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     

Lack of rapid initiating, promoting or sequential syncarcinogenic effects of di(2-ethylhexyl)phthalate in rat liver carcinogenesis

The effect of prolonged dietary administration of the peroxisomeproliferating plasticizer di(2-ethylhexy1)phthalate (DEHP wasstudied on liver carcinogenesis initiated by N-2- fltuorenylmxhmide(FAA) and with that of the neoplasm-promoter phenobarbital (PB).Also, DEHP was studied as an initiator by giving it in placeof FAA before PB. Male rats were fed FAA for 7 weeks to inducebepatocellular altered foci, and were subsequently given nochemical, 12 000 p.p.m. DEHP or 500 p.p.m. PB for 24 weeks inthe diet. In the rats fed DEHP, substantial hepatomegaly andperoxisome proliferation were induced. No evidence of indudionof hepatacellular altered foci or hepatic neoplasms was foundeither when DEHP was given alone for 24 weeks or for 7 weeksfollowed by PB. Also, DEHP fed for 24 weeks had no promotingeffect on liver altered foci that were induced by FAA and producedlittle or no enhancement of the occurrence of FAA-induced liverneoplasms. In contrast, PB exerted a marked enhancing effecton foci and substantially increased the incidence and multiplicityof liver neoplasms. Thus, the findings demonstrate that DEHPdid not have either a rapid initiating activity, a significantsequential syncarcinogenic activity, or a promoting effect onliver carcinogenesis under conditions in which numerous agentswith such activities have been identified.     

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     

Methyl tertiary butyl ether lacks tumor-promoting activity in N-nitrosodiethylamine-initiated B6C3F1 female mouse liver

Methyl tertiary butyl ether (MTBE) is an additive in some formulationsof unleaded gasoline (UG) that enhances octane and reduces carbonmonoxide emissions from motor vehicles. MTBE in CD-I mice andUG in B6C3F1 mice increased the incidence of liver tumors selectivelyin female mice in their chronic bioassays. Both agents werenegative in in vitro tests of genotoxicity, and exhibit similarhepatic microsomal cytochrome P450 activity and hepatocyte proliferationafter short-term exposure. We previously demonstrated that UGhas hepatic tumor-promoting activity in DEN-initiated femaleB6C3F1 mice. Thus, we hypothesized that MTBE would have hepatictumor-promoting activity in the same initiation-promotion modelsystem in which UG was a hepatic tumor promoter. Twelve-day-oldfemale B6C3F1 mice were initiated with a single i.p. injectionof the mutagen N-nitrosodiethylamine (DEN) (5 mg DEN/kg, 7.1ml/kg body weight) or saline. Beginning at 8 weeks of age, micewere exposed to 0 ppm or the hepatocarcinogenic dose of approximately8000 ppm MTBE. After subchronic exposure, MTBE significantlyincreased liver weight and hepatic microsomal cytochrome P450activity without hepa-totoxicity or an increase in non-focalhepatocyte DNA synthesis. These are subchronic effects similarto those produced by UG. However, MTBE did not significantlyincrease the mean size of hepatic foci and volume fraction ofthe liver occupied by foci as compared to DEN-initiated controlsat either 16 or 32 weeks. The lack of tumor-promoting abilityof MTBE in DEN-initiated female mouse liver was unexpected andsuggests that MTBE does not produce liver tumors through a tumor-promotingmechanism similar to that of UG.     

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.     

Loss of tumor-promoting activity of unleaded gasoline in N- nitrosodiethylamine-initiated ovariectomized B6C3F1 mouse liver

Unleaded gasoline (UG) vapor (2056 ppm) increased the incidence of liver tumors in a chronic bioassay and exhibited tumor-promoting activity in N-nitrosodiethylamine (DEN)-initiated female mouse liver. Estrogen inhibited mouse liver tumor development and the hepatocarcinogenic and tumor-promoting dose of UG produced uterine changes suggestive of estrogen antagonism. To directly test the hypothesis that UG-induced tumor-promoting ability is secondary to its interaction with the mouse liver tumor inhibitor, estrogen, we compared the tumor-promoting ability of UG in ovariectomized (Ovex) mice with the hepatic tumor-promoting ability of UG in intact mice. Ovaries were surgically removed at 4 weeks of age. Exposure to wholly vaporized UG (2018 ppm) under bioassay and tumor-promoting conditions began at 8 weeks of age. After 4 months of exposure, UG increased relative liver weight and hepatic microsomal cytochrome P450 pentoxyresourfin-O- dealkylase and ethoxyresorufin-O-deethylase activity to a similar extent in intact and Ovex mice. Non-focal hepatocyte proliferation, as measured by the incorporation of bromo-deoxyuridine, was not changed by UG exposure and was similar in all treatment groups. After 4 months of exposure to DEN-initiated mice, UG significantly increased the volume fraction of liver occupied by foci (three-fold) as compared to control intact mice. As expected, volume of foci was elevated in DEN/Ovex/control mice as compared to DEN/intact/control mice. In DEN/Ovex mice UG did not significantly increase the focal volume fraction. Thus, the tumor promoting activity of UG, as demonstrated by increased volume fraction of liver occupied by hepatic foci in intact mice, is greatly attenuated in Ovex mice. The volume fraction data in Ovex mice support the hypothesis that the tumor promoting activity of UG is dependent upon the interaction of UG with ovarian hormones. These data also indicate that hepatic microsomal cytochrome P450 PROD and EROD induction, hepatomegaly and non-focal hepatic LI are not specific markers of hepatic tumor promoting activity of UG.      

Butylated hydroxytoluene lacks the activity of phenobarbital in enhancing diethylnitrosamine-induced mouse liver carcinogenesis

The effect of the food additive butylated hydroxytoluene (BHT) as an enhancer of liver carcinogenesis in mice was investigated. Liver carcinogenesis was initiated by intraperitoneal injection of diethylnitrosamine (DEN) in male B6C3F1 mice at 100 or 200 mumol/kg body weight once a week for 10 weeks (total exposure 1000 or 2000 mumol/kg body weight). After an exposure-free recovery interval of 4 weeks, groups of mice were fed either basal diet or diets containing either 5000 ppm BHT or 500 ppm phenobarbital (PB), as a positive control, for 24 weeks. Exposure to the initiating doses of DEN alone induced no liver foci at 10 weeks or at 14 weeks after the recovery period, but at termination at 38 weeks, foci and adenomas were present in a dose-related incidence. In the groups given BHT after DEN/recovery, the incidence and the multiplicity of liver foci and adenomas were not different from those in mice given only DEN/recovery, whereas, in the groups given PB after DEN, liver lesions were increased by 1.7-3.0-fold. In conclusion, BHT had no promoting or syncarcinogenic effect on DEN-induced mouse liver carcinogenesis, whereas under the same conditions, PB acted as an enhancer.     

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.     

Interstrain differences in susceptibility to liver carcinogenesis initiated by N-nitrosodiethylamine and its promotion by phenobarbital in C57BL/6NCr, C3H/HeNCrMTV- and DBA/2NCr mice

Selected inbred strains of mice were compared with respect totheir susceptibility to two-stage liver carcinogenesis. Five-week-oldmale mice of strains C57BL/6NCr (C57), C3H/HeNCr^MTV- (C3H) andDBA/2NCr (DBA) were given a single i.p. injection of N-nitrosodiethylamine(DEN, 90 mg/kg body weight) or the solvent tricaprylin (10 ml/kg).Beginning 2 weeks later, half of the DEN-treated and half ofthe control mice were given drinking water containing 0.05%phenobarbital (PB). Ten mice from each treatment group werekilled at 12, 24, 36 and 52 weeks of age (5, 17, 29 and 45 weeksexposure to PB). PB significantly increased both the numberof hepatocellular foci/cm² and the incidence of hepatocellulartumors after 17 weeks of treatment in 24-week-old DEN-initiatedmice of strains C3H (0.11 ± 0.07 versus 2.9 ±0.3 foci/cm² and 20 versus 70% incidence of hepatocellular tumors)and DBA (0.09 ± 0.09 versus 3.72 ± 0.6 foci/cm²and 0 versus 90% incidence of hepatocellular tumors) but wasineffective in C57 mice (0.04 ± 0.04 versus 0.07 ±0.07 foci/cm²). At 36 weeks of age the incidence of liver celltumors in mice given DEN but not PB was 10 (DBA), 10 (C57) and50% (C3H); the incidence was increased by PB to 90% in DBA and100% in C3H mice, but there was no increase in C57 mice. Evenat 52 weeks, the low incidence of hepatocellular tumors in C57mice given DEN only (20%) was not significantly increased bysubsequent exposure to PB. Serum PB levels observed at 12, 24and 36 weeks of age were significantly higher in DBA mice thanin C57 or C3H mice. Similar results were observed in a separatestudy in which PB was administered in drinking water to 7-week-oldmale mice of these three strains for 20 days, during which periodserum PB levels were measured at shorter intervals. DBA micethus appear to be unable to metabolize PB, which itself ratherthan its metabolites is probably responsible for tumor-promotingeffects. DBA mice were especially sensitive, while C57 micewere refractory to promotion of hepatocar-cinogenesis by PB.These two strains, which differ with respect to other significantparameters for chemical carcinogenesis including inducibilityfor aryl hydrocarbon hydroxylase and susceptibility to promotionof hydrocarbon-initiated skin tumors by 12-O-tetradecanoyl-phorbol-13-acetate,thus also provide a means for analysis of the pharmacogeneticsof susceptibility to hepatocellular tumor promotion.     

Rapid growth of preneoplastic lesions in hepatocarcinogen-Sensitive C3H/HeJ male mice relative to C57BL/6J male mice

We have previously shown that C3H/HeJ male mice are {small tilde}20-fold more susceptible to the induction of liver tumors byN-ethyl-N-nitrosourea (ENU) than are C57BL/6J male mice andthat this difference in sensitivity is largely determined bya single genetic locus (Hcs, hepatocarcinogen sensitivity).In order to determine whether the Hcs locus affects initiationor promotion of hepatocarcinogenesis, we studied the developmentof putatively preneoplastic hepatic lesions that are deficientin glucose-6-phosphatase (G6Pase) in mice treated at 12 daysof age with ENU. In ENU-treated male mice of both strains, thenumber and size of G6Pase-deficient hepatic foci increased overtime between 12 and 24 weeks of age. However, the rate of growthof the lesions was 1.7 times faster for C3H/HeJ male mice (volumedoubling time 2.0 ± 0.1 weeks) than for C57BL/6J mice(3.4 ± 0.4 weeks). Although the number and size of G6Pase-deficientfoci induced by ENU treatment of female C3H/HeJ and C57BL/6Jmice were smaller than for foci in similarly treated male mice,there was no significant difference between the growth ratesof the foci in female C3H/HeJ and C57BL/6J mice. Thus, the phenotypiceffect of the Hcs locus appears to be dependent on promotionof liver tumor induction by the male hormonal environment. Inagreement with studies on the growth rate of the foci in malemice, the [₃H]thymidine labeling index of G6Pase-deficient hepatocytesin C3H/HeJ males (12%) was 1.5-fold higher than in C57BL/6Jmale mice (8.0%) at 20 weeks and 1.2-fold higher at 28 weeks(11% versus 9.5%). The labeling index of histochemically normalhepato cytes in C3H/HeJ male mice (0.38%) was 2.6-fold higherthan in C57BL/6J mice (0.15%) The Hcs locus may affect the promotionphase of hepatocarcinogenesis in male mice by increasing theproliferative rate of both normal and preneoplastic hepatocytes.     

Facilitated expression of adaptive responses to phenobarbital in putative pre-stages of liver cancer

Female rats received N-nitrosomorpholine to produce alteredcell foci (potential cancer pre-stages) in the liver, followedby phenobarbital (PB) for 2 weeks. As indicators of adaptiveresponses we measured DNA synthesis cumulatively by infusionof [₃H]thymidine for the entire period of PB treatment, andcytochrome P450-PB by immunocytochemistry on histo-logical liversections. Altered cell foci were identified by expression of-glutamyltransferase (-GT), and/or altered morphology. The followingresults were obtained, (i) In normal parts of the liver PB inducedDNA replication only in association with expression of P450-PB;both responses were restricted to the pericentral area of theliver lobule, (ii) In foci, PB treatment led to enhanced DNAsynthesis. Furthermore, PB caused a 3-fold increase in the numberof foci expressing cytochrome P450-PB, -GT or both. Cumulativedetermination of DNA synthesis showed that this increase didnot result from selective proliferation of single initiatedcells. It is concluded that in foci also PB can induce expressionof the adaptive increases in cytochrome P450-PB and DNA synthesis,(iii) Foci show the responses to PB more readily than normalhepatocytes, and irrespective of their position within the liverlobule. These findings suggest that expression of adaptive responsesto inducing tumor promoters is facilitated in altered foci.     

K-ras mutations in lung tumors from A/J and A/JxTSG-p53 F1 mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and phenethyl isothiocyanate

The purpose of this study was to evaluate the effects of theloss of a p53 allele and phenethyl isothiocyanate (PEITC) pre-treatmenton the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) and K-ras mutation frequency in a hybrid mouse model.Male TSG-p53 ‘knock-out’ mice were bred with A/Jfemale mice to produce (A/JxTSG-p53) F₁ mice either homozygous(p53+/+) or heterozygous (p53+/–) for p53 alleles. Thesemice, together with female A/J mice, were treated at 6–8weeks of age with NNK or dosed with PEITC prior to administrationof NNK. The A/J mice treated with NNK had a 100% incidence oflung tumors, with 9.7 ± 3.4 tumors/mouse. A/J mice pre-treatedwith PEITC prior to NNK administration had 3.5 ± 2.1lung tumors/animal, although the incidence remained at 100%.In (A/JxTSG-p53 F₁ mice with either the p53(+/–) or p53(+/+)genotype PEITC pre-treatment significantly decreased tumor incidence(100 to 40 and 36%, respectively) and multiplicity (2.0 ±0.5 to 0.5 ± 0.4 and 2.1 ± 0.5 to 0.5 ±0.4, respectively), indicating that PEITC is an effective chemopreventiveagent in both A/J mice and (A/JxTSG-p53) F₁ mice. Analysis oflung tumor DNA from A/J mice treated with NNK or NNK/PEITC indicatedthat 15 of 17 (88%) and 20 of 23 (87%) of the tumors, respectively,contained G     

Lowered antioxidant enzymes in spontaneously transformed embryonic mouse liver cells in culture

Normal embryonal mouse liver cells in culture were shown toundergo spontaneous transformation during prolonged subculture.The spontaneously transformed cells lost their anchorage dependence,as measured by a soft agar assay, and gave rise to tumors innude mice. Accompanying this transformation, the antioxidantenzymes, copper-and zinc-containing superoxide dismutase (CuZnSOD),manganese superoxide dismutase (MnSOD), catalase (CAT) and glutathionereductase, decreased significantly in activity; the declinein enzymatic activity of CuZnSOD, MnSOD and CAT was due to adecline in the levels of immunoreactive protein. These spontaneouslytransformed high passage in vitro liver cells appeared similarin morphology, antioxidant enzyme activity and tumorigenicityto their counterparts transformed by N-methyl-N-nitro-N-nitrosoguanidineand Simian virus 40. These data provide experimental evidencethat changes in antioxidant enzymes are associated with spontaneousin vitro cellular transformation of mouse embryonal liver cells.     

Characterization of p53 mutations in methylene chloride-induced lung tumors from B6C3F1 mice

Mutations of the p53 tumor suppressor gene are the most commondefined genetic alterations seen in a wide variety of humancancers. In contrast, little is known about the importance ofthe p53 gene in chemically induced tumors of rodents, whichare widely used as models for the evaluation of human healthrisks. In this study we examined 54 methylene chloride-inducedand seven spontaneously arising lung tumors from female B6C3F1mice for losses of heterozygosity (LOH) at markers near thep53 gene on chromosome 11. LOH was detected in seven methylenechloride-induced lung carcinomas by Southern analysis of a restrictionfragment length polymorphism and PCR analysis of five simplesequence length polymorphisms. In each case allele loss wasobserved at all six markers; thus, these chromosomal alterationswere likely to have resulted from mitotic nondisjunction. Incontrast, LOH was not detected in 20 liver tumors from methylenechloride-treated mice at the Acrb locus, which is tightly linkedto the p53 gene on chromosome 11. In addition single strandconformation polymorphism analysis was performed to screen formutations in the most conserved regions of the p53 gene (exons5 to 8). Consequently, potential mutations identified by directsequencing, were only detected in four of the seven tumor sampleswith LOH, but not in any of the remaining lung tumors. Overexpressionof the p53 protein by immuno-histochemical staining was detectedonly in the four tumors that contained p53 point mutations andin a focal area of another tumor. Finally, using a simple sequencelength polymorphism within the retinoblastoma tumor suppressorgene, LOH on mouse chromosome 14 was also detected in threelung carcinomas and one liver tumor. Inactivation of p53 andpossibly the retinoblastoma tumor suppressor gene appear tobe infrequent events in lung and liver tumors from methylenechloride treated mice.     

Comparative formation of chlorophenol metabolites from hexachlorocyclohexane in mouse and rat in vivo and in vitro

Comparative formation of chlorophenol metabolites were evaluatedin hexachlorocyclohexane (HCH) fed mouse and rat livers by h.p.l.c.No significant species differences were noted in the formationof chlorophenol metabolites studied both in vivo and in vitro.The results indicate that the marked species difference thatis observed in tumor induction between mice and rats by HCHmay not be due to the different rate of formation of chlorophenolmetabolites.     

Amplified response to phenobarbital promotion of hepatotumorigenesis in obese yellow Avy/A (C3H x VY) F-l hybrid mice

Mottled yellow A^vy/A and agouti ^A/a (C3H x VY) F-l hybrid malemice were fed untreated control diet or diet with a target doseof 500 p.p.m. sodium phenobarbital (PB) for 17–19 months.No differences in prevalence of hepatocellular adenomas or carcinomaswere found between untreated yellow and agouti mice. PB treatmentincreased prevalence of adenomas but decreased prevalence ofcarcinomas. No difference in enhancement of adenoma formationby PB was observed between yellow and agouti mice bearing singleadenomas. However, the proportion of PB-treated yellow micebearing multiple adenomas (66%) was much greater than the proportionof analogous agouti mice (18%). Fatty changes in the peri-portalarea of the liver and focal cytoplasmic vacuolization were inducedto a much greater extent in PB-treated yellow mice than amongtreated agoutis. PB increased the prevalence and severity offocal areas of chronic inflammation in the liver considerablymore in agouti than in yellow mice. The possible relation ofthis finding to the altered immune responses of obese yellowmice remains to be determined. The results of this study suggestthat the use of yellow A^vy/A and agouti A/a (C3H x VY) F-l hybridmice in carcinogenicity asays make make it possible to differentiatebetween weak and strong promoters as well as between promotersand complete carcinogens. Weak promoters should induce hepatocellularadenomas in yellow mice even if they fail to do so in agoutimice. Promoting substances which act similarly to PB may beidentified in this system by simultaneously increasing adenomaprevalence and decreasing carcinoma prevalence. Complete carcinogensshould increase carcinoma prevalence in the yellow mice evenat low dose levels.     

The influence of food intake on the development of diethylnitrosamine-induced liver tumours in mice

The aim of this study was to determine whether a dietary restriction,which significantly decreases body weight gain, also influencesthe development of diethylnitrosamine (DEN) induced liver tumoursin Swiss mice. At birth, mice were in jected i.p. with a singledose of DEN (0.4 µmol/g body wt); negative control micewere sham injected. After weaning the animals received a stockdiet either ad libitum (control group) or at 30% restriction(restricted group). A positive control group was fed ad libitumand received phenobarbital (500 p.p.m.) in their drinking water.At 12 weeks of age, glucose-6-phosphatase (G6Pase}-deficientfoci were present in the liver of 80% of the control animalsbut only in 32% of the restricted group. Quantitatively, restrictedanimals had fewer foci per unit volume liver and these weresmaller than in the control animals. By 36 weeks of age, hepatocarcinomaswere seen in 100% of the control mice while in the restrictedgroup there were no such mallgnant lesions and only 32% showedadenomas. The results clearly show that restriction of foodintake inhibits promotion and progression of induced liver tumours.Amongst other uses, this model permits the study of the effectof dietary restriction on liver tumours, at an early stage.     

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.     

Comparative tumorigenicity of N-nitroso-2-hydroxymorpholine, N-nitrosodiethanolamine and N-nitrosomorpholine in A/J mice and F344 rats

N-Nitroso-2-hydroxymorpholine (NHMOR), a genotoxic metaboliteof the environmental carcinogens N-nitrosomor-pholine (NMOR)and N-nitrosodiethanolaraine (NDELA), was assayed for tumorigenidtyin A/J mice and F344 rats. Groups of female mice were givenNHMOR, NMOR or NDELA in the drinking water over a 10-week period;total doses were 53–55 µmol/mouse. The experimentwas terminated after 30 weeks. Whereas NMOR was a potent tumorigen,inducing 20.3 lung tumors/mouse, NHMOR and NDELA were only weaklytumorigenic, giving 1.2 and 1.4 lung tumors/mouse respectively.Groups of female F344 rats were also given these three nitrosaminesin drinking water for 50 weeks, as follows: NHMOR, total dose0.6 mmol/rat; NHMOR, 1.2 mmol; NMOR, 1.1 mmol and NDELA, 5.6mmol. The experiment was terminated after 120 weeks. NMOR wasa potent carcinogen, inducing liver tumors in 100% of the rats.NDELA gave hepatocelhilar tumors in 70% of the rats. NHMOR wasinactive even at the higher dose. The results of this studydo not support the hypothesis that NHMOR is a proximate carcinogenof NDELA or NMOR.     

Evidence for in vivo non-mutagenicity of the carcinogen hydrazine sulfate in target tissues of lacZ transgenic mice

Transgenic mouse models permit the confirmation of in vitromutagenicity in vivo without the constraints in the selectionof tissues imposed by other in vivo assays. This feature isof particular importance in the determination of mutagenicityin the target tissues of carcinogens, especially those thatare in vitro mutagens. Such information is critical in the determinationof whether a chemical is carcinogenic via a genotoxic or non-genotoxicmechanism. Hydrazine sulfate is an in vitro mutagen that induceslung and liver tumours in mice. Transgenic mice from strain40.6 (Muta^TMmouse) were administered single oral doses up toa toxic concentration (400 mg/kg). No dose induced any lacZmutations in lung, liver or bone marrow. Since the highest singledose used is higher than the cumulative dose that induced tumoursin previous studies, it may be that either hydrazine sulfateis genotoxic in target tissues in vivo only when given in multipledoses or that it is a nongenotoxic carcinogen.