92. Mehendale H.M., Roth R.A., Gandolfi A.J., Klaunig J.E., Lemasters J.J., Curtis L.R. (1994). Novel mechanisms in chemically induced hepatotoxicity. FASEB J. 1994 Dec;8(15):1285-95. PMID: 8001741.

Abstract. This review focuses on cellular events that modulate hepatotoxicity subsequent to initial liver insult. Cellular events that determine the nature and extent of hepatotoxic injury and the ultimate outcome of that injury are also discussed. The roles of cell types other than hepatocytes, hepatocyte organelle-specific processes, and regeneration in progression or recovery from liver injury are emphasized. Leukocyte activities are key events in two distinct hepatotoxicities. Neutrophil-mediated, periportal inflammation appears to play a primary role in progression of alpha-naphthylisothiocyanate-induced cholangiolitic hepatitis. However, a humorally mediated autoimmune response to protein adducts that occurs after anesthesia is critical in onset of halothane-induced hepatitis. New insights into specific events at the hepatocyte level are also emerging. Although reducing gap junctional communication between hepatocytes can protect against progression of liver injury, down-regulation of the subunit proteins (connexins) can isolate neoplastic cells from growth regulation. Acidic intracellular pH characteristic of hypoxia is protective against both hypoxic and toxicant-induced cell injury. In oxidative injury, a pH-mediated mitochondrial permeability transition causes mitochondrial uncoupling and ATP loss and leads to cell death. The ultimate outcome of hepatotoxic injury depends on the extent of tissue repair. Stimulation of tissue repair after a sublethal dose of CCl4 appears to be the central mechanism in protection against death from a subsequent large dose. Taken together, these examples illustrate the importance of events subsequent to initial liver injury as determinants of extent of liver damage.

91. Kwiatkowski A.P., Baker T.K., Klaunig J.E. (1994). Comparison of glucocorticoid-mediated changes in the expression and function of rat hepatocyte gap junctional proteins. Carcinogenesis. 1994 Aug;15(8):1753-7. PMID: 8055659.

Abstract. Gap junctional intercellular communication (GJIC) is often modulated by chemical carcinogens and during carcinogenesis, in part, through changes in gap junction mRNA levels. However, the mechanisms by which gap junction mRNA levels are altered in either normal or cancer cells are largely unknown. Since glucocorticoids are potent modulators of gene expression and stability, we have investigated the effects of these hormones on GJIC and gap junction mRNA expression in rat hepatocytes cultured in three different media. Addition of dexamethasone to cultures of rat hepatocytes resulted in a maintenance of GJIC and both major liver gap junctional mRNAs, connexin (Cx)26 and Cx32, at levels above those in hepatocytes cultured in glucocorticoid-free media. In addition, hepatocytes cultured without dexamethasone for 24 h could be induced to communicate and increase Cx mRNA levels by the addition of dexamethasone to their medium. These media-independent changes in GJIC and gap junction mRNA levels by dexamethasone warrant further investigations into their mechanisms of action and the potential therapeutic value of glucocorticoids in the treatment of cancer.


90. Klaunig J.E. (1993). Selective induction of DNA synthesis in mouse preneoplastic and neoplastic hepatic lesions after exposure to phenobarbital. Environ Health Perspect. 1993 Dec;101 Suppl 5:235-9. PMID: 8013413.

Abstract. Recent evidence has suggested that the induction of DNA synthesis by nongenotoxic chemical carcinogens plays an important role in the formation of cancer. The present study examined the effect of a nongenotoxic carcinogen, phenobarbital, (PB) on the induction of DNA synthesis in preneoplastic foci and adenomas in B6C3F1 mice. Male mice were treated with diethylnitrosamine at 30 days of age. After 6 months, mice had both hepatic foci and adenomas. Mice were divided into three groups at random and treated with PB in the drinking water and examined for DNA labeling by autoradiography. Before sacrifice, each mouse received an osmotic minipump containing [3H] thymidine. Results showed a PB dose-dependent increase in DNA synthesis in hepatic foci. Adenomas were unresponsive to the DNA synthesis-enhancing effect of PB, maintaining a level of 20-25%. The normal surrounding liver showed an increase in DNA synthesis (10-15% labeling index) at the 7-day sampling, which returned to normal control levels by 28 and 45 days. The foci showed a heterogeneity in response, with some foci showing an increase (20-30% labeling index), and others maintaining control DNA synthesis levels (4-6% labeling index). These results show that preneoplastic foci in the mouse respond preferentially to the induction of DNA synthesis by PB, that this response is dose dependent, and that it is maintained as long as the treatment continues.

89. Ruch R.J., Madhukar B.V., Trosko J.E., Klaunig J.E. (1993). Reversal of ras-induced inhibition of gap-junctional intercellular communication, transformation, and tumorigenesis by lovastatin. Mol Carcinog. 1993;7(1):50-9. PMID: 8435109.

Abstract. The plasma-membrane association and transforming activity of the ras oncoprotein p21 are dependent upon posttranslational farnesylation. Farnesyl synthesis and p21 ras farnesylation are inhibited by hydroxymethylglutaryl-CoA reductase inhibitors such as lovastatin. In this study, we examined whether lovastatin could reverse the transformed phenotype of a v-Ha-ras-transformed rat liver epithelial cell line (WB-ras cells) and if changes were associated with the enhancement of gap-junctional intercellular communication (GJIC). WB-ras cells grow in soft agar, have reduced GJIC, and are highly tumorigenic. Membrane association of p21 ras in these cells was inhibited after in vitro treatment with lovastatin (0.1-0.5 microM) for 48 h. Concomitantly, the cells displayed a more normal morphology, decreased growth in soft agar, and enhanced GJIC. These changes were prevented by cotreatment with mevalonic acid. The morphology and GJIC of rat liver epithelial cells transformed with other oncogenes (src, neu, and raf/myc) were not affected by lovastatin. Intrahepatic WB-ras tumors were induced in male rats by intraportal-vein injection of WB-ras cells. The size and DNA labeling index of these tumors were decreased approximately 75% by administration of lovastatin (5 mg/kg orally twice daily for 2 wk). These results suggest that lovastatin reversed the transformed phenotype of WB-ras cells by inhibiting p21 ras plasma membrane association. Furthermore, the concomitant enhancement of GJIC in lovastatin-treated cells suggests a role for reduced GJIC in the expression of the transformed phenotype.


88. Ruch R.J., Klaunig J.E. (1992). Enhancement of Rodent Hepatocyte Gap Junctional Intercellular Communication by Dexamethasone. In Vitro Toxicology, 5(2), 103-111.


87. Klaunig J.E. (1992). Chemopreventive effects of green tea components on hepatic carcinogenesis. Prev Med. 1992 Jul;21(4):510-9. PMID: 1409492.

Abstract. Catechin components of green tea have been shown to possess anticarcinogenic properties possible related to their antioxidant activity. In the present study, a catechin containing green tea extract (GTE) was examined for its effect on three previously defined properties of liver tumor promoters, induction of cytolethality, inhibition of gap junctional intercellular communication, and induction of cell proliferation. Hepatocytes from male B6C3F1 mice were isolated and placed in primary culture. The effects of GTE of oxygen free radical-induced cytolethality was examined by coincubating GTE with the oxygen radical generating compounds paraquat, glucose oxidase (GO), and xanthine oxidase (XO). GTE prevented the induction of hepatocyte cytolethality by GO, XO, and paraquat in a dose-responsive manner. Similarly, GTE prevented the inhibition of gap junctional-mediated intercellular communication (measured by lucifer yellow dye coupling) by phenobarbital, lindane, and paraquat in a dose-dependent manner. The effect of GTE on hepatocyte DNA synthesis was examined in male mice containing preneoplastic liver lesions induced by diethylnitrosamine. GTE significantly decreased the labeling index in hepatic preneoplastic foci from animals treated with phenobarbital for 7 days. These studies suggest that the previous reported anticarcinogenic activity of green tea may be related to its effect on the tumor promotion stage of the cancer process.


86. Siglin J.C., Weghorst C.M., Klaunig J.E. (1991). Role of hepatocyte proliferation in alpha-hexachlorocyclohexane and phenobarbital tumor promotion in B6C3F1 mice. Prog Clin Biol Res. 1991;369:407-16. PMID: 1719565.


85. Ruch R.J., Bandyopadhyay S., Somani P., Klaunig J.E. (1991). Evaluation of amiodarone free radical toxicity in rat hepatocytes. Toxicol Lett. 1991 Apr;56(1-2):117-26. PMID: 2017769.

Abstract. The possible roles of free radicals and lipid peroxidation in the mechanism of toxicity of amiodarone (AD) [2-butyl-3-(3′,5′-diiodo-4′ alpha-diethylaminoethoxybenzoyl)benzofuran] and its principle metabolite, desethylamiodarone (DE), were examined in primary cultured Sprague-Dawley male rat hepatocytes. AD (20 and 40 micrograms/ml) and DE (10 and 25 micrograms/ml) killed hepatocytes in concentration- and time-dependent fashions. Several antioxidants [Cu,Zn-superoxide dismutase (200 U/ml), catalase (200 U/ml), N,N’-diphenylphenylenediamine (DPPD; 25 microM), butylated hydroxytoluene (0.1 mM), and N-acetylcysteine (5 mM)] were incapable of preventing AD and DE hepatocyte toxicity. Only vitamin E (VE, d,l-alpha-tocopherol acetate; 20-200 microM) prevented AD and DE toxicity. No correlation between the onset of hepatocyte death by AD and DE and hepatocyte lipid peroxidation was seen. Both drugs inhibited NADPH-dependent rat liver microsomal superoxide production. These results, excluding the preventive effects of VE, do not support a free radical/lipid peroxidation mechanism of hepatocyte toxicity by AD and DE. VE may have prevented hepatocyte toxicity through non-antioxidant effects.

84. Klaunig J.E., Siglin J.C., Schafer L.D., Hartnett J.A., Weghorst C.M., Olson M.J., Hampton J.A. (1991). Correlation between species and tissue sensitivity to chemical carcinogenesis in rodents and the induction of DNA synthesis. Prog Clin Biol Res. 1991;369:185-94. PMID: 1946517.

83. Klaunig J.E. (1991). Alterations in intercellular communication during the stage of promotion. Proc Soc Exp Biol Med. 1991 Nov;198(2):688-92. PMID: 1924405.

Abstract. The promotion stage is a crucial step in the process of carcinogenesis. During this stage, the initiated cell population is clonally expanded to morphologically discriminable forms. Exogenous or endogenous agents that influence this clonal expansion have tumor-promoting activity. Inhibition of gap junctional intercellular communication is one of a number of cellular changes seen in cells after exposure to promoting agents. GJIC can be inhibited through either modification of intracellular control mechanism or through transcriptional or translational down-expression of the gap junction protein. Through either mechanism, the net effect is a decrease in GJIC by tumor promoters. This decrease in GJIC, while occurring in normal cells and preneoplastic cells alike, appears to be more efficacious in the preneoplastic cells, and appears to prevent GJIC between the preneoplastic cells and the normal surrounding hepatocytes. This isolation of the preneoplastic cells by hepatic tumor promoters from the normal surrounding hepatocytes may separate the preneoplastic cells from growth regulatory control of the normal liver, thus allowing the preneoplastic cells to clonally expand by cell proliferation. Whether the disruption of GJIC and down-regulation of gap junction protein expression seen in hepatic foci by exposure to tumor promoters are causes or effects of the resulting cell proliferation remains to be determined. Certainly, the modification of GJIC and the expression of the gap junction protein by tumor promoters are important cellular changes that produce a phenotypically altered population of hepatocytes.

82. Goodman J.I., Ward J.M., Popp J.A., Klaunig J.E., Fox T.R. (1991). Mouse liver carcinogenesis: mechanisms and relevance. Fundam Appl Toxicol. 1991 Nov;17(4):651-65. PMID: 1685715.


81. Klaunig J.E., Hartnett J.A., Ruch R.J., Weghorst C.M., Hampton J.A., Schafer L.D.(1990). Gap junctional intercellular communication in hepatic carcinogenesis. Prog Clin Biol  Res. 1990;340D:165-74. PMID: 2371293.

80. Klaunig J.E., Ruch R.J., Hampton J.A., Weghorst C.M., Hartnett J.A. (1990). Gap-junctional intercellular communication and murine hepatic carcinogenesis. Prog Clin Biol Res. 1990;331:277-91. PMID: 2315344.

79. Ruch R.J., Fransson R., Flodstrom S., Warngard L., Klaunig J.E. (1990). Inhibition of hepatocyte gap junctional intercellular communication by endosulfan, chlordane and heptachlor. Carcinogenesis. 1990 Jul;11(7):1097-101. PMID: 2372869.

Abstract. The cyclodiene pesticides endosulfan, chlordane and heptachlor have been reported to be non-genotoxic rodent hepatocarcinogens. These three compounds and several metabolites of endosulfan (endosulfan sulfate, endosulfan ether and endosulfan lactone) were examined for their effects on gap junctional intercellular communication (GJIC) in primary cultured male F344 rat hepatocytes and B6C3F1 mouse hepatocytes. GJIC was evaluated by Lucifer Yellow CH dye-coupling. Endosulfan and endosulfan sulfate inhibited rat and mouse hepatocyte GJIC in a dose-responsive manner (50-200 microM) after 4 h treatment. Endosulfan ether inhibited rat hepatocyte GJIC only at 200 microM and had no effect on mouse hepatocytes. Endosulfan lactone did not affect rat or mouse hepatocyte GJIC. Chlordane and heptachlor inhibited both mouse and rat hepatocyte GJIC at concentrations of 50-200 microM. The inhibition of GJIC by the cyclodienes showed similar dose-response relationships and kinetics of onset of inhibition and reversibility for both mouse and rat hepatocytes. Concomitant treatment of the cells with inhibitors of cytochrome P450 monooxygenases (SKF-525A, piperonyl butoxide or carbon monoxide) did not alter the inhibition of GJIC by the cyclodienes, suggesting that cytochrome P450 metabolism was not involved in the inhibitory mechanism. Dibutyryl cyclic AMP (0.5 mM), however, decreased the inhibition of GJIC by the cyclodienes and may indicate that these compounds inhibit intercellular communication through a cAMP-dependent process. The inhibition of mouse and rat hepatocyte GJIC by the cyclodienes correlated with previous reports indicating that these compounds are non-genotoxic rodent liver carcinogens.

78. Ruch R.J., Klaunig J.E., Kerckaert G.A., LeBoeuf R.A. (1990). Modification of gap junctional intercellular communication by changes in extracellular pH in Syrian hamster embryo cells. Carcinogenesis. 1990 Jun;11(6):909-13. PMID: 2347066.

Abstract. Studies were conducted to determine the effect of culture medium pH on gap junctional intercellular communication (GJIC) in early passage Syrian hamster embryo (SHE) cells. Previous studies have demonstrated that SHE cells cultured at a clonal density at pH 6.70 are morphologically transformed by carcinogens at a significantly higher frequency than cells cultured in media of higher pH. Several other cell characteristics consistent with promotion-like effects are observed with pH 6.70 culture of SHE cells. It was postulated that the promotion-like effects observed in SHE cells cultured at acidic pH are mediated in part by a reduction of GJIC. In this study, we evaluated GJIC in SHE cells by fluorescent dye coupling. Results from this study indicate that GJIC decreased as a function of decreased extracellular pH. Cells cultured at pH 6.70, 7.15 or 7.35 exhibited 47, 75 and 85% coupled cells respectively. The decrease in dye coupling occurred by 24 h after switching the cells from pH 7.15 to 6.70 medium. The decreased GJIC observed at pH 6.70 was not due to changes in cell proliferation and was reversible within 24 h when pH 6.70 cultures were refed with pH 7.15 medium. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited SHE cell GJIC in a pH-dependent manner with cells at pH 6.70 exhibiting the greatest inhibition by TPA and cells at pH 7.35 being unresponsive. The effect of pH on GJIC in SHE cells is consistent with the pH-dependent response to chemically induced morphological transformation and may be mechanistically related to this phenomenon.

77. De Feijter A.W., Ray J.S., Weghorst C.M., Klaunig J.E., Goodman J.I., Chang C.C., Ruch R.J., Trosko J.E. (1990). Infection of rat liver epithelial cells with v-Ha-ras: correlation between oncogene expression, gap junctional communication, and tumorigenicity. Mol Carcinog. 1990;3(2):54-67. PMID: 2346586.

Abstract. The role of v-Ha-ras oncogene in tumorigenesis in an in vitro/in vivo model system was studied by investigating the expression of the Ha-ras gene, gap junctional intercellular communication, and tumorigenicity as endpoints. Infection of a Fischer 344 rat liver epithelial cell line (WB 344) with a retrovirus containing the v-Ha-ras oncogene resulted in altered cell morphology and decreased contact sensitivity. Gap junctional intercellular communication in v-Ha-ras infected WB cells (WBHa-ras), assessed by fluorescence redistribution after photobleaching (FRAP), microinjection/dye transfer, and scrape-loading/dye transfer techniques, was markedly decreased compared with the level in control WB cells. Injection of 10(7) WBHa-ras cells into the portal vein of male F344 rats caused multiple focal hepatic lesions within 1 and 2 wk, merging to large invading tumors after 3 and 4 wk. Examination of the methylation pattern of the Ha-ras gene in WBHa-ras and control WB cells showed that the infected Ha-ras gene was relatively hypomethylated in comparison to the normal cellular Ha-ras gene, indicating a greater potential for expression. There was an increased level of Ha-ras mRNA in hepatomas as compared with both adjacent nontumor liver tissue and liver tissue obtained from normal animals. Three cell lines derived from three different primary hepatic tumors induced by an injection of WBHa-ras cells in a F344 rat displayed similar growth characteristics, levels of gap junctional communication, and methylation patterns as the original WBHa-ras cells. The results of these studies have established a strong positive correlation between expression of the Ha-ras oncogene, reduced gap junctional intercellular communication, decreased contact sensitivity, and tumorigenicity of the v-Ha-ras-infected rat liver epithelial cells.

76. Klaunig J.E., Ruch R.J., Weghorst C.M. (1990). Comparative effects of phenobarbital, DDT, and lindane on mouse hepatocyte gap junctional intercellular communication. Toxicol Appl Pharmacol. 1990 Mar 1;102(3):553-63. PMID: 1690460.

Abstract. Gap junctional intercellular communication appears to be important in the regulation of cellular homeostasis, differentiated cell functions, and growth control in adult tissues. Interruption of intercellular communication by chemical compounds has been shown to be a sublethal response to a number of tumor promoters. The mechanism by which tumor promoters inhibit intercellular communication remains unresolved. In the present study the kinetics of inhibition of mouse hepatocyte gap junctional intercellular communication (measured by dye coupling) by three well-established hepatic tumor promoters [phenobarbital, 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT), and gamma-hexachlorocyclohexane (lindane)] are compared. All three compounds inhibited intercellular communication in a time- and dose-dependent manner in both freshly plated and 24-hr-old hepatocyte cultures. Following removal of the tumor promoters from the culture medium, intercellular communication was reestablished within 0.5 hr (phenobarbital) to 1.5 hr (DDT and lindane). Prolonged treatment of hepatocytes for up to 48 hr with the three promoters resulted in the continued inhibition of intercellular communication by lindane and DDT, but the development of refractoriness to phenobarbital-induced inhibition of intercellular communication. Concomitant treatment with combinations of the three promoters showed an additive effect of the compounds on inhibition of intercellular communication. Inhibition of intercellular communication by phenobarbital was prevented by addition of the cytochrome P450 enzyme inhibitor SKF-525A. SKF-525A had no effect on the inhibition of intercellular communication induced by lindane or DDT. Coincubation of the three promoters with the cAMP analog 8-bromo-cAMP prevented the promoter-induced inhibition of intercellular communication.

75. Klaunig J.E., Ruch R.J. (1990). Role of inhibition of intercellular communication in carcinogenesis. Lab Invest. 1990 Feb;62(2):135-46. PMID: 2406502.

74. Klaunig, J.E., Ruch, R.J., Weghorst, C.M., Hampton, J.A. (1990). Role of Inhibition of Intercellular Communication in Hepatic Tumor Promotion. In Vitro Toxicology, 3 (1), 91-107.

73. Trosko J.E., Chang C.C., Madhukar B.V., Klaunig J.E. (1990). Chemical, oncogene and growth  factor inhibition gap junctional intercellular communication: an integrative hypothesis of carcinogenesis. Pathobiology. 1990;58(5):265-78. PMID: 2076190.

Abstract. Most, if not all, cancer cells have some dysfunction in gap-junction-mediated intercellular communication, either because of defects in cell adhesion or inability to have functional gap junctional communication. In addition, most, if not all, tumor-promoting chemicals and conditions down-regulate gap junction function, while some antitumor-promoting chemicals can up-regulate gap junctional communication. Several oncogenes are associated with down-regulation of gap junction function and several hormone and growth regulators, known to be tumor promoters, are also able to down-regulate gap junction function. On the other hand, some tumor suppressor genes have been linked to the up-regulation of gap junctions. Based on these observations, it is hypothesized that, if a progenitor cell is unable to perform gap junctional intercellular communication, normal growth control and cell differentiation would not be possible, thereby favoring the development of malignant neoplasia.

72. Bandyopadhyay S., Klaunig J.E., Somani P. (1990). Cytotoxic interactions of cardioactive cationic amphiphilic compounds in primary rat hepatocytes in culture. Hepatology. 1990 Jul;12(1):48-58. PMID: 2373484.

Abstract. Hepatocytes from adult male Sprague-Dawley rats were isolated by the two-stage collagenase perfusion technique; 1 x 10(6) cells/plate were incubated in primary cell culture in Leibovitz’s L-15 medium for 24 hr with or without various concentrations (12.5 to 400 mumol/L) of cardioactive cationic amphiphilic compounds such as propranolol, verapamil, sotalol, atenolol and procainamide. Propranolol and verapamil caused a significant release of lactate dehydrogenase (used as cytotoxic index in this study) in the culture media in a concentration-dependent manner, with LC50 values of 220 +/- 10 and 224 +/- 7 mumol/L, respectively. Atenolol, sotalol and procainamide had no effect on lactate dehydrogenase release. Electron microscopy of the hepatocytes showed that subtoxic concentrations of propranolol (12.5 to 125 mumol/L) and verapamil (12.5 to 100 mumol/L) induced multilamellar inclusion bodies after 24 hr of incubation. The two higher concentrations of propranolol (50 and 125 mumol/L) and 100 mumol/L of verapamil produced a significant decrease in the percentage of volume density of the mitochondria as quantitated by morphometrical analysis. An unusual feature of the electron microscopical changes with propranolol and verapamil was the presence of mitochondria within the multilamellar inclusion bodies. When these two drugs were used together or with subtoxic concentrations of amiodarone or desethylamiodarone, release of lactate dehydrogenase was significantly enhanced. No correlation was evident between the cytotoxic response and the volume density of cellular inclusions in hepatocytes treated with different concentrations of propranolol, verapamil, amiodarone or desethylamiodarone. Sotalol, atenolol and procainamide in concentrations up to 400 mumol/L did not produce any ultrastructural changes in hepatocytes after 24 hr of incubation. These results show that (a) cationic amphiphilic structure per se is not the only requirement for induction of multilamellar inclusions, (b) propranolol and verapamil can induce the formation of multilamellar inclusion bodies and cause a concentration-dependent release of lactate dehydrogenase from hepatocytes and (c) combination of different cationic amphiphiles in subtoxic concentrations can enhance cytotoxicity and increase the volume density of multilamellar inclusions.

71. Somani P., Bandyopadhyay S., Klaunig J.E., Gross S.A. (1990). Amiodarone- and desethylamiodarone-induced myelinoid inclusion bodies and toxicity in cultured rat hepatocytes. Hepatology. 1990 Jan;11(1):81-92. PMID: 2153095.

Abstract. Hepatocytes isolated from Sprague-Dawley rats were incubated with various concentrations of either amiodarone or desethylamiodarone for 0 to 96 hr. Both drugs produced a concentration-dependent increase of lactate dehydrogenase release in the culture medium, which correlated well with cell death as measured by trypan blue exclusion test. Desethylamiodarone was more toxic than amiodarone in the cultured hepatocytes. Incubation with subtoxic concentrations of either amiodarone (7.6 microM) or desethylamiodarone (8 microM) for 24 hr resulted in the development of myelinoid inclusion bodies in the hepatocytes without any excess release of lactate dehydrogenase. In experimental protocols where the hepatocytes were exposed to either amiodarone or desethylamiodarone for up to 96 hr, there was an increase in lactate dehydrogenase and the percent volume-density of multilamellar inclusion bodies with cumulative drug exposure with time. A linear correlation between hepatocyte drug concentration and multilamellar inclusion bodies was found for both amiodarone and desethylamiodarone. These results demonstrate that both amiodarone and its major metabolite, desethylamiodarone, induce lysosomal inclusions, which, under appropriate conditions, can be dissociated from cell death. Withdrawal of the drug after 24 hr exposure did not result in disappearance of the inclusion bodies from the hepatocytes for up to 96 hr of tissue culture. The concentrations at which amiodarone- or desethylamiodarone-induced electron microscopic changes and hepatotoxicity were only two to five times as high as the usual serum drug levels in patients given antiarrhythmic therapy with amiodarone.

70. Xie Z.J., Wang Y.H., Askari A., Huang W.H., Klaunig J.E., Askari A. (1990). Studies on the specificity of the effects of oxygen metabolites on cardiac sodium pump. J Mol Cell Cardiol. 1990 Aug;22(8):911-20. PMID: 2172559.

Abstract. Isolated myocytes of rat heart, and sealed sarcolemmal vesicles of bovine heart, were used to examine the selectivity of the effects of partially reduced oxygen species (generated by a mixture of xanthine and xanthine oxidase) on cardiac sodium pump and several other ion transporters of the plasma membrane. When myocytes were exposed to xanthine plus xanthine oxidase, there were time-dependent inhibitions of ouabain-sensitive 86Rb+ uptake and (Na+ + K+)-ATPase activity that could be prevented by allopurinol, or by catalase and superoxide dismutase; suggesting the involvements of H2O2 or oxygen free radicals in the inhibition of the pump. This inhibition preceded any significant decrease in cellular ATP or in the number of viable cells. While ouabain increased 45Ca2+ uptake by myocytes as expected, exposure to xanthine plus xanthine oxidase decreased 45Ca2+ uptake; suggesting that the Na+, Ca2(+)-exchanger of the intact myocytes is also inhibited by oxygen metabolites. Simultaneous inhibitions of the pump, the Na+, Ca2(+)-exchange, the Na+, H(+)-exchange, and the Na+, Pi-cotransport activities also occurred in sarcolemmal vesicles that were treated with xanthine plus xanthine oxidase. These findings indicate that inactivations of the sodium pump and other sarcolemmal ion carriers are early events in the oxidant-induced damage to the cardiomyocyte. In the rat heart myocytes, a fraction of (Na+ + K+)-ATPase that seems to be more sensitive to ouabain, was inactivated more rapidly upon exposure of myocytes to xanthine plus xanthine oxidase; raising the possibility of the existence of different pump populations with different sensitivities to extracellularly generated oxygen metabolites.

69. Nigrovic V., Segal F., Klaunig J.E., Fry K. (1990). The Site and Mechanism of the Cytotoxic Effect of Atracurium In Vitro. European Journal of Anesthesiology, 7, 123-131.

68. Klaunig J.E., Weghorst T.R., Weghorst C.M. (1990). Liver tumor promoting ability of corn oil gavage in B6C3F1 male mice. Cancer Lett. 1990 Apr 30;50(3):215-9. PMID: 2322934.

Abstract. In chronic carcinogenic bioassays, chemicals being tested with low water solubility have been administered via corn oil gavage. The present study examined the effect of chronic corn oil gavage on hepatic tumor formation in the B6C3F1 male mouse. Mice were initiated with diethylnitrosamine (DENA) either at 15 days of age with a single i.p. injection (5 micrograms/gbw) (protocol 1) or at 4 weeks of age via the drinking water (15 mg/l) for a duration of 3 weeks (protocol 2). At weaning (protocol 1) or 8 weeks of age (protocol 2) initiated and untreated mice were administered either corn oil at a dose of 0.15 ml via gavage (once a day, 5 days/wk) or saline (0.15 ml via gavage, once a day 5 days/wk). All mice were killed at 28 weeks of age and hepatic lesions were quantitated. Only mice exposed to DENA demonstrated hepatic tumors. Mice treated with DENA (at 15 days of age) and corn oil gavage exhibited a significant decrease in the number of hepatic adenomas compared with DENA (at 15 days of age) only treated mice. No difference was noted in the number of hepatic adenomas between mice treated with DENA (at 4 wks of age) and corn oil gavage and mice exposed to DENA (at 4 wks of age) only.

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