[CANCER RESEARCH 43, 5761-5767, December 1983] Metabolism of Pancreatic Carcinogens N-Nitroso-2,6-dimethylmorpholine and A/-Nitrosobis(2-oxopropyl)amine by Microsomes and Cytosol of Hamster Pancreas and Liver1 Demetri M. Kokkinakis, Dante G. Scarpelli,2 M. Sambasiva Rao, and Paul F. Hollenberg Department of Pathology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 ABSTRACT Liver preparations from Syrian golden hamsters catalyze the metabolism of the pancreatic carcinogen A/-nitroso-2,6-dimethyl- morpholine largely to A/-nitroso(2-hydroxypropylX2-oxopropyl)- amine (HPOP). This reaction is catalyzed by a mixed-function oxidase in the presence of reduced nicotinamide adenine dinu- cleotide phosphate and oxygen at a rate of 3.8 nmol/min/mg of protein, and it is inhibited by known cytochrome P-450-specific inhibitors. A second potent pancreatic carcinogen A/-nitrosobis(2- oxopropyljamine (BOP) is converted to HPOP by hamster liver in which two enzyme systems appear to be involved. The first is a reducÃ-aseassociated with microsomes which reduces BOP to HPOP in the presence of reduced nicotinamide adenine dinucleo- tide at a rate of 9.1 nmol/min/mg of protein. The second enzyme is a cytosolic one which catalyzes the same reaction at a slower rate (2.3 nmol/min/mg of protein) and is more effective with reduced nicotinamide adenine dinucleotide phosphate as cofac- tor. Based on the amount of protein in hepatic cytosol and endoplasmic reticulum, the two enzymes may be involved to a similar extent in the reduction of BOP to HPOP in the liver. Pancreas, on the other hand, lacks the microsomal reducÃ-ase for BOP but contains a cytosolic enzyme which catalyzes its reduction in the presence of reduced nicotinamide adenine di nucleotide phosphate at a rate of 0.35 nmol/min/mg of protein. Since both pancreatic carcinogens A/-nitroso-2,6-dimethylmor- pholine and BOP are metabolized to HPOP in the liver at rates much higher than those observed in the target organ pancreas, it is suggested that the liver may play an important role in pancreatic carcinogenesis in the hamster by these compounds. INTRODUCTION Sensitivity of the pancreas of the Syrian golden hamster to a variety of A/-nitrosamine carcinogens has led to the development of an animal model (11,12,15,16, 20) of considerable utility for experimental studies of the pathogenesis (11, 15, 24), biology (18, 21), and chemotherapy (1) of ductal adenocarcinoma. NNDM3 and BOP and their metabolites HPOP, BHP, MOP, and MHP are potent carcinogens with considerable organotropy for Received August 30,1982; accepted August 31,1983. 1This work was supported in part by the Edith Patterson and Marie A. Fleming cancer research funds, Northwestern University, and by Grants CA34051 and CA16954 from the National Cancer Institute, USPHS. 1 To whom requests for reprints should be addressed. 'The abbreviations used are: NNDM, A/-nitroso-2,6-dimethylmorpholine; BOP, W-nitrosobis(2-oxopropyl)amine; HPOP, N-nitroso(2-hydroxypropyl) (2-oxopropyl)- amine; BHP, N-nitrosobis(2-hydroxypropyl)amine; MOP, W-nitrosomethyl(2-oxopro- pyl)amine; MHP, W-nitrosomethyl(2-hydroxypropyl)amine; HPLC, high-pressure liq uid chrornatography; S-9, 9000 x g supernatant of a tissue homogenate; NMR, nuclear magnetic resonance. Received August 30, 1982; accepted August 31, 1983. the pancreas. Accordingly, this class of carcinogens has been the focus of numerous studies during the past several years. Their mutagenicity has been examined with the Ames assay (9, 22) and Chinese hamster V79 cells (9) and their metabolic degradation has been followed with considerable detail (2-7,21, 26, 28). With few exceptions (21, 24, 28), however, the majority of metabolic studies have involved whole animals and were aimed at elucidating metabolic pathways by the identification of various metabolites in body fluids as well as describing the kinetics of their appearance and disappearance. Although these have contributed substantially to our understanding of the met abolic degradation of certain nitrosamines, they have not pro vided the infomation needed for elucidation of the sequence of enzymatic reactions leading to formation of the proximate car cinogen, nor have they identified the sites of metabolism and their relation to the target organ. It is significant that our understanding of the metabolic events by which other carcinogens such as benzo(a)pyrene, acetylami- nofluorene.and aflatoxin Bi are activated to their ultimate carci nogenic forms was enhanced greatly by studies of their metab olism by target tissues in vitro rather than by in vivo studies. Full exploitation of the hamster model of pancreatic cancer for the details of the chemical and cellular events of A/-nitrosamine carcinogenesis will no doubt also require a greater understanding of the role of the pancreas in the metabolism of this class of carcinogens. The present studies are part of an ongoing effort toward establishing the details of W-nitrosamine metabolism by the pancreas. In this instance, pancreatic metabolism of NNDM and BOP is characterized and compared with that by the hamster liver. MATERIALS AND METHODS Chemicals. NNDM, BOP, HPOP, BHP, MOP, MHP, and [3H]NNDM (labeled at the «positions; specific activity, 1.68 mCi/mmol) were sup plied generously by Dr. William Lijinsky of the Frederick Cancer Research Center. [14C]HPOP (specific activity, 4 mCi/mw) was obtained from Amersham Corp., Chicago, III. The NADPH, NADH, NADP+, and NAD+ were obtained from the Sigma Chemical Co. (St. Louis, Mo.). Acetonitrile (gold label) was obtained from Aldrich Chemical Co. Double-distilled water used for HPLC analysis was adjusted to pH 7.4 with sodium bicarbonate and filtered through a 0.42-Mm pore size Millipore filter (Millipore Corp., Bedford, Mass.). All other chemicals used were analytical reagent grade from commercial suppliers. Isolation of S-9 and Microsomes from Liver and Pancreas. Male Syrian golden hamsters (Chartes River Breeding Laboratories, Inc., Wil mington, Mass.) weighing 100 to 120 g were maintained on a hamster diet (Teklad test diets; Teklad, Madison, Wis.) and starved 20 hr before killing. Livers were removed, weighed, minced, placed in 4 volumes of buffer (0.1 M Tris:0.15 M KCI:1 mw EDTA, pH 7.4), and homogenized (10 to 15 strokes) in a Potter-Elvehjem homogenizer with Teflon pestle. DECEMBER 1983 5761 Research. on November 28, 2021. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from