Effect of phenobarbital on the development of liver tumors in juvenile and adult mice.

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.

Kinetics of phenobarbital inhibition of intercellular communication in mouse hepatocytes.

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.

A nonviral, virus strain-specific antigen expressed on rat cells transformed by avian sarcoma virus.

Five of six rat sarcomas, induced by the Schmidt-Ruppin (SR) strain of avian tumor virus, expressed a Mr 60,000 tumor cell surface antigen (TSA), immunoprecipitable from non-ionic detergent extracts. Expression of the antigen was exclusive to rat cells transformed by the SR virus strain. Moreover, expression of TSA appeared restricted by cell type. The five TSA-positive SR-transformed rat cell lines tested were apparently of fibroblastic origin, but cultured rat cerebral endothelial cells (RCE-T1), transformed by SR virus, showed no expression of TSA. However, the antigen emerged on cultured tumors obtained after histoincompatible transplantation of these cells into newborn rats of another strain (tumor digest cells). Investigation of TSA for immunological relationship to viral structural antigens and the src gene product indicated that the TSA is distinct from any of these and more probably derives from a virus-directed alteration in a host molecule.

Liver cell-specific transcriptional regulation of connexin32.

Gap junctional intercellular communication facilitates liver homeostasis and growth control in the liver. The major gap junction protein expressed by hepatocytes is connexin32 (Cx32) and non-parenchymal hepatic cells do not express this gene. We investigated the regulation of Cx32 transcription by trans-activating factors in liver cells. Transient transfection assays using deletions of the rat Cx32 promoter (nt -753 to -33) linked to the luciferase gene were performed in MH1C1 rat hepatoma cells that express endogenous Cx32 compared with WB-F344 rat liver epithelial cells that do not. The basal promoter element was located within nt -134 to -33 and was 1.4-fold more active in MH1C1 cells than WB-F344 cells whereas the entire promoter fragment (nt -754 to -33) was four-fold more active in MH1C1 cells. Specific nuclear protein-DNA complexes that bound to Sp1 consensus sites within the basal promoter were formed using nuclear extracts from both types of cells. Additional promoter sequences increased promoter activity more strongly in MH1C1 cells than WB-F344 cells and this was correlated with the binding of hepatocyte nuclear factor-1 (HNF-1) to two HNF-1 consensus sites centered at -187 and -736. Expression of HNF-1 and binding to these elements was only observed with MH1C1 cells. Other specific protein-DNA complexes were formed, however, that included YY-1- and NF-1-containing complexes, but these were not related to promoter activity. Dexamethasone increased Cx32 promoter activity and expression in MH1C1 cells, but had little effect in WB-F344 cells and did not alter protein-DNA complex formation. These data suggest that Sp1 is responsible for Cx32 promoter basal activity, that HNF-1 determines the cell-specific expression of Cx32, and that dexamethasone increases Cx32 expression through other mechanisms.

Differential ganciclovir-mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase.

The two human colon carcinoma cell lines HT-29 and SW620, which stably express herpes simplex virus thymidine kinase (HSV-TK), are sensitized to the cytotoxic effects of the antiviral drug ganciclovir (GCV). Compared with HT-29 cells, SW620 cells were more sensitive to lower GCV concentrations (<1 microM), accumulated GCV triphosphate more rapidly, and incorporated higher levels of GCV into DNA. Following a 24-hr exposure to 10 microM GCV, bystander killing was as much as sixfold greater in SW620 cells than HT-29 cells. This bystander effect was dependent on the level of HSV-TK expression, the number of cells expressing HSV-TK, and the overall confluency of the cells. However, bystander killing did not correlate with gap junctional intercellular communication as determined by microinjection of Lucifer Yellow fluorescent dye. SW620 cells were coupled to <3% adjacent cells (compared with >50% for HT-29 cells), but were still able to transfer phosphorylated GCV to bystander cells as soon as 4 hr after drug was added. These results emphasize the importance of cell-specific metabolism in HSV-TK/GCV-mediated cytotoxicity and may suggest a novel mechanism for bystander killing.

Cell-cell communication in carcinogenesis.

To explain the complex carcinogenic process by which a single normal cell in human beings can be converted to an invasive and metastatic cancer cell, a number of experimental findings, epidemiological observations and their associated hypothesis/theories have been integrated in this review. All cancers have been generally viewed as the result of a disruption of the homeostatic regulation of a cell's ability to respond appropriately to extra-cellular signals of the body which trigger intra-cellular signal transducting mechanisms which modulate gap junctional intercellular communication between the cells within a tissue. Normal homeostatic control of these three forms of cell communication determines whether: (a) the cell remains quiescent (Go); (b) enters into the cell proliferation phase; (c) is induced to differentiate; (d) is committed to apoptose; or (e) if it is already differentiated, it can adaptively respond. During the evolution from single cell organisms to multicellular organisms, new cellular/biological functions appeared, namely, the control of cell proliferation ("contact inhibition"), the appearance of the process of differentiation from committed stem cells of the various tissues and the need for programmed cell death or apoptosis. Interestingly, cancer cells have been characterized as cells: (a) having been derived from a stem-like cell; (b) without their ability to control cell growth or without the ability to contact inhibit; (c) which can not terminally differentiate under normal conditions; and (d) having altered ability to apoptosis under normal conditions. During that evolutionary transition from the single cell organism to the multicellular organism, many new genes appeared to accompany these new cellular functions. One of these new genes was the gene coding for a membrane associated protein channel (the gap junction) which between coupled cells, allowed the passive transfer on ions and small molecular weight molecules. A family of over a dozen of these highly evolutionarily-conserved genes (the connexin genes) coded for the connexin proteins. A hexameric unit of these connexins in one cell (a connexon) couples with a corresponding connexon in a contiguous cell to join the cytoplasms. This serves to synchronize either the metabolic or electrotonic functions of cells within a tissue. Most normal cells within solid tissues have functional gap junctional intercellular communication (GJIC) (exceptions are free-standing cells such as red blood cells, neutrophils, and several, if not all, the stem cells). On the other hand, the cancer cells of solid tissues appear to have either dysfunctional homologous or heterologous GJIC. Therefore, among the many differences between a cancer cell and its normal parental cell, the carcinogenic process involves the transition from a normal, GJIC-competent cell to one that is defective in GJIC. The review examines how GJIC can be either transiently or stably modulated by endogenous or exogenesis chemicals or by oncogenes and tumor suppressor genes at the transcriptional, translational, or posttranslational levels. It also uses the gap junction as the biological structure to facilitate cellular/tissue homeostasis to be the integrator for the "stem cell" theory, "disease of differentiation theory", "initiation/promotion/progression" concepts, nature and nurture concept of carcinogenesis, the mutation/ epigenetic theories of carcinogenesis, and the oncogene/ tumor suppressor gene theories of carcinogenesis. From this background, implications to cancer prevention and cancer therapy are generated.

Gap junctions as targets for cancer chemoprevention and chemotherapy.

The development of the most efficacious strategy for the prevention and treatment of cancers is based on understanding the underlying mechanisms of carcinogenesis. This includes the knowledge that the carcinogenic process is a multi-step, multi-mechanism process and that no two cancers are alike, in spite of some apparent universal characteristics, such as their inability to have growth control, to terminally differentiate, to apoptose abnormally and to have an apparent extended or immortalized life span. The multi-step process, involving the "initiation" of a single cell via some irreversible process, with the clonal expansion of this initiated cell by suppressing growth control and inhibiting apoptosis (promotion step), leads to a situation whereby additional genetic and epigenetic events can take place (progression step) to confer the necessary phenotypes of invasiveness, and metasis (neoplastic stage). While it is clear that, in principle, prevention of each of these three steps is possible, in practical terms, while it would make sense to minimize the initiation step, one can never reduce this step to zero. On the other hand, since the promotion step is the rate-limiting step of carcinogenesis, intervening to block this step makes the most sense. Also, by understanding the ultimate biological function that confers growth control, terminal differentiation or apoptosis for cells, there is even some hope of treating some, but not all, malignant cells such that they can regain some ability to perform these vital cellular functions. Gap junctional intercellular communication (GJIC) has been speculated to be a necessary, if not sufficient, biological function of metazoan cells for the regulation of growth control, differentiation and apoptosis of normal progenitor cells. Normal, contact-inhibited fibroblast and epithelial cells have functional GJIC, while most, if not all, tumor cells have dysfunctional homologous or heterologous GJIC. Cancer cells are characterized by the lack of growth control, inability to terminally differentiate or apoptose under normal conditions and have extended or immortalized life spans. Chemical tumor promoters, growth factors and hormones have been shown to inhibit GJIC. Several oncogenes and anti-sense connexin genes have been shown to down-regulate GJIC function. Anti-oncogene drugs, anti-tumor promoting natural and synthetic chemicals, as well as GJIC-deficient neoplastic cells, transfected with various connexin genes, have been shown to re-gain GJIC and growth control with the loss of tumorigenicity. Therefore, the hypothesis for a rational approach to identify anti-tumor promoting chemopreventive drugs and anti-carcinogenic treatments is to use the prevention of the down regulation of GJIC by the tumor promoters and the restoration of GJIC in neoplastic cells. While previous and many current strategies for chemoprevention and therapy have been based on treating specific oncogene products or cell signalling mechanisms, as well as advance molecular modifications of older strategies, none have specifically used the prevention of GJIC by agents during the rate limiting step of carcinogenesis or the restoration of GJIC in neoplastic cells which are deficient in GJIC. Since there are multiple mechanisms by which GJIC is down regulated during the tumor promotion phase and since stable GJIC deficiencies in neoplastic cells can be the result of transcriptional, translational or posttranslational mechanisms, it should be clear there would not be one "golden bullet" approach to resolve either the chemoprevention or therapeutic approach. Even with the hypothesis that GJIC, which depends on the transcription of normal connexin genes, their normal translation, trafficking, assembly and function, it should be clear that cells with normal connexin genes and potentially normal GJIC might not have functional GJIC because of dysfunction of other defects in cancer cells, namely cell-adhesion or cell-matrix problems (both of which are necessary for GJIC to occur). In essence, if dietary or chemopreventive/therapy is to be effective, the strategy must either ameliorate the growth stimulatory effects of exogenous chemicals, growth factors or hormones, that trigger various mitogenic/anti-apoptotic signal transducing systems that inhibit GJIC.

Strain and species effects on the inhibition of hepatocyte intercellular communication by liver tumor promoters.

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.

Antioxidant prevention of tumor promoter induced inhibition of mouse hepatocyte intercellular communication.

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.

Enhancement of gap junctional intercellular communication in tumor promoter-treated cells by components of green tea.

Green tea (Camellia sinensis) has been reported to inhibit tumor promotion in vivo and in vitro. Many tumor promoters inhibit gap junctional intercellular communication (GJIC) which may be an important mechanism of promotion. In the present study, we hypothesized that green tea would enhance GJIC in promoter-treated cells. An aqueous extract of green tea (GTE) and several of its constituents were tested for their effects on GJIC in p,p'-dichlorodiphenyltrichloroethane (DDT)-, 12-O-tetradecanoylphorbol-13-acetate (TPA)- and dieldrin-treated WB-F344 rat liver epithelial cells. All three promoters inhibited GJIC in a dose-responsive manner at non-cytolethal concentrations. (GTE (10-80 gamma/ml) enhanced GJIC 20-80% in promoter-treated cells. (-)-Epigallocatechin gallate and (-)-epicatechin gallate also enhanced GJIC in DDT-treated cells, but no effects were seen with (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, caffeine, or theobromine. These data suggest GTE may inhibit tumor promotion by enhancing GJIC and that the most active components are the catechin gallates.

Lack of growth inhibition or enhancement of gap junctional intercellular communication and connexin43 expression by beta-carotene in murine lung epithelial cells in vitro.

The carotenoid, beta-carotene, has been examined in human trials as a possible lung cancer chemopreventive agent, but initial results indicate that the compound is ineffective. Here we have considered whether beta-carotene could enhance gap junctional intercellular communication (GJIC) and affect the growth of lung epithelial cells, since these effects may be involved in the carotenoid's chemopreventive actions. In accordance with its lack of lung cancer chemopreventive activity, beta-carotene (1-10 microM; 1-5 days treatment durations) did not affect GJIC, gap junction protein (connexin43; Cx43) expression, or growth in vitro of non-transformed (C10) or neoplastic (E9 and 82-132) murine lung epithelial cells. beta-Carotene enhanced GJIC and Cx43 expression and reduced the growth of C3H10T1/2 murine fibroblasts, however. These data indicate that the effects of beta-carotene on GJIC and growth are cell-specific which may partly explain why the carotenoid is an ineffective lung cancer chemopreventive agent.

Selective resistance to cytotoxic agents in hepatocytes isolated from partially hepatectomized and neoplastic mouse liver.

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.

Inhibition of mouse hepatocyte intercellular communication by phthalate monoesters.

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.

Inhibition of lung tumor cell growth in vitro and mouse lung tumor formation by lovastatin.

The HMG-CoA reductase inhibitor, lovastatin (LOV), has been reported to inhibit Ras farnesylation and the growth of Ras-transformed cells. Mouse lung tumors and human lung adenocarcinomas often have activating mutations in K-ras alleles. In the present study, we determined whether LOV inhibited the growth in vitro of mouse (C10, E9, LM1, LM2, and 82-132) and human (NCl-H125, H292, H441, H460, and H661) nor-transformed and neoplastically transformed lung epithelial cells and whether growth inhibition was related to cell transformation or K-ras activation. LOV inhibited the growth of mouse and human lung cells, but cell sensitivities were unrelated to neoplastic transformation or K-ras mutation. In addition, we evaluated whether LOV could inhibit the formation of lung adenomas induced by the tobacco-specific nitrosamine, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. LOV was administered in the diet at 0, 40, 160, or 400 ppm ad libitum to male strain A/J mice beginning 1 week after lung tumor induction with NNK (10 mumol/mouse). Mice were euthanized 6 months later. Enumeration of lung tumors revealed that LOV did not affect tumor incidence or size, but significantly reduced tumor multiplicity in a dose-related manner. These data suggest that LOV can suppress the formation of NNK-induced lung tumors, possibly at an early promotional stage. This suppression does not appear to be related to either the presence of mutated K-ras or to changes in K-ras expression.

Cooperative effects of v-myc and c-Ha-ras oncogenes on gap junctional intercellular communication and tumorigenicity in rat liver epithelial cells.

The objective of this study was to isolate and partially characterize several rat liver epithelial cell clones containing myc, ras and myc/ras oncogenes in order to study their roles in apoptosis and to test the hypothesis that gap junctional intercellular communication is necessary for apoptosis in solid tissues and that the loss of junctional communication leads to tumorigenesis. The co-transfection of the myc and ras oncogenes in the normal rat liver epithelial cell line (WB-F344) resulted in a loss of functional channels and normal growth regulation; cell-cell communication was significantly decreased and tumorigenicity determined in adult male F344 rats was induced. We examined cell growth properties, gap junctional intercellular communication (GJIC), using the scrape-loading-dye transfer and fluorescence-redistribution-after-photobleaching assays, and tumorigenicity in a series of normal and v-myc-, c-Ha-ras- and v-myc/c-Ha-ras-transfected WB-F344 cell lines. The c-Ha-ras- and the v-myc/c-Ha-ras-transduced cell lines appeared distinctly different from the other lines, having spindle-shaped morphology, shorter generation time and contact insensitivity. On the other hand, the normal WB-F344 cell line and the v-myc-transduced cell line showed excellent GJIC. Moreover, the c-Ha-ras-transduced cell lines displayed decreasing levels of GJIC associated with their increasing tumorigenicity. The v-myc/c-Ha-ras-transformed cell lines showed the lowest levels of GJIC and were also the most tumorigenic. These findings suggest that the reduction of GJIC in c-Ha-ras- and v-myc/c-Ha-ras-transformed WB-F344 cells is linked to their tumorigenic potential. These cell lines should provide valuable tools to study the role of GJIC in apoptosis during tumorigenesis.

Gap junctional intercellular communication in mouse lung epithelial cell lines: effects of cell transformation and tumor promoters.

Gap junctional intercellular communication (GJIC) is reduced by neoplastic transformation and treatment with tumor promoters in many types of cells but few data exist for the lung. GJIC was therefore evaluated in non-transformed (C10) and transformed (E9, 82-132, and PCC4) mouse lung epithelial cell lines and in C10 cells treated with tumor promoters. GJIC was assessed by fluorescent dye microinjection (dye-coupling). Dye-coupling levels were highest in C10 cells (85-90% communicating cells) followed by 82-132 cells (40-50%), E9 cells (15-20%), and PCC4 cells (3-10%). Indirect immunofluorescent staining with anti-gap junction protein (connexin) antibodies revealed that C10 cells expressed gap junctions comprised of connexin43, but not connexin32 or connexin26. The tumor promoters, butylated hydroxytoluene (BHT), 12-O-tetradecanoylphorbol-13-acetate (TPA), and p,p'-dichlorodiphenyltrichloroethane (DDT), inhibited dye-coupling in C10 cells but phenobarbital (PB) did not. BHT promotes mouse lung tumor formation, PB does not, while the effects of TPA and DDT on lung tumor development have not been reported. These data indicate that cell transformation and certain tumor promoters reduce GJIC in mouse lung epithelial cells and demonstrate correlations between the in vitro inhibition of GJIC and lung tumor promotion.

Carcinogenicity of chlorinated methane and ethane compounds administered in drinking water to mice.

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.

Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes.

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.

The role of oval cells and gap junctional intercellular communication in hepatocarcinogenesis.

The role of oval cells, and Gap Junctional Intercellular Communication (GJIC) in hepatic differentiation and neoplasia is controversial. Oval cells accumulate in great number when hepatocyte regeneration is blocked following massive hepatotoxicity or after treatment with some hepatocarcinogens. This suggests oval cells are facultative stem cells or close progeny of liver stem cells that are activated only under specific conditions. Studies with oval cell lines clearly indicate that they can differentiate into hepatocytes and that neoplastic derivatives of oval cells can produce hepatocellular and biliary neoplasms. Because hepatocytes express Cx32 and biliary cells express Cx43, the differentiation of oval cells into hepatocytes or In addition, because Cx32 hemichannels and Cx43 hemichannels cannot form heterotypic patent channels, the type of connexin expressed by the differentiating oval cell will determine whether it communicates with hepatocytes or biliary epithelial cells, respectively. This communication may be necessary for the further differentiation and regulated growth of the differentiating oval cells and impairment of this GJIC may contribute to the formation of hepatocellular and cholangiocellular neoplasms. The type of connexin expressed may also determine the susceptibility of the differentiating oval cells to the various types of rodent liver tumor promoters. Thus, three major points have been developed here. First, Cx32 or Cx43 expression and GJIC with hepatocytes or biliary epithelial cells, respectively, may determine the final differentiated fate of oval cells. Secondly, blocked GJIC may determine whether oval cells progress to hepatocellular or cholangiocellular carcinoma. Lastly, the ability of tumor promoters to block Cx32 or Cx43-mediated GJIC in differentiating oval cells may determine whether these agents promote the formation of hepatocellular or cholangiocellular carcinomas. Thus, GJIC may be the key factor in the differentiation of oval cells and blocked GJIC may promote their neoplastic transformation in a lineage-specific manner. In this chapter, we have outlined several new hypotheses on the role of oval cells and GJIC in hepatocarcinogenesis. We hope that other investigators will consider our ideas, but realize these views will be contentious to many. Our intent, however, was to stimulate discussion and debate, even argument, because truth often arises amidst controversy and may be found in the most peculiar places.

Enhancement of gap junctional intercellular communication by dibutyryl cyclic AMP in lung epithelial cells.

Reduced gap junctional intercellular communication (GJIC) occurs in neoplastic cells and contributes to their phenotype. Cyclic AMP agonists inhibit lung cancer cell growth and enhance GIIC in other cell types, but little is known about their effects on lung epithelial cell gap junctions. We have examined whether N6, 2'-O-dibutyryladenosine 3':5'-cyclic mono-phosphate (DBcAMP) affected GJIC, gap junction protein (connexin43) expression, and the growth of non-transformed and neoplastic mouse lung epithelial cells. DBcAMP (0.01.1 mM) stimulated GJIC (assayed by fluorescent dye microinjection) and connexin43 expression (assessed by Northern and Western blotting) and reduced their proliferation. These results suggest an association between cAMP growth inhibition and enhanced GJIC in lung epithelial cells.