COMPARATIVE METABOLISM OF THE CIS AND T R A N S ISOMERS OF N-NITROSO-2,6-DIMETHYLMORPHOLINE IN RATS, HAMSTERS AND GUINEA PIGS*
BRENDA UNDERWOOD and WILLIAM LIJINSKY
Basic Research Program-LBI, Chemical Carcinogenesis Laboratory, Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.)
(Received August 12th, 1983) (Revision received February 21st, 1984) (Accepted March 1st, 1984)
SUMMARY
The in vivo metabolism of the cis and trans isomers of N-[3,5-3H] nitroso- 2,6-dimethylmorpholine (NDMM) was studied in female Fischer rats, Syrian golden hamsters and guinea pigs by analysis of urinary metabolites using high pressure liquid chromatography (HPLC). Animals were treated by gavage with 12 mg/kg body wt. of NDMM, composed of both isomers and 12 ~Ci/ kg body wt. of either of the separated radioactive isomers (cis or trans). Control animals received 12 mg, 12 IzCi/kg body wt. NDMM with both iso- mers labeled in their natural proportion.
There was a substantial increase in the excretion of a particular metabol- ite, 2-(2-hydroxyl-methyl)ethoxy propanoic acid, in the urine of rats, ham- sters and guinea pigs 24 h after received the trans isomer (24, 22 and 13% of the total dose excreted, respectively). A minor metabolite was determined to be 2,6-dimethylmorpholine-3-one, another product of a-oxidation. The metabolite 1-amino-2-hydroxypropanol was identified, indicating that NDMM was metabolized by both a- and ~-oxidation.
In all three species, animals administered the cis isomer excreted larger amounts of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitroso-bis(2-hydroxypropyl)amine (BHP) products of beta oxidation, than those treated with the trans isomer. Hamsters and guinea pigs treated with the more carcinogenic cis isomer in these species, also excreted twice as
*Research sponsored by the National Cancer Institute, DHHS, under contract No. NO1- CO-23909 with Lit ton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Abbreviations: BHP, N-nitrosobis(2-hydroxypropyl)amine; HPLC, high-pressure liquid chromatography; HPOP, N-nitroso-N-(2-hydroxypropyl)-N-(2-oxopropyl)amine; NDMM, N-nitroso-2,6-dimethylmorpholine.
much of two other metabolites than was found in the urine of animals given the trans isomer.
The trans isomer of NDMM appeared to be preferentially metabolized by a-oxidation and from earlier studies this metabolic pathway seemed to be important in carcinogenesis by NDMM in the rat. The cis isomer might be in a conformation more favorable for ~-oxidation and this pathway may be of primary importance in carcinogenesis by NDMM in hamsters and guinea pigs.
Key words: Metabolism -- N-Nitroso-2,6-dimethylmorpholine -- High pressure liquid chromatography -- Metabolites -- Rat -- Hamster -- Guinea pig
INTRODUCTION
The discovery of the marked difference in carcinogenic potency between the cis and trans isomers of nitroso-2,6-dimethylmorpholine in rats, Syrian hamsters and guinea pigs let us to consider that the differences might be due to differences in metabolism of the two isomers. This possibility was empha- sized because of the disparity between the comparative potencies of the isomers in rats, and either hamsters or guinea pigs. In Fischer rats the trans isomer was more potent than the c/s [1], whereas in both hamsters [2] and guinea pigs [3] the cis was more potent than the trans. This pointed to dif- ferent mechanisms of activation of the carcinogen, possibly by enzyme systems having different affinities for the two isomers. That there are pro- found differences in metabolism of NDMM related to tumor induction is also shown by the effect of deuterium labeling in the a(3 and 5)- or ~(2 and 6)-positions on carcinogenic potency, which is precisely the opposite in rats [4] and in hamsters [2]. The patterns of urinary metabolites of NDMM did not differ greatly among the three species [5] and therefore perhaps did not reflect differences in metabolic pathways that might underlie the very dif- ferent organ specificities of the carcinogen in these animals, esophagus in the rat [1,4,6], pancreas and liver in the hamster [2,7,8], and liver angiosar- comas in the guinea pig [3,9,10]. However, those small differences that were seen might be revealing. These differences might be amplified by comparing the metabolites formed from the two isomers in each of the three species and thereby assist in elucidating the mechanisms by which NDMM induces such different types of tumor in rats, hamster and guinea pigs. The results of these experiments with the cis and trans isomers are presented here.
MATERIALS AND METHODS
Instrumentat ion Mass spectra were obtained from a VG Micromass ZAB-2F mass spectro-
meter equipped with a VG 2035 data system (VG Micromass, Cheshire,
177
U.K.). Gas chromatographic analysis was carried out using a Perkin-Elmer Model Sigma 3 gas chromatograph coupled to the mass spectrometer (Perkin-Elmer Corp., Norwalk, CT). Columns used were 1.83 m X 2 mm containing either Texax G/C (Alltech Assoc., Deerfield, IL) or Ultrabond 20M (RFR Corp., Hope, RI). HPLC was performed on a Waters Associates system (Waters Associates, Inc., Milford, MA) using either a 4.6 mm X 25 cm Dupont Zorbax-ODS C18 (Dupont Corp., Wilmington, DE) or a What- man Partisil 10 ODS-2 column (Whatman, Inc., Clifton, NH). A 3.9 mm X 30 cm pPorasil (Waters Associates, Inc., Milford, MA) column was used to determine the purity and specific activity of the control, trans and cis labeled stock solutions. A 9.4 mm X 25 cm Whatman Patisil-10 ODS-2 column was used for large scale preparations. Radioactivity was monitored with a Packard Model 3225 liquid scintillation counter (Packard Instrument Company, Inc., Downers Grove, IL).
Chemicals
NDMM was prepared as previously described [6] and contained the cis and trans isomers in the approximate ratio 2 : 1. The cis and trans isomers of NDMM were separated [ 12] and labeled with tritium by exchange with triti- ated water in the presence of base [5,11] . The normal mixture of isomers of NDMM was also labeled with tritium for use as a control. The isotope was incorporated only in the 3 and 5 positions, that is those positions alpha to the nitroso function [13] and both labeled isomers were 99% radiochemi- cally homogeneous. The solutions of the labeled cis and trans isomers were prepared by adding the labeled isomer to the non-radioactive mixture of isomers. All three solutions (labeled isomers and labeled mixture were di- luted to the same specific activity.
2-Hydroxypropyl benzamide and HPOP were provided by Dr. Joseph Saavedra (NCI-Frederick Cancer Research Facility, Frederick, MD). BHP was prepared as described previously [ 14].
Benzoyl chloride was obtained from Aldrich Chemical Co. (Milwaukee, WI). Sephadex G-10 and LH-20 column packing material were purchased from Pharmacia AB (Uppsala, Sweden). PCS (Amersham Corp., Arlington Height, IL) was used as the liquid scintillation cocktail. Glass-distilled sol- vents routinely used for HPLC were UV--grade methanol (Burdick and Jack- son Laboratories, Inc., Muskegon, MI). Other solvents used are reagent grade purchased from Fisher Scientific Company (Silver Spring, MD).
A n i m a l t rea tmen t s Three female F 344 rats, three female Syrian golden hamsters and three
female guinea pigs approx. 8 weeks old were used for each experiment. Two animals of each species were given the tritium labeled mixture of isomers. All animals were maintained in plastic metabolism cages without food but were given water ad libitum.
After 18 h of food deprivation, the test animals were treated by gavage
178
with 12 mg/kg body wt. of NDMM composed of both isomers and 12 pCi/kg body wt. of either of the radioactive isomers (cis or trans) in corn oil.
Because both isomers probably compete for the same enzyme system, it was decided to make the experiments as comparable as possible by maintain- ing the ratios in each sample administered. Therefore, each of the labeled isomers was added to a mixture of the two isomers so that they were in con- stant proportion (2 cis : 1 trans) in the solution with which the animals were treated.
As a control, urine was also collected from animals receiving 12 mg, 12 pCi/kg [3,5-3H2] NDMM containing both isomers in their normal propor- tions.
Analysis of urine At 8 and 24 h urine samples were collected and frozen; 10-~l aliquots
were mixed with 10 ml PCS scintillation cocktail and assayed. The propor- t ion of radioactivity excreted in urine was determined at both time points. Sodium chloride was added to the bulk of the urine to a final concentration of 10 mM; 3 vol. methanol were added at 0°C and the suspension was centri- fuged at 8000 X g for 15 min. The pellet was washed with methanol/water (3: 1) and the combined supernatants were evaporated in a stream of nitro- gen at 0°C. Recovery of urinary radioactivity in the methanol supernatants was 90--95%. Aliquots of 100 or 150 gl, containing approx. 40 000 cpm were applied to a Dupont Zorbax-ODS column or a Whatman Partisil-10 ODS-2 (used for hamster urine samples) developed at 1.5 ml/min using the program previously described [ 5 ].
For larger scale isolation of metabolites groups of ten hamsters received 8 mg, 8 ~Ci NDMM (containing both isomers in their normal proportions) by gavage. The methanol supernatant from the urine was evaporated and reprecipitated three times. The final supernatant was evaporated to approx. 1.5 ml in nitrogen then applied to a Sephadex G-lC, 'olumn (2.6 X 100 cm) and eluted with a buffer containing 1 mM Tris (pH 7.5) and 2 mM NaCl. After chromatography on Sephadex G-10, the separated peak areas were pooled and concentrated. Each fraction (approx. 1.5 ml) was chromato- graphed on a Sephadex LH-20 column (2.6 X 100 cm) eluted with 2 mM sodium acetate buffer (pH 4.5). Fractions were 4 ml each.
The metabolites were further purified and identified as outlined below: Fraction A (2-[2-hydroxyl-l-methyl] ethoxy propanoic acid). The approx-
imate peak fraction containing metabolite A on Sephadex G-10 was fraction number 59 (retention time 11.3 h) and fraction number 77 (retention time 9.3 h) on Sephadex LH-20. When A was acidified in 0.001 M HC1 its charge was neutralized and it became more firmly bound to a Whatman Partisil-10 ODS-2 eluted with 0.001 M HCI/4% methanol. Solutions of A were extract- ed with ethyl acetate, blown to dryness, then taken up in methanol. Next they were treated with diazomethane to form the methyl ester of the acid. The derivative was admitted to the mass spectrometer by use of a Texax GC column.
179
Fract ion E (1-amino-2-propanol) . Compound E eluted in fraction 70 (re- tention time 13.4 h) on Sephadex G-10 and fraction 89 (retention t ime 10.8 h) on Sephadex LH-20. The final steps of purification of metaboli te E involved use of a Whatman Partisil 10-ODS-2 column eluted with 0.01 M ace- tate buffer (pH 5.6), then deionized water. The benzoyl chloride derivative of fraction E was made by dissolving approx. 100 ~g of the compound in 50 ~l acetonitrile, adding 1 pl benzoyl chloride and 3 #1 pyridine and then heating overnight at 60°C in a sealed vial. After evaporation the residue was resuspended in 0.5--1.0 ml of 2% NaHCo3 and extracted three times with ethyl acetate. The organic phase was then washed twice with 0.1 N HC1, con- centrated and resuspended in ethanol.
Isopropanolamine was treated with benzoyl chloride as described. The derivatives were further purified on a Whatman ODS-2 column and coeluted with 50% methanol and deionized water in 260 s. The derivatives were ad- mit ted to the mass spectrometer on the direct probe and heated gradually to give adequate vaporization.
Fract ion K. Compound K eluted approximately in fraction 83 (retention time 15.9 h) on Sephadex G-10 and fraction 100 (retention time 12 h) on Sephadex LH-20. After LH-20 chromatography, compound K was further purified on a 9.4 mm X 25 cm Magnum Whatman Partisil-10 ODS-2 column eluted at 7 min with 50% MeOH-deionized water at 3.0 ml/min. The com- pound was subjected to mass spectral analysis using an Ultra-bond GC column at 120°C.
RESULTS
HPLC analysis of rat, hamster and guinea pig urine samples revealed 12 peaks of radioactivity, as was previously reported [ 5 ], which were tentative- ly assumed to be individual metabolites. The proport ions of these fractions in each sample of urine are summarized in Table I. Fractions I, J and L were chromatographically identical with BHP, HPOP and NDMM, respectively [5]. Animals which received the trans isomer on the average excreted 1.5 times more radioactivity in 24 h than those given the cis isomer. The pattern of urinary metaboli tes from animals treated with the two isomers differed quantitatively in all three species. Substantial differences were seen in the rat 0--8 h cis and trans urine samples (Fig. 1). Fraction A in the trans urinary chromatogram represented approx. 31% of the radioactivity eluting from the column, while there was 3% of A in the cis chromatogram. In contrast, frac- tion B was 25% of the radioactivity in the c/s chromatogram as opposed to 7% in the trans. Similar differences were observed for 8--24 h urine samples (data not shown}. Fractions I and J were consistently greater in the cis samples in both the 0--8 h and 8--24 h urinary samples.
In the hamster 0--8 h trans urinary sample there was nine times more of fraction A and slightly more K than from the cis isomer. There were increas- ed amounts of fractions B,C,D,I and J in the cis urinary sample compared with the trans samples (Fig. 2).
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181
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Fig. 1. HPLC c h r o m a t o g r a m s o f ra t c o n t r o l ( m i x t u r e of c/s and trans), trans and cis urin- ary me tabo l i t e s a t 0- -8 h. A l iquo t s of app rox . 40 000 c p m were appl ied to a D u p o n t Zor- bax-ODS c o l u m n a n d e lu ted 0---5 m i n wi th 0.0Z M sod ium ace ta t e buf fe r (pH 5.6); 5--6 m i n wi th 0 - -10% m e t h a n o l in ace t a t e buf fe r ; 6 - -17 rain wi th 10% m e t h a n o l in ace ta t e buf fe r ; 17 - -20 rain wi th 10- -60% m e t h a n o l in ace ta t e bu f f e r ; 20- -27 ra in wi th 60% m e t h a n o l in ace t a t e bu f f e r at 1.5 m l / m i n . The t op inset shows the e lu t ion pos i t ion of k n o w n so lu t ions o f BHP, HPOP and NDMM. The ra t con t ro l 0- -8 h u r ine r ep r e sen t ed 31% of the to ta l dose given, t h e trans, 24% and t h e c/s 38% of t he to ta l dose.
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183
Differences were also observed in the guinea pig urinary samples. In the 0--8 h samples, fraction B represents only 18% of the radioactivity in the trans urinary sample in comparison to 54% in the cis sample. The cis 0--8 h urinary sample also contained four times more I and J than was seen in the trans sample (Fig. 3).
Fraction A. Although a parent ion was not observed in the mass spectrum of the methyl ester of fraction A (the extracted radioactive material follow- ing t reatment with diazomethane), an ion was present corresponding in exact mass t o the lactam 2,6-dimethyl-3-oxo-l,4-dioxane. This would be expected from cyclization of the acid or of the methyl ester. By comparison of the characteristics of fraction A with analogous metabolites formed from other cyclic nitrosamines in rats, we believe it to be 2-(2-hydroxy-l-methyl)- e thoxypropanoic acid [ 5].
Fraction E. Mass spectral data for both the synthesized benzoyl derivative of 1-amino-2-propanol (top) and the benzoyl derivative of fraction E (bot tom) are shown in Fig. 4. Fraction E is assumed to be 1-amino-2-propan- ol.
Fraction K. Since this fraction eluted near NDMM on HPLC, it was thought that compound K might also be a cyclic compound. On gas chroma- tography it separated into two major peaks, X and Y. The mass spectra of these (Fig. 5) were identical and are assumed to be of the cis and trans iso- mers of 2,6-dimethylmorpholine-3-one, having a molecular ion at m/z 129 and the fragmentation was consistent with the expected fragmentation of 2,6<limethylmorpholine-3-one.
DISCUSSION
There appeared to be a relationship between the conformation of NDMM and its carcinogenicity. The metabolism of each isomer was similar in all three species and different from the other isomer, but the relative carcino- genic potency of the two isomers was not the same in ,rats and in hamsters and guinea pigs. On the other hand, Gingell et al. reported similar amounts of both BHP and HPOP in the 24 h urine of hamsters treated with cis- or trans-NDMM [ 15].
The major urinary product from F344 rats given the trans isomer, which was more carcinogenic than the cis in this species, was 2-(2-hydroxyl-1- methyl )e thoxy propanoic acid (Fraction A). Metabolite A is a product of s-oxidation. Several other investigators have observed analogous hydroxy
Fig. 2. HPLC chromatograms of hamster control (mixture of c/8 and trans), trans and c/s urinary metabolites at 0--8 h. Aliquots of approx. 40 000 cpm were applied to a Whatman Partisil-10 ODS-2 column and eluted to 0--6 min with 0.01 M sodium acetate buffer (pH 5.6); 6--7 min with 0--10% methanol in acetate buffer; 7--17 min with 10% methanol in acetate buffer; 17--20 mln with 10--60% methanol in acetate buffer; 20--27 min with 60% methanol in acetate buffer at 1.5 ml/min. The hamster 0--8 h control urine sample represented 54% of the total dose given, while the trans equaled 40% and the cis, 47% of the dosage.
Fig. 4. Mass spectra of 2-hydroxypropylbenzamide (benzoyl derivative of 1-amino-2-pro- panol) and the benzoyl derivative of metabolite E.
acids formed in vivo and in vitro by a -ox idat ion o f cycl ic nitrosamines [ 1 6 - - 1 9 ] . Metabol i te K, 2 ,6 -d imethy lmorphol ine -3 -one , also arises from a-oxidat ion . Other investigators have isolated analogous lactams from cycl ic ni trosamine metabo l i sm [ 1 6 , 2 0 ] .
The format ion o f metabol i tes A and K indicated that a -ox idat ion o f NDMM is a significant pa t hway o f metabol i sm o f the ni trosamine in the rat. That a -ox idat ion is related to carcinogenesis o f N D M M in the rat is suggested
Fig. 3. HPLC chromatograms of guinea pig control (mixture c/s and trans), trans and c/s urinary metabolites at 0--8 h. Aliquots of approx. 40 000 cpm were supplied to a Dupont Zorbax ODS column and eluted 0--5 min with 0.01 M sodium acetate buffer (pH 5.6); 5--6 min with 0--10% methanol; 6--17 rain with 10% methanol in acetate buffer; 17--20 rain with 10--60% methanol in acetate buffer; 20--27 rain with 60% methanol in acetate buffer at 1.5 ml/min. The guinea pig control 0--8 h urine sample equaled 32% of the total dose given, while the trans urine sample was 54% of the total dose and the c/s 28%.
186
1 0 0 ,
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60 100
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Fig. 5. Mass spectrum of metabolite K. This analysis proved that peak X and Y from the GC were identical. The proposed structure for K is shown at the top of the chromatogram and is 2,6-dimethylmorpholine-3-one.
by the observation that substitution of deuter ium for hydrogen in the ~- positions (3 and 5) to the nitroso funct ion decreased carcinogenic po tency [4]. Rats receiving the cis isomer excreted very little of metaboli te A, but excreted more of metaboli te B and of HPOP, which is a product of ~-oxi- dation.
The urines from the hamster and guinea pig given the trans isomer also contained large amounts of metaboli te A, yet the trans isomer was less car- cinogenic than the cis in these species. Hamsters and guinea pigs t reated with the cis isomer which was more carcinogenic in these species excreted double the amount of metabolites C or B, respectively, than was excreted by ani- mals given the trans isomer. Also, hamsters and guinea pigs t reated with the
187
cis isomer excreted more BHP and HPOP. Deuterium substi tuted for hydro- gen in the 2 and 6 positions (~ to the nitroso function) decreased the poten- cy of NDMM in the hamster [2], which suggested that ~-oxidation was the important pathway of carcinogenesis in hamsters. Possibly a-oxidation was not a relevant metabolic pathway in hamster carcinogenesis by NDMM. The presence of metaboli te E, 1-amino-2-propanol, in the urine of all three species treated with either isomer indicated that NDMM was metabolized by both a- and ~-oxidation inal l three species.
These results suggest that the trans isomer is preferentially metabolized by a-oxidation and is, therefore, the more carcinogenic isomer in the rat. In- creased amounts of BHP and HPOP in the urine of animals administered the cis isomer of NDMM compared with those given the trans isomer support Gingell's proposal that the cis isomer may be in a conformation more favor- able for ~-oxidation [15]. In the hamster [2] and the guinea pig, initial ~- oxidation seems to be of primary importance in NDMM carcinogenesis.
Identification of all urinary metaboli tes of NDMM, in particular meta- bolites C and B, should provide further information pointing to those specif- ic pathways of metabolism of NDMM that are involved in carcinogenesis in the three species.
ACKNOWLEDGEMENTS
The authors thank Dr. S.S. Singer for the isolation and purification of the cis and trans isomers of NDMM, Dr. J.E. Tomaszewski for the preparation of the tritium labeled isomers and Dr.G.A. McClusky for the mass spectral analyses.
REFERENCES
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