Mutagenicity of 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid treated with nitrite in the presence of alcohols

Genetic Toxicology

ELSEVIER

Mutation Research 367 (1996) 43-49

Mutagenicity of 1-methyl- 1,2,3,4-tetrahydro-/3-carboline-3-carboxylic acid treated with nitrite in the presence of alcohols Minoru Higashimoto a Takehito Yamamoto a Takemi Kinouchi b H i s a o M a t s u m o t o a, Y o s h i n a r i O h n i s h i b,, a Department of Hygienic Chemistry. Faculty of Pharmaceutical Sciences, Tokushima Bunri Unit'ersity, Yamashiro-cho, Tokushima 770,. Japan Department of Bacteriology, School of Medicine, The Unit,ersity of Tokushima, Kuramoto-cho, Tokushima 770, Japan

Received 1 August 1995; revised 25 September 1995; accepted 25 September 1995

Abstract The mutagenicity of a product produced from 1-methyl-l,2,3,4-tetrahydro-/3-carboline-3-carboxylic acid (MTCCA), which is a component in soy sauce, after treatment with 50 mM nitrite at pH 3, 37°C, for 60 rain in the presence of 7.5% ethanol was much higher than that in the absence of ethanol during the nitrite treatment. The enhancement of the mutagenicity of nitrite-treated MTCCA by ethanol required simultaneous treatment of MTCCA with nitrite and ethanol. The mutagenicity of MTCCA treated with nitrite in the presence and absence of ethanol was detected in the same fractions on HPLC and was highest for Salmonella o'phimurium strain YG1029 possessing elevated O-acetyltransferase activity among the several Salmonella test strains, suggesting that the same mutagen belonging to aromatic compounds was produced both in the presence and absence of ethanol. Methanol, n-propanol and isopropanol as well as ethanol were also observed to have an augmenting effect. However, the sugars glucose and sucrose had no effect. When MTCCA was treated with nitrite in the presence of commercial alcoholic beverages equivalent to 1.25-10% ethanol, Japanese 'sake' and 'shochu' were demonstrated to have a highly augmenting effect and beer, wine, whisky and brandy to have a mildly augmenting effect. Keywords: fl-Carboline; Nitrite treatment; Alcohol; Ames Salmonella mutagenicity test; Enhancement

1. Introduction 1- M e t h y l - 1 , 2 , 3 , 4 - t e t r a h y d r o - / 3 - c a r b o l i n e - 3 carboxylic acid ( M T C C A , Fig. 1), a condensation

* Corresponding author. Tel.: +81 886-33-7069; Fax: +81 886-33-0771 ; E-mail: ohnishi @basic.med.tokushima-u.ac.jp.

product of tryptophan with acetaldehyde during the b r e w i n g process (Herraiz and Ough, 1993; Oshita et al., 1993), is contained in fermentation products such as soy sauce, soybean paste, vinegar, beer, wine and Japanese sake (Bosin et al., 1986; Adachi et al., 1991). The m e a n a m o u n t of M T C C A in soy sauce is 4 8 7 / _ t g / m l (Higashimoto et al., 1988). Wakabayashi et al. (1983) reported that M T C C A reacts with nitrite

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M. Hi~,,ashimoto et al. / Mutation Research 367 (1996) 43-49

44

~~'~ ~

COOH

I ~ 1 ' ~ NH H CH:~ Fig. 1. Structure of MTCCA.

in acidic medium and produces a mutagen and that the mutagenicity can partly account for the mutagenicity of nitrite-treated soy sauce. We have studied the effect of alcohols on nitrite treatment of soy sauce. We previously reported that the mutagenicity of soy sauce or tyramine treated with nitrite was reduced in the presence of alcohols and that soy sauce might contain an effective component other than tyramine because the reduction effect of ethanol on the mutagenicity of nitrite-treated soy sauce was clearly weaker than that on the mutagenicity of nitrite-treated tyramine which is a major mutagen precursor in soy sauce (Higashimoto et al., 1995). Herein, we show that the mutagenicity of MTCCA is strongly increased after nitrite treatment in the presence of alcohols.

Wako Pure Chemical Industries (Osaka, Japan). The other chemicals were of reagent grade. 2.2. Nitrite treatment The chemicals were dissolved in sterilized water. Unless otherwise stated, aqueous solutions of 1.9 ml containing 0.3 mg of MTCCA and 0-0.2 ml of alcohol or alcoholic beverage equivalent to 0-0.2 ml of ethanol were mixed in brown tubes with 0.1 ml of 1 M sodium nitrite and adjusted to pH 3.0 with 6 N HC1. The doses of alcoholic beverages were calculated with the labeled proof. And the concentrations of alcohols were shown as those in the nitrite-treated reaction mixtures. The reaction mixtures were incubated at 37°C for 60 min in the dark. The nitrosation was stopped by addition of 1 ml of 0.1 M ammonium sulfamate to decompose the residual nitrite (Takeda and Kanaya, 1982) as previously reported (Higashimoto et al., 1988, 1993). The reaction mixture was used immediately or after high-performance liquid chromatography (HPLC) for the mutation assay. All the experiments were performed more than two times. 2.3. Mutation test

2. Materials and methods 2.1. Alcoholic beverages and chemicals Three to five brands of six kinds of alcoholic beverage commercially available in Tokushima were used. Four brands of beer made by three makers, three brands of sake, a Japanese liquor, made by three makers, three brands of red wine, a brand of rose wine and a brand of white wine made by three makers, and a brand of synthetic 'shochu', a brand of shochu brewed with buckwheat and a brand of shocbu brewed with barley made by three makers were all produced in Japan. Two brands of whisky were produced in Japan and one in Scotland. One brand of brandy was produced in Japan and two brands in France. The indicated ethanol concentrations of beer, sake, wine, shochu, whisky and brandy were 5-6, 13.5-15.5, 13-14, 25-35, 43 and 40%, respectively. MTCCA (CAS No. 5470-37-1) was purchased from Sigma Chemicals (St. Louis, MO, USA). Ethanol, methanol, n-propanol and isopropanol were from

The mutation test with Salmonella typhimurium strains TAI00, TA1535, YGI026, YG1029 and YG7108 was conducted twice for each experiment with two plates per sample by the preincubation procedure of Maron and Ames (1983) in the absence of $9 mix. Strains YGI026, YG1029 and YG7108 are nitroreductase-increased, O-acetyltransferase-increased mutants of strain TA100 and an O 6-methylguanine-DNA methyltransferase-less ada- ogtdouble mutant of strain TA1535, respectively (Watanabe et al., 1989, 1990; Yamada et al., 1993, 1995). Unless otherwise stated, 0.1 ml of the nitritetreated samples or eluates from HPLC was mixed with 0.5 ml of the buffer solution (pH 7.4) and 0.1 ml of overnight culture of a test strain. The mixture was then preincubated at 37°C for 20 rain and plated on the agar plates. Although ethanol is known to be bactericidal, the toxicity of ethanol in the nitritetreated reaction mixture which is finally diluted 10.5-fold to contact with the test bacteria must be negligible because the reduction of viable cells at the

45

M. H igashimot o et al. / Mutation Research 367 (1996) 43 49

end of the preincubation of the assay was not observed when we used the nitrite-treated samples containing less than 20% ethanol as previously reported (Higashimoto et al., 1995). The mutation test for fractions in HPLC was conducted with 1.64 × 109 CFU per plate of strain YG1029 which were suspended in saline after centrifugation of the overnight culture at 4000 rpm for 30 min. The number of spontaneous revertants was subtracted in all the data on mutagenicity. The numbers of spontaneous revertants (means _+ S.D.) from strains TA100, TA1535, YG1026, YG1029, YG7108 and concentrated YG1029 were 12l -t- 14, 1 l +_ 3, 163 +_ 33, 122 +_ 10, 19 _+ 3 and 185 _+ 3, respectively.

2.4. High-performance (HPLC)

liquid

chromatography

HPLC was performed with a Shimadzu LC-6A instrument equipped with a UV monitor at 270 nm. The column oven was maintained at 50°C. The analysis was conducted with a column (8 × 250 ram) packed with Nucleosil 100-5C18 (Macherey-Nagel, Diiren, Germany) using 10 mM sodium phosphate buffer/100 mM NaC104 (pH 2.6) in 50% methanol at a flow rate of 3 ml/min.

3. Results

3.1. Mutagenici~ of MTCCA treated with nitrite in the presence and absence of ethanol Since the optimum pH of the reaction of MTCCA and nitrite was about 3 as shown in Fig. 2, nitrite

o~ o

~

1000

o

Oo0 o

0

o

0

2

4

5

6

pH

Fig. 2. Mutagenicity of 50 /xg of MTCCA treated with nitrite in the pH range of 1.17-6.15. Aqueous solutions containing 1.5 mg of MTCCA were mixed with nitrite and adjusted to appropriate pH with 6 N HCI. Number of spontaneous revertants was 118.

i

,

10

o

o

i

,

15

20

treatment was carried out at pH 3, which was consistent with the conditions in the nitrite treatment of soy sauce and tyramine in the previous paper (Higashimoto et al., 1995). The mutagenicity of MTCCA treated with 50 mM nitrite in the absence of ethanol was increased in the presence of ethanol in proportion to the concentration up to 7.5% at which concentration the sample induced 1520 everrants per 10 /zg of MTCCA as shown in Fig. 3. Good correlations were observed between the mutagenicity and the dose of MTCCA treated with nitrite both in the presence and absence of 7.5% ethanol except for the low doses in the absence of ethanol as shown in Fig. 4. The slope of dose-response curve of the former was 12.2 times steeper than that of the latter. However, there is no enhancement of the mutagenicity of nitrite-treated MTCCA when the culture of strain TA 100 was plated with nitrite-treated MTCCA combined with nitrite-treated ethanol or ethanol (treatments 5 and 6 in Fig. 5). The enhancement of the mutagenicity of nitrite-treated MTCCA required simultaneous treatment of MTCCA with nitrite and ethanol (treatment 4 in Fig. 5).

1000

3

,

Fig. 3. Mutagenicity of l0 /xg of MTCCA treated with nitrite in the presence of 0-20% ethanol. Number of spontaneous revertants was 127. Symbols are the means of duplicate experiments.

o 1

,

5

Ethanol(%)

o

>=

o

o r~

2000

looo

°o o

0

~ y = 183X +

0

25

3.5

y = 15,0X - 75,7

50

75

100

MTCCA(.ug)

Fig. 4. Mutagenicity of MTCCA treated with nitrite in the presence ( O ) and absence of ( 0 ) 7.5% ethanol. Aqueous solutions containing 0.075-3 mg of MTCCA were treated with nitrite. Number of spontaneous revertants was 118. Symbols are the means of duplicate experiments.

M. Higashimotoet al. / Mutation Research367 (1996) 43-49

46

3.3. HPLC chromatogram and mutagenici~ of HPLC .fractions of nitrite-treated MTCCA

o~ 6o0 ,00

1

2

3

4

5

6

7

Treatment

Fig. 5. Mutagenicity of 5 /xg of MTCCA treated with nitrite before and after addition of 7.5% ethanol. Treatment: 1, intact MTCCA; 2, nitrite-treated MTCCA; 3, nitrite-treated ethanol; 4, nitrite-treated (MTCCA+ethanol); 5, nitrite-treated MTCCA+ nitrite-treated ethanol; 6, nitrite-treated MTCCA+ethanol; 7, MTCCA+ nitrite-treated ethanol. Fifty microliter of each sample combined with 50/zl of water or another sample were assayed for mutagenicity. Number of spontaneous revertants was 118.

3.2. Mutagenici~ of MTCCA treated with nitrite in the absence and presence of ethanol for the five Salmonella test strains The mutagenicity of M T C C A treated with nitrite in the absence and presence of ethanol was studied for the YG test strains YG7108, YG1026 or YG1029 (Table 1) which had different sensitivity for alkylating agents, nitroarenes or aromatic amines, respectively (Watanabe et al., 1989, 1990; Yamada et al., 1993, 1995). Each of the two doses of M T C C A treated with nitrite in the absence or presence of 7.5% ethanol was highly mutagenic for strain Y G I 0 2 9 compared with the parental strain TA100 as shown in Table 1.

The products of M T C C A treated with nitrite in the presence and absence of 7.5% ethanol at 37°C for 60 min were fractionated by HPLC at one-rain intervals for 40 min as shown in Fig. 6a and b, respectively. Although the mutagenicity of eluates from HPLC which contained 50% methanol could not be measured by the method with normal cell number of test strain Y G I 0 2 9 , it was qualitatively measured with increased cell number. The large peak at the retention time of 9.1 rain corresponding to the main product, N-2 nitroso-substituted M T C C A , was not mutagenic as reported by Wakabayashi et al. (1983). Strong mutagenicities were similarly observed in the fractions of retention time 19-21 rain, corresponding to the extremely small peaks of both chromatograms although the product sample of M T C C A in the absence of ethanol was applied to the column 5-fold more than that in the presence of ethanol. The recoveries of the mutagenicity in their fractions were 64.0 and 75.2% in the presence and absence of ethanol, respectively. The amount of the mutagen formed was small and it was chemically unstable. To confirm that the peak at retention time 19-21 rain corresponds to the major mutagen in the nitrite-treated M T C C A , the nitrite-treated M T C C A was left at the ambient temperature and the mutagenicity and the peak area on HPLC were examined over a time course of 8 h. The

Table 1 Mutagenicity of MTCCA treated with nitrite in the absence and presence of 7.5% ethanol for Salmonel& typhimurium strains TA1535, YG7108, TA100, YGI026 and YG1029 Sample MTCCA MTCCA + ethanol

MTCCA (/Lg/plate)

TA1535

His + revertants/plate YG7108

TAI00

YGI026

YGI029

10 ~ 20 ~ 1b 2b

0 0 0 2

2 4 0 0

66 95 138 309

35 (0.53) 40 (0.42) 76 (0.55) 155 (0.50)

676 1120 1750 3120

(1.0) (1.0) (1.0) (1.0)

(10.2) (11.8) (12.7) (10.1)

Numbers of spontaneous revertants for strains TAI535, YG7108, TA100, YG1026 and YGI029 were 10, 19, 104, 165 and 142, respectively. The ratio to the revertants for strain TAI00 is shown in parentheses. a.b 100 and 200/zl aliquots of nitrite-treated samples were assayed for mutagenicity immediately(a) or after 10-fold dilution with water (b).

M. Higashimoto et al. /Mutation Research 367 (1996) 43-49

47

6OO a 40o

T v

200

E tO

0

600

b ffl

(3 e-

E

t~ O

> n"

<

2OO

0

~

Retention time (min) Fig. 6. HPLC chromatogram and mutagenicity of HPLC fractions of 20 /xg (a) and 100 /xg (b) of MTCCA treated with nitrite in the presence (a) and absence (b) of 7.5% ethanol, respectively. Aqueous solutions containing 0.2 mg (a) and 1.0 mg (b) of MTCCA were treated with nitrite. Each one tenth of nitrite-treated sample was analyzed by HPLC. Chromatograms were drawn at the range of attenuation = 10 before 19 min and then attenuation = 4. One hundred microliter aliquots of the e|uates fractionated each one min were assayed for mutagenicity. Cell number of strain YG1029 used for the mutation test was 1.64 × 109 cells per plate. Number of spontaneous revertants was 186.

small peak detected at 20 min in the HPLC chromatogram seemed to contain the major mutagen because the peak area and the mutagenicity of the nitrite-treated MTCCA decreased with a very good correlation with the lapse of time as shown in Fig. 7. 3.4. Mutagenicity of MTCCA treated with nitrite in the presence of other alcohols or alcoholic be~'erages The mutagenicity of nitrite-treated MTCCA was strongly increased in the presence of n-propanol and

isopropanol and more strongly increased in the presence of methanol as shown in Fig. 8. However, the sugars glucose or sucrose had no augmenting effect in the same conditions. The augmenting effect of representative alcoholic beverages usually taken by Japanese on the nitrite treatment of MTCCA was studied. The mutagenicity of MTCCA treated with 50 mM nitrite was strongly increased in the presence of sake and shochu, whereas the effect of beer, wine, whisky and brandy was not so strong, as is shown in Fig. 9. The mutagenicity reached the maximum in

1000 standing time: 0 hr

o.

=o

y = 0.60x + 92 r = 0.989 ~

0

i 5O0

i IOO0

I

Peak area (/zV-sec)

Fig. 7. Correlation of a peak area at retention time of 19-21 rain on HPLC of 5 /xg of MTCCA treated with nitrite in the presence of 7.5% ethanol and the mutagenicity of the nitrite-treated MTCCA after standing for various times at ambient temperature. Fifty microliter aliquots of nitrite-treated sample were assayed for mutagenicity. Number of spontaneous revertants was 126.

0

5

10

15

C o n c e n t r a t i o n of alcohol (%)

Fig. 8. Mutagenicity of 10 /xg of MTCCA treated with nitrite in the presence of 0-15% methanol (O), n-propanol (zx), isopropanol (13), glucose ( . ) or sucrose ( • ) . Number of spontaneous revertants was 129.

M. Higashimoto et al. / Mutation Research 367 (1996) 43-49

48

2000

Beer 12 Beer 13

~

---am

Sake 21 Sake 22 Sake 23

1000

OJ

0

0

2

4

6

8

10

• & •

Wine 31 Wine 32 Wine 33 Wine 34 Wine 35

0

2

4

6

8

10

3000 00" 2000 ~

~

Shochu 41 Shochu 42 Shochu 43 Q

0

2

4

6

8

10

3000 [

~

Whisky51

2000

--C---

Whisky 52 Whisky 53

2

4

6

8

10

Brandy 61

Brandy63 Brandy 63

1000

0

2

4

6

8

10

2

4

6

8

10

Concentration of ethanol (%) Fig, 9. Mutagenicity of l0 /xg of MTCCA treated with nitrite in the presence of several alcoholic beverages. Number of spontaneous revertants were 112-118.

the presence of the alcoholic beverages equivalent to 5-7.5% ethanol.

4. Discussion We previously reported that the moderate consumption of alcoholic beverages might be good for our health because the mutagenicity of soy sauce was reduced by treatment with nitrite in the presence of ethanol or alcoholic beverages (Higashimoto et al., 1995). However, we must recognize again the undesirable side reaction that ethanol and some alcoholic beverages have a strong stimulative effect on the nitrosation of MTCCA as described here. Among the commercial alcoholic beverages used here, sake and shochu produced mutagen-forming effects as strong as ethanol, but the other beverages were not so effective. Furthermore, the augmenting effect of some brands of sake and shochu is stronger than that of pure ethanol, partially because they may contain MTCCA. Sake is a fermented liquor, while shochu is a distilled liquor. Whisky and brandy, both distilled liquors, containing a few impurities strangely have a

weak augmenting effect, suggesting that the nitrosation of MTCCA or the formation of ethyl nitrite will be affected by the impurities. In any case, beverages such as beer, wine, whisky and brandy may contain ingredients that inhibit the formation of a mutagen from MTCCA treated with nitrite in the presence of ethanol. The mutagens produced from MTCCA that was treated with nitrite in the presence and absence of ethanol were detected in the same fractions of 19-21 min on HPLC (Fig. 6) and were highly active for strain YG1029 (Table 1). Therefore the same mutagenic compound seem to be produced from MTCCA both in the presence and absence of ethanol. However, since the fractions of 19-21 rain had very low absorbance, it could not be chemically identified. The major product of nitrite-treated MTCCA in the fraction of 9 rain on HPLC must be N-2 nitroso MTCCA which is nitroso-substituted at the nitrogen in the six-membered ring of pyridine moiety (Wakabayashi et al., 1983). Although the compound is stable, it has no mutagenicity (Fig. 6). On the other hand, it was reported to produce an unstable and mutagenic nitroso compound by nitrosation of the nitrogen in the five-membered ring of indole acetonitrile (Wakabayashi et al., 1989). Therefore, it can be inferred that the unstable mutagen produced from nitrite-treated MTCCA is N-9 nitroso compound which is nitroso-substituted at the nitrogen in the five-membered ring of the indole moiety in Fig. 1. The separation and isolation of this unknown compound is now in progress. The average contributions of tyramine and MTCCA on the mutagenicity of nitrite-treated 25 brands of soy sauce have been estimated as 97.0 and 2.9%, respectively, in the absence of ethanol (Higashimoto et al., 1988). However, it was suggested that MTCCA as well as tyramine is a significant mutagen precursor in soy sauce when it is treated with nitrite in the presence of alcohols. Although soy sauce contains 1-methyl-l,2,3,4-tetrahydro-/3-carboline (MTC), a precursor of a weak mutagen (Higasbimoto et al., 1987, 1988), the influence of MTC on the mutagenicity of nitrite-treated soy sauce in the presence of ethanol will be negligible because the mutagenicity of nitrite-treated MTC is weak itself and little affected by the presence of ethanol (data not shown). The reason why the reduc-

M. Higashimoto et a l . / Mutation Research 367 (1996) 43-49 ing e f f e c t o f e t h a n o l o n t h e m u t a g e n i c i t y o f n i t r i t e treated soy sauce was obviously weaker than that on the mutagenicity of nitrite-treated tyramine ( H i g a s h i m o t o et al., 1995) c o u l d b e b e c a u s e o f t h e existence of MTCCA

which strongly increased the

m u t a g e n i c i t y in t h e p r e s e n c e o f e t h a n o l ,

Acknowledgements W e are g r a t e f u l to Dr. T. N o h m i f o r s u p p l y i n g Salmonella o'phimurium strains YG1026, YGI029 and YG7108.

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49

spices and a medicinal plant in Thailand, Mutation Res., 303, 135-142. Higashimoto, M.+ T. Yamamoto, T. Kinouchi, Y. Handa, H. Matsumoto and Y. Ohnishi (1995) Mutagenicity of soy sauce treated with nitrite in the presence of ethanol or alcoholic beverages, Mutation Res., 345, 155-166. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Oshita, I., H. Kanamori, M. Mizuta and I. Sakamoto (1993) Formation of /3-carboline derivatives during the soy sauce manufacturing process and their changes during storage, J. Jpn. Soc. Nutr. Food Sci., 46+ 179-182 (in Japanese). Takeda, Y. and H. Kanaya (1982) A screening procedure for the formation of nitroso derivatives and mutagen by drug-nitrite interaction, Chem. Pharm. Bull., 30, 3399-3404. Wakabayashi, K., M. Ochiai, H. Saito, M. Tsuda, Y. Suwa+ M. Nagao and T. Sugimura (1983) Presence of l-methyl-l,2+3,4tetrahydro-/3-carboline-3-carboxylic acid, a precursor of a mutagenic nitroso compound+ in soy sauce, Proc. Natl. Acad. Sci. USA, 80, 2912-2916. Wakabayashi, K., M. Nagao and T. Sugimura (1989) Mutagens and carcinogens produced by the reaction of environmental aromatic compounds with nitrite, Cancer Surveys, 8, 385-399. Watanabe, M., M. Ishidate Jr. and T. Nohmi (1989) A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella O'phimurium strains TA98 and TAI00, Mutation Res.+ 216, 211-220. Watanabe, M., M. lshidate Jr. and T. Nohmi (1990) Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels, Mutation Res., 234, 337-348. Yamada, M., A. Hakura, T. Sofuni and T. Nohmi (1993) New method for gene disruption in Salmonella typhimurium: construction and characterization of an ada-deletion derivative of Salmonella t3phimurium TA1535, J. Bacteriol., 175, 55395547. Yamada, M., B. Sedgwick, T. Sofuni and T. Nohmi (1995) Sonstruction and characterization of Salmonella O'pymirium deficient in DNA repair of O6-methylguanine, J. Bacteriol., 177, 1511-1519.