two volunteer study groups (one in China and the other in United States). Green tea consumption in both study groups decreased oxidative DNA damage (8-OHdG in white blood cells and urine), lipid peroxidation (MDA in urine), and free radical generation (2, 3-DHBA in urine) in smokers. Nonsmokers (US study group) also exhibited a decrease in overall oxidative stress. 


121. Watkins J.B. 3rd, Klaunig J.E., Smith H.M., Cornwell P., Sanders R.A. (1998). Streptozotocin-induced diabetes increases gamma-glutamyltranspeptidase activity but not expression in rat liver. J Biochem Mol Toxicol. 1998;12(4):219-25. PMID: 9580874.

Abstract. Earlier work describing increased biliary excretion of the acetaminophen-cysteine conjugate advanced the hypothesis that streptozotocin-induced diabetes increases gamma-glutamyltranspeptidase (GGT) expression in Sprague-Dawley rats. To test this hypothesis, rats were divided into control, diabetic, and insulin-treated diabetic groups. Diabetes was induced by intravenous injection of 45 mg streptozotocin/kg body weight and was effectively controlled by insulin treatment in the appropriate group. Densitometric quantification demonstrated that hepatic GGT activity in diabetic rats was significantly increased when compared to normal and insulin-treated diabetic controls. Histochemical staining of liver was greater in female than in male rats, and staining increased in female rat liver as the duration of diabetes lengthened from 30 to 90 days. GGT activity was increased by diabetes in liver canalicular-enriched and basolateral-enriched membrane preparations, and it was unchanged in renal brush border-enriched membranes. Total mRNA isolated from diabetic and insulin-treated diabetic rat livers did not conclusively demonstrate an elevation of GGT mRNA relative to normal. Western blot analysis showed no differences in the amount of GGT in diabetic versus normal rat livers. These data indicate that streptozotocin-induced diabetes does not alter the expression of, but does increase the activity of, GGT in liver.

120. Kolaja K.L., Xu Y., Walborg E.F. Jr, Stevenson D.E., Klaunig J.E. (1998). Vitamin E modulation of dieldrin-induced hepatic focal lesion growth in mice. J Toxicol Environ Health A. 1998 Mar 27;53(6):479-92. PMID: 9537283.

Abstract. The effect of vitamin E on dieldrin-induced hepatic focal lesion growth in male B6C3F1 mice previously treated with diethylnitrosamine (DEN) was investigated. After hepatic focal lesions were formed, mice were placed into one of the following treatment groups: Group 1, 50 mg vitamin E/kg diet (control NIH-07 diet); Group 2, 10 mg dieldrin/kg NIH-07 diet; Group 3, 10 mg dieldrin and 450 mg vitamin E/kg NIH-07 diet; and Group 4, 450 mg vitamin E/kg NIH-07 diet. Mice were killed and necropsied after 30 and 60 d of dietary treatment. The effect of treatment on lesion growth was examined by measuring the number of focal lesions per liver and the relative hepatic focal lesion volume. In addition, the possible cellular mechanism of focal hepatocyte growth was investigated by examining both focal DNA synthesis and apoptosis. Dieldrin treatment alone (Group 2) increased the focal lesion volume, focal lesion number, and focal lesion labeling index. Supplementation with vitamin E (Group 3) blocked this effect. Vitamin E supplementation to the diet alone (Group 4) also enhanced focal lesion growth and increased the number of lesions per liver, the relative focal volume, and the labeling index in hepatic focal lesions. Interestingly, vitamin E supplementation inhibited apoptosis in normal liver but did not produce an observable decrease in apoptosis in hepatic focal lesions. The present study showed that dieldrin (Group 2) or vitamin E supplementation alone (Group 4) promoted the growth of hepatic focal lesions in mice. However, when vitamin E is supplemented to dieldrin-fed mice (Group 3), there is an inhibition of hepatic focal lesion growth.

119. Dragan Y., Klaunig J., Maronpot R., Goldsworthy T. (1998). Mechanisms of susceptibility to mouse liver carcinogenesis. Toxicol Sci. 1998 Jan;41(1):3-7. PMID: 9520336.

118. Crouch D.J., Frank J.F., Farrell L.J., Karsch H.M., Klaunig J.E. (1998). A multiple-site laboratory evaluation of three on-site urinalysis drug-testing devices. J Anal Toxicol. 1998 Oct;22(6):493-502. PMID: 9788525.

Abstract. Presented are findings from a multisite laboratory evaluation comparing on-site urinalysis drug-test results to results from Syva EMIT immunoassay and gas chromatography-mass spectrometry (GC-MS). Three laboratories participated in the NHTSA-funded project. Specimens were tested for amphetamines, benzodiazepines, cocaine, cannabinoids, and opiates. Each laboratory selected 20 urines that tested positive for a single drug/drug class and 20 that tested negative to challenge the on-site drug-testing devices. Qualitative and quantitative GC-MS confirmations were performed to ensure that all positive samples contained the target drug(s)/metabolite(s) and that all negative samples did not contain the target analytes. EZ-SCREEN, ONTRAK, and TRIAGE on-site test kits were selected for evaluation. On-site false-positive results, in which GC-MS-verified negative urine samples gave positive on-site results, were rare. Two such errors were recorded with both EZ-SCREEN and TRIAGE. Cross-reactivity from samples containing GC-MS-verified high concentrations of alternate drugs was also rare. One cross-reactive error was recorded while testing for cocaine with EZ-SCREEN, a second while testing for benzodiazepines with ONTRAK, and a third while testing for cocaine with ONTRAK. The EZ-SCREEN kit did not appear to adhere to a cutoff concentration as demonstrated by the number of samples that contained low concentrations of the target drugs that tested positive with this device. A significant finding of this study was that comparing on-site test device results with those of EMIT for samples with drug concentrations near the reporting cutoff was very complex. It required a thorough knowledge of the performance of each device, EMIT, and GC-MS. It also required an investigation of each discrepant result-a consideration not taken in many previous evaluations of on-site testing devices. Compared with current federal guidelines for workplace urinalysis testing, more donor samples would screen positive for cannabinoids and cocaine by the on-site devices than by EMIT immunoassay. However, fewer would be reported as positive because most contained GC-MS-determined drug concentrations lower than the federal confirmation and reporting limits.

117. Jiang J., Xu Y., Klaunig J.E. (1998). Induction of oxidative stress in rat brain by acrylonitrile (ACN). Toxicol Sci. 1998 Dec;46(2):333-41. PMID: 10048137.

Abstract. Chronic treatment with acrylonitrile (ACN) has been shown to produce a dose-related increase in glial cell tumors (astrocytomas) in rats. The mechanism(s) for ACN-induced carcinogenicity remains unclear. While ACN has been reported to induce DNA damage in a number of short-term systems, evidence for a genotoxic mechanism of tumor induction is the brain is not strong. Other toxic mechanisms appear to participate in the induction of tumor or induce the astrocytomas solely. In particular, nongenotoxic mechanisms of carcinogen induction have been implicated in this ACN-induced carcinogenic effect in the rat brain. One major pathway of ACN metabolism is through glutathione (GSH) conjugation. Extensive utilization and depletion of GSH, an important intracellular antioxidant, by ACN may lead to cellular oxidative stress. The present study examined the ability of ACN to induce oxidative stress in male Sprague-Dawley rats. Rats were administered ACN at concentrations of 0, 5, 10, 100, or 200 ppm in the drinking water and sampled after 14, 28, or 90 days of continuous treatment. Oxidative DNA damage indicated by the presence of 8-hydroxy-2′-deoxyguanosine (OH8dG) and lipid peroxidation indicated by the presence of malondialdehyde (MDA), a lipid peroxidation product, in rat brains and livers were examined. The levels of reactive oxygen species (ROS) were also determined in different rat tissues. Both the levels of nonenzymatic antioxidants (GSH, vitamin E) and the activities of enzymatic antioxidants (catalase, superoxide dismutase, glutathione peroxidase) in rat brains and livers were measured. Increased levels of OH8dG, MDA, and ROS were found in the brains of ACN-treated rats. Decreased levels of GSH and activities of catalase and SOD were also observed in the brains of ACN-treated rats compared to the control group. Interestingly, there were no changes of these indicators of oxidative stress in the livers of ACN-treated rats. Rat liver is not a target for ACN-induced carcinogenesis. These data indicate that ACN selectively induces oxidative stress in rat brain at doses that produce carcinogenesis in chronic treatment studies.

116. Klaunig J.E., Xu Y., Isenberg J.S., Bachowski S., Kolaja K.L., Jiang J., Stevenson D.E., Walborg E.F. Jr. (1998). The role of oxidative stress in chemical carcinogenesis. Environ Health Perspect. 1998 Feb;106 Suppl 1:289-95. Review. PMID: 9539021.

Abstract. Oxidative stress results when the balance between the production of reactive oxygen species (ROS) overrides the antioxidant capability of the target cell; oxidative damage from the interaction of reactive oxygen with critical cellular macromolecules may occur. ROS may interact with and modify cellular protein, lipid, and DNA, which results in altered target cell function. The accumulation of oxidative damage has been implicated in both acute and chronic cell injury including possible participation in the formation of cancer. Acute oxidative injury may produce selective cell death and a compensatory increase in cell proliferation. This stimulus may result in the formation of newly initiated preneoplastic cells and/or enhance the selective clonal expansion of latent initiated preneoplastic cells. Similarly, sublethal acute oxidative injury may produce unrepaired DNA damage and result in the formation of new mutations and, potentially, new initiated cells. In contrast, sustained chronic oxidative injury may lead to a nonlethal modification of normal cellular growth control mechanisms. Cellular oxidative stress can modify intercellular communication, protein kinase activity, membrane structure and function, and gene expression, and result in modulation of cell growth. We examined the role of oxidative stress as a possible mechanism by which nongenotoxic carcinogens may function. In studies with the selective mouse liver carcinogen dieldrin, a species-specific and dose-dependent decrease in liver antioxidant concentrations with a concomitant increase in ROS formation and oxidative damage was seen. This increase in oxidative stress correlated with an increase in hepatocyte DNA synthesis. Antioxidant supplementation prevented the dieldrin-induced cellular changes. Our findings suggest that the effect of nongenotoxic carcinogens (if they function through oxidative mechanisms) may be amplified in rodents but not in primates because of rodents’ greater sensitivity to ROS. These results and findings reported by others support a potential role for oxidative-induced injury in the cancer process specifically during the promotion stage.

115. Bachowski S., Xu Y., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1998). Role of oxidative stress in the selective toxicity of dieldrin in the mouse liver. Toxicol Appl Pharmacol. 1998 Jun;150(2):301-9. PMID: 9653061.

Abstract.  Dieldrin, an organochlorine insecticide, induces hepatic tumors in mice but not in rats. Although the mechanism(s) responsible for this species specificity is not fully understood, accumulating evidence indicates that oxidative stress may be involved. This study examined the association of dieldrin-induced hepatic DNA synthesis with the modulation of biomarkers of oxidative damage to lipids (malondialdehyde [MDA]) and DNA (8-hydroxy-2-deoxyguanosine [oh8dG]), in male B6C3F1 mice and F344 rats fed dieldrin (0.1, 1.0, or 10 mg/kg diet) for 7, 14, 28, and 90 days. The nonenzymatic components of the antioxidant defense system (ascorbic acid, glutathione, and alpha-tocopherol) were also examined. Increased urinary MDA was observed in mice fed 0.1, 1.0, or 10 mg dieldrin/kg diet for 7, 14, 28, and 90 days; while increased hepatic MDA was seen only after 7 days in mice fed 0.1, 1.0, or 10 mg dieldrin/kg diet and after 14 days in mice fed 10 mg/kg diet. In rats, dieldrin had no effect on either hepatic MDA or urine MDA levels after 7, 14, and 28 days of treatment. A dose-dependent increase in urinary MDA was observed in rats at the 90-day sampling time. The only significant elevation in urinary or hepatic oh8dG content was limited to urinary oh8dG in mice fed 10 mg/kg dieldrin diet for 14 days. Dietary dieldrin produced sustained decreases in hepatic and serum alpha-tocopherol and sustained elevations in hepatic ascorbic acid in both mice and rats. Rats, however, possessed a three- to four-fold higher content of endogenous or basal (control) hepatic alpha-tocopherol; and, even when fed 10 mg dieldrin/kg diet, the levels of hepatic alpha-tocopherol were maintained at higher levels than those of mice fed control diet. In both rats and mice fed dieldrin, transient (14 and 28 days on diet) elevations in hepatic glutathione were observed. These data support the hypothesis that the species specificity of dieldrin-induced hepatotoxicity may be related to dieldrin’s ability to induce oxidative stress in the liver of mice, but not in rats. Only in mice fed dieldrin was a temporal association of increases in hepatic MDA content and hepatic DNA synthesis seen, suggesting that oxidative damage (shown by increased lipid peroxidation) may be involved in early events in dieldrin-induced hepatocarcinogenesis. Rats may be protected from dieldrin-induced oxidative stress by a more effective antioxidant defense system, characterized by higher basal levels of hepatic alpha-tocopherol and ascorbic acid than that seen in the mouse.


114. Bachowski S., Kolaja K.L., Xu Y., Ketcham C.A., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1997). Role of oxidative stress in the mechanism of dieldrin’s hepatotoxicity. Ann Clin Lab Sci. 1997 May-Jun;27(3):196-209. PMID: 9142372.

Abstract. The production of reactive oxygen species (ROS) by toxic chemicals has been implicated in acute and chronic disease states, including cancer. This increase in cellular ROS can lead to a state of oxidative stress. Many compounds selectively induce hepatic tumors in mice but not rats. The mechanism for the induction of hepatic cancer by these compounds and the observed species selectivity of this effect are not known but may be related to the induction of oxidative stress. Dieldrin is one such compound and is used in the present study to characterize the relationship between oxidative stress and the observed selective hepatotoxicity of dieldrin in mice. It was found that dieldrin induced oxidative stress in the mouse but not the rat, and the observed oxidative stress correlated with the induction of DNA S-phase synthesis. This evidence suggests that the induction of oxidative stress may be a mechanism by which dieldrin and other mouse specific compounds selectively induce their hepatic toxic effects in mice.

113. Isfort, R.J., Cody, D.B., Doersen, C, Richards, W.G., Yoder, B.K., Wilkinson, J.E., Kier, L.D., Jirtle, R.L, Isenberg, J.S., Klaunig, J.E., Woychik, R.P. (1997). The Tetratricopeptide Repeat Containing Tg737 Gene is Liver Neoplasis Tumor Suppressor Gene. Oncogene, 15, 1797-1803. PMID: 9362446

Abstract. The Tg737 gene was investigated for gross alterations in a series of rodent/human liver tumors and human tumorigenic cell lines. The Tg737 gene was found to be altered in approximately 40% of the rodent chemically-induced liver tumors, 40% of the human liver tumors, and in liver, kidney and pancreatic human tumor cell lines. Ectopic re-expression of the Tg737 gene in aTg737 deleted mouse liver tumor cell line resulted in suppression of tumorigenic growth, without altering in vitro cell culture growth. Treatment of mice which are either homozygous normal or heterozygous deleted at the Tg737 locus with the carcinogen diethylnitrosamine resulted in an increase in preneoplastic foci formation in the Tg737 heterozygous deleted mice. Ectopic expression of the Tg737 gene results in multinucleated cells, loss of Tg737 gene expression results in the proliferation of liver stem cells (oval cells) without concomitant differentiation, and reexpression of the Tg737 gene reestablished responsiveness to external differentiation factors. We believe this is the first report demonstrating tumor suppression activity for a tetratricopeptide repeat gene family member and provides insights into the function of this family of genes in mammalian cells 

112. Isenberg J.S., Kolaja K.L., Ayoubi S.A., Watkins J.B. 3rd, Klaunig J.E. (1997). Inhibition of  WY-14,643 induced hepatic lesion growth in mice by rotenone. Carcinogenesis. 1997 Aug;18(8):1511-9. PMID: 9276624.

Abstract.  The effect of rotenone treatment on [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY-14,643) hepatic lesion growth in male B6C3F1 mice was investigated. Following induction of hepatic focal lesions by diethylnitrosamine (DEN) 35 mg/kg twice a week for 8 weeks, mice were placed into one of the four treatment groups: group I, control NIH-07 diet (control diet), group II, rotenone (600 mg/kg diet), group III NIH-07 diet containing WY-14,643 (1000 mg/kg diet), and group IV, NIH-07 diet containing WY-14,643 (1000 mg/kg diet) and rotenone (600 mg/ kg diet). Mice were killed after 30 and 60 days of dietary treatment. The effect of treatment with WY-14,643 and rotenone on hepatic lesion growth was examined by estimating the number of focal lesions per liver and the relative volume of focal lesions. WY-14,643 (group III) increased both the number and the volume of focal lesions. In particular, an increase in number and volume of basophilic lesions was seen. Co-treatment with WY-14,643 and rotenone (group IV) decreased both the number and the volume of the total number of focal lesions and basophilic foci compared with WY-14,643 treatment alone (group II). Alterations in the growth of hepatic focal lesions was further investigated by examining DNA synthesis and apoptosis within individual lesions. WY-14,643 (group III) treatment increased the DNA synthetic labeling index in all foci. Co-treatment of rotenone and WY-14,643 (group IV) decreased focal DNA synthesis and mitosis and increased the incidence of apoptotic hepatocytes. These data suggest that rotenone’s ability to inhibit WY-14,643-induced hepatic focal lesion growth was mediated through a decrease in hepatic focal proliferation and an increase in focal apoptosis.

111. Kolaja K.L., Klaunig J.E. (1997). Vitamin E modulation of hepatic focal lesion growth in mice. Toxicol Appl Pharmacol. 1997 Apr;143(2):380-7. PMID: 9144454.

Abstract. The effect of DL-alpha-tocopherol acetate (vitamin E) on hepatic focal lesion growth in male B6C3F1 mice previously treated with diethylnitrosamine (DEN) was investigated. After hepatic focal lesions were formed, mice were placed into one of the following dose groups: 0 mg vitamin E/kg NIH-07 diet, 50 mg vitamin E/kg NIH-07 diet (control diet), 250 mg vitamin E/kg NIH-07 diet, and 450 mg vitamin E/kg NIH-07 diet. Mice were euthanized after either 30 or 60 days of dietary treatment. In normal (nonlesion) liver, vitamin E deficiency (0 mg/kg diet) increased hepatic DNA synthesis. In addition, vitamin E supplementation (450 mg/kg diet) decreased the incidence of hepatic apoptosis, while vitamin E deficiency (0 mg/kg diet) increased the incidence of hepatic apoptosis. The effect of vitamin E-induced lesion growth was examined by measuring the number of focal lesions per liver and the relative focal lesion volume. High-dose vitamin E supplementation (450 mg/kg diet) appeared to enhance the growth of hepatic focal lesions. In particular, basophilic lesions appeared to be the most sensitive to high-dose vitamin E modulation (450 mg/kg diet) as evidenced by increased number, volume, and labeling index of hepatic focal lesions. Vitamin E deficiency also appeared to enhance the growth of hepatic focal lesions, though to a lesser extent than vitamin E supplementation (450 mg/kg diet). In the present study, both vitamin E supplementation (450 mg/kg diet) and deficiency (0 mg/kg diet) appeared to enhance focal lesion growth albeit neither treatment enhanced lesion growth as dramatically as known nongenotoxic hepatocarcinogens (e.g., phenobarbital and dieldrin). The data presented here suggest that oxidative stress in focal hepatocytes may be a component of the liver tumor promotion process.


110. Sargent L., Dragan Y.P., Babcock K., Wiley J., Klaunig J., Pitot H.C. (1996). Cytogenetic analysis of three rat liver epithelial cell lines (WBneo, WBHa-ras, and WBrasIIa) and correlation of an early chromosomal alteration with insulin-like growth factor II expression. Cancer Res. 1996 Jul 1;56(13):2992-7. PMID: 8674053.

Abstract. Cytogenetic changes that occur during the progression of rat hepatocarcinogenesis were assessed with three rat liver epithelial cell lines derived from WB cells. Previously characterized WBneo, WBras, and WBrasIIa cells were grown in culture and analyzed for structural and numerical chromosomal integrity by banded karyotype analysis. The WBneo cells had a low level of aneuploidy with a consistent loss of the Y chromosome by passage 7. The ras-transfected cell line selected for growth in soft agar, WBras, had acquired a loss of chromosome 3 (12%) or 3p (34%), a trisomy of chromosome 1, as well as the chromosome Y loss. The cell line produced from tumors generated by injection of the WBras cells into a syngeneic F344 rat, WBrasIIa, contained additional chromosomal changes. The WBrasIIa line comprised cells retaining a trisomy of chromosome 1 (55%) and cells with two copies of chromosome 1, with a minimal duplication of 1q3.7 to 1q4.3 (45%). This tumor-derived cell line contained, in addition, a higher percentage of cells with a loss of all or part of chromosomes 3 and 6, indicating the possible presence of tumor suppressor genes in this region. The smallest region of duplication of chromosome 1 was bands 1q3.7-4.3. The insulin-like growth factor II (IGF-II) gene is located within the region of duplication on chromosome 1. Because IGF-II is both a rat liver mitogen and an inhibitor of apoptosis, its expression was examined in these three rat liver epithelial cell lines. Northern blot analysis demonstrated an increase in IGF-II mRNA expression in the WBras and WBrasIIa cell lines relative to the WBneo control cell line. Several IGF-II transcripts analogous to those detected in fetal rat liver were observed. An additional IGF-II transcript that migrates above the 28S ribosomal marker was also observed. These results were confirmed at the protein level by immunohistochemical and Western blot analysis. This increased expression of IGF-II may confer a selective growth advantage to rat liver epithelial cells with a duplication of 1q3.7-4.3. This growth advantage may be enhanced by the further sequential loss of putative tumor suppressor genes on chromosomes 3 and 6

109. Steinmetz K.L., Klaunig J.E. (1996). Transforming growth factor-alpha in carcinogen-induced F344 rat hepatic foci. Toxicol Appl Pharmacol. 1996 Sep;140(1):131-45. PMID: 8806879.

Abstract. Transforming growth factor-alpha (TGF alpha) is a positive growth regulator in epithelial cells, including hepatocytes. Overexpression of TGF alpha has been associated with increased growth and malignancy of end-stage cancers in humans and rodents. The overall aim of this study was to characterize TGF alpha staining in diethylnitrosamine-induced hepatic foci from male F344 rats with the hematoxylin and eosin (H and E) histological phenotype. The association between the individual focal DNA synthesis labeling index and the presence of TGF alpha was also examined. Hepatic foci were identified as eosinophilic, basophilic, clear cell, or mixed cell. Of these foci, 37.5% labeled positive for TGF alpha. There were distinct differences in the pattern of TGF alpha labeling between the different H and E histological phenotypes. Intense, uniform TGF alpha labeling was observed in eosinophilic foci. Basophilic foci labeled for TGF alpha diffusely uniform throughout the cytoplasm. In clear-cell foci, TGF alpha labeling occurred primarily along the periphery of the cell membrane. In mixed-cell foci, labeling occurred both along the periphery and diffusely throughout the cytoplasm. On those slides stained, glutathione-S-transferase (placental; GSTP) was detected in almost all eosinophilic and mixed-cell foci, whereas approximately half of the basophilic and clear-cell foci stained for GSTP. The presence of GSTP in a focus was not always associated with the presence of increased TGF alpha protein. All rat hepatic adenomas and the one carcinoma labeled positive for TGF alpha. Increased levels of TGF alpha protein were associated with increased DNA synthesis labeling index. The number of TGF alpha-positive foci with the highest DNA synthesis labeling indices were statistically higher than those with lower levels of DNA synthesis labeling. Although characteristic staining patterns for TGF alpha were associated with specific histological subtype, the role that TGF alpha plays in the progression of focal lesions to neoplasia requires further definition. High levels of TGF alpha protein appear to be acquired sometime during the hepatocarcinogenic process. It may be that early lesions that acquire high levels of TGF alpha are the ones to develop into hepatocellular carcinoma (e.g., hepatocellular carcinoma is determined very early in the carcinogenic process). It is apparent that further work is needed to delineate the role of TGF alpha in both rodent and human hepatocarcinogenesis.

108. Kolaja K.L., Bunting K.A., Klaunig J.E. (1996). Inhibition of tumor promotion and hepatocellular growth by dietary restriction in mice. Carcinogenesis. 1996 Aug;17(8):1657-64. PMID: 8761422.

Abstract. The effects of dietary restriction on the growth of hepatic focal lesions in phenobarbital (PB) promoted mice were examined. Dietary restriction which can inhibit many age-related diseases in rodents including hepatic cancer also decreases cell proliferation and increases apoptosis in the liver. In contrast, PB, a non-genotoxic rodent hepatocarcinogen, enhances the growth of hepatic focal lesions in mice and rats by increasing cell proliferation and inhibiting apoptosis. The present study examined the impact of dietary restriction on PB-induced hepatic tumor promotion. Focal lesions were produced by diethylnitrosamine (DEN) treatment (35 mg DEN/kg body weight injections, twice per week for 8 weeks). After lesions were produced, mice were placed into one of the following four groups: NIH-07 control diet/no PB (group 1); NIH-07 diet/500 mg PB per liter of drinking water (group 2); dietary restricted NIH-07 diet/no PB (group 3); and dietary restricted NIH-7 diet/ 500 mg PB per liter of drinking water (group 4). In this study, PB (500 mg/l) treatment to ad libitum-fed mice (group 2) enhanced focal lesion volume, number, and labeling index compared with group 1. In addition, PB treatment (group 2) inhibited apoptosis in normal and focal hepatocytes compared with untreated control mice (group 1). In contrast, in dietary restricted mice treated with PB (group 4) a significantly lower focal lesion volume, number and labeling index were seen compared with the ad libitum-fed/PB treatment group (group 2). PB treatment in dietary restricted mice (group 4) did not inhibit focal apoptosis, in fact, the incidence of focal apoptosis was increased in these mice compared with ad libitum and PB-treated mice (group 2). In dietary restricted mice treated with PB (group 4), the ability of PB to promote the growth of preneoplastic focal lesions was inhibited. These results show that dietary restriction can ablate the tumor promotional effects of PB in hepatic focal lesions and suggest that inhibition of focal lesion DNA synthesis and enhancement of apoptosis may be a mechanism for this effect.

107. Kolaja K.L., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1996). Dose dependence of phenobarbital promotion of preneoplastic hepatic lesions in F344 rats and B6C3F1 mice: effects on DNA synthesis and apoptosis. Carcinogenesis. 1996 May;17(5):947-54. PMID: 8640942.

Abstract. Phenobarbital (PB), a non-genotoxic hepatocarcinogen in rodents, has been studied extensively but its mechanism of carcinogenic action is unclear. PB appears to function as a tumor promoter by selectively inducing the growth of preneoplastic hepatocytes. In the present study, the comparative effects of PB at tumor-promoting and non-promoting doses were examined in male B6C3F1 mice and male F344 rats. In addition, the mechanism by which PB produced the selective induction of preneoplastic cell growth (increased DNA synthesis/cell proliferation and/or decreased apoptosis) was investigated. Preneoplastic focal lesions were produced using diethylnitrosamine (DEN). After the lesions were histologically apparent, mice and rats were fed PB (10, 100, or 500 mg/kg NIH-07 diet) or control diet and sampled after 7, 30 and 60 days of treatment In both mice and rats, 100 and 500 mg PB/kg increased the number and the relative volume of focal lesions. In rats and mice, 10 mg PB/kg did not enhance focal lesion growth. The preneoplastic lesions that clonally expanded due to phenobarbital treatment were predominantly eosinophilic in appearance. In addition, DNA synthesis in focal hepatocytes was significantly increased in the 100 and 500 mg PB/kg diet. In PB-treated mice and rats, there also was a significant decrease in the rates of apoptosis in focal hepatocytes. Therefore, our data showed that PB at doses of 100 and 500 mg/kg diet promoted focal hepatic lesion growth both by increasing DNA synthesis and cell proliferation and by decreasing the rate of apoptosis.

106. Kolaja K.L., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1996). Selective dieldrin promotion of hepatic focal lesions in mice. Carcinogenesis. 1996 Jun;17(6):1243-50. PMID: 8681438.

Abstract. Chronic exposure to a number of chlorinated pesticides, including dieldrin, results in an increased incidence and/or multiplicity of hepatocellular neoplasia in mice, with no such effect in similarly treated rats. One possible explanation of this observed selective carcinogenicity is species-specific hepatic tumor promotion. In the present study we examined the dose-response effect of dieldrin (at several doses) on focal lesion growth (tumor promotion), hepatocyte apoptosis and DNA synthesis in rat and mouse liver. Preneoplastic focal hepatic lesions were produced by diethylnitrosamine (DEN). After the lesions developed, mice and rats were placed into one of the following dose groups: control (NIH-07 diet) or 0.1, 1.0 or 10.0 mg dieldrin/kg diet. Increased focal lesion volume, number of foci per liver and focal DNA synthetic labeling index were observed in 10 mg dieldrin/kg diet-treated mice, but not in similarly treated rats. Dieldrin at dietary concentrations of 0.1 and 1.0 mg/kg diet produced an increase in the number of preneoplastic lesions (0.1 mg/kg diet at 7 days only) and focal volume (0.1 mg/kg diet at 7 and 30 days, 1.0 mg/kg diet at 30 days), but these concentrations did not increase focal DNA labeling index. At dietary concentrations of 0.1, 1.0 and 10 mg dieldrin/kg diet no significant change in lesion percent volume, number of preneoplastic lesions per liver or preneoplastic lesion DNA labeling index was seen in treated rats compared with control rats. Apoptosis, a form of programed cell death, was not decreased in foci by any concentration of dieldrin in either rats or mice. Thus our results suggest that dieldrin may function as a mouse-specific tumor promoter through increased lesion DNA synthesis.

105. Kolaja K.L., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1996). Reversibility of promoter induced hepatic focal lesion growth in mice. Carcinogenesis. 1996 Jul;17(7):1403-9. PMID: 8706241.

Abstract. The effect of cessation of phenobarbital and dieldren treatment on hepatic focal lesion growth in male B6C3F1 mice was investigated. Following induction of lesions by diethylnitrosamine, mice were placed on control NIH-07 diet (control diet) or NIH-07 diet containing either dieldrin (10.0 mg/kg diet) or phenobarbital (500 mg/kg diet). Mice were sacrificed after 30 and 60 days of dietary treatment. Two additional groups of mice were fed either the dieldren- or phenobarbital-containing diet for 30 days followed by feeding of NIH-07-only diet for an additional 30 days. The effect of treatment and removal of dieldrin or phenobarbital on lesion growth was examined by measuring both the number of focal lesions per liver and the relative volume of focal lesions. In addition, the rate of cell proliferation and programmed cell death in focal lesion growth was investigated by examining DNA synthesis and apoptosis in the focal lesions. Dietary dieldrin or phenobarbital increased the number of focal lesions and the focal lesion volume. In both dieldrin- and phenobarbital-treated mice, an increased number of eosinophilic lesions were seen. The focal lesion volume was increased in both eosinophilic and basophilic lesions. Dieldrin and phenobarbital treatment also increased the DNA synthetic labeling index in both eosinophilic and basophilic lesions. Removal of dieldrin or phenobarbital from the diet after 30 days of promoter treatment decreased the total number and volume of hepatic focal lesions. The labeling index of the focal lesions was also decreased in these mice. At the terminal sacrifice, the percentage of apoptotic cells in focal lesions was higher in mice fed dieldrin- or phenobarbital-containing diets for the entire 60 days than in mice returned to control diet for the last 30 days. Eosinophilic lesions were more dependent on the presence of a promoting stimulus than the basophilic lesions. These data indicate that induction and maintenance of the growth of some preneoplastic lesions in the mouse may be dependent upon continuous tumor promoter treatment.

104. Cao J., Xu Y., Chen J., Klaunig J.E. (1996). Chemopreventive effects of green and black tea on pulmonary and hepatic carcinogenesis. Fundam Appl Toxicol. 1996 Feb;29(2):244-50. PMID: 8742322.

Abstract. The chemopreventive effects of decaffeinated green and black tea treatment on liver and lung tumorigenesis were examined in carcinogen-treated mice. Male C3H mice were given decaffeinated green or decaffeinated black tea in their drinking water prior to, during, and after treatment with diethylnitrosamine (50 micrograms/kg bw, i.p., once per week for 8 weeks). After 40 weeks of tea treatment, mice were sampled and examined for pulmonary and hepatic tumors. Mice treated with both DENA and tea displayed a significant decrease in the mean number of lung and liver tumors compared to DENA-only treated animals. Mice that received 0.63 or 1.25% green tea or 1.25% black tea exhibited a reduction in liver tumor numbers of 54, 50, and 63%, respectively from that seen in the DENA-only treated mice. Tea treatment also significantly decreased the multiplicity of lung adenomas. Mice receiving DENA and either 0.63 or 1.25% green tea or 1.25% black tea showed a decrease in the mean number of lung tumors of 40, 46, and 34%, respectively, from DENA-only treated mice. While a possible association between the chemopreventive activity of tea on lung tumor response and the concentration of (-) epigallocatechin gallate (EGCG) in the tea was suggested, no apparent relationship between EGCG concentration and liver tumor response was seen, however. These results show a dose-dependent chemoprevention of both lung and liver tumors by both black and green tea in diethylnitrosamine-treated C3H mice.

103. Kolaja K.L., Stevenson D.E., Johnson J.T., Walborg E.F. Jr, Klaunig J.E. (1996). Subchronic effects of dieldrin and phenobarbital on hepatic DNA synthesis in mice and rats. Fundam Appl Toxicol. 1996 Feb;29(2):219-28. PMID: 8742319.

Abstract. Dieldrin, an organochlorine pesticide, has been shown to be hepatocarcinogenic in mice but not rats. Phenobarbital, in contrast, induces hepatic tumors in both mice and rats. Previous studies have shown that acute dietary exposure of rats or mice to either dieldrin or phenobarbital produces several liver changes, including centrilobular hypertrophy, induction of hepatic cytochrome P450, and increased liver weight. The present study examined the subchronic effect of dieldrin (0.1, 1.0, 3.0, 10.0 mg dieldrin/kg diet) and phenobarbital (10, 50, 100, 500 mg phenobarbital/kg diet) on the induction of hepatic DNA synthesis and hepatocyte lethality in male B6C3F1 mice and male F344 rats. Eight-week-old animals were treated as above and evaluated for hepatic DNA synthesis after 7, 14, 21, 28, and 90 days of continual treatment to dieldrin or phenobarbital. Maximal induction of hepatic DNA synthesis in mice was seen at the 14-, 21-, and 28-day sampling times. In rats, no significant increase in hepatic DNA synthesis or hepatocyte lethality was observed at any dose of dieldrin investigated. Phenobarbital produced a significant increase in hepatic DNA synthesis in both rat and mouse liver following 7 days of treatment. The induction of DNA synthesis in rat liver was transient, with the labeling index returning to control levels by 14 days of treatment. In contrast, mice treated with phenobarbital showed a significant increase in hepatic DNA synthesis throughout the treatment. In both mice and rats, dieldrin and phenobarbital induced hepatic DNA synthesis selectively in the centrilobular region of the hepatic lobule. The lack of an increase in serum enzymes indicative of hepatic damage and the absence of liver histopathology in mice or rats fed dieldrin or phenobarbital indicate that the induction of DNA synthesis was not mediated by a cytolethal, compensatory hyperplastic response, suggesting a mitogenic mechanism. Therefore, the species-specific induction of hepatic DNA synthesis by either dieldrin or phenobarbital correlated with the previously observed species-specific induction of hepatic cancer by these two compounds.


102. Klaunig J.E., Xu Y., Bachowski S., Ketcham C.A., Isenberg J.S., Kolaja K.L., Baker T.K., Walborg E.F. Jr, Stevenson D.E. (1995). Oxidative stress in nongenotoxic carcinogenesis. Toxicol Lett. 1995 Dec;82-83:683-91. PMID: 8597127.

Abstract. The induction of oxidative stress in the target tissue has been proposed as a possible mechanism of action for nongenotoxic carcinogens. A variety of nongenotoxic hepatocarcinogens including peroxisome proliferators, organochlorines, barbiturates, and metals have been shown to produce an increase in reactive oxygen species (ROS) in the liver. Our group has examined the induction of oxidative stress by the organochlorine mouse hepatic carcinogen, dieldrin. Using a salicylate spin trap assay, dieldrin was found to produce mouse liver-specific increases in ROS in cultured hepatocytes. Increased amounts of hepatic 8-hydroxy-2′-deoxyguanosine and malondialdehyde (MDA) and decreased levels of cellular antioxidants were also seen in cultured mouse hepatocytes following dieldrin treatment. In subchronically dieldrin-treated mice and rats, hepatic vitamin E (Vit E) was decreased correlated with dieldrin dose. While Vit E levels were decreased in both rats and mice, the normal lower levels of Vit E in the mouse resulted in a subsequent oxidative stress, evidenced by an increase in MDA formation in the mouse liver. Dieldrin also produced a dose-dependent increase in DNA synthesis in the mouse (not the rat) following subchronic treatment. These effects seen in both cells in culture and in vivo were species specific, organ specific, and dose dependent which directly correlated with the observed pattern of cancer induction for dieldrin in rodents (mouse liver-specific). These findings support a possible role for the induction of oxidative stress in nongenotoxic hepatic carcinogenesis possibly through modulation of gene expression.

101. Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1995). The species specificity of dieldrin- or phenobarbital- induced hepatocarcinogenesis: case studies with implications for human health risk assessment. Prog Clin Biol Res. 1995;391:337-45. PMID: 8532726.

100. Kolaja K.L., Stevenson D.E., Johnson J.T., Walborg E.F. Jr, Klaunig J.E. (1995). Hepatic effects of dieldrin and phenobarbital in male B6C3F1 mice and Fisher 344 rats: species selective induction of DNA synthesis. Prog Clin Biol Res. 1995;391:397-408. PMID: 8532732.

99. Stevenson D.E., Kehrer J.P., Kolaja K.L., Walborg E.F. Jr, Klaunig J.E. (1995). Effect of dietary antioxidants on dieldrin-induced hepatotoxicity in mice. Toxicol Lett. 1995 Jan;75(1-3):177-83. PMID: 7863524.

Abstract. An increasing number of reports suggest that oxidative stress plays a role in the toxicity of various xenobiotics, including organochlorine pesticides and drugs such as phenobarbital. Antioxidants appear to be protective against the damage induced by an acute dose of endrin, supporting the theory of a role for reactive oxygen in the toxicity of this class of compounds. The current study examined the effects of the dietary administration of vitamin C (400 mg/kg diet) or vitamin E (200 mg DL-alpha-tocopherol acetate/kg diet) on hepatotoxicity induced by subchronic (7 or 28 days) feeding of dieldrin (1, 3 and 10 mg/kg diet) to male B6C3F1 mice. Hepatoxicity induced by feeding of dieldrin for 28 days was evidenced by liver enlargement, hypertrophy of centrolobular hepatocytes, induction of hepatic ethoxyresorufin O-deethylase activity, and increased DNA synthesis in hepatocytes, particularly in centrolobular hepatocytes. Neither vitamin inhibited the dose-dependent increase in liver/body weight ratios, hypertrophy of centrolobular hepatocytes, or induction of hepatic ethoxyresorufin O-deethylase. Vitamin E, however, inhibited hepatic DNA synthesis at all dietary intakes of dieldrin, while vitamin C was inhibitory at 1 and 3, but stimulatory at 10 mg dieldrin per kg diet. The major changes in DNA labeling occurred in the centrolobular zones, but were not consistently inhibited by vitamins C or E. The ability of antioxidant vitamins to inhibit dieldrin-induced hepatic DNA synthesis suggests oxidative stress is involved in the toxicity of this compound; however, the inability of these vitamins to prevent all hepatotoxic changes indicates other factors are also involved

98. Bachowski S., Xu Y., Baker T.K., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1995). The potential role of oxidative stress in nongenotoxic carcinogenesis in the mouse liver. Prog Clin Biol Res. 1995;391:385-96. Review. PMID: 8532730.

97. Baker T.K., Bachowski S., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1995). Modulation of gap junctional intercellular communication in rodent, monkey and human hepatocyte by nongenotoxic compounds. Prog Clin Biol Res. 1995;391:71-80. PMID: 8532738.

96. Baker T.K., Kwiatkowski A.P., Madhukar B.V., Klaunig J.E. (1995). Inhibition of gap junctional intercellular communication by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat hepatocytes. Carcinogenesis. 1995 Oct;16(10):2321-6. PMID: 7586129.

Abstract. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent rodent hepatic tumor promoter. Unlike observations with the majority of tumor promoting chemicals studied to date, most investigations have failed to demonstrate down-regulation of gap junctional intercellular communication (GJIC) in cultured cells by TCDD. The present study examined the effect of TCDD on GJIC in rat hepatocytes in primary culture. At non-cytolethal doses TCDD inhibited GJIC in a time- (1, 4, 24 and 48 h) and concentration (1 x 10(-8) – 1 x 10(-14) M)-dependent manner. This inhibition occurred within 4 h of treatment at doses of 1 x 10(-8) – 1 x 10(-12) M TCDD and persisted for up to 48 h, despite removal of TCDD. Treatment of rat hepatocytes with TCDD resulted in a decrease in hepatocyte connexin 32 mRNA, but had no apparent effect on connexin 26 mRNA. Co-incubation of rat hepatocytes with TCDD and alpha-napthoflavone abolished down-regulation of GJIC by TCDD. Similarly, co-treatment with a cAMP analog (8-bromoadenosine 3′,5′-cyclic monophosphate) prevented down-regulation of GJIC by TCDD. The results of this investigation demonstrated, for the first time, that TCDD inhibits GJIC in the in vivo target of its tumor promoting effect and that this effect may, in part, be mediated through the Ah receptor. In addition, this study showed that inhibition of GJIC by TCDD may be due to transcriptional down-regulation or stability of the connexin 32 gap junction mRNA.

95. Kolaja K.L., Stevenson D.E., Walborg E.F. Jr, Klaunig J.E. (1995). The effect of dieldrin and phenobarbital on preneoplastic hepatic lesion growth in male F344 rat and B6C3F1 mouse. Prog Clin Biol Res. 1995;391:409-23. PMID: 8532733.

94. Siglin J.C., Weghorst C.M., Rodwell D.E., Klaunig J.E. (1995). Gender-dependent differences in hepatic tumor promotion in diethylnitrosamine initiated infant B6C3F1 mice by  alpha-hexachlorocyclohexane. J Toxicol Environ Health. 1995 Feb;44(2):235-45. PMID: 7531777.

Abstract. Chronic exposure of B6C3F1 mice to phenobarbital (PB), subsequent to a single initiating dose of diethylnitrosamine (DENA) at 15 d of age, has been previously shown to inhibit hepatic tumorigenesis in male mice, while promoting hepatic tumor formation in female mice (Weghorst & Klaunig, 1989). In the present study, the effects of another hepatic tumor promoter, alpha-hexachlorocyclohexane (alpha-HCH), in similarly initiated B6C3F1 mice was investigated. Male and female mice received a single intraperitoneal (ip) injection of either DENA or saline at 15 d of age. Beginning at 28 d of age, the mice received either alpha-HCH in the diet (250 ppm) or untreated basal diet. Like PB, alpha-HCH inhibited hepatic tumorigenesis in male mice, while promoting hepatic tumor formation in female mice following chronic exposure. In an additional experiment, already formed preneoplastic hepatic foci in male and female B6C3F1 mice were examined for their responsiveness to the induction of DNA synthesis by alpha-HCH treatment. The mice received a single ip injection of DENA at 15 d of age to induce hepatocellular foci. Beginning at 24 wk of age, mice received either basal diet or diet containing 250 ppm alpha-HCH for 7 consecutive d. DNA synthesis was assessed by continuous [3H]thymidine infusion via subcutaneously implanted osmotic minipumps. In female mice treated with alpha-HCH, DNA synthesis in hepatocellular foci was increased substantially compared to untreated females. In contrast, male mice receiving alpha-HCH showed no increase in DNA synthesis in hepatocellular foci from that seen in non-alpha-HCH-treated males. Based on these results, we postulate that the gender-dependent differences in hepatic tumorigenesis observed in B6C3F1 mice initiated during infancy may be related to chemical tumor promoter modulation of the normal hormonal environment, or to differences in the ability of hepatocellular foci to respond to the induction of DNA synthesis by the tumor promoter.

93. Jou Y.S., Layhe B., Matesic D.F., Chang C.C., de Feijter A.W., Lockwood L., Welsch C.W., Klaunig J.E., Trosko J.E. (1995). Inhibition of gap junctional intercellular communication and malignant transformation of rat liver epithelial cells by neu oncogene. Carcinogenesis. 1995 Feb;16(2):311-7. PMID: 7859363.

Abstract. A retrovirus containing a neu oncogene was introduced into a Fischer F344 rat liver epithelial cell line (WB-F344) to study the effect of the expression of neu oncoprotein on gap junctional intercellular communication (GJIC), the ability to form colonies in soft agar and the ability to form tumors in rat liver by these cells. After viral infection, five different neu-transduced epithelial clones were randomly selected for further analysis. Southern blot analysis of HindIII-digested genomic DNA hybridized with a neu-specific probe indicated that the neu oncogene carried by the retrovirus was integrated into different chromosomal locations in the five different neu-transduced WB cell lines. Using the fluorescence recovery after photobleaching (FRAP) assay, we found that GJIC was significantly reduced in neu-transduced WB clones, compared with control virus-infected and parental WB cells. Western blot analysis of connexin 43 in the neu-transduced cell lines showed altered phosphorylation patterns compared with the normal WB-rat liver cell line. Confocal image analysis of the neu-transduced cells showed that the connexin 43 protein, as detected by fluorescent immunostaining, was localized in the cell nucleus. The neu-transduced WB cell lines also acquired the ability to grow in soft agar. Furthermore, cells from three of the five neu-transduced cell lines, when injected into the liver of Fischer F344 rats through the portal vein, were highly tumorigenic (multiple focal hepatic tumors developed within 2 weeks). Cells derived from the tumor were shown to be G-418 resistant, demonstrating that the tumor was derived from the injected WB-neu cells. The results of this study demonstrate that the expression of the neu oncogene is able to block GJIC and to induce tumorigenicity in the rat liver WB-F344 cell line.

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