Fischer, W. H.

Lutz, Werner K.

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Carclnogenesb voi.IS oo.1 pp.129-131, 1994 SHORT COMMUNICATION Mouse skin papilloma formation by chronic dermal application of 7, 12-d.imethylbenz[ a ]anthracene is not reduced by diet restriction Wolfgang H.Fischer and Werner K.Lutz1 Institute of Toxicology, Swiss Federal Institute of Tedmology (ETH), and University of Zürich, CH-8603 Schwerzenbacb, Switzerland 1To wbom correspondence should be ~ DJet restriction bas repeatedly been shown to reduce the incldence of spontaneous and chem.ically induced tumors in rodents. However, no conclus:ive data are avallable to show whether ca.rclnogenesfs by chronic exposure to a genotoxic agent can also be retarded. In this study, diet restriction to 70% was lnvestigated for a protective effect on the formation of skin papilloma in male NMRI mlce treated twice weekly with 20 nmol 7,12-dimethylbenz[a]anthracene (DMBA). Rather surprisingly, no protection was seen. Both time of onset of papilloma formation (13 weeks in both groups) and time of 50% cumulative incldence {t50; 17.5 and 18 weeks) were similar in the unrestricted and the restricted group. In contrast, a clearly protective eft'ect was found in mice initiated with 100 nmol DMBA and promoted twice weekly with 2.5 nmol U-0-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 exposure to a genotoxic carcinogen. lt cannot be considered a universal measure of cancer prevention. A large number of experimental studies have demonstrated that diet restriction markedly reduces both spontaneous and chemically induced tumor fonnation in mice and rats ( 1 - 17). A number of reviews are also available ( 18-21). Reports on experiments where restriction had no protective effect are rare ( 1 0). It therefore appears as if reduction of food intake could be regarded as a universal cancer-preventive measure. In the majority of the investigations on chemically induced tumor induction, the carcinogen was given as a single dose (10,12-14) or for up to 4 weeks (5,7,10,15). In few studies, the treatment with the carcinogen Iasted Ionger but was stopped either before the appearance of the first twnor (2-4) or when the first tumor appeared in the unrestricted group ( 1). It therefore remains to be shown whether the process of carcinogenesis can be retarded by diet restriction under conditions of chronic administration of a genotoxic agent. To answer this question, the skin twnor model with NMRI mice and chronic administration of7,12-dimethylbenz[a]anthracene (DMBA*) was used (22-24). Restrietion was by feed reduction to 70%, a Ievel chosenon the basis of the findings that underfeeding at a Ievel of 60% was well tolerated and indistinguishable from caloric (carbohydrate) restriction to 60% with respect to body weight and tumor incidence ( 16). Male NMRI mice [Crl:NMRI BR] were obtained from Charles River Savo, Kisslegg, Gennany, at 6 weeks of age. The mice • Abbrevladons: DMBA, 7, 12-di.mcthylbenz[a ]81'11hraceoe; TP A, 12-<J..tetra. decaooylphorbol-13-aa:tate; B[a l P, benzo[a ]pyrcne. © Oxford Universiry Press were housed individually at 21 ± 1 °C with a 12 h light/dark cycle in Macrolon cages type ll. Feed (Nafag 890 from Nafag AG, Gossau, Switzerland) and water were given ad libitum to all mice from 6 to 8 weeks of age. The backs of the mice were shaved at week 7 (a swatch 4 cm long, 3 cm wide; Wella Minicut). One week later, restriction and treatmentwas started. The restricted mice were given weighed feed portians amounting to 70% of the feed eaten by the unrestricted group. Every evening at 6.30 p.m., i.e. 30 m.in before the beginning ofthe dark phase, a rotaring wheel delivered the weighed portions into the feed compartment of the cages. The size of the portions was adjusted weekly on the basis ofthe weigbed feed intake by the unrestticted group. Treatment A (chronic administration of DMBA): starring at week 0, 60 mice (30 ad übitum, 30 restricted) received twice weekly a dermal application of 20 nmol DMBA (Sigma) in 0.1 ml acetone on the shaved part of the back until 4 days before they were killed. Application solution was prepared biweekly and was stored at - 20°C. One mouse in each group died spontaneously during the experiment. Treatment B (initiation with DMBA, promotion with 12-0-tetradecanoylphorbol-13-acetate [TPA]; positive control for a protective effect of diet restriction): twenty-eight mice (14 ad libitum, 14 restricted) were treated once with 100 nmol DMBA in 0.1 ml acetone ( = week 0). Treatment with 2.5 nmol TPA (LC Services Corporation, Wobum, MA), twice weekly in 0.1 ml acetone, was started 1 week later. A TPA stock solution (8 x) was prepared once, divided into 5 ml aliquots and stored at -20°C. Two mice of the restricted group died spontaneously during the experiment. Controls: two groups of mice (9 ad libitum, 9 restricted) were treated twice weekly with 0. 1 ml acetone. Allmice were visually inspected and palpated for papillomas twice weekly and were weighed biweekly. They were killed 2 weeks after showing the first, persistent papilloma with diameter >1 mm. Body weights are given in Figure 1. Diet restriction resulted in a significant reduction in body weight under both treatments. In the initiation-promotion model (Figure 1 ; treatment B), the initial decrease appeared to be faster, probably because of some high-dose effect ofthe initiating 100 nmol DMBA. The initiation might also be the reason why the weight gain in the unrestricted group oftreatment B was slower than in treatment A. 1be weight curves for the control groups treated with acetone alone were not different from those seen with treatment A (data not shown). The cumulative skin-papilloma incidence is showo in Figure 2. Unexpectedly, diet restriction had no protective effect when the mice were treated chronically with DMBA (Figure 2A). Both time of onset of papilloma formation (13 weeks) and time of 50% cumulative incidence (t50 ; 17.5 and 18 weeks) were similar in both groups. A papilloma incidence of 100% was reacbed after 25 and 28 weeks in unrestricted and restricted animals respectively. In contrast, and as expected from the literature (16), diet restriction bad a clearly proteelive effect in the initiation-129 W.H.Flscher and W.K.Lutz 55 A 50 0 hJibi!Jm • Re ltltc:*S 45 i: 130 25 20 15;+-.~ ......... ~-,....,r-r-T"""""""T""""~ ........... '"'O"""T-.--r-r-.,....,...., -2 0 2 4 6 8 10 12 14 16 18 20 22 55 B 50 0 Mlbllum • RellltcDd 25 20 W8ek of axpertment 15~~~~~~~~.-......... ~~r-~ -2 0 2 4 8 8 10 12 14 16 18 20 22 Y$ek crf axperlment Jila. 1. Body weightJ of male NMRI mice fed od libitum (opcn cin:les) or restricted to 70% feed in1akc (full circles). Treatment A (group size 81 statt 30): 20 nmol DMBA twice wcddy, starting 81 weck 0. Treatment B (group size at start: 14): initiation wilh 100 nmol DMBA 81 week 0, followed by promotioo with 2., mnol TPA twice wcckly, .startin& 81 weck 1. Data are given as mcan weigbts : 1 SD. A - hJibllum - - - Reeltlc:l8d Flg. 2. Cumulative iDcideDce of skin papillomas in male NMRI mice fcd od libitllm (solid linc) or ~ to 70% feed intake (cWhed lioe). Treatment A: 20 nmol DMBA twice wccldy, starting 81 weck 0. Treatment 8: initiation with 100 nmol DMBA 81 week 0, followed by promotion wilh 2.S omol TP A twice wceldy, slartiog 81 week I. 130 promotion model (Figure 2B). The onset of papilloma fonnation increased from 7 to 11.5 weeks, the t50 was shifted from 8.5 to 19 weeks. The papilloma incidence in the unrestricted group was 100% after 13 weeks and it would have taken much Ionger to resch 100% in the restricted group. Histological examination of the papillomas did not reveal any observable differences between tbe four groups. In the acetone control groups, no tumor was recorded within 28 weeks of observation. For controls treated only with DMBA (1 x 100 nmol) or TPA (10 nmol twice weekly), the papilloma incidence within 24 weeks bad been reported to be 0 or 4% (22). Our negative data are in contrast to Tannenbaum' s early findings mentioned above (1-4). The discrepancy could be due to the use of different carcinogens and different dose Ievels. The 5 1'8 DMBA dose used here and tbe 60 ~ benzo[a]pyrene (B[a]P} dose used by Tannenbaum (1,3) can be considered equicarcino-genic (25 ,26) but the relative importance of genotoxic versus promoting activity associated with the repeated administration of the two carcinogens could be different. For DMBA, carcinogenicity might be dominated by genotoxicity while the 10-fold higher chemical dose of B[a]P could include a more pronounced 'promotional' activity. Such an explanation would also be in line with recent work by Birt and co-workers who showed that diet restriction bad a beneficial effect only when applied during tumor promotion but not during initiation (16). It could be argued that a dose of 20 nmol DMBA twice weekly was too high to allow restriction to come into play. The possibility that high doses of carcinogen could override the effects of diet restric:tion was mentioned by Tarmmbaum and Silverstone in tbeir review article ( 18). However, the dose used here did not result in a maximum rate of the process of carcinogenesis. While we observed a median latency time of 17.5 weeks, dose Ievels of 25, 50 and 100 nmol resulted in tso values of 16, 12.5 and 11 weeks respectively (24). Furthermore, the initiation-promotion protocol used in treatment B resulted in an even faster appearance of the papillomas in the unrestricted group and still allowed diet restriction to be protective. The question of whether diet restric:tion can retard the process of papilloma fonnation therefore strongly depends on the mechanism of action. It appears as if clonal expansion of initiated cells during tumor promotion could be reduced by diet restriction but that the rate of accumulation of critical mutations from chronic exposure to a genotoxic agent is not affected. Our results suggest that diet restriction is not proteelive under all circwnstances of chronic exposure to a carcinogen. Whether or not there is an effect depends upon the specific mechanism of carcinogenesis. Fora situation of chronic higb-dose exposure of mouse skin to a genotoxic agent the fonnation of papilloma could not be reduced. Although the datJl do not give any information on the process of tumor progression to carcinoma, they strongly indicate that diet restric:tion should not be considered a universal cancer-preventive measure. References 1. Tannenhmrrn.A. (1940) Tbc initiation and growtb of tumors. I. Effects of underfccding. Am. J. Cancer, 38, 335-3SO. 2. TJIJIDC'flhaum A. (1942) The gmesis aod growdl oftumoiS. 0. Effeas ofcaloric n:strittioD per se. Cancer Res., 2, 460-461. 3. Tannenbmrm.A. (1944) Tbc dcpaden e of dJc gencsis of i:nduced skin tumors on tbe caloric intake during d.ifferem srages or carcinogeoesis. Cancu RD., 4, 673-671. 4. Tannrnbmrm A. (1945) Tbc defJeuclrnr; ofiUIDOr formabon 011 tbc CXlD'lp''6ilion of the calorie-rcstricted diet as well as on the dcgrce of rcstriction. CmJcer Ru., 5, 616-625. S. Tannenbawn,A. and Silventone,H. (1949) Tbc influence of the degrce of caloric restriction oo tbe formatioo of skin rumors and bepatomas in mice. CtJnur Rn., 9, 724-727. 6. Ross,M.H. and Bras,G. (1971) Lasting influence of caloric restriction on prevalence of neoplasms in the rat. J. Na1l. Cancer Irrst. , 47, 1095 - 1113. 7. Newbeme,P.M., Weigen,J. and Kula,N. (1979) Effeas of dietary fat on bepatic mixed-function Oxidsses and bepatocdlular carciooma induced by aflatoxin BI in rats. Canar Rn., 39, 3986-3991. 8. Tucker,MJ. (1979) The effect of long-term food restriction on tumours in rodems. 1111. J. Cancer, 23, 803-807. 9. Weindruch,R. and Walford,R.L. (1982) Dicwy restriction in mice beginning at 1 year of age: effect on life-span and spontancous cancer iDcidence. Science, 215, 1415-1417. 10. Pollard,M. m1 l...ucmt,P .H. 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(1972) Aryl hydrocarbon bydroxylase and polycyclic hyd.rocatbon tumorigenesis: effect of the enzyme inhibitor 7 ,8-benzoflavone on tumorigenesis and macromolecule binding. Proc. Na1L Acad. Sei. USA, 69, 824-828. 26. Slaga,T.J. and Fischer,S.M. (1983) Strain differences and solvent effcctS in mouse s1cin carcinogenesis experiments using carcinogens, tumor initiators and promottrS. Prog. E:xp. Tumor Ru., 26, 85-109. RtceivtJ on July 26, 1993: revised 071 Octobtr 1, 1993: accepud 071 Octobtr 7, 1993 131 

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

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

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