The effect of lithocholic acid on proliferation and apoptosis during the early stages of colon carcinogenesis: differential effect on apoptosis in the presence of a colon carcinogen

Lithocholic acid (LCA) is implicated in human and experimentalanimal carcinogenesis. Its effect on apoptosis and proliferationof the colonic epithelium was studied in a 1,2-dimethylhydrazine(DMH)-induced murine carcinogenesis model. Four groups of mice,control, LCA, DMH and DMH+LCA, were studied for 4 weeks, a periodcorresponding to early stages of carcinogenesis. Apoptosis (AI)and proliferation (PI) indices in the colon were determinedby immunohistochemistry. LCA stimulated apoptosis [AI = 1.2± 0.3% (all values are the mean ± SEM) versuscontrol 0.5 ± 0.1%, P < 0.05], as did DMH (4.3 ±0.8%, P < 0.02). DMH increased apoptosis at the base of thecrypt nearly 50-fold, with no effect at the lumenal third. Inmice receiving DMH, LCA suppressed apoptosis almost completely(0.1 ± 0.03%); this suppression was complete at the lowertwo-thirds of the crypt (AI = 0) and 60% at the lumenal third.LCA increased proliferation (PI = 22.2 ± 4.6% versus15.4 ± 1% in controls), but this did not reach statisticalsignificance. DMH increased proliferation (PI = 34.6 ±2.3%, P < 0.01). In mice receiving DMH, proliferation (41± 2.9%) was about two-thirds of the additive effect.LCA affected proliferation, mainly in the middle third of thecrypt; DMH's effect was similar in distribution, but more pronounced.In mice receiving DMH, LCA shifts proliferation upward, extendingit to the lumenal third of the crypt. LCA's main cell kineticeffect in the colon is on apoptosis; this effect differs innormal (stimulation) and pre-malignant colon (nearly completesuppression). LCA does not significantly stimulate proliferationin either normal or pre-malignant colon. The differential effectof LCA on apoptosis in the presence of a carcinogen partiallyexplains its effect as a promoter on colon carcinogenesis inanimal models, and may have important implications for humancarcinogenesis.     

Increased induction of aberrant crypt foci by 1,2-dimethylhydrazine in rats fed diets containing purified genistein or genistein-rich soya protein

The isoflavonoid genistein inhibits mitosis and increases apoptosisin a variety of tumour cell lines in vitro, and may exert anticarcinogeniceffects in vivo. To assess its effects on the colon, rats werefed a semi-synthetic control diet, or similar diets enrichedwith genistein (0.25 g/kg), either as the pure isoflavone oras part of a soya protein isolate, for 7 days before receivingsubcutaneous injections of saline or 1,2-dimethylhydrazine (DMH).After 48 h, rats given saline were killed and samples of theirsmall and large intestinal mucosa were obtained for assessmentof crypt cell mitosis and apoptosis by visual analysis of isolatedintact crypts. Rats given DMH were fed control diet and killedafter 48 h for assessment of crypt cytokinetics or maintainedfor 42 days then killed and their colonic mucosa analysed foraberrant crypt foci (ACF). Two further groups were given controldiet before DMH, followed by the genistein or soya-based dietfor 42 days before assessment of ACF. Neither genistein norsoya protein isolate had a significant effect on crypt cellmitosis or apoptosis in untreated rats, or on the proliferativeresponse to treatment with DMH. However, consumption of puregenistein or the soya protein isolate before treatment withDMH was associated with a 3-fold (P < 0.001) or 2-fold (P< 0.05) increase, respectively, in ACF in the distal colon.There was no significant effect of genistein or soya proteinisolate given after DMH treatment. We conclude that genisteinhas no detectable effect on colonic crypt mitosis or apoptosisin the rat in vivo, but that it promotes induction of ACF byan as yet undefined mechanism when fed immediately before treatmentwith DMH.     

Inhibition by dietary curcumin of azoxymethane-induced ornithine decarboxylase, tyrosine protein kinase, arachidonic acid metabolism and aberrant crypt foci formation in the rat colon

The present study was designed to investigate the modulatoryrole of dietary curcumin on (i) azoxymethane (AOM)-induced ornithinedecarboxylase (ODC), tyrosine protein kinase (TPK) and arachidonicadd metabolism in liver and colonic mucosa of male F344 rats,(ii) in vitro arachidonic add metabolism in the liver and colonicmucosa and (iii) AOM-induced aberrant crypt foci (ACF) formationin the colon of F344 rats. At 5 weeks of age groups of animalswere fed one of the experimental diets containing 0 or 2000p.p.m. curcumin. Two weeks later all the animals except thevehicle-treated groups were given s.c. injections of AOM, 15mg/kg body wt, once weekly for 2 weeks. The animals intendedfor biochemical study were killed 5 days later and the colonicmucosa and liver were analyzed for ODC, TPK, lipoxygenase andcyclo-oxygenase metabolites. The animals intended for ACF studywere killed 9 weeks later and analyzed for ACF in the colon.The results indicated that in saline-treated animals dietarycurcumin significantly inhibited the ODC (P<0.001) and TPK(P<0.05) activities in the liver and colonic mucosa. Dietarycurcumin significantly decreased the levels of AOM-induced ODCactivity in the liver and colon (P< 0.0001) and TPK activityin the liver and colon (P<0.01–0.0001) and the formationof 5(S)-, 8(S)-, 12(S)-and 15(S)-hydroxyeicosatetraenoic acids(HETEs) in the liver and colon (P< 0.0001). Also, curcuminsuppressed AOM-induced prostaglandin (PG) and thromboxane (Tx)formation in the liver (PGE₂, PGF₂     

Carcinogenicity of naturally occurring 1-hydroxyanthraquinone in rats: induction of large bowel, liver and stomach neoplasms

The carcinogenic potential of 1-hydroxyanthraquinone (HA), anaturally occurring compound, was examined. A total of 60 maleACI/IN rats, 1.5 months old at the commencement were dividedinto two groups. Group 1 (30 rats) were fed the diet containingHA at a concentration of 1% throughout the experIment (480 days).Group 2(30 rats) served as the control given a basal diet alone.Twenty-five of 29 effective animals in group 1 developed adenomasor adenocarcinomas in the cecum or upper portion of the colon,the mean number of large bowel tumors/tumor bearing rat being2.3. In addition to these intestinal tumors, liver neoplasms(neoplastic nodules and hepatocellular carcinomas) were observedin 12 rats and benign stomach tumors were obtained in five animals;no rats of group 2 demonstrating development of any of thesetumor types. The incidences of the large bowel, liver and stomachneoplasms in group 1 were all significant as compared with group2 (P < 2 x 10^–13, P < 5 x 10^–5 and P <3 x 10^–2 respectively) clearly indicating that HA is carcinogenicin rats.     

Inhibition of hepatocellular carcinoma by glycyrrhizin in diethylnitrosamine-treated mice

Glycyrrhizin (GL) is widely used in Japan as a therapeutic agentfor chronic active liver diseases. However, its action on hepatocarcinogenesisremains to be elucidated. To clarify its effect, mice treatedwith diethylnitrosamine (NDEA) with or without GL were analyzed.Five-week-old male BALB/c mice were divided into two groups,GL (n = 50) and C (n = 47). Mice in the GL group intramuscularlyreceived 2 mg of GL 3 days a week, and mice in the C group receivedthe same volume of saline in the same way. After 2 weeks, themice were treated with an i.p. injection of 75 mg/kg body wtof NDEA weekly for 3 weeks and 100 mg/kg body wt of NDEA weeklyfor the following 3 weeks. Thirty additional mice that did notreceive NDEA treatment were divided into two groups, GC (n =15) and SC (n = 15). They received GL or saline, respectively.Mice in the 4 groups were killed every 5 weeks after the lastinjection of NDEA from 7 weeks to 32 weeks. Liver function testssuch as AST and albumin were significantly improved in the GLgroup compared with the C group (P < 0.05, each). Althoughliver nodules appeared in the C group at 22 weeks, they werenot observed until 32 weeks in the GL group. At 32 weeks, themean number of liver tumors, composed of adenoma and hepatocellularcarcinoma (HCC), in the GL group was 0.71, which was significantlydecreased compared with 1.64 of the C group (P < 0.05). Themean number of HCC in the GL group was 0.29/liver, which waslower than 0.82/liver in the C group (P < 0.05). The incidencerate of HCC at 32 weeks was 64% in the C group and 21% in theGL group (P < 0.05, C versus GL group). Our results suggestthat GL treatment inhibits the occurrence of HCC.     

Selenium and difluoromethylornithine additively inhibit DMH-induced distal colon tumor formation in rats fed a fiber-free diet

We investigated the effects of difluoromethylornithine, an inhibitorof ornithine decarboxylase (ODC) and selenium supplementationon tumor formation induced by the carcinogen 1, 2-dimethylhydrazine(DMH) in Sprague-Dawley rats. A biochemical link between polyaminebiosynthesis and selenium metabolism to its cancer preventativeform has been suggested by the common requirement of S-adenosylmethio-nine.One-hundred and twenty male Sprague-Dawley rats were dividedinto experimental (n = 80) and control (n = 40) groups. Experimentalanimals received DMH 20 mg/kg s.c. for 20 weeks. Animals werefed either a regular diet (selenium content 0.2 p.p.m.) or ahigh selenium diet (5 p.p.m.) with or without 0.2% DFMO in thedrinking water. At death, week 30, animal weights within experimentalor control groups were not different between the four diet treatmentgroups. Tumor number and incidence in the proximal colon wasnot affected by DFMO treatment, selenium supplementation orthe combined treatment. In contrast, in the distal colon, 19tumors developed in the DFMO treated group, 22 tumors in thehigh selenium group and only 12 tumors in the combined highselenium/DFMO treatment group compared to 32 tumors in the regulardiet group. Similarly, tumor incidence was decreased by DFMOand selenium supplementation and their effects were additive.In control animals, ODC activity was decreased by DFMO treatmentand selenium supplementation in the distal colon and liver,but not the proximal colon. ODC activity of tumor tissue wasgreater than normal colon tissue from diet paired animals forproximal and distal colon, except for distal colonic tumorsin the high selenium/DFMO treatment group. Polyamine content,however, did not correlate with ODC activity in normal or neoplastictissue. In general, S-adenosylmethionine levels from normalcolon and liver tissue were unaffected by diet treatment. Seleniumsupplementation in combination with DFMO treatment selectivelyinhibited distal colon tumor formation in rats fed a fiber-freediet.     

Evaluation of chemopreventive effect of dietary selenium-rich egg on mouse skin tumor induced by 2'-(4-nitrophenoxy)oxirane and 12-O-tetradecanoylphorbol-13-acetate

Chemopreventive effect of dietary selenium (as sodium seleniteor as Se-rich egg) on mouse skin tumor induced by topical applicationof 2'-(4-nitrophenoxy)oxirane (NPO) as tumor initiator and 12-O-tetradecanoylphorbol-13-acet-ate(TPA) as tumor promoter was evaluated in relation to the dietarysource and levels of selenium. Selenium supplementation (0.3p.p.m.) to the basal diet (0.07 p.p.m. Se) as sodium seleniteor as Se-rich egg brought about a 40 or 37% reduction respectively,in the incidence of papilloma formation at 12 weeks after NPOtreatment. Tumor yield (number of papillomas per mouse) at 14weeks after NPO treatment in the basal diet group, basal dietsupplemented with 0.3 p.p.m. Se as sodium selenite group andbasal diet supplemented with 0.3 p.p.m. Se as Se-rich egg groupwere 7.5 ± 2.1, 2.7 ± 2.3 and 4.1 ± 3.5respectively. Dietary supplementation of 1.0 p.p.m. Se as Se-richegg to the basal diet reduced the incidence and the multiplicityof papillomas during the early phase of promotion (11 weeks)but its antitumor activity decreased thereafter, indicatingthat the accumulation of tissue selenium above the saturatedlevel may not be beneficial. Selenium concentrations in blood,liver and skin tissue of mice in basal diet group (0.33 ±0.02, 0.54 ± 0.10 and 0.21 ± 0.03 p.p.m. respectively)increased significantly (P<0.05) by the supplementation of0.3 p.p.m. Se as selenite (0.58 ± 0.02, 1.17 ±0.10 and 0.31 ± 0.05 p.p.m. respectively), 0.3 p.p.m.Se as Se-rich egg (0.59 ± 0.02, 1.25 ± 0.11 and0.33 ± 0.06 p.p.m. respectively) and 1.0 p.p.m. Se asSe-rich egg (1.20 ± 0.05, 2.32 ± 0.28 and 0.51± 0.01 p.p.m. respectively). Glutathione peroxidase activityin blood of mice of the basal diet group (2.4 ± 0.4 EU/mg) increased significantly (P<0.05) by dietary seleniumsupplementation (3.8 ± 0.6 EU/mg in 0.3 p.p.m. Selenite;3.7 ± 0.8 EU/mg in 0.3 p.p.m. Se as Se-rich egg; 4.9±0.9 EU/mg in 1.0 p.p.m. Se as Se-rich egg) and the enzymeactivities in liver and skin tissue also increased by 0.3 p.p.m.Se (as selenite or Se-rich egg) supplementation, but no furtherincreases in their activities were obtained by 1.0 p.p.m. Se(as Se-rich egg). It is, therefore, concluded that a moderatelevel of dietary selenium (0.3 p.p.m.) has an efficient chemopreventiveactivity at the promotional stage of carcinogenesis and thatdietary selenium rich egg as well as dietary selenite exertedantitumor activity.     

Interaction of dietary fat and route of carcinogen administration on 1,2-dimethylhydrazine-induced colon tumorigenesis in rats

Since the results of an earlier study indicating no effect ofdietary fat on dimethylhydrazine (DMH)-induced colon cancerin rats differed from those of other investigators, the presentstudy was initiated to determine if the modulating effect offat intake on colon tumorigenesis was dependent on the routeof DMH administration. Male weanling Sprague-Dawley rats (160)were fed one of two nutritionally balanced diets containing5% or 24% corn oil (CO). Following 3 weeks adaptation to theirrespective diets, 40 rats from each diet group were treatedwith five doses of DMH (30 mg/kg) by intragastric i.g.) gavageor subcutaneous (s.c.) injection, over a 3 week period. Ratswere sacrificed when they showed clinical signs of colon tumorand surviving animals were killed 51 weeks after the initialDMH treatment. The cumulative probability of death with coloncarcinoma did not differ between the dietary or treatment groups.There was no effect of route of administration or dietary faton total intestinal tumor incidence. The number of rats withcolon carcinoma was: 5%CO.IG=25; 24%CO.IG=27; 5%CO.SC=23; 24%CO.SC=19.Polypoid tumor incidence was significantly higher in the 24%CO.SCgroup (12/40) compared to the 5%CO.SC group (3/40) (Chi-squared= 5.25; p <0.03) while sessile tumor incidence was the inverse.Marginally significant differences in tumor morphology werenoted between the IG groups.     

Carcinogenicity of chrysazin in large intestine and liver of mice

The carcinogenicity of chrysazin (1,8-dihydroxy-9,10-anthracenedione) was examined by dietary administration to C3H/HeN mice. All of the effective mice (17) which were given 0.2% chrysazin diet and which survived more than 500 days developed adenomatous hyperplasia with cystic glands of the cecum. Similar lesions were also seen in the colon of mice in this group. These intestinal lesions were not obtained in any effective mouse (19) of the control group. The incidence of hepatocellular carcinoma of mice given chrysazin (4/17) was significantly higher than that of the controls (0/19). These results indicate that chrysazin is carcinogenic in mice as well as in rats. Some mechanistic aspects of the causation of these intestinal lesions and liver neoplasms are also discussed.     

Chemoprevention of azoxymethane-induced intestinal carcinogenesis by a novel synthesized retinoidal butenolide, 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone, in rats

The present study was designed to investigate the modifyingeffects of dietary 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone(KYN-54), a new synthetic retinoidal butenolide, during thepost-initiation phase on azoxymethane (AOM)-induced rat intestinalcarcinogenesis. The number of aberrant crypt foci (ACF) in ratcolon, colonic ornithine decarboxylase (ODC) activity and bromodeoxy-uridine(BrdUrd) labeling index in rat colonic epithelium were alsoassessed. At 7 weeks of age, male F344 rats (except the KYN-54alone and control groups) were given weekly s.c. injectionsof AOM at 15 mg/kg body wt for 3 weeks. Starting 1 week afterthe last injection of AOM, rats (except the control group) werefed a diet containing KYN-54 at concentrations of 100 or 200p.p.m. throughout the experiment All animals were necropsiedat 32 weeks after the start of the experiment. Compared withthe AOM alone group, KYN-54 at both doses reduced the incidenceand multiplicity of tumors in entire intestine (small and largeintestines). In the 200 p.p.m. KYN-54 fed group especially,tumor incidence and multiplicity in the entire intestine werelower compared with the AOM alone group (P < 0.005 and P< 0.05 respectively). Also, the number of ACF/cm² colon inthe groups of rats treated with AOM and KYN-54 at both doseswere significantly lower than that of rats treated with AOMalone (^P < 0.05). Colonic ODC activity and BrdUrd labelingindex in the groups of rats treated with AOM and KYN-54 at bothdoses were slightly lower than those treated with AOM alone.KYN-54 at 200 p.p.m. significantly lowered BrdUrd labeling indexinduced by AOM (P < 0.005). These results suggest that KYN-54might be a promising chemopreventive agent for intestinal neoplasia.     

Concurrent chemo- and radiotherapy in patients with locally advanced carcinoma of the cervix

BACKGROUND:: The feasibility of concurrent chemotherapy and radiotherapyfor advanced primary carcinoma of the cervix was evaluated andthe results were compared to historical controls. PATIENTS AND METHODS:: In a single institution study, patients (n=74) with primarycervical carcinoma received 3 cycles carboplatin/5-fluorouracilconcurrent with radiotherapy, followed by salvage hysterectomy(group I). Treatment results were compared with those of a historicalcontrol group (n=39) (group II), treated similarly but withoutchemotherapy. RESULTS:: In group I median follow-up is 28 months (12–68+) andin group II 23 months (14–90+ months). The 5-year overallsurvival, progression-free survival and local recurrence freesurvival for group I and II are, respectively, 69% versus 38%(P<0.003), 67% versus 38% (P<0.005) and 84% versus 43%(P<0.0001). Two patients in each group developed posttreatmententeritis. CONCLUSIONS:: Radiotherapy with concurrent carboplatin and 5-fluorouracilresulted in a better overall survival, disease free survivaland local disease free survival compared to historical controls.The toxicity of this schedule did not exceed that of radiationalone in historical controls. carboplatin, cervical cancer, chemotherapy, 5-fluorouracil, radiotherapy     

Positive association between dietary fat intake and risk of gastric stump carcinoma in rats

Effect of high- and low-fat diets on gastric stump carcino-genesiswas experimentally investigated. A total of 130 Wistar malerats weighing 250–300 g received either sham operationor Billroth II partial gastrectomy, the resection of the distaltwo-thirds glandular stomach and reconstruction of gastro-jejunostomy.After surgery, each group of rats was switched from a standarddiet (CRF-1) to a special diet containing either 15% soybeanoil (high-fat) or 0.5% soybean (low-fat), fed ad libitum andtap water, and were killed 50 weeks after surgery. Gastric tumourswere observed only in the animals that underwent gastrectomy,while no tumours were detected in the animals following thesham operation. Tumours located invariably at the gastrojejunostoma,were carcinomas or adenomas in histology. Carcinomas developedin 12 of 29 gastrectomy animals (41%) fed the high-fat dietand 4 of 27 gastrectomy animals (15%) fed the low-fat diet.The difference was significant (P < 0.05). The incidenceof adenoma was also significantly higher in the gastrectomyanimals fed the high-fat diet (38%) than that in those fed thelow-fat diet (15%) (P < 0.05). A daily faecal output of bileacids was significantly greater in the gastrectomy animals fedthe high-fat diet (19.0 ± 16.4 µmol/day) than thatin those fed the low-fat diet (11.2 ± 6.2 µmol/day;P < 0.05). This study suggests that increased fat intakeis associated with a high risk of gastric stump carcinoma.     

Mutational and LOH analyses of p53 alleles in colon tumors induced by 1,2-dimethylhydrazine in F1 hybrid mice

To elucidate the role of p53 in colon tumorigenesis in mice,we examined allele loss and mutational alteration of the p53gene in colon tumors induced by 1,2-dimethylhydrazine (DMH)in F₁ hybrid mice. Intragenic polymorphism of the p53 gene amongparental strains enabled us to assess allele loss of the p53gene and also to determine parental origin of mutated and/orlost alleles. Allele loss was detected in two of 163 tumorsheterozygous for the p53 gene. Polymerase chain reaction-single-strandconformation polymorphism analysis of p53 exons 5–8 revealed33 mutations in 20 of 182 colon tumors, the incidence beinglower than that in human colon cancers. The majority of thesemutations were of transition type: G: A transitions at non-CpGsites were most prevalent, while those at CpG sites were lesscommon. Distribution of the mutations along p53 amino acid sequencerevealed a difference in the location of ‘hot spots’between mice and humans. Incidence of p53 alterations did notdiffer among alleles of different parental origins, suggestingthat genetic changes in DMH-induced mouse colon tumors had occurredindependently of parental origin and DMH susceptibility. Detailedanalysis of p53 mutations on each allele revealed intratumoralheterogeneity in mouse colon tumors. The low incidence of p53mutations and rare allele loss suggest that p53 alteration playsonly a minor role in colon tumorigenesis in mice.     

Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation in CD-1 mice

In previous studies, we have shown that inositol hexaphosphate(InsP₆), a constituent of cereal diet, inhibited azoxymethane-inducedexperimental large intestinal cancer (LIC) in Fischer 344 rats.We now report a similar antineoplastic action of InsP₆ in CD-1mice injected with 1,2-dimethylhydrazine (DMH). We had hypothesizedthat InsP₆ may bring about this effect by undergoing dephosphorylationto lower phosphorylated forms; the ready availability of Ins,to react with phosphates, may increase the total amount of thelower phosphorylated Ins and potentiate the action of InsP₆.LIC induced by DMH (15 mg/kg/week ? 13) in mice given a mixtureof 1% InsP₆ + 1% Ins show a significant reduction (P <0.005)in LIC prevalence over InsP₆ treatment. Surprisingly, Ins, anin vitro growth promoting agent also caused a significant (P< 0.001) suppression of LIC. InsP₆ ? Ins also showed a concomitantreduction in the mitotic rate in the non-neoplastic epithelium.Body weight data did not suggest any overt toxic effect of long-termadministration of InsP₆, Ins or InsP₆ ? Ins. Since InsP₆ isantineoplastic in two species of experimental animals, it should,in combination with Ins, be considered in our strategies forprevention of large intestinal cancer.     

Transgenic expression of human MGMT protects against azoxymethane-induced aberrant crypt foci and G to A mutations in the K-ras oncogene of mouse colon

Transgenic mice over-expressing MGMT, which codes for the humanprotein O⁶-alkylguanine-DNA alkyltransferase, are protectedfrom methylating agent-induced thymic lymphomas. In this studywe evaluated the ability of transgenic overexpression of MGMTin the colon to protect mice from the development of azoxymethane(AOM)-inducedaberrant crypt foci (ACF) and mutations in K-ras. Colonic alkyltransferasein MGMT+ transgenic mice was > 5-fold higher than in nontransgenics:10.5 ± 1.1 vs 2.2 ± 1.1 fmol/µg DNA, P => 0.0001. Mice received 20 mg AOM/kg i.p. at 6 weeks or 15mg AOM/kg at 6 and 7 weeks of age, and 8 wks later colons wereexamined for ACF. A significant protective effect of MGMT wasseen in mice given single dose of 20 mg AOM/kg. The incidenceof ACF/colon was lower in MGMT+ mice (2.0 ± 1.2) thanin nontransgenic mice (3.9 ± 1.8, P = 0.02). G to A mutationsin codon 12 of K-ras were detected by PCR-RFLP in ACF and inrandom samples of normal appearing mucosa. The incidence ofACF with mutant K-ras in MGMT transgenic mice (0.6 ±0.7/colon) was significantly reduced compared to nontransgenicmice (2.3 ± 1.7/colon, P = 0.02). We propose that AOMinduces at least two overlapping but not identical premalignantlesions (aberrant crypt foci and K-ras mutations) which canbe prevented by over-expression of MGMT. Thus, MGMT may protectcolonic mucosa from carcinogenesis involving methylating agentssuch as AOM.     

Inhibitory effect of chlorophyllin on PhIP-induced mammary carcinogenesis in female F344 rats

Chlorophyll and chlorophyllin, a water-soluble salt of chlorophyll,have been reported to inhibit carcinogen—DNA binding andexert antimutagenic activity for some carcinogenic heterocyclicamines and aflatoxins. In the present experiment, the possibleinhibitory effects of chlorophyllin on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenicity were investigated. FemaleF344 rats were administered both PhIP, 0.02% in the diet, andchlorophyllin, 1% in the diet (group 1), or either PhIP (group2) or chlorophyllin (group 3) alone for 54 weeks. The incidenceof mammary adenocarcinomas induced by PhIP was reduced by chlorophyllinco-administration from 40 % (8/20 rats) to 15% (3/20). Whilethe difference was not statistically significant, the multiplicityof adenocarcinomas was significantly (P < 0.05) reduced bychlorophyllin co-administration from 0.50 per animal to 0.15.On the other hand, incidence of colon adenomas was slightly,but not significantly, increased from 10% to 20%. Neither mammarynor colon adenocarcinomas were observed in group 3. Thus, chlorophyllinreduced PhIP mammary carcinogenesis, suggesting that chlorophyllinis an effective chemopreventor when ingested simultaneouslywith the carcinogen.     

SHORT COMMUNICATION: Inhibitory effects of d-limonene on the development of colonic aberrant crypt foci induced by azoxymethane in F344 rats

The modifying effect of the monoterpenoid d-limonene in drinkingwater on the development of azoxymethane (AOM)-induced colonicaberrant crypt foci (ACF) was investigated in male F344 rats.The effects of d-limonene intake on ornithine decarboxylase(ODC) activity and on the silver stained nucleolar organizerregion protein (AgNOR) count in the colonic mucosa were alsoestimated. Animals were given 3 weekly s.c. injections of AOM(15mg/kg body wt) to induce ACF. These rats were treated withor without 0.5% d-limonene in the drinking water, starting 1week before the first dosing with AOM. All rats were killed2 weeks after the last AOM injection, to measure the numberof ACF, ODC activity and AgNOR count/ nucleus in the colon.In rats given AOM and d-limonene the frequencies of ACF andaberrant crypts/colon, and aberrant crypts/focus were significantlydecreased compared with those of rats given AOM alone (P <0.001, P < 0.001 and P < 0.001 respectively). Number ofAgNOR counts/nucleus of rats treated with AOM and d-limonenewas significantly smaller than that of rats treated with AOMalone (P < 0.001). These results suggest that the monoterpenoidd-limonene might be a chemopreventive agent for colonic carcinogenesisin rats.     

Protection against 12-O-tetradecanoylphorbol-13-acetate-caused inflammation in SENCAR mouse ear skin by polyphenolic fraction isolated from green tea

Earlier studies conducted in our laboratory have shown thata polyphenolic fraction isolated from green tea (GTP) possessesanti-skin tumor initiating and anti-skin tumor promoting activityin the two-stage skin tumorigenesis protocol in SENCAR mouse.We have also shown that topical application of GTP inhibitstumor promoter-caused induction of epidermal ornithine decarboxylaseactivity in SENCAR mice in a dose-dependent manner, and thatits oral feeding in drinking water to SKH-1 hairless mice enhancesantioxidant and phase II enzyme activity in liver, lung, smallbowel and skin. In this study, we show that single or multipleapplications of GTP on SENCAR mouse ear prior to or after theapplication of 12-O-tetradecanoylphorbol-13-acetate (TPA) affordsignificant protection (P < 0.05) against TPA-induced edema.Pre-application of GTP also afforded significant protectionagainst TPA-induced hyperplasia in the ear skin. The percentageprotection by GTP both in terms of epidermal thickness and verticalcell layers was 75 and 90% respectively (P < 0.005). In furtherstudies, we assessed the protective effect of GTP against TPA-causedinfiltration of neutrophils in the ear skin of SENCAR mouse,by determining a naturally occurring constituent of neutrophils,myeloperoxidase, as a quantitative marker of tissue neutrophilcontent. Prior application of GTP resulted in significant protectionagainst TPA-caused infiltration of neutrophils (P < 0.005).These results suggest that GTP possesses potential as a cancerchemopreventive agent against stage I tumor promotion.     

Biphasic Modifying Effect of Indole-3-carbinol on Diethylnitrosamine-induced Preneoplastic Glutathione S-Transferase Placental Form-positive Liver Cell Foci in Sprague-Dawley Rats

The biphasic modifying effects of indole-3-carbinol (I3C), a naturally occurring constituent of edible cruciferous vegetables, on the development of glutathione S-transferase placental form (GST-P)-positive liver cell foci were investigated by using a medium-term liver bioassay system and a newborn rat hepatocarcinogenesis system. In Experiment 1, a total of 65 male Sprague-Dawley (SD) rats were divided into 5 groups. Animals were given a single intraperitoneal (i.p.) injection of 200 mg/kg diethylnitrosamine (DEN) dissolved in saline for groups 1, 2, and 3 or a single i.p. injection of saline for groups 4 and 5. Group 1 was given the diet containing 0.25% I3C for 2 weeks prior to DEN initiation and then basal diet for 8 weeks. Group 2 was given basal diet for 4 weeks prior to and after DEN initiation and then the diet containing 0.25% I3C for 6 weeks. The rats of group 3 were placed on basal diet during the experiment. Animals of groups 4 and 5 were treated in the same manner as those of groups 1 and 2 except for injection with saline instead of DEN solution. All rats were subjected to two-thirds partial hepatectomy at week 3 and were killed at week 8 after DEN or saline injection. In Experiment 2, a total of 45 female SD rats were dosed with DEN (100 mg/kg, i.p.) or saline at 24 h after birth. After weaning at week 3, the rats were fed diet containing 0.25% I3C for 9 weeks and then were killed at week 12. In Experiment 1, preinitiation exposure to 0.25% I3C caused a significant decrease in numbers of GST-P-positive liver cell foci (P<0.05), while postinitiation exposure to 0.25% I3C caused significant increases in both number (No./cm²) and area (mm²/cm²) of GST-P-positive liver cell foci (P<0.05 or 0.01). In Experiment 2, the relative liver weight in the DEN + I3C group was significantly increased (P<0.001). The numbers and areas of GST-P-positive liver cell foci in the DEN + I3C group were significantly increased as compared to the values of the DEN-alone group (P<0.001). These results clearly demonstrated that I3C exerts a promoting effect on the postinitiation stage as well as an inhibitory effect on the preinitiation stage in the medium-term liver bioassay.