APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1983, p. 651-6570099-2240/83/020651-07$02.00/0Copyright 0 1983, American Society for Microbiology

Vol. 45, No. 2

Activation of Cycasin to a Mutagen for Saccharomycescerevisiae by Rat Intestinal Flora

VERNON W. MAYER* AND CAROL J. GOINGenetic Toxicology Branch, Division of Toxicology, Food and Drug Administration, Washington, D.C. 20204

Received 15 July 1982/Accepted 11 November 1982

Genetic test systems involving microorganisms and liver enzyme preparationsmay be insufficient to detect compounds that require breakdown by enzymesprovided by the microbial flora of the intestinal tract. A method is described forproviding such activation and for simultaneously testing the potential geneticactivity of breakdown products in an indicator organism. Parabiotic chamberscontaining Saccharomyces cerevisiae genetic test organisms in one chamber wereseparated by a membrane filter from rat cecal organisms and test chemicalcontained in the other chamber. The genetic activities of cycasin breakdownproducts for mutation, gene conversion, and mitotic crossing-over in samplesincubated aerobically are reported. Samples containing cycasin alone had a smallbut clearly increased frequency of genetic damage. Samples containing rat cecalorganisms without cycasin showed no increase in genetic activity. Anaerobicincubation resulted in no increase in genetic activity in any of the samples.

The detection of genetic activity of chemicalmutagens relies to a great extent on assaysinvolving various procaryotic and eucaryoticmicroorganisms. Commonly, indicator strains ofmicroorganisms are exposed to a test chemicalin the presence of liver microsomal enzymepreparations that act upon the chemical to con-vert it to one or more breakdown products.These breakdown products may then be geneti-cally active in the indicator organism. Enzymetreatment methodologies were developed in re-sponse to evidence that microorganisms used ingenetic screening tests lacked, in many in-stances, the requisite enzymes to activate chem-ical mutagens.There is growing concern, however, that ge-

netic tests in microorganisms with liver micro-somal enzyme preparations may not be suffi-cient in all situations to accurately predict thepotential genetic activity of a compound forhumans. Compounds that are ingested, for ex-ample, are exposed first to conditions prevailingin the gastrointestinal tract, including the resi-dent microbial flora. Numerous metabolic con-versions that result from the action of microbialflora on xenobiotics have been characterized(reviewed in references 4 and 10). Among anaer-obic processes, azo and nitro dyes are reducedby a number of bacterial species found in thegastrointestinal tract (5, 15), and a number ofthese reduction products are mutagenic in bacte-rial tests (13, 14, 20, 21). Cell-free extracts ofselected intestinal anaerobic bacteria activate2-aminofluorene to render it a mutagen (34).4-Isothiocyanate-4'-nitrodiphenylamine, an anti-

schistosomal drug, is not mutagenic in bacterialtests (9); however, urine from rodent and mam-malian species treated with the compound con-tains a mutagenic metabolite that is apparentlynot formed in germ-free animals or with coad-ministration of an antibiotic (2, 3). In othersituations, the genetic test organism itself maycarry out the reduction, resulting in the forma-tion of a mutagenic product (18, 23, 31, 41). Afurther potentially complicating factor is sug-gested by experiments in which reductive acti-vation by intestinal anaerobes was not sufficientto produce a mutagenic compound unless fur-ther metabolic conversion was provided by mi-crosomal preparations from either the liver orintestinal mucosa (2, 3, 13, 21, 33). There is alsoevidence that mutagens are formed by someintestinal bacteria, but not by others, duringtheir normal growth in media in pure culture (13,43) and in mixed culture from human fecalmaterial (25). However, this effect has not beenconsistently observed (23, 33, 34).Another relevant activity of intestinal tract

flora is the conversion of glycosides of numer-ous compounds to their corresponding agly-cones (4). There are a number of indications thatmany of these aglycones are genetically activeand that the particular reaction is not carried outby the common test organisms (6-8, 28, 35, 44).One such compound, cycasin, has received con-siderable attention over a number of years, andits genotoxicity, attributable to its conversion tomethylazoxymethanol by ,-glucosidase en-zymes of the intestinal microbial flora of mam-mals, has been well characterized (24, 40). Cyca-

651

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Nov

embe

r 20

21 b

y 59

.16.

0.12

5.

652 MAYER AND GOIN

sin, per se, is not mutagenic for Salmonellatyphimurium, but its metabolite, methylazoxy-methanol, is mutagenic either when tested di-rectly (32, 39) or when formed by the action ofintestinal organisms (17) or their extracts (28,44). For these reasons, this compound was usedin our work as a model compound for thedevelopment of tests in Saccharomyces cerevisi-ae to study compounds requiring alternativeactivation methods.

MATERIALS AND METHODSGenetic indicator organisms. Two diploid S. cerevisi-

ae strains, D3 (48) and D7 (47), were used in thisstudy. Strain D3 was used for the detection of red-sectored colonies, which are attributable to a varietyof genetic damage processes, including mitotic geneconversion and mitotic crossing-over, as well as muta-tion, nondisjunction, and chromosomal deletions.Thus, this strain serves as a useful screening organismfor mutagens that might induce one or more of avariety of genetic endpoints. The details of the geneticmarkers contained in strain D3, its cultivation, anddetection of genetic damage at the heterozygous ade2locus were previously described (29, 48, 49).

Diploid S. cerevisiae strain D7 was also used toprovide further assurance that the observed geneticeffects were not strain dependent, as seen in otherstudies (30), and to permit more certain identificationof the nature of the genetic endpoint measured. StrainD7 contains the heteroallelic pair trp5-12, trpS-27 fordetection of mitotic gene conversion, the homoallelicilvl-92 for detection of reverse mutation and allelenonspecific suppressor mutation, and the heteroallelicpair ade240, ade2-119 for the detection of mitoticcrossing-over (47). Strain D7 therefore requires isoleu-cine and tryptophane for growth, but it will growwithout adenine because the two ade2 alleles comple-ment to produce adenine prototrophy and white colo-nies. The methods of maintenance, cultivation, anddetection of genetic damage at the various loci werepreviously described (45-47).Media and chemicals. Both S. cerevisiae strains

were cultivated routinely on a semisynthetic completemedium (37) solidified with 2% agar. For aerobictreatment conditions, a growth medium consisting of0.1 M sodium phosphate buffer (pH 7.0) containing0.5% glucose, 0.5% yeast extract, and 0.5% peptonewas used. For anaerobic treatment conditions, thesame growth medium was used, but 140 mg of ergos-terol, 14.3 ml of Tween 80, and 19 ml of ethanol wereadded per liter. This anaerobic medium is based on theformulation and procedures described by Andreasenand Stier (1) and by Harris (19). After preparation, theanaerobic medium was stored for several days beforeuse in a GasPak anaerobic system (BBL MicrobiologySystems). A minimal medium (26) was supplementedwith either tryptophane at 20 mg/liter to test forisoleucine prototrophs or isoleucine at 30 mg/liter totest for tryptophane prototrophs.Cycasin (methylazoxymethanol-p-D-glucoside) in

cycad flour was provided by Stanford Research Insti-tute, Menlo Park, Calif. In some experiments, thecycad flour was used directly for the treatment condi-tions, and in other experiments a sample of purified

cycasin, provided by H. Matsumoto, College of Agri-culture, University of Hawaii, was used.

Preparation of rat cecum contents. Adult male ran-dom breed albino rats were maintained on laboratoryrat chow. As required for each experiment, a rat waskilled by CO2 asphyxiation, the cecum was removed,and its contents were deposited into a tared beaker.Three volumes of growth medium per gram of cecalmaterial was added and mixed. The larger particulatematerial was allowed to settle to the bottom of thebeaker, and the liquid was decanted into a flask. Thesuspension was further diluted 100-fold in growthmedium for the experiments carried out aerobically.For anaerobic experiments, anaerobically stored me-dium was used, and preparation of the cecal materialwas carried out under a flow of CO2 to the extentpossible.

Preparation of the S. cerevisiae indicator organisms.S. cerevisiae cells of either strain D3 or D7 were grownon semisynthetic complete medium for 2 to 3 days, bywhich time a confluent lawn of cells had formed. Thecells were rinsed from the agar surface with 5 ml of0.85% saline and diluted appropriately for counting ina hemocytometer. The S. cerevisiae cells were thendiluted in growth medium to the desired concentra-tion. Experiments with strain D3 were initiated withapproximately 5 x 106 cells per ml, and those withstrain D7 were initiated with 5 x 107 cells per ml.Experimental design. Treatments were conducted by

using a parabiotic chamber (Bellco Glass, stock no.1945-00030) as shown in Fig. 1. The left and right glasstubes, separated by a triacetate Metricel membranefilter having a 0.2-p.m pore size and a diameter of 25mm (Gelman, type GA-8), were clamped securelytogether. The apparatus was autoclaved before use.Various mixtures were added to either side of fourparabiotic chambers to serve as treatment and controlsamples. In the first chamber, 4 ml of S. cerevisiaesuspension was added to one side and 4 ml of mediumalone was added to the other side; this chamber servedfor taking S. cerevisiae cell samples to monitor thespontaneous frequencies of the genetic endpoints. Thesecond chamber contained 4 ml of S. cerevisiae sus-pension on one side and 4 ml of diluted cecal suspen-sion on the other; this chamber served for monitoringany effect of the presence of cecal material on thefrequencies of the measured genetic endpoints. Thethird chamber contained 4 ml of the S. cerevisiae cellsuspension in one tube and 4 ml of growth mediumcontaining cycad flour at 12.5 ,ug/ml or purified cyca-sin at 0.25 jig/ml in the opposite tube to determinewhether cycasin alone, without cecal material, hadany effect on the indicator organisms. The fourthchamber contained 4 ml of S. cerevisiae cell suspen-sion in one tube and 4 ml of diluted cecum suspensionwith either cycad flour (12.5 p.g/ml) or purified cycasin(0.25 ,ug/ml) in the other. This chamber served toindicate the effect of the combination of cecal materialand cycasin on the genetic endpoints of the S. cerevisi-ae indicator organisms. For aerobic conditions, thescrew caps were fitted loosely, and the apparatus wassecured to a reciprocal shaker and incubated at either20 or 30°C. For anaerobic experiments, the tubes wereflushed with nitrogen before the screw caps weretightened, and the entire apparatus was placed in theGasPak anaerobic system and incubated at 30°C. Inone experiment, either cycad flour or purified cycasin

APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Nov

embe

r 20

21 b

y 59

.16.

0.12

5.

CYCASIN ACTIVATION BY RAT INTESTINAL FLORA 653

500 r

YeastGenetic CecalIndicator OrganismsOrganism and Cycasin

0.2pMFilter PressureMembrane Clamp

FIG. 1. Parabiotic chamber apparatus.

was added directly to the S. cerevisiae cell suspensionso that a more intimate contact between S. cerevisiaeindicator organism and the cycasin was achieved thancould be obtained in the parabiotic chambers.Sampling and determination of genetic event frequen-

cies. At time intervals of 0, 6, 24, 48, and 72 h, samplesof the S. cerevisiae cell suspension were removedfrom the parabiotic chambers. The cells were appro-priately diluted and counted with a hemocytometer.The suspension was then further diluted so that thecell numbers were sufficient for plating on the varioussolid media for enumeration of the appropriate typecolonies. Semisynthetic complete plates containingstrain D3 cells were incubated for 3 days at 30°C, and

1200 K

1000 F-

600

400

200

obL mmp-CWI3

A Purified Cycasin* Cecal Organisms* Yeast Cells Only° Cycad Flour

//

Purified Cycasin /+ g

Cecal Organisms/

//

/ o

/ ,o' ~~Cycad Flour

-- g/Cecal Organisms

~__S' ,,r,

6 24 48

Hours Incubation at 20°CFIG. 2. Induction of red-sectored colonies in S.

cerevisiae D3 incubated aerobically at 20°C with vari-ous cycasin-cecal organism combinations in parabioticchambers.

400 I-Ca

0

a

i0(AIcc)

Purified Cycasin /+ /

Cecal Organisms// p

/

/ / Cycad Flour

I , Cecal Organisms

I,Co Cycad FlourA Purified Cycasin03 Cecal Organisms* Yeast Cells Only

100k

o"fz ',.IT v0 6 24 48 72

Hours Incubation at 30'CFIG. 3. Incubation of red-sectored colonies in S.

cerevisiae D3 incubated aerobically at 300C with vari-ous cycasin-cecal organism combinations in parabioticchambers.

colonies were enumerated for population and for thenumber of red-sectored colonies (29). Strain D7 cellswere plated onto minimal medium with tryptophanefor enumeration of isoleucine prototrophs, onto mini-mal medium with isoleucine for enumeration of trypto-phane prototrophs, and onto semisynthetic completemedium for enumeration of the population and thevarious red-pink-sectored colonies. All petri plateswere incubated at 300C. Isoleucine prototrophic colo-nies were scored after 6 days of growth; tryptophaneprototrophic colonies and those colonies on the nonse-lective semisynthetic complete medium were scoredafter 3 days of growth.

RESULTSThe frequencies of red-sectored colonies in S.

cerevisiae D3 cells incubated aerobically at 200Cwith cecal organisms and cycasin are shown inFig. 2. Only the samples containing both cycasinand cecal organisms had an increase in thefrequency of red-sectored colonies with increas-ing incubation time. No increase in sector fre-quency was observed over the 72-h incubationperiod in samples of S. cerevisiae cells incubat-ed alone, cycasin without cecal organisms, or S.cerevisiae cells with cecal organisms. A similarpattern was observed when the experiment wasperformed at 30°C (Fig. 3), although the re-sponse of the S. cerevisiae in the two cycasinsamples with cecal organisms was lower than at20°C. The sector frequently was slightly in-creased in both samples containing cycasinalone (Fig. 3). This observation was furtherexplored by incubating samples of cycasinmixed directly with the growing S. cerevisiae

00._

0'aa)0

(-

S

SA.

0

VOL. 45, 1983

c

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Nov

embe

r 20

21 b

y 59

.16.

0.12

5.

654 MAYER AND GOIN

200 r

0._

-E000

0

150 F-

pPurifiedCycasin

/,,/ Cycad

P,< / Flour/- ,1

,If */

Yeast Cells Only

1001-

50

0 6 24 48

00.

00.CL

0

+0.

72

Hours Incubaton at 30°C

FIG. 4. Induction of red-sectored colonies in S.cerevisiae D3 incubated aerobically with either cycadflour or purified cycasin without cecal organisms.

cells (Fig. 4). Under these conditions, cycasincaused a substantial increase in sector frequen-cy, although it was noticeably less than thatobserved in samples containing cycasin and ce-cal organisms together.

Similar experiments were performed with theparabiotic chambers, using strain D7 becausethe genetic markers in this strain are betterdefined. Strain D7 was also used to ensure thatthe effects observed were not due to someunique feature of strain D3. The results of incu-bating strain D7 aerobically at 30°C with cecal

30 A/

25 eP5urified Cycasin

Cecal Organisms

1 /20_/

15 /

-I /'10

/ & Purified Cycasin5 0 Cecal Organisms

/ 0 Yeast Cells Only

_/___...........

0 6 24 48

Hours Incubation at 300CFIG. 5. Induction of mutation in S. cerevisiae D7

incubated aerobically with various purified cycasin-cecal organism combinations in parabiotic chambers.

72

Hours Incubation at 30°CFIG. 6. Induction of mitotic gene conversion in S.

cerevisiae D7 incubated aerobically with various puri-fied cycasin-cecal organism combinations in parabioticchambers.

organisms and cycasin are shown in Fig. 5 to 7.Purified cycasin mixed with cecal organismsinduced an increase in the frequency of eachgenetic endpoint: mutation (Fig. 5), mitotic geneconversion (Fig. 6), and mitotic crossing-over(Fig. 7). In addition to the red and pink sectorswhich are diagnostic for mitotic crossing-over instrain D7, other aberrant colonies were ob-served among the normal white colonies, name-ly, red, pink, red-white, and pink-white. Thegenetic origin of these types is uncertain; theycould arise as a result of a variety of processes,including mutation, nondisjunction, and geneconversion. These colony types were enumerat-ed and increased with time of exposure to thepurified cycasin-cecal organism mixture (Fig. 8).None of the genetic endpoints measured in sam-ples of S. cerevisiae cells alone, cycasin alone,or cecal organisms alone showed increases infrequency of genetic events over the time ofexposure.

Yeast, as a facultative organism, can groweither aerobically or anaerobically. Advantagewas taken of this characteristic to determinewhether cycasin could be activated to genetical-ly active breakdown products under anaerobicconditions. An experiment was performed underanaerobic conditions as described above foraerobic cultivation, and samples were handledappropriately to measure each genetic endpointat 72 h. No notable increase in frequency wasseen with any of the genetic endpoints measured(Table 1). From cell counts of the various sam-ples at each time interval (data not shown), itwas apparent that the S. cerevisiae cells in-creased in number between zero time and 24 to48 h, by which time the stationary phase was

04--Mif- - "Illl-::::I§; ...................

APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Nov

embe

r 20

21 b

y 59

.16.

0.12

5.

CYCASIN ACTIVATION BY RAT INTESTINAL FLORA 655

c0._

._

q

C

0

._

Co

0

0

000C'a

*0

0

40 r

30K

20 _

10 _

O

/ Purified Cycasin

Cecal Organisms

/// A Purified Cycasin

0 Yeast Cells Only

// 0 Cecal Organisms

/I_0 6 24 48

Hours Incubation at 30°CFIG. 7. Induction of mitotic crossing-over in S.

cerevisiae D7 incubated aerobically with various puri-fied cycasin-cecal organism combinations in parabioticchambers.

achieved. There was no obvious or consistenteffect of the various treatment conditions on S.cerevisiae culture growth. As expected, anaero-bic cultures grew more slowly than aerobiccultures and achieved a cell titer only slightlylower than those under aerobic conditions.

DISCUSSIONThe results of the experiment involving either

S. cerevisiae indicator strain with the cycasinand cecal content treatment conditions indicatethat cycasin is being acted upon by enzymessupplied by the cecal content. The genetic activ-ity found in the cycad flour and with purifiedcycasin samples without cecal organisms wasprobably not due to some level of contaminationwith preformed methylazoxymethanol, becausethe activity at 6 and 24 h would be expected tobe greater than that observed. It seems morelikely that cycasin is being autonomously hydro-lyzed at a low rate under the incubation condi-tions in these experiments or that the S. cerevisi-ae cells themselves can carry out the reaction.This latter possibility is supported by evidencethat a 3-glucosidase is inducible in S. cerevisiae(16). Activation of some other compounds bymetabolic processes of eucaryotic microorga-nisms under appropriate conditions has beenpreviously observed (11, 12, 36).

Earlier experiments were performed at 20°Csimply for convenience. Later experiments wereperformed at 30°C, the optimum temperature for

the S. cerevisiae indicator organism, and thistemperature was expected to increase the meta-bolic activities of the cecal organisms as well.However, the genetic activity was reduced atthe higher incubation temperature, which is incontrast to reports with other chemical muta-gens (30, 38). The reason for this effect isunknown, but the decrease could be due tofurther metabolism of methylazoxymethanol toan inactive or less active form, accelerated at thehigher temperature, by the cecal organisms orthe S. cerevisiae.

Results between experiments did not showany variation that could be attributed to varia-tion in cycasin activation by the cecal contentsfrom different rats. The rats were maintainedunder identical conditions of housing and feed-ing to minimize the possibility of such variation.Samples from the various experiments that

contained only the S. cerevisiae indicator orga-nisms and the cecal organisms together in theparabiotic chamber showed no indication thatproducts of the cecal organisms increased thefrequency of the genetic endpoints measured.Although our experiments were not designedspecifically for this purpose, such an effect, ifpresent, probably could have been detected.Our results are therefore consistent with someobservations (23, 33, 34) and are in contrast withothers (25, 43).The observation that genetic activity occurred

with cycad flour containing cycasin is of some

c0

10.

a-

o '00

H:0.5S

0

150

:PurifiedCycasin\Cocal Organisms a

100 /

75/

50 j * Purified Cycasin* Yeast Cells Only

/ 0 Cecal Organisms

050 6 24 48 72

1

Hours Incubation at 30°CFIG. 8. Induction of other red- or pink-sectored

colonies in S. cerevisiae D7 incubated aerobically withvarious purified cycasin-cecal organism combinationsin parabiotic chambers.

VOL. 45, 1983

1

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Nov

embe

r 20

21 b

y 59

.16.

0.12

5.

TABLE 1. Lack of genetic activity of cycasin for S. cerevisiae under anaerobic conditionsFrequencies of genetic endpoints

Sample and time of incubation ILV+ per 106 TRP+ per 105 Cross-overs per Other sectors percolonies colonies 10' colonies 10' colonies

Yeast cell control, 0 h 7.6 7.9 0.76 4.9Yeast cell control, 72 h 7.9 9.2 2.1 9.5Cecal organisms control, 72 h 11.0 15.2 2.2 9.4Purified cycasin control, 72 h 10.0 7.9 1.0 7.5Purified cycasin + cecal organisms, 72 h 15.5 11.6 3.1 13.4

importance. Increasing consideration is beinggiven to the detection of genetically active com-pounds that are contained in complex mixtures,and some results indicate that detection is notalways possible (42). With judicious applicationof our method, it should be possible to testcompounds that are normally ingested for theirgenetic hazard potential under conditions thatmore closely resemble the normal exposure con-ditions of the gastrointestinal tract.Samples consisting of cycasin and cecal orga-

nisms were genetically active under aerobic butnot anaerobic conditions in our investigations.Previous studies on the mutagenicity of cycasinand other glycoside compounds have been per-formed under aerobic conditions with cell-freeextracts of intestinal organisms (6-8, 44), andour findings are consistent with these results.The particular enzyme, ,-glucosidase, that isresponsible for converting cycasin to methylaz-oxymethanol (24, 28, 40) is ubiquitous in manyintestinal aerobic microbes, including bacteria(22, 40) and fungi (16, 27), and it is found inintestinal anaerobes as well (22). Therefore,further studies should be conducted to deter-mine the reason for the inability to detect geneticactivity of cycasin under anaerobic conditions.

ACKNOWLEDGMENTSWe are indebted to Albert T. Sheldon and Ib Knudsen for

their original suggestion to use parabiotic chambers to test forintestinal organism activation of mutagens and for their helpfuldiscussions.

LITERATURE CITED1. Andreasen, A. A., and T. J. B. Stier. 1953. Anaerobic

nutrition of Saccharomyces cerevisiae. I. Ergosterol re-quirement for growth in a defined medium. J. Cell. Comp.Physiol. 41:23-36.

2. Batzinger, R. P., E. Bueding, K. Crawford, and J. Bruce.1979. Prevention of the mutagenic activation of an anti-schistosomal isothiocyanate in primates by an antibiotic.Environ. Mutagen. 1:353-360.

3. Batzinger, R. P., E. Bueding, B. J. Reddy, and J. H.Welsburger. 1978. Formation of a mutagenic drug metabo-lite by intestinal microorganisms. Cancer Res. 38:608-612.

4. Brown, J. P. 1977. Role of gut bacterial flora in nutritionand health: a review of recent advances in bacteriologicaltechniques, metabolism and factors affecting flora compo-sition. Crit. Rev. Food Sci. Nutr. 8:229-336.

5. Brown, J. P. 1981. Reduction of polymeric azo and nitro

dyes by intestinal bacteria. Appl. Environ. Microbiol.41:1283-1286.

6. Brown, J. P., and P. S. Dietrich. 1979. Mutagenicity ofanthraquinone and benzanthrone derivatives in the Sal-monella/microsome test: activation of anthraquinone gly-cosides by enzymic extracts of rat cecal bacteria. Mutat.Res. 66:9-24.

7. Brown, J. P., and P. S. Dietrich. 1979. Mutagenicity ofplant flavonols in the Salmonella/mammalian microsometest. Activation of flavonol glycosides by mixed glycosi-dases from rat cecal bacteria and other sources. Mutat.Res. 66:223-240.

8. Brown, J. P., P. S. Dietrkch, and R. J. Brown. 1977.Frameshift mutagenicity of certain naturally occurringphenolic compounds in the Salmonella/microsome test:activation of anthraquinone and flavonol glycosides by gutbacterial enzymes. Biochem. Soc. Trans. 5:1489-1492.

9. Bueding, E., R. Batzinger, and G. Petterson. 1976. Anti-schistosomal and some toxicological properties of a nitro-diphenylaminoisothiocyanate. Experientia 32:604-606.

10. Callen, D. F. 1978. A review of the metabolism of xeno-biotics by microorganisms with relation to short-term testsystems for environmental carcinogens. Mutat. Res.55:153-163.

11. Callen, D. F., and R. M. Philpot. 1977. Cytochrome P-450and the activation of promutagens in Saccharomycescerevisiae. Mutat. Res. 45:309-324.

12. Callen, D. F., C. R. Wolf, and R. M. Philpot. 1980. Cyto-chrome P-450 mediated genetic activity and cytotoxicityof seven halogenated aliphatic hydrocarbons in Saccharo-myces cerevisiae. Mutat. Res. 77:55-63.

13. Chung, K.-T., G. E. Fulk, and A. W. Andrews. 1978. Themutagenicity of methyl orange and metabolites producedby intestinal anaerobes. Mutat. Res. 58:375-379.

14. Chung, K.-T., G. E. Fulk, and A. W. Andrews. 1981.Mutagenicity testing of some commonly used dyes. Appl.Environ. Microbiol. 42:641-648.

15. Chung, K.-T., G. E. Fulk, and M. Egan. 1978. Reductionof azo dyes by intestinal anaerobes. Appl. Environ.Microbiol. 35:558-562.

16. Duerksen, J. D., and H. Halvorson. 1959. The specificityof induction of 3-glucosidase in Saccharomyces cerevisi-ae. Biochim. Biophys. Acta 36:47-55.

17. Gabridge, M. G., A. Denunzio, and M. S. Legator. 1969.Cycasin, detection of associated mutagenic activity invivo. Science 163:689-691.

18. Garner, R. C., and C. A. Nutman. 1977. Testing of someazo dyes and their reduction products for mutagenicityusing Salmonella typhimurium TA1538. Mutat. Res. 44:9-19.

19. Harris, M. 1956. Occurrence of respiration deficient mu-tants in baker's yeast cultivated anaerobically. J. Cell.Comp. Physiol. 48:95-112.

20. Hartman, C. P., A. W. Andrews, and K.-T. Chung. 1979.Production of a mutagen from Ponceau 3R by a humanintestinal anaerobe. Infect. Immun. 23:686-689.

21. HartNan, C. P., G. E. Fulk, and A. W. Andrews. 1978.Azo reduction of trypan blue to a known carcinogen by acell free extract of a human intestinal anaerobe. Mutat.Res. 58:125-132.

APPL. ENVIRON. MICROBIOL.656 MAYER AND GOIN

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Nov

embe

r 20

21 b

y 59

.16.

0.12

5.

CYCASIN ACTIVATION BY RAT INTESTINAL FLORA 657

22. Hawksworth, G., B. S. Drmr, and M. J. Hill. 1971.Intestinal bacteria and the hydrolysis of glycosidic bonds.J. Med. Microbiol. 4:451-459.

23. Karplnsky, G. E., and H. S. Rosenkranz. 1980. The anaer-obic-mediated mutagenicity of 2-nitrofluorene and 2-aminofluorene for Salmonella typhimurium. Environ. Mu-tagen. 2:353-358.

24. Laqueur, G. L., and M. Spatz. 1968. Toxicology of cyca-sin. Cancer Res. 28:2262-2267.

25. L n, M., R. VanTsell, S. E. H. West, M. F. Eb-rich, and T. D. Wikins. 1980. In vitro production ofhuman fecal mutagen. Mutat. Res. 79:115-124.

26. Magni, G. E., and R. C. vonBorstel. 1962. Different ratesof spontaneous mutation during mitosis and meiosis inyeast. Genetics 47:1097-1108.

27. Mahadevan, P. R., and B. Eberhart. 1964. The 0-glucosi-dase system of Neurospora crassa. II. Purification andcharacterization of aryl 0-glucosidase. Arch. Biochem.Biophys. 108:22-29.

28. Matsushima, T., H. Matsumato, A. Shiral, M. Sawumura,and T. Suglmura. 1979. Mutagenicity of the naturallyoccurring carcinogen cycasin and synthetic methylazoxy-methanol conjugates in Salmonella typhimurium. CancerRes. 39:3780-3782.

29. Mayer, V. W. 1973. Induction of mitotic crossing over inSaccharomyces cerevisiae by breakdown products ofdimethylnitrosamine, diethylnitrosamine, 1-naphthyl-amine and 2-naphthylamine formed by an in vitro hydrox-ylation system. Genetics 74:433-442.

30. Mayer, V. W., C. J. Hybner, and D. J. Brusick. 1976.Genetic effects induced in Saccharomyces cerevisiae bycyclophosphamide in vitro without liver enzyme prepara-tions. Mutat. Res. 37:201-212.

31. McCalla, D. R., and D. Voutsinos. 1974. On the mutage-nicity of nitrofurans. Mutat. Res. 26:3-16.

32. McCann, J., E. Chol, E. Yanasaki, and B. N. Ames. 1975.Detection of carcinogens as mutagens in the Salmonellalmicrosome test: assay of 300 chemicals. Proc. Natl. Acad.Sci. U.S.A. 72:5135-5139.

33. McCoy, E. C., L. A. Petrullo, and H. S. Rosenkranz.1979. The demonstration of a cooperative action of bacte-rial and intestinal mucosa enzymes in the activation ofmutagens. Biochem. Biophys. Res. Commun. 89:859-862.

34. McCoy, E. C., W. T. Speck, and H. S. Rosenkranz. 1977.Activation of a procarcinogen to a mutagen by cell-freeextracts of anaerobic bacteria. Mutat. Res. 46:261-264.

35. Napo, M., N. Morita, T. Yahagi, M. Shimuzu, M. Kuro-yanagi, M. Fukuoka, K. Yoshihira, S. Nator{, T. Fujino,

and T. Sugimura. 1981. Mutagenesis of 61 flavonoids and11 related compounds. Environ. Mutagen. 3:401-419.

36. Ong, T.-M., and F. J. deSerres. 1972. Mutagenicity ofchemical carcinogens in Neurospora crassa. Cancer Res.32:1890-1893.

37. Plttman, D., J. M. Webb, A. Rosmfanesh, and L. E.Coker. 1960. Evidence for the genetic control of photore-activation. Genetics 45:1023-1037.

38. Schwaier, R., F. K. Z rann, and U. V. Laer. 1965.The effect of temperature on the mutation induction inyeast by N-alkylnitrosamides and nitrous acid. Z. Verer-bungsl. 97:72-74.

39. Smith, D. W. E. 1966. Mutagenicity of cycasin aglycone(methylazoxymethanol), a naturally occurring carcinogen.Science 152:1273-1274.

40. Spatz, M., D. W. E. Smith, E. G. McDaniel, and G. L.Laqueur. 1967. Role of intestinal microorganisms in deter-mining cycasin toxicity. Proc. Soc. Exp. Biol. Med.124:691-697.

41. Speck, W. T., and H. S. Rosenkranz. 1976. Mutagenicityof azathioprine. Cancer Res. 36:108-109.

42. Stolz, D. R., B. Stavric, F. Iverson, R. Bendall, and R.K_assen. 1979. Suppression of naphthylamine mutagenic-ity by amaranth. Mutat. Res. 60:391-393.

43. Suzuki, K., and T. Mltsaoka. 1978. Mutagenicity of intes-tinal bacteria. Mutat. Res. 50:295-299.

44. Tamura, G., C. Gold, A. Ferro-Luzzi, and B. N. Ames.1980. Fecalase: a model for activation of dietary glyco-sides to mutagens by intestinal flora. Proc. Nati. Acad.Sci. U.S.A. 77:4961-4965.

45. Zimmermann, F. K. 1973. Detection of genetically activechemicals using various yeast systems, p. 209-239. In A.Hollaender (ed.), Chemical mutagens. Plenum Press, NewYork.

46. Zlmmemann, F. K. 1975. Procedures used in the induc-tion of mitotic recombination and mutation in the yeastSaccharomyces cerevisiae. Mutat. Res. 31:71-86.

47. Zh rmann, F. K., R. Kern, and H. Rasenberger. 1975.A yeast strain for simultaneous detection of inducedmitotic crossing-over, mitotic gene conversion and re-verse mutation. Mutat. Res. 28:381-388.

48. Z smmermann, F. K., and R. Schwaler. 1967. A geneticeffect of symmetric dimethylhydrazine: induction of mi-totic recombination. Naturwissenschaften 54:251.

49. Zimmerman, F. K., R. Schwaler, and U. V. Laer. 1966.Mitotic recombination induced in Saccharomyces cerevi-siae with nitrous acid, diethylsulfate and carcinogenicalkylating nitrosamides. Z. Vererbungsl. 98:230-246.

VOL. 45, 1983

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Nov

embe

r 20

21 b

y 59

.16.

0.12

5.