Mutagenicity of 2-hydroxyalkyl-N-nitrosothiazolidines

Fd Chem. Toxic. Vol. 22, No. 4, pp. 253 259, 1984

Printed in Great Britain. All rights reserved

Copyright (

0278-6915/84 $3.00+0.00 1984 Pergamon Press Ltd

MUTAGENICITY OF 2-HYDROXYALKYL-NNITROSOTHIAZOLIDINES K. UMANO and T. SttlBAMOTO Department of Environmental Toxicology, University of California, Davis, CA 95616 and S. Y . FERNANDO and C.-I. WEI Department of Food Science and Human Nutrition, University of Florida, Gainesville, FL 326l 1, USA

(Receired 7 July 1983)

Abstrac~The mutagenicity of 2-hydroxyalkyl-N-nitrosothiazolidines was tested using Salmonella typhimurium strains TA98 and TA100. The N-nitrosothiazolidines tested were unsubstituted N-nitrosothiazolidine (NT), N-nitrosothiazolidine-4-carboxylic acid (NTC), 2-hydroxymethyl-N-nitrosothiazolidine (HMNT), 2-(l,2,3,4-tetrahydroxybutyl)-N-nitrosothiazolidine, 2-(l,2,3,4-tetrahydroxypentyl)-Nnitrosothiazolidine, 2-(1,2,3,4,5-pentahydroxypentyl)-N-nitrosothiazolidine (PHPNT) and 2-(l,2,3,4,5pentahydroxypentyl)-N-nitrosothiazolidine-4-carboxylic acid. Among the N-nitrosothiazolidines tested, only HMNT and PHPNT exhibited clear dose-response mutagenicity toward strain TAI00 with or without metabolic activation. None of the 2-hydroxyalkyl-N-nitrosothiazolidines were mutagenic to strain TA98. NT exhibited much stronger mutagenicity than either HMNT or PHPNT. Mutagenic activities of NT and PHPNT were eliminated by carboxyl substitution in lhe position ~ to the N-nitroso group.

INTRODUCTION

Since the discovery of carcinogenic N-nitrosamines (Magee & Barnes, 1967) amines have received attention as precursors of N-nitrosamines in foods (Crosby, 1976; Scanlan, 1975). Generally, mutagenic or carcinogenic N-nitrosamines are formed when a secondary amine such as dimethylamine, pyrrolidine or piperazine reacts with nitrite under acidic conditions. Amines or amine precursors such as proteins, amino acids and phospholipids are present in most foods and may be available for reaction with nitrite. Several years ago, thiazolidine derivatives, which contain nitrosatable nitrogen in their heterocyclic ring, were reported to have formed in a browning model system (Coughlin, 1979: Sakaguchi & Shibamoto, 1978). Sakaguchi & Shibamoto (1978) reported that four alkylthiazolidines were formed in a model system in which cysteamine was reacted with o-glucose, acetaldehyde or glyoxal. Once thiazolidines are formed in foods, they may react with nitrites from a nitrite-rich food such as a cured meat. This reaction may result in the formation of mutagenic or carcinogenic N-nitrosamines. Sakaguchi & Shibamoto (1979) isolated N-nitroso-2-methylthiazolidine as a major volatile product from a cysteamine acetaldehyde nitrite model system under

Abbreviations: HMNT = 2-Hydroxymethyl-N-nitrosothiazolidine: NT = N-nitrosothiazolidine; NTC - Nnitrosothiazolidine-4-carboxylic acid: PHPNT - 2( 1,2, 3,4, 5 - pentahydroxypentyl- N - nitrosothiazolidine ; PHPNTC = 2-(1,2,3,4,5-pentahydroxypentyl)-N-nitrosothiazolidine-4-carboxylic acid; THBNT = 2-(1,2,3,4tetrahydroxybutyl-N-nitrosothiazolidine; THPNT = 2( 1,2,3,4-tet rahydroxypentyl-N-nitrosot hiazolidine. F('T 224 A

simulated cooking conditions; Mihara & Shibamoto (1980) identified N-nitroso-2-methylthiazolidine and N-nitroso-2-ethylthiazolidine in a reaction mixture obtained from a cysteamine D-glucos~nitrite model system. Mihara & Shibamoto (1980) and Sekizawa & Shibamoto (1980) tested several purified Nnitrosothiazolidines for mutagenic activity using the Ames assay: N-nitrosothiazolidine and several of its 2-alkyl derivatives were mutagenic in Salmonella O,phimurium strain TA98 with S-9 mix, strongly mutagenic in strain TA100 without S-9 mix, mutagenic in strain TA100 with S-9 mix and not mutagenic in strain TA98 without S-9 mix. Further, the mutagenic activity of the unsubstituted parent nitrosamine and its 2-alkyl derivatives decreased in the following order: unsubstituted > isopropyl > propyl > ethyl > butyl > isobutyl > methyl. Although many thiazoles and thiazolines (dehydrogenated compounds of thiazolidines) have been found in foods (Mussinan & Walradt, 1974; Tonsbeek, Copier & Plancken, 1971; Wilson, Mussinan, Katz & Sanderson, 1973), thiazolidine derivatives had never been found in food until the reports of Gray, Reddy, Price et al. (1982) and Kimoto, Pensabene & Fiddler (1982), w h o found Nnitrosothiazolidine (NT) in fried bacon at levels of up to c. 5 ppb. These studies proved the formation of thiazolidines in cooked foods. It has been postulated that mutagenic volatile 2-alkyl-N-nitrosothiazolidines form from nonvolatile 2-(1,2,3,4, 5-pentahydroxypentyl)-N-nitrosothiazolidine (PHPNT) in cooked food (Kitamura, Wei & Shibamoto, 1981). 2-(l,2,3,4,5-Pentahydroxypentyl)thiazolidine, which is an Amadori-type compound (an early product of a glucose-decarboxylated cysteine browning reaction), undergoes N-nitrosation 253

254

K. UMANOet al. Table 1. Thiazolidines tested for mutagenicity

R2

Thiazolidine N-Nitrosothiazolidine N-Nit rosothiazolidine-4-carboxylic acid 2-Hydroxymethyl-N-nitrosothiazolidine 2-(1,2,3,4-Tetrahydroxybutyl)-N-nitrosothiazolidine 2-(1,2,3,4-Tetrahydroxypentyl)-N-nitrosothiazolidine 2-(l,2,3,4,5-Pentahydroxypentyl)-N-nitrosothiazolidine 2-( 1,2,3,4,5-Pentahydroxypentyl)-N -nit rosot hiazolidine4-carboxylic acid

N- NO

Abbreviation

R 1

NT NTC HMNT THBNT THPNT PHPNT

H H

CH2OH CH(OH)CH(OH)CH(OH)CH2OH CH(OH)CH(OH)CH(OH)CH(OH)CH~ CH(OH)CH(OH)CH(OH)CH(OH)CH2OH

R2 H COOH H H H H

PHPNTC

CH(OH)CH(OH)CH(OH)CH(OH)CH2OH

COOH

readily under mild conditions in the presence of nitrite (Coughlin, 1979). In this study, the mutagenicity of PHPNT and its possible heat-breakdown products were investigated using the Ames assay.

EXPERIMENTAL

Materials Thiazolidine and its 2-alkylhydroxy derivatives were prepared from cysteamine hydrochloride (Sigma Chemical Co., St Louis, MO) and the corresponding fatty aldehydes. The thiazolidines synthesized are listed in Table 1. NT. NT was prepared as described by Ray (1978). N-Nitrosothiazolidine-4-carboxylic acid (NTC). To prepare NTC, thioproline (2g, Sigma Chemical Co.) was dissolved in 23 ml 1 N-HC1 by stirring in a 100-ml Erlenmeyer flask held in ice-NaCl-cooled water. Sodium nitrite solution (1.24 g in 20 ml water) was added to this thioproline solution over a 30-rain period. The solution was stirred for 24 hr at room temperature. The reaction mixture was twice extracted with 50ml dichloromethane. The extract was washed with 30 ml water and the dichloromethane solution was dried over anhydrous sodium sulphate for 12 hr. The solvent was removed under reduced pressure. The solid material obtained was recrystallized from a dichloromethane pentane (3: 1, v/v) solution (668 rag, yield % = 34). 2-Hydroxymethyl-N-nitrosothiazolidine ( H M N T ) . To prepare HMNT, an aqueous solution (8ml) containing cysteamine hydrochloride (1136 mg) and glycoaldehyde (600 mg, Sigma Chemical Co.) was refluxed for 1 hr. The reaction mixture was cooled to room temperature and 7 ml 2 N-NaOH was added. The solution was twice extracted with 40ml dichloromethane. After the solvent had been removed, 2-hydroxymethylthiazolidine remained as a yellow oily liquid (729 mg). 2-Hydroxymethylthiazolidine (729 mg) was dissolved in 2 ml water and 9 ml 1 N-HCI was added at 0°C. Sodium nitrite solution (507 mg in 5 ml water) was added dropwise to the above solution. The reaction mixture was stirred for 24 hr at room temperature. The solution was twice extracted with 60ml dichloromethane. The extract was washed with 15 ml water and dried over anhydrous sodium sulphate. After removal of sol-

vent, H M N T (350 mg, yield ~ = 24) remained as a yellow oil. 2- ( 1, 2, 3, 4- Tetrahydroxybutyl) - N - nitrosothiazolidine ( T H B N T ) . To prepare THBNT, a 22-ml solution containing cysteamine hydrochloride (5.30g) and L-(+ )-arabinose (7.01 g, Sigma Chemical Co.) was refluxed for 3 hr. The reaction mixture was cooled to room temperature and 7.1 g sodium bicarbonate was added while the solution was stirred. Ethanol (500 ml) was added to the solution, and a white precipitate was filtered off. The filtrate was reduced to 100ml in vacuo and a white precipitate was filtered off. The white precipitate was washed with cold ethanol to give tetrahydroxybutylthiazolidine. Crude tetrahydroxybutylthiazolidine was recrystallized with ethanol (473mg, yield °/~o= 48). Tetrahydroxybutylthiazolidine (836 mg) was dissolved in 8 ml water, and the solution was cooled to ffC. After l N-HCI had been added with stirring, sodium nitrite solution (414mg in 6ml water) was added dropwise to the solution. The reaction mixture was stirred for 24 hr. After water had been removed in vacuo, a dark brown solid was obtained. The solid was dissolved in 40 ml methanol, and a white solid precipitate was filtered off. The filtrate was concentrated in vacuo to give a yellow oil. This yellow oil was dissolved in 30 ml methanol and the filtrate was concentrated two more times. The yellow oil obtained was dried at 80~C under reduced pressure, and a light yellow solid THBHT was obtained (730 mg, yield o/ o/-_ 77). 2-(1,2, 3,4-Tetrahydroxypentyl)-N-nitrosothiazolidine ( T H P N T ) and 2-(1,2,3,4,5-pentahydroxypentyl)N-nitrosothiazolidine (PHPNT). THPNT and PHPNT were prepared using the method described for THBNT. 2-(1,2,3,4-Tetrahydroxypentyl)thiazolidine (800mg) produced TH PN T (783mg, yield = 86), and 2-(l,2,3,4,5-pentahydroxypentyl)thiazolidine (1434 mg) produced PHPNT (1225 mg, yield ~ = 76). 2- ( 1, 2, 3,4,5-PentahydroxypenO, I)-N-nitrosothiazo lidine-4-carboxylic acid ( P H P N T C ) . To prepare PHPNTC an aqueous solution (5 ml) of c-cysteine (615mg, Sigma Chemical Co.) and D-glucose (900mg, Aldrich Chemical, Milwaukee, WI) was refluxed for 3 hr. After the reaction mixture had cooled to room temperature, water was removed in vacuo. The residue was dissolved in 60 ml methanol and the solution was refluxed for 1 hr. White solid

Mutagenicity of 2-hydroxyalkyl-N-nitrosothiazolidines

255

precipitates were filtered off and the solid was washed with ethanol to give 793mg 2-(1,2,3,4,5pentahydroxypentyl)-thiazolidine-4-carboxylic acid (yield ~o = 65), of which 500 mg was nitrosated, using the method described for THBNT, to obtain PHPNTC (392 mg, yield ~o = 71). The N-nitrosothiazolidine derivatives synthesized were characterized using proton nuclear magnetic resonance spectroscopy and mass spectroscopy.

with 2ml top agar at 4 5 C , and poured onto a bottom agar plate. The plates were incubated at 3 7 C for 2 days before the colonies were counted. Microscopic examination of the background bacterial lawn was also performed to check the toxicity of the compounds to the bacteria: a sparse bacterial lawn with pinpoint colonies indicated that levels were toxic.

Mutagenicity testing Bacteria. S. typhimurium tester strains TA98 and TAI00 were provided by Dr B. N. Ames (Biochemistry Department, University of California, Berkeley). Both strains were examined for genetic properties before use. Liver-enzyme induction and medium preparation. The agar medium and the liver S-9 were prepared by the method described by Ames, McCann & Yamasaki (1975). Induction of liver enzymes was achieved by the intraperitoneal injection of Aroclor 1254 (diluted in corn oil to 200mg/ml) at a dosage of 500 mg/kg body weight into male Sprague-Dawley rats (body weight c. 200 g; Charles River Breeding Laboratories, Wilmington, MA) 5 days before they were killed. S-9mix. The S-9 mix contained per ml: 0.1 ml rat liver S-9, 33/~mol KCI, 8/~mol MgC1, 5~mol glucose-6-phosphate, 4¢tmol NADP, and 100/~mol sodium phosphate buffer (pH 7.4). In the 0°/o S-9 mix preparation, rat liver S-9 was replaced by 0.25 M-potassium phosphate buffer (pH 7.4). Positive control. Known mutagens were tested concurrently to confirm the revertant properties of the tester strains used in each experiment (Ames et al. 1975; de Serres & Shelby, 1979). 2-Aminofluorene was used for both strains in plates containing S-9 mix. For those plates containing no S-9 mix, 2-nitrofluorene was used for strain TA98 and methylmethane sulphonate was used for TA100. Genotypic characteristics and spontaneous reversion. The genotypic characteristics of the tester strains were checked following the procedures outlined by Ames et al. (1975). The rate of spontaneous mutations in the tester strains was also checked. The numbers of spontaneous revertants on control plates fell within the range of spontaneous revertant numbers of the tester strains as indicated by de Serres & Shelby (1979). Testing procedure. Samples were dissolved in spectrophotometric-grade dimethylsulphoxide (Schwarz/Mann, Orangebury, NY) and then serially diluted with dimethylsulphoxide. Plate-incorporation and preincubation assays were applied to test the mutagenic potencies of the samples (Ames et al. 1975; Yahagi, Degawa, Seino et al. 1975). The plate incorporation assay was performed by adding to 2 ml of top agar 0.1 ml of a culture of the S. typhimurium tester strain, 25/~1 of the solution to be tested, and 0.5 ml of the 10~ or 0 H S-9 mix. The mixture was poured onto a bottom agar plate. For the preincubation assay, 25/~l of the solution to be tested and 0.5 ml of the 10~ or 0~o S-9 mix were added to test tubes containing 0.1 ml of a culture of the S. typhimurium tester strain. The mixture was preincubated at 37°C for 20 rain in a water bath, mixed

RESUI.TS AND DISCUSSION The results of mutagenicity testing of the thiazolidine derivatives are shown in Table 2. Unsubstituted N-nitrosothiazolidine (NT) was also tested K)r mutagenicity for comparison with 2-hydroxyalkyl-N-nitrosothiazolidines. The chemicals that exhibited a clear dose effect on strain TAI00 were unsubstituted NT, HMNT and PHPNT. Corresponding dose-response curves are shown in Fig. 1 (plate-incorporation method) and Fig. 2 (preincubation method). None of the 2-hydroxyalkyl-N-nitrosothiazolidines were mutagenic in TA98. Unsubstituted NT is a strong mutagen. This finding is consistent with previous research (Sekizawa & Shibamoto, 1980). However, the mutagenic activity of NT was eliminated by carboxyl substitution in the position ~ to the N-nitroso (NTC). This same phenomenon has been reported regarding the mutagenicity and carcinogenicity of N-nitrosopyrrolidine and N-nitrosoproline. The mutagenic or carcinogenic activities disappeared as a result of carboxyl substitution (Rao, Cox, Allen et al. 1981). PHPNT showed weak mutagenicity toward TA 100 in both the plate-incorporation assay and the preincubation assay. PHPNT exhibited mutagenicity at the levels of 2.5 and 5 rag/plate with and without S-9 mix, respectively. These levels of mutagenicity are somewha! low compared with those of 2-alkyl-N-nitrosothiazolidines. For example, 2-isopropyl-N-nitrosothiazolidine is mutagenic to TA100 without S-9 mix at the level of 65 ~g/plate (Sekizawa & Shibamoto, 1980). Also, when a carboxyl group was introduced to the position :~ to the N-nitroso group (PHPTC), mutagenicity was eliminated. Among the 2-hydroxyalkyl-nitrosothiazolines, HMNT showed the strongest mutagenicity toward TA100 in both the plate-incorporation assay and the preincubation assay. In the preincubation assay (Fig. 2), the mutagenicity of HMNT was suppressed by adding S-9 mix up to the level of 250#g HMNT/plate, whereas mutagenicity was increased by the addition of S-9 mix at 500#g HMNT/plate; HMNT produced similar results in the plateincorporation assay (Fig. 1). The suppression of mutagenicity by the addition of S-9 mix has also been observed with 2-alkyl-N-nitrosothiazolidines (Sekizawa & Shibamoto, 1980). As previously mentioned, PHPNTC is an Amadori-type compound (Schubert, 1939; Weitzel, Engelman & Fretzdorfl', 1959). Although Amadori compounds occur widely in foods, beverages and tobacco (Coughlin, 1979), the occurrence of Nnitroso Amadori compounds in foods has not yet been reported. Heyns, Roper, Roper & Bernd (1979) tested a series of N-nitroso Amadori compounds for mutagenicity and found that none of the compounds

HMNT

NTC

NT

Thiazolidine*

-S-9

102_+9 101._7 112._ 10 549 ._ 71 1008._ 147 1892 ._ 47 toxic toxic toxic toxic toxic 119 + 5 101 ._ 15 144 + 14 129+15 1 2 9 + 18 121 ._ 15 139 ._ 10 110 -- 1 100 ._ 9 182_+15 427 ± 21 771 + 32 toxic

Concn (~g/plate)

0(SR) 0(+DMSO) 1 2.5 5 10 20 40 100 500 2500 5 10 20 100 500 2500 5000 5 10 20 100 500 750

TAI00

125_+7 114._5 114._ 14 291 ._ 16 346._26 368 ._ 28 751 ._ 62 toxic toxic toxic toxic 137 ._ 15 110 ._ 13 141 ._ I 1 115._3 140._ 10 122 + 7 toxic 119 -- 9 133 ._ 25 131 ._9 316 ± 20 1(173 ._ 109 toxic

+S-9

NT 31 ._9 27 ._ 2 29 ._ 6 toxic toxic toxic toxic 27 ._ 4 29 ._ 2 22 ._ 4 26._6 22._4 24 + 5 24 + 5 29 ._ 5 25 ._ 5 26+7 29 ._ 8 25 ._ 7 13 + 2

27_+5 25._5

-S-9

Plate-incorporation assay TA98

NT 54._ 11 59 ._ 12 86 ._ 9 26 ._ 9 toxic toxic toxic 35 ._ 8 31 ._ 2 32 ._ 7 28_+4 29._7 25 + 7 toxic 34 ._ 4 36 ._ 7 32_+4 32 _+ 5 37 ._ 6 38 -- I

35_+3 33._4

+S-9

1027 ._ 135 NT 430 ._ 72 150 ._ 25 NT NT NT NT NT NT NT 110._15 124_+ 12 179 + 15 toxic NT NT 182--41 446 ._ 23 667 ._ 50 NT

115._9 107._ 14

-S-9

No. of revertants/plate

TAI00

121 ._ 12 NT 620 ._ 39 1622 ._ 323 NT NT NT NT NT NT NT 126._15 127._8 158 + 27 toxic NT NT 155+10 247 + 44 1028 ._ 63 NT

115._11 119._ 19

+S-9

26 + 4 21 + 4 toxic toxic NT NT NT NT NT NT NT 24+5 17+6 26._ 6 38 -- 10 NT NT 10+4 19 ._ 6 20 ._ 2 NT

21 ._4 15._2

S-9

Preincubation assay

Table 2. Results o f mutagenicity tests of thiazolidines using Salmonella typhimurium strains TA98 and T A I 0 0

TA98 +S-9

24 ._ 5 29._4 32 + 9 35 + 6 NT NT NT NT NT NT NT 31+3 28 ._5 29._ 6 toxic NT NT 29--7 28 ._ 7 29 4- 9 NT

32._2 25+6

> z © 2

5 10 20 100 250 500 2500 5 10 20 100 250 500 2500 5 10 20 100 500 2500 5000 5 10 20 100 250 50O 2500

*For definitions see p. 253 or Table 1. Values are means ± ISD for three runs.

PHPNTC

PHPNT

THPNT

THBNT

116_+6 121 + 15 177+15 283 ± 15 103 ± 14 99 ± 9 100 ± 7 101 ± 7 106 ± 10 119±10 NT SR = Spontaneous revertants

144_+ 13 143 ± 15 131±15 123 ± 17 156± 11 NT 131 ± 16 117± 19 138± 16 125±18 134+ II 130 + 16 NT 121 ± 1 0 115_+ 13 130_+ 5 130±9 157+ 12 233±16 300 ± 31 96 _+8 118 ± 3 126 + 5 121 ± 1 ! 104 ± 12 138±8 NT

112+5 128 ± 18 130± 13 110 ± 24 139± 14 NT 118± 13 95±8 121±6 101±12 116±9 104 ± 7 NT 110±7 123_+ I1 110 + 9

137 ± 9

114 + 9

28+5 34+4 38±4 38 ± 5 32 ± 7 36 ± 4 32 ± 5 37_+4 NT 37±5 32 _+4

29 + 7

36+8 30±8 33±5 NT 32±5 33 k 2 32±8 29±7 27±5 33±8 NT 33 ± 4 24 ± 6 38±5 34_+5

26 + 4

DMSO = Dimethylsulphoxide

29_+6 23 _+3 30±9 NT 31 + 9 toxic 28±6 28±2 23+3 25_+5 NT 26 ± 6 toxic 28_+2 23_+5 23 ± 5 22±5 23 ± 3 24±4 23 _+4 26 ± 4 25 ± 4 26 + 3 27±4 NT 19±4 20 ± 3

24 + 5

N T = N o t tested

NT NT 91 _+5 105±6 149 ± 4 171 ± 16 NT NT NT 100+12 125+8 144±6 169 ± 12 NT NT NT NT 126± 10 122± 10 178±13 219 +_ 18 NT NT NT 107± 17 119 ± 21 170_+16 NT

20±2 23 ± 1 NT NT NT NT 19±3 25±8 25±5 toxic NT NT 16 ± 3 24± 1 27 ± 5 17_+2 NT

27±6 NT NT NT 20±5 21±3

151 + 8 NT NT NT 130±6 121±11

148±9

30 + 4

124 + 9

480 ± 19 NT NT NT NT 121 ± 3 124±21 198±6 246 + 31 NT NT NT 112±1 131 ± 18 152± 10 NT

NT NT 21 ± 3 24±6

NT NT 140± 19 131± 12 29±3 NT NT NT 24±4 25±3 28+5 25 + 5 NT NT NT NT 33_+2 22+3 23±1 25 ± 5 NT NT 20 ± 5 23±6 19 ± 6 28+8 NT

29 ± 5

NT NT 27_+ 3 31±5

-,,,,i

:e

v.,

m-" .,<

v<

258

K. UMANOet al. 2000 •

1000" Q,.

\

800

g oo 600

~_ 400

200

I

0

.__L____/,~_ I

_ _ _ L

1 O0

/,,_..1

10

20

500

1

Concn

of test compound ( f i g / p l a t e ) .....

[

l

2 5

5

{mg/ptate)

Fig. 1. Mutagenicity in plate-incorporation assays of N-nitrosothiazolidine (A,LS.), 2-hydroxymethyIN-nitrosothiazolidine ( I , [ ] ) and 2-(I,2,3,4,5-pentahydroxyl~entyl)-N-nitrosothiazolidine(Q,O), in Salmonella typhimurium strain TA100, with (solid symbols) and without (open symbols) the addition of S-9 mix. Each point is the average of three runs.

tested showed mutagenic activity, On the other hand, Coughbn (1979) reported that a heterocyclic Areadoff compound, N-nitroso-fructose-l-tryptophan, was mutagenic in the Ames assay in both strain TA98 and TA100 in the absence of S-9 mix. Further studies on the biological activity of N-nitrosated Amadori compounds are required, THBNT and THPNT did not show mutagenic activity toward TA98 and TA100 with or without S-9 mix. Generally, hydroxylation of the alkyl group on C-2 in the thiazolidine ring reduces the mutagenicity. 2-Alkyl-N-nitrosothiazolidines (2-ethyl-, 2-propyl-, 2-isopropyl-, 2-isobutyl-) exhibit muta-

2000 1

genicity (Mihara & Shibamoto 1980; Sekizawa & Shibamoto, 1980), but THBNT and THPNT do not. The suppression or elimination of mutagenicity by hydroxylation or carboxylation may be due to water solubili% It has been found that when 2-(l,2,3,4,5pentahydroxypentyl)thiazolidine is heated in an aqueous solution, 2-hydroxymethylthiazolidine is produced (Kitamura et al. 1981), and when cysteamine and ~)-glucose are heated together in an aqueous solution, unsubstituted thiazolidine is produced (Sakaguchi & Shibamoto, 1978). These results indicate that 2-(1,2,3,4,5-pentahydroxy-

•

~_ 800

-~ 600

•

J/

"6 o 400

Z

200

k'/ll

0

I

0

I

, /

1

- ~

I

~

I

10 20 100 2~'.'.'.'.'.'.'.'.'.~) 500 Concn of compound (,u.g / plate ] . . . . .

"

_~,.,~

I _ _ J

I

2 5 (mg / plate )

5

Fig. 2. Mutagenicity in preincubation assays of N-nitrosothiazolidine (A,A), 2-hydroxymethylN-nitrosothiazolidine (ll,F3) and 2-(l,2,3,4,5-pentahydroxypentyl)-N-nitrosothiazolidine (Q,O), in Salmonella typhimurium strain TAI00, with (solid symbols) and without (open symbols) the addition of S-9 mix.

Mutagenicity of 2-hydroxyalkyl-N-nitrosothiazolidines pentyl)thiazolidine, which is one of the early products of b r o w n i n g reactions in cooked foods, is a possible precursor of mutagenic N-nitrosothiazolidines. This investigation was supported in part by the Center of Natural and Environmental Resources Program, IFAS, University of Florida. Acknowledgement

REFERENCES

Ames B. N., McCann J. & Yamasaki E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test. Mutation Res. 31, 347. Coughlin J. R. (1979). Formation of N-nitrosamines from Maillard browning reaction products in the presence of nitrite. Ph.D. Dissertation, University of California, Davis. Crosby N. T. (1976). Nitrosamines in foodstuffs. In Residue Reviews. Residues o f Pesticides and Other Contaminants in the Total Environment. Edited by F. A. Gunther. Vol. 64.

p. 77. Springer-Verlag, New York. de Serres F. J. & Shelby M. D. (1979). Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Res. 64, t 59. Gray J. I., Reddy S. K., Price J. F., Mandagere A. & Wilkins W. F. (1982). Inhibition of N-nitrosamines in bacon. Fd Technol. 36, 39. Heyns K., Roper S., Roper H. & Bernd M. (1979). NNitroso sugar amino acids. Angew. Chem. 19, 878. Kimoto W. I., Pensabene J. W. & Fiddler W. (1982). Isolation and identification of N-nitrosothiazolidine in fried bacon. J. Agric. Fd Chem. 30, 757. Kitamura K., Wei C.-I. & Shibamoto T. (1981). Watersoluble thiazolidines formed in a cysteamine o-glucose browning model system. J. Agric. Fd Chem. 29, 378. Magee P. N. & Barnes J. M. (1967). Carcinogenic nitroso compounds. Adv. Cancer Res. 10, 163. Mihara S. & Shibamoto T. (1980). Mutagenicity of products

259

obtained from cysteamin~glucose browning model systems. J. Agric, Fd Chem. 28, 62. Mussinan C. J. & Walradt J. P. (1974). Volatile constituents of pressure cooked pork liver. J. Agric. Fd Chem. 22, 827. Rao T. K., Cox J. T., Allen B. E., Epler J. L. & Lijinsky W. ([ 98 l). Mutagenicity of N-nitrosopyrrolidine derivatives in Salmonella (Ames) and Escherichia coli K-12 (343/113) assays. Mutation Res. 89, 35. Ray S. (1978). Direct gas chromatographic analysis of cyclic N-nitrosamines. J. Chromat. 153, 173. Sakaguchi M. & Shibamoto T. (1978), Formation of heterocyclic compounds from the reaction of cysteamine and D-glucose, acetaldehyde, or glyoxal. J. Agric. Fd Chem. 26, 1179. Sakaguchi M. & Shibarnoto T. (1979). Isolation of N-nitroso-2-methyl-thiazolidine from a cysteamineacetaldehyde-sodium nitrite model system. Agric. biol. Chem. 43, 667. Scanlan R. A. (1975). N-Nitrosamines in foods. C R C Crit. Rev. Fd Technol 5, 357. Schubert M. P. (1939). The combination of cysteine with sugars..1. Biol. Chem. 130, 601. Sekizawa J. & Shibamoto T. (1980). Mutagenicity of 2-alkyl-N-nitrosothiazolidines. J. Agric. Fd Chem. 28, 781. Tonsbeek C. H. T., Copier H. & Plancken A. J. (1971). Isolation of 2-acetyl-2-thiazoline from beef broth. J. Agric. Fd Chem. 19, 1014. Weitzel G., Engelmann J. & Fretzdorff A. M. (1959). Darstellung und Bildungstenderz yon Zucker-CysteinVerbindungen und ihren Komplexalzen. Stabilit~itskonstanten der Zink-und Kobalt (II)-Komplexe. Z. Physiol. Chem. 315, 236. Wilson R A., Mussinan C. J., Katz I. & Sanderson A. (1973). Isolation and identification of some sulfur chemicals present in pressure-cooked beef. J. Agric. Fd Chem. 21, 873. Yahagi T., Degawa M., Seino Y., Matsushima T., Nagao M., Sugimura T. & Hashimoto T. (1975). Mutagenicity of carcinogenic azo dyes and their derivatives. Cancer Lett. 1, 91.