Antimutagenicity of cell fractions of microorganisms on potent mutagenic pyrolysates

Mutation Research, 298 (1993) 247-253

247

© 1993Elsevier Science PublishersB.V. All rights reserved 0165-1218/93/$06.00

MUTGEN 01851

Antimutagenicity of cell fractions of microorganisms on potent mutagenic pyrolysates Xue Bin Zhang and Yoshiyuki Ohta Laboratory for Microbial Biochemistry, Faculty of Applied Biological Science, Hiroshima University, Higashi, Hiroshima 724, Japan

(Received 8 October 1991) (Revisionreceived 13 August1992) (Accepted 20 August1992) Keywords: Desmutagenicity;Binding;Bio-antimutagenicityand cell fractions

Summary The inactivation of 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) by binding of mutagenic pyrolysate to fractions of microorgan-

isms (their desmutagenic and bio-antimutagenic activity) was investigated. All strains bound Trp-P-1 effectively, but Glu-P-1 to a lesser extent. The Gram-negative bacteria (GNB) could bind about 10-20 ~ g / m g of Trp-P-1 more than the Gram-positive bacteria (GPB), and about 50-60/xg/mg more than the yeasts. The cell wall skeletons of all strains tested had great binding ability, but in the cytoplasm of all strains tested it was lower. The peptidoglycan, outer membrane, and glucan isolate from the cell wall skeletons showed the highest binding ability to Trp-P-1. The cell wall skeletons of the tested strains greatly inhibited the mutagenicity induced by Trp-P-1, and to a lower extent that of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx). Although the cultured broth and solution of cells extracted by phosphate buffer (pH 7.0) showed antimutagenicity against Trp-P-1, this activity was lower than the binding of Trp-P-1 to the cells. The cultured broth and freeze-dried cytoplasms of yeast cells showed bio-antimutagenicity towards Trp-P-1, but those of all bacteria tested did not.

Much attention has recently been paid to antimutagenic factors that reduce the rates of spontaneous and induced mutagenesis. Antimutagens

Correspondence: Dr. X.B. Zhang, Laboratoryfor Microbial Biochemistry, Faculty of Applied Biological Science, Hiroshima University,Higashi-Hiroshima724, Japan. Abbreviations: Trp-P-1, 3-amino-l,4-dimethyl-5H-pyrido[4,3-

b]indole; Glu-P-1, 2-amino-6-rnethyldipyrido[1,2-a:3',2'-d]imidazole; MeIQx, 2-amino-3,8-dimethyl-imidazo[4,5-flquinoxaline; PTG, peptidoglycan; GNB, Gram-negative bacteria; GPB, Gram-positivebacteria.

are classified into 2 categories: desmutagens and bio-antimutagens. The former inactivates mutagens by chemical or enzymatic interaction before they can attack genes. The latter suppresses the process of mutagenesis (mutation fixation) after DNA is damaged by mutagens (Kada et al., 1982). Many studies clearly show that vegetables, microorganisms and their fractions inhibit the mutagenicity and carcinogenicity of chemical compounds. The fibers of many kinds of vegetables inactivate Trp-P-1 and Trp-P-2 by adsorbing them (Kada et al., 1984), and the dialysates from vegetables and fruits inhibited the mutagenicity of

248 Trp-P-1, Trp-P-2, benzo[a]pyrene, and aflatoxin B 1 toward S. typhimurium TA100 (Shinohara et al., 1988). Mammalian placental extracts showed strong antimutagenic activities for reverse mutations induced in Escherichia coli by UV radiation, y-rays and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Mochizuki and Kada, 1982). We previously reported that cells of microorganisms bound effectively with Trp-P-1 and Trp-P-2, and that cells which were killed at 120°C for 15 rain (Zhang et al., 1990, 1991) or in human gastric juice (Zhang and Ohta, 1991a) showed considerable binding activity. We also found that cell walls of microorganisms bound many kinds of carcinogenic heterocyclic amines and N-nitrosodimethylamine (Zhang and Ohta, 1991b; Zhang et al., 1991). There are no reports on the mechanism of antimutagenicity of cell fractions of microorganisms toward heterocyclic amines. In this report, we investigated what the mechanism of antimutagenicity of microorganisms could be against the mutagenicity induced by heterocyclic amines, and what the differences are between Gram-positive, Gram-negative bacteria and yeasts on inactivation of heterocyclic amines. Materials and methods

Culture strains of IFO were purchased from the Institute of Fermentation, Osaka (IFO). Escherichia coli 1592 and 231, Pseudomonas putida 01, and Streptococcus cremoris Z-25 are stock cultures in our laboratory. The yeast strains Saccharomyces cerevisiae 50, Torulospora dellbrueckii 22, Candida sp. 13 and Hansenula sp. 12 were isolated from Pito (Demuyakor and Ohta, 1991). The Gram-negative bacteria and Staphylococcus aureus IFO12722 were grown at 37°C for 24 h in PYM broth (Zhang and Ohta, 1992). Lactobacillus acidophillis IFO13951 and Bifidobacterium bifidum IFO14252 were grown anaerobically at 37°C for 24 h in LC broth, and S. cremoris Z-25 was maintained in GPY broth (Zhang and Ohta, 1991a). The yeasts were grown at 30°C for 24 h in MPYG broth (Zhang et al., 1991). Salmonella typhimurium TA1535/psk1002 was obtained from the Prefectural Institute of Public Health, Osaka. This strain was grown at 37°C in LB broth or TGA medium (1% Bacto tryptone, 0.5% NaC1

and 0.2% glucose) supplemented with ampicillin 20/~g/ml and was used for the umu-test (Oda et al., 1985).

Chemicals Chemical sources were as follows: Trp-P-1, Glu-P-1 and the enzyme substrate o-nitrophenyl/3-o-galactopyranoside were purchased from Wako Pure Chemicals (Osaka); MelQx was a gift from Dr. K. Wakabayashi of the National Cancer Research Institute (Tokyo). Preparation of fractions of bacterial cells The freeze-dried cell wall skeletons (CWS) and cytoplasms of bacteria were prepared as described previously (Zhang and Ohta, 1991b, 1992). Preparation of peptidoglycan (PTG), glucan and mannan Glucan and mannan from CWS and peptidoglycan were prepared according to the method of Hosoi et al. (1974) or Hirai et al. (1987). The preparations were freeze-dried until use. Preparation of out membrane and cytoplasmic membrane of E. coli IF014249 The preparation of the outer membrane and the cytoplasmic membrane of E. coli IFO14249 was carried out according to the methods of Yomato et al. (1975). The preparations were freeze-dried until use. Heat treatment Harvested cells were killed by heating at 120°C or at 100°C for 15 min. Viable cells were not found with a plate count method. These killed cells and untreated viable cells were freeze-dried. Preparations of cultured broth and PB extracts Sterilized broth (200 ml) was inoculated with 1 ml of the subculture and incubated at 30°C (for yeast) or 37°C (for bacteria) for 24 h. Cells were harvested by centrifugation at 1500 x g for 20 min and washed twice with 0.1 M phosphate buffer (pH 7) (PB). The supernatant of the culture broth was used for the umu-test as cultured broth. Harvested cells were suspended in PB. After keeping the suspension at 4°C for 24 h, the cells were removed by centrifugation at 1500 x g

249 for 20 min. T h e s u p e r n a t a n t was u s e d for t h e u m u - t e s t as PB extracts.

In vitro binding assay by H P L C T h e in vitro b i n d i n g assay was p e r f o r m e d as d e s c r i b e d in a p r e v i o u s p a p e r ( Z h a n g et al., 1990). 5 m g o f l y o p h i l i z e d b a c t e r i a l fractions (cells, C W S , cytoplasm, P T G , glucan, o r m a n n a n ) w e r e s u s p e n d e d in 0.95 ml o f distilled w a t e r (A-suspension). T o t h e s e A - s u s p e n s i o n s o r to 0.95 ml o f t h e filtrate o f c u l t u r e d b r o t h or PB extracts (Bsuspension), a d i m e t h y l sulfoxide ( D M S O ) solution o f m u t a g e n s ( 5 0 / x g of T r p - P - 1 a n d Glu-P-1, o r 1 0 / z g o f M e I Q x , e a c h in 0.05 ml) was a d d e d , a n d t h e m i x t u r e s w e r e i n c u b a t e d at 37°C for 10 min for A - s u s p e n s i o n o r for 30 min for B - s u s p e n sion. T h e n t h e r e a c t i o n mixtures w e r e centrifuged. T h e s u p e r n a t a n t was c o l l e c t e d a n d ult r a f i l t e r e d ( m e m b r a n e filter with a p o r e size of 0.45 /zm, T o y o A d v a n t e c , J a p a n ) . 20 /xl o f t h e

filtrate was a p p l i e d to a H P L C a p p a r a t u s for estimation of the amount of unbound mutagens, a n d 100 /xl of t h e filtrates was u s e d for t h e umu-test.

Umu-test f o r assay o f antimutagenicity T h e o v e r n i g h t c u l t u r e of t h e t e s t e r b a c t e r i a l strain (S. typhimurium T A 1 5 3 5 / p s k 1 0 0 2 ) was dil u t e d 50-fold into T G A m e d i u m a n d it was incub a t e d at 37°C until t h e b a c t e r i a l density r e a c h e d an OD600 of 0.25-0.3. T h e b a c t e r i a l c u l t u r e was s u b d i v i d e d into 2.4-ml p o r t i o n s in test tubes, a n d 0.1 ml o f m u t a g e n i c s o l u t i o n a n d t h e test s a m p l e was a d d e d to e a c h tube. T h e n e i t h e r 0.5 ml o f 0.1 M PB ( p H 7.4) o r $9 mixture c o n t a i n i n g 5 0 / x l of $9 m i c r o s o m a l fraction for m e t a b o l i c activation was a d d e d . A f t e r 2 h o f i n c u b a t i o n at 37°C with shaking, t h e level of /3-galactosidase activity in t h e cells was assayed. /3-Galactosidase activity ( / 3 - G T D A ) was m e a s u r e d by M i l l e r ' s m e t h o d

TABLE 1 EFFECT OF HEAT TREATMENT ON THE BINDING OF Trp-P-1 AND GIu-P-1 TO CELLS OF MICROORGANISMS Bound mutagen (%) Trp-P-1

Glu-P-1

Cells

IO0°C

120°C

Cells

100°C

120°C

Gram-negative bacteria Escherichia coli IFO3301 IFO14249 1592 231 Citrobacterfreumidii IFO13547 Pseudomonas putida 01

94 99 96 97 96 97

90 83 82 95 94 96

76 82 77 59 58 65

13 15 6 15 14 17

12 13 4 14 14 16

12 10 3 14 12 12

Gram-positive bacteria Bacillus cereus IFO3457 Staphylococcus aureus IFO12722 Streptococcus cremoris Z-25 Lactobacillus acidophilis IFO13951 Bifidobacterium bifidum IFO14252

99 98 98 94 97

99 98 98 94 96

99 76 96 94 94

16 14 25 14 18

10 14 25 13 16

15 14 23 10 16

Yeasts Saccharomyces cerevisiae 50 Torulospora dellbrueckii 22 Candida sp. 13 Hansenula sp. 12

98 95 93 94

90 54 67 66

57 58 67 64

6 8 9 12

5 7 9 11

5 6 7 10

All assays were performed in duplicate. Values are means. The SDs for duplicate samples were less than 10%.

25O modified slightly by Oda et al. (1985). The enzyme activity was calculated according to Miller (1972) and Oda et al. (1985).

/3-galactosidase activity of the mixture was measured as above. The bio-antimutagenicity was calculated as follows:

/3-galactosidase activity(unit)

Inhibition of mutagenicity (%) [/3-GTDA of control -/3-GTDA of test ( + sample)] [/3-GTDA of control]

= [ 1000(OD420 - 1.75OD550]/ [t.v.OD60o] Antimutagenicity (%)

XlO0

[fl-GTDA of control -/3-GTDA of test ( + sample)] [/3-GTDA of control]

Results x

100

Inactivation of Trp-P-1 and Glu-P-1 by freeze-dried cells or heat-treated cells Assay of bio-antimutagenicity The culture of S. typhimurium

TA1535/ pskl002 obtained as above was subdivided into 3.4-ml portions in test tubes and 0.1 ml of the mutagenic solution (50 / z g / m l ) was added to each tube. Then 0.5 ml of PB or $9 mixture for metabolic activation was added. After 1 h of incubation at 37°C with shaking, the mixture was washed twice with PB by centrifugation to remove mutagen retained in suspension (1500 x g). The bacterial cells were harvested and suspended in 2 ml of PB or PB containing 10 mg of freezedried cytoplasm and cultured broth. The suspensions were incubated at 37°C for 30 min, then washed twice with PB by centrifugation to remove the samples retained in suspension. The precipitate was suspended in 3 ml of PB. The

As shown in Table 1, all strains showed high binding ability of Trp-P-1, but lower binding ability of Glu-P-1. Through heating at 120°C for 15 rain, the binding ability decreased by about 1740% in GNB and by 3 0 - 4 0 % in yeast. In GPB binding activity did not decrease with heating, except for S. aureus IFO12722 on Trp-P-1. The same results were obtained with GIu-P-1.

Comparison of microorganisms for binding of TrpP-1 We used Trp-P-1 at a high concentration (1000 / z g / m l ) to compare the binding abilities of bacteria and yeasts. From Fig. 1 it can be observed that the two GNB strains had higher binding ability of Trp-P-1 than other strains. The binding of Trp-P-1 by G N B was 10-20 / z g / m g higher

TABLE 2 THE BINDING OF Trp-P-1 (50 ~g/ml) TO CELL FRACTIONS OF BACTERIA AND YEASTS Strain

Binding of Trp-P-1 (%) CWS Cell CTP

GC

MN

PTG

OM

CM

99

65

Lactobacillusacidophilus IFO13951

91

94

14

96

98

9

92

98

20

100

Eschench~ co~ IFO14249

Saccharomycescerev~e 50

100

96

All assays were performed in duplicate. Values are means. The SDs for duplicate samples were less than 10%. CWS, cell wall skeleton; CTP, cytoplasm;GC, glucan; MN, mannan; PTG, peptidoglycan; OM, outer membrane; CM, cytoplasmic membrane.

251

The binding of Trp-P-1 to fractions of microorganisms The data described above confirmed the binding of Trp-P-1 to freeze-dried fractions of bacterial cells (Table 2). The CWS and cells of all strains could bind more than 90% of Trp-P-1. The cytoplasm (CTP) of yeast bofind 20% of Trp-P-1, E. coli IFO14249 and L. acidophilus IFO13951 only 9-14%. The glucan and mannan of yeast, the outer membrane, cytoplasmic membrane of E. coli IFO14249 and PTG of L. acidophilus IFO13951 bound more Trp-P-1. It may be thought that the binding of mutagens comes from the glucan, mannan, outer membrane, cytoplasmic membrane and PTG of CWS.

Z i20 E go

i T _. _

Fig. 1. Binding of Trp-P-1 to various microorganisms. Freezedried cells (5 mg) were suspended in 0.95 ml of distilled water. Trp-P-1 (1000 /zg in 0.05 ml of methanol) was added to the suspensions, which were then incubated at 37°C for 10 min. After centrifugation, the amount of unbound Trp-P-1 in the supernatant was recorded by HPLC. A: Lactobacillus acidophilus IFO13951; B: Bifidobacterium bifidum IFO14254; C: Escherichia coli IFO3301; D: Citrobacter freumidii IFO13547; E: Saccharomyces cerevisiae 50; F: Candida sp. 13.

Inhibition of mutagenicity induced by Trp-P-1 and MelQX As shown in Table 3, the CWS of all strains greatly inhibited the mutagenicity induced by Trp-P-1, but that of MelQX to a lower extent. Their cytoplasms had low inhibitory effect against Trp-P-1, and almost none against MelQX. The binding ability of both mutagens to ceils of the tested strains was not markedly different from

than GPB, and 50-60 tzg/mg higher than yeast. E. coli IFO3301 shows the highest binding of Trp-P-1.

TABLE 3 INHIBITION OF MUTAGENICITY INDUCED BY Trp-P-1 AND MelQx BY FRACTIONS OF CELLS OF BACTERIA AND YEAST MelQx (10/~g/ml)

Trp-P-1 (50/zg/ml) CWS

Cytoplasm

A

B

A

81

84

6

80

92

65 92 92 95

CWS B

Cytoplasm

A

B

A

B

9

11

34

2

3

8

5

29

28

0

8

90

11

8

10

11

0

0

96 97 95

40 25 28

10 20 16

9 29 14

10 25 29

0 2 9

1 0 0

Gram-negative bacteria

Escherichia coli IFO3301

Citrobacter freumidii IFO13547 Gram-positive bacteria

Bacillus cereus IFO3457

Lactobacillus acidophilus IFO13951

Saccharornyces cerevisiae 50 Hansenula sp. 12

All assays were performed in duplicate. Values are means. The SDs for duplicate samples were less than 10%. A: the inhibition of the mutagenicity induced by the mutagen (%). B: binding of the mutagenic pyrolysates of microorganisms. CWS: cell wall skeleton.

252

their ability to inhibit the mutagenicity induced by both mutagens. It may be inferred that the desmutagenicity of microorganisms mostly comes from the binding of mutagens by their CWS.

Antimutagenicity of cultured broth and PB extracts of cells Table 4 shows that the two strains of GPB or yeasts gave 20-29% inhibition of Trp-P-1, except with two strains of GNB. There was no binding of Trp-P-1 in culture broth; therefore, it is suggested that a mechanism other than binding exists for inhibiting the mutagenicity of Trp-P-1. PB extracts had a lower inhibiting effect on the mutagenicity induced by Trp-P-1 than culture broth.

The bio-antimutagenicity of cultured broth and freeze-dried cytoplasms of microorganisms on TrpP-1 In Table 5, we show the bio-antimutagenicity of cultured broth and cytoplasm isolated from several bacteria and yeasts. It was found that cultured broths and cytoplasms of all strains of bacteria tested did not show bio-antimutagenicity against mutagenicity induced by Trp-P-1, but that

TABLE 4 ANTIMUTAGENICITY OF C U L T U R E D BROTH AND PHOSPHATE BUFFER EXTRACT OF VARIOUS BACTERIA AND YEASTS ON Trp-P-1 (50/xg/ml) Strain

Streptococcus cremoris Z-25 Lactobacillus acidophilus IFO13951 Escherichia coli IFO3301 Citrobacter freumidff IFO3547 Saccharomyces cerevisiae 50 Hansenula sp. 12 Saccharornyces cerevisiae 28

Antimutagenicity and binding (%) Cultured broth

PB extract

IHT

Binding

IHT

22

0

7

8

23

0

2

4

2

0

9

0

0

0

12

11

20 18

0 0

12 13

3 3

29

0

14

2

TABLE 5 T H E B I O - A N T I M U T A G E N I C I T Y OF C U L T U R E D BROTH (CB) AND FREEZE-DRIED CYTOPLASM (5 mg) (FDC) OF MICROORGANISMS ON Trp-P-1 (50 # g / m l ) CB

FDC

Gram-positive bacteria

Bacillus cereus IFO3457 Staphylococcus aureus IFO12722 Streptococcus cremoris Z-25 Lactobacillus acidophilus IFO13951 Yeasts Saccharomyces cerevisiae 28 50

+ +

Candida sp. 13 Hansenula sp. 12

+ +

Gram-negative bacteria

Escherichia coli IFO3301 IFO14249 231

Citrobacter freumidii IFO13547 Pseudomonas putida 01 +, inhibitory effect was over 20%; - , there was no inhibition.

of yeasts did. Their bio-antimutagenicity was only about 20% lower than their binding.

Binding

Discussion

All assays were performed in duplicate. Values are means. The SDs for duplicate samples were less than 10%. IHT: inhibition of the mutagenicity induced by Trp-P-1.

We previously reported that lactic acid bacteria, yeast and intestinal bacteria (GNB) strongly inactivated Trp-P-1 and Trp-P-2 by binding both mutagcns (Zhang et al., 1990, 1991; Zhang and Ohta, 1991b). From the data shown in Table 1, we observed that all strains tested bound more than 90% of Trp-P-1 at a concentration of 50 ~ g / m l , but at a concentration of 1000 /~g/ml Trp-P-1, the binding ability of bacteria was higher than that of yeast cells (Fig. 1). It is clear that binding of bacteria and yeast mostly occurs in their CWS, further mainly to PTG (GPB), glucan

253

and mannan (yeast), and outer and cytoplasmic membrane (GNB) (Table 2). Trp-P-1 and Trp-P-2 bound to bacteria and yeast are not able to cause mutation under the assay condition (Zhang et al., 1990; Asahara, 1991). It may be that there is a mechanism(s) other than binding for inhibiting the mutagenicity of Trp-P-1 and Trp-P-2. Using S. typhimurium TA1535/psk1002 (umu-test), we found that the cultured broth and PB extracts of GPB or yeast showed antimutagenicity against Trp-P-1 (Table 4). This finding suggests that the tested GPB and yeasts also inhibited the mutagenicity of Trp-P-1, apart from binding it. However, this inhibition was 20% lower as compared with binding. Table 5 shows that the cultured broth and freeze-dried cytoplasm of yeast had bio-antimutagenicity against the mutagenesis induced by Trp-P-1, whereas that of bacteria had not. From the above results, we can suggest that the tested bacteria only have desmutagenicity including binding activity and some other mechanism. The yeasts not only have desmutagenicity, but also have bio-antimutagenicity against Trp-P-1. The antimutagenic effect of bacteria and yeast on Trp-P-1 is due mainly to their binding activity. We also found that cultures of the tested yeasts inhibited mutation of base-pair substitution induced by mutagens and showed anticlastogenic activity with the micronucleus test (data not shown). In further studies, we are concentrating on isolating and finding the structures of ingredients showing antimutagenicity in cultured broth and cytoplasms. References Arima, K., and I. Takano (1979) Protoplast fusion in microorganisms, Soc. Fermen. Tech. Japan, 57, 380-386. Asahara, N., X.B. Zhang and Y. Ohta (1991) Antimutagenicity and binding of mutagenic pyrolysate by microorganism isolated from Japanese miso, J. Sci. Food Agric., 58, 395-401.

Demuyakor, B., and Y. Ohta (1991) Characteristics of pito yeasts from Ghana, Food Microbiol., 8, 183-193. Hirai, O., T. Fujitsu, J. Mori, H. Kikuchi, S. Koda, M. Fujioka and Y. Morimoto (1987) Antitumour activity of purified arabinogalactan-peptidoglycan complex of the cell wall skeleton of Rhodococcus lentifragmentus, J. Gen. Microbiol., 133, 369-373. Hosoi, M., Y. Nagakawa, N. Oshikata and N. Tanabe (1972) Comparative studies on the inhibition of tumor growth by yeast cell walls and carboxymethylpachymaran, Daiichi Yakka Daigaku Nenpo, 2, 37-44. Kada, T., T. Inoue and M. Namiki (1982) Environmental desmutagens and antimutagens. In: Klekowaki (Ed.), Environmental Mutagenesis, Carcinogenesis and Plant Biology, Praeger Scientific, New York, pp. 133-152. Kada, T., M. Kato, K. Aikawa and S. Kiriyama (1984) Adsorption of pyrolysate mutagens by vegetable fibers, Mutation Res., 141, 149-152. Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 352-355. Mochizuki, H., and T. Kada (1982) Antimutagenic action of mammalian placental extracts on mutations induced in Escherichia coli by UV radiation, y-rays and N-methylN'-nitro-N-nitrosoguanidine, Mutation Res., 95, 457-474. Oda, Y., S. Nakamura, I. Oki, T. Kato and H. Shinagawa (1985) Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens, Mutation Res., 147, 219-229. Shinohara, K., S. Kuroki, M. Miwa, Z.L. Kong and H. Hosoda (1988) Antimutagenicity of dialyzates of vegetables and fruits, J. Sci. Food Agric., 52, 1369-1375. Yamato, L., Y. Anraku and K. Hirosawa (1975) Cytoplasmic membrane vesicles of Escherichia coli, J. Biochem., 77, 705-718. Zhang, X.B., Y. Ohta and A. Hosono (1990) Antimutagenicity and binding of lactic acid bacteria from a Chinese cheese to mutagenic pyrolysates, J. Dairy Sci., 73, 2702-2710. Zhang, X.B., and Y. Ohta (1991a) In vitro binding of mutagenic pyrolyzates to lactic acid bacterial cells in human gastric juice, J. Dairy Sci., 74, 752-757. Zhang, X.B., and Y. Ohta (1991b) Binding of mutagens by fractions of the cell wall skeleton of lactic acid bacteria on mutagens, J. Dairy Sci., 74, 1477-1481. Zhang, X.B., and Y. Ohta (1992) Binding of mutagenic pyrolysate to fractions of intestinal bacterial cells, Can. J. Microbiol., in press. Zhang, X.B., Y. Ohta, B. Demuyakor and N. Asahara (1991) the binding of heterocyclic amines by yeast cells and their fractions, J. Sci. Food Agric., 57, 253-262.