Fd Chem. Toxic. Vol. 29, No. 4, pp. 243-247, 1991 0278-6915/91 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1991 Pergamon Press pie

EXCRETION AND DISTRIBUTION OF NITRITE-TREATED OR UNTREATED 1-METHYL- 1,2,3,4-TETRAHYDRO-

fl-CARBOLINE-3-CARBOXYLIC ACID IN RATS

A. NAGAHARA and S. KUMAGAI Department of Biomedical Research on Food, National Institute of Health, Shinagawa-ku,

Tokyo 141, Japan

(Received 2 August 1990; revisions received 26 November 1990)

Abstract--The excretion and distribution of 14C were studied in rats given by oral intubation 14C-labelled (-)-(1S,3S)-l-methyl-l,2,3,4-tetrahydro-fl-carboline-3-carboxylic acid and its stereoisomer, (-)-(1R,3S)-l-methyl-l,2,3,4-tetrahydro-fl-carboline-3-carboxylic acid (MTCAs), compounds that are known to become mutagenic on reaction with nitrite. For comparison, another group of rats was given by oral intubation products of the in vitro treatment of [~4C]MTCAs with nitrite. When rats were given the untreated ['4C]MTCAs, most of the 14C was excreted in the faeces within 24 hr and was noted only in the intestine on autoradiograms of rats killed 30 min and 6 hr after dosing. In contrast, when rats were given the nitrite-treated [~4C]MTCAs, ~4C was mainly excreted in the urine within 24 hr and was noted not only in the intestine but also in the liver and kidney on autoradiogram. These results suggest that the majority of MTCAs pass rapidly through the gastro-intestinal tract, while the bulk of the reaction products of MTCAs with nitrite are absorbed from the gastro-intestinal tract and excreted in the urine.

INTRODUCTION

( - )-(1S,3S)-l-Methyl-l,2,3,4-tetrahydro-fl-carboline- 3-carboxylic acid and its stereoisomer (-)-(1R,3S)-1- methyl- 1,2,3,4-tetrahydro-fl-carboline-3-carboxylic acid (MTCAs) are mutagen precursors isolated from soy sauce (Wakabayashi et al., 1983). These compounds become direct-acting mutagens when nitrosated in vitro with high levels of sodium nitrite under acidic conditions (Wakabayashi et al., 1983). Since human saliva contains nitrite, ingestion of MTCAs was suspected to be a cause of human gastric cancer (Wakabayashi et al., 1983). However, more recently Nagahara et al. (1986) have shown that the in vitro reaction of soy sauce with nitrite made the soy sauce mutagenic only when the nitrite concentration was much higher than that seen in human saliva. Furthermore, micronucleus tests in vivo have shown no mutagenicity of the nitrite-treated soy sauce when given orally to mice (Nagahara et al., 1991).

To assess the effect of nitrosation of MTCAs on their fate in the animal body, we studied the excretion and distribution in rats of oral doses of ['4C]MTCAs, both with and without pretreatment with nitrite in vitro, in rats.

MATERIALS AND METHODS

Chemicals. MTCAs (harmane-l,2,3,4-tetrahydro- 3-carboxylic acid) were purchased from Sigma Chemical Co. (St Louis, MO, USA). [14C]MTCAs (4-14C-MTCAs, 12.2mCi/mmol, Fig. 1) were syn-

Abbreviations: MNTCA = 1-methyl-2-nitroso-l,2,3,4-tetra- hydro-fl-carboline-3-carboxylic acid; MTCAs = ( - ) - ( 1 S,3S)- 1-methyl- 1,2,3,4-tetrahydro-fl-carboline-3-carb- oxylic acid and its stereoisomer (-)-(1R,3S)-l-methyl- 1,2,3,4-tetrahydro-fl-carboline-3-carboxylic acid.

243

thesized from L-tryptophan-sidechain-3-[J4C] by Tokai Institute of Daiichi Pure Chemicals Co. Ltd (Ibaragi-ken, Japan). Nitrite treatment of MTCAs was by incubating MTCAs containing [t4C]MTCAs (17 mU as MTCAs) with NaNO 2 (386 mM) in 0.1 M- citric acid buffer (pH 3.0) at 37°C for 1 hr. The reaction mixture was used for animal experiments without purification of nitrosated MTCAs.

Animals and administration o f [14C]MTCAs. Male SD rats (Crj:CD) purchased from Charles River Japan, Inc. (Atsugi-shi, Japan) were used at 3 wk of age for autoradiography and excretion experiments, and at 5 wk of age for the other experiments, after overnight withdrawal of food. Rats received by gas- tric intubation [~4C]MTCAs diluted with MTCAs (3.0mg MTCAs and 15.5#Ci [t4C]MTCAs in 1 ml citric acid buffer) at a dose of 14 mg MTCAs/kg body weight, or MTCAs treated with nitrite at the same dose, for autoradiography and for studying the ~4C excretion into urine, faeces and expired air. For studying the 14C excretion into bile and changes in 14C concentrations in blood and plasma, doses of 3.0 mg MTCAs mixed with 1.55/~Ci [~4C]MTCAs in 1 ml citric buffer were used.

Collection o f excreta. After oral intubation with the nitrite-treated or untreated MTCAs, rats were housed, one per cage, in glass metabolism cages (Natsume Seisakusho Co. Ltd, Tokyo, Japan). Urine

~ _ _ _ ~ . ~ COOH

~ , , ) ' ~ , N ,,)~',,,,,/NH H i

CH3

Fig. 1. Structure of p4C]MTCAs. *Position of '4C.

244 A. NAOAHARA and S. KUMAGAI

Table 1. Excretion of 14C in the urine and faeces of rats dosed orally with nitrite-treated or untreated MTCAs

Untreated MTCAs Nitrite-treated MTCAs (% of administered J4C (% of administered 14C

Time after excreted in:) excreted in:) dosing

(br) Urine Faeces Urine Faeces

0-24 11.6 + 0.60 74.5 + 7.37 81.6 + 2.86 12.5 __. 0.77 24-48 0.41 + 0.11 1.97 + 0.86 1.42 __. 0.41 0.30 _ 0.036 48-72 0.094 + 0.026 0.035 + 0.027 0.33 __. 0.015 0.088 __. 0.025

Values are means + SD for four rats.

and faeces were collected separately in glass con- tainers at the bot tom of the cage at 24-hr intervals. Ethanolamine containing expired CO2 was removed at 10-60-min intervals from a gas trap, through which expired air was removed from the cages by a vacuum pump.

Autoradiography. Rats were rapidly frozen in dry ice-acetone under ether anaesthesia 30 min or 6 hr after dosing. Sagital 20-/~m sections through the whole frozen body were cut and dried at - 2 0 ° C . Autoradiograms were made by pressing the dried sections against X-ray films (Fuji X-ray film no. 150; Fuji Film Co., Tokyo, Japan) for 2 wk.

Collection of bile. Rats were laparotomized under light ether anaesthesia, and a bile duct was cannu- lated with a polyethylene tube. After the abdominal incision was closed, each conscious rat was given the nitrite-treated or untreated [~4C]MTCAs and was then placed in a restraining cage (Sugiyamagen Co., Tokyo, Japan). Bile was collected through the cannulae at l -hr intervals for 8 hr.

Collection of blood. Under ether anaesthesia, the carotid artery was brought to the exterior through a skin incision made in the neck, and was cannulated with a polyethylene tube for blood sampling. After the incision was closed, each conscious rat was given the nitrite-treated or untreated [t4C]MTCAs. Two to

E

(g

8 o.z5 10

1 o

F_ E O.50 o

~ o.2~

/ ', i

,/ " \

\ b

\

13 --0-

I I I I I I I I I f

10 50 60 90 120 t50 180 240 :500

Time o f te r dosing ( ra in)

Fig. 2. Mean concentrations of ~4C in the plasma (O) and whole blood (O) of two to four rats who were given orally the nitrite-treated [t4C]MTCAs, and those in the plasma (n ) and whole blood (1 ) of two to four rats who were given

orally the untreated [t4C]MTCAs.

four drops of blood were collected at 10- to 60-min intervals for 6 hr by loosening the clamp round the carotid artery.

Determination of radioactivity. Plasma, urine and ethanolamine were mixed with ACS II (Amersham Corp., IL, USA) and then counted in a scintillation counter (Type LS-200B, Beckman). Homogenate of faeces and whole blood were mixed with perchloric acid and hydrogen peroxide, warmed in the oven at 70°C for 60 min, mixed with ACS II, and counted.

Statistics. The statistical significance of differences between means was determined by Student's t-test.

RESULTS

When the [14C]MTCAs, not treated with nitrite, were given to rats, 1 4 C w a s excreted mainly in the faeces, mostly during the first 24hr (Table 1). In contrast, when the nitrite-treated [~4C]MTCAs were given, 14C was excreted largely in the urine, again mainly during the first 24 hr (Table 1). Very low levels of radioactivity (lower than 0.008% of the admin- istered dose) were detected in the expired air during the first 24 hr, but thereafter none could be detected, regardless of whether the rats received the untreated or treated [~4C]MTCAs.

The ]4C concentration in the plasma increased after the administration of the nitrite-treated [14C]MTCAs, reached a maximum after 60 min, and then decreased (Fig. 2). The 14C concentrations in whole blood were

c

"1o o

"6

c 0.5 .2

0-1 2-5 4 - 5 t - 2 5 -4 5 - 6

Time o f t e r dosing (h r )

Fig. 3. Excretion of ~4C into the bile of rats who were given orally nitrite-treated MTCAs (0) or untreated MTCAs (O). Points and vertical bars indicate means +SD for

groups of four rats,

Plate 1. Autoradiograms of rats killed (a) 30 min or (b) 6 hr after receiving orally untreated [~4C]MTCAs, or (c) 30 min or (d) 6 hr after receiving orally nitrite-treated [14C]MTCAs.

245

Fate of MTCAs in rats 247

roughly half of the concentrations in the plasma for most of the experimental period, indicating that ~4C distributes in plasma but not in blood cells. In rats given the untreated [14C]MTCA, the ~4C concen- tration in plasma and in whole blood remained nearly constant at a level below that seen in the rats given the treated [~4C]MTCAs, for most of the observation period (Fig. 2). The rate of biliary excretion of 14C was higher in rats that received the nitrite-treated [~4C]MTCAs than in those that received the untreated ones (Fig. 3), as might be expected from the ~4C concentrations in the plasma.

Autoradiographic studies revealed that ~4C is dis- tributed not only in the intestine but also in the liver and kidney 30 min after dosing, but is found only in the intestine after 6 hr, in the rats that received the nitrite-treated [~4C]MTCAs (Plate 1). When rats received the untreated [14C]MTCA, [14C] was found only in the intestine either 30 min or 6 hr after dosing (Plate 1).

(Wakabayashi et al., 1983). MNTCA is less polar than MTCAs, because the nitroso group is hydro- phobic while the charged amine is hydrophilic. Since cell membranes are permeable to lipophilic com- pounds, MNTCA would transfer across the epithelial cells of the gastro-intestinal tract more easily than MTCAs. Thus the more efficient absorption of nitrite-treated than untreated MTCAs may be due to nitrosation of the secondary amine of MTCAs.

Endogenous formation of N-nitroso compounds from ingested food components has been well docu- mented (Massey et al., 1988; Meier-Bratschi et al., 1983; Rounbehler et al., 1977). The observed differ- ence in the fate of the nitrite-treated and untreated MTCAs suggests that N-nitroso compounds formed by nitrosation of secondary amines could be absorbed from the gastro-intestinal tract more readily than the parent compounds containing the amine group.

DISCUSSION

When rats received the untreated [~4C]MTCAs, a large part of the administered 14C was excreted into the faeces within 24 hr. No clear increase in the plasma concentration of 14C was noted after dosing. Furthermore, the 14C was observed only in the intes- tine on autoradiogram 30 min and 6 hr after dosing. These results suggest that a large part of the untreated MTCAs pass rapidly through the gastro-intestinal tract without being absorbed and are excreted in the faeces.

In contrast, urine was the major excretion route in rats that received the nitrite-treated [~4C]MTCAs. Autoradiography and the pattern of the changes in the plasma concentration of 14C showed that after being absorbed very rapidly from the gastro- intestinal tract into the blood, a large part of the ~4C transferred to the liver and kidney within 6 hr.

It has been observed that nearly 80% of MTCAs are converted in vitro to the non-mutagenic com- pound 1-methyl-2-nitroso- 1,2,3,4-tetrahydro-fl-car- boline-3-carboxylic acid (MNTCA) by incubation with nitrite under in vitro conditions similar to those used for the nitrite pretreatment in the present study

REFERENCES

Massey R. C., Key P. E., Mallett A. K. and Rowland I. R. (1988) An investigation of the endogenous formation of apparent total N*nitroso compounds in conven- tional microflora and germ-free rats. Food and Chemical Toxicology 26, 595-600.

Meier-Bratschi A., Lutz W. K. and Schlatter Ch. (1983) Methylation of liver DNA of rat and mouse by N-nitro- sodimethylamine formed in viva from dimethylamine and nitrite. Food and Chemical Toxicology 21, 285-289.

Nagahara A., Ohshita K. and Nasuno S. (1986) Relation of nitrite concentration to mutagen formation in soy sauce. Food and Chemical Toxicology 24, 13-15.

Nagahara A., Ohshita K. and Nasuno S. (1991) Investi- gation of soy sauce treated with nitrite in chromosomal aberration test in vitro and micronucleus test in vivo. Mutation Research Letters. In press.

Rounbehler D. P., Ross R., Fine D. H., Iqbal Z. M. and Epstein S. S. (1977) Quantitation of dimethylnitrosamine in the whole mouse after biosynthesis in vivo from trace levels of precursors. Science, New York 197, 917.

Wakabayashi K., Ochiai M., Saito H., Tsuda M., Suwa Y., Nagao M. and Sugimura T. (1983) Presence of 1-methyl- 1,2,3,4-tetrahydro-fl-carboline-3-carboxylic acid, a pre- cursor of a mutagenic nitroso compound, in soy sauce. Proceedings of the National Academy of Sciences of the U.S.A. 80, 2912.