[CANCER RESEARCH 55, 2293-2298, June I, 1995] Dietary Fat, Fiber, and Carcinogen Alter Fecal Diacylglycerol Composition and Mass1 Jennifer S. Pickering, Joanne R. Lupton, and Robert S. Chapkin2 Faculty of Nutrition, Molecular and Ceil Biology Croup, Texas A&M University, College Station, Texas 77843-247ÃOE ABSTRACT Fecal diacylglycerols (DAGs) are known activators of protein kinase C (PKC), which in turn modulates colonie epithelial cell growth programs and, therefore, could play a role in the malignant transformation process. However, the effects of physiological modifiers such as diet and carcinogen on fecal DAG mass and composition have not been reported. We therefore designed a 2 x 2 x 2 factorial study (2 fats: corn oil and fish oil; 2 fibers: pectin and cellulose; with and without carcinogen). Rats were provided with diets for 5 weeks. Three weeks after the second injection of azoxymethane, feces were collected from 10 rats/treatment in = 80 total) and analyzed for DAG mass and fatty acyl composition by combined TLC and gas chromatography. Dietary fat had a significant effect on the mol % fatty acyl composition of fecal DAG. Greater amounts of long chain n-3 polyunsaturated fatty acids (20:Sn-3, 22:Sn-3, and 22:6n-3) were detected in fecal DAG of fish oil-fed animals relative to corn oil (/' < 0.001). In contrast, corn oil resulted in a higher mol % of 18:2n-6 relative to fish oil (P < 0.016). The most salient effect of fiber was on total production (nmol/day) of DAG, which was 2.5 times higher with cellulose than pectin supplementation. In addition, there was an effect of fiber on both mol % and concentration of 22:6n-3, with cellulose producing higher amounts relative to pectin (P < 0.04). A significant interaction between fat and fiber was observed with nmols of 17:0 excreted in 24 h, with fish oil/cellulose producing 94.2 nmol as compared to 3.5 seen with corn oil/pectin (P < 0.02). There was a significant interaction between fat and carcinogen on all of the DAG n-3 fatty acids, which were elevated with carcinogen/fish oil treatment. These data show that fat, fiber, and carcinogen can modu late the fatty acyl composition and mass of fecal DAG. Since the produc tion of fecal DAG, an activator of PKC, may alter colonie mucosa! cell proliferation, our data offer insight into a mechanism by which diet may modify the risk of colon cancer development. INTRODUCTION PKC'1 consists of a family of serine/threonine kinases that upon activation can phosphorylate proteins that regulate short- and long- term cellular events, including cell proliferation and differentiation. It is now clear that PKC expression and activation are involved in the regulation of colonie mucosa! proliferation (1-4). This is noteworthy because the elevation of colonie cell proliferation has been associated with the incidence of colon cancer (5). DAGs link extracellular signals with intracellular responses through the activation of PKC (6). Intracellular DAG produced from inositol and choline phospholipid hydrolysis, coupled with increased Ca2+ levels, can activate PKC to influence cellular processes (7). It is interesting that several studies have recently shown that rat and human feces contain appreciable levels of DAG (8-11). This is significant Received 1/4/95; accepted 4/4/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by grants from the American Institute for Cancer Research, the Texas A&M Interdisciplinary Research Initiatives Program, and NIH Grants CA59034 and CA61750. 2 To whom requests for reprints should be addressed, at 442 Kleberg Center, Texas A&M University, College Station, TX 77843-2471. 3 The abbreviations used are: PKC, protein kinase C; DAG, 1,2-diacyl-JTi-glycerol; AAG, l-O-alkyl-2-acyl-sn-glycerol; A'AG, l-O-alkenyl-2-acyl-jn-glycerol; ANS, anilino-1-naphthalene sulphonic acid ammonium salt; AOM, azoxymethane; PUFA, polyunsaturated fatty acid. because DAG in the colonie lumen is capable of entering mucosal cells of the colon (9), where it can activate the PKC pathway (12). In addition, extracellular DAGs of the chain length found in feces can enhance the growth of benign colonie tumors and some carcinomas, while normal cells are not affected (13). These studies suggest that fecal intraluminal DAG may have implications in the development of colon cancer. DAG has been detected in foods at appreciable levels, although only negligible amounts of dietary DAG reach the colon intact (9). Sources of DAG in the lumen have not been elucidated, although it may be derived partly from bacterial action (8). As a result of these observations, interest has focused recently on the ability of dietary constituents to modulate intraluminal DAG levels. For example, Reddy et al. (10) have demonstrated that the type of fiber consumed can modify fecal DAG mass and composition. Dietary fat and fiber have interactive effects in the colon (14) and are capable of influencing colonie PKC isoform expression (15). However, the combined effects of dietary fat and fiber on fecal DAG mass and composition have not been examined. Therefore, the present study was designed to investigate the effects of dietary fat and fiber on the mass and fatty acyl composition of fecal DAG in the rat experi mental colon carcinogen model. In addition, fecal diglyceride sub classes were characterized to determine aliphatic linkage at the sn-\ chemical position. MATERIALS AND METHODS Materials. l,2-diheneicosanoyl-.vn-glycero-phosphocholine (21:0-21:0-PC) was purchased from Avanti Polar Lipids (Alabaster, AL). Phospholipase C (Type V, Bacillus Cereus) and azoxymethane were from Sigma Chemical Co. (St. Louis, MO). Silica gel 60 plates (20 x 20 cm, 0.25-mm thickness) for TLC were from Merck (Darmstadt, Germany). Bakerbond silica gel columns were purchased from J. T. Baker, Inc. (Phillipsburg, NJ). Dextrose was from HarÃ-anTeklad (Madison, WI). Cellulose, casein, DL-methionine, choline bitartrate, AIN-76 vitamin, and AIN-76 mineral mix were purchased from Bio-Serve (Frenchtown, NJ). Corn oil was kindly provided by Traco Labs (Seymour, IL). Vacuum-deodorized Menha den fish oil was provided by the NIH Fish Oil Test Material Program, Southeast Fisheries Center (Charleston, SC). High methoxylated pectin was from Grindsted (Industrial Airport, KS). Animals and Diets. The animal use protocol for these experiments was approved by the University Animal Care Committee at Texas A&M Univer sity. All animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NRC 1985). Eighty male weanling Spraguc- Dawley rats (HarÃ-an, Houston, TX) were housed individually in cages in a temperature- and humidity-controlled facility with a 12-h light/dark cycle. After a 3-day acclimation period of consuming a nonpurified diet, rats were assigned randomly to one of 8 treatments (10 rats/treatment) ina2x2x2 factorial design with 2 fats, 2 fibers, with or without carcinogen. The diet compositions are shown in Table 1. The two fats were fish oil (11.5 g/100 g diet) and corn oil (15 g/100 g diet). The fish oil diet contained 3.5 g corn oil/100 g diet to ensure that essential fatty acid requirements were met (14). Corn oil and fish oil contained identical levels of tert-butyl-hydroquinone (0.025%) and vitamin E (1.5 mg/g a-tocopherol and 1.0 mg/g y-tocopherol). The two fibers were pectin and cellulose (6 g/100 g diet). Each group was divided further into + or - carcinogen treatment. Food and water were available freely. Carcinogen Administration. After 1 week of consuming semipurified diets, the rats were given injections 2 times at 1-week intervals with 2293 Research. on December 9, 2015. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from