Comparative tumor-promoting activities of phenobarbital, amobarbital, barbital sodium, and barbituric acid on livers and other organs of male F344/NCr rats following initiation with N-nitrosodiethylamine

Tumor-promoting abilities of four barbiturates, phenobarbital [(PB) CAS: 50-06-6], amobarbital [(AB) CAS: 57-43-2], barbital sodium [(BB) CAS: 144-02-5], and barbituric acid [(BA) CAS: 67-52-7], on the development of neoplasms in livers and other organs of rats following initiation with N-nitrosodiethylamine [(DENA) CAS: 55-18-5] were compared. Four-week-old F344/NCr male rats were given a single ip injection of 75 mg DENA/kg body weight. Beginning 2 weeks later, they were given either tap water (group 1) or drinking water containing 500 ppm of PB (group 2), the sodium salt of BB (group 3), AB (group 4), or BA (group 5) for the remaining experimental period. Control groups (groups 6-10) received an ip injection of saline alone and 2 weeks later were given either tap water or drinking water containing barbiturates as listed above. Animals were sacrificed at either 52 weeks or 78 weeks. None of the barbiturates altered the growth and survival of animals. PB and BB increased liver weights and significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 weeks and hepatocellular foci, hepatocellular adenomas, and trabecular carcinomas at 78 weeks in DENA-treated rats. No such enhancing effects were observed with AB or BA. PB or BB did not significantly enhance the incidence of nonhepatic tumors at 52 weeks. However, at 78 weeks BB significantly enhanced the development of renal tubular adenomas and carcinomas, while PB enhanced the development of thyroid follicular cell neoplasms in DENA-treated rats. These results clearly showed that barbiturates exhibited structure-promoting activity relationships and that their promoting abilities were not restricted to liver alone. Substitution of both hydrogen atoms at the C-5 position of the pyrimidine ring by alkyl or aryl groups appears to be essential but not sufficient for tumor-promoting activity of barbiturates.     

Effects of phenobarbital and carbazole on carcinogenesis of the lung, thyroid, kidney, and bladder of rats pretreated with N-bis(2-hydroxypropyl)nitrosamine

Studies were made on potential modifying effects of phenobarbital (PB) and carbazole on tumor development induced by N-bis(2-hydroxypropyl)nitrosamine (DHPN), a wide-spectrum carcinogen in rats. Effects on the lung, thyroid, kidney, bladder and liver were investigated. Male F344 rats were given 0.2% DHPN in their drinking water for 1 week and then 0.05% PB or 0.6% carbazole in their diet for 50 weeks. Control animals were treated with either DHPN or PB or carbazole only. Neither PB nor carbazole affected the incidence or histology of lung tumors. However, PB promoted the development of thyroid tumors and preneoplastic lesions of the liver, while carbazole promoted the induction of renal pelvic tumors.     

Effects of Phenobarbital and Carbazole on Carcinogenesis of the Lung, Thyroid, Kidney, and Bladder of Rats Pretreated with N-Bis(2-hydroxypropyl)nitrosamine

Studies were made on potential modifying effects of phenobarbital (PB) and carbazole on tumor development induced by N-bis(2-hydroxypropyl)nitrosamine (DHPN), a wide-spectrum carcinogen in rats. Effects on the lung, thyroid, kidney, bladder and liver were investigated. Male F344 rats were given 0.2% DHPN in their drinking water for 1 week and then 0.05% PB or 0.6% carbazole in their diet for 50 weeks. Control animals were treated with either DHPN or PB or carbazole only. Neither PB nor carbazole affected the incidence or histology of lung tumors. However, PB promoted the development of thyroid tumors and preneoplastic lesions of the liver, while carbazole promoted the induction of renal pelvic tumors.     

Modification by sodium L-ascorbate, butylated hydroxytoluene, phenobarbital and pepleomycin of lesion development in a wide-spectrum initiation rat model

Rats were treated for 1 week each with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), 0.2% N-bis(2-hydroxypropyl)-nitrosamine (DHPN) and 0.2% N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the drinking water, and then administered diet containing 5% sodium L-ascorbate (Na-AsA), 1% butylated hydroxytoluene (BHT) or 0.05% phenobarbital (PB), or weekly intraperitoneal injections of 2 mg of pepleomycin per kg body weight until week 36. Histopathological examination revealed that all exerted significant modulation effects on tumor development in the various target organs. Na-AsA was found to inhibit liver but promote renal pelvis and bladder carcinogenesis. BHT similarly decreased liver and enhanced bladder lesion development. PB, in contrast promoted hepatocarcinogenesis. However both PB and BHT were associated with increased incidences of adenomas and adenocarcinomas of the thyroid. Thus the wide-spectrum initiation model allowed confirmation of site-specific modification potential and in addition demonstrated potentiation of kidney and bladder carcinogenesis promotion by pepleomycin.     

Organ-specific promoting effect of phenobarbital and saccharin in induction of thyroid, liver, and urinary bladder tumors in rats after initiation with N-nitrosomethylurea

Tumor-promoting effects of phenobarbital (PB) and sodium saccharin (SS) were tested in rats pretreated with N-nitrosomethylurea (NMU) with special reference to the site of their action. Male F344 rats were initially given injections of NMU (20 mg/kg i.p.) twice a week for 4 weeks, then given basal diet containing 0.05% PB or 5% SS for the next 32 weeks, and then killed. Appropriate control studies were also done. Histological examination of whole organs of the rats showed that PB promoted thyroid carcinogenesis whereas SS did not. A significant increase in the incidences of total tumors as well as papillary adenocarcinoma of the thyroid was observed in the group given PB after NMU (p less than 0.001). The incidence of papillary adenoma with or without papillary adenocarcinomas was also high in the NMU-PB-treated group. The organ-specific promoting effect of PB in the induction of preneoplastic lesions, as demonstrated by development of gamma-glutamyl transpeptidase-positive foci in the liver and of SS in papillary or nodular hyperplasia in the urinary bladder, as reported previously, was also confirmed. The incidences of papillomas in the forestomach were similar in groups treated with NMU-PB, NMU-SS, or NMU alone. The results indicate that PB is a tumor promoter in the liver and thyroid and that SS is a tumor promoter in the urinary bladder of rats.     

Promoting effects of phenobarbital and barbital on development of thyroid tumors in rats treated with N-bis(2-hydroxypropyl)nitrosamine

Phenobarbital (PB) and barbital (BB) promoted the developmentof thyroid tumors in rats treated with a sub-effective doseof N-bis(2-hydroxypropyl)nitrosamine (DHPN) for thyroid tumorigenesis.Rats were given s.c. injections of 70 mg DHPN/100 g body weightonce a week for 4 or 6 weeks with or without diet containing500 p.p.m. PB or BB for the next 12 weeks. The incidences ofthyroid tumors at the end of week 20 of the experiment were66% in rats given DHPN for 4 weeks and then PB, 23% in ratsgiven DHPN for 4 weeks and then BB, 100% in rats given DHPNfor 6 weeks and then PB, 45% in rats given DHPN for 6 weeksand then BB, and 23% in rats given DHPN for 6 weeks. Rats givenonly DHPN for 4 weeks or only PB or BB had no thyroid tumorsafter 20 weeks.     

Inhibition by pentachlorophenol of the initiating and promoting activities of 1'-hydroxysafrole for the formation of enzyme-altered foci and tumors in rat liver

The hepatocarcinogen 1'-hydroxysafrole (HOS) exhibited weakinitiating activity and strong promoting activity for the inductionof enzyme-altered foci and tumors in rat liver. Thus, administrationof a single dose of HOS to rats 18 h after a 70% hepatectomy,followed by administration of phenobar-bital (PB) in the dietfor 6 months, induced a low, but statistically significant,number of foci of enzyme-altered cells. This treatment did notresult in gross liver tumors, even when the PB treatment wascontinued for 16 months. Large numbers of enzyme-altered focideveloped when HOS was administered in the diet at levels of0.05–0.25% to rats previously administered a single doseof N,N-diethylnitros-amine (DEN) 24 h after a 70% hepatectomy.Similarly, rats given a single dose of DEN 24 h after a partialhepatectomy and then fed 0.10 or 0.25% of HOS in the diet for10 months developed a high incidence of hepatocellular carcinomas.In the absence of pretreatment with DEN, dietary administrationfor at least 4 months of 0.10 or 0.25% of HOS induced significantnumbers of enzyme-altered foci; these data and liver tumor inductionby continuous feeding of HOS, in the absence of pretreatmentwith DEN, provide additional evidence for an initiating, aswell as a promoting, activity of HOS in rat liver. Concurrentadministration of the hepatic sulfotransferase inhibitor pentachlorophenolwith HOS in each of the above assays almost completely inhibitedthe initiating and promoting activities of HOS for the formationof enzyme-altered foci and tumors; these data strongly suggestthat both the initiating and promoting activities are mediatedby the sulfuric acid ester, 1'-sulfooxysafrole. HOS also exhibitedinitiating activity in adult mouse liver. Thus, dietary administrationof 0.25% of HOS for only 1 month, followed by administrationof the hepatic tumor promoter 1,4-bis[2-(3,5-dichloropyridyloxy)]benzeneresulted in a high incidence and multiplicity of hepatomas by10 months. In the absence of the promoter, administration ofHOS for only 1 month induced no hepatomas; 1,4-bis[2-(3,5-dichloropyridyloxy)]benzenealone induced only a low incidence. In mice not given the promoter,continuous administration of HOS     

The influence of subsequent dehydroepiandrosterone,diaminopropane, phenobarbital,butylated hydroxyanisole and butylated hydroxytoluene treatment on the development of preneoplastic and neoplastic lesions in the rat initiated with di-hydroxy-di-N-propyl nitrosamine

The comparative modifying potential of dehydroepiandrosterone (DHEA), diaminopropane (DAP), phenobarbital (PB), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the development of lesions initiated by dihydroxy-di-n-propyl nitrosamine (DHPN) in F344 rats were investigated. DHEA, BHA and BHT were all associated with significant reduction in numbers of glutathione-S-transferase P form (GST-P) positive foci in the liver whereas PB brought about their enhanced development. BHT and PB exerted promoting activity on the incidence of thyroid adenomas while DAP similarly increased lung adenoma formation. The results illustrate the advantages to be gained from two stage experiments using broad spectrum carcinogen initiation for comparative analysis of ‘modifiers’ of the neoplastic process and suggest that studies of enzyme alteration within putative preneoplastic lesions may be directly relevant to elucidation of mechanisms underlying such modification.     

In vitro and in vivo response of hepatocytes from hepatic nodules to the mitoinhibitory effects of phenobarbital

One of the proposed mechanisms by which phenobarbital (PB) promoteshepatocarcinogenesis in the rat is by differential mitoinhibition.However, our earlier studies indicated that PB inhibited DNAsynthesis in vitro in hepatocytes isolated from both surroundingnon-nodular liver and hepatic nodules promoted by orotic acid(OA). Since nodules generated by one promoter need not necessarilybe resistant to another promoter, the present study was undertakento determine whether foci/nodules promoted by PB itself areresistant to the mitoinhibitory effects of PB. Accordingly,rats were initiated with diethylnitrosamine (DENA, 200 mg/kgi.p) and promoted with PB (0.07% of PB as its sodium salt) intheir drinking water for 16 or 33 weeks. In vitro studies indicatedthat PB (3–5 mM) inhibited DNA synthesis induced by epidermalgrowth factor (EGF) in hepatocytes from surrounding non-nodularliver as well as from nodules promoted by PB for 33 weeks. Inanother experiment, initiated rats exposed to PB for 33 weekswere subjected to either two-thirds partial hepatectomy (PH)or sham hepatectomy. Hepatocytes were labelled with tritiatedthymidine in vivo for 48 h. Autoradiographic analysis indicatedthat in the presence of PB, the hepatocytes from both foci/nodulesand the surrounding non-nodular liver responded to PH to thesame extent. In addition, they both responded to PH less efficientlyas compared to the corresponding controls. Further, initiatedrats exposed to PB for 16 weeks when subjected to PH and killed4 weeks thereafter, the percentage area occupied by     

Enhancement of dimethylnitrosamine-initiated hepatocarcinogenesis in hamsters by subsequent administration of carbon tetrachloride but not phenobarbital or p,p'-dichlorodiphenyltrichloroethane

The effect of phenobarbital (PB), p,p'-dichlorodiphenyl-trichloethane(DDT) or carbon tetrachloride (CCl₄) on dimethylnitrosamine(DMN)-induced hepatocarcinogenesis in Syrian golden hamsterswas examined. Hamsters were given a single injection of DMN(6 mg/kg body wt) followed by either PB or DDT in the diet orrepeated CCl₄ gavage for 30 weeks. The numbers of both alteredliver cell foci and hepatocellular neoplasms in the hamstersgiven 0.1 ml CCl₄, i.g. once every 2 weeks after the DMN weresignificantly higher than in the animals given DMN alone. PBor DDT (500 p.p.m. in the diet) after DMN did not produce asignificantly higher incidence of altered foci or hepatocellularneoplasms compared to DMN alone. Thus, an enhancing effect ofCCl₄ on DMN-induced hepatocarcinogenesis in the hamster wasdemonstrated, but neither PB or DDT—both liver neoplasmpromoters in rats and mice—displayed promoting activityunder the conditions of study.     

Inhibition by indomethacin and 5,8,11,14-eicosatetraynoic acid of the induction of rat hepatic ornithine decarboxylase by the tumor promoters phenobarbital and 12-O-tetradecanoylphorbol-13-acetate in vivo

The involvement of arachidonate metabolism in the inductionof rat hepatic ornithine decarboxylase (ODC) activity by thetumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) andphenobarbital (PB) was investigated. Pretreatment of the ratswith indomethacin or 5,8,11,14-eicosateraynoic acid dose dependentlyinhibited the induction of ODC by both tumor promoters. Bothinhibitors were more potent inhibitors of PB induction thanTPA induction of ODC. The data are consistent with an involvementof arachidonate cyclooxygenase products in the induction ofrat hepatic ODC by the tumor promoters.     

Metabolism and DNA adduct formation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in fish cell lines in culture

The metabolic activation of the carcinogens benzo[a]anthracene(BP) and 7,12-dimethylbenz[a]anthracene (DMBA) was examinedin cell lines derived from bluegill fry (BF-2), rainbow trout(RTG-2) and brown bullhead (BB). All three cell lines metabolizedBP (0.5 µg/ml medium) almost completely to water-solublemetabolites within 120 h, but the maximum amount of BP boundto DNA ranged from only 5 pmol/mg DNA in the BF-2 cells to 17in the BB cells and 44 in the RTG-2 cells. The major BP-DNAadduct in the BB and BF-2 cells was that formed by reactionof (+)-anti-BP-7,8-diol-9,10 epoxide [(+)anti-BPDE] with deoxyguanosine.This adduct was also present in the RTG-2 cell DNA, but therewere larger amounts of unidentified polar BP-DNA adducts. Exposureof the cells to [³H]BP-7,8-diol, a metabolic precursor of (+)anti-BPDE,resulted in binding of 1.5, 12 and 35 pmol BP per mg DNA inthe BF-2, BB and RTG-2 cells, respectively. More than 90% ofthe BP-7,8-diol added to the BF-2 cultures was recovered asa glucuronic acid conjugate, but the RTG-2 cells formed moreglutathione conjugates than glucuronide conjugates. The BB cellsformed both types of conjugates at a slower rate for more than75% of the 7,8-diol was recovered unchanged after 24 h. Thethree cell lines differed in the proportion of a 0.1 µg/mldose of DMBA metabolized in 48 h: the values ranged from 47%in the BF-2 cells to 78% in the BB cells and 97% in the RTG-2cells. The amount of DMBA bound to DNA ranged from 4.7 to 8.6pmol/mg DNA in the three cell lines: DMBA-3,4-diol-1,2-epoxide(DMBADE) adducts were present in the BB cell DNA, but no significantamounts of DMBADE-DNA adducts were detected in the RTG-2 orBF-2 cell DNA. These results demonstrate that fish cell culturescan activate BP to an ultimate carcinogenic metabolite, (+)anti-BPDE,but the level of binding of this metabolite to DNA is much lowerthan that which occurs in rodent embryo cell cultures. In BF-2cell cultures formation of BP-7,8-diol-glucuronide effectivelyprevents the activation of this diol to (+)anti-BPDE. A substantialproportion of the BP-7,8-diol is also metabolized to glucuromdeand glutathione conjugates in BB and RTG-2 cells. DMBA alsobinds to DNA at very low levels in these fish cell cultures.Thus effective conjugation of diols and their metabolites byfish cell lines appears to greatly reduce metabolic activationof hydrocarbons through the bay-region diol epoxide pathwaythat predominates in mammalian cell cultures.     

Possible involvement of poly ADP-ribosylation in phenobarbital promotion of rat hepatocarcinogenesis

The effects of inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide(ABA), luminol and 3-methoxybenzamide (MBA) on the rat livertumor promotion activity of phenobarbital (PB) were assessed.Fischer 344 male rats were initiated with N-nitrosodiethylamine(200 mg/kg) and placed on either basal diet, diet containing0.05% PB, diet containing various doses of the inhibitors aloneor diet containing 0.05% PB plus various doses of inhibitorsfor 10 weeks, and then killed. Quantitation of the developmentof glutathione S-transferase placental form-positive foci revealedthat ABA at doses of 2 and 1.5, but not 1%, significantly inhibitedthe PB promotion activity. Luminol dose-dependently reducedPB promotion at doses of 3 and 6% but exerted no effects atthe 1 and 2% levels. MBA also demonstrated a dose-dependentinhibitory influence at doses of 1 and 2%. The results are thusstrongly suggestive of an involvement of poly ADP-ribosylationin the mechanisms underlying liver tumor promotion by PB.     

P-450 enzyme induction by 5-ethyl-5-phenylhydantoin and 5,5-diethylhydantoin, analogues of barbiturate tumor promoters phenobarbital and barbital, and promotion of liver and thyroid carcinogenesis initiated by N-nitrosodiethylamine in rats

Male F344/NCr rats, 6 wk old, were fed 500 ppm of phenobarbital (PB) or equimolar doses of either 5-ethyl-5-phenylhydantoin (EPH) or 5,5-diethylhydantoin (EEH) in diet for 2 wk and hepatic cytochrome P-450-mediated alkoxyresorufin O-dealkylase and aminopyrine N-demethylase activities were determined. Both PB and EPH greatly increased P-450-mediated enzyme activities in rat liver while EEH was ineffective. To evaluate the hydantoins as tumor promoters, 5-wk-old male F344 rats were given a single i.p. injection of 75 mg N-nitrosodiethylamine/kg body weight. Beginning 2 wk later, they were placed either on normal diet or diet containing 500 ppm of PB or equimolar doses of EPH or EEH for the remaining experimental period. Control groups received an i.p. injection of saline followed by each of the test diets. Animals were sacrificed at either 52 or 78 wk. PB and EPH significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 wk and hepatocellular carcinomas at 78 wk in N-nitrosodiethylamine-initiated rats. Neither the incidence of hepatocellular neoplasms nor the number and size of hepatocellular foci was significantly increased by EEH. At 78 wk, both PB and EPH enhanced the development of thyroid follicular cell neoplasms in N-nitrosodiethylamine-initiated rats while no such enhancement was observed with EEH. Thus, EPH, a long-acting sedative/anticonvulsant, like the structurally similar PB, promoted hepatocellular and thyroid follicular cell carcinogenesis and induced the PB-inducible form(s) of cytochrome P-450 (P-450b) in rats. In contrast, EEH unlike barbital failed to promote hepatocellular and thyroid follicular cell carcinogenesis and also failed to induce PB-inducible form(s) of cytochrome P-450 in rats.     

Enhancement of thyroid and hepatocarcinogenesis by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene in rats at doses that cause maximal induction of CYP2B

To investigate the promoting effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene(TCPOBOP) on liver and thyroid carcinogenesis of rats at dosesthat cause maximal induction of hepatic CYP2B, 5-week-old maleF344 rats were given either a single i.p. dose of 75 mg N-nitrosodiethylamine(NDEA)/kg body wt in saline or saline alone. After 2 weeks therats were fed control diet or a diet containing 330 or 1000p.p.m. TCPOBOP or 500 p.p.m. phenobarbital (PB; a positive controlgroup). A total of four sequential sacrifices (9, 30, 52 and79 weeks of age) was performed. At 30 weeks the mean volume(mm³) of hepatocellular foci in NDEA-initiated rats exposedto either dose of TCPOBOP or to PB was significantly increasedas compared with rats exposed to NDEA followed by control diet(P < 0.05). In addition, the volume percentage of liver occupiedby foci was significantly greater in NDEA-initiated/1000 p.p.m.TCPOBOP-promoted rats as compared with rats exposed to NDEAalone (P < 0.05, n = 6). At 52 weeks of age the incidences(and multiplicities, in units of tumors per tumor-bearing rat)of hepatocellular adenomas were 0, 83 (2.6 ± 1.3), 100(3.4 ± 2.1) or 67% (2.5 ± 1.9) in rats exposedto NEDA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOPor 500 p.p.m. PB respectively (n = 12). Hepatocellular carcinomaswere found only in rats given 1000 p.p.m. TCPOBOP (17% incidence)or PB (8% incidence) following NDEA initiation. The incidencesof thyroid follicular cell adenomas were 0, 17, 33 or 8% inrats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m.TCPOBOP or 500 p.p.m. PB respectively. Between 53 and 79 weeksof age 38% of rats treated with NDEA alone developed multiple(1.5 ± 0.8) hepatocellular adenomas. This incidence wasenhanced to 100% in rats exposed to NDEA followed by either330 or 1000 p.p.m. TCPOBOP. Multiplicities of hepatocellularadenomas were also increased significantly (10.5 ± 3.9,10.4 ± 7.0 and 10.1 ± 6.7 respectively) in ratspromoted with 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB. Noneof the rats exposed to NDEA alone developed hepatocellular carcinomas,while multiple hepatocellular carcinomas occurred in 38% ofthe rats exposed to 330 p.p.m. and 78% of the rats given 1000p.p.m. TCPOBOP following NDEA initiation. Thyroid follicularcell tumors occurred at 79 weeks in more than 40 and 50% incidencesin rats exposed to NDEA followed by 330 or 1000 p.p.m. TCPOBOPrespectively. Also, a significant decrease in serum levels oftriiodothyronine and thyroxine were observed in non-initiated79-week-old rats fed 1000 p.p.m. TCPOBOP, compared with age-matcheduntreated controls (n = 6). Increases in hepatic CYPC2B-mediatedbenzyloxy-resorufin O-dealkylase activity detected in rats exposedto 330 and 1000 p.p.m. TCPOBOP for 2 or 23 weeks were similarin magnitude to those caused by 500 p.p.m. PB. Thus TCPOBOPat maximal CYP2B induction doses exhibits a strong promotingactivity for both liver and thyroid of rats.     

Possible involvement of arachidonic acid metabolism in phenobarbital promotion of hepatocarcinogenesis

The effects of inhibitors of arachidonic acid metabolism andantioxidants on the rat liver tumor promotion activity of phenobarbital(PB) were assessed using the enzyme-altered focus as the end-pointlesion. Fischer 344 male rats were initiated with N-nitrosodiethylamine(200 mg/kg) and then divided into five groups placed on basaldiet, diet containing 0.05% PB, diet containing 0.05% PB plus0.75%, 1% or 1.5% levels of various inhibitors of arachidonicacid metabolism or antioxidants, or diet containing 1% or 1.5%inhibitors or antioxidants alone for 10 weeks, and then killed.-Bromo phenacyl bromide, an inhibitor of phospholipase A₂ significantly inhibited the promotion activity of PB at dose levelsof 0.75% and 1.5%, reaching plateau at 0.75%. Both quercetin,an inhibitor of lipoxygenase, and morin, a dual inhibitor oflipoxygenase-cyclooxygenase, significantly reduced the promotionactivity of PB at the 1.5% but not 0.75% dose levels. Moreover,acetylsalicylic acid, an inhibitor of cyclooxygenase dose-dependentlyinhibited the promotion activity of PB. Among the antioxidantsinvestigated, vitamin E did not affect, but n-propyl gallateand ethoxyquin exerted a dose-dependent inhibition of PB promotion.These results are strongly suggestive of an involvement of phospholipaseA₂ lipoxygenase and cyclooxygenase arachidonic acid metabolicpathways in the mechanisms underlying PB enhancement of hepatocarcinogenesis.     

Differential renal tumor response to N-ethylnitrosourea and dimethylnitrosamine in the Nb rat: basis for a new rodent model of nephroblastoma

Previous studies have indicated that the Nb rat has a higherpredisposition to the spontaneous development of nephro-blastomathan most other strains of laboratory rat. In order to determinewhether this inherent susceptibility could be exploited as amodel for Wilms' tumor, Nb rats were treated with various chemicalsin regimens known to be associated with renal tumor inductionin the young rat. Nb rats were either exposed in utero to variousdose levels of N-ethylnitrosourea (ENU) on day 18 ± 1of gestation, injected s.c. with one 25 mg/kg dose of dimethylnitrosamine(DMN) as neonates or dosed twice orally with 7,12-dimethylbenz[a]anthracene(DMBA) shortly after ovariectomy. Transplancental ENU at a doseof 60 mg/kg resulted in an average of 50% frequency of unequivocalnephroblastomas in both sexes with no renal mesenchymal tumors.In contrast, DMN administered to neonates produced a similarincidence of renal mesenchymal tumors but no nephroblastomas.DMBA in the ovariectomized female was not a successful strategyfor inducing primary renal tumors in the Nb rat (0 nephroblastomas,1 renal mesenchymal tumor in 16 effective survivors), althoughthe kidney was sometimes the site for metastatic invasion bytumors originating at other locations. The induction of nephroblastomasand renal mesenchymal tumors by ENU and DMN in parallel experimentsemphasized the many differences which establish them as distinctand unrelated tumor entities. The relatively high incidenceof nephroblastoma in the Nb rat using transplacentally administeredENU appears to represent a suitable basis for developing a rodentmodel of human nephroblastoma or Wilms' tumor.     

The use of initiated cells as a test system for the detection of inhibitors of gap junctional intercellular communication

The effects of five non-mutagenic carcinogens—Aroclor1260, benzoyl peroxide (BP), phenobarbital (PB), 12-O-tetradecanoyl-phorbol-13-acetate(TPA) and 1, 1'-(2, 2, 2-trichloroethylidene)bis[4-chlorobenzene](DDT)—on gap junctional intercellular communication (GJIC)were tested in a cell line consisting of initiated cells (3PC).Four agents suspected of tumor promotion activity—O-anisidine,clofibrate, L-ethionone and d-limonene—were also testedfor their effects on GJIC. Finally sodium fluoride (NaF), whosecarcinogenic property is still unclear, was tested for its effectson GJIC in the 3PC cell line. Four of the five selected tumorpromoters (Aroclor 1260, BP, DDT and TPA) decreased GJIC betweenthese initiated epidermal cells. The four non-mutagenic carcinogenswith tumor-promoting activity in vivo (o-anisidine, clofibrate,L-ethionine and d-limonene) all inhibited GJIC, whereas NaFhad no effect. Seven compounds (o-anisidine, Aroclor 1260, BP,DDT, L-ethionine, d-limonene and TPA) had a dose-dependent aswell as time-dependent inhibitory effect on GJIC. Under theexperimental conditions used, clofibrate showed only a dose-relatedinhibition of GJIC. PB showed no inhibitory effect on GJIC inthe 3PC cell line. In order to determine the role of biotransformationin the tumor-promoting activity of PB, its effect on GJIC wasalso examined in the presence of an Aroclor 1254-induced ratliver homogenate (S9 mix) and in the hepatoma cell line HepG2.In the presence of rat liver homogenate PB decreased GJIC inthe 3PC cell line, whereas in the HepG2 cells PB showed a time-anddose-dependent inhibitory effect. To study the potential differencesin susceptibility of cells representing different stages inthe process of tumor formation, the effect of the selected tumorpromoters on GJIC was also investigated in primary mouse keratinocytesand in a mouse skin carcinoma-derived cell line (CA3/7). Primarykeratinocytes were sometimes more (BP and clofibrate) and sometimesless sensitive (ethionineand limonene) for inhibitory effectson GJIC compared to the effects in the cell line 3PC. Exceptfor TPA and anisidin, GJIC between the CA3/7 cells was lessaffected by the selected agents compared to the 3PC cell line.These results show that, during the process of tumor formationthe susceptibility of cells to inhibition of GJIC by tumor promotersis variable. Overall the CA3/7 cells are less sensitive comparedto 3PC cells. The susceptibility of primary keratinocytes isvariable compared to 3PC cells, depending on the agent used.These results also show that GJIC is a valid parameter for testingthe tumor-promoting activity of compounds. Finally, this studydemonstrates that mouse keratinocyte cell lines could serveas an in vitro model for the detection of non-mutagenic carcinogenswith diverse target organs in vivo. For this use the cell lineconsisting of initiated cells (3PC) is more sensitive than thecarcinoma-derived cell line CA3/7.     

Anti-4-1BB antibody-based combination therapy augments antitumor immunity by enhancing CD11c+CD8+ T cells in renal cell carcinoma

To improve the potential treatment strategies of incurable renal cell carcinoma (RCC), which is highly resistant to chemotherapy and radiotherapy, the present study established a combination therapy with immunostimulatory factor (ISTF) and anti-4-1BB monoclonal antibodies (mAbs) to augment the antitumor response in a murine RCC model. ISTF isolated from Actinobacillus actinomycetemcomitans stimulates macrophages, dendritic cells and B cells to produce IL-6, TNF-α, nitric oxide and major histocompatibility complex class II expression. 4-1BB (CD137) is expressed in activated immune cells, including activated T cells, and is a promising target for cancer immunotherapy. The administration of anti-4-1BB mAbs promoted antitumor immunity via enhancing CD11c⁺CD8⁺ T cells. The CD11c⁺CD8⁺ T cells were characterized by high killing activity and IFN-γ-producing ability, representing a phenotype of active effector cytotoxic T lymphocytes. The present study showed that combination therapy with ISTF and anti-4-1BB mAbs promoted partial tumor regression with established RCC, but monotherapy with ISTF or anti-4-1BB mAbs did not. These effects were speculated to be caused by the increase in CD11c⁺CD8⁺ T cells in the spleen and tumor, and IFN-γ production. These insights into the effector mechanisms of the combination of ISTF and anti-4-1BB mAbs may be useful for targeting incurable RCC.     

Inhibition of intercellular communication in rat hepatocytes by phenobarbital, 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane (DDT) and {gamma}-hexachlorocyclohexane (lindane): modification by antioxidants and inhibitors of cyclo-oxygenase

Several tumour promoting chemicals have been shown to inhibitintercellular communication (IC) through gap junctions in cellcultures. In the present investigation we studied the effectof the hepatic tumour promoters pheno-barbital (PB), 1, 1, 1-trichloro-2,2-(p-chlorophenyl)ethane (DDT) and