Liver Tumor Promoter Research Articles

Stimulation of tumor growth in vitro and in vivo by suramin on the VX2 model.

Suramin is an antitrypanosomal compound with confirmed efficacy against several human malignancies. It is generally assumed that its mechanism of action includes the interaction with different growth factors, unlike most of the anticancer drugs. Its anticancer activity has not been tested in vivo against squamous cell carcinoma. The purpose of this study was to assess the efficacy and toxicity of suramin in vivo and in vitro on the VX2 tumor model at therapeutic monitored plasma concentrations. We determined the pharmacokinetics of suramin in rabbits, and modelized its administration in order to obtain plasma concentrations between 150 and 300 micrograms/ml throughout the treatment course of 3 weeks. Under these conditions, antitumor effects of suramin were evaluated in vivo by comparing liver tumor involvement in suramin-treated and control rabbits. Liver involvement was quantified by image analysis and in vitro effects were also determined at the same concentrations. In vivo, suramin promoted liver tumor growth significantly (p < 0.05), compared to untreated controls. In vitro, suramin significantly stimulated tumor cell growth at concentrations above 200 micrograms/ml (p < 0.01). Suramin may have stimulatory effects on tumor growth in squamous cell carcinoma at relevant plasma drug concentrations. Caution should be taken in further trials in patients with squamous cell carcinomas.

Promotion of hepatic preneoplastic lesions in male B6C3F1 mice by unleaded gasoline.

In previous studies, unleaded gasoline (UG) vapor was found to be a liver tumor promoter and hepatocarcinogen in female mice, but UG was not a hepatocarcinogen in male mice. However, UG vapor had similar transient mitogenic effects in nonlesioned liver of both male and female mice under the conditions of the cancer bioassay. We used an initiation-promotion protocol to determine whether UG vapor acts as a liver tumor promoter in male mice and to examine proliferative effects that may be critical to tumor development. Twelve-day-old male B6C3F1 mice were injected with N-nitrosodiethylamine (DEN; 5 mg/kg, intraperitoneally) or vehicle. Starting at 5-7 weeks of age, mice were exposed by inhalation 6 hr/day, 5 days/week for 16 weeks to 0 or 2046 ppm of PS-6 blend UG. UG treatment caused a significant 2.3-fold increase in the number of macroscopic hepatic masses in DEN-initiated mice, whereas no macroscopic masses were observed in non-initiated mice. Altered hepatic foci (AHF), which were predominantly basophilic in phenotype, were found almost exclusively in DEN-initiated mice. UG treatment significantly increased both the mean volume (threefold) and the volume fraction (twofold) of the AHF without increasing the number of AHF per unit area. UG also induced hepatic pentoxyresorufin-O-dealkylase (PROD) activity, a marker of CYP2B, by more than 12-fold over control with or without DEN cotreatment. To study hepatocyte proliferative effects of UG, we treated mice with 5-bromo-2'-deoxyuridine (BrdU) via osmotic pump for 3 days before necropsy and measured hepatocyte BrdU labeling index (LI) in AHF and nonlesioned liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Promotion of N-nitrosodimethylamine-initiated mouse lung tumors following single or multiple low dose exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is highly toxic to several rodent species and may have adverse health effects in exposed human populations. Further, TCDD has been shown to be a potent liver tumor promoter in the rat after repeated administration. These studies were conducted to determine the tumor promoting capability of TCDD in the Swiss mouse following single or multiple exposures. Following tumor initiation with N-nitrosodimethylamine (NDMA; 25 mg/kg), animals were given either a single dose (1.6, 16 or 48 micrograms/kg) or repeated injections (0.05 microgram/kg/week for 20 weeks) of TCDD and sacrificed at 52 weeks of age. Neither NDMA nor TCDD caused an increase in incidence of liver tumors. NDMA induced lung tumors in 100% of animals, with 12 +/- 0.1 tumors/mouse. The multiplicity of lung tumors was significantly increased by low dose TCDD treatment, with 20 +/- 2.6 tumors/mouse following a single 1.6 micrograms/kg dose (P = 0.016) and 18 +/- 1.7 (P = 0.031) following repeated 0.05 microgram/kg doses (x 20). Higher doses of TCDD did not increase multiplicity of lung tumors and, in fact, may have been toxic to the lungs of NDMA-treated mice, as evidenced by the infiltration of pigmented macrophages. These data demonstrate the potent tumor promoting capability of TCDD in mouse lung.

Down-regulation by butylated hydroxytoluene of the number and function of gap junctions in epithelial cell lines derived from mouse lung and rat liver.

The mouse pneumotoxicant and lung and liver tumor promoter butylated hydroxytoluene (BHT) was examined for its effects on gap junctional intercellular communication (GJIC) in mouse lung epithelial (C10) and rat liver epithelial (WB-F344) cell lines. GJIC, as measured by fluorescent dye microinjection, was inhibited in both types of cells by BHT in dose- and time-dependent fashions. Inhibition was detected in WB-F344 cells at BHT concentrations > or = 62.5 microM and in C10 cells at concentrations > or = 150 microM after 4 h treatment. Inhibition occurred within 15-30 min and was reversed by removing BHT from the culture medium. The highly toxic BHT metabolite 6-t-butyl-2-(hydroxy-t-butyl)-4-methylphenol (BHTOH) and the non-toxic BHT metabolite, 2,6-di-t-butyl-4-hydroxymethylphenol (BHTBzOH) were also tested. In both cell lines BHTOH was a more potent inhibitor of GJIC than BHT, whereas BHTBzOH was ineffective. The mechanisms of inhibition of GJIC by BHT were also examined. The initial rapid inhibition detected within 15-30 min may have been due to gap junction channel closure or blockage, since no changes in gap junction number, connexin (Cx) 43 levels or Cx43 phosphorylation were observed. By 2-4 h, however, gap junctions were internalized into the cytoplasm, the number of immunodetectable plasma membrane gap junctions was reduced and phosphorylated Cx43-P2 was decreased. Treatment of the cells for 24 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) prevented inhibition of GJIC by TPA, but not by BHT. Western blot analyses of TPA-treated WB-F344 or C10 cells revealed the presence of a hyperphosphorylated form of Cx43 (Cx43-P3) and no reduction in Cx43-P2, in contrast to BHT-treated cells. These data suggest that BHT and TPA inhibit lung and liver epithelial cell GJIC through distinct mechanisms.

Prototype liver tumour promoters suppress 2-acetylaminofluorene-induced apoptosis in rat hepatocytes

Prototype liver tumour promoters suppress 2-acetylaminofluorene-induced apoptosis in rat hepatocytes

Active cell death: role in hepatocarcinogenesis and subtypes.

Active cell death, the genetically programmed self-destruction of a cell, is now recognized to be a widespread phenomenon in biology that counterbalances mitosis to preserve tissue homeostasis. It is subject to the control of the growth regulatory networks in tissues. Close examination of the morphology of dying cells in liver and other tissues suggests that there are a number of morphological types of active cell death, ranging from forms dominated by nuclear changes and without signs of autophagy ("classical" apoptosis), e.g., in thymocytes and the liver, to those dominated by autophagic degradation of cytoplasm, e.g., in the mammary gland. The induction of gene expression in these diverse types of cell death is anticipated to be different. Here we review the data regarding the regulation of apoptosis in the liver by liver tumor promoter, transforming growth factor beta-1 and related peptides as well as nutrition. In the course of hepatocarcinogenesis, initiated cells as well as preneoplastic and neoplastic cell populations showed enhanced cell replication, but also enhanced apoptosis. Tumor promoter shift the balance between birth and death by increasing the rate of cell replication and by decreasing the rate of apoptosis. Thereby, liver tumor formation is accelerated. Food restriction exhibits the opposite effect and consequently, provides protection from carcinogenesis.

Identification of hepatic mitogenic and cytochrome P-450-inducing fractions of unleaded gasoline in B6C3F1 mice.

Unleaded gasoline (UG), a complex mixture of over 300 hydrocarbons, induced liver tumors selectively in female mice and exhibited liver tumor promoting activity. UG also induced cell proliferation and cytochrome P-450-related enzyme activities in mouse liver, properties commonly associated with liver tumor promoters. To determine if the mitogenic and/or cytochrome P-450-inducing properties of UG reside in individual fractions of UG, UG was separated into four fractions on the basis of boiling point (BP): fraction 1, BP < 66 degrees C; fraction 2, 66 degrees C < BP < 100 degrees C; fraction 3, 100 degrees C < BP < 132 degrees C; fraction 4, BP > 132 degrees C. Fractions 1 and 2 were combined to form "light UG" (BP < 100 degrees C), and fractions 3 and 4 were combined to form "heavy UG" (BP > 100 degrees C). Female B6C3F1 mice were implanted with osmotic pumps containing 5-bromo-2'-deoxyuridine (BrdU) on d 1, treated by intragastric intubation with corn oil or 3000 mg/kg/d of light, heavy, or whole UG on d 2-4, and euthanized on d 5. Pentoxyresorufin O-dealkylase (PROD) and ethoxyresorufin O-deethylase (EROD) activities were assayed in hepatic microsomes, and hepatocyte BrdU labeling index (LI) was determined in liver sections. Whole UG and heavy UG caused comparable increases in hepatic PROD and EROD activities and the hepatocyte LI. Light UG caused relatively small increases in hepatic PROD and EROD activities and did not increase the hepatocyte LI. When fractions 3 and 4 were tested separately in the above treatment protocol, both fractions strongly induced hepatic PROD and weakly induced hepatic EROD activities. However, only fraction 3 increased the hepatocyte LI. To isolate mitogenic components in fraction 3, equimolar doses of individual chemicals in fraction 3 were tested in the above treatment protocol. Toluene did not increase the hepatocyte LI, whereas 2,2,3-trimethylpentane (TMP), 2,2,4-TMP, and 2,3,4-TMP all dramatically increased the hepatocyte LI. Thus, while the hepatic cytochrome P-450-inducing activity of UG was concentrated in components of UG with BPs > 100 degrees C, this activity apparently resides in UG components with a wide range of BPs. The mitogenic activity of UG, in contrast, was highly concentrated in components of UG with BPs ranging from approximately 100 to 132 degrees C, and quite possibly in specific TMPs.

Two significant aspects of microcystin-LR: specific binding and liver specificity.

Microcystin-LR is a unique and potent liver tumor promoter, belonging to the okadaic acid class compounds. Although microcystin-LR is a potent inhibitor of protein phosphatases 1 and 2A, as is okadaic acid, microcystin-LR has liver specificity dominance. Two significant aspects, specific binding and liver specificity of [3H]dihydromicrocystin-LR, a reduced form of microcystin-LR, were studied and compared with those of [3H]okadaic acid. [3H]-Dihydromicrocystin-LR had higher affinity for the receptors in both the particulate and cytosolic fractions of rat liver and various tissues than had [3H]okadaic acid. Intraperitoneal administration of [3H]dihydromicrocystin-LR into mice resulted in the highest uptake into the liver, 71.5 +/- 6.9% of the total administered radioactivity, whereas p.o. administration resulted in less than 1% uptake into the liver, suggesting that the mechanisms of the incorporation of microcystin-LR into the liver by i.p. and p.o. administrations are greatly different. The presence of associated forms of [3H]dihydromicrocystin-LR with macromolecules in the liver indicates a need for further investigation.

Effects of a synthetic polyprenoic acid (E-5166) on the gap junction of rat hepatocytes treated with liver tumor promoters, phenobarbital, and p,p'-dichlorodiphenyltrichloroethane.

Effects of in vivo exposure to the synthetic polyprenoic acid, 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid (E-5166) using 6 groups of rats treated simultaneously or individually with phenobarbital (PB) or with p,p'-dichlorodiphenyltrichloroethane (DDT) on the gap junctions of hepatocytes were examined by freeze-fracture analysis. Experimental groups were as follows: Group 1, basal diet alone; Group 2, E-5166 treatment; Group 3, 0.05% PB diet; Group 4, PB diet and E-5166 treatment; Group 5, 0.05% DDT diet; and Group 6, DDT diet and E-5166 treatment. E-5166 was given by gavage (40 mg/kg body weight, 3 times/wk). Experimental diets and E-5166 were started at 6 wk of age. All animals were sacrificed at 2 wk after the start of the experiments. The density of gap junctions in PB- or DDT-treated rats was higher and that in E-5166-treated rats was lower than that in controls. The density of gap junctions in rats given PB and E-5166-/or DDT and E-5166-treated rats was slightly increased when compared to controls. The average area of gap junctions in DDT- or PB and E-5166-treated rats was significantly smaller than in controls (p < 0.005), although no statistical differences were found among PB-, E-5166-, and DDT and E-5166-treated rats and controls. Unit area of gap junctions to membrane area in rats given E-5166, DDT, or PB and E-5166 was lower than in controls. However, the unit area of gap junctions in DDT and E-5166-treated rats did not differ from in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Blue-green algae toxins and liver cancer

Microcystins (MCYSTs) isolated from blue-green algae, are hepatotoxic polypeptides. It will induce severe intrahepatic hemorrhage and liver necrosis at low concentrations in rats and mice. MCYST-LR is one of MCYSTs which consists of 2 variable L- amino acids (leucine and arginine), 3 D-amino acids and 2 unusual amino acids (including Adda). MCYSTs bind to protein phosphatase 1 and 2A, and strongly inhibit their activities. The resultant increase of phosphoprotein was referred to be involved in tumor-promoting activity in liver. According to the above results and animal study, MCYST-LR is a potent liver tumor-promoter. There were 9 positive from 30 samples of pond-ditch water in high endemic county-Haimen by high-peformance liquid chromatograph and 3 already confirmed by liquid chromograph/mass spectrometer. The quantities of MCYSTs were different between drinking water of liver cancer cases and controls groups (0.122±0.057 and 0.072±0.044 μg/200ml respectively) by ELISA. It is not easy to remove by conventional water treatment procedures. The relationship between MCYSTs and oncogenes and anti-oncogenes are under studying.

Effect of 4-acetylaminofluorene and other tumour promoters on hepatocellular growth and binucleation.

The complete liver carcinogen 2-acetylaminofluorene (2-AAF) promoted the outgrowth of large neoplastic liver nodules and hepatocellular carcinomas in diethylnitrosamine-treated rats. 2-AAF did not alter the overall proliferative activity of normal hepatocytes, but suppressed binucleation and induced, on a long-term basis, an increase in proliferative activity and in the fraction of diploid hepatocytes relative to control animals. The analogue 4-acetylaminofluorene (4-AAF) was much less effective than 2-AAF as a promoter of large nodules and carcinomas, but promoted the outgrowth of medium-sized nodules (1 < 2.5 mm). While 2-AAF specifically stimulated the growth of cells in enzyme-altered foci, the cells responding to 4-AAF were more randomly distributed throughout the liver tissue. In contrast to 2-AAF, 4-AAF strongly stimulated the growth (DNA synthesis) of normal hepatocytes, but like 2-AAF it suppressed binucleation and caused a long-term increase in the proliferative activity and in the fraction of diploid hepatocytes. Other liver tumour promoters (cyproterone acetate, alpha-hexachlorocyclohexane, methylclofenapate) likewise stimulated the growth and suppressed the binucleation of normal hepatocytes. All hepatocellular ploidy classes were affected virtually equally by mitogenic stimulation, but at low proliferation rates the mononuclear cells were more proliferative than the binuclear cells. Since this difference could be eliminated by increasing the mitogen dose, it would seem that mononuclear cells may be somewhat more sensitive towards mitogens than binuclear cells. In contrast to previously reported results [Styles et al. (1990) Carcinogenesis, 11, 1149-1152], methylclofenapate was not found to specifically stimulate binuclear hepatocytes. Our results indicate that liver tumour promoters in general tend to induce a non-binucleating, non-polyploidizing hepatocellular growth pattern, similar to that observed during liver regeneration. 4-AAF is confirmed to be, at best, a very weak promoter of liver carcinogenesis, but appears to be an effective promoter of benign tumours.

Regulation of mannose 6-phosphate/insulin-like growth factor-II receptors and transforming growth factor beta during liver tumor promotion with phenobarbital.

Chronic exposure of rats to the liver tumor promoter phenobarbital (PB) significantly reduces the ability of normal hepatocytes, but not of initiated hepatocytes, to respond to mitogenic stimuli. This reduced proliferative ability of normal hepatocytes was correlated with a marked elevation in hepatic concentration of the potent mito-inhibitory factor, transforming growth factor-beta 1 (TGF-beta 1). PB also increased the mannose 6-phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor concentration in hepatocytes, with a concomitant up-regulation in gene expression. Since the M6P/IGF-II receptor facilitates the proteolytic activation of TGF-beta 1, this suggests that PB increases the capacity of normal hepatocytes to activate TGF-beta 1. In contrast, a subset of preneoplastic lesions induced with N-nitrosodiethylamine did not demonstrate elevated levels of the M6P/IGF-II receptor or TGF-beta 1 in response to PB. These findings emphasize the potential importance of TGF-beta 1 during liver tumor promotion with PB and suggest that reduction of M6P/IGF-II receptor levels in liver tumors may provide the tumor cells with an important selective growth advantage.

Enhancement of hepatocarcinogenesis and induction of specific cytochrome P450-dependent monooxygenase activities by the barbiturates allobarbital, aprobarbital, pentobarbital, secobarbital and 5-phenyl- and 5-ethylbarbituric acids.

To test predictions that barbiturates which are long-acting sedatives and/or strong inducers of CYP2B-mediated monooxygenase activities would be effective promoters of hepatocarcinogenesis, a series of clinically-useful barbiturates and structural analogs were tested for ability to promote hepatocellular carcinogenesis in male F344/NCr rats initiated with N-nitrosodiethylamine and for efficacy as inducers of CYP2B activity in non-initiated rats of the same sex and strain. The barbiturates were administered in the diet at concentrations equimolar to 500 p.p.m. of the known liver tumor promoter phenobarbital, which served as the positive control for this study. Phenobarbital, which has the longest duration of sedative action of this series of compounds, caused the greatest induction of CYP2B activity, and displayed strong liver tumor promoting effects. Allobarbital and aprobarbital, two intermediate-duration sedatives, were found to promote hepatocarcinogenesis, with allobarbital proving to be as effective as phenobarbital in this respect and aprobarbital being somewhat weaker as a promoter. These intermediate-duration sedatives were each relatively weak CYP2B-type inducers, causing approximately 25% of the induction displayed by phenobarbital. The nonsedatives, 5-phenyl- and 5-ethyl-barbituric acids, were essentially inactive as CYP2B-type inducers and were also found to be relatively inactive as promoters of hepatocarcinogenesis. Of the shorter-duration sedatives, pentobarbital was found to promote, and was relatively effective as a CYP2B-type inducer, while secobarbital showed little or no promoting activity and was less effective as an inducer of CYP2B activities. Pentobarbital thus proved an important exception to our hypothesis that only long-acting sedative barbiturates would promote hepatocarcinogenesis. Although both the durations of sedative action and the degrees of CYP2B-type induction exhibited by these compounds correlate with a quantitative parameter for liver tumor-promoting activity (relative promotion index), neither parameter appears to be sufficient, by itself, as a predictor of promoting activity for rat liver.

Promotion of hepatocellular foci and adenomas by di(2-ethylhexyl) phthalate and phenobarbital in C3H/HeNCr mice following exposure to N-nitrosodiethylamine at 15 days of age.

Previous studies have shown that phenobarbital (PB), as well as another known liver tumor promoter, alpha-hexachlorocyclohexane (HCH), inhibits hepatic tumor formation in infant N-nitrosodiethylamine (NDEA)-initiated C57BL/6 x C3H/He (B6C3F1) male mice. These inconsistencies in detecting PB and HCH as tumor promoters have raised important questions on the mechanism of tumor promotion in mice, as well as the reliability of the infant B6C3F1 mouse as an initiation model in two-stage carcinogenesis experiments. Therefore, in an effort to avoid the inconsistencies associated with the B6C3F1 mouse, the present study evaluated the ability of two known hepatic liver tumor promoters, di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, and phenobarbital (PB), a barbiturate, to promote hepatocellular tumorigenesis in mice of the C3H/HeNCr strain initiated during infancy. At 15 days of age, male and female C3H/HeNCr mice received either a single ip injection of NDEA (5 micrograms/g body weight) or saline. At weaning (4 weeks of age), mice were divided into 3 groups and treated with either DEHP in the diet (12,000 ppm), PB in the drinking water (500 ppm), or control drinking water and diet for 24 weeks. All mice were killed at 28 weeks of age and the number and size of hepatic foci and adenomas were evaluated. Mice exposed to NDEA+DEHP or NDEA+PB showed significant increases in the number and size of hepatic tumors compared to those receiving NDEA alone. DEHP treatment in males yielded larger adenomas than those seen in PB-treated males.(ABSTRACT TRUNCATED AT 250 WORDS)

Loss of gap junctions from DDT-treated rat liver epithelial cells.

The mechanism by which the liver tumor promoter 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) inhibits gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells could involve gap junction loss and/or decreased gap junction channel permeability. We examined these two possibilities in the present study. Immunohistochemical studies using antibodies specific to connexin43, the major gap junction protein expressed by these cells, revealed that gap junction number and size were reduced during exposure to DDT. The reductions in gap junctions (33-91%) correlated with dose-dependent (1-10 microM) and time-dependent (0.5-4 h) decreases in cell-to-cell fluorescent dye-coupling (64-85%), as well as cellular levels of phosphorylated connexin43. These effects were reversible following removal of the tumor promoter from the culture medium, although cycloheximide reduced the level of gap junction reformation. The losses in gap junctions were not due to decreased connexin43 gene expression since steady-state levels of connexin43 mRNA were not similarly affected by DDT. Fenarimol (10 microM), a structural analog of DDT, did not inhibit GJIC and had no effect on gap junction structure or connexin43 expression. These data suggest that the inhibition of GJIC by DDT resulted from the removal of gap junctions from the plasma membrane and their degradation rather than simply a decrease in their permeability.

Dose-response effects of malachite green on free radical formation, lipid peroxidation and DNA damage in Syrian hamster embryo cells and their modulation by antioxidants.

Malachite green (MG), consisting of green crystals with a metallic lustre, is very soluble in water and is highly cytotoxic to mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the possible involvement of reactive free radicals in morphological transformation of Syrian hamster embryo (SHE) cells by MG. In this study we have studied the dose-response effects of MG on free radical formation, lipid peroxidation and DNA damage in SHE cells. Electron spin resonance analysis with 5,5-dimethyl-1-pyrroline-N-oxide as a spin-trapping agent was used to study the production of free radicals from MG. Exposure of SHE cells to MG demonstrated a dose-dependent increase in the generation of free radicals, lipid peroxidation and DNA damage. Treatment of SHE cells with antioxidant enzymes such as catalase (CAT) and glutathione peroxidase (GPx) prior to MG exposure decreased lipid peroxidation and DNA damage, with CAT being more effective than GPx. Since metabolism of MG leads to the generation of free radicals, and CAT and GPx decreased MG-induced lipid peroxidation and DNA damage, the present study confirms the possible relationship between the genotoxicity of SHE cells by MG and the involvement of reactive free radical formation.

Tumor necrosis factor-alpha, a new tumor promoter, engendered by biochemical studies of okadaic acid.

Okadaic acid is a potent tumor promoter on mouse skin and in rat glandular stomach, and an inhibitor of PP-1 and PP-2A. How okadaic acid biochemically induces tumor promotion in these tissues was reviewed. Okadaic acid bound to a catalytic subunit of PP-1 and PP-2A and induced hyperphosphorylation of proteins, such as vimentin, cytokeratins, HSP 27, and tumor suppressor gene products. Since one of the okadaic acid class compounds, microcystin-LR, induced tumor promotion in rat liver, the okadaic acid pathway mediated through inhibition of PP-1 and PP-2A is seen to be a general biochemical process of tumor promotion in various organs. The biochemical mimicry of okadaic acid by TNF-alpha led us to find that TNF-alpha is an endogenous tumor promoter. The study of tumor promotion in two-stage carcinogenesis experiments with the okadaic acid class of compounds engendered a new tumor promoter applicable to human cancer development.

2,3,7,8-Tetrachlorodibenzo-p-dioxin as growth modulator in mouse hepatocytes with high and low affinity Ah receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a highly potent tumor promoter in rodent liver, has been shown to increase epidermal growth factor (EGF)-stimulated DNA synthesis in rat hepatocytes in primary culture, a comitogenic effect possibly linked to the mechanism of liver tumor promotion. In hepatocytes isolated from male congenic mice (C57BL/6J) with high-affinity (AhbAhb) or low-affinity (AhdAhd) Ah receptor the association of the comitogenic action of TCDD with the Ah receptor was investigated. The potency of TCDD as inducer of CYP1A-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity was approximately 10-fold lower in AhdAhd compared to AhbAhb cells. In both cell types, TCDD did not stimulate DNA synthesis in the absence of EGF. When added together with EGF, TCDD exhibited two opposing effects on DNA synthesis, measured as incorporation of [3H]thymidine into DNA: (i) At 3 x 10(-14) M, TCDD increased EGF-stimulated DNA synthesis approximately 1.4-fold in AhbAhb but not in AhdAhd cells at a plating density of 35,000 cells/cm2. In hepatocytes from AhdAhd mice, 3 x 10(-12) M TCDD was required to elicit a similar comitogenic response. (ii) At a density of 10,000 cells/cm2, 3 x 10(-12) M TCDD had a pronounced inhibitory effect on EGF-stimulated DNA synthesis in AhbAhb but not in AhdAhd cells. Essentially similar results were obtained by counting of 5-bromo-2'-deoxyuridine-labeled nuclei. These findings demonstrate that TCDD can enhance or antagonize EGF-stimulated DNA synthesis in mouse hepatocytes modified by the cell density. The different concentration-response relationships in hepatocytes from both strains suggest that the Ah receptor regulates these responses.

Evaluation of the tumor-promoting activity of two beta-adrenoreceptor blocking agents, propranolol and atenolol, in liver of Fischer 344 rats.

The tumor-promoting activity of two beta-adrenoreceptor blocking agents, propranolol and atenolol, was tested in a two-stage protocol of hepatocarcinogenesis in male and female Fischer 344 rats. Propranolol is a lipophilic non-selective beta-blocker mainly eliminated via the liver; atenolol is a hydrophilic beta 1-selective blocking agent, mainly eliminated via the kidney. Animals were initiated with a single dose of diethylnitrosamine (DEN, 200 mg/kg, i.p.) and, after 17 days of recovery, were continuously treated with propranolol (75-100 mg/kg) or atenolol (300 mg/kg) by gavage for up to 21 months. Rats given phenobarbital (0.05% in the diet) were used as positive controls. After 2, 4 and 8 months of promotion, preneoplastic lesions were quantified by staining sections of liver for gamma-glutamyltranspeptidase (GGT). In non-initiated rats, neither propranolol nor atenolol influenced the development of spontaneous preneoplastic or neoplastic liver lesions. The results obtained in DEN-initiated rats given propranolol cannot be unequivocally interpreted. In the male, propranolol seemed to be ineffective. In the female, there was weak enhancement of DEN-induced GGT foci at 4 and 8 months and of neoplastic lesions thereafter. However, there was great interindividual variability in focus and tumor yields. Unfortunately, due to the high incidence of liver tumors in rats given DEN alone and the small number of propranolol-treated rats that survived until the end of the experiment, no definite conclusion can be drawn about the modifying potential of this beta-blocker on liver carcinogenesis. There was no evidence of liver tumor promotion in DEN-initiated rats of either sex given atenolol.

Isolation of microcystin‐LR from amicrocystis(cyanobacteria) waterbloom collected in the drinking water reservoir for porto, Portugal

Abstract Plankton tows collected over the water intake towers on Crestuma reservoir, which is the drinking water supply for about 2,000,000 persons in the Porto, Vila Nova de Gaia, and Gondomar regions near the city of Porto, were found to be hepatotoxic using the mouse intraperi‐toneal bioassay. Phytoplankton in these samples was dominated by the cyanobacteria Microcystis aeruginosa (95%). The lethal dose for 50% of the animals tested of lyophilized bloom material was approximately 30 mg/kg (dry cell weight/animal weight). Using HPLC separation, a single fraction was obtained. Isolation and purification of this fraction resulted in a toxin that was shown to be microcystin‐LR by amino acid and MS analyses. Since microcystin‐LR is a potent hepatotoxin and liver tumor promoter, and since high cell densities of Microcystis aeruginosa producing microcystin‐LR were found near the water intake lines, it shows that a regular monitoring of microcystin(s) should be developed in water used for human consumption to ...