Susceptibility to phorbol ester skin tumor promotion in (C57BL/6xDBA/2) F1 mice is inherited as an incomplete dominant trait: evidence for multi-locus involvement

Since current evidence suggests that the tumor promotion stageis a primary determinant in susceptibility to multistage carcinogenesis,we have characterized the genetics of susceptibility to phorbolester skin tumor promotion in inbred mice. Susceptibility ofhybrids (B6D2F₁), beween DBA/2 (sensitive) and C57BL/6 (resistant)parents, initiated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and promoted with 12-O-tetradecanoylphorbol-13-acetate(TPA) was similar to DBA/2 mice at doses of 13.6 nmol per mousebut clearly less when doses of 1.7–6.8 nmol per mousewere used. In addition, no significant differences were observedbetween male andfemale B6D2F₁ mice in terms of tumor incidencealthough some differences were observed in tumor multiplicitiesbetween male and female F₁ mice at the highest TPA dose. ReciprocalF₁ mice initiated with DMBA (i.e. D2B6F₁) were also responsiveto TPA. Female D2B6F₁ mice were of lower sensitivity at lowerdoses of TPA, compared to female DBA–2, a finding similarto that observed with B6D2F₁ mice initiated with MNNG. Furtheranalyses of the susceptibility of B6D2F₂ and B6D2F₁xC57BL–6backcross mice to TPA promotion indicated that more than onedominant genetic locus must account for the differences in promotionsensitivity between DBA/2 and C57BL/6 mice. To understand furtherthe genes responsible for promotion sensitivity, histologkalevaluations were performed on DBA/2, C57BL/6 and B6D2F₁ mice.Histological examination revealed that the epidermis of DBA/2mice showed a marked hyperplasia and the presence of a muchgreater number of dark basal keratinocytes (DCs) compared withC57BL/6 mice 48 h after the last of four applications of TPA(doses 3.4 nmol). A marked dermal infiltration of polymorphonuclearleukocytes (PMNs) was observed in DBA/2 mice, whereas littleinfiltration was observed in the skin of C57BL/6 mice. The hyperplasiain the skin of B6D2F₁ mice was intermediate between DBA/2 andC57BL/6 mice at all TPA doses examined except the lowest dose(1.7 nmol), whereas the DC response, although significantlylower at doses of 6.8 nmol or below, was similar to DBA/2 miceat higher TPA doses (13.6 and 17.0 nmol). The infiltration ofPMNs in the dermis of B6D2F₁ mice was similar to or greaterthan DBA/2 mice at all doses of TPA tested. Our results suggestthat (i) susceptibility to TPA promotion is inherited as anincomplete dominant trait; (ii) neither cytoplasmic geneticdeterminants nor the X-chromosome appear to play a significantrole in susceptibility to TPA; and (iii) the degree of sustainedepidermal hyperplasia and especially the induction of DCs aftermultiple applications of TPA show an excellent correlation withinherited susceptibility to promotion. Our data are consistentwith a model where allelic differences at more than one geneticlocus contribute to the sensitivity of DBA/2 mice to phorbolester promotion.     

Possible dissociation of the phorbol ester-induced oxidant response and tumor promotion in the F1 offspring of SSIN x C57BL/6J mice

The F₁ progeny of a cross between 12-O-tetradecanoyl-phor-bol-13-acetate(TPA) tumor promotion-sensitive SSIN mice and TPA promotion-resistantC57BL/6J mice were found to be sensitive to TPA as a tumor promoter.The tumor response was substantial, with an average of 15 papillomasper mouse and a 100% incidence following initiation with 400nmol dimethylbenz[a]anthracene and promotion with 6.5 nmol (4µg) TPA. To determine whether tumor promotability of theF₁ mice correlates with other parameters believed to be associatedwith TPA responsiveness, oxidant generation, epidermal hyperplasiaand edema were compared in the parents and F₁ hybrids. The SSINproduced a strong hyperplastic response to TPA, the C57BL/6Ja negligible response and the F₁ hybrids a moderate response.In the SSIN, 6.5 nmol (4 µg) TPA caused an 18% increasein the water content of the skin (edema) while no change wasseen for either the C57BL/6J or F₁ hybrids. The oxidant responseof the F₁ hybrids to either TPA or phospholipase C was markedlyless than that observed for the SSIN and was similar to theresponse previously observed for the C57BL/6J mice. These findingssuggest that the oxidant response may not be an essential aspectof TPA tumor promotion. It may be related to the edema response,suggesting that at least this aspect of inflammation is notnecessary for promotion.     

Sodium o-phenylphenate (OPP-Na) promotes skin carcinogenesis in CD-1 female mice initiated with 7, 12-dethylbenz[a]anthracene

Sodium o-phenylphenate (OPP-Na) was applied at a dose levelof 5.0 mg/animal dermally to the fur-clipped dorsal area offemale CD-I mice twice weekly for 47 weeks after applicationsof 10 µg of 7, 12-dimethylbenz[a]anthracene (DMBA) asan initiator twice weekly for 5 weeks. A total of 25 skin tumors(21 papillomas and four carcinomas) developed in 15 out of 20mice (75%). The incidence and yield of skin tumors in this DMBA/OPP-Nagroup were significantly higher than in the DMBA/acetone-treatedgroup where only six tumors (five papillomas and one carcinoma)were observed in five out of 20 mice (25%). Only one papillomadeveloped in OPP-Na/12-o-tetradecanoylphorbol 13-acetate (TPA)treated animals (initiation testing group), and no tumors wereobserved in mice receiving OPP-Na/acetone. OPP-Na elevated 5-bromodeoxyuridine(BrdU)incorporation in basal cells of mouse epidermis dose-relatedlyand the thickness of the skin in the treated group was alsoincreased to ? 2- or 3-fold that of controls. In addition, highdose application (20 mg/mouse) caused ukeration. O-Phenylphenol(OPP) administration was associated with extensive corrosiveeffects. The data suggest that OPP-Na is an ulcerogenk agentwhich induces epidermal proliferation and which can act as apromoter, but not as an initiator or a complete carcinogen,in two-stage mouse skin carcinogenesis.     

Tumorigenesis and carcinogenesis in mouse skin treated with hyperthermia during stage I or stage II of tumor promotion

The action of hyperthermia treatments on tumor promotion separatedinto a two-stage protocol has been investigated. 7,12-Dimethylbenz[a]anthracene(DMBA) initiated dorsal skin of female SENCAR mice was promotedwith either H₂O₂ or 12-O-tetradecanoylphorbol-13-acetate (TPA)(4 applications, 2 times/week) as the first stage of promotion,followed by promotion with mezerein (28 applications, 2 times/week)as the second stage. Hyperthermia (44°C, 30 min) treatmentof the skin at the time of stage II promotion only (just beforeeach mezerein application) suppressed 100% of papillomas whenH₂O₂ was used as a first-stage promoter and 96% when TPA wasused as the first stage, as compared to unheated control animals.The same hyperthermia treatment given only at stage I of promotionhad similar results. Hyperthermia treatments just before stageI TPA promotion (4 treatments only) followed by mezerein asthe second stage reduced papilloma formation by 92%. When H₂O₂was used as the first stage promoter and again mezerein as thesecond, papilloma frequency was reduced by 74%, as comparedto unheated controls. This antipromotion activity of hyperthermiacould not be linked to an inhibition of skin protease activity.Although papilloma frequency was markedly suppressed by hyperthermiaduring stage I promotion only, carcinoma formation was not.A similar number of carcinomas appeared in the groups of micereceiving hyperthermia with either H₂O₂ or TPA as first-stagepromoters, as in comparable groups receiving no hyperthermia.In contrast, when hyperthermia treatments were given duringstage II promolion with mezerein (using either H₂O₂ or TPA asstage I promoters), carcinomas (as well as papillomas) weremarkedly reduced. The results suggest that DMBA initiation createstwo types of promotion-dependent cells, a majority with relativelylow progression probability and a minority with relatively highprogression probability. The former require both stage I andII promotion while the latter require only stage II promotionto form tumors. Hyperthermia treatments given during stage IIpromotion protected against promotion and progression of bothtypes of initiated cells, but similar treatments only duringstage I did not protect against promotion and progression ofthe latter. Although promotion was required for expression,relative progression probability appeared linked to initiationand not promotion events. These findings suggest that hyperthermiatreatment of persons exposed to tumor-promoting agents may reducethe risk of induced tumorigenesis.     

FVB/N mice: an inbred strain sensitive to the chemical induction of squamous cell carcinomas in the skin

The widespread use of FVB/N mice for the establishment of transgeniclines containing active oncogenes suggested the importance oftesting the parent FVB/N mice for sensitivity to experimentalcarcinogenesis. After initiation of mouse skin by a single treatmentwith 7, 12-dimethylbenz[a]anthracene (DMBA) and promotion by20 weekly applications of 12-O-tetradecanoylphorbol-13-acetate(TPA), the skin tumor incidence was compared in FVB/N mice,TPA-sensitive (SENCAR and CD-I) and TPA-resistant mice (BALB/cand C57BL/6). Initiation by 25 µg DMBA followed by promotionwith a low dose of TPA (2 µg/week) induced one or morepapillomas in only 25% of FVB/N mice, compared with 100% inSENCAR, 53% in CD-I, 17% in BALB/c and 0% in C57BL/6 mice. Ata more effective dose of TPA (5 /ig/week), FVB/N mice initiatedby 5, 25 or 100µg DMBA developed 3.4, 6.9 and 11.8 papillomasper mouse. In contrast, the incidence of squamous cell carcinomas(SCCs) (17–18/30 mice) did not increase with DMBA dose.TPA promotion of non-initiated mice induced only six papillomas,but three progressed to SCCs, a high rate of malignant conversion.Skin tumor induction by 20 weekly treatments with 10 µgDMBA produced few papillomas, but 50.0% of the papillomas progressedto carcinomas in FVB/N mice, compared with 9.15% in SENCAR,37.5% in CD-I, 23.1% in BALB/c and 15.0% in C57CL/6 mice. Thefirst carcinomas appeared after 14 weeks in FVB/N, 24 weeksin SENCAR, 26 weeks in CD-I and C57BL/6 and 34 weeks in BALB/cmice. Thus, FVB/N mice develop an unusually high incidence ofSCCs after treatment with repeated DMBA, DMBA initiation-TPApromotion and even TPA alone.     

Inhibition of two-stage skin carcinogenesis as well as complete skin carcinogenesis by oral administration of TMK688, a potent lipoxygenase inhibitor

1-{[5'-(3'-methoxy-4'-ethoxycarbonyloxyphenyl)-2',4'-penta-dienoyl]aminoethyl}-4-diphenylmethoxypiperidine(TMK688) is a potent and orally active 5-lipoxygenase inhibitorhaving anti-histamine activity in its moiety. Recently we havefound that TMK688 also inhibits epidermal cyclooxygenase activitywith a potency similar to its inhibiting 5-lipoxygenase. Oraladministration of 30 mg/kg TMK688, a dose which markedly inhibitstumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulatedLTB₄ formation in mouse skin, markedly inhibited both TPA-promotedand a non-TPA-type tumor promoter anthralin-promoted skin tumorformation in 7,12-dimethylbenz[a]anthracene (DMBA)-initiatedCD-1 mice. The inhibitory effect of TMK688 was not due to anydamage inflicted on the initiated cells but due to its anti-tumor-promotingactivity. TMK688 not only inhibited two-stage skin carcinogenesisbut also inhibited benzo[a]pyrene-caused complete skin carcinogenesis.Throughout the tumorigenesis experiment, the survival rate ofanimals was 100% and the TMK688-treated mice looked healthy.The body weight gain of TMK688-treated mice was not significantlydifferent from that of non-treated mice. Both TMK688 and 1-{[5'-(3'-methoxy-4'-hydroxyphenyl)-2',4'-pentadienoyl]aminoethyl}-4-diphenylmethoxypiperidine(TMK777), an active metabolite of TMK688 having more potent5-lipoxygenase inhibitory activity and less potent cyclooxygenaseinhibitory activity than TMK688, inhibited epidermal 8-lipoxygenaseactivity induced by a topical application of TPA to mouse skin.The 8-lipoxygenase inhibitory activity of TMK777 was     

Mouse skin papilloma formation by chronic dermal application of 7, 12-dimethylbenz[a] anthracene is not reduced by diet restriction

Diet restriction has repeatedly been shown to reduce the incidenceof spontaneous and chemically induced tumors in rodents. However,no conclusive data are available to show whether carcinogenesisby chronic exposure to a genotoxic agent can also be retarded.In this study, diet restriction to 70% was investigated fora protective effect on the formation of skin papilloma in maleNMRI mice treated twice weekly with 20 nmol 7,12-dimethylbenz(a)anthracene(DMBA). Rather surprisingly, no protection was seen. Both timeof onset of papilloma formation (13 weeks in both groups) andtime of 50% cumulative incidence (t₅₀; 17.5 and 18 weeks) weresimilar in the unrestricted and the restricted group. In contrast,a clearly protective elTect was found in mice initiated with100 nmol DMBA and promoted twice weekly with 2.5 nmol 12-O-tetradecanoylphorbol-13-acetate:the onset of papilloma formation increased from 7 to 11.5 weeks,the t₅₀ was shifted from 8.5 to 19 weeks. Diet restriction,therefore, was not protective under conditions of chronic exposureto a genotoxic carcinogen. It cannot be considered a universalmeasure of cancer prevention.     

Chemopreventive effect of 4'-demethyl epipodophyllotoxin on DMBA/TPA-induced mouse skin carcinogenesis

The chemopreventive effect of topical application of 4'-demethylepipodophyllotoxin (DMEP), an antimitotic agent, on a two-stageskin carcinogenesis model in Swiss Albino mice induced by 9,10-dimethylbenz[a]anthracene(DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated.Two topical applications with 0.24% DMBA over 1 week, followedlater by 5 nmol of TPA twice weekly produced 100% incidenceof tumors in these animals by 18 weeks. Treatment of animalswith DMEP (until the end of the experiment), 30 min before TPAtreatment, significantly reduced the tumor incidence, tumorvolume and the conversion efficiency of papillomas to squamouscell carcinomas. The tumor formation and growth was also delayedby DMEP pre-treatment. Application of DMEP protected againstthe losses provoked in levels of glutathione and activity ofcatalase and superoxide dismutase in skin and liver of animalsby the application of DMBA/TPA. Thus, DMEP might possibly beexerting its chemopreventive activity by acting as an antioxidant. Abbreviations: CAT, catalase; DMBA, 9,10-dimethylbenz[a]anthracene; DMEP, 4'-demethyl epipodophyllotoxin; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; SOD, superoxide dismutase; TPA, 12-O-tetradecanoylphorbol-13-acetate. ² To whom correspondence should be addressed Email: neeta{at}medinst.ernet.in     

Enhanced pancreatic and skin tumorigenesis in cabbage-fed hamsters and mice

Studies were conduded to evaluate the ability of dietary driedcabbage supplements to inhibit pancreatic carcinogenesis inhamsters and skin tumorigenesis in mice. Pancreatic cancer wasinduced by treatment with 40 mg/kg body wt N-nitrosobis-(2oxopropyl)amine(BOP). Cabbage was fed from before carcinogen treatment in lowfat diet and, beginning 1 week after BOP treatment, cabbagewas given in low fat and high fat diets in comparison with therespective non-cabbage containing diets. Dried cabbage was incorporatedat 9 and 11% levels into the low and high fat diets. Feedingcabbage in the high fat diet elevated the yield of BOP-inducedpancreatic ductular cardnoma (1.6 carcinomas/effedive animal)in comparison with that observed in hamsters fed cabbage ina low fat diet or in those given a high fat diet without cabbage, 0.6–0.8 carcinomaa/effedive animal (P 0.05). Furthermore,the incidence of BOP-induced gall bladder adenocadnomm was elevatedin cabbage-fed hamsters irrespedve of dietary fat intake. Effetsof dietary fat and cabbage on food consumption, body weight,and serum T₃ and T₄ values are described. Skin tumorigenesiswas induced in SENCAR mice by 10 nmd 7,12 dlmethylbenz[a]anthracene(DMBA) and promoted beginning 1 week later with twice weeklyapplications of 2 µg 12-O-tetradecanoyl-13-phorbol acetate(TPA). Dried cabbage was incorporated into AIN semi-purifieddiets from before DMBA treatment and throughout TPA treatment.Skin papilloma yield was elevated in DMBA-initiated TPA-promotedmice that were fed diets containing 10% cabbage. Mice fed cabbagedeveloped an average of 8.45 papillomas per mouse following22 weeks of promotion while mice given control diet developed7.25 papillomas per mouse (P < 0.001). Cabbage feeding didnot influence survival, food consumption or body weight of themice. These results suggest the need for further research onthe use of cabbage as a chemopreventive measure.     

Diacylglycerol is an effector of the clonal expansion of cells containing activated Ha-ras genes

Diacylglycerols (DAG) are lipid second messengers which aregenerated during phospholipase-catalyzed hydrolysis of phospholipids.The model DAG, sn-1, 2-didecanoylglycerol (DIC₁₀), is an effectivetopical tumor promoter in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiatedmouse skin. We now report that 11/12 of DMBA-initiated/DIC₁₀-promotedpapillomas examined contain an A — T mutation in the 61stcodon of the Ha-ras gene, suggesting that DAGs affect the clonalexpansion of activated Ha-ras-containing cells. To explore furtherthe DIC₁₀-induced clonal expansion of activated Ha-ras-containingcells, we have examined the tumor-promoting effect of DIC₁₀in the skin of transgenic TG.AC mice, which harbor a v-Ha-rastransgene. By 9 weeks of promotion, 100% of the TG.AC mice developedsquamous papillomas and by 15 weeks these mice developed>20papillomas/mouse. Because fatty acids are known to participatein signal transduction pathways, and since cellular lipasescould cleave the fatty acid side chains present in DIC₁₀, wehave examined the tumor promoting activity of n-decanoic acidto verify the specificity of promotional activity of DIC₁₀.n-Decanoic acid did not function as a tumor promoter. Thesedata implicate DAG as an effector of the clonal expansion ofmutated Ha-ras-containing cells, and support a mechanism wherebyan increase in endogenous DAG could contribute to the clonalexpansion of cells containing a Ha-ras oncogene.     

Three-stage tumorigenesis in mouse skin: DNA synthesis as a prerequisite for the conversion stage induced by TPA prior to initiation

Recent evidence shows that stage I of tumor promotion in NMRI-mouseskin induced by a single dose of 12-O-tetradecanoylphorbol-13-acetate(TPA) can be effected not only after initiation by 7,12-dimethylbenz(a)anthracene(DMBA) but also several weeks prior to initiation. In view ofthis partial inversion of the initiation-promotion sequencewe proposed to replace the term ‘stage I of promotion’by the term ‘conversion’. The convertogenic effectivityof a single dose of TPA applied after DMBA can be suppressedin a rather characteristic and non-toxic fashion by hydroxyurea(HU). In the present study we asked whether HU might also interferewith the converting effectivity of a single dose of TPA givenprior to DMBA. NMRI mice received a single dose of TPA 3 weeksprior to initiation by DMBA which was followed by twice weeklyapplication of skin irritant 12-O-retinoylphor-bol-13-acetate(RPA) in order to effect stage II of promotion. A single doseof HU given i.p. at different times after TPA was found to interferewith tumor formation exhibiting an almost complete inhibitionif administered 18 h after TPA. This inhibition did not preventa subsequent promotion by repetitive TPA treatment. The dataindicate that conversion can be inhibited by HU in the samecharacteristic fashion regardless of whether TPA was administeredafter or prior to initiation. The data also support the autonomouscharacter of the conversion process for which epidermal DNAsynthesis appears to be obligatory.     

A study on skin tumour formation in mice with 50 Hz magnetic field exposure

In order to test the possibility that magnetic fields (MF) actas a tumour promoter, a long-term skin carcinogenicity studyof 50 Hz sinusoidal MF with flux densities of 50 µT and0.5 mT was performed in female NMRI mice. 7, 12–dimethylbenz[a]anthracene(DMBA) in acetone was applied to the dorsal skin, as an initiator,and exposure to MF was performed for 19 (weekdays) or 21 h/day(weekends and holidays) for 103 weeks starting one week afterthe initiator treatment. The phorbol ester 12–O–tetradecanoylphorbol-13-acetate(TPA) was used as a positive control for skin tumour promotingactivity. MF was also evaluated for complete carcinogenic actionin groups of mice that were treated with acetone only. Six animalsfrom each group were taken for skin hyperplasia analysis andwere killed after 9, 26 and 52 weeks. The appearance of skinlesions were carefully followed and histopathological diagnosiswas made for all neoplasms present at death. The statisticalanalyses on occurrence of skin tumour bearing animals and cumulatedskin tumours, with corrections for survival, did not reveala difference between the controls and the MF exposed groups.The epithelial thickness of DMBA + MF-treated animals was ofthe same magnitude as for DMBA-treated animals. Leukaemia wasa little more frequent among animals exposed to 0.5 mT MF comparedto the control animals. However this difference was not statisticallysignificant.     

Genetics of chemical carcinogenesis--III. Tissue-specificity of the genes controlling susceptibility and resistance to skin carcinogenesis in the mouse

Carcinogenesis-resistant (Car-R) and carcinogenesis-susceptible(Car-S) mice have been obtained by the method of bi-directionalselective breeding. After 10 generations of selection Car-Rand Car-S mice show a remarkable difference in their responseto chemical carcinogenesis. Car-R and Car-S mice, initiatedand promoted by skin application of 9, 10-dimethyl-1,2-benzanthracene(DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) reacha tumour multiplicity of 0.05 and 6.2, respectively, after 49days of promotion. When benzo[a]pyrene (B[a]P) is topicallyapplied for initiation, followed by TPA promotion, Car-R andCar-S mice maintain a large difference in sensitivity to skintumour induction. Car-S mice are also more susceptible thanCar-R mice to complete carcinogenesis produced by single orrepeated applications of DMBA only. On the contrary, when DMBAor B[a]P are administered by subcutaneous injection rather thanby topical application, no significant difference in tumourincidence is observed between the two lines. All tumours inducedby topical administration of carcinogens on the skin are ofepithelial origin, whereas the tumours produced by subcutaneousinjection are of connectival origin. These observations suggesta tissue-specific effect of the selected genes, probably restrictedat the skin level.     

Effects of dose and duration of treatment with the tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate on mouse skin carcinogenesis

The effects of dose and duration of treatment with the potenttumor-promoting agent 12-O-tetra-decanoylphorbol-13-acetate(TPA) on the formation of skin tumors in Charles River CD-1mice was studied. Mice were initiated with a single applicationof 0.2 µmol of 7, 12-dimethylbenz[a]anthracene (DMBA)in 0.2 ml acetone. Beginning two weeks after initiation, micewere treated twice weekly with various doses (0.01 – 20nmol) of TPA in 0.2ml acetone. Application of either 0.01 or0.1 nmol of TPA did not elcity tumors during the 50 weeks durationof treatment. A dose-dependent increase in the number of papillomaswas observed through the range of 1 to 10 nmol of TPA. Twiceweekly applications of 20nmol of TPA did not further enhancethe papilloma incidence. A good correlation was observed betweenthe induction of ornithine decarboxylase (ODC) activity andthe formation of skin tumors by various doses of TPA. To determine the effect of promotion duration on the incidenceof papillomas and carcinomas, mice were treated with 10 nmolof TPA for various durations (6,12,18,24,30, or 36 weeks) beginning2 weeks after initiation with 0.2 µmol of DMBA. Mice promotedfor only 6 weeks developed papilllomas and carcinomas afterpromotion had been discontinued. There was an intermediate incidenceof tumors in the group treated for 12 weeks. Promotion for 18,24, 30, or 36 weeks elicited virtually identical yields of papillomas.The incidence of carcinomas was proportional to promotion durationtimes of 6, 12, and 18 weeks, but carcinoma incidence was lessthan maximal in mice promoted for 24 weeks or longer. The results indicate that a) the incidence of papillomas servesas a rapid (18 weeks) index for subsequent appearance of carcinomas,b) twice weekly applications of 10 nmol of TPA for 18 weeksfollowing initiation of female CD-1 mice with 0.2 µmnolof DMBA is an appropriate protocol for maximum tumor yield ininitiation-promotion experiments, and c) ODC induction may bean important component of the mechanism of skin tumor promotionby TPA.     

Initiating activity in a two-stage mouse skin model of nine mutagenic pyrolysates of amino acids, soybean globulin and proteinaceous food

Trp-P-1, Trp-P-2, MeAC, AC, Glu-P-1, Glu-P-2, Lys-P-1, IQ andPhe-P-1 were tested for tumour initiating activity in a two-stageskin carcinogenesis model using 12-O-tetradecanoylphorbol-13-acetate(TPA) as the promoter. The total initiating doses were 20 mgfor Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2, 40 mg for MeAC andAC, 5 mg for Lys-P-1, 7.5 mg for IQ and 100 mg for Phe-P-1.7,12-Dimethylbenz[a]anthracene was used as a positive controlcompound at a total dose of 100 µg. All compounds weretopically applied twice weekly for 5 weeks on the dorsal skin,and then followed by similar TPA administration for 47 weeks.Trp-P-2 induced skin tumours in 30% of the mice (0.35 tumours/mouse),and Trp-P-1, MeAC and Phe-P-1 in 10–20% (0.20–0.25tumours/mouse). The smaller amounts of Lys-P-1 and IQ appliedinduced tumours at an incidence of 10 and 5% respectively. Notumours appeared in the groups treated with test chemicals aloneor TPA alone. Statistical analysis according to either the Fisherexact test or Peto trend test revealed significant differencesfor tumour appearance in the Trp-P-1, Trp-P-2, MeAC and Phe-P-1followed by TPA groups as compared with that given TPA alone.The data were used to generate ID₅₀ (50% initiating dose) valuesfor each of the compounds.     

Sandalwood oil prevent skin tumour development in CD1 mice.

Sandalwood oil (SW oil) has been used for the treatment of inflammatory and eruptive skin diseases. In the present study, the chemopreventive effects of SW oil on 7,12-dimethylbenz(a)-anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate(TPA)-promoted skin tumour development and TPA-induced ornithine decarboxylase (ODC) activity in CD1 mice were investigated. Female CD1 mice (5-6 weeks old) were divided in different groups, having 30 mice in each group. One week after topical application of DMBA (200 nmole in 100 microl acetone) alone or with SW oil at different concentrations (100 microl, 1.25, 2.5, 3.75, 5% in acetone), at different times (0.5, 1, 2 h) before DMBA, the mice were treated topically with TPA (5 nmole in 100 microl acetone) alone or with SW oil at different concentrations (100 microl, 1.25, 2.5, 3.75, 5% in acetone) at different times (0.5, 1, 2 h) before TPA applications twice a week for 20 weeks. The mice were weighed and papillomas counted weekly. The results indicate that SW oil pre-treatment decreased the papilloma incidence and multiplicity in a concentration and time-dependent manner. The pre-treatment with 5% SW oil (100 microl) 1 h before DMBA and TPA treatments provided a maximum of 67% and 96% decrease in papilloma incidence and multiplicity, respectively, after 20 weeks of promotion. The mice pre-treated with SW oil at all concentrations and time period before TPA had significantly lower ODC activity than the group treated with TPA alone. The data suggest that SW oil could be an effective chemopreventive agent against chemically-induced skin cancer.     

Ionizing radiation enhances malignant progression of mouse skin tumors

Chemical carcinogenesis in mouse skin has been divided intothe process of initiation, promotion and progression. Recentlywe have shown that ionizing radiation acts as an initiator inthis model system. In this paper we describe a three-stage experimentusing ionizing radiation in the third stage of mouse skin carcinogenesis.CD-1 mice were initiated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) followed by biweekly promotion with 12-O-tetradecanoylphorbol-13-acetate(TPA). After 20 weeks of promotion, the animals were treatedwith either acetone, TPA (twice a week for 2 weeks) or eightfractions of 1 MeV electrons (1 Gy/fraction over a period of10 days). The conversion of papillomas to squamous cell carcinomaswas 80% for animals treated with ionizing radiation comparedwith 25% for tumor-bearing animals treated with TPA. Ionizingradiation increased the number of cumulative carcinomas pergroup. The lack of an increase in the number of cumulative papillomasper group due to ionizing radiation suggests that the dose andfractionation protocol used in this study enhanced the progressionof pre-existing papillomas.     

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     

New strains of inbred SENCAR mice with increased susceptibility to induction of papillomas and squamous cell carcinomas in skin

To develop mouse strains useful for studies of susceptibility and resistance to the induction of skin tumors, three new inbred SENCAR strains were independently derived by random inbreeding of outbred SENCAR mice. Characterization of these mice for sensitivity to skin tumor development indicated that mice of all three strains displayed increased sensitivity to initiation by 7,12-dimethylbenz[a]anthracene (DMBA), urethane, or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Promotion by mezerein as well as carcinogenesis by repeated treatment with DMBA or MNNG produced papillomas with a high frequency of conversion to squamous cell carcinomas (SCCs). Compared with outbred SENCAR mice, development of both squamous papillomas and carcinomas was increased at least two-fold by all protocols tested. The F₁ hybrid between SENCARA/Pt males and resistant BALB/cAnPt females was resistant to the induction of both papillomas and SCCs after initiation by 2 μg of DMBA and promotion by 20 weekly applications of 2 μg of TPA. Papillomas developed in all of the SENCARA/Pt mice, none of the BALB/cAnPt mice, and 12% of the F₁ progeny. Thus, at these doses of initiator and promoter, resistance was incompletely dominant in the F₁ hybrid. However, the responsiveness of the F₁ mice could be increased substantially by increasing the dose of the promoter. Mol. Carcinog. 20:143–150, 1997. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Tumor initiating activity of 9- and 10-fluoro-7,12-dimethylbenz[a]-anthracene (DMBA) and the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on tumor initiation by monofluoro derivatives of DMBA in SENCAR mice

We have determined the skin tumor initiating activity in SENCARmice of 6 monofluoro derivatives of 7,12-dimethylbenz[a]anthracene(DMBA). 9-Fluoro-DMBA (9-F-DMBA) was approximately as active,and 10-F-DMBA was more active than the parent hydrocarbon, DMBA.The difference between DMBA and 10-F-DMBA was most dramaticat the highest initiating doses of 10-F-DMBA tested. 4-F-DMBA,which was only weakly active as an initiator, was also testedas a complete carcinogen on mouse skin; after 30 weeks of treatment,50- and 100-nmol weekly doses failed to elicit papillomas orcarcinomas. Animals treated with 50 nmol of DMBA weekly exhibiteda 100% papiloma incidence and a 42% carcinoma incidence. Pretreatmentwith 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) effectivelyinhibited tumor initiation with all of the monofluoro derivativesof DMBA tested. The ED₅₀ (dose of TCDD producing half-maximalinhibition) for the inhibition of DMBA initiation in SENCARmice was determined to be 1.8 x 10^–3 µg/mouse (5.6pmol). The results indicate that the introduction of a fluorineatom in ring D of DMBA has no effect (positions 9 and 11) orenhances (position 10) tumor initiating activity. We believe10-F-DMBA to be the first example of a hydrocarbon with a fluorosubstituent giving rise to increased tumor initiating activity.The results also indicate that structural modifications thatalter tumor initiating activity do not alter the ability ofTCDD to inhibit tumorigenesis by DMBA.