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[CANCER RESEARCH 39, 2026-2030, June 197910008-5472/79/0039-0000$02.OO
Effect of 2-Nitrofluorene, 1,2-Dimethylhydrazine, and Azoxymethaneon Salmonella typhimurium Mutants in the GastrointestinalTract of Gnotobiotic Rats'
John H. Carter,2 Martha A. McLafterty, and Peter Goldman3
Department of Pharmacology, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215
structurally related compounds which are not mutagenic to thebacteria in vitro and for which no evidence of carcinogenicityexists(6).
This host-mediated assay offers a possible means of studyingthe relationship of the bacterial flora to the formation of reactiveintermediates that may be related to gastrointestinal cancinogenesis (5, 6).
After the ingestion of the gastrointestinal carcinogen 2-nitmofluorene (4), the revertant response of strain TA1538 in thefeces is ordinarily quite striking. The response is considerablydiminished, however, when 2 constituents of the normal ratflora, Lactobacillus plantarum and Bacteroides vulgatus, areassociated in addition to strain TA1538 (5). The diminishedrevertant response might be explained by certain chamactenistics of these bacteria. For example, B. vulgatus is capable ofreducing 2-nitrofluomeneto 2-aminofluorene (5) and thus mightdiminish the response by decreasing the exposure of theSalmonella tester strain to the more potent mutagen. On theother hand, colonization of various sites within the gastmomntestinal tract by either B. vulgatus onL. plantarum might displacestrain TA1538 from the site(s) where mutagenesis occurs.These possibilities suggest that an examination of the responseto 2-nitmofluomenein the presence of only one of the 2 additionalbacteria may elucidate the mechanism of the blunted mevertantresponse.
This paper reports studies on the effect of these additionalbacteria on the response of the strain TA1538-associated ratto 2-nitnofluomene.In addition, studies are reported of the useof other Ames Salmonella tester strains in this host-mediatedassay to detect mutagenic activity with experimental coloncarcinogens, such as 1,2-dimethylhydrazine and azoxymethane.
MATERIALS AND METHODS
Animals and Microbial Methods. Germ-free Sprague-Dawley rats (45 to 47 days old) and steam-sterilized rat-mouse 7RFdiet were purchased from Charles River Breeding Laboratories(Wilmington, Mass.). Germ-free rats were associated with S.typhimurium strains TA1 538, TA98, or TA1 00 (obtained fromDr. Bruce Ames, University of California, Berkeley, Calif.) andwere housed in stainless steel metabolism cages maintained ina sterile, isolated environment (6).
Germ-free rats were associated with strain TA1538 and 14days later with either L. plantarum (V.P.l. 051 6) or B. vulgatus(V.P.l. 9520), at which time the concentration of revertants inthe feces of the animals was less than 200 revertants/g. Someanimals associated with strain TA1538 and L. plantarum werealso associated with B. vulgatus 8 days after their prior expo
2026 CANCER RESEARCH VOL. 39
ABSTRACT
Gnotobiotic Sprague-Dawley rats, associated with Salmonella typhimurium strain TA1538, may also be associated witheither Lactobacillus plantarum or Bacteroides vulgatus as wellas with both of these strains. After the ingestion of 2-nitmofluomene(3.4 mg), the fecal concentration of his@mevertantsisgreatly elevated in all groups of rats except those associatedwith strain TA1538 and B. vulgatus. The concentrations of thevarious bacteria were measured at eight sites in the gastrointestinal tract. B. vulgatus achieved high concentrations in thestomach when it was associated with strain TA1538, but itsconcentration was not as high when it was associated with L.plantarum together with strain TA1538. The concentration ofB. vulgatus in individual rats correlated negatively with theirresponse to 2-nitrofluomene. B. vulgatus readily reduces 2-nitrofluorene to 2-aminofluorene, a reaction which is negligiblein cultures of L. plantarum and strain TA1538. Since 2-aminofluomeneis less mutagenic than 2-nitmofluomene,B. vulgatusappears to diminish the revertant response by removing themore potent mutagen from within the gastrointestinal tract.
Other Ames Salmonella tester strains (TA1535, TA100, andTA98) can also be maintained in association with otherwisegerm-free rats. The feces of animals associated with strainsTA1535 and TA100, however, show a variable increase in theconcentration of his@mevertantsin response to the ingestion ofexperimental colon carcinogens, e.g. , 1,2-dimethylhydrazine(21 mg/kg) and azoxymethane (19 mg/kg).
INTRODUCTION
An association of the histidine auxotmophof Salmonella typhimurium (strain TA1 538) can be maintained for severalmonths within the gastrointestinal tract of otherwise germ-freerats. The bacteria achieve concentrations of greater than 10@/g of contents in the forestomach and greater than 108/g ofcontents of the lower bowel and in the feces. Carcinogenswhich cause strain TA1538 to revert to histidine independencein the in vitro assays developed by Ames will, when ingested,cause an increased concentration of histidine-independent mevertants to appear in the feces. In contrast, the number ofmevertants in the feces is not increased by the ingestion of
@ These studies were supported by Grants GA21681 from the National LargeBowel Cancer Project and CAl 5260 from the National Cancer Institute.
2 Recipient of Postdoctoral Fellowship 1 F2 CAO5434 from the National
Cancer Institute.3 To whom requests for reprints should be addressed, at the Department of
Pharmacology, Beth Israel Hospital, 330 Brookline Avenue, Boston, Mass.02215.
Received November 30, 1978; accepted February 22, 1979.
on May 18, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Colon Carcinogens and Mutagenesis in Gnotobiotic Rats
sure to L. plantarum (5). Samples of feces were collected daily,and both the his mutants and the his@mevertantswere enumematedon the day of collection (6). The feces of gnotobioticanimals were also assayed daily on selective media (8) toenumerate L. plantarum and B. vulgatus. Fourteen days afterthe ingestion of the carcinogen, the matswere sacrificed, andthe bacteria were enumerated at various sites in the gastrointestinal tract (6, 8).
Feeding of Chemicals. 2-Nitrofluorene (Eastman OrganicChemical Co., Rochester, N. Y.) was dissolved in acetone andsterilized by filtration through a 0.2-@smFluoropore filter (Mmliipore Filter Corp., Bedford, Mass.) before it was added to thegerm-free diet. The acetone was permitted to evaporate for 1hr before the mixture was given to the animals. 1,2-Dimethylhydrazine dihydrochlonide (Aldrich Chemical Co., Milwaukee,Wis.) was dissolved in aqueous 1 mM EDTA and neutralized topH 6.8 with NaOH prior to dilution to the specified concentration. This solution and aqueous solutions of azoxymethane(Ash Stevens, Inc., Detroit, Mich.) and methyl(acetoxymethyl)nitmosammne(a gift of Dr. P. P. Roller, Carcinogen Metabolism and Toxicology Branch, National Cancer Institute,Bethesda, Md.) were filtered through a [email protected] filter(Millipome)before passage into the isolators. Samples (1.0 ml)of solutions of dimethylhydrazmne, azoxymethane, andmethyl(acetoxymethyl)nitrosammne were administered by gastnic intubation. This procedure was greatly facilitated by retarding the reactions of the rats by exposing them to Fluothanevapor (AyemstLabs, Inc., New York, N. Y.) for approximately10 sec priortointubation.
Statistical comparison of means was based on Student'stest. The correlations were evaluated using the Pearson product-moment test.
RESULTS
The Effect of Additional Bacteria on the Mutagenic Response of S. typhimurlumTAI 538 in Rats Ingesting 2-Nitrofluorene. When gnotobiotic rats previously associated withSalmonella strain TA1538 were additionally associated witheither L. plantarum or B. vulgatus, the fecal concentration ofSalmonella remained between 108 and 1O°/g,as it had in thepresence of both strains on in their absence (5). Both L.plantarum and B. vulgatus achieved concentrations in the fecesofbetween 10°and 1010/g.
Each group of gnotobiotic rats, except those associated withstrain TA1538 and B. vulgatus, showed a significant increasein the fecal concentration of his―revertants after the ingestionof a single dose of 3.4 mg of 2-nitrofluorene (Chart 1). Evenafter a larger challenge with 2-nitmofluomene(34.0 mg, administemed8 days later), the matsassociated with strain TA1538and B. vulgatus showed only a weak response, in no casegreater than 1400 revertants/g of feces. Previous studies hadshown that the higher dose of 2-nitrofluomenewas capable ofprovoking a substantial revertant response in animals associated with L. plantarum in addition to B. vulgatus and strainTA1538 (5).
One explanation for the lack of a response in these animalsis that B. vulgatus metabolizes 2-nitmofluomeneand thus decreases the exposure of the Salmonella tester strain to themutagen. In vitro experiments show that B. vulgatus, but notstrain TA1538 or L. plantarum, can reduce 2-nitrofluorene to
2-aminofluonene (5). Chart 2 compares the response of strainTA1538 to 2-nitrofluorene and 2-aminofluorene. Only with 2-aminofluomeneis mutagenic activity dependent upon the presence of a liver microsomal preparation. This observation suggests that in the gastrointestinal tract B. vulgatus converts 2-nitmofluorene to 2-aminofluomene which, at this site, is lessmutagenic, resulting in a decreased revertant response ofstrain TA1538 in vivo.
This hypothesis, however, does not explain why those animals associated with L. plantarum in addition to B. vulgatusand strain TA1538 show a high nevertant response. A possibleexplanation is that L. plantarum establishes itself within thegastrointestinal tract in a way that displaces B. vulgatus fromthe location(s) where it metabolizes 2-nitmofluoreneto the lesspotent 2-aminofluorene. This possibility was examined by detemminingthe distribution of L. plantarum and B. vulgatus at
0
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Germ-freeratswereassociatedwithstrainTA1538 andadditionallywith the other bacteria indicated. Strain TA1 538 was enumerated dailyin the feces, and that value recorded below is from the samples whichyielded the highest concentration of his@ revertants following theingestion of 3.4 mg of 2-nitrofluorene.Salmonella
strain TA1538 (log,0)/gBacteria associatedfecesTA1538
8.24 ±0.81eTA1538 + L. plantarum 8.45 ±0.44TA1538 + L. plantarum+ B. vulgatus 8.20 ±0.47TA1538 + B. vulgatus 8.18 ±0.12a
Mean values from 6 rats in each group ± 5.0.
J. H. Carter et al.
various levels of the gastrointestinal tract of sacrificed animalswith different bacterial associations (Chart 3). Only in thestomach was the concentration of B. vulgatus significantlyhigher in unresponsive mats(associated with strain TA1538 andB. vulgatus) than in the responsive ones (additionally associated with L. plantarum) (p < 0.01 ). The concentrations of B.vulgatus in the stomachs of individual matswere examined tosee if they correlated with the revertant response that hadearlier been observed in the same rats. Chart 4 shows theexistence of a significant inverse correlation (coefficient ofcorrelation, r = —0.69;p < 0.05).
The concentrations of B. vulgatus in the cecum and colonwere also examined to see if these concentrations correlatedwith the revertant response. Neither rats associated with strainTA1538 and B. vulgatus nor those additionally associated withL. plantarum showed such a correlation. This correlation maynot have been revealed under these circumstances, however,
because matswere sacrificed 8 days after the mevertantmesponse had been observed. A day-to-day variation is found inthe concentration of B. vulgatus in the feces, and such vamiation, if it occurred in the colon, might obscure the relationship.
A second possible explanation for the effect of B. vulgatuson the mevertantresponse is that this strain displaces strainTA1538 from the location in the gastrointestinal tract wherecontact between mutagen and tester strain makes possible thereverse mutational response. If this were the case, then it mightbe reflected in a lowered concentration of strain TA1538 in thefeces of rats associated with B. vulgatus in comparison withthat of other mats.As indicated in Table 1, however, there is nodifference in the fecal concentration of strain TA1538 amongthe various groups of rats after the ingestion of 2-nitrofluonene.The possibility that strain TA1538 was displaced by B. vulgatuswas also examined by comparing the salmonella concentrations at various levels of the gastrointestinal tract in the differentgroups of matswhich were sacrificed 14 days after the ingestionof 2-nitmofluorene (Chart 5). At no site above the colon doesthe association with B. vulgatus alone cause a significantdepression of the concentration of strain TA1538. In the colon,however, the concentration of strain TA1538 is lowest in theanimals associated with strain TA1538 and B. vulgatus (p <0.01 compared to the animals associated only with strainTA1538 on additionally with B. vulgatus and L. plantarum, butp > 0.5 compared to the animals associated with strain TA1538and L. plantarum). Even when statistically significant, this decreased concentration of strain TA1538 does not appear to beof sufficient magnitude to account for the approximately 10- to100-fold decrease that had been observed earlier in the revertant response.
Table 1
The fecal concentration of S. typhimurium TA 1538 in rats after theingestion of 2-nitrofluorene
LaJI-.C.) @10s.
Chart 3. The concentration of L. plantarum or B. vulgatus in the gastrointestinal tract of germ-free rats associated additionally with strain TA1538 (0), strainTA1538 and L. plantarum, or strain TA1538 and B. vulgatus. The values presented are the mean ±S.D. for 6 animals in each group. The locations in thegastrointestinal tract are: A, forestomach; B, stomach; C, duodenum; D, E, andF, variouslevelsin thesmallintestine(6);G,cecum;andH,colon.
1010
=lv
=
;io@
NUMBEROF His•REVERTANTS
Chart 4. The relationship between the highest concentration of his@revertantsobserved during the 4-day period after ingestion of a single dose of 3.4 mg of 2-nitrofiuorene and the concentration at sacrifice of B. vulgatus in stomach contents. Six rats were associated with strain TA1538 and B. vulgatus (0), and 4rats were associated with strain TA1538, B. vulgatus, and L. plantarum (s).
1)
U
Chart 5. The concentration of S. typhimurium strain TA1538 in the gastrointestinal tract of germ-free rats associated only with strain TA1538 (0) or withstrain TA1538 in addition to L. plantarum (c), B. vulgatus (s), or L. plantarumand B. vulgatus (s). The locations in the gastrointestinal tract are as noted in thelegend to Chart 3.
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Colon Carcinogens and Mutagenesis in Gnotobiotic Rats
Furthermore, if the action of B. vulgatus were the result of itsdisplacement of strain TA1538 from the colon, then a cormelation might be expected between the concentration of strainTA1538 in the colon of any animal and the mevertantresponsein that animal. A correlation of this kind was not observed foranimals additionally associated with B. vulgatus. Of course, theinterpretation of this result is subject to the possibility that acorrelation might be obscured by variations in the bacterialconcentrations that occur in the interval between the ingestionof the carcinogen and the time that the rats are sacrificed.
The Associationof Germ-free Ratswith Salmonella StrainsTAI 535, TAI 00, and TA98 and the Response of TheseStrains to the Feeding of Various Colon Carcinogens. AmesSalmonella tester strains TA1535, TA100, and TA98 wereeach found to associate with otherwise germ-free rats and toreach concentrations throughout the gastrointestinal tract andin the feces comparable to those observed previously for ratsassociated with strain TA1538.
In matsassociated with strain TA1535, the revertant responsefollowing the administration of 2-nitrofluomeneand the expenimental colon carcinogens 1,2-dimethylhydrazmneand azoxymethane is shown in Chart 6. No response was observed afterthe ingestion of 2-nitrofluonene, and the responses to 1,2-dimethylhydrazine and azoxymethane were variable. The expeniment conducted initially showed an increased mevertantresponse after each ingestion of 1,2-dimethylhydrazmne.However, this result was not reproducible in a second group of ratsassociated with strain TA1535 as indicated in Chart 6.
The response to these carcinogens in 3 different groups ofrats associated with strain TA100 is presented in Chart 7. Thefairly uniform response of the first group of matsto 1,2-dimethylhydrazmneand azoxymethane was not confirmed, however, ina second group of rats associated with strain TA100. A thirdgroup of matsappeared to respond to the colon carcinogenmethyl(acetoxymethyl)nitnosammne.
When 6 rats were associated with strain TA98, the fecalconcentration of his―revertants remained above 1000/g, thehighest concentration of mevertantsobserved with any Salmo
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S
cowt 241 iii thu IChart 7. The concentration of his@revertants in rats associated with strain
TA100 in response to the ingestion of various carcinogens. One group of 6 rats(•)wasgiven3.4mgof2-nitrofluorene(2-NF),followed3weekslaterbyasingledose of 1,2-dimethylhydrazine (OMH; 21 mg/kg), and finally 3 weeks later by asingle dose of azoxymethane (AOM; 18 mg/kg). A second group of rats (t@)wasgiven a single dose of 1,2-dimethylhydrazine (21 mg/kg) and 3 weeks later asingle dose of azoxymethane (19 mg/kg), while a third group (0) received asingle dose of methyl(acetoxymethyl)nitrosamine (M; 12.5 mg/kg). The greatestconcentration of his@revertants in a sample of feces collected at 24-hr intervalsfor 4 days following administration of the carcinogen is plotted. The number ofrevertants in a sample taken 1 day prior to the feeding is the control (CONT.).
nella strain prior to feeding a carcinogen. After the ingestion of3.4 mg of 2-nitrofluomene, all matsshowed a response of between 2 x 1O@and 5 x 1O@mevertants/g. No significantresponse was observed after these matsingested either 1,2-dimethylhydmazineor azoxymethane.
These data show that strains TA1535 and TA100, but notstrain TA98, may be responsive to 3 colon carcinogens, butthat the magnitudes of these responses are variable. Thesensitivities of strains TA98, TA100, and TA1535 to sodiumdeoxycholate and to crystal violet were not altered during theseexperiments, each of which required approximately 12 weeks.Strains TA98 and TA100 also retained their plasmids throughout the experiments as shown' by their continued resistance toampicillin.
The Determination of Mutageniclty of 1,2-Dimethylhydrazinc In Vitro. Tissue homogenates and S-9 microsomal preparations (1) of the liver, colon, small intestine, and stomachwere prepared from matswith the following characteristics: (a)germ-free; (b) germ-free matsexcept that the matshad beenpreviously administered 2 single doses of 1,2-dimethylhydmazine (21 mg/kg) by gastric intubation 5 and 10 days prior tosacrifice; and (c) germ-free rats given 4 doses of 1,2-dimethylhydrazine over a 2-week period prior to sacrifice.
Whole-tissue homogenates and 5-9 microsomal preparations of liver, colon, and stomach were mixed in varying combinations and ratios in order to test their ability to activate 1,2-dimethylhydrazine in the pour-plate assay. This assay wasperformed with concentrations of 1,2-dimethylhydmazinewhichranged from 0.2 to 200 @gand in the presence or absence ofan NADPH-genenating system (1). Strain TA1535 was used forthese assays since its spontaneous reversion matein vitro isconsiderably less than that for strain TA100. No reliable mesponse was detected under any of approximately 75 assayconditions.
DISCUSSION
When the matassociated with strain TA1538 ingests 2-nitrofluorene, the concentration of his@mevertantsappearing in the
10@
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i@.
S
S S
SS S
S
coin: 2-NF DM11-iDMII-2DMH-3 AOM
Chart 6. The concentration of his@revertants in the feces of rats associatedwith strain TA1535 In response to the Ingestion of various carcinogens. Onegroup of 6 rats (•)was given 3.4 mg of 2-nitrofluorene (2-NF), followed by 3single doses of 1,2-dlmethylhydrazine (21 mg/kg) at 2 weeks (DMH-1), 5 weeks(DMH-2), and 8 weeks (DMH-3) after the initial ingestion of 2-nitrofiuorene.Azoxymethane (AOM; 18 mg/kg) was administered 3 weeks after the final doseof 1,2-dimethylhydrazlne. A second group of 6 rats (ti) was given 2 single dosesof 1,2-dimethylhydrazine (21 mg/kg) separated by a 2-week interval and 3weeks after the second dose received a single dose of azoxymethane (18 mg/kg). Plotted are the greatest concentrations of his@revertants in the fecescollected during 24-hr intervals for the 4 days following ingestion of the carcinogen. The number of revertants in a fecal sample taken 1 day prior to the feedingIs the control (CONT.).
JUNE 1979 2029
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J. H. Carter et al.
feces undergoes an increase. This increase is greatly diminished, however, when the matsare associated with B. vulgatusin addition to strain TA1 538. When the association also includes L. plantarum, the revertant response to 2-nitmofluomeneis indistinguishable from that when only strain TA1 538 is associated. The explanation for these observations apparentlyrests with the ability of B. vulgatus to reduce the more mutagenic 2-nitnofluomeneto the less mutagenic 2-aminofluorene(5), a metabolic reaction that is negligible with either strainTA1538 on L. plantarum. An additional observation, that L.plantarum tends to displace B. vulgatus from the stomach,accounts for the finding that the nevertant response is normalwhen both L. plantarum and B. vulgatus are associated withstrain TAI 538. The distribution of the various bacteria in thestomach and the capacity of the various bacteria to reduce 2-nitmofluoreneto 2-aminofluonene are compatible with the viewthat B. vulgatus decreases the mevertantresponse by metabolizing 2-nitnofluomeneto the less potent mutagen, 2-aminofluonene. Furthermore, the presence of B. vulgatus (B. fragiisssp. vulgatus) within the gastrointestinal tract should also bluntmutagenesis by 2-aminofluonene if it is formed in vivo, since B.vulgatus reduces the N-hydmoxyderivatives (7) which are intemmediatesin the activation of this kind of carcinogen by theliver (3).
The site of mutagenesis in the gastrointestinal tract is stillunclear. The lag of at least 24 hr between the ingestion of 2-nitrofluonene and the appearance of nevertants in the feces iscompatible with the time required for mevertantsformed in thestomach to traverse the gastrointestinal tract. The occurrenceof mutagenesis in the stomach is also consistent with theobservation that ingested mutagens, which do not requiremammalian enzyme activation, e.g. , 2-nitrofluorene, are morepotent in provoking the fecal revertant response than thosethat require mammalian activation , e.g. , 2-acetylaminofluomene.4The occurrence of mutagenesis in the stomach or elsewhere in the upper bowel as a result of the ingestion of 2-
4 Unpublished observations.
nitrofluorene is also compatible with the observation that 2-nitmofluoneneis a carcinogen for the upper but not the lowerbowel.
Base-pair mutants such as strain TA100 and TA1535 arelike strain TA1538 in having the capacity to associate with thegerm-free mat.Unlike strain TA1538, however, these strainsmay be responsive to colon carcinogens such as azoxymethane and 1,2-dimethylhydrazine. Unfortunately, the mesponse to these carcinogens is of small magnitude and vamiable. It remains to be seen whether these strains can provide aresponse which is reliable enough to use as an approach tostudying mutagenesis in response to these colon carcinogens.
ACKNOWLEDGMENTS
The authors wish to thank Or. Joseph A. Ingelfinger for his critical analysis ofthiswork.
REFERENCES
1. Ames, B. N., McCann, J. C., and Yamasaki, E. Methods for detectingcarcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31: 347—364,1975.
2. Carter, J. H., Wheeler, L. A., Ingelfinger, J. A., Soderberg, F. B., andGoldman, P. Association of Salmonella mutants with germ-free rats: Characterization of the reverse mutational response to 2-nitrofluorene. Mutat.Res., 41: 209—216,1976.
3. Miller, J. A. Carcinogenesis by chemicals: An Overview—G.H. A. ClowesMemorial Lecture. Cancer Res., 30: 559—576,1970.
4. Miller, J. A., Sandin, A. B., Miller, E. C., and Rusch, H. P. Carcinogenecityof compounds related to 2-acetylaminofluorene, II. Variations in the bridgesand the 2-substituent. Cancer Res., 15: 118—199, 1955.
5. Wheeler, L. A., Carter, J. H., Ingelfinger, J. A., Soderberg, F. B., andGoldman, P. The effects of additional flora on the response of Salmonellamutants lodged in the gastrointestinal tract. Cancer Res., 37: 451—455,1977.
6. Wheeler, L. A., Carter, J. H., Soderberg, F. B., and Goldman, P. Associationof Salmonella mutants with germfree rats: Site specific model to detectcarcinogens as mutagens. Proc. NatI. Acad. Sci. U. S. A., 72: 4607—4611,1975.
7. Wheeler, L. A., Soderberg, F. B., and Goldman, P. The reduction of N-hydroxy-4-acetylaminobiphenyl by the intestinal microflora of the rat. CancerRes., 35: 2962-2968, 1975.
8. Wheeler, L. A., Soderberg, F. B., and Goldman, P. The relationship betweennitro group reduction and the intestinal microflora. J. Pharmacol. Exp. Ther.,194: 135—144, 1975.
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1979;39:2026-2030. Cancer Res John H. Carter, Martha A. McLafferty and Peter Goldman Gastrointestinal Tract of Gnotobiotic Rats
Mutants in theSalmonella typhimuriumAzoxymethane on Effect of 2-Nitrofluorene, 1,2-Dimethylhydrazine, and
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