Isolation and characterization of active metabolites of tryptophan-pyrolysate mutagen, TRP-P-2, formed by rat liver microsomes

Chem.-Biol. Interactions, 30 (1980) 125--138

125

© Elsevier/North-Holland Scientific Publishers Ltd.

ISOLATION AND CHARACTERIZATION OF ACTIVE METABOLITES OF TRYPTOPHAN-PYROLYSATE MUTAGEN, TRP-P-2, FORMED BY RAT LIVER MICROSOMES

YASUSHI YAMAZOE, KENJI ISHII, TETSUYA KAMATAKI,RYUICHI KATO and TAKASHI SUGIMURA Department of Pharmacology, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160 and (T.S.) National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104 (Japan)

(Received October 15th, 1979) (Revision received January 2nd, 1980) (Accepted January 4th, 1980)

SUMMARY The mutagenic compound derived from the pyrolysis of tryptophan, 3amino-l-methyl-5H-pyri~o-[4,3b]indole (Trp-P-2) was metabolized by rat liver microsomes to more than four metabolites, separable by high performance liquid chromatography. Among these metabolites, two metabolites, M-3 and M-4 were directly active in increasing the frequency of mutation in Salmonella t y p h i m u r i u m TA98. Treatments of rats with polychlorinated biphenyl (PCB) or 3-methylcholanthrene dramatically induced the activity of liver microsomes to form these active metabolites, while treatment with phenobarbital was without effect. A major active metabolite (M-3) formed the pentacyano-ammine ferroate, which is known to be formed by reaction of sodium pentacyano-ammine ferroate with some hydroxylamines. Further this metabolite was oxidized to the minor active metabolite (M-4) with potassium ferricyanide or 7-manganese dioxide, and was reduced back to Trp-P-2 with titanium trichloride. These results indicated that the major active metabolite of Trp-P-2, which is formed by cytochrome P-450, is the 3-hydroxyamino derivative. INTRODUCTION It has been well known that smoke condensate and charred meat contain mutagenic compounds. Analyzing tryptophan pyrolysates, Sugimura et al. [1] and Kosuge et al. [2] found that 3-amino-l-methyl-5H-[4,3b]indole (Trip-P-2) as well as 3-amino-l,4-dimethyl-5H-[4,3b]indole (Trp-P-1) exerts the mutagenic activity after undergoing metabolic activation by an enzyme(s) in liver 9000 X g supernatant fraction. The mutagenicity of Trp-P-2 was

126

demonstrated to be very potent; the mutagenicity as tested by a modified Ames method using Salmonella typhimurium TA98 was approx. 700 times as high as benzo[a]pyrene. The carcinogenic nature of Trp-P-2 has also been demonstrated using an in vitro carcinogenesis bio-assay system of b~mgter embryo cells [3]. The binding of an active metabolite(s) of tritium labeled Trp-P-2 to DNA has also been reported [4]. Concerning the enzyme catalyzing the metabolic activation of Trp-P-2, recent studies from this laboratory have shown that the metabolic activation of Trp-P-2 to the ultimate mutagen is mediated by cytochrome P-450 (K. Ishii and coworkers, unpublished data). Despite these studies, the chemical and physical nature of the active metabolite of Trp-P-2 has not been fully understood. Therefore, in the present paper we would like to report on the isolation and characterization of a higldy mutagenic product of Trp-P-2 formed by the incubation in the presence of liver microsomes of PCB-treated rats and NADPH. MATERIALS Trp-P-2 acetate was synthesized by the method as reported previously [5]. Potassium ferricyanide, 3-methylcholanthrene, titanium trichloride were purchased from Wako Pure Chemicals, Osaka, Japan. Sodium phenobarbital was obtained from Fujinaga Pharmaceutical Co., Tokyo, Japan. Acetonitrile and monobasic potassium phosphate were obtained from Kanto Chemicals Co., Tokyo, Japan. Sodium pentacyano-ammi~e ferroate were purchased from Nakarai Chemicals Ltd., Kyoto, Japan. 7-Manganese dioxide was prepared by the method described by Vershchgin et al. [6] using potassium permanganate and manganese sulfate. NADP, glucose 6-phosphate and glucose 6-phosphate dehydrogenase are products of Oriental Yeast Co., Osaka, Japan.

METHODS

Preparation of liver microsomes Male rats of Sprague--Dawley strain weighing 140--180 g were used throughout this study. PCB (KC 500) dissolved in corn oil was given intraperitoneally at a dose of 500 mg/kg; the animals were sacrificed seven days after the injection. Sodium phenobarbital dissolved in saline and 3-methylcholanthrene dissolved in corn oil were given intraperitoneally at a daily dose of 80 and 40 mg/kg, respectively, for three consecutive days; the animals were sacrificed approx. 24 h after the last injections. All animals were fasted for about 18 h prior to sacrifice. Liver microsomes were prepared as described previously [7], and the microsomes were washed once by homogenization with 1.15% potassium chloride and centrifugation at 105 000 × g for 30 min. The washed microsomes were stored at -80°C under an atmosphere of nitrogen until use. Microsomal protein was quantitated by the method of Lowry et al. [8]. Cytochrome P-450 was determined by the method described by

129 A

FL

B Trp- P-2

M-2_3•

~Trp-P-2

Trp-P-2

ulll L

M-1 UV

J

L.J

,___.._.

0 2 ~, 6 8 10 12min 0 2 4 6 8 10 12rain

Fig. 1. Reverse phase liquid chromatograms of Trp-P-2 and its metabolites before (A) and after 30 rain of incubation (B). Incubation mixture is the same as described in Materials and Methods. the incubation mixture was extracted before incubation. Trp-P-2 was eluted with the retention time of a b o u t 5.5 min. Fig. 1-B shows the elution pattern o f the extract of the incubation mixture incubated at 37°C for 30 min. The peak height due to Trp-P-2 was decreased to a b o u t 30% by the incubation. Four UV peaks probably resulted from the formation of metabolites of Trp-P-2 appeared. The four fractions, namely M-l, M-2, M-3 and M-4, had the retention times of 3, 4, 6 and 8 rain, respectively. These metabolites eluted in these four fractions did n o t fluoresce. At least t w o other metabolites in addition to these four fractions have appeared to be formed since very faint peaks were observed when the concentrations o f acetonitrile in the mobile phase were increased to 60%.

Time course o f Trp-P-2 metabolism and its relation to the mutagenic product formation and pentacyano-ammine ferroate formation To k n o w which one of these metabolites are ultimate mutagen, the time course of the metabolite formation and Trp-P-2 metabolism were measured (Fig. 2A). The concentration o f Trp-P-2 was decreased rapidly with incubation time; the rate of the metabolism o f Trp-P-2 in the initial phase was approx. 10 nmol/mg protein/rain and only a b o u t 16% o f the added Trp-P-2 remained after 45 rain of incubation. The formation of M-1 was also rapid, however, the concentration of M-1 reached a plateau level within 5 rain of incubation. The formation o f M-2 and M-3 were linear with incubation time

130 E~200

M-2

\x

100

(A)

Q

J¢

~

50~

100

04

r

& &

tQ

LI-.-

5

.5

10

15

t~.2

25

30

(B)

45rain v

110 mutagenic activity

.4

o

20

A

ornrnineferr0ute complex

I

s X o

5 1() 15 20 25 30 4Smin Fig. 2. A: disappeanmce of Txp-P-2 and formation of the metabolites as a function of incubation time. B: formation of mutagenic product(s) and pentocyano-ammine tertoate as a function of incubation time. See Materials and Methods for experimental conditions.

until about 20 rain while the concentration of M-3 decreased after 30 rain of incubation. The fornmtion of M-4 was rather anomalous compared to the other metabolites. The M-4 formation became more rapid after about 20 rain of incubation, probably suggesting that M-4 is not formed directly from Txp-P-2 but from a metabolite of Trp-P-2. A possible N-hydcoxylated product of Trp-P-2 was determined by pentacyano-ammine ferroate complex formation simultaneously with the mutation assay. The results showing the close relationship between the formation of pentacyano~mmine ferroate complex and mutagenic activity are represented in Fig. 2B. From the results that the time course pattern to form M-2 and M-3 are similar to the formation of pentacyano~mmine ferroate complex and increase in the mutation frequency, it was suggested that M-2 or M-3 contained ultimate mutagen(s), probably including N-hydroxy-Trp-P-2.

Detection of mutagenic metabolites To further clarify which one of the metabolites eluted from HPLC col-mu is the ultimate mutagen, the mutagenic activity of the eluate was tested using S. typhimurium TA98 as the test strain. The elution profile of the metahalites and the mutagenicity of each fraction are shown in Fig. 3. As can be

127 Omura and Sato [9] except that 0.2% Emulgen 913 and 20% glycerol were added to the microsomal suspension.

Incubation Unless otherwise stated, incubation raixture consisted of 20 mg protein of microsomes from PCB-treated rats, 0.8 mM NADP, 8 mM glucose 6-phosphate, 6 mM MgC12, 20 units of glucose 6-phosphate dehydrogenase, 50 mM Na, K-phosphate (pH 7.4) and 0.2 mM Trp-P-2 in a final volume of 20 ml. The incubations were carried out at 37°C aerobically and were started by the addition of NADPH~enemting system including NADP, glucose 6-phosphate, glucoseS-phosphate dehydrogenase and MgCI2. Aliquots of the incubation mixture were taken at the desired time after starting the incubation for the colorimetric and chromatographic analysis of the metabolites and for the mutation assay.

Colorimetric assay of the metabolite of Trp-P-2 A possible N-hydroxylated product of Trp-P-2 was estimated by determining the pentacyano-amraine ferroate formation [10]. A 1-ml portion of the incubation mixture was transferred into a tube containing the equal amount of 0.04% sodium pentacyano-amraine ferroate in 60% ethanol. The reaction was carried out at room temperature for 15 rain, then the tube was centrifuged at 2500 X g for 10 rain. The formation of pentacyano-amraine ferroate was measured by reading the absorbance at 540 nm, using a Shiraadzu UV-VIS spectrophotometer, Model 150-2.

Separation and quantitation of Trp-P-2 metabolites Separation and quantitation of TrI~P-2 metabolites were conducted using a high performance liquid chromatograph (HPLC, ALC/GPC, Model 204, Waters Assoc.) equipped with UV absorbance detector (Model 440), fluorescence detector (Model 420), automatic injector (WISP, Model 710), and a reverse phase column, IzBondapak C18 (4 mm ID X 30 cm). A mobile phase of acetonitrile--0.02 M potassium phosphate monobasic (45 : 55) was utilized at a flow rate of 1--1.2 ml/rain. An aliquot (100-~1) of the incubation mixture was transferred to another tube containing an equal amount of acetonitrile. The mixture was mixed wen, then centrifuged at 2500 X g for 10 rain. The resulting supematant (30 ~1) was injected to the HPLC by means of WISP and the chromatograms were recorded by UV absorbance at 280 nm and the fluorescence with the emission at 395 nm and the excitation at 343 nm usinga dual-pen recorder (Model U-225M, Nippon Densikagaku).

Mutation assay The mutation assay for the metabolite of Trp-P-2 was carried out as described by Ames et al. [11] with some minor modifications. A 200-/al portion of the incubation mixture was filtered through a membrane filter (railipore, type HA, 0.45/am) to eliminate further reaction, and an aliquot (20-~1) of

128 the filtrate was transferred to a t u b e containing 0.4 ml of 100 mM sodium phosphate (pH 7.4) containing 8 mM MgC12 and 33 mM potassium chloride, then 0.1 ml of a histidine requiring bacterial tester strvSn S. typhirnuriurn TA98, kindly supplied by Dr. B.N. Ames, University of California, Berkeley, CA, was added. The bacteria was suspended in a nutrient broth. After shaking the tube at 37°C for 10 rain, 2 ml of molten soft agar was added and mixed. The mixture was poured gently on a minimal glucose agar plate containing 0.1 p m o l of L-histidine and 0.1 ~mol of biotin. The number o f His+ revertant colonies was counted after incubation at 37°C for approx. 48 h. Control assays were also conducted and the n u m b e r of spontaneous mutation was subtracted from each experimental number of revertants. The data represent the mean o f at least duplicate determinations. The mutagenicity o f the meta0olites of Trp-P-2 which were eluted from HPLC column was also assayed. The fractions eluted from the column (1.2 ml each) were evaporated under the current of nitrogen to remove the organic solvent, then distilled water previously bubbled with oxygen-free nitrogen gas was added to adjust the volume to 1.0 ml. A 100-~1 portion of the resultant solution was subjected for the mutation assay. The mutation assay was carried o u t as mentioned above.

Mass spectrometry o f the ether extract The incubation mixture (20 ml) prepared as described above was treated with 300/~1 of CaC12, and extracted with 10 ml of diethylether. After organic layer was discarded, the remaining aqueous layer was alkalized with 20% sodium carbonate, then the layer was re,extracted with 10 ml of diethylether. The organic layer was transferred to a tube and was evaporated to dryness under the current of nitrogen gas. N,O-Bis-trimethylsilylacetamide (100 ~l) was added, then the sample was reacted at 50°C for 15 min. The mass spectra of the metabolites were obtained b y gas chromatography mass spectrometry using a JEOL JMS-D 300 instrument equipped with a 20K gas chromatograph fitted with a glass column (1 m × 2 mm i.d.) of 3% OV-1 on chromosorb W HP (80/100 mesh) at 220°C with helium as the carrier gas (30 ml/min). The instrument was operated in the e.i. m o d e (electron energy 70 eV, ionizing current 250 ~A, ion source pressure 2 × 10 -v Torr, ion source temperature 250°C. RESULTS

Separation o f Trp-P-2 and its metabolites by HPLC The elution profiles from the HPLC of Trp-P-2 and its metabolites formed during incubation with PCB-treated rat liver microsomes in the presence of NADPH are shown in Fig. 1. The elution profile was monitored b y changes in the optical density at 280 nm and the fluorescence as indicated b y UV and FL in the figures, respectively. As can be seen in Fig. 1A, no significant UV absorption and fluorescence increases were observed in the void fractions except those due to NADPH and u n k n o w n components of microsomes wheh

131 .O6

25

2O

.04, 15

"~.03 O

.01

0.5

I " " O' Ft'.

0 234567891011

Fig. 3. Separation of Trp-P-2 metabolites and their relation to mutagenic activity. The incubation mixture prepared as described in Materials and Methods, was treated with an equivalent vol. of acetonitrile after 45 min of incubation. After centrifugation of the mixture, the resultant supernatant (100 ~l) was injected o n t o the column.

seen clearly, the highest mutagenic activity was found in fraction 5, followed by fractions 7 and 6. These results indicate that mutagenic product(s) was contained in M-3 fraction but not in M-2 fraction. The recovery of the mutagenic activity in the eluates was about 95%, indicating that no mutagenic products were lost during HPLC treatments.

Effect of treatments of rats with inducers on microsomal Trp-P-2 metabolizing activity It has been shown that the metabolic activation of Trp-P-2 by microsomes or 9000 X g supematant fraction of rat livers as determined by mutation assay was enhanced when rats were pretreated with PCB or 3-methylcholanthrene (K. Ishii et al., unpublished data). Despite these mutation experiments, little has been done on the effects of these inducers on the rate of metabolism of Trp-P-2. Thus, the effects of pretreatment of rats with phenobarbital, 3-methylcholanthrene and PCB on the metabolism of Trp-P-2 were examined. The rate of metabolism of Trp-P-2 was measured by disappearance of substmte and by the formation of M-3. As shown in Fig. 4A, Trp-P-2 was not metabolized to significant extents when microsomes from untreated and phenobarbital-treated rats were employed. The metabolic rate of Trp-P-2 was, on the other hand, drsmatically induced by treatment of rats with either PCB or 3-methylcholanthrene. PCB was more effective than 3-methylcholanthrene in inducing the Trp-P-2 metabolizing activity. Since M-3 fraction eluted from HPLC contained mutagenic metabolite(s), the effects of pretreatment with inducers on the microsomal activity to form M-3 from Trp-P-2 were also examined. The pretreatment with phenobarbital enhanced the activity to some degree, however, pretreatment with PCB or 3-methylcholantluene resulted in a more marked increase in the M-3 formation activity (Fig. 4B). Thus the rate of metabolism of Trp-P-2 determined

132

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(A)

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PCB

0 10 Peak height 50

20

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4"0min

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40

PCB

30 MC 20 10 0

10

20

30

PB control 40min

Fig. 4, A: effect of pretreatments of rat with phenobarbital (PB), 3-methylcholanthrene (MC) and PCB on the rate of Trp-P-2 metabolism. The incubation mixtures containing 3 mg protein of microsomes, 0.8 mM NADP, 8 mM glucose 6-phosphate, 6 mM MgCI=, 3 I.U. of glucose-6-phosphate dehydrogenase, 50 mM Na,K-phosphate (pH 7.4) and 0.2 mM Trp-P-2 acetate in a final volume of 3 ml were incubated at 37°(3. At the time indicated, aliquots of the incubation mixture were extracted and treated with an equivalent vol. of acetonitrile. Other details of the procedures are the same as described in Materials and Methods. B: effect of pretreatments of rats with phenobarbital (PB), 3-methylcholanthrene (MC) and PCB on the rate of M-3 formation. Experimental conditions are as described in (A) and Materials and Methods. as t h e d e c r e a s e in t h e s u b s t r a t e c o n c e n t r a t i o n a n d t h e f o r m a t i o n o f M-3 w e r e i n d u c e d b y PCB a n d 3 - m e t h y l c h o l a n t h r e n e in a m a n n e r similar t o t h e increase in t h e m u t a t i o n f r e q u e n c y in 8. typhimurium T A - 9 8 (K. Ishii e t al., unpublished data).

Effects of oxidizing and reducing agents on the metabolites of Trp-P-2 As s h o w n in Fig. 2B, it has b e e n suggested t h a t t h e u l t i m a t e m u t a g e n o f Trp-P-2 is t h e N - h y d r o x y - T r p - P - 2 . T o f u r t h e r s u p p o r t this idea, t h e e f f e c t s o f o x i d i z i n g a n d r e d u c i n g agents o n t h e m e t a b o l i t e s w e r e e x a m i n e d . P o t a s s i u m f e r r i c y a n i d e is k n o w n t o oxidize a n a r o m a t i c h y d r o x y l a m i n e t o f o r m a n i t r o s o derivative [ 1 2 , 1 3 ] . As was e x p e c t e d , t h e a d d i t i o n o f this r e a g e n t t o t h e ex-

133

tract of the incubation mixture at a concentration of 5 mM resulted in a disappearance of M-3 peak within 1 rain, and alternatively an increase in the peak height of M-4 fraction was observed (Figs. 5A,5B). Thus, it was assumed that the M-3 fraction contained the hydroxylamine derivative and M-4 fraction nitroso derivative of Trp-P-2. The addition of potassium ferricyzmide did not affect the peak heights of M-l, M-2 and Trp-P-2. Similar results were also obtained when 40 mM 7-manganese dioxide was used as an oxidizing agent instead of potassium ferricyanide [14]. Such N-oxygenated compounds as hydroxylamino, nitroso and tertiary amine N~xide derivatives are known to be reduced to corresponding amines by the treatment with titanium trichloride [15--17]. Addition of this reagent at a concentration of 40 mM to the extract of the incubation mixture caused the disappearance of the metabolites of M-3 and M-4 fractions and the increase of the amount of Trp-P-2, while the peak heights of M-1 and M-2 remained unchanged (Fig. 5C). This evidence again supported the view that the M-3 and the M-4 fractions contain the hydroxylamino and nitroso derivatives of Trp-P-2.

Mutagenicity of the metabolite in M-4 fraction To compare the mutagenicity of M-3 with M-4, mutagenicity assays of these fractions were conducted. To prevent the conversion from M-3 to M-4

A

FL .

B

FL

°

Trp-P-2

M-3

[

.M-4

uv

0 2 4 6 8 1012min

0 2 4 6 8 1012rain

0

2 4 6 8 1012min

Fig. 5. Effects of potassium ferricyanide and titanium trichloride on the metabolitas of Trp-P-2. The extracted supematant obtained as described in Fig. 3 was used for these experiments. Potassium ferricyanide (10 mM) or titanium trichloride (40 raM) was added to the same volume (100/zl) of thesupernatants. After standing for a few minutes, aliquots (30 ~1) of these reaction mixtures were subjected to HPLC. Chromatograms after treatm e n t with water (control), potassium ferricyanide and titanium trichloride were shown in A, B and C, respectively. Potassium ferricyanide was eluted with a retention time of 2 min (Fig. B) and titanium trichloride with a retention time of 9.5 min (Fig. C), respectively. Details of HPLC conditions are the same as described in Materials and Methods.

134 before the */-manganese treatment, ascorbic acid (1 mM) was added to the incubation mixture. The addition of ascorbic acid completely inhibited the formation of the M-4 w i t h o u t lowering the formation rate of the M-3. After 30 rain of incubation, about 72% of the Trp-P-2 was metabolized. A portion of the extract of the incubation mixture was treated with 7-manganese dioxide and the resultant M 4 fraction was isolated by I-IPLC. Thus, the mutagenicity o f M-4 fraction was compared to M-3 fraction which was isolated from the extract w i t h o u t treatment with 7-manganese dioxide. The results shown in Table I indicate t h a t the mutagenicity o f M-4 is comparable to t h a t o f M-3 (Table I).

UV spectra o f Trp-P-2 and its metabolites Trp-P-2 and the active metabolites, M-3 and M-4, were isolated by preparative HPLC. The UV spectra of these metabolites are shown in Fig. 6. M-3 showed the spectrum similar to Trp-P-2 except t h a t an absorption m a x i m u m at about 260 nm was shifted somewhat to a shorter wavelength. M-4 showed a peak at about 260 nm and a shoulder at 250 nm. In addition, M-4 had a peak at 380 nm which was n o t seen in the spectra of Trp-P-2 and M-3. Trp-P-2 and M-3 had a small peak or shoulder at about 310 nm.

Mass spectrum o f the metabolite The mass spectrum of the main metabolite peak in the chromatogram of the trimethylsflylated extract is shown in Fig. 7. In this mass spectrum, the molecular ion peak was observed at m/e 357. Further, the fragment ion peaks, at m/e 342 (M-15), m/e 285 (M-72) and m/e 269 (M-88) which are characteristic for trimethylsilylated c o m p o u n d were also detected. From this mass spectrum, it was indicated that the main metabolite was a h y d r o x y l a t e d metabolite of Trp-P-2. This mass spectrum could n o t be demonstrated after treatment of the extract with -/-manganese dioxide. TABLE I M U T A G E N I C A C T I V I T I E S O F Trp-P-2 M E T A B O L I T E S Sample

Rev./plate

%

Incubation mixture

42675

100

(15 ~1~) M-3 f r a c t i o n b M-4 fractionc

41680 39680

97.7 93.0

a T h e a m o u n t o f t h e i n c u b a t i o n m i x t u r e w h i c h c o n t a i n e d 0.77 ~g o f Trp-P-2 a c e t a t e b e f o r e t h e r e a c t i o n was s t a r t e d . b A f t e r t h e t r e a t m e n t o f t h e i n c u b a t i o n m i x t u r e ( 1 0 0 ~1) w i t h same v o l u m e o f a c e t o n i t r i l e as d e s c r i b e d in Materials a n d M e t h o d s , 3 0 ul o f t h e e x t r a c t (eqv. t o 15 ul o f t h e i n c u b a t i o n m i x t u r e ) was i n j e c t e d i n t o t h e c o l u m n , t h e n t h e f r a c t i o n c o n t a i n i n g M-3 was s u b j e c t e d f o r t h e m u t a t i o n assay. CThe same e x t r a c t d e s c r i b e d a b o v e b u t a f t e r ~,-manganese d i o x i d e t r e a t m e n t was s u b j e c t e d for HPLC a n d m u t a t i o n assay.

135 A~ .5

.•

Trp-P-2

~liM

.2

~

4

/~ ~ M-3

200

300

..

400

5(}0nm

Fig. 6. Absorption spectra of M-3, M-4 and Trp-P-2. The same supernatant as described in Fig. 5 was injected onto HPLC and the fractions containing M-3 or Trp-P-2 were collected. T o o b t a i n M-4, the supernatant was treated w i t h *f-manganese d i o x i d e and the resultant supernatant was subjected t o I-~LC. A f t e r the fractions containing M-3, M-4 o r Trp-P-2 were rechromatographed on HPLC, the absorption spectra o f the fractions were measured p r o m p t l y .

leee

>, E-, I-4 (/)

z¢d

,!

Z

8

SO

ISle

ISe

,±t

~

U}

z

t~ z

Fig. 7. Mass spectrum of the main metabolite in the chromatogram of the trimethylsilylated extract. The experimental details are described in Materials and Methods.

136 DISCUSSION

It has been established that Trp-P-2 is metabolically activated to a mutagenic product(s) by an NADPH
14 R -NH 2

Trp-P-2

R-NHOH M-3 R -NO

M-4

137 r e d u c e d t o t h e p a r e n t c o m p o u n d , Trp-P-2, b y t h e t r e a t m e n t w i t h t i t a n i u m t r i c h l o r i d e (Fig. 5C). F u r t h e r m o r e , t h e shift o f t h e a b s o r p t i o n m a x i m u m in t h e U V s p e c t r a f r o m a b o u t 3 1 0 n m t o 3 8 0 u m associated w i t h t h e o x i d a t i o n o f M-3 t o M ~ suggests t h a t a m i n o g r o u p o f Trp-P-2 was c o n v e r t e d t o a b a t h o c h r o m i c f u n c t i o n a l g r o u p (Fig. 6). F r o m t h e s e results, it can be conf i r m e d t h a t M-4 is t h e n i t r o s o derivative o f Trp-P-2 (Fig. 8). Studies f o r f u r t h e r c o n f i r m a t i o n o f these m e t a b o l i t e s are n o w in progress. ACKNOWLEDGEMENT This w o r k was s u p p o r t e d b y a Grant-in-Aid f o r C a n c e r R e s e a r c h f r o m t h e Ministry o f E d u c a t i o n , Science a n d Culture. REFERENCES

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