[CANCER RESEARCH 35, 2278-2283, August 1975]

Summary

Male Sprague-Dawley rats were fed a diet containing 500ppm of cyclopropenoid fatty acids (CPFA). After 2 weeks,mitotic activity and [3H]thymidine incorporation into DNAwere significantly increased in pancreatic acinar cells.Continued feeding of the diet for 4 and 8 weeks led todecreasing mitotic activity which was still significantlyincreased over that of control animals. Focal necrosis ofacinar cells was histologically apparent after 8 weeks ofCPFA ingestion. Ultrastructural evidence of focal cytoplasmic injury was detected in acinar epithelial cells as early as 2weeks after feeding of the CPFA diet was begun. Adifference in dose response to CPFA appears to existbetween Sprague-Dawley and Fischer F344 rats in that thelatter, a dose of CPFA (5 mg/l00 g body weight), did notevoke a mitogenic response.

Infroduction

CPFA2 are unusual C18and C19compounds indigenous toplants of the order Malvales which include several important sources of food for domestic animals and man and areknown to exert adverse biological effects (15, 16, 19).Recently, it has been shown that CPFA and the methyl esterof one of these, sterulic acid, are capable of inducing mitosisin hepatocytes of rainbow trout and rat (21). During thecourse of this earlier study, it appeared that CPFA alsostimulated mitosis in epithelial cells of the exocrine pancreas. This communication is a preliminary report of theextent to which CPFA are able to induce mitosis in thesecells.

Materials and Methods

Fifteen male Sprague-Dawley rats weighing 50 g eachwere fed Purina rat chow containing 500 ppm CPFAsupplied in Stercu!ia foetida oil (41% sterculic and 7%malvalic acids); 15 male rats of comparable weight werepair fed on Purina rat chow alone and servedas controls.

1 Presented at the symposium “Current Progress in Pancreatic Car

cinogenesis Research,―February 4, 1975, Orlando, Fla., as part of theThird Annual Carcinogenesis Conference. Supported by USPHS ContractNOI-CP-23271 from the Division of Cancer Cause and Prevention,National Cancer Institute.

2 The abbreviation used is: CPFA, cyclopropenoid fatty acids.

Five rats each were killed after 2, 4, and 8 weeks of feeding.In experiments designed to study the relation of rat strainand dosage of CPFA to the induction of mitosis, fifteenl00-g Sprague-Dawley and Fischer F344 male rats weredivided into 3 groups of 5 animals each and given CPFA asS.foetida oil by stomach tube as a single dose (10, 5, and 2.5mg/ 100 g body weight, respectively). After 24 hr theanimals were sacrificed. The pancreas was carefully dissected, fixed in buffered formalin, embedded in paraffin,and prepared for light microscopy; only cells in metaphase(disappearance of nuclear membrane) or later stages ofmitosis were considered as being in division. Five thousandcells were counted in each animal. Samples of liver fromrandomly selected animals were minced into 0.5- to 1-mmthick fragments and fixed in cold 2.5% glutaraldehyde in 0.1M phosphate buffer at pH 7.55. These were embedded in

Epon and thin sections were prepared with an LKBultramicrotome. These were stained with uranyl acetate andlead citrate (17) and examined in a Hitachi HU-l I-Celectron microscope. Sections 0.5 to I sm thick were stainedwith toluidine blue (25) for light microscopic study.

The effect of CPFA on DNA synthesis was studied asfollows. Five rats fed diet containing CPFA for 2 weeks and5 controlratspair fedPurinarat chowfor 2 weeksweregiven i.p. injections of [3H]thymidine (Schwarz/Mann,Orangeberg,N.Y.; specific activity, 13Ci/mmole) at a doseof 1 jsCi/g of body weight 1 hr before they were killed. Thepancreas was excised, with care being taken to remove allthe tissue in the duodenal loop, extending to the splenichilus; this included the duodenal, gastric, and splenicsegments of the gland. Pancreas weights and pancreaticDNA were determined. Approximately 250 mg of pancreaswere homogenized in cold 0.9% NaC1 solution with aPotter-Elvehjem homogenizer to make a 20% homogenate(w/v). The DNA was extracted by a modification of theSchmidt-Thannhauser-Schneider method (8) and separatedfrom RNA by differential acid solubility (22, 23). The DNAcontent was determined colorimetrically (24). Specific activity of [3H]thymidine incorporated into DNA was measuredwith a Packard Tri-Carb liquid scintillation spectrometer byuse of a mixture of 2,5-bis(5'-tert-butylbenzoxazoyl)-2'-thiophene (Packard Instrument Company, LaGrange, Ill.) andtoluene. The internal standard, [3H]toluene (Packard), hada counting rate of 2.26 x 106 dpm/ml. After percentageefficiency was determined, the counting rates of the samplesand specific activity of DNA were calculated.

2278 CANCER RESEARCH VOL. 35

Preliminary Observations on the Mitogenic Effect of CyclopropenoidFatty Acids on Rat Pancreas'

Dante G. Scarpelli

Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, Kansas 66103

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DoseofCPFAa

(mg/l00g bodyMitotic

index/ I000 acinarcellsSprague

wt)DawleypFischerp00.2±0.3―0.1±0.32.53.9

±2.0<0.0014.8 ±1.7<0.0015.05.3±1.9<0.001010.06.8

±1.5<0.0010

CPFA (ppm)Wkof

feedingMitoticindexP020.5±0.6b50025.7±2.0<0.001040.3±0.450043.2±0.9<0.001080.1±0.350081.9±1.00.01

No.Groupof

animalsPancreas wt (g)pDNA

(mg/gpancreas)DNA

specificactivity

(dpm/mgDNA)pControl551.6

±2.96a5.08 ±0.49b12,768 ±1,230―CPFAC555.2±1.92@O.057.32 ±0.7764,361 ±7,975<0.005

CPFA and Pancreatic Acinar Ce!ls

Results

Mitotic Indices. Chronic ingestion of low levels of CPFAleads to an increased number of mitosis in acinar cells of ratpancreas (Fig. 1; Table 1). The mitogenic effect became lessmarked with continued feeding of CPFA and in animals ofincreasing age. Mitoses in ductal epithelium were rarelyencountered and no differences were found between animalsfed CPFA and controls.

The relationship between the dosage of CPFA and ratstrain and its mitogenic effect is shown in Table 2. While themitogenic effect of CPFA appeared to be dose dependent, adose of 5 mg/ 100 which caused further stimulation ofmitosis in pancreatic acinar cells of Sprague-Dawley ratsdid not stimulate mitosis in Fischer F344 rats.

Incorporation of [3H]Thymidine into Nuclear DNA.Ingestion of a diet containing 500 ppm ofCPFA for 2 weekswas reflected by an increase in pancreatic wet weight whichwas significant and by a greater incorporation of [3H]thymidine into nuclear DNA of rat pancreas, as compared to thatseen in pair-fed controls (Table 3).

Light and Electron Microscopy. After 8 weeks of feedinga diet containing CPFA, focal areas of acinar cell injurywere seen. These were often limited to several cells peracinus (Fig. 2); not infrequently, mitoses were present innearby acinar cells (Fig. 3).

The earliest ultrastructural changes suggestive of injuryfollowing CPFA feeding were encountered after 2 weeks;these consisted of focal alterations of the cytoplasm such assequestered islands of rough-surfaced endoplasmic reticulum, membrane whorls, and accumulations of amorphousmaterial in vacuoles (Fig. 4). Not all acinar cells showedevidence of injury; many had large nucleoli and a cytoplasmdevoid of secretory granules. These cytological changes are

Table I

Chronic feeding of CPFAa and mitosis ofpancreatic acinar cells

consistent with enhanced nucleoprotein and protein synthesis (Fig. 5). Several acinar cells with the characteristictightly packed arrays of endoplasmic reticulum and densezymogen granules from a pair-fed control rat are shown inFig. 6.

Discussion

In the immediate postnatal period of somatic growth,epithelial cells of the pancreas, like those of liver, kidney,adrenal, and thyroid, are capable of considerable sustainedproliferation, a property that decreases with increasing ageto levels of about 0. 1% or less in the adult animal. Theseare referred to as expanding cell populations and appear tolie intermediate between so-called static populations suchas neurons, which lose their capacity to divide very shortlyafter birth and normally persist through the life of theanimal, and renewing cell populations, in which there is acontinuous replacement of cells, the prime example beingepithelium of the digestive tract (6, 11).

Although expanding cell populations in the adult animalremain quiescent or nondividing for protracted periods oftime with the appropriate stimulus, they are capable ofreentering the@ or presynthetic phase of the cell cycle andthey ultimately undergo mitosis. The most effective stimulus for directing quiescent cells to enter the cell cycle is areduction of the cell population secondary to subtotal orunilateral surgical resection of the organ (1 , 3, 9, 10) or tocell death following toxic or ischemic injury (5, 7, 12, 14).Thus, it is significant that CPFA are capable of stimulatingacinar epithelial cells of the rat pancreas to divide, especially since this appears initially to occur in the absence ofcell death. In fact, after 8 weeks on the CPFA diet when

Table 2

Relationship between dosage of CPFA and rat strain and mitosis ofpancreatic acinar cells

a CPFA, 500 ppm, administered in diet.

b Mean ± S.D.

a CPFA administered as single dose, animals sacrificed 30 hr later.

b Mean ± S.D.

Table 3

Incorporation of [3H]thymidine into DNA of rat pancreas

a Mean ± S.D.

b Mean ± SE.

C 500 ppm for 2 weeks.

AUGUST 1975 2279

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D. G.Scarpel!i

focal necrosis of pancreatic acinar cells became evident, themitotic index was lower than it had been previously at 2 and4 weeks of feeding.

The mitogenic effect on pancreatic acinar cells appears tobe less potent than that observed for hepatocytes (21);whether this is due to the lower concentration of CPFAwhich would be expected to be delivered to the pancreas ascompared to liver or whether acinar cells are less sensitive tothe fatty acids is not known. Such possibilities are furthersupported by the modest increase in pancreatic wet weightafter 2 weeks of feeding CPFA. These results corroborateearlier studies by Fitzgerald et a!. (7) and Lehv andFitzgerald (I 3) who pointed out that pancreatic regeneration, especially after partial resection, was quite slow ascompared to liver. The mechanism(s) by which CPFAaugment cell division remain to be elucidated; interestingpoints in this regard are that these fatty acids appear to beincorporated into tissue lipids (20) and cell membranes(unpublished observations). In view ofthe central role of cellmembranes, especially the plasma membrane in the regulation of mitosis (2, 4, 18), it may be that the stimulation ofcell division by CPFA is the result of some qualitativeand/or quantitative alteration in their lipid composition.

References

I. Abercrombie, M., and Harkness, R. D. The Growth of Cell Populations and the Properties in Tissue Culture of Regenerating Liver of theRat. Proc. Roy. Soc., London 5cr. B, 138: 544-561, 1951.

2. Baserga, R., Costlow, M., and Rovera, G. Changes in MembraneFunction and Chromatin Template Activity in Diploid and Transformed Cells in Culture. Federation Proc., 32: 2115—2118, 1973.

3. Bucher, N. L. R., and Malt, R. A. Regeneration of Liver and Kidney.Boston: Little Brown and Co., 1971.

4. Burger, M. M. Proteolytic Enzymes Initiating Cell Division andEscape from Contact Inhibition of Growth. Nature, 227: 170—171,1970.

5. Cuppage, F. E., Cunningham, N., and Tate, A. Nucleic Acid Synthesisin the Regenerating Nephron following Injury with Mercuric Chloride.Lab.Invest.,21:449-457,1969.

6. Enesco, M., and Leblond, C. P. Increase in Cell Number as a Factor inthe Growth of the Organs and Tissues of the Young Male Rat. J.Embryol. Exptl. Morphol., 10: 530-562, 1962.

7. Fitzgerald, P. J., Herman, L., Carol, B., Roque, A., Marsh, W. H.,Rosenstock, L., Richards, C., and Perl, D. Pancreatic Acinar CellRegeneration. I. Cytologic, Cytochemical, and Pancreatic Weight

Changes. Am. J. Pathol., 52: 983-1011, 1968.8. Fleck, A., and Munro, H. N. The Precision of Ultraviolet Absorption

Measurements in the Schmidt-Thannhauser Procedure for NucleicAcid Estimation. Biochim. Biophys. Acta, 55: 571 -583, 1962.

9. Harkness, R. D. Regeneration of Liver. Brit. Med. Bull., 13: 87-93,1957.

10. Higgins, G. M., and Anderson, R. M. Experimental Pathology of theLiver. I. Restoration of the Liver of the White Rat following PartialSurgical Removal. A. M. A. Arch. Pathol., 12: 186-202, 1931.

I 1. Leblond, C. P. Classification of Cell Populations on the Basis of TheirProliferative Behavior. Nail Cancer Inst. Monograph, 14: 119-150,1964.

12. Leevy, C. M., Hollister, R. M., Schmid, R., MacDonald, R. A., andDavidson, C. S. Liver Regeneration in Experimental Carbon Tetrachloride Intoxication. Proc. Soc. Exptl. Biol. Med., 102: 672-675,1959.

13. Lehv, M., and Fitzgerald, P. J. Pancreatic Acinar Cell Regeneration.IV. Regeneration after Surgical Resection. Am. J. Pathol., 53:513—535,1968.

14. Longnecker, D. S., Crawford, B. G., and Nadler, D. J. Recovery ofPancreas from Mild Puromycin-induced Injury. Arch. Pathol., 99:5-10, 1975.

15. Mattson, F. H. Cyclopropenoid Fatty Acids. In: Toxicants OccurringNaturally in Food, Ed. 2, pp. 196- 198. Washington, D. C.: NationalAcademy of Sciences, 1973.

16. Mickelsen, 0., and Yang, M. G. Naturally Occurring Toxicants inFoods. Federation Proc., 25: 104-123, 1966.

17. Millonig, G. A. Modified Procedure for Lead Staining of ThinSections. J. Biophys. Biochem. Cytol., 11: 736-739, 1961.

18. Pardee, A. B. The Surface Membrane as a Regulator of Animal CellDivision. In Vitro, 7: 95—104, 1971.

19. Phelps, R. A., Shenstone, F. S., Kemmerer, A. R., and Evans, R. J. AReview of Cyclopropenoid Compounds: Biological Effects of SomeDerivatives. Poultry Sci., 44: 358-394, 1965.

20. Roehm, J. N., Lee, D. J., Sinnhuber, R. 0., and Polityka, S. D.Deposition of Cyclopropenoids in the Tissue Lipids of Rainbow Troutfed Methyl Sterculate. Lipids, 6: 426—430,1971.

2 1. Scarpelli, D. G. Mitogenic Activity of Sterculic Acid, a Cyclopropenoid Fatty Acid. Science, 185: 958-960, 1974.

22. Schmidt, G., and Thannhauser, S. J. A Method for the Determinationof DesoxyribonucleicAcid, RibonucleicAcid, and PhosphoproteinsinAnimal Tissues. J. Biol. Chem., 161: 83—89,1945.

23. Schneider, W. C. Extraction and Estimation of Desoxynucleic Acidand of Pentose Nucleic Acid. J. Biol. Chem., 161: 293-303, 1945.

24. Schneider, W. C. Determination of Nucleic Acids in Tissues byPentose Analysis. Methods Enzymol., 3: 680-684, 1957.

25. Trump, B. F., Smuckler, E. A., and Benditt, E. P. A Method forStaining Epoxy Sections for Light Microscopy. J. Ultrastruct. Res., 5:343-348, 1961.

Fig. 1. Rat pancreas after 2 weeks on the C.PFA diet. Five acinar cells are in mitosis (arrows); note the absence of acinar cell necrosis. x 300.Fig. 2. Rat pancreas after 8 weeks on the CPFA diet. Focal necrosis of acinar cells is evident (arrow). x 750.Fig. 3. Rat pancreas after 8 weeks on the CPFA diet. There is considerable alteration of acinar structure, individual cell necrosis (arrow); a mitotic

figure is present (lower left). x 750.Fig. 4. Normal rat pancreas showing the tightly packed arrays of rough-surfaced endoplasmic reticulum and zymogen granules characteristic of

acinar cells. Note the nuclear and nucleolar profiles. x 6,000.Fig. 5. Rat pancreas after 2 weeks on the CPFA diet. The nucleolus is enlarged and occupies a considerable portion ofthe nuclear volume. x 6,500.Fig. 6. Higher magnification of the cytoplasm of an acinar cell after 2 weeks of CPFA feeding. There are numerous whorls of membranous material

associated with islands of rough-surfaced endoplasmic reticulum, changes suggestive of focal cytoplasmic injury. x 17,600.

CANCER RESEARCH VOL. 352280

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Dr. Weisburger: Dr. Scarpelli, we, of course, know you for

your participation on the important elucidation of thefactors that bore on the induction of hepatoma 10 or 15years ago. It is interesting that you now come back to I ofthe elements that seem to be involved at this time, and this isreally very encouraging. Did you imply that the decreasingeffect as you fed sterculic acid in these studies was related tothe increasing age? If so, did you use older animals at thehigher doses and find it was possible to compensate for thiseffect?

Dr. Scarpelli: Yes, there is a decreasing mitogenic effect ofcyclopropenoid fatty acids with increasing age of theanimals both with liver and pancreas. Animals weighing 500or 600 g will develop a fatty liver but no mitogenic response.

Dr. Weisburger: One is always intrigued with chemicalswhich have double bonds in them. Do you by chance haveaccess to the corresponding epoxide, because it could be thatthe effect of age may relate to the fact that the older animalsnot only have a decreased rate of metabolism generally, butit could also be that they have lower formation of an active

metabolite such as the epoxide or, reversely, increaseddetoxification, through epoxide hydrase or glutathionetransferase to detoxified products?

Dr. Scarpelli: That's a very good point. I do not have accessto such information. Dr. Sinnhuber of Oregon StateUniversity is studying the metabolism of cyclopropenoidfatty acids.

Dr. Longnecker: Do the pancreases achieve a larger size?

Dr. Scarpelli: Yes, they do, It's of course limited, stoppingafter about 2 months of feeding.

Dr. Longnecker: We have recently observed the effects oftreating animals between the weights of 50 and 250 g withthe effects of starting with animals that weigh 250 g. Wehave many fewer nodules in the older rats.

Dr. Alarif: I was just wondering about the type of compound you used. Was it the methyl ester or the free acid?

2282 CANCER RESEARCH VOL. 35

‘S

Informal Discussion following the Paper by Scarpelli

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1975;35:2278-2282. Cancer Res   Dante G. Scarpelli  Cyclopropenoid Fatty Acids on Rat PancreasPreliminary Observations on the Mitogenic Effect of

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