Bile Acids: Co-mutagenic Activity in the Salmonella-Mammalian-Microsome Mutagenicity Test: Brief Communication 1.2 S. J. Silverman 3.4 and A. W. Andrews 5.6.
7
ABSTRACT-Of 30 bile acids tested, none was mutagenic in the Salmonella-mammalian·microsome test with indicator strains G46, TA1530, TA1535, TA1536, TA1537, TA1538, TA98, or TA100. However, when lithocholic acid or one of its conjugates was tested with suboptimal amounts of 2·aminoanthracene and phenobarbital·stimulated rat liver homogenate, enhancement and co·mutagenesis were observed if TA1538 was the indicator strain.-J Natl Cancer Inst 59: 1557-1559,1977.
Cook and Haslewood (1) and Fieser and Newman (2) sug· gested the possible role of acid sterols as precursors of car· cinogenic hydrocarbons when they synthesized MCA from bile acids. Shear et al. (3) were unable to induce a tumor response by injection of cholic or deoxycholic acids into mice and concluded that results obtained by previous in· vestigators were due to the solvent vehicle. Interest declined until Hill et al. (4) reported the effects of diet on both fecal flora and sterols. Feces of people in Western countries, where the populations consume large amounts of meat, contained greater quantities of both neutral and acid steroids than did the stools obtained from Asians whose diets consist primarily of vegetables, grains, and fish. Degradation of the steroids was more extensive in the stools of those on the mixed Western diets than was observed with the specimens from those on a more vegetarian diet. On the basis of these findings and the postulated pathways of metabolic degrada· tion, they suggested that bacterial degradation of the fecal steroids results in the production of carcinogenic polycyclic hydrocarbons. Narisawa et al. (5) reported that lithocholic and deoxycholic acids acted as enhancing agents when instilled in· trarectally into rats previously exposed to MNNG by the same route. The bile acids alone induced no tumors. Most tumors formed were polypoid adenomas, a few of which showed signs of malignant changes. Because a synergistic effect exists in animals, the enhancement should also be demonstrable in vitro.
MATERIALS AND METHODS Using the Salmonella-mammalian-microsome test devised by Ames et al. (6), we observed that several of the 30 bile acids acted as co-mutagens when strain T A1538 was exposed to suboptimum levels of 2-AA activated by a specific phenobarbital. stimulated rat liver homogenate (S9)' The test chemicals were dissolved in DMSO. The final concen· tration of the bile acids was 5.0 mg/ml, and the 2-AA final concentration was 0.02 mg/ml. The testing was performed essentially as described by Ames. However, we did modify the system by adding 0.2 ml instead of 0.1 ml of a 14-hour bacterial culture in nutrient broth to the top agar and by us· ing plates each containing 15 ml rather than 30 ml of VBE medium (7). Analysis of variance was by Dunnett's test of multiple ranges (8, 9). VOL. 59, NO.5. NOVEMBER 1977
1557
In some experiments, suspensions of the indicator strain were washed in a salt solution similar to that used in VBE .~ledium and exposed to either 2 JJg of 2-AA plus liver homogenate or 500 JJg of lithocholic acid at 37° C for 15 minutes. The cells were then recentrifuged and resuspended in the VBE salt solution containing histidine and biotin and resuspended to the original volume with the salt-biotinhistidine solution. Cells exposed to 2·AA and S9 mix were plated: with lithocholic acid added to the top agar; the bactena previously exposed to lithocholic acid were plated with 2-AA and S9 mix added to the top agar.
RESULTS AND DISCUSSION Generally, less than 20 spontaneous revertants were ob· served on plates inoculated with strain T A1538 and bile acids, 2·AA, or S9 mix. A few spontaneous revertants were also noted when the bacteria were incubated with a mixture of the bile acid and 2 -AA or bile acid and S9 mix. The bile acids tested were not mutagenic (table 1). Those showing a co-mutagenic effect with activated 2-AA were lithocholic (3a-hydroxy-5fJ-cholanic) acid, its conjugates with the amino acids glycine and taurine, or lithocholic acid dehydroxylated at the 3-carbon atom to form 3·keto·5fJcholanic acid (table 2). The dose responses were determined only for bile acids that showed the co-mutagenic effect. 3a, 12a -Dih ydroxy -5fJ -cholanic (deoxycholic), 3a, 12 a ·di . hydroxy·5fJ-chol·8(14)·en·24-oic (apocholic), and 3fJ·ace· toxy·bisnor-A5·cholenic acids failed to act as mutagens or co-mutagens, although they have been reported to induce tumors (11-13). Table 3 shows that to be effective the cells must be ex· posed to lithocholic acid prior to, or simultaneously with,
ABBREVIATIONS USED: MCA = 3-methylcholanthrene; MNNG = N-methyl2-AA = 2-aminoanthracene; DMSO = dimethyl sulfoxide; VBE = Vogel-Bonner E; AAF = N-2-acetylaminofluorene; BP = benzo[aJpyrene.
N' -nitro-N-nitrosoguanidine;
1 Received December 6. 1976; revised June 16, 1977; accepted June 27, 1977. 2 Supported.by Public Health Service (PHS) contract NOI-C025423 with Litton Bionetics, Inc .. from the Office of the Director, National Cancer Institute (NCI). S P.O. Box B. NCI Frederick Cancer Research Center, Frederick. Md. 21701. 4 Present address: Department of Biology, Hood College. Frederick, Md. 21701. 5 Chemical Carcinogenesis, P.O. Box B. NCI Frederick Cancer Research Center. 6 Address reprint requests to A. W. Andrews. 7 We acknowledge the excellent technical support of C. R. Valentine and Corinthia E. Brown. and we thank C. W. Riggs for the statistical analysis.
J NA TL CANCER INST
1558
SILVERMAN AND ANDREWS
TABLE I.-Mutagenicity and co-mutagenicity of bile acids a in the Salmonella-mammalian-microsome b test Strains C
Bile acids 5(3-Cholanic acid 3-Keto-5(3-cholanic acid 12a-Hydroxy-3-oxo-5(3-cholanic acid 3a-Hydroxy-7-oxo-5(3-cholanic acid 3a-Hydroxy-12-oxo-5(3-cholanic acid 3a-Hydroxy-6-oxo-5(3-cholanic acid 3a,6a-DihydroxY-5(3-cholanic acid 30',7 (3-DihydroxY-5 (3-cholanic acid 3,6-Diketo-5(3-cholanic acid 3,12-Diketo-5 (3-cholanic acid 3,7,12-Triketo-5(3-cholanic acid 7, 12-Diketo-3a-hydroxY-5(3-cholanic acid 30',7 a-Dihydroxy-12-keto-5(3-cholanic acid 3a,12a-Dihydroxy-7 -keto-5(3-cholanic acid 30',7 a,12a-Trihydroxy-5(3-cholanic acid 3a,12a-Dihydroxy-5(3-cholanic acid 3a,7a-DihydroxY-5(3-cholanic acid 30',7 a,12a-Trihydroxy-5(3-cholanic acid-24-glycine 3a,7a-DihydroxY-5(3-cholanic acid-24-glycine 30',7 a,12a-Trihydroxy-5(3-cholanic acid-24-taurine 3a,12a-Dihydroxy-5(3-cholanic acid-24-taurine 3a,7a-DihydroxY-5(3-cholanic acid-24-taurine 3a,12a-Dihydroxy-5(3-chol-S(14)-en-24-oic acid 23,24-Bisnor-3(3-acetoxy-5(3-cholen-22-oic acid 3a-Hydroxy-5(3-cholanic acid 30'- Hydroxy-5(3-cholanic acid, glycine ester 3a-Hydroxy-5(3-cholanic acid, taurine ester 3(3-Hydroxy-5a-cholanic acid 3a-Hydroxy-6-keto-5a-cholanic acid 3(3-Hydroxy-5-cholen-24-oic acid
Mutagenicity
Co-mutagenicity, with2-AA+S9
5,S,9,0 S,0,5,6,7,9 S 5,6,7,S,9,0 5,6,7,S,9,0
+
S
5,S 5,S 5,S 5,S 5,S 5,8 5,S 5,8 5,6,7,8,9,0 5,6,7,S,9,0 5,6,7,S,9,0 5,8 5,S,9,0 5,6,7,S 5,6,7,S 5,S,9,0 5,6,7,S,9,0 5,6,7,S,9,0 8,3,4,5,6,7,9,0 S,3,4,5,9,0 S,5,6,7,9 5,S,0 5,S 5,S
+ + +
All bile acids tested at 500 iJg/plate. Phenobarbital-stimulated microsomes. C Coding used by McCann et al. (l0):3=TA1530, 4=G46, 5=TA1535, 6=TA1536, 7=TA1537, S=TA1538, 9=TA98, 0=TA100. a
b
TABLE 2.-Titration of bile acids with undiluted phenobarbitalstimulated rat liver homogenate and 2 iJg 2-AA
Test compound DMSO Lithocholic acid Taurolithocholic acid 3-Keto-5(3-cholanic acid Glycolithocholic acid
Geometric mean a of the number Concen- of revertant colonies with strain tration TA153S, with additive: iJg None 2-AA S9 2-AA+S 9b 100 200 500 100 250 500 100 250 500 100 250 500
8.4 13.0 10.3 15.5 S.6 10.6 4.6 12.7 10.4 10.9 5.7 9.6 10.5
20.6 19.8 23.7 22.2 16.1 lS.0 14.2 19.2 10.5 lS.0 12.4 11.0 13.0
17.4 13.6 11.3 13.5 19.3 15.2 14.0 16.0 15.2 12.1 13.9 12.9 12.3
1,509.7 2,055.7 4,203.6 4,970.3 2,2S4.0 3,221.6 2,724.2 2,161.4 3,040.2 2,699.9 1,679.5 3,565.S 3,8S7.9
No.=3. b Values above 2,S6S.4 revertant colonies are significantly different. a
activated 2-AA. So far the co-mutagenic effect of these bile acids has been observed only with activated 2-AA. When tested with Sg mix and MNNG (with strain TA1535), AAF, MeA, or BP, the steroids did not increase the number of revertant colonies significantly beyond that of the controls. The number of revertants was actually decreased when the bile acids were tested with AAF (table 4).
J
NATL CANCER INST
TABLE 3.-Number of revertant colonies of strain TA1538 when exposed to the test compounds in various sequences Exposure (15 min) to: Nothing
2-AA+S, Lithocholic acid 2-AA+S,+ lithocholic acid a b
Plated with Nothing Lithocholic acid 2-AA+S, 2-AA+S,+ lithocholic acid Nothing Lithocholic acid Nothing 2-AA+S, Nothing
Number of revertants a
Mutation frequencyb
9±0 5±1.5 880±102.8
1.19X10-6 6.63X10- 7 1.17X10-4
2,088± 163.5 59±16.1 20±5.5 9±2.1 923±124.7
2.77XlO- 4 7.62X1O-6 2.58XlO-6 1.34X10-6 1.37 X 10-4
59±17.5
S.19X10-6
Mean±SD (3 expts). Based on viable counts after 15-min exposure.
Whereas Reddy et al. (14) found that both lithocholic and dexoycholic acids showed an adjuvant effect when administered to rats previously exposed to MNNG, we did not observe lithocholic acid to be active with MNNG in vitro; deoxycholic acid was ineffective with 2-AA or the other carcinogens. When the liver homogenate was obtained from SpragueDawley male rats challenged with Aroclor 1254 instead of phenobarbital sodium, some enhancement but no statistically significant co-mutagenic effect occurred (table 5). Alvares et al. (15) indicate that phenobarbital induces VOL. 59. NO.5. NOVEMBER 1977
BILE ACIDS: CO-MUTAGENIC ACTIVITY IN THE AMES ASSAY TABLE
4.-Co-mutagenic effect of bile acids and some known carcinogens-mutagens a
Bile acids or controls Cells only Cells with DMSO Cells with lithocholic acid Cells with taurolithocholic acid Cells with 3-keto-5(J-cholonic acid Cells with glycolithocholic acid
Number of revertant colonies produced with carcinogens-mutagens: b MNNG 2-AA AAF MCA BP 2/lg 2/lg 5/lg 5/lg 5/lg 3.247 2.007 1.538
2.545 2.154 4.847
1,421 2.049 96
50 40 45
153 86 180
2.807
3.008
50
127
145
2.585
4.879
33
25
183
2.858
4.041
49
70
224
Bile acids tested at 500 /lg/plate. Tester strain TA1535 was used for MNNG; TA1538 was used for 2-AA. AAF. MCA. and BP. Number of tests: MNNG. 5; 2-AA. 7; AAF. 2; MCA. 1; and BP. 2. Values represent geometric means. a
b
5.-Number of revertant colonies expressed as geometric means a for co-mutagenic effect of Aroclor 1254- or phenobarbitalinduced microsomes
TABLE
Treatment
0' 2/lg2-AA 8g 2/lg2-AA+S g a
Aroclor 1254 with: Lithocholic acid DMSO 500/lg 10.6 12.3 17.7 29.9 50.1 20.2 1.351.1 2.204.4
Phenobarbital with: Lithocholic acid DMSO 500/lg 8.2 5.8 13.8 10.6 15.7 10.0 2.324.0 5.610.8
No.=3.
cytochrome P-450 and Aroclor 1254 induces a type of cytochrome P-448. If this is so. microsomal metabolism and activation of the co-mutagenic effect are possibly cytochrome P-450-dependent. The mechanism for the enhancing effect is speculative. The acid steroids are surface-active agents and may alter the permeability of the cell membrane, or they may interfere with DNA repair. Lithocholic acid is toxic for mammalian cells, but its effect on bacteria has not been reported. In these studies. the steroids did not reduce the number of spontaneous mutations or inhibit the growth of the organisms on nutrient agar. Neither alteration of membrane
VOL. 59. NO.5. NOVEMBER 1977
1559
permeability nor inhibition of DNA repair accounted for the specificity of the enhancing effect with 2 -AA only. The metabolic activity of the intestinal bacteria produces changes in the bile acids and might modify carcinogens present in this milieu. Therefore, correlation between in vitro and in vivo tests would not necessarily be expected. It would be of interest to test the mutagenicity of these agents after anaerobic incubation in a suspension of fecal organisms.
REFERENCES (1) COOK JW. HASLEWOOD GA: Conversion of a bile acid into a hydrocarbon derived from 1.2 benzanthracene. Chern Ind Rev 11:758-759.1935 (2) FIESER LF. NEWMAN MS: Methyl cholanthrene from cholic acid. J Am Chern Soc 57:961. 1955 (3) SHEAR MJ. LEITER J. PERRAULT A: Studies in carcinogenesis. XV. Compounds related to 20-methylcholanthrene. J Natl Cancer Inst 2:99-115.1941 (4) HILL MJ. DRASER BS. ARIES V. et al: Bacteria and aetiology of canceroflarge bowel. Lancet 1:95-100,1971 (.5) NARISAWA T, MAGADIA NE, WEISBURGER JH. et al: Promoting effect of bile acids on colon carcinogenesis after intrarectal instillation of N-methyl-N' -nitro-N-nitrosoguanidine in rats. J Natl Cancer Inst 55:1093-1097,1974 (6) AMES BN. MCCANN J. YAMASAKI E: Methods for detecting carcinogenicity and mutagens with the Salmonella/mammalianmicrosome mutagenicity test. Mutat Res 31 :347-364. 1975 (7) VOGEL HJ. BONNER DM: Acetylomithinase of E. coli: Partial purification and some properties. J BioI Chern 218:97-106, 1956 (8) DUNNETT CWo A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc 50: 1096-1121, 1955 (9) - - - : New tables for multiple comparison with a control. Biometrics 20:482-491. 1964 (10) MCCANN J, CHOI E, YAMASAKI E. et al: Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc NatlAcadSci USA 72:5135-5139.1975 (11) COOK JW, KENNAWAY EL, KENNAWAY NM: Production of tumours in mice by deoxycholic acid. Nature 145:627. 1940 (12) LACASSAGNE A, Buu-HOI NP. ZAJDELA F: Carcinogenic activity of apocholic acid. Nature 190:1007-1008.1961 (13) - - - : Carcinogenic activity in situ of further steroid compounds. Nature 209:1026-1027. 1966 (14) REDDY BS, WEISBURGER JH, NARISAWA T, et al: Colon carcinogenesis in germ-free rats with 1 ,2-dimethylhydrazine and N-methyl-N'nitro-N-nitrosoguanidine. Cancer Res 34:2368-2372. 1974 (1.5) ALVARES AP. BICKERS DR, KAPPAS A: Polychlorinated biphenyls: A new type of inducer of cytochrome P-448 in the liver. Proc Natl Acad Sci USA 70:1321-1325. 1973
J NATL CANCER INST
7
ABSTRACT-Of 30 bile acids tested, none was mutagenic in the Salmonella-mammalian·microsome test with indicator strains G46, TA1530, TA1535, TA1536, TA1537, TA1538, TA98, or TA100. However, when lithocholic acid or one of its conjugates was tested with suboptimal amounts of 2·aminoanthracene and phenobarbital·stimulated rat liver homogenate, enhancement and co·mutagenesis were observed if TA1538 was the indicator strain.-J Natl Cancer Inst 59: 1557-1559,1977.
Cook and Haslewood (1) and Fieser and Newman (2) sug· gested the possible role of acid sterols as precursors of car· cinogenic hydrocarbons when they synthesized MCA from bile acids. Shear et al. (3) were unable to induce a tumor response by injection of cholic or deoxycholic acids into mice and concluded that results obtained by previous in· vestigators were due to the solvent vehicle. Interest declined until Hill et al. (4) reported the effects of diet on both fecal flora and sterols. Feces of people in Western countries, where the populations consume large amounts of meat, contained greater quantities of both neutral and acid steroids than did the stools obtained from Asians whose diets consist primarily of vegetables, grains, and fish. Degradation of the steroids was more extensive in the stools of those on the mixed Western diets than was observed with the specimens from those on a more vegetarian diet. On the basis of these findings and the postulated pathways of metabolic degrada· tion, they suggested that bacterial degradation of the fecal steroids results in the production of carcinogenic polycyclic hydrocarbons. Narisawa et al. (5) reported that lithocholic and deoxycholic acids acted as enhancing agents when instilled in· trarectally into rats previously exposed to MNNG by the same route. The bile acids alone induced no tumors. Most tumors formed were polypoid adenomas, a few of which showed signs of malignant changes. Because a synergistic effect exists in animals, the enhancement should also be demonstrable in vitro.
MATERIALS AND METHODS Using the Salmonella-mammalian-microsome test devised by Ames et al. (6), we observed that several of the 30 bile acids acted as co-mutagens when strain T A1538 was exposed to suboptimum levels of 2-AA activated by a specific phenobarbital. stimulated rat liver homogenate (S9)' The test chemicals were dissolved in DMSO. The final concen· tration of the bile acids was 5.0 mg/ml, and the 2-AA final concentration was 0.02 mg/ml. The testing was performed essentially as described by Ames. However, we did modify the system by adding 0.2 ml instead of 0.1 ml of a 14-hour bacterial culture in nutrient broth to the top agar and by us· ing plates each containing 15 ml rather than 30 ml of VBE medium (7). Analysis of variance was by Dunnett's test of multiple ranges (8, 9). VOL. 59, NO.5. NOVEMBER 1977
1557
In some experiments, suspensions of the indicator strain were washed in a salt solution similar to that used in VBE .~ledium and exposed to either 2 JJg of 2-AA plus liver homogenate or 500 JJg of lithocholic acid at 37° C for 15 minutes. The cells were then recentrifuged and resuspended in the VBE salt solution containing histidine and biotin and resuspended to the original volume with the salt-biotinhistidine solution. Cells exposed to 2·AA and S9 mix were plated: with lithocholic acid added to the top agar; the bactena previously exposed to lithocholic acid were plated with 2-AA and S9 mix added to the top agar.
RESULTS AND DISCUSSION Generally, less than 20 spontaneous revertants were ob· served on plates inoculated with strain T A1538 and bile acids, 2·AA, or S9 mix. A few spontaneous revertants were also noted when the bacteria were incubated with a mixture of the bile acid and 2 -AA or bile acid and S9 mix. The bile acids tested were not mutagenic (table 1). Those showing a co-mutagenic effect with activated 2-AA were lithocholic (3a-hydroxy-5fJ-cholanic) acid, its conjugates with the amino acids glycine and taurine, or lithocholic acid dehydroxylated at the 3-carbon atom to form 3·keto·5fJcholanic acid (table 2). The dose responses were determined only for bile acids that showed the co-mutagenic effect. 3a, 12a -Dih ydroxy -5fJ -cholanic (deoxycholic), 3a, 12 a ·di . hydroxy·5fJ-chol·8(14)·en·24-oic (apocholic), and 3fJ·ace· toxy·bisnor-A5·cholenic acids failed to act as mutagens or co-mutagens, although they have been reported to induce tumors (11-13). Table 3 shows that to be effective the cells must be ex· posed to lithocholic acid prior to, or simultaneously with,
ABBREVIATIONS USED: MCA = 3-methylcholanthrene; MNNG = N-methyl2-AA = 2-aminoanthracene; DMSO = dimethyl sulfoxide; VBE = Vogel-Bonner E; AAF = N-2-acetylaminofluorene; BP = benzo[aJpyrene.
N' -nitro-N-nitrosoguanidine;
1 Received December 6. 1976; revised June 16, 1977; accepted June 27, 1977. 2 Supported.by Public Health Service (PHS) contract NOI-C025423 with Litton Bionetics, Inc .. from the Office of the Director, National Cancer Institute (NCI). S P.O. Box B. NCI Frederick Cancer Research Center, Frederick. Md. 21701. 4 Present address: Department of Biology, Hood College. Frederick, Md. 21701. 5 Chemical Carcinogenesis, P.O. Box B. NCI Frederick Cancer Research Center. 6 Address reprint requests to A. W. Andrews. 7 We acknowledge the excellent technical support of C. R. Valentine and Corinthia E. Brown. and we thank C. W. Riggs for the statistical analysis.
J NA TL CANCER INST
1558
SILVERMAN AND ANDREWS
TABLE I.-Mutagenicity and co-mutagenicity of bile acids a in the Salmonella-mammalian-microsome b test Strains C
Bile acids 5(3-Cholanic acid 3-Keto-5(3-cholanic acid 12a-Hydroxy-3-oxo-5(3-cholanic acid 3a-Hydroxy-7-oxo-5(3-cholanic acid 3a-Hydroxy-12-oxo-5(3-cholanic acid 3a-Hydroxy-6-oxo-5(3-cholanic acid 3a,6a-DihydroxY-5(3-cholanic acid 30',7 (3-DihydroxY-5 (3-cholanic acid 3,6-Diketo-5(3-cholanic acid 3,12-Diketo-5 (3-cholanic acid 3,7,12-Triketo-5(3-cholanic acid 7, 12-Diketo-3a-hydroxY-5(3-cholanic acid 30',7 a-Dihydroxy-12-keto-5(3-cholanic acid 3a,12a-Dihydroxy-7 -keto-5(3-cholanic acid 30',7 a,12a-Trihydroxy-5(3-cholanic acid 3a,12a-Dihydroxy-5(3-cholanic acid 3a,7a-DihydroxY-5(3-cholanic acid 30',7 a,12a-Trihydroxy-5(3-cholanic acid-24-glycine 3a,7a-DihydroxY-5(3-cholanic acid-24-glycine 30',7 a,12a-Trihydroxy-5(3-cholanic acid-24-taurine 3a,12a-Dihydroxy-5(3-cholanic acid-24-taurine 3a,7a-DihydroxY-5(3-cholanic acid-24-taurine 3a,12a-Dihydroxy-5(3-chol-S(14)-en-24-oic acid 23,24-Bisnor-3(3-acetoxy-5(3-cholen-22-oic acid 3a-Hydroxy-5(3-cholanic acid 30'- Hydroxy-5(3-cholanic acid, glycine ester 3a-Hydroxy-5(3-cholanic acid, taurine ester 3(3-Hydroxy-5a-cholanic acid 3a-Hydroxy-6-keto-5a-cholanic acid 3(3-Hydroxy-5-cholen-24-oic acid
Mutagenicity
Co-mutagenicity, with2-AA+S9
5,S,9,0 S,0,5,6,7,9 S 5,6,7,S,9,0 5,6,7,S,9,0
+
S
5,S 5,S 5,S 5,S 5,S 5,8 5,S 5,8 5,6,7,8,9,0 5,6,7,S,9,0 5,6,7,S,9,0 5,8 5,S,9,0 5,6,7,S 5,6,7,S 5,S,9,0 5,6,7,S,9,0 5,6,7,S,9,0 8,3,4,5,6,7,9,0 S,3,4,5,9,0 S,5,6,7,9 5,S,0 5,S 5,S
+ + +
All bile acids tested at 500 iJg/plate. Phenobarbital-stimulated microsomes. C Coding used by McCann et al. (l0):3=TA1530, 4=G46, 5=TA1535, 6=TA1536, 7=TA1537, S=TA1538, 9=TA98, 0=TA100. a
b
TABLE 2.-Titration of bile acids with undiluted phenobarbitalstimulated rat liver homogenate and 2 iJg 2-AA
Test compound DMSO Lithocholic acid Taurolithocholic acid 3-Keto-5(3-cholanic acid Glycolithocholic acid
Geometric mean a of the number Concen- of revertant colonies with strain tration TA153S, with additive: iJg None 2-AA S9 2-AA+S 9b 100 200 500 100 250 500 100 250 500 100 250 500
8.4 13.0 10.3 15.5 S.6 10.6 4.6 12.7 10.4 10.9 5.7 9.6 10.5
20.6 19.8 23.7 22.2 16.1 lS.0 14.2 19.2 10.5 lS.0 12.4 11.0 13.0
17.4 13.6 11.3 13.5 19.3 15.2 14.0 16.0 15.2 12.1 13.9 12.9 12.3
1,509.7 2,055.7 4,203.6 4,970.3 2,2S4.0 3,221.6 2,724.2 2,161.4 3,040.2 2,699.9 1,679.5 3,565.S 3,8S7.9
No.=3. b Values above 2,S6S.4 revertant colonies are significantly different. a
activated 2-AA. So far the co-mutagenic effect of these bile acids has been observed only with activated 2-AA. When tested with Sg mix and MNNG (with strain TA1535), AAF, MeA, or BP, the steroids did not increase the number of revertant colonies significantly beyond that of the controls. The number of revertants was actually decreased when the bile acids were tested with AAF (table 4).
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NATL CANCER INST
TABLE 3.-Number of revertant colonies of strain TA1538 when exposed to the test compounds in various sequences Exposure (15 min) to: Nothing
2-AA+S, Lithocholic acid 2-AA+S,+ lithocholic acid a b
Plated with Nothing Lithocholic acid 2-AA+S, 2-AA+S,+ lithocholic acid Nothing Lithocholic acid Nothing 2-AA+S, Nothing
Number of revertants a
Mutation frequencyb
9±0 5±1.5 880±102.8
1.19X10-6 6.63X10- 7 1.17X10-4
2,088± 163.5 59±16.1 20±5.5 9±2.1 923±124.7
2.77XlO- 4 7.62X1O-6 2.58XlO-6 1.34X10-6 1.37 X 10-4
59±17.5
S.19X10-6
Mean±SD (3 expts). Based on viable counts after 15-min exposure.
Whereas Reddy et al. (14) found that both lithocholic and dexoycholic acids showed an adjuvant effect when administered to rats previously exposed to MNNG, we did not observe lithocholic acid to be active with MNNG in vitro; deoxycholic acid was ineffective with 2-AA or the other carcinogens. When the liver homogenate was obtained from SpragueDawley male rats challenged with Aroclor 1254 instead of phenobarbital sodium, some enhancement but no statistically significant co-mutagenic effect occurred (table 5). Alvares et al. (15) indicate that phenobarbital induces VOL. 59. NO.5. NOVEMBER 1977
BILE ACIDS: CO-MUTAGENIC ACTIVITY IN THE AMES ASSAY TABLE
4.-Co-mutagenic effect of bile acids and some known carcinogens-mutagens a
Bile acids or controls Cells only Cells with DMSO Cells with lithocholic acid Cells with taurolithocholic acid Cells with 3-keto-5(J-cholonic acid Cells with glycolithocholic acid
Number of revertant colonies produced with carcinogens-mutagens: b MNNG 2-AA AAF MCA BP 2/lg 2/lg 5/lg 5/lg 5/lg 3.247 2.007 1.538
2.545 2.154 4.847
1,421 2.049 96
50 40 45
153 86 180
2.807
3.008
50
127
145
2.585
4.879
33
25
183
2.858
4.041
49
70
224
Bile acids tested at 500 /lg/plate. Tester strain TA1535 was used for MNNG; TA1538 was used for 2-AA. AAF. MCA. and BP. Number of tests: MNNG. 5; 2-AA. 7; AAF. 2; MCA. 1; and BP. 2. Values represent geometric means. a
b
5.-Number of revertant colonies expressed as geometric means a for co-mutagenic effect of Aroclor 1254- or phenobarbitalinduced microsomes
TABLE
Treatment
0' 2/lg2-AA 8g 2/lg2-AA+S g a
Aroclor 1254 with: Lithocholic acid DMSO 500/lg 10.6 12.3 17.7 29.9 50.1 20.2 1.351.1 2.204.4
Phenobarbital with: Lithocholic acid DMSO 500/lg 8.2 5.8 13.8 10.6 15.7 10.0 2.324.0 5.610.8
No.=3.
cytochrome P-450 and Aroclor 1254 induces a type of cytochrome P-448. If this is so. microsomal metabolism and activation of the co-mutagenic effect are possibly cytochrome P-450-dependent. The mechanism for the enhancing effect is speculative. The acid steroids are surface-active agents and may alter the permeability of the cell membrane, or they may interfere with DNA repair. Lithocholic acid is toxic for mammalian cells, but its effect on bacteria has not been reported. In these studies. the steroids did not reduce the number of spontaneous mutations or inhibit the growth of the organisms on nutrient agar. Neither alteration of membrane
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permeability nor inhibition of DNA repair accounted for the specificity of the enhancing effect with 2 -AA only. The metabolic activity of the intestinal bacteria produces changes in the bile acids and might modify carcinogens present in this milieu. Therefore, correlation between in vitro and in vivo tests would not necessarily be expected. It would be of interest to test the mutagenicity of these agents after anaerobic incubation in a suspension of fecal organisms.
REFERENCES (1) COOK JW. HASLEWOOD GA: Conversion of a bile acid into a hydrocarbon derived from 1.2 benzanthracene. Chern Ind Rev 11:758-759.1935 (2) FIESER LF. NEWMAN MS: Methyl cholanthrene from cholic acid. J Am Chern Soc 57:961. 1955 (3) SHEAR MJ. LEITER J. PERRAULT A: Studies in carcinogenesis. XV. Compounds related to 20-methylcholanthrene. J Natl Cancer Inst 2:99-115.1941 (4) HILL MJ. DRASER BS. ARIES V. et al: Bacteria and aetiology of canceroflarge bowel. Lancet 1:95-100,1971 (.5) NARISAWA T, MAGADIA NE, WEISBURGER JH. et al: Promoting effect of bile acids on colon carcinogenesis after intrarectal instillation of N-methyl-N' -nitro-N-nitrosoguanidine in rats. J Natl Cancer Inst 55:1093-1097,1974 (6) AMES BN. MCCANN J. YAMASAKI E: Methods for detecting carcinogenicity and mutagens with the Salmonella/mammalianmicrosome mutagenicity test. Mutat Res 31 :347-364. 1975 (7) VOGEL HJ. BONNER DM: Acetylomithinase of E. coli: Partial purification and some properties. J BioI Chern 218:97-106, 1956 (8) DUNNETT CWo A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc 50: 1096-1121, 1955 (9) - - - : New tables for multiple comparison with a control. Biometrics 20:482-491. 1964 (10) MCCANN J, CHOI E, YAMASAKI E. et al: Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc NatlAcadSci USA 72:5135-5139.1975 (11) COOK JW, KENNAWAY EL, KENNAWAY NM: Production of tumours in mice by deoxycholic acid. Nature 145:627. 1940 (12) LACASSAGNE A, Buu-HOI NP. ZAJDELA F: Carcinogenic activity of apocholic acid. Nature 190:1007-1008.1961 (13) - - - : Carcinogenic activity in situ of further steroid compounds. Nature 209:1026-1027. 1966 (14) REDDY BS, WEISBURGER JH, NARISAWA T, et al: Colon carcinogenesis in germ-free rats with 1 ,2-dimethylhydrazine and N-methyl-N'nitro-N-nitrosoguanidine. Cancer Res 34:2368-2372. 1974 (1.5) ALVARES AP. BICKERS DR, KAPPAS A: Polychlorinated biphenyls: A new type of inducer of cytochrome P-448 in the liver. Proc Natl Acad Sci USA 70:1321-1325. 1973
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