Metabolism of 3-Methylcholanthrene in Rat Pancreas

OWEN BLACK, JR, PhD, EVELYN MURRILL, PhD, and CINDY FANSKA

In vitro and in vivo studies revealed that pancreases of Long-Evans male rats metabo- lized 3-methylcholanthrene (3MC) principally at the 1- and 2-carbon positions. The pan- creatic metabolizing capability was not induced by pretreatment with either ben- zo(a)pyrene (BP) or phenobarbital (PB). During the period of observation from 5 to 48 hr, the maximum tissue content of radioactivity observed in the in vivo studies occurred at 16 hr after injection of 3MC in both liver and pancreas. Pancreatic tissue retained about four times more carcinogen per gram of tissue than liver tissues at all time periods.

A major research effort is presently underway to study the nature and causes of pancreatic cancer. The incidence of pancreatic cancer is rising in the United States; it is now the fourth leading cause of cancer deaths (1). The etiology of pancreatic cancer is not as well understood as some other organ can- cers. The incidence of pancreatic cancer in workers exposed to industrial chemicals was five times high- er than expected (2). For this reason, it is important to examine the link between pancreatic cancer and exposure to carcinogens.

It has been established that a large number of car- cinogens, such as polycyclic hydrocarbons, require metabolic activation to become carcinogenic. A few

Manuscript received June 17, 1980; revised manuscript re- ceived October 13, 1980; accepted October 13, 1980.

From the Cancer Research Laboratory, Medical Research Service, Veterans Administration Medical Center (Downtown Division); Departments of Medicine and Cell and Molecular Bi- ology, Medical College of Georgia, Augusta, Georgia; and Mid- west Research Institute, Kansas City, Missouri.

A portion of this work was presented at Southeastern Section, American Association for Cancer Research, Atlanta, Georgia, October 7, 1976, and in abstract form at the meetings of the Fed- eration of American Societies for Experimental Biology, Chi- cago, Illinois, April 1-7, 1977.

This work was supported by the Medical Research Service of the Veterans Administration and Contract N01-CP-55656 with the National Cancer Institute.

Address for reprint requests: Owen Black, Jr., 509/15 (DD), Cancer Research Laboratory, Veterans Administration Medical Center, Augusta, Georgia 30910.

studies have demonstrated that pancreatic tissue is capable of metabolizing drugs and carcinogens (3- 5). These studies have shown that the capability of pancreatic tissue to biotransform drugs is much less than the liver; however, few mechanisms of this process have been demonstrated. The extent to which the pancreas is able to produce carcinogens in situ will, in part, determine the risk for sub- sequent tumor development in the tissue.

The pancreas, unlike skin and lungs, is normally not directly exposed to carcinogens. A recent his- tofluorescence study suggested that pancreatic duct cells received a significant exposure to carcinogen metabolites (6). Thus, not only is it important to un- derstand the mechanism of action of carcinogens in pancreatic tissue, it is important to determine the temporal exposure of pancreatic tissue to exoge- nously and endogenously produced metabolites. The metabolism of 3MC by the pancreas has not been described previously.

This report is one of a series of investigations to determine whether the pancreas is capable of me- tabolizing certain carcinogens. Knowledge of which carcinogens are metabolized by pancreatic tissue and which are not will serve to characterize, in part, the pancreatic metabolic system. The purpose of the studies reported herein is threefold. We wished (1) to determine the capability of the pancreas to metabolize 3-methylcholanthrene (3MC) and to de-

358 Digestive Diseases and Sciences, Vol. 26, No. 4 (April 1981) 0163-2116/81/0401~0358503.00/1 �9 1981 Digestive Disease Systems, Inc.

METABOLISM OF 3MC IN RAT PANCREAS

termine if the drug-metabolizing system can be in- duced; (2) to elucidate the metabolic pathways in- Volved in the pancreas by examining the products of metabolism; and (3) to determine the temporal ex- posure by measuring the relative amounts of radio- activity appearing in pancreas, liver, bile, and blood for periods up to 36 hr following injection of radio- active 3MC.

MATERIALS AND METHODS

Long-Evans male rats, 350-400 g in weight, were used for these studies. The animals were maintained in a tem- perature- and light-controlled environment and had free access to Wayne Lab-Blox food and water. Non- radioactive 3MC and benzo(a)pyrene (BP) were pur- chased from Eastman Kodak Co. (Rochester, New York) while radioactive 3MC was purchased from New England

,Nuclear Co. (Boston, Massachusetts) (6-~4C; 60.2 mCi/ raM). Radiochemical purity of the sample was verified as being 99% by thin-layer chromatography using benzene- ethanol (9:1) solvent and was used without further purifi- cation. Chromatographic standards of ll-hydroxy 3MC (lbOH); 11, 12 oxide 3MC; 1-hydroxy 3MC (1-OH); 2- hydroxy 3MC (2-OH), and 11,12-quinone 3MC were fur- nished by the Information and Resources Segment of the National Cancer Institute. Induced rats were treated with either 20 mg/kg BP in DMSO or 75 mg/kg phenobarbital (PB) in saline once daily for four days. The animals were killed 24 hr after the last injection by cervical dislocation.

In Vitro Methods. The following methods are those de- veloped by Sims (7). The pancreases and livers from con- trol or pretreated rats were removed and chilled, trimmed, weighed and homogenized in cold 1.15% KCI as a 20% homogenate.

The homogenate was centrifuged in a Sorvall centrifuge at 1500g for 20 min. The supernatant from this spin was used for the incubation so as to include what soluble con- jugation enzymes were present. Experiments using tryp- sin inhibitor at 0.1 mg/ml and 0.5 mg/ml final concentra- tions indicated that pancreatic protease activity had little effect on the results. Three ml of the supernatant (10-15 mgtml protein) were incubated with an equal volume of 0.1 M phosphate buffer (pH 7.4), so that the final solution contained 0.2 mM NADP, 4.4 mM glucose-6-phosphate, and 0.03/xmol [~4C]3MC. Preliminary experiments estab- lished that the above conditions were optimal for pancre- atic activity in regard to tissue concentration and in- cubation component requirements. Time-dependence studies revealed that a 1-hr incubation was necessary to observe products that were at least 1% of the total radio- activity. The mixture was incubated in duplicate at 37 ~ C. Tissue samples that had been boiled 10 min were used as blanks. The incubation mixtures were extracted seven times with four volumes of ethyl acetate. The organic or ethyl acetate phase was reduced to dryness in a rotary evaporator and resuspended to 5 ml with ethyl acetate. Volumes of the aqueous fractions were brought up to 10 ml. Aliquots of both phases were examined for radio- activity by scintillation counting.

In Vivo Methods. Animals were anesthetized with an intraperitoneal injection of 40 mg/kg phenobarbital. A tracheotomy was routinely performed. A cannula was in- serted into the pancreatobiliary duct to collect bile exclu- sive of pancreatic juice. Collections were started 5, 10, 16, 22, 36, and 48 hr after intraperitoneal injection of 10 /zCi [14C]3MC (0.17 tzmol suspended in DMSO). These time periods were selected from previous studies (3). Af- ter the collection period (up to 3 hr), a sample of blood was removed from the heart and the animals were killed. The pancreases and livers were excised, rinsed in saline, trimmed, and weighed. A 20% homogenate was prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were extracted with ethyl acetate as described in the pre- ceding in vitro section.

High-Pressure Liquid Chromatographic Methods. Or- ganic fraction samples were taken to dryness and redis- solved by sonication in methanol with a known mixture of unlabeled 3MC standards. The samples were injected in a Waters high-pressure liquid chromatograph (HPLC) which had a Cz8/xBondapak (300 x 4 min ID) column. A linear elution program was used with an initial solvent composition of methanol-water (62:38) and a final compo- sition of methanol-water (78:22) run over a 50-min time period. Metabolites in the fractions were detected with a 254- and 313-nm ultraviolet light detector and collected for scintillation counting. Metabolite identification was based both on comparison of retention time and radio- active peaks to retention times of known unlabeled stan- dards present in the samples, and on absorbance ratios of the sample peaks compared to standards.

Statistical procedures used to determine mean and standard errors and statistical significance were taken from Steel and Torrie (8).

RESULTS

The quantities of metabolic products which parti- tioned to the aqueous phase of the extraction mix- ture are present in Table 1, while the products ap- pearing in the organic phase are presented in the HPLC profiles in Figure 1. The pancreas metabo- lized 1.7% of the 3MC to water-soluble metabolites. Liver converted the same percentage of 3MC to wa- ter-soluble products. The pancreatic capability to produce water-soluble products was not increased when rats were pretreated with either BP or PB, whereas liver increased the water-soluble radio- activity more than twofold. There was a slight but not statistically significant (Student 's t test) drop in water-soluble products in pancreatic samples fol- lowing BP or PB pretreatment.

Figure 1A presents the H P L C profile of the me- tabolites in the organic extract from in vitro in- cubated pancreatic homogenates. A major site of metabolism of 3MC in the pancreas is at the 1,2 po- sition. This is evidenced by the presence of 1-OH/2- OH (peak B), and 1-one/2-one (peak E), and 1,2-diol

Digestive Diseases and Sciences, Vol. 26, No, 4 (April 1981) 359

BLACK ET AL

TABLE 1. PARTITION OF CARCINOGEN BETWEEN ORGANIC AND AQUEOUS PHASES FROM TISSUES OF RATS PRETREATED WITH BP OR PB AND INCUBATED In Vitro*

Radioactivity (dpm/g tissue • 10 -3)

Pancreas Liver

Aqueous Aqueous Organic Aqueous % of Total Organic Aqueous % of Total

Control 2,435 +- 192 47 -+ 9 i.7 -+ 0.4 2,208 _+ 106 45 _+ 6 1,8 _+ 0 . 3 BPinduced 2,435 _+ 68 26 -+- 3 0.6 -+ 0.1 2,265 _+ 328 112 _+ 7 4.5 _+ 0.9 PBinduced 2,125 + 266 29 _+ 2 0.9 -+ 0.1 2,131 -+ 22 66 _+ 15 4.1 -+ 0.4

*Control values are the means + SE of the means of four experiments. Induced animals received 20 mg/kg BP or 75 mg/kg PB intraperitoneally for 4 days prior to sacrifice. Control animals received identical volumes of vehicles. The aqueous content in experi- ments with boiled tissue, expressed as aqueous % of total, have been subtracted from the aqueous % of total values shown above (the pancreatic aqueous values were 470 dpm/g of pancreas and the liver aqueous values were 900 dpm/g pancreas).

5600

12,000

840

480

]20(

~ 15,60C

12,00C

8400

4800

!: F

A

B c

B

A

SAMPLE FRACTION

F

Fig 1. High-pressure liquid chromatograph of organic extracts of tissue incubated in vitro with [14C]3MC. By comparison to known standards obtained from NCI present in the sam- ples, the following peaks were identified in part A (pancreas): peak A is cis-1,2-diol; peak B is l- and 2-OH; peak C is unknown; peak D is Unknown; peak E is 1- and 2-one; peak F is 3MC. Part B contains extracts from hepatic tissue.

360 Digestive Diseases and Sciences, Vol. 26, No. 4 (April 1981)

METABOLISM OF 3MC IN RAT PANCREAS

9oA I 8O ...... BItE , - - - - BLOOD

70 t'V'<'

vbX 60 I l' II'llilll kL ............. PANCREAS

(nLJ~ 5 0 ,/;" '

: :

30 ." ', / , r--, , I ,: ' , -

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40 ~.~

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0 '0 " 1 2 2'4 3'6 4'8 T I M E ( h r s )

Fig 2. Concentration of radiolabeled 3MC as a function of time following a single injection of 3MC. Data are the mean values ___ SE of the means presented as dpm/mg pancreas and liver and dpm/ml for bile and blood. Each rat was injected with 10 p.Ci p4C]3MC. Animals were sacrificed following a 3-hr collection period which began at the indicated time. (A) Total radioactivity is the sum of the organic and aqueous phase components; (B) percent aqueous is the percent of total radioactivity in the aqueous phase expressed as a percent of the total radioactivity recovered (organic + aqueous)'.

peaks (peak A). Although the methanol elution sys- tem did not distinguish the 1- from the 2-OH and the 1- from the 2-one peaks, an acetonitrile elution sys- tem of Stoming and Geradot (9) resolved the iso- mers. Identity of peaksC and D is presently uncer- tain. It is not known whether peaks C and D repre- sent products that are unique to the pancreas or products that were not removed by the pancreas as well as they possibly were by the liver.

Figure 1B shows the HPLC profile of 3MC me- tabolites in the organic extracts from in vitro in- cubated liver homogenates. This profile is qualita- tively Similar to that of the pancreas (Figure 1A). Metabolism at the 1 and 2 positions predominated.

Figure 2A presents data showing the partition of the label among pancreas, liver, bile, and blood over the periods of observation. Total radioactivity is the sum of the organic and aqueous phase com- ponents. Of the time periods observed, the level of radioactivity was highest in both pancreas and liver at 16 hr. This' observation confirmed the findings of Bresnick (10) and Levine and Singer (11) for the liver, The level of radioactivity in bile reached a peak at 10 hr; the level in blood peaked at 16 hr. By 22 hr, the levels in these Samples reached a plateau. It is interesting to note that over the six time periods observed, the pancreas contained nearly four times more radioactivity per gi-am than did the liver. The

Digestive Diseases and Sciences, Vol. 26, No. 4 (Apri! 1981) 361

pancreatic tissue-to-blood ratio ranged from 2 to 36 times higher than the liver-to-blood ratio during the periods of observation.

Radioactivity recovered in the aqueous phase is presented in Figure 2B as a percent of total radio- activity. The percent of radioactivity in pancreas and liver rose and fell in parallel. The maximum lev- el in the liver reached 50%, while the level in the pancreas reached 20%. At 5 hr 93% of the bile ra- dioactivity was water-soluble metabolites;this per- centage remained high over the period of observa- tion. The percent of water-soluble metabolites in blood leveled off somewhat by 10 hr.

It was apparent from HPLC of in vivo samples (not shown) that, as in the in vitro studies, the prin- cipal metabolism occurred at the 1 and 2 positions of 3MC. The products of in vivo metabolism were qualitatively similar to the in vitro products.

DISCUSSION

There are few reports in the literature which dis- cuss biotransformation of drugs in the pancreas. Lavigne and Marchand reported that the pancreas was capable of metabolizing hexobarbital, zoxa- zolamine, and ethylmorphine (4). More recently, Iqbal et al demonstrated that benzo(a)pyrene hy- droxylase activity was present in pancreatic micro- somes and had an activity less than 1% of the liver activity (5). In a preliminary study which measured the uptake of 3MC in pancreas and other tissues, we reported that the pancreas did not appear to exten- sively metabolize 3MC (3). The present studies con- firmed the above observations and demonstrated that pancreatic tissue biotransformed 3MC to wa- ter- and ethyl acetate-Soluble metabolites which were qualitatively similar to liver products. The rel- ative quantities of products isolated from pancreas in the ethyl acetate extracts suggested that the prin- cipal route of metabolism was via the 1 and 2 posi- tions. The 60-rain in vitro incubations undoubtedly allowed further metabolism of the primary products of pancreatic metabolism, Stoming et al reported that 2-hydroxy metabolites were predominant liver products (12). Others have shown that i- and 2-po- sition products are formed from hepatic metabolism of 3MC (13, 14). A closer examination of the pan- creatic products of metabolism from micr0somal studies is necessary to determine the ultimate or penultimate pancreatic metabolic products.

Our data indicated that pancreatic metabolism of 3MC was not inducible with either BP or PB pre-

BLACK ET AL

treatment under conditions commonly used to in- duce metabolism in other systems. It is widely ac- cepted that aryl-hydrocarbon hydroxlase (AHH) enzyme activity and its inducibility are genetically determined and that both the activity and induc- ibility will vary widely with species, strain, sex, tis- sue, nutritional regimen, and other factors (15, 16). Iqbal et al reported that the in vitro metabolism of BP was induced by pretreatment (5). However, un- der conditions of the present experiments, given the particular strain, sex, nutrition, etc, conditions which are commonly used and which will induce he- patic metabolism, pancreatic metabolism of 3MC was not induced by either BP or PB. This observa- tion serves to differentiate pancreatic and hepatic metabolite mechanisms under these experimental conditions.

The total amount of 3MC in pancreatic tissue in vivo was nearly four times greater than the amount in hepatic tissue at all time periods observed. This was surprising since the liver generally Shows great- er levels of carcinogen than other tissues on a per gram basis, Chuang and Bresnick reported 5-10 times higher levels of radioactivity in liver than in lung or mammary tissues (17). The reason for the difference in pancreatic and hepatic content of the drug is unknown. Smith and Hagopian demon- strated in the prostate that the content of 3MC was 20-fold higher than liver 26 hr after intraperitoneal injection of 3MC (18). It could be interpreted that hepatic clearance was much greater than pancreatic clearance.

In summary, the evidence indicated that pancre- atic tissue was capable of metabolizing 3MC princi- pally via the 1 and 2 positions. The metabolizing system was not induced by BP or PB pretreatment. Finally, pancreatic tissue contained more 3MC per gram of tissue over the observation period than liver for reasons not presently understood. These studies suggest that, in addition to the exposure of pancreatic tissue to hepatically produced carcino- gens, there is an additional exposure to carcinogen- ic products produced in situ by the pancreas.

ACKNOWLEDGMENTS

The authors wish to acknowledge the expert technical assistance of St-Paul Gaffney, Jr., and the editorial assist- ance of Ms. Hazel G. Wall and Mrs. Helen K. Clary.

REFERENCES

1. NCHS. Vital Statistics of the United States. U.S. Depart- ment of Health, Education, ~ind Welfare, Public Health

362 Digestive Diseases and Sciences, Vol. 26, No. 4 (April 1981)

M E T A B O L I S M O F 3MC I N R A T P A N C R E A S

Service, National Center of Health Statistics. Rockville, Maryland (annual editions through 1968)

2. Mancusco TF, El-Attar AA: Cohort study of workers ex- posed to betanaphthylamine and benzidine. J Occup Med 9:277-285, 1967

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4. Lavigne JG, Marchand C: Effects of phenobarbital pre- treatment on rat pancreas. Am J Physiol 222:360-364, 1972

5. Iqbal ZM, Varnes ME, Yoshida A, Epstein SS: Metabolism of benz(a)pyrene by guinea pig pancreatic microsomes. Can- cer Res 27:1011-1015, 1977

6. Black O: Histofluorescence of 3-methylcholanthrene metab- olites in the rat pancreas. Gastroenterology 75:438-444, 1978

7. Sims P: The metabolism of 3-methylcholanthrene and some related compounds by rat-liver homogenates. Biochem J 98:215-228, 1966

8. Steel RGD and Torrie JH: Principles and Procedures of Statistics. McGraw-Hill, New York, 1960, p 73

9. Stoming TA, Geradot ILl: High pressure liquid chromato- graphic sepa ra t ion of the metabo l i t e s of 3-methyl- cholanthrene. Life Sci 20:113-116, 1977

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11. Levine WG, Singer RW: Hepatic intracellular distribution of foreign compounds in relation to their biliary excretion. J Pharmacol Exp Ther 183:411-419, 1972

12. Stoming TA, Bornstein W, Bresnick E: The metabolism of 3- methylcholanthrene by rat liver microsomes--a reinvestiga- tion. Biochem Biophys Res Commun 79:461-469, 1977

13. Takahashi G, Yashuhira K: Excretion and conversion of 3- methylcholanthrene rnetabolites in the intestinal tract of the mouse. Cancer Res 32:710-715, 1972

14. B/.irki K, Seibert RA, Bresnick E: Induction of benzpyrene hydroxylase activity in fetal rat liver implants. I. Metabolism of 3-methylcholanthrene and differential effects of its deriva- tive on benzpyrene hydroxylase activity. Biochim Biophys Acta 260:98-110, 1972

15. Nebert DW, Gelboin HW: The in vivo and in vitro induction of aryl hydroxylase in mammalian cells of different species, tissues, strains, and developmental and hormonal states. Arch Biochem Biophys 134:76-89, 1969

16. B/irki K, Liebelt AG, Bresnick E: Expression of aryl hydro- carbon hydroxylase induction in mouse tissues in vivo and in organ culture. Arch Biochem Biophys 158:641-649, 1973

17. Chuang AHL, Bresnick E: Aryl hydrocarbon hydroxylase in mouse mammary gland. Cancer Res 36:4125-4129, 1976

18. Smith ER, Hagopian M: The uptake and secretion of 3- methylcholanthrene by the prostate gland of the rat and dog. J Natl Cancer Inst. 59:119-122, 1977

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