67. Lin E.L., Klaunig J.E., Mattox J.K., Weghorst C.M., McFarland B.H., Pereira M.A. (1989). Comparison of the effects of acute and subacute treatment of phenobarbital in different strains of mice. Cancer Lett. 1989 Nov 15;48(1):43-51. PMID: 2819695.

Abstract. A strain specificity has been demonstrated for the effect of subsequent administration of phenobarbital (PB), in which diethylnitrosamine (DENA)-initiated hepatocarcinogenesis was promoted in C3H mice, inhibited in B6C3F1 (C57BL x C3H) and not affected in C57BL mice. A correlation has been established between the ability of barbiturates and hydantoins to promote tumor formation and their ability to induce liver growth, hepatic DNA synthesis and mixed function oxidase activities. Therefore, we examined in these 3 strains of mice and in C3B6F1 (C3H x C57BL) mice the effect of PB administered in their drinking water for 4 days or 28 days. The liver weight to body weight ratio was increased by PB in all types of mice. Microsomal protein concentrations were increased in C57BL mice after 28 days of treatment, in C3H after both 4 days and 28 days and in B6C3F1 after 4 days of treatment. No effect upon microsomal protein content was observed in C3B6F1 mice. DNA content was increased in C3H mice, both in the 4-day and 28-day treatment groups, while the other strains showed either a decrease or no difference from control. DNA synthesis was elevated in all strains of mice after 4 days of treatment with PB, however, after 28 days of treatment there was either a much reduced increase (C57BL and C3B6F1) or no difference (C3H and B6C3F1) from controls. In all 4 types of mice after 4 and 28 days of treatment, PB increased the concentration of cytochrome P-450, the activity of aminopyrine-N-demethylase (AmDm) and 7-ethoxyresorufin-O-deethylase (ErDe) and the oxidation of testosterone (T). The oxidative metabolites of T were similar in the 4 types of mice.

66. Ruch R.J., Crist K.A., Klaunig J.E. (1989). Effects of culture duration on hydrogen peroxide-induced hepatocyte toxicity. Toxicol Appl Pharmacol. 1989 Sep 15;100(3):451-64. PMID: 2781569.

Abstract. The effects of culture duration on primary cultured mouse hepatocyte antioxidant levels (superoxide dismutase, catalase, glutathione peroxidase, vitamin E, and glutathione) and susceptibility to glucose oxidase (GO)- and hydrogen peroxide (H2O2)-induced cell killing and lipid peroxidation were examined. Membrane fatty acid composition was also evaluated. Adult male B6C3F1/CrlBR mouse hepatocytes were isolated by collagenase perfusion of the liver and cultured on 60-mm plastic dishes in Leibovitz’s L-15 medium supplemented with glucose (1 mg/ml), dexamethasone (1 microM), fetal bovine serum (10%, v/v), and gentamicin sulfate (50 micrograms/ml) for 0 hr (freshly isolated cells) to 96 hr. Hepatocyte toxicity (determined by lactate dehydrogenase release and lipid peroxidation) after a 2-hr exposure to GO (0.8-80 micrograms/ml) or H2O2 (1-5 mM) decreased with increased time in culture. This decreased hepatocyte sensitivity to GO and H2O2 toxicity was not related to antioxidant enzyme activity since superoxide dismutase, catalase, and glutathione peroxidase declined during the 96-hr culture period. In contrast, glutathione and vitamin E levels in the cultured hepatocytes rose to 274.9 +/- 8.3% and 220.6 +/- 18.6% of the levels in freshly isolated cells (129.6 +/- 11.5 nmol and 0.10 +/- 0.01 nmol per 10(6) hepatocytes, respectively). The percentage of polyunsaturated fatty acids in hepatocyte phospholipids and triglycerides decreased with culture duration while the percentage of oleic acid increased in esterified and free fatty acid pools after 2 hr in culture. Total fatty acids were not affected by time in culture. These results suggest that the decreased hepatocyte susceptibility to the toxic effects of hydrogen peroxide may have been due to elevations in cellular GSH and vitamin E levels and decreases in membrane polyunsaturated fatty acids. The data also indicate that hepatocytes in primary culture undergo changes in antioxidant levels and fatty acid composition that may affect free radical toxicity at different times in culture.

65. Diwan B.A., Lubet R.A., Nims R.W., Klaunig J.E., Weghorst C.M., Henneman J.R., Ward J.M., Rice J.M. (1989). Lack of promoting effect of clonazepam on the development of N-nitrosodiethylamine-initiated hepatocellular tumors in mice is correlated with its inability to inhibit cell-to-cell communication in mouse hepatocytes. Carcinogenesis. 1989 Sep;10(9):1719-24. PMID: 2766464.

Abstract. The tumor-promoting ability of clonazepam (CZP), a widely used benzodiazepine anticonvulsant, was investigated in an in vivo mouse liver tumor promotion assay and an in vitro mouse hepatocyte intercellular communication assay. The development of preneoplastic hepatocellular foci of cellular alteration and hepatocellular neoplasms was studied in male B6C3F1 mice initiated, at 5 weeks of age, with a single i.p. injection of N-nitrosodiethylamine (NDEA; 90 mg/kg body weight) in tricaprylin, followed by administration of either phenobarbital (PB; 0.05%) or CZP (0.068% or 0.136%) in diet beginning 2 weeks after carcinogen injection and continuing to 60 weeks of age. Several mice from each group were killed after 9, 21, 33 or 53 weeks on test diet, and portions of liver and other organs were fixed in formalin and examined histologically. Unlike PB, CZP did not promote the development of preneoplastic hepatocellular foci or neoplasms (adenomas and carcinomas) in NDEA-initiated mice. Following limited (2 weeks) dietary exposure at 0.15%, CZP was a potent inducer of hepatic P450IIB1-mediated alkoxyresorufin O-dealkylase activities. In contrast, the degree of induction in hepatic tissue from mice fed 0.136% CZP for 53 weeks was markedly lower than that in mice fed 0.05% PB for 53 weeks. In the in vitro assay, diazepam, a strong tumor promoter in mouse liver, significantly inhibited mouse hepatocyte gap junctional intercellular communication, while CZP had no significant effect on this parameter. Thus, CZP, a drug structurally related to diazepam, is inactive as a liver tumor promoter in mice.

64. Weghorst C.M., Pereira M.A., Klaunig J.E. (1989). Strain differences in hepatic tumor promotion by phenobarbital in diethylnitrosamine- and dimethylnitrosamine-initiated infant male mice. Carcinogenesis. 1989 Aug;10(8):1409-12. PMID: 2752514.

Abstract. The effects of phenobarbital (PB) on hepatocellular carcinogenesis in three strains of nitrosamine-initiated infant male mice were evaluated. Fifteen-day-old C57Bl/6NCrlBR (C57Bl), C3H/HeNCr1BR (C3H) and B6C3F1 mice were treated with a single i.p. injection of either diethylnitrosamine (DENA) (5 micrograms/body wt), dimethylnitrosamine (DMNA) (5 micrograms/body wt) or saline. One-half of the treated mice received PB via the drinking water (500 mg/l) for 24 weeks. The remaining treated mice were given deionized drinking water. Mice were killed at 28 weeks of age and hepatic lesions were evaluated. Only animals that received DENA or DMNA exhibited tumors. C3H mice treated with DENA + PB demonstrated a significant increase in hepatic adenoma number compared to C3H mice exposed to DENA only. Conversely, B6C3F1 males treated with DENA + PB exhibited a significant decrease in the number of hepatic adenomas compared to B6C3F1 males treated with DENA alone. No change was noted in adenoma size in B6C3F1 mice treated with DENA + PB from those receiving DENA only. Chronic PB exposure of C57Bl males previously treated with DENA had no effect on hepatic adenoma number or size. C3H mice treated with DMNA + PB displayed an increase in both adenoma size and adenoma number compared to C3H mice receiving DMNA only. Similarly, in B6C3F1 mice, PB treatment increased both the adenoma incidence and adenoma number in DMNA initiated mice. PB had no effect on hepatic adenoma incidence or number in DMNA-treated C57Bl mice. These data suggest that the ability of PB to promote hepatic tumorigenesis in the 15-day-old initiated mouse is dependent on both the strain of the mouse and the initiating chemical carcinogen.

63. Weghorst C.M., Klaunig J.E. (1989). Phenobarbital promotion in diethylnitrosamine-initiated infant B6C3F1 mice: influence of gender. Carcinogenesis. 1989 Mar;10(3):609-12. PMID: 2924405.

Abstract. Phenobarbital (PB) promotes hepatic tumorigenesis when chronically administered to male B6C3F1 mice after initiation with diethylnitrosamine (DENA) at 30 days of age. In contrast, when male B6C3F1 mice were initiated with DENA at 15 days of age, an inhibition of hepatic tumorigenesis occurred. The present study was undertaken to evaluate the influence of gender on the inhibiting ability of PB in the 15 day old DENA-initiated B6C3F1 mouse. Mice were injected with either DENA (5 micrograms/g) or saline at 15 days of age. At weaning mice were given either PB (500 p.p.m.) containing drinking water or deionized drinking water for 24 weeks. Male mice treated with DENA and PB demonstrated a significant decrease in the number of hepatocellular adenomas compared to males receiving DENA only. In contrast, females exposed to DENA and PB exhibited an enhancement of hepatic adenoma number compared to those receiving only DENA. In an additional experiment, individual preneoplastic foci from male and female B6C3F1 mice initiated with DENA at 15 days of age were examined for their responsiveness to the mitogenic stimuli of PB. Mice were exposed to either PB-containing or PB-free drinking water for 7 days. In non-PB treated males and females, preneoplastic hepatocytes demonstrated higher rates of DNA synthetic labelling compared to normal hepatocytes with no gender difference noted. Males exposed to PB exhibited increased levels of DNA synthesis in normal cells but not in preneoplastic foci. Females treated with PB, however, demonstrated significant increases in DNA synthesis in both preneoplastic and normal hepatocytes compared to non-PB treated females and PB-treated males. These findings suggest that in male mice initiated with DENA at 15 days of age, the preneoplastic foci are refractory to the proliferative effects of PB which may account for the observed inhibition of hepatic tumorigenesis by PB in this mouse strain.

62. Gross S.A., Bandyopadhyay S., Klaunig J.E., Somani P. (1989). Amiodarone and desethylamiodarone toxicity in isolated hepatocytes in culture. Proc Soc Exp Biol Med. 1989 Feb;190(2):163-9. PMID: 2536944.

Abstract. Amiodarone, a class III antiarrhythmic drug, has been found to be effective in the management of patients with life-threatening ventricular arrhythmias. Recent reports describe the presence of myelinoid inclusion bodies following amiodarone therapy in liver, myocardium, white blood cells, lung, cornea, skin, and lymph nodes; their relationship to toxicity is unclear. The exact role of desethylamiodarone, the major metabolite, of amiodarone in systemic toxicity of the parent drug is not known. Concentration-response relationships for amiodarone and desethylamiodarone were investigated by adding 1-50 micrograms/ml of the compounds of dimethyl sulfoxide (controls) to hepatocytes isolated from Sprague-Dawley rats and cultured in Leibovitz L-15 medium. Using lactate dehydrogenase release into the medium to quantitate cell death, both drugs were found to cause cell death in a concentration-dependent manner within 24 hr of incubation; this data showed desethylamiodarone to be significantly more toxic than amiodarone. In experiments with 50-micrograms/ml concentrations of amiodarone or desethylamiodarone, we found desethylamiodarone to produce a significantly greater release of lactate dehydrogenase as compared with amiodarone within 2-4 hr. Electron microscopic studies indicated the presence of myelinoid inclusion bodies at early culture stages followed by progressive swelling of mitochondria and rough endoplasmic reticula, disruption of membranes, aggregation of subcellular structures, and ultimately cell death. Ultrastructural changes occurred sooner in the hepatocytes treated with desethylamiodarone than with amiodarone. These data demonstrate that (i) desethylamiodarone is more toxic than amiodarone; (ii) acute toxicity of desethylamiodarone and amiodarone can be quantitated by lactate dehydrogenase release; (iii) both desethylamiodarone and amiodarone can induce myelinoid inclusion bodies in cultured hepatocytes; and (iv) toxicity is characterized by progressive subcellular changes leading to cell death.

61. Birkhahn R.H., Awad S., Klaunig J.E., Thomford N.R. (1989). Interaction of ketosis and liver regeneration in the rat. J Surg Res. 1989 Nov;47(5):427-32. PMID: 2509817.

Abstract. Monoacetoacetin, the monoglyceride of acetoacetate, was investigated as a nutritional support for the regenerating liver. Following partial hepatectomy, rats were either fed an oral diet ad libitum or administered by total parenteral feeding glucose alone, monoacetoacetin-glucose mixture, or lipid emulsion-glucose for the nonprotein calories. Five rats from each treatment were killed at 6-hr intervals beginning 12 hr after partial hepatectomy and ending at 72 hr. The number of cells synthesizing DNA and the number of cells in mitosis were compared. Rats fed orally or infused with glucose alone or with lipid emulsion had similar parameters throughout. Rats infused with monoacetoacetin had approximately double the number of cells in mitosis and DNA synthesis compared to the other treatments. This stimulation by monoacetoacetin persisted 72 hr. It was concluded from the data that acetoacetate was the agent responsible for increased DNA synthesis and mitosis, but the mechanism for the stimulation was not identified.

60. Klaunig J.E., Ruch R.J., Lin E.L. (1989). Effects of trichloroethylene and its metabolites on rodent hepatocyte intercellular communication. Toxicol Appl Pharmacol. 1989 Jul;99(3):454-65. PMID: 2749732.

Abstract. Chronic exposure to trichloroethylene (TCE) results in hepatocellular cancer in mice but not rats. The induction of hepatic tumors by TCE appears to be mediated through nongenotoxic or tumor promotion mechanisms. One cellular effect exhibited by a number of nongenotoxic carcinogens and tumor promoters is the inhibition of gap junction mediated intercellular communication. In the present study, the effects of trichloroethylene (TCE) and its metabolites, trichloracetic acid (TCA), trichloroethanol (TCEth), and chloral hydrate (CH) on gap junction mediated intercellular communication in cultured B6C3F1 mouse and F344 rat hepatocytes were assessed. TCE and TCA inhibited intercellular communication in mouse hepatocytes but not in rat hepatocytes. TCEth and CH had no effect on hepatocyte intercellular communication in either rat or mouse cells. TCE and TCA inhibited intercellular communication in both 24-hr-old and freshly plated mouse hepatocytes. Both compounds produced greater inhibition of intercellular communication in freshly plated cells when compared to 24-hr-old cultures. TCE appeared to require cytochrome P450 metabolism by the mouse hepatocytes to exhibit its inhibitory effect on dye coupling since treatment with SKF-525A prevented the inhibition of intercellular communication by TCE. The inhibitory effect of TCA on intercellular communication was unaffected by treatment with SKF-525A. While the species dependent effect of TCE on intercellular communication may be correlated with different rates and extent of metabolism of TCE by rat and mouse hepatocytes, the inhibiting effect of TCA only on mouse hepatocytes suggests that other intrinsic factors in the male mouse make this species more susceptible to the effects of TCE and TCA on gap junction mediated intercellular communication. These findings may account, in part, for the observed species difference in susceptibility to TCE induced liver carcinogenesis.

59. Ruch R.J., Cheng S.J., Klaunig J.E. (1989). Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis. 1989 Jun;10(6):1003-8. PMID: 2470525.

Abstract. An antioxidant fraction of Chinese green tea (green tea antioxidant; GTA), containing several catechins, has been previously shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. In the present study, GTA was shown to have antioxidative activity toward hydrogen peroxide (H2O2) and the superoxide radical (O2-). GTA also prevented oxygen radical and H2O2-induced cytotoxicity and inhibition of intercellular communication in cultured B6C3F1 mouse hepatocytes and human keratinocytes (NHEK cells). GTA (0.05-50 micrograms/ml) prevented the killing of hepatocytes (measured by lactate dehydrogenase release) by paraquat (1-10 mM) and glucose oxidase (0.8-40 micrograms/ml) in a concentration-dependent fashion. GTA (50 micrograms/ml) also prevented the inhibition of hepatocyte intercellular communication by paraquat (5 mM), glucose oxidase (0.8 micrograms/ml), and phenobarbital (500 micrograms/ml). In addition, GTA (50 micrograms/ml) prevented the inhibition of intercellular communication in human keratinocytes by TPA (100 ng/ml). Cytotoxicity and inhibition of intercellular communication, two possible mechanisms by which tumor promoters may produce their promoting effects were therefore prevented by GTA. The inhibition of these two effects of pro-oxidant compounds may suggest a mechanism by which GTA inhibits tumor promotion in vivo.

58. Nigrovic V., Pandya J.B., Klaunig J.E., Fry K. (1989). Reactivity and toxicity of atracurium and its metabolites in vitro. Can J Anaesth. 1989 May;36(3 Pt 1):262-8. PMID: 2720863.

Abstract. Cytotoxicity of atracurium and of its metabolites was tested in vitro. Exposure of isolated rat hepatocytes to atracurium produced cellular damage evidenced by extrusion of an intracellular enzyme, lactate dehydrogenase (LDH), into the incubation medium. Leakage of LDH was directly related to the concentration of atracurium in the medium (250 to 800 microM). If the spontaneous degradation of atracurium (presumably via Hofmann elimination) was first carried out in vitro and the degradation products subsequently added to the isolated hepatocytes, the leakage of LDH was also dose-dependent but larger than that observed after the addition of the parent drug. When l-cysteine was admixed to the products of the spontaneous degradation of atracurium prior to their addition to the liver cells, no leakage of LDH was observed. The results are compatible with the working hypothesis that atracurium itself and, even more so, acrylates formed in Hofmann elimination of atracurium, are reactive toward nucleophiles and damage the cells by alkylating nucleophiles present in cellular membranes. Antecedent covalent binding of acrylates to the nucleophile cysteine, i.e., the formation of acrylate-cysteine adducts, saturated the reactive capacity of acrylates for nucleophiles and thus prevented the reactive metabolites from alkylating the endogenous nucleophiles. Possible clinical consequences resulting from in vivo generation of reactive metabolites are not clear at the present time but are projected to be related to (a) the dose of atracurium administered, (b) the amount of acrylates generated, (c) the functional importance of the endogenous nucleophiles alkylated, and (d) the pathway and the speed of detoxification of atracurium and its metabolites.


57. Klaunig J.E., Ruch R.J., DeAngelo A.B., Kaylor W.H. (1988). Inhibition of mouse hepatocyte intercellular communication by phthalate monoesters. Cancer Lett. 1988 Dec 1;43(1-2):65-71. PMID: 3203332.

Abstract. A series of straight and branched chain phthalate monoesters were examined for their effects on hepatocyte intercellular communication in male B6C3F1 mouse hepatocytes. Intercellular communication was determined autoradiographically following the passage and incorporation of [5-3H]uridine nucleotides from pre-labelled hepatocytes into non-labelled hepatocytes. Intercellular communication was evaluated in hepatocytes after 8 h treatment of straight and branched chain phthalate esters at sublethal concentrations. Straight chain phthalate monoesters (mono(ethyl)phthalate, mono(n-butyl) phthalate, mono(n-hexyl)phthalate, mono(n-octyl)phthalate, mono(n-nonyl)phthalate and mono(isononyl)phthalate) had no effect on hepatocyte intercellular communication. Branched chain phthalate monoesters that contained an ethylalkyl moiety (i.e. mono(2-ethylpropyl) phthalate, mono(2-ethylbutyl)phthalate, mono(2-ethylpentyl)-phthalate and mono(2-ethylhexyl)phthalate) inhibited intercellular communication. These results show a structure-activity relationship in the ability of phthalate monoesters to inhibit intercellular communication in mouse hepatocytes. Based upon previous correlations between inhibition of intercellular communication in hepatocytes and hepatocarcinogenicity, these data suggest that branched chain phthalate esters may be liver carcinogens in male B6C3F1 mice.

56. Klaunig J.E., Pereira M.A., Ruch R.J., Weghorst C.M. (1988). Dose-response relationship of diethylnitrosamine-initiated tumors in neonatal balb/c mice: effect of phenobarbital promotion. Toxicol Pathol. 1988;16(3):381-5. PMID: 3194660.

Abstract. The dose-response of diethylnitrosamine (DENA) initiation of hepatocarcinogenesis was determined in infant Balb/c male mice with and without subsequent phenobarbital treatment. Male Balb/c mice received a single intraperitoneal injection of DENA (0, 2.5, 10.0, 25.0 or 50.0 micrograms/gbw) in saline on day 15 of age. Ninety mice were treated at each dose level. At weaning, mice received either deionized drinking water (45 mice per group) or deionized drinking water containing 500 mg/L sodium phenobarbital (PB) (45 mice per group). Mice from each group were sacrificed 12, 24, and 40 weeks post-weaning. Liver and lung tumors were found in DENA-only-treated and DENA + PB-treated mice. In DENA-only-treated mice, the incidence and number of hepatic adenomas were similar (not dose-dependent) at DENA doses of 10, 25, and 50 micrograms/gbw at each of the 3 sampling times. DENA-only-treated mice did display a time-related increase in hepatic adenoma incidence and number at each dose. In PB-treated mice, the hepatic adenoma number was dependent upon the dose of DENA between 2.5 and 50 micrograms/gbw. PB treatment following DENA administration resulted in a decrease in the time required for the detection of hepatic adenomas and increased the number of hepatic adenomas at most sampling times compared to the mice that received DENA only. Hepatocellular carcinomas (HPC) were detected in mice receiving the highest DENA doses (25 and 50 micrograms/gbw). PB treatment increased the number and incidence of HPC and decreased the time of first detection of HPC.

55. Klaunig J.E., Weghorst C.M., Pereira M.A. (1988). Effect of phenobarbital on diethylnitrosamine and dimethylnitrosamine induced hepatocellular tumors in male B6C3F1 mice. Cancer Lett. 1988 Sep-Oct;42(1-2):133-9. PMID: 3180032.

Abstract. The effect of the type of carcinogen initiator on the ability of phenobarbital (PB) to promote hepatic tumor formation in 15-day-old initiated male B6C3F1 mice was evaluated. Fifteen-day-old male B6C3F1 mice were divided into 6 groups of 10 mice each. Groups 1 and 2 received a single intraperitoneal (i.p.) injection of diethylnitrosamine (DENA) (5 micrograms/body wt). Groups 3 and 4 received a single i.p. injection of diethylnitrosamine (DENA) (5 micrograms/g body wt). Groups 3 and 4 received a single i.p. injection of dimethylnitrosamine (DMNA) (5 micrograms/g body wt). Groups 5 and 6 received a single i.p. injection of saline. At weaning (28 days of age), mice in groups 2, 4 and 6 received PB (500 mg/ml) in their drinking water. Mice in groups 1, 3 and 5 received deionized drinking water. Drinking water treatment continued for 24 weeks at which time mice were sampled. At sampling, mice were examined for hepatic tumors by histology. Mice in groups 5 (no treatment) and 6 (PB only) did not exhibit hepatic tumors. Groups 2 (DENA + PB) displayed a decrease in hepatic adenomas from that of group 1 (DENA only), confirming previous observations. Treatment with DMNA and PB (group 4), however, resulted in a significant increase in both hepatic adenoma incidence and number over that of DMNA only (group 3) treated mice. The promoted adenomas appeared to be predominantly eosinophilic in appearance. The type of initiator therefore appears important in determining if 15-day-old initiated male B6C3F1 mice respond to the promotion effects of PB.

54. Klaunig J.E., Barut B.A. (1988). Influence of transplantation site on metastatic ability of mouse bladder carcinoma sublines. J Urol. 1988 Oct;140(4):844-7. PMID: 3418820.

Abstract. The influence of the primary implantation site on the metastatic behavior of a murine transitional cell carcinoma line (MBT-2) and three metastatic sublines (L3F1, L3F2, and L3F3) was studied. The parent MBT-2 cell line produced a low incidence of lung metastasis after intravenous injection and no metastases from the primary tumor when injected either subcutaneously in the right hind flank or in the footpad. Intramuscular implantation of the MBT-2 cells in the right hind flank resulted in a significant increase over the subcutaneous, footpad, and intravenous sites in the incidence and number of lung metastases. Three in vivo/in vitro selected metastatic sublines (L3F1, L3F2, and L3F3) were highly metastatic when injected subcutaneously, intramuscularly, and intravenously. A low number of pulmonary metastases was observed after footpad implantation of the three sublines. This study demonstrated a definite implantation site-influence on the metastatic ability of the parent MBT-2 line and the three selected sublines. Intramuscular implantation was the most permissive implantation site for the development of spontaneous metastasis for the MBT-2 line and the L3F1, L3F2, and L3F3 sublines.

53. Ruch R.J., Klaunig J.E. (1988). Inhibition of mouse hepatocyte intercellular communication by paraquat-generated oxygen free radicals. Toxicol Appl Pharmacol. 1988 Jul;94(3):427-36. PMID: 3400094.

Abstract. Intercellular communication through gap junctions functions in electrical synapsing, homeostasis, hormonal response, embryogenesis, and growth control. Many neurotoxicants, teratogens, and carcinogens are capable of inhibiting gap junctional intercellular communication and this effect may be related to their toxic activity. In addition, many of these toxic agents are capable of stimulating oxygen free radical production in cells. The purpose of this study was to determine if oxygen free radicals at noncytotoxic levels could inhibit intercellular communication in primary cultured mouse hepatocytes. Intercellular communication was evaluated in 24-hr-old cultures of male B6C3F1/Cr1BR mouse hepatocytes by microinjection of fluorescent Lucifer Yellow CH dye and visualization of dye spread to adjacent hepatocytes (dye-coupling). Dye-coupling was rapidly established in freshly plated primary cultured hepatocytes reaching a level of over 90% after 24 hr of culture. After 24 hr, dye-coupling paralleled hepatocyte survival. Treatment of hepatocyte cultures with noncytotoxic concentrations of paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride; PQ) (0.5-5 mM), hydrogen peroxide (0.5-2 mM), glucose oxidase (0.1 U/ml), or xanthine oxidase (0.2 U/ml plus 1 mM xanthine) for exposure durations of 2-8 hr resulted in concentration-dependent decreases in dye-coupling. Addition of the antioxidants DPPD (N,N-diphenyl-p-phenylenediamine; 25 microM) and vitamin E (D,L-alpha-tocopherol acetate; 100 microM) decreased the inhibitory effect of PQ on dye-coupling. In contrast, addition of the catalase inhibitor 3-amino-1,2,4-triazole or the glutathione depletor diethylmaleate to PQ-treated cultures potentiated PQ-induced inhibition of dye-coupling. PQ stimulated NADPH-dependent mouse liver microsomal superoxide radical production. Thus, one effect of prooxidant compounds appears to be the inhibition of IC. This effect may be important in the sublethal toxicity of oxygen radical generating compounds.

52. Ruch R.J., Klaunig J.E. (1988). Kinetics of phenobarbital inhibition of intercellular communication in mouse hepatocytes. Cancer Res. 1988 May 1;48(9):2519-23. PMID: 3356013.

Abstract. Gap junction-mediated intercellular communication in untreated and phenobarbital-treated C57BL/6 X C3H F1 mouse hepatocytes was evaluated by microinjection of fluorescent Lucifer Yellow CH dye. Intercellular communication (dye coupling) was detected in untreated hepatocytes after 0.5 h in culture, reached a maximum level in 24- and 48-h-old cultures (85.2%), and then decreased over the next 72 h. Phenobarbital (20-500 micrograms/ml) decreased dye coupling in a dose-related manner when added to freshly plated cultures. This inhibitory effect was evident during 0.5-12 h of treatment but was not seen in cultures treated for 24 h. Phenobarbital also decreased dye coupling within 30 min when added to established (24-h-old) hepatocyte cultures. This effect was maximal after 2 h treatment. In these cultures, dye coupling recovered within 15 min after removal of the promoter. Hepatocytes, pretreated with phenobarbital for 24 h, did not show inhibition of dye coupling after reapplication of phenobarbital. Thus, phenobarbital inhibited mouse hepatocyte dye coupling rapidly and reversibly, and the cells became refractory to the inhibitory effect after prolonged treatment.

51. Klaunig J.E., Barut B.A. (1988). Role of the implantation site on metastatic ability of the murine MBT-2 transitional cell carcinoma. Urol Res. 1988;16(1):19-21. PMID: 3344561.

Abstract. The influence of implantation site on the metastatic behavior of a murine transitional cell carcinoma line (MBT-2) was examined. MBT-2 cells were injected into one of four anatomic sites; subcutaneously, intramuscularly, intravenously or into the footpad, to evaluate the influence of implantation site on the formation and number of metastases. The MBT-2 cell line produced a low incidence of lung metastases after intravenous injection with a mean of 1.1 lung tumors per mouse. Injection of MBT-2 cells into the footpad or subcutaneously did not produce metastases from the primary tumor. Intramuscular implantation, however, resulted in a sixty percent incidence of metastasis with a mean of 8.2 lung nodules per mouse. This study demonstrated a definite implantation site influence on the metastatic ability of the MBT-2 line.


50. Klaunig J.E., Ruch R.J. (1987). Role of cyclic AMP in the inhibition of mouse hepatocyte intercellular communication by liver tumor promoters. Toxicol Appl Pharmacol. 1987 Nov;91(2):159-70. PMID: 2823418.

Abstract. The liver tumor promoters phenobarbital (PB) (20-500 micrograms/ml) and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) (1-10 micrograms/ml) inhibited intercellular communication between primary cultured B6C3F1 mouse hepatocytes after 8 hr of treatment. Intercellular communication was detected autoradiographically as the passage and incorporation of [5-3H]uridine nucleotides from prelabeled donor hepatocytes to recipient hepatocytes. The addition of either dibutyryl cyclic AMP (N6,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) (0.001-0.1 mM) or caffeine (0.01-1 mM) decreased or completely abolished the inhibitory effects of PB and DDT on intercellular communication. Cyclic AMP (adenosine 3′:5′-cyclic monophosphate; cAMP) in primary cultured mouse hepatocytes was measured by radioimmunoassay. Cyclic AMP in nontreated, freshly plated cultures declined from 4.2 +/- 0.7 pmol/mg protein after 1 hr in culture to 2.4 +/- 0.5 pmol/mg protein after 8 hr in culture. Phenobarbital at 250 and 500 micrograms/ml significantly decreased cyclic AMP below control values after 1 hr of treatment. However, no difference in the amount of cyclic AMP was detected between control and PB-treated cultures after 2, 4, and 8 hr in culture or with lower PB concentrations. DDT at 10 micrograms/ml decreased cAMP levels in the hepatocytes after 1, 2, 4, and 8 hr of treatment. No effects were seen after 8 hr of treatment or with lower DDT concentrations. DDT (10 micrograms/ml) also decreased cAMP levels in 24-hr-old cultures while PB (500 micrograms/ml) had no effect. Addition of dibutyryl cAMP (0.1 mM) or caffeine (1.0 mM) to freshly plated cultures elevated cAMP levels 50-fold and twofold, respectively. These data suggest that the inhibition of mouse hepatocyte intercellular communication by PB and DDT at the highest concentrations tested may be mediated by transient decreases in intercellular cAMP levels.

49. Klaunig J.E., Barut B.A. (1987). The efficacy of chemotherapeutic agents against murine bladder metastasis. J Urol. 1987 Dec;138(6):1471-3. PMID: 3682079.

Abstract. Three chemotherapeutic agents, methotrexate, cyclophosphamide and cis-diamminedichloro-platinum (cis-platinum), were examined for their effectiveness against metastases in a murine transitional cell carcinoma model. Systemic treatment of the drugs was applied against a MBT-2 derived subline which generates 100% incidence of lung metastases in C3H mice by five weeks. The drugs were examined for their effect against the number of metastases, incidence of metastasis and size of the subcutaneously implanted primary tumor. All three compounds significantly reduced both the number of lung metastases and the incidence when compared to untreated animals. None of the agents proved 100% effective against metastatic tumors. These results suggest the existence of a chemotherapeutic resistant population of metastatic cells. Administration of methotrexate and cis-platinum effectively reduced the size of the primary tumor as compared to untreated animals. Cyclophosphamide did not significantly affect primary tumor size. The response of the antineoplastic agents against the metastatic tumor cells indicates that the L3F2 metastatic cell line is an effective model to study agents against metastatic bladder cancer.

48. Klaunig J.E., Weghorst C.M., Pereira M.A. (1987). Effect of the age of B6C3F1 mice on phenobarbital promotion of diethylnitrosamine-initiated liver tumors. Toxicol Appl Pharmacol. 1987 Aug;90(1):79-85. PMID: 3629593.

Abstract. Chronic exposure to phenobarbital (PB) in the drinking water of male B6C3F1 mice starting at 4 weeks of age and subsequent to a single (ip) injection of diethylnitrosamine (DENA) administered on Day 15 of age has been shown to result in the inhibition of hepatic tumor formation. In this study, we varied the time of onset of PB administration to determine if sexual maturity would affect liver tumor formation and progression. Male B6C3F1 mice were divided into eight groups. Groups 1-4 received a single (ip) dose of 5 mg/kg DENA at 15 days of age while mice in groups 5-8 received saline. At weaning (4 weeks of age), groups 1 and 5 received deionized drinking water (DDW) for 24 weeks; groups 2 and 6 received PB (500 ppm) in the drinking water (PB DW) for 16 weeks followed by DDW for 8 weeks; groups 3 and 7 received DDW for 4 weeks, PB DW for 16 weeks, and then DDW for 4 weeks; and groups 4 and 8 received DDW for 8 weeks and PB DW for 16 weeks. Mice were killed at 28 weeks of age and hepatic lesions were evaluated. Mice which did not receive DENA (groups 5-8) exhibited no liver tumors. Animals in groups 1-4 exhibited hepatocellular foci and adenomas. PB treatment in groups 2, 3, or 4 resulted in a significant decrease in the incidence of DENA-initiated hepatocellular foci and adenomas when compared to those observed in group 1. The number of foci in group 4 was significantly decreased compared to those in groups 2 and 3. There was no significant difference in the adenoma incidence among groups 2, 3, and 4. No significant differences were observed in the sizes of foci or adenomas among groups 1-4. Data from this study suggest that the inhibition of hepatocellular tumorigenesis by PB remains intact even when the start of the administration of PB is withheld up to 12 weeks of age.

47. Klaunig J.E., Ruch R.J. (1987). Strain and species effects on the inhibition of hepatocyte intercellular communication by liver tumor promoters. Cancer Lett. 1987 Aug;36(2):161-8. PMID: 3621148.

Abstract. The effect of the liver tumor promoters phenobarbital (PB), 1,1-bis(4-chlorophenyl)-2,2,2-trichlorethane (DDT), and dieldrin on gap junction-mediated intercellular communication between primary cultured hepatocytes from male mice (B6C3F1), C3H, C57BL, and Balb/c strains) and male F344 rats was determined. Intercellular communication was detected autoradiographically as the passage and incorporation of [5-3H]uridine nucleotides from prelabelled donor hepatocytes to donor-contacting recipient hepatocytes. At non-toxic concentrations, PB (20-500 micrograms/ml) inhibited intercellular communication between B6C3F1, C3H, and Balb/c mouse hepatocytes and F344 rat hepatocytes, but not between C57BL mouse hepatocytes. DDT (1-10 micrograms/ml) inhibited intercellular communication between hepatocytes from all 4 strains of mice and the F344 rat. Dieldrin (1-10 micrograms/ml) inhibited intercellular communication between hepatocytes from the 4 strains of mice but not between rat hepatocytes. These findings showed a good correlation with the in vivo liver tumor promoting/hepatocarcinogenic actions of PB, DDT and dieldrin in the 4 mouse strains and the F344 rat strain.

46. Hampton J.A., Klaunig J.E., Goldblatt P.J. (1987). Resident sinusoidal macrophages in the liver of the brown bullhead (Ictalurus nebulosus): an ultrastructural, functional and cytochemical study. Anat Rec. 1987 Dec;219(4):338-46. PMID: 3448951.

Abstract. Ultrastructural, functional, and cytochemical characteristics of resident sinusoidal macrophages (RSM) in brown bullhead (Ictalurus nebulosus) liver were examined. Following perfusion fixation of the hepatic vascular bed, light micrographs revealed RSM that possessed multiple elongate cytoplasmic processes and frequently contained erythrocytes in various stages of degradation. Following brief perfusion fixation, light microscope examination of vibratome sections of bullhead liver reacted for peroxidase revealed intensely positive RSM. By transmission electron microscopy, peroxidase activity was localized to the nuclear envelope and cytoplasmic granules of RSM and in endothelial and perisinusoidal fat-storing cells. In cryostat sections of fresh-frozen liver, glucose-6-phosphate dehydrogenase (G-6-PDH) was uniformly distributed over hepatocytes, whereas intensely positive punctate staining for G-6-PDH was localized over RSM. To test for phagocytosis by RSM, latex beads (0.81 micron) were injected into a tributary of the hepatic portal vein 2 min prior to perfusion fixation. Latex beads appeared either singly or in dense aggregates within RSM. Ultrastructurally, RSM were characterized by an irregularly shaped, eccentrically located nucleus, electron-dense vacuoles, small patches of granular endoplasmic reticulum, a well-developed Golgi apparatus, elongated mitochondria, desmosomes or desmosome-like densities that served as a source of attachment to endothelial cells, and a centriole with radiating microtubules. Invaginations of the plasma membrane (vermiform processes) characteristic of mammalian Kupffer cells were not observed in bullhead RSM. The results indicated a resident cell population of sinusoidal macrophages in the bullhead liver with properties that partially resembled mammalian Kupffer cells. These results are important for the identification of the normal resident cells in the bullhead liver.

45. Goldblatt P.J., Hampton J.A., DiDio L.N., Skeel K.A., Klaunig J.E. (1987). Morphologic and histochemical analysis of the newt (Notophthalmus viridescens) liver. Anat Rec. 1987 Apr;217(4):328-38. PMID: 3035962.

Abstract. Architectural arrangement, ultrastructure, and selected histochemical properties of the newt (Notophthalmus viridescens) liver were examined. Although hematopoietic tissue (1-4 cells thick) invested the liver, direct vascular communication between this tissue and hepatic parenchyma was not observed. The liver was intensely positive when stained with Oil-red-O and periodic acid-Schiff reagent and connective tissue was limited to large vascular channels and the capsule. A distinctive polarity was observed in the hepatic vascular system when lobes were viewed in cross section. Dorsally, portal venules accompanied arterioles and branches of the biliary system, while tributaries of hepatic veins were observed ventrally. Following perfusion fixation, hepatocytes appeared as sheets of cells 1-5 cells thick; however, lobules as defined in adult mammalian liver were absent. Hepatocytes contained abundant smooth endoplasmic reticulum, mitochondria, electron-dense lysosomes, patches of granular endoplasmic reticulum, and lipid droplets. Continuous endothelial cells lined sinusoids and exhibited fenestrae organized into structures similar to sieve plates observed in mammalian liver. Variable numbers of melanin-containing macrophages and subendothelial macrophages were observed; however, Kupffer cells and lipid containing perisinusoidal fat-storing cells were not seen. Patterns of reaction product for glucose-6-phosphatase (G-6-Pase), glucose-6-phosphate dehydrogenase (G-6-PDH), and succinic dehydrogenase (SDH) were localized in the newt liver. All enzymes exhibited a uniform distribution pattern; however, small punctate regions of intensely positive G-6-PDH cells were noted within hepatic parenchyma. Cells comprising the hematopoietic tissue were intensely positive for G-6-Pase, G-6-PHD, and negative for SDH.

44. Nigrovic V., Klaunig J.E., Smith S.L., Schultz N.E. (1987). Potentiation of atracurium toxicity in isolated rat hepatocytes by inhibition of its hydrolytic degradation pathway. Anesth Analg. 1987 Jun;66(6):512-6. PMID: 3578863.

Abstract. This study tested the hypothesis that the esters of acrylic acid might be responsible for the previously observed cytotoxic effect of atracurium. Rats were pretreated with triorthotolyl phosphate (TOTP), an inhibitor of the hydrolytic degradation of atracurium. Because hydrolysis of acrylates is also inhibited by TOTP and because the hydrolysis represents a detoxification pathway for these esters, we postulated that the leak of lactic dehydrogenase (LDH) induced by atracurium would be enhanced in hepatocytes harvested from rats pretreated with TOTP. Hepatocytes isolated from rats previously treated with TOTP (25 or 50 mg/kg intraperitoneally, 20 hr before induced death) were incubated for 4 hr in the absence of muscle relaxants or in the presence of either atracurium (0.008-0.8 mM) or metocurine (0.015-0.85 mM). Atracurium produced a concentration-dependent leakage of LDH. The leakage out of cells obtained from TOTP-pretreated rats was greater than was the leakage out of hepatocytes harvested from animals pretreated only with corn oil (a vehicle for TOTP). Metocurine did not produce a leak of LDH. It is concluded that the LDH leakage was produced by ester-type products of atracurium degradation. Acrylates appear to be the toxic agent.

43. Ruch R.J., Klaunig J.E., Pereira M.A. (1987). Inhibition of intercellular communication between mouse hepatocytes by tumor promoters. Toxicol Appl Pharmacol. 1987 Jan;87(1):111-20. PMID: 2432692.

Abstract. Tumor promoters can inhibit gap junction-mediated intercellular communication in cultured cells. Since intercellular communication is thought to be important in normal cellular growth control, inhibition of intercellular communication by tumor promoters may be an important mechanism by which preneoplastic cells escape normal growth regulation and progress towards neoplasia. We have evaluated the effects of tumor promoters on intercellular communication between B6C3F1 mouse hepatocytes in primary culture. “Donor” hepatocytes were labeled with 4 microCi[5-3H]uridine/ml for 4 hr. Nonlabeled “recipient” hepatocytes were then plated onto and cocultured with the labeled donors. Hepatocyte cultures were then treated with either the tumor promoters or the solvent vehicle. After 2-24 hr, the cells were fixed and processed for autoradiography. Intercellular communication between donor and recipient hepatocytes was detected as an increase in autoradiographic grains over recipient cells in contact with donor cells, indicating the passage of labeled nucleotides from donor to recipient hepatocytes. Autoradiographic grains were not observed over recipient hepatocytes not in contact with donor cells thus indicating negligible transfer of labeled nucleotide through the medium. Intercellular communication in untreated and solvent vehicle treated hepatocytes was detected in approximately 80% of the donor-contacting recipients after 8-12 hr culture. Phenobarbital, DDT (1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane), Aroclor 1254, lindane (1,2,3,4,5,6-hexachlorocyclohexane, gamma-isomer), and TPA (12-O-tetradecanoyl phorbol-13-acetate), at noncytotoxic concentrations, significantly decreased hepatocyte intercellular communication in a dose-dependent manner.


42. Ruch R.J., Klaunig J.E. (1986). Effects of tumor promoters, genotoxic carcinogens and hepatocytotoxins on mouse hepatocyte intercellular communication. Cell Biol Toxicol. 1986 Dec;2(4):469-83. PMID: 2477123.  

Abstract. Intercellular communication via gap junctions may be an important mechanism of cellular growth control. Tumor promoters can inhibit intercellular communication between cultured cells, while genotoxic carcinogens apparently lack this capability. The inhibition of intercellular communication by tumor promoters may be an essential mechanism by which tumor promotion occurs in vivo. In this study, the liver tumor promoters phenobarbital, lindane (1,2,3,4,5,6-hexachlorocyclohexane, gamma-isomer), DDT (1,1-Bis[4-chlorophenyl]-2,2,2-trichloroethane), Aroclor 1254 (a polychlorinated biphenyl mixture) and dieldrin inhibited intercellular communication between male B6C3F1 mouse hepatocytes in primary culture. Intercellular communication was detected as the passage of [5-3H]uridine nucleotides from pre-labelled donor hepatocytes to non-labelled recipient hepatocytes. Mouse hepatocyte intercellular communication was also inhibited by the skin tumor promoter TPA (12-0-tetradecanoyl-phorbol-13-acetate), but not by the bladder tumor promoter saccharin. The genotoxic hepatocarcinogens dimethylnitrosamine, diethylnitrosamine, benzo[a]pyrene and 2-acetylaminofluorene, and the hepatocytotoxins bromobenzene, acetaminophen, carbon tetrachloride, chloroform and methotrexate had no effect on mouse hepatocyte intercellular communication at non-cytotoxic levels. These results suggest that the ability to inhibit mouse hepatocyte intercellular communication is an effect specific to tumor promoters. 

41. Nigrovic V., Klaunig J.E., Smith S.L., Schultz N.E., Wajskol A. (1986). Comparative toxicity of atracurium and metocurine in isolated rat hepatocytes. Anesth Analg.1986 Nov;65(11):1107-11. PMID: 3767007.

Abstract. Primary cultures of liver cells isolated from seven rats were used to study the possible toxicity of atracurium and metocurine. The muscle relaxants were separately added to the culture medium and the cells then incubated for 4 hr. The amount of lactic dehydrogenase (LDH) that leaked into the culture medium was determined at the end of incubation. The customary assumption was made that the exudation of LDH reflects the toxic effects of the relaxants. In untreated dishes, approximately 11% of the total intracellular LDH leaked out during the incubation. The net leakage of LDH produced by the relaxants was obtained by subtracting this amount from the LDH activity determined in the media of dishes with the relaxants added. On this basis, metocurine, in concentrations of 12-850 X 10(-6)M, did not cause a net leak of LDH. On the other hand, atracurium, in similar molar concentrations, caused a statistically significant and concentration-dependent leak of LDH that, at its maximum, amounted to more than one half of the intracellular LDH. The results are interpreted in terms of damage to cellular membranes produced by atracurium or its metabolites. Although the exact biochemical process was not identified, we hypothesize that acrylates–produced by Hofmann elimination from atracurium–might be the likely toxic species.

40. Ruch R.J., Klaunig J.E. (1986). Antioxidant prevention of tumor promoter induced inhibition of mouse hepatocyte intercellular communication. Cancer Lett. 1986 Nov;33(2):137-50. PMID: 2431762.

Abstract. The liver tumor promoters, phenobarbital (20-500 micrograms/ml), lindane (1,2,3,4,5,6-hexachlorocyclohexane, gamma-isomer; 0.1-5.0 micrograms/ml), and DDT (1,1-bis[4-chlorophenyl]-2,2,2-trichloroethane; 0.5-10.0 micrograms/ml), and the hydrogen peroxide-generating enzyme, glucose oxidase (0.01-0.10 units/ml) inhibited gap junctional intercellular communication between B6C3F1 mouse hepatocytes in primary culture. Addition of the antioxidants, superoxide dismutase (100 units/ml), DPPD (N,N’-diphenyl-1,4-phenylenediamine; 25 microM), and vitamin E (DL-alpha-tocopherol acetate; 100 microM), to tumor promoter-treated cultures prevented the inhibition of hepatocyte intercellular communication. DPPD and vitamin E, prevented the inhibition of hepatocyte intercellular communication by glucose oxidase. Superoxide dismutase had no effect on the inhibition of intercellular communication caused by glucose oxidase. These results suggest that activated oxygen species are produced during liver tumor promoter treatment of cultured mouse hepatocytes and are responsible for the inhibition of mouse hepatocyte intercellular communication by the promoters.

39. Ruch R.J., Klaunig J.E., Schultz N.E., Askari A.B., Lacher D.A., Pereira M.A., Goldblatt P.J. (1986). Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes. Environ Health Perspect. 1986 Nov;69:301-5. PMID: 3816733.

Abstract. Mechanisms of chloroform (CHCl3) and carbon tetrachloride (CCl4) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl3 and CCl4 was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl3 (5 mM) and CCl4 (2.5 mM) used and with the longest duration of treatment (20 hr). CCl4 was approximately 16 times more toxic than CHCl3 to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25 microM) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl3 and CCl4 toxicity. The toxicity of CHCl3 and CCl4 could also be decreased by adding the antioxidants N,N’-diphenyl-p-phenylenediamine (DPPD) (25 microM), alpha-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl3 and CCl4 are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl3 and CCl4; and free radicals may be important mediators of the toxicity of these two halomethanes.

38. Klaunig J.E., Ruch R.J., Pereira M.A. (1986). Carcinogenicity of chlorinated methane and ethane compounds administered in drinking water to mice. Environ Health Perspect. 1986 Nov;69:89-95. PMID: 3816740.

Abstract. The chlorinated hydrocarbons chloroform (CHCl3), 1,1-dichlorethane (1,1-DCE) and 1,2-dichloroethane (1,2-DCE) have been detected in finished drinking water. When administered to B6C3F1 mice by gavage in corn oil, these compounds have been shown to induce hepatic tumors. The present study examines the effect on liver tumor incidence of continuous treatment of CHCl3 (600 mg/L and 1800 mg/L), 1,1-DCE (835 mg/L and 2500 mg/L), and 1,2-DCE (835 mg/L and 2500 mg/L) administered in drinking water to male B6C3F1 mice using a two-stage (initiation/promotion) treatment protocol. Seventy 4-week-old male B6C3F1 mice constituted each treatment group. Of these mice, 35 were initiated by treatment with diethylnitrosamine (DENA) (10 mg/L) in the drinking water for 4 weeks. The remaining 35 received deionized drinking water. Each group was subsequently treated with one of two concentrations of CHCl3, 1,1-DCE, or 1,2-DCE in drinking water for 52 weeks. An additional group received phenobarbital (PB) (500 mg/L) and served as the positive control for liver tumor promotion. Mice were sampled after 24 weeks (10 mice) and 52 weeks (25 mice). At sampling, liver and lung tumors were detected. None of the compounds increased the number or incidence of lung or liver tumors by themselves. PB promoted liver tumor formation (but not lung tumors) in the DENA-initiated mice. 1,1-DCE and 1,2-DCE did not affect the incidence or number of liver or lung tumors in the DENA-initiated animals. CHCl3, however, inhibited liver and lung tumorigenesis in the DENA-initiated mice.

37. Dixit R., Schut H.A., Klaunig J.E., Stoner G.D.  (1986). Metabolism and DNA binding of 2,6-dinitrotoluene in Fischer-344 rats and A/J mice. Toxicol Appl Pharmacol. 1986 Jan;82(1):53-61. PMID: 3945944.

Abstract. 2,6-Dinitrotoluene (2,6-DNT) is a potent hepatocarcinogen in Fischer-344 rats, while its 2,4-isomer is believed to be noncarcinogenic. Neither 2,6-DNT nor 2,4-DNT is carcinogenic in the strain A mouse lung tumor bioassay. To explore the possible reasons for these differences in tumor responses, we have studied the in vitro metabolism and DNA binding of 2,6-DNT in cultured hepatocytes of the Fischer-344 rat and the A/J mouse, and have also investigated the in vivo DNA binding of 2,6-DNT and 2,4-DNT in these two species. In vitro metabolism of 2,6-DNT by rat and mouse hepatocytes was similar and resulted mainly in the formation of 2,6-dinitrobenzyl alcohol, either unconjugated or as a glucuronide (57.5 to 85.5% of the total per fraction), with smaller amounts of polar, acidic metabolites (8.4 to 38.7%) and minor amounts (1.2 to 5.3%) of 2-amino-6-nitrotoluene. Anaerobic metabolism of 2,6-DNT by an extract of rat or mouse cecal contents resulted mainly in the formation of 2-amino-6-nitrotoluene and 2-(N-acetylamino)-6-nitrotoluene, and minor amounts of 2,6-diaminotoluene. Ip administration of 2,6-DNT or 2,4-DNT (150 mg/kg each) to Fischer-344 rats resulted, after 24 hr, in covalent binding to DNA of the liver (131.1 to 259.9 pmol 2,6-DNT/mg DNA; 215.4 to 226.8 pmol 2,4-DNT/mg DNA), and lower binding to DNA of the lungs and the intestine (14.9 to 22.7 pmol 2,6-DNT/mg DNA; 45.0 to 75.0 pmol 2,4-DNT/mg DNA). Similar treatment of A/J mice resulted in lower binding in the liver (25.9 to 31.9 pmol 2,6-DNT/mg DNA; 42.6 to 58.9 pmol 2,4-DNT/mg DNA), no detectable binding of 2,6-DNT in extrahepatic tissues and low amounts of binding of 2,4-DNT to lung and intestinal DNA (9.7 to 39.0 pmol/mg DNA). In vitro binding of 2,6-DNT to DNA of cultured hepatocytes from both A/J mice and Fischer-344 rats required prior metabolism of 2,6-DNT by the respective extracts from cecal contents. DNA binding was non-detectable in hepatocytes incubated with 2,6-DNT only. It is concluded that binding of 2,6-DNT to liver DNA requires its prior reductive metabolism, probably by intestinal microorganisms, and that the higher binding of 2,6-DNT in the Fischer-344 rat than in the A/J mouse may, in part, be responsible for the high susceptibility of the Fischer-344 rat to 2,6-DNT carcinogenesis.

36. Burns R.A., Klaunig J.E., Shulok J.R., Davis W.J., Goldblatt P.J. (1986). Tumor-localizing and photosensitizing properties of hematoporphyrin derivative in hamster buccal pouch carcinoma. Oral Surg Oral Med Oral Pathol. 1986 Apr;61(4):368-72. PMID: 2939386.

Abstract. The tumor-localizing and photochemotherapeutic properties of hematoporphyrin derivative (HPD) were examined in 7, 12 dimethylbenzanthracene (DMBA)-induced oral cancers in the Syrian hamster. Oral tumors in hamsters injected with HPD (50 micrograms per gram of body weight) exhibited bright salmon pink fluorescence when exposed to long-wave ultraviolet light 24 hours after intraperitoneal HPD injection. Adjacent tumor-free mucosa did not fluoresce. Similarly, tumors not treated with HPD, normal mucosa treated with HPD, and normal mucosa not treated with HPD did not fluoresce. Tumors in animals that received HPD and photochemotherapy (PCT) were examined for gross and microscopic pathologic changes following the phototreatment. Tumors displayed edema, hemorrhage, and cellular necrosis that progressed with the time of sampling after photochemotherapy. Complete tumor necrosis was evident in the majority of oral tumors 24 hours after HPD PCT.

35. Barut B.A., Klaunig J.E. (1986). Isolation and characterization of metastatic sublines  from a murine transitional cell bladder carcinoma. Clin Exp Metastasis. 1986 Jan-Mar;4(1):1-11. PMID: 3698364.

Abstract. Four sublines of a murine N-[4-(5-nitro-2-furyl)-2-thiazolyl]-foramide (FANFT)-induced transitional cell carcinoma (MBT-2) possessing spontaneous metastatic ability were isolated via in vivo/in vitro serial selection of metastatic lung lesions. Subcutaneous inoculation of the parent cell line (MBT-2) produced primary tumors when injected into C3H mice. These primary tumors rarely metastasize. A subline designated L3F1 was established from 1 MBT-2 pulmonary metastatic tumor. Further in vivo/in vitro selections established three additional sublines designated L3F2, L3F3 and L3F4. Serial selection resulted in MBT-2 sublines of greater metastatic potential in terms of both incidence of metastasis and the number of metastatic tumors per lung. The parent line differed from the four sublines in metastatic potential, in vitro cell morphology, and in vitro growth parameters. The L3F2 subline was examined for the time of onset of metastasis by removal of the primary tumor. Metastasis of the subcutaneously transplanted tumor occurred between 14 and 21 days after injection of the L3F2 subline. The L3F2 primary tumors and lung metastases were morphologically characterized by light and electron microscopy.

34. Pereira M.A., Klaunig J.E., Herren-Freund S.L., Ruch R.J. (1986). Effect of phenobarbital on the development of liver tumors in juvenile and adult mice. J Natl Cancer Inst. 1986 Aug;77(2):449-52. PMID: 3461205.

Abstract. The effect of long-term exposure to phenobarbital (CAS: 50-06-6) subsequent to tumor initiation on the development of liver tumors in BALB/c and (C57BL/6 X C3H/Anf)F1 (B6C3F1) mice was determined. In male B6C3F1 mice that received either 15 or 45 ppm diethylnitrosamine [(DENA) CAS: 55-18-5] between 6 and 10 weeks of age, subsequent treatment with 500 ppm sodium phenobarbital in the drinking water resulted in the promotion of liver tumors. However, in male B6C3F1 mice initiated on day 15 of age with 25 mg DENA/kg, beginning long-term treatment of 500 ppm sodium phenobarbital at 4 weeks of age inhibited the development of liver tumors, whereas in male BALB/c mice initiated with 25 mg DENA/kg on day 15 of age, beginning the long-term treatment with 500 ppm sodium phenobarbital at 4 weeks of age promoted the development of liver tumors. Hence phenobarbital can either enhance or inhibit the formation of liver tumors, depending both on the mouse strain used and the animal’s age at the start of exposure.

33. Shulok J.R., Klaunig J.E., Selman S.H., Schafer P.J., Goldblatt P.J. (1986). Cellular effects of hematoporphyrin derivative photodynamic therapy on normal and neoplastic rat bladder cells. Am J Pathol. 1986 Feb;122(2):277-83. PMID: 2936252.

Abstract. HPD is known to localize in neoplastic cells and when exposed to the appropriate wavelength of light causes cytotoxicity. The authors have established a rat urothelial cell model for use in comparing and contrasting the effects of HPD photodynamic therapy (PDT) in normal (RBL-01) and transitional cell carcinoma (AY27) bladder cell lines. Uptake, toxicity, and morphologic damage following exposure to HPD PDT were evaluated. Trypan blue exclusion was used for determination of the toxicity of several HPD concentrations (1, 10, 25, and 50 micrograms/ml) with increasing duration of incubation with HPD (0, 1, 2, 4, 12, 24, and 48 hours). Both cell lines displayed increased toxicity with higher concentrations of HPD; however, the AY27 cells were more susceptible to the toxic effects of HPD PDT than the RBL-01 cells at the higher HPD doses studied (25 and 50 micrograms/ml). Viability decreased with increased duration of HPD incubation in RBL-01 cells up until 4 hours, after which it showed a steady increase. Viability decreased in the AY27 cells with increased duration of HPD incubation. An increase in serum concentration in the medium resulted in an increase in viability for both cell lines. Both cell lines demonstrated fast initial uptake of HPD followed by slower uptake over the time studied. By 24 and 48 hours the AY27 cells contained twice the amount of methanol-extractable porphyrins as the RBL-01 cells. The initial morphologic change following HPD PDT was damage to mitochondria. Mitochondrial damage occurred immediately after PDT in the AY27 cells and 30 minutes after PDT in the RBL-01 cells. Both cell lines exhibited a similar progression of cell injury; however, morphologic damage was observed earlier after PDT and appeared more extensive in the AY27 cells.


32. Goldblatt P.J., Gunning W.T., Klaunig J.E. (1985). Light and Electron Microscopy of Peripheral Blood. Micron and Microscopic Acta, 16(3), 195-198.

31. Ruch R.J., Klaunig J.E., Pereira M.A. (1985). Selective resistance to cytotoxic agents in hepatocytes isolated from partially hepatectomized and neoplastic mouse liver. Cancer Lett. 1985 Apr;26(3):295-301. PMID: 2581690.

Abstract. Hepatocytes were isolated from B6C3F1 male mouse neoplastic livers (containing hepatocellular adenomas and carcinomas) or two-thirds partially hepatectomized livers and tested in primary culture for their cytotoxic response to hepatotoxins. Partially hepatectomized mouse hepatocytes were less sensitive to lindane, methotrexate, diethylnitrosamine and adriamycin, and more sensitive to cycloheximide compared to normal mouse hepatocytes. Neoplastic hepatocytes were less sensitive to lindane and methotrexate, did not differ in cytotoxic response to diethylnitrosamine and adriamycin and were more sensitive to cycloheximide compared to normal mouse hepatocytes.

30. Selman S.H., Goldblatt P.J., Klaunig J.E., Keck R.W., Kreimer-Birnbaum M. (1985). Localization of hematoporphyrin derivative in injured bladder mucosa. An experimental study. J Urol. 1985 Jun;133(6):1104-7. PMID: 3158750.

Abstract. Hematoporphyrin derivative, a fluorescent mixture of porphyrins, has the putative property of being retained in neoplastic tissue after systemic administration. This preferential retention is the basis for the use of this agent as a tumor localizer and tumor photosensitizer. The retention of hematoporphyrin derivative in non-neoplastic but regenerating urothelium has not been reported. In this study, thermal urothelial injury was induced in Fischer 344 rats. Animals were then injected intravenously with hematoporphyrin derivative at 3 days, 1, 2, 3 and 6 weeks after injury. Photography under ultraviolet illumination was used to detect porphyrin fluorescence in the bladder mucosa. Up to 3 weeks after injury porphyrin fluorescence was detectable in areas of inflammation and hyperplasia around the area of injury. This study suggests that in this experimental model HpD fluorescence is not specific for neoplasia.

29. Chang L.W., Pereira M.A., Klaunig J.E. (1985). Cytotoxicity of halogenated alkanes in primary cultures of rat hepatocytes from normal, partial hepatectomized, and preneoplastic/neoplastic liver. Toxicol Appl Pharmacol. 1985 Sep 15;80(2):274-83. PMID: 2862719.

Abstract. Six halogenated hydrocarbons, chloroform, 1,2-dibromoethane (1,2-DBE), 1,1-dichloroethane (1,1-DCE), 1,2-dichloroethane (1,2-DCE), 1,1,1-trichloroethane (1,1,1-TCE), and 1,1,2-trichloroethane (1,1,2-TCE), were evaluated for their cytotoxicity in primary cultures of rat hepatocytes isolated from normal, partially hepatectomized, and preneoplastic/neoplastic rat livers. Preneoplastic/neoplastic lesions of phenotypically altered foci and hepatocyte nodules were induced by either (1) initiation by diethylnitrosamine (DENA) followed by 2 weeks of 0.02% 2-acetylaminofluorene (2-AAF) in the diet and a single gavage dose of carbon tetrachloride 1 week after the start of the 2-AAF diet or (2) initiation by DENA followed by promotion with 500 ppm sodium phenobarbital in the drinking water for 24 weeks. The hepatocytes containing preneoplastic/neoplastic cells isolated from animals treated with either protocol, compared to hepatocytes isolated from normal liver, were resistant to the cytotoxicity of aflatoxin B1 (AFB1). None of the six halogenated alkanes exhibited any difference in their cytotoxicity toward hepatocytes isolated from normal liver or from liver containing preneoplastic/neoplastic lesions induced by either procedure. Hepatocytes isolated from partially hepatectomized animals were resistant to the cytotoxicity of AFB1 and chloroform but not to the cytotoxicity of 1,2-DBE or 1,2-DCE. The ranking of relative cytotoxicity in hepatocytes from untreated rats was 1,2-DBE much greater than 1,2-DCE greater than 1,1,2-TCE greater than 1,1,1-TCE greater than chloroform greater than 1,1-DCE. Treatment with SKF-525A protected the hepatocytes from the cytotoxicity of AFB1 while increasing the cytotoxicity of all six halogenated alkanes. Treatment with diethyl maleate increased the cytotoxicity of AFB1 and all six halogenated alkanes. These observations suggest that preneoplastic/neoplastic rat hepatocytes are not resistant to the cytotoxicity of the six halogenated alkanes because their toxicity might be mediated by a cytochrome P-450 species which is not inhibited by SKF-525A and is not decreased in preneoplastic/neoplastic lesions.

28. Klaunig J.E., Selman S.H., Shulok J.R., Schafer P.J., Britton S.L., Goldblatt P.J. (1985). Morphologic studies of bladder tumors treated with hematoporphyrin derivative photochemotherapy. Am J Pathol. 1985 May;119(2):236-43. PMID: 3158208.

Abstract. The morphologic changes that occurred in transplanted rat bladder tumors after treatment with hematoporphyrin derivative (HPD) and/or phototherapy were investigated. Transitional cell bladder tumors were initiated subcutaneously in male F344 rats by injection of AY27 cells. When tumors reached 1 cm in diameter, the rats received either HPD (10 mg/kg body weight) photochemotherapy, HPD only, phototherapy only, or no treatment. Tumors were sampled immediately (0 time), 1/2, 1, 2, 4, and 24 hours after phototreatment for light and electron microscopy. Tumors receiving HPD-photochemotherapy displayed progressive injury to both tumor cells and endothelial cells. Early changes (0-2 hours) included focal tumor and endothelial cell vacuolation and swelling as well as sloughing of tumor cells into papillary spaces. Tumor cells and endothelial cells displayed vacuolization and damage to cell mitochondria immediately after phototreatment. Intercellular spaces also increased in size. Lethally injured cells were apparent in papillary spaces. At 4 hours after phototherapy, tumor cells and endothelial cells exhibited extensive cell damage, including mitochondrial destruction, endoplasmic reticulum swelling, polyribosome disaggregation, and plasma membrane blebbing. By 24 hours after phototherapy, the majority of cells within the tumor were necrotic. Untreated tumors and those treated with phototherapy-only did not exhibit these changes. Tumors that received HPD only exhibited focal areas of cell swelling and focal mitochondrial vacuolization in both tumor and endothelial cells. These changes, unlike the HPD-light-treated group did not progress and were reversible.

27. Klaunig J.E., Ruch R.J., Goldblatt P.J. (1985). Trout hepatocyte culture: isolation and primary culture. In Vitro Cell Dev Biol. 1985 Apr;21(4):221-8. PMID: 4008436.

Abstract. Rainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92 X 10(6) liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochrome p450 activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d of culture. Culture temperature also influenced the p450 activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and the appearance of bundles of intermediate filaments also were observed with increased time in culture.

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