Effects in Rats of Cyclopropenoid Fatty Acids from Cottonseed Oil

Transcription

1 48 JoumalofFoodProtecton, VoL 45, No.1, Pages (January 1982) Copyrght, Internatonal Assocaton of Mlk, Food. and Envronmental Santarans Effects n Rats of Cyclopropenod Fatty Acds from Cottonseed Ol LUCY L. FAN*, BARNEY W. HILTON and ALAN WOHLMAN Research Department, Frto Lay, Inc., 9 N. Loop 12, Irvng. Texas 7561 (Receved for publcaton February 17, 1981) ABSTRACT The effects of cyclopropenod fatty acds naturally present n cottonseed ol have been nvestgated n rats by feedng dets contanng, 6, 3, 6 and 3,,..g of cyclopropenod fatty acds/g for 3 and 6 months. At the end of 3 and 6 months of feedng, rats were sacrfced for hstopathologcal and bochem cal evaluatons. Body weght, food consumpton, physcal appearance, organ weght, hematologcal profles, hstopatho logcal ndcators, lver mcrosomal proten, actvty of NADPH-cytochrome (P-45) reductase, and the hepatc contents of cytochromes P-45 and b 5 were not dstnctly dfferent among the fve groups of rats. However, there was a postve correlaton between the amount of cyclopropenod fatty acds deposted n the body fat and content of these fatty acds n the dets. The sgnfcance of these results s dscussed. Cyclopropenod fatty acds (CPF A) occur n lpds of plants belongng to the order Malvales (3), ncludng the economcally mportant cotton plant, and have the followng general structure: CH 3 - (CH 2 h cchze- (CHz)n COOH Cottonseed ol wthout refnng contans from 1 to 2% CPFA wth malvalc acd (MA, n = 6) consttutng.7 to 1.5% and sterculc acd (SA, n = 7).3 to.5% (25). By far the rchest source of CPF A s Stercula foetda seed ol (SFO) whch contans SO to 7% CPFA (29), but ths ol has not been known to be used for human consumpton. CPF A has been shown to be responsble for a varety of bologcal effects, ncludng retarded growth n rats and chcks, ncreased lver sze n rats, delayed sexual development n female rats, altered fatty acd metabolsm, pnk dscoloraton n avan egg whtes (21), and ncreased lver cancer ncdences when fed n conjuncton wth aflatoxn B 1 or M 1 n ranbow trout (12,13,26.27). However, the U.S. Food and Drug Admnstraton thnks that trout beng a cold-blooded anmal, has many metabolc and physologcal features dfferng sgnfcantly from mammalan bology and that conclusons based on trout may not be applcable to mammals (11). On the other hand, CPFA has faled to demonstrate a cocarcnogenc actvty wth aflatoxn when fed to Fsher and Wstar strans of rats (19). Thus the co-carcnogenc response as observed n ranbow trout may ndeed be due to a dfference n metabolc pattern. Most of the studes on detary effects of CPF A have been performed by feedng expermental rats wth SFO at to 3% n the det, whch s an extremely hgh dose of CPF A. It s thus necessary to conduct a mammalan feedng study based on a normal detary ntake of CPF A assocated wth a commonly consumed ol, e.g. cottonseed ol. The current Unted States det (8) derves 42o/oof calorc energy from fat; ths s approxmately equal to 24% n the det, by weght. Commercal cottonseed ol s reported (II) to contan.2 to.45% CPFA. If one assumes that all the detary fat consumed by the Amercan publc wee cottonseed ol, t could be estmated that 48 to 1,8 1-Lg CPFA/g would be present n U.S. dets. Therefore, the experment was carred out n rats, usng cottonseed ol as the source of CPFA at to 3, 1-1glg n the dets for 3 and 6 months to evaluate any possble effects. MATERIALS Al\"D METHODS Expermental anmals Sprague-Dawley albno out-bred rats, about 3 weeks old, weghng 4 to SO g. were obtaned from Harlan-Sprague-Dawley, Madson, Wsconsn. These rats were mantaned on Purna rat chow and tap water ad lbtum for 2 weeks. Durng ths perod. ther physcal appearances and weght gans were recorded daly. At the end of acclmaton, 8 rats were selected and dvded nto fve groups (8 males and 8 females per group). based on body weght dstrbuton. The assgnment was randomzed by usng a table of random numbers and computer. They were housed ndvdually and fed wth fve test dets ad lbtum. At the end of the Jrd month, 4 rats randomly selected from the fve groups (4 males and 4 females per group) were sacrfced and the other 4 rats were sacrfced at the end of the 6th month.

2 CYCLOPROPENOID FATTY ACIDS FED TO RATS 49 Test dets Fve test dets were formulated and prepared n powder form by the Ralston Purna Co., St. Lous, Mo., employng the usual method of the normal rat chow preparaton at that company. The compostons of these dets are presented n Table 1. They were kept n the freezer, and the contents of CPFA were assayed before feedng. Food consumpton was calculated by subtractng the amount left-over from the amount gven. Any vsble splls on the pad beneath the cages were collected and taken nto consderaton. Halphen reacton Halphen (9) ntroduced a color reacton to detect cottonseed ol n ol mxtures. The concentraton of CPFA was determned spectrophotometrcally accordng to a modfed Halphen procedure (4), usng a standard curve of methyl sterculate from Supelco Inc., Bellefonte, PA., under the same condtons. Hematologcal determnaton After recordng body weghts of 12-h pre-fasted rats, blood samples were obtaned from the rat tal just before sacrfcng, and were submtted for hematologcal evaluaton by Southwest Medcal Clnc n Irvng, Texas. Hemograms ncludng hemoglobn, hematocrt, red and whte cell dfferental counts, and the SMAC profles (fechncon Co., TABLE 1. Det compostons. Ingredents Dets A B c D E Cottonseed ol a 2 Improved cottonseed olb 2 Corn olc Casen (vtamn-free) Sucrose Non-nutrtve fberd 3D 3D 3D 3D 3D Dextrn DL-Methonne RP Vtamn mxturee Cholne chlorde RP Mneral mxture #11 f Energy (calores per 1 g) Calculated CPF A {flg/ g n dets) acommercal cottonseed ol was purchased from Levelland Vegetable Ol Company (Lubbock, Texas) and s a specally rocessed ol contanng a hgh amount of 1.19o/o CPFA. Specally deodorzed ol contanng.12% CPFA was also obtaned from Levelland Vegetable Ol Co. ccorn ol was provded by Ralston Purna Company, St. Lous, Mssour. dsolka-floc, non-nutrtve fber, a cellulose product, was obtaned from Brown Company, Berln, New Hampshre 357. ethe vtamn mxture was provded by the Ralston Purna Company n the amount per klogram of det whch ncludes the followng: Thamn hydrochlorde 2 mg, Rboflavn 2 mg, Nacn 9 mg, Pyrdoxn hydrochlorde 2 mg, D-Calcum pantothenate 6 mg, Folc acd 4 mg, D-Botn.4 mg, Inostal 2 mg, Vtamn B 12 2 J-Ig, Menadonesodum bsulfte 2 mg, Vtamn D I.U. per g, Vtamn A acetate 22 I.U. per g, DL-Alpha-tocopherol acetate SO I.U. per kg. fthe mneral mxture was provded by the Ralston Purna Company whch ncludes the followng: Calcum.75%, Phosphorus.45%, Potassum.46%, Sodum.29%, Chlorde.29%, Magnesum.65%, Manganese 65 mg per kg, Iron 6 mg, per kg, Znc 2 mg per kg, Copper 15 mg per kg, Fluorde 5 mg per kg, Cobalt 3.2 mg per kg, Chromum 3 mg per kg, Iodne.6 mg per kg, Molybdenum.8 mg per kg, and Selenum.2 mg per kg. ncludng serum aspartate transamnase, serum alanne transamnase, blrubn, blood urea ntrogen, serum creatnne, serum calcum, cholesterol, and trglycerdes) were determned and evaluated separately for each group of rats, after 3 or 6 months offeedng. Hstopathologcal examnaton Followng tal blood collecton, rats were mmedately sacrfced by decaptaton and a gross necropsy was undertaken. Lver, kdney and lung, were ether quck-frozen for subsequent fat stan or mmedately fxed n Carson's fxatve (2) for lght mcroscopy. Frozen sectons were cut on an IEC-CTD cryostat and staned wth ol red for lpd. Tssues for routne lght mcroscopy were processed n a Techncon Ultra and staned wth hematoxyln and eosn. In some nstances, rats showng mlk desquamatve ntersttal pneumonts were examned by the perodc acd Schff reacton, Gomor methenamne slver, and a tssue gram stan. Tssue preparaton and bochemcal assay Lver mcrosomes were solated as descrbed by Remmer et a!. (22). Proten was determned by the method of Lowry eta!. (14), usng bovne serum albumn as a standard. NADPH-cytochrome_. _(P-45) reductase was assayed as descrbed by Masters eta!. (IS) by measurng the rate of cytochrome_s reducton at 55 nm, usng the mllmolar extncton coeffcent of 21 mm- 1 em., n a Beckman Model35 spectrophotometer. Cytochromes P-45 and b 5 were determned by the method of Estabrook et a!. (7), usng the mllmolar extncton coeffcents of 1 and 185 mm' 1 em for the dfferences n absorbance at 45 nm mnus 49 nm and 424 nm mnus 49 nm, respectvely, n a Varan Super Scan 3 spectrophotometer. Statstcs method Statstcal evaluaton of the data was made by Duncan's New Multple-range Test (28). The level of sgnfcance was chosen as P<.5. RESULTS Growth rate and food consumpton In general, the rats were determned to be healthy throughout the entre feedng study, as judged by daly physcal observatons. The growth rates of male and female rats durng the frst 3 months of feedng are shown n Fg. 1. Daly food consumpton rates are shown n Table 2. As can be seen from these data, male rats grew faster and consumed more food than the female rats. However, there were no dstnct dfferences among the fve feedng groups, regardless of the ol content, ol nature or amount of CPF A n the detary fats. CPFA Depost n body fat Body fats removed from the abdomnal adpose tssues were melted at a temperature less than 1 C (at ths temperature, CPFA does not decompose) and subjected to the Halphen reacton to measure the CPFA content. The results are presented n Table 3. The amount of CPF A deposted n rat body fat was postvely correlated (r = +.982) wth the content of CPF A n ts det. Hematologcal examnatons The hematologcal values for the fve feedng groups were not sgnfcantly dfferent from each other and were wthn the normal range for rats (17).

3 5 FAN, HILTON AND WOHLMAN., E 4 Male Rats 2....: 3t ::r : >- ID -;;; E "' 1'! 1: : 2 3t >- f ID tjt Female Rats c WEEKS 8 t II 1!t :! 12! Hstopathologcal nvestgatons Hematoxyln and eosn-staned sectons of lvers were examned and graded on a scale of zero to four for fatty change, polyplody, nflammaton, fbross, and Kupffer cell hyperplasa. No apparent dfferences were found among the fve feedng groups. Lkewse, fat stans of lver were all essentally negatve and no nter group dfferences were detected. Kdneys were examned, usng hematoxyln and eosn sectons, for nephrocalcnoss, ntersttal nflammaton, fatty changes n tubules and ntersttal fatty nfltraton; no dstnct dfferences were found relatve to the content of CPF A n the dets. However, those females ngestng ether corn ol (group E) or cottonseed ol (group B and D) at the 2So/o detary level dd have an ncrease n occurrence of nephrocalcnoss, as shown n Fg. 2. Fat stans on kdney sectons WEEKS Fgure 1. Growth rates of rats. Body weghts are plotted aganst age. The symbol (e) represents group A contanng So/o cottonseed ol and 6 1-1glg CPFA n the det; <<» represents group B contanng 25% cottonseed ol and 3, 1-1glg CPFA n the det; (D) represents group C contanng 5% mproved cottonseed ol and 6 1-1g/g CPFA n the det; (.) represents group D contanng 25o/o mproved cottonseed ol and 3 1-1g/g CPF A n the det; (A) represents group E contanng 25% corn ol and 1-1g/ g CPFA n the det. TABLE 2. Comparson ofdaly.food consumpton. Det Grams of food consumed per dayd CPFA 4;g/g) group Ol Male rats Female rats 6 A 5%csoa ± ± c 5%1-csob 2.62 ± ± 1.1 3, B 25%csoa ± ± D 25%1-csob ± ± 1.39 E 25%CQC ± ()4 ± 1.36 acottonseed ol contanng 1.19%CPFA, the same as n Table 1. blmproved cottonseed ol contanng.12% CPFA, the same as n Table 1. ccorn ol. d Daly food consumpton, average± standard devaton. TABLE 3. CPFA CPF A content Fgure 2. Kdney wth nephrocalcnoss, 44 tmes magnfca ton. Note the dark rregular masses representng calcum deposts at the cortcomedullary juncton. In det 4;g/ g) bol (wt. o/o) Male O.D , Female Male Female O.D2 O.D

4 CYCLOPROPENOID FATTY ACIDS FED TO RATS 51 showed no apparent dfferences n the lpd content among the groups. The hematoxyln and eosn sectons of lung demonstrated some cases of focal ntersttal pneumonts wth occasonal hemorrhagc pneumonts. These changes agan dd not concde wth partcular detary groups and were rrespectve of the amount of detary CPFA or the detary fat. In summary, these results ndcate that there were no dstnct CPFA-related effects n the hstopathologcal evaluatons of lver, kdney, and lung. Body and organ weght The body weght, organ weght and the rato of organ to body weght were determned after 3 months of feedng (fable 4) and after 6 months of feedng (fable 5). No sgnfcant dfferences among the fve groups were detected. Bochemcal assys of lver mcrosomes NADPH-cytochrome(P-45) reductase actvty, and cytochromes P-45 and b 5 concentratons were determned n solated lver mcrosomes of rats after 3 and 6 months of feedng (fable 6). Agan, there was no sgnfcant dfference when rats of the same sex and feedng perod were compared. DISCUSSION In constrast to the present fndng that there was no dstnct dfference n growth rate wth respect to CPF A content, t has been shown (18,24) that growth rates were retarded when hgher levels of CPF A (1, - 25, IJ.g/g derved from SFO) were fed to rats. It s nterestng to note that rats on 25% dets consumed less food than those on So/o-fat dets and yet had a smlar weght gan wthn each sex group. Ths s predctable because hgh fat dets suppled more energy per unt weght bass. It would be nterestng to know what s the control mechansm for food ntake. Even though female rats exposed to 25% fat had a hgher frequency of nephrocalcnoss (Fg. 2), ths appears to be unrelated to serum levels of calcum (based on SMAC data). The reason for nephrocalcnoss s unknown. Agan, the hstopathologcal fmdngs are dfferent from that of hgh dose CPFA (SFO)-fed rats (18), whch exhbted pathologcal abnormalty. The present study ndcates that there were no detrelated dfferences n body weghts or organ weghts, whch were smlar to those of cottonseed ol fed rats (1 9), but are dfferent from those of SFO-fed rats (18). TABLE 4. Body and organ weghfl (after 3 months of feedng). CPFA n det Det 4;g/g) Sex group Body weght Lver weght Male E 451 ± 22b 11.8 ± Male c 458 ± ± 1. 3 Male D 432 ± ± 2. 6 Male A 449 ± ± 1.4 3, Male B 462 ± ± 2.1 Female E 264 ± ±.9 6 Female c 25 ± ±.4 3 Female D 263 ± ±.3 6 Female A 256 ± ±.7 3, Female B 282 ± ±.9 Kdney weght Lver wt./body vvt. Kdney wt./body wt. 3.1 ±.2.62 ± ± ±.2.67 ±.33.7 ±.3 3. ±..73 ± ±. 3.3 ±.2.84 ± ± ±.4.74 ± ±.7 2. ±.3.5 ±..75 ± ±.3.5 ±..75 ±.7 2. ±.2.8 ±.1.74 ± ±.1.6 ±..73 ±.1 2. ±.2.7 ±.3.7 ±.1 a No sgnfcant dfference wth respect to males (2 data) and females (2 data), separately, based on Duncan's test. beach value represents mean± S.D. from 4 rats per male and female group, respectvely. TABLE 5. CPFA n det Det 4;g/g) Sex group Body weght Lver weght Kdney weght Lver wt.!body wt. Male E 572 ±JOb 14.2 ± ±.2.49 ±.1 6 Male c 49 ± ± ±.3.72 ±.1 3 Male D 5 ± ± ±.3.39 ±.16 6 Male A 517 ± ± ±.2.53 ±.7 3, Male B 556 ± ± ±.4.44 ±.16 Female E 31 ± ± ±.3.34 ±.2 6 Female c 263 ± ± ±.3.56 ±.1 3 Female D 273 ± ± ±.3.75 ±.5 6 Female A 271 ± ± ±.1.35 ±.1 3, Female B 34 ± ± ±.1.45 ±.7 a No sgnfcant dfference wth respect to males (2 data) and females ( data), separately, based on Duncan's test. beach value represents mean± S.D. from 4 rats per male and female group, respectvely. Kdney wt./body wt..64 ±.3.72 ±.5.65 ±.5.69 ±.3.65 ±.4.62 ±..7 ±.7.77 ± ±.5.68 ±.3

5 52 FAN. HILTON AND WOHLMAN TABLE 6. Bochemcal assays of lver mcrosomal fracton a. Mllgrams of CPFA mcrosomal proten NADPH-cytochrome c n det per gram of lver Cytochrome P-45b Cytochrome b5b reductasec (J.;g/g) Sex 3Months 6 Months 3 Months 6Months 3 Months 6 Months 3Months 6 Months Male 9.29 ± ± ± ± ±.9.33 ± ± ± Male 1.77 ± ± ±.1 LOS± ±.7.52 ± ± ± Male 1.48 ± ± ± ± O.o9.54 ±.12.5 ± ± ± 1 6 Male 9.83 ± ± ±.2.95 ±.1.44 ±.1.48 ± ± ± Male ± ± ±.9.92 ±.6.47 ±.8.47 ± O.ot 86.7 ± ± 15.4 Female ± ± ±.1.83 ±.8.41 ±.5.56 ± ± ± Female 1.68 ± ± ±.5.79 ±.6.35 ±.1.55 ± ± ± Female 9.91 ± ± ±.5.82 ±.1.37 ±.9.53 ± ± ± Female 1.47 ± ±.7.71 ±.5.7 ±.5.36 ±.6.46 ± ± ± Female 12.5 ± ± ±.8.75 ±.1.4 ±.7.51 ± ± ± 21.8 ano sgnfcant dfference wth respect to males (2 data) and females (2 data), separately, ether after 3 months or 6 months. bnanomoles of enzymes per mllgram of mcrosomal proten. cnanomoles cytochrome c reduced per mnute per mllgram of mcrosomal proten. The enzyme NADPH-cytochrome. (P-45) reductase as well as cytochromes P-45 and b 5 are components of the mcrosomal electron transport systems that partcpate n the metabolsm of endogenous substrates such as sterods and fatty acds, and n the metabolsm of many exogeneous substrates, ncludng varous drugs and carcnogens (23). Several cytochrome P-45 soenzymes have been reported recently; each has a slghtly dfferent absorpton maxmum. Baley et al. (1) showed that the actvty of lver mcrosomal NADPH-cytochrome _!;_ (P-45) reductase and the hepatc content of cytochrome P-45 were altered followng feedng of SA or MA to ranbow trout. In the present study, the spectra of the hepatc cytochrome P-45 system from both CPF A-free and CPFA-fed groups were dentcal, and the quanttes of cytochrome P-45 were smlar n both groups. The alteratons of the hepatc mcrosomal system, as observed n ranbow trout under dfferent condtons, dd not appear to occur n rats. The only detectable dfference among the fve feedng groups was the amount of CPFA deposted n body fat (Table 3). Ths dose-related response s n agreement wth a prevous fndng (19). The metabolsm of CPFA has been nvestgated by Snnhuber and hs assocates, utlzng radoactvely labeled sterculc acd (5,6,2). They suggested that the CPF A contaned n fat s readly absorbed, and degraded products are excreted n the urne as short chan dcarboxylc acds. However, the metabolc pathways of CPFA are not well understood. One mght speculate that the adverse bologcal effects resultng from hgh doses of detary CPF A may be caused by elevated CPF A levels deposted n body fat. Hendrcks et al. (1), Snnhuber et al. (27) and Baley et al. (J) conducted smlar studes on ranbow trout. Ther data suggest that CPF A may be carcnogens and co-carcnogens wth aflatoxns; and SA has been shown to be more actve than MA. The major dfference between cottonseed ol and SFO s that MA s the major CPF A n the former ol, whle SA s the major CPFA n SFO. SFO has not been known to be used as a detary fat. The results of the present subchronc feedng study ndcate that there are no detrmental effects of small amounts of CPF A occurrng naturally n cottonseed ol. The long human experence wth cottonseed ol as a source of detary fat lends addtonal support to ths concluson (16). ACKNOW'LEDGMENTS The authors thank Ms. Gal Womack for her excellent techncal assstance and Dr. Joseph D. Lynn for hs valuable hstopathologcal consultaton. Many thanks to Dr. Randall Wood of Texas A & M Unversty, College Staton, Texas, for hs nformaton on methyl sterculate; to Drs. Russell Snnhuber and Joseph Nxon of Oregon State Unversty, Corvalls. Oregon, for useful nformaton; to Drs. Stuart Cagen. Ronald Carlott, and Bob Ln of Frto-Lay, Inc., and Dr. Bette Sue Sler Masters of the Unversty of Texas Health Scence Center at Dallas for ther crtcal revews of ths manuscrpt. REFERENCES 1. Baley, M. L.. J. E. Nxon, J.D. Hendrcks, N. E. Pawlowsk, P. M. Loveland, and R.. Snnhuber Comparatve effects of sterculc and malvalc acds on the hepatc cytochrome P-45 system and tumor nducton n ranbow trout. Fed. Proc. 38: Carson, F. L., J. H. Martn, and J. A. Lynn Formaln fxaton for electron mcroscopy: a re-evaluaton. Am. J. Cln. Pathol. 59: Carter, F. L., and V. L. Frampton Revew of the chemstry of cyclopropene compounds. Chern. Rev. 64: Deutschman, A. J. Jr., and I. S. Klaus Spectrophotometrc determnaton of sterculc acd. Anal. Chern. 32: T. A. Esele, J. K. Yoss. J. E. Nxon, N. E. Pawlowsk, L. M. Lbbey, and R.. Snnhuber Rat unnary metaboltes of [9, 1-methylene- 14 C] sterculc acd. Bochm. Bophys. Acta 488: Esele, T. A., R. S. Parker, J. K. Yoss, J. E. Nxon, N. E. Pawlowsk, and R.. Snnhuber Incorporaton of label from [9, 1-methylene 14 C] sterculc acd n ranbow trout, Salmo gardner. Lpds 14: Estabrook, R. W.. J. Peterson. J. Baron, and A. Hldebrandt The spectrophotometrc measurement of turbd suspensons of cytochromes assocated wth drug metabolsm. pp In C. F. Chgnell (ed.) Methods n pharmacology. Meredth Corporaton. New York.

6 CYCLOPROPENOID FATTY ACIDS FED TO RATS Frend, B., Changes n nutrents n the U.S. det caused by alteratons n food ntake patterns. Agrcultural Research Servce, U.S. Department of Agrculture. p Halphen, G A characterstc reacton of cotton ol. Analyst 22: Hendrcks, J.., R.. Snnhuber, P. M. Loveland, H. E. Pawlowsk, and J. E. Nxon Hepatocarcnogencty of glandless cottonseeeds and cottonseed ol to ranbow trout (Salmo gardner). Scence 28: Hle, J. P., Modfed cottonseed products ntended for human consumpton. Fed. Reg. 43: Lee, D. J., J. H. Wales, and R.. Snnhuber Synergsm between cyclopropenod fatty acds and chemcal carcnogens n ranbow trout (Salmo gardnen). Cancer Res. 28: Lee, D.J., J. H. Wales, and R.. Snnhuber Promoton of aflatoxn-nduced hepatoma growth n trout by methyl malvalate and terculate. Cancer Res. 31: Lowry,. H., N.J. Rosebrough, A. L. Farr, and R. j_ Randall Proten measurement wth the Foln phenol reagent. J. Bo. Chem.193: IS. Masters, B.S. S., C. H. Wllams, Jr., and H. Kamn The preparaton and propertes of mcrosomal TPNH-cytochrome C reductase from pg lver. pp In R. W. Estabrook and M. E. Pullman (ed.) Methods n enzymology, Vol. X. Academc Press, New York. 16. Mattson, F. H., Potental toxcty of food lpds. pp Commttee on Food Protecton, Food and Nutrton Board, Natonal Research Councl (ed.) Toxcants occurrng naturally n foods. Natonal Academy of Scences, Washngton, D.C. 17. Mtruka, B., and M. Rownsley Clncal bochemcal and hematologcal reference values n normal expermental anmals. pp Nxon, 1. E., T.A. Esele, 1. H. Wales, and R.. Snnhuber Effect of subacute toxc levels of detary cyclopropenod fatty acds upon membrane functon and fatty acd composton n the rat. Lpds 9: Nxon,J. E., R.. Snnhuber, D.J. Lee, M. K. Landers, and J. R. Harr Effect of cyclopropenod compounds on the carcnogenc actvty of dethylntrosamne and aflatoxn B 1 n rats. J. Nat!. Cancer Inst. 53: Nxon, J. E., J. K. Yoss, T. A. Esele, N. E. Pawlowsk, and R.. Snnhuber Metabolsm and tssue dstrbuton of label from [9, 1-methylene- 14 C] sterculc acd n the rat. Lpds 12: Phelps, R. A., F. S. Shenstone, A. R. Kemmerer, and R. J. Evans A revew of cyclopropenod compounds: bologcal effects of some dervatves. Poult. Sc. 44: Remmer, M., H. Grem, J. B. Schenkman, and R. W. Estabrook Methods for the elevaton of hepatc mcrosomal mxed functon oxdase levels and cytochrome P-45. pp In R. W. Estabrook and M. E. Pullman (ed.) Methods n enzymology, Vol. X. Academc Press, New York. 23. Sato, R., and T. Omura Cytochrome P-45. Academc Press, New York. 24. Schneder, D. L., E. T. Sheehan, M. G. Vevch, and A. R. Kemmerer Effect of Stercula foetda ol on weanlng rat growth and survval. J. Agr. Food Chern. 16: Sheostone, F. S., and J. R. Vckery Occurrence of cyclopropene acd n seom plants of the order Malvales. Nature 19: Snnhuber, R.., D. J. Lee, J. H. Wales. and J. L. Ayres Detary factors and hepatoma n ranbow trout (Salmo gardnen} II. Co-carcnogeness by cyclopropenod fatty acds and the effect of gossypol and altered lpds on aflatoxn-nduced lver cancer. J. Nat. Cancer Inst. 41: Snnhuber, R.., D. J. Lee, J. H. Wales, M. K. Landers, and A. C. Kcyl Hepatc carcnogeness of aflatoxn M 1 n ranbow trout (Salmo gardnen) and ts enhancement by cyclopropenod fatty acds. J. Nat. Cancer Inst. 53: Steel, R. G. D., and 1. H. Torre Analyss of varance 1: the one-way classfcaton. pp In Prncpals and procedures of statstcs. McGraw-Hll Book Company, Inc., New York, Toronto, London. 29. Yoss, J. K Metabolsm of 14 C-sterculc acd n rats and ranbow trout (Salmo gardnen). Publshed on demand by Unversty Mcroflms Internatonal, Ann Arbor, Mchgan, U.S.A., London, England. JOURNAL OF FOOD PROTECTION, VOL 45, JANUARY 1982