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Bile salt/acid induction of DNA damage in bacterial and mammalian cells: Implications for colon cancer a
Risa L. Kandell & Carol Bernstein
a
a
Department of Microbiology and Immunology, College of Medicine , University of Arizona , Tucson, AZ, 85724 Published online: 04 Aug 2009.
To cite this article: Risa L. Kandell & Carol Bernstein (1991) Bile salt/acid induction of DNA damage in bacterial and mammalian cells: Implications for colon cancer, Nutrition and Cancer, 16:3-4, 227-238, DOI: 10.1080/01635589109514161 To link to this article: http://dx.doi.org/10.1080/01635589109514161
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Bile Salt/Acid Induction of DNA Damage in Bacterial and Mammalian Cells: Implications for Colon Cancer Downloaded by [New York University] at 21:27 22 May 2015
Risa L. Kandell and Carol Bernstein
Abstract Two bile salts, sodium chenodeoxycholate and sodium deoxycholate, induced a DNA repair response in the bacterium Escherichia coli. Similarly, a bile acid and a bile salt, chenodeoxycholic acid and sodium deoxycholate, induced DNA repair (indicated by unscheduled DNA synthesis) in human foreskin fibroblasts. Also, DNA repair-deficient Chinese hamster ovary (CHO) cells were found to be more sensitive than normal cells to killing by bile salts. In particular, mutant UV4 CHO cells, defective in DNA excision repair and DNA cross-link removal, were more sensitive to sodium chenodeoxycholate, and mutant EM9 CHO cells, defective in strand-break rejoining, were more sensitive to sodium deoxycholate than wild-type cells. These results indicate that bile salts/acid damage DNA of both bacterial and mammalian cells in vivo. Previous epidemiological studies have shown that colon cancer incidence correlates with fecal bile acid levels. The findings reported here support the hypothesis that bile salts/acids have an etiologic role in colon cancer by causing DNA damage. (Nutr Cancer 16, 227-238, 1991)
Introduction
Epidemiological evidence has long pointed to dietary factors or intracolonic factors as important in colon cancer (1-4). As reviewed by Cheah (5), bile acids, dietary (oxidized) fat, fecal mutagens, and ketosteroids have been the four most widely studied potential etiologic agents in colorectal cancer. Cheah (5) concluded that bile acids are the most strongly implicated on the basis of evidence in 87 studies relating to six different areas: 1) population comparisons, 2) case control comparisons, 3) animal experiments, 4) bacterial experiments, 5) mammalian cell line experiments, and 6) DNA damage experiments. The primary bile acid chenodeoxycholic acid constitutes about 35% of the bile acids in the gallbladder (6). It is usually modified by bacteria of the intestine, so that it constitutes only 0.5-1.0% of the bile acids in the feces. An important secondary bile acid is deoxycholic acid, The authors are affiliated with the Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
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which constitutes about 52% of the bile acids in the feces (6). Because it is recirculated through the enterohepatic shunt, it is also present as about 20% of the bile acids in the gallbladder. These two bile acids were chosen for study because one is eliminated from the colon by bacterial modification and the other is introduced in the colon because of bacterial action. The two bile acids might have pointed to a role of bacterial toxification or detoxification of the bile acids in genotoxic activity. However, no great difference in their level of DNA-damaging effects was found in our study. Although a distinction between bile acids and bile salts should be made, organic chemists have traditionally employed the term bile acid as a generic name for this cholanic class of biological compounds (7). However, bile acids have an aqueous solubility of 10~8 to 10~3 M, and bile salts have an aqueous solubility of 1-2 M (7). In the intestinal content, bile salts are the major form present. However, as noted by Carey and Cahalane (8), "whenever the gut luminal pH lies below the precipitation values of bile salts, the latter may precipitate from solution as the sparingly soluble undissociated bile acids. This occurs under normal physiological conditions in both stomach and colon." Thus, both bile acids and bile salts could be important in the etiology of colon cancer. In describing our work, we specifically indicate whether a bile acid or salt was used. A number of tests are widely employed to assess DNA-damaging abilities of compounds. In Escherichia coli, DNA-damaging agents induce a set of gene functions known collectively as the SOS response (9). The SOS chromotest was devised with E. coli to measure DNA repair (10) by use of a strain in which there is an operon fusion placing lacZ, the structural gene for /3-galactosidase, under the control of the SOS gene sulA or sfiA. In this strain, DNA damage results in turn-on of sulA and also lacZ. The /3-galactosidase produced can cleave 5-bromo-4-chloro-3-indoIyl-/3-D-galactoside (Xgal) to form a blue product. We used a modification of the standard SOS chromotest to detect the DNA repair response to DNA damage caused by bile salts in E. coli. One method for investigating DNA repair in mammalian cells is to measure unscheduled DNA synthesis (UDS). This method assays the limited DNA synthesis that occurs during excision repair (for review, see Ref. 11). DNA damage in mammalian cells can also be detected by differential cytotoxicity of DNA repair-deficient lines of Chinese hamster ovary (CHO) cells, compared with wild-type CHO cells. We used both UDS and differential cytotoxicity to detect DNA damage caused by bile acids or salts in mammalian cells. Although bile acids have been shown to be mutagenic to bacterial cells in a modified Ames Salmonella test (12,13), indicating that bile acids cause DNA damage in bacteria, the results were not dramatic and should be confirmed. Other results showing that bile acids can act as mammalian tumor promoters and cocarcinogens (14-17) or can damage bacteriophage DNA in vitro (18) did not address the question of whether bile acids can cause DNA damage in mammalian cells. Our results indicate that bile salts/acids can directly cause DNA damage in vivo both in bacteria and mammalian cells. This is consistent with the hypothesis that bile salts/acid are important in the etiology of colon cancer, perhaps as carcinogenic agents. Materials and Methods
Bacterial and Mammalian Strains E. coli strain JL1705 (kindly provided by Dr. J. W. Little, University of Arizona) has the structural gene for lacZ, /3-galactosidase, under the control of sulA (sfiA) and was constructed from E. coli JL1047 and plasmid pJWL184 (19). Human foreskin fibroblasts were kindly provided by Dr. M. R. Haussler (University of Arizona). The CHO wild-type strain AA8 and mutant strains EM9 and UV4 were kindly provided by Dr. C. A. Hoy (20).
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Growth and Maintenance of Cells E. coli JL1705 cells were grown as overnight cultures in Luria broth, and working stocks of the strain were maintained on trypticase plates, where the trypticase agar consisted of 10 g trypticase peptone (Baxter Healthcare, Tempe, AZ), 5 g NaCl, and 10 g Bacto-agar (Difco, Detroit, MI) in 1 liter of H2O, adjusted to pH 7.2. The test for SOS induction was carried out on trypticase plates to which Xgal (80 /tg/ml) and ampicillin (50 jtg/ml) were added; bacteria were suspended in a soft agar overlay (21) with ampicillin (100 fig/ml) added. Melted agar was cooled to 42 °C before the addition of Xgal or ampicillin. Xgal was diluted in A7,Ar-dimethylformamide at a concentration of 20 mg/ml and stored at —20°C. Human foreskin fibroblasts were grown as a monolayer culture in filter-sterilized McCoy's 5A medium (modified, Sigma Chemical, St. Louis, MO) at pH 7.5 and supplemented with 0.5% solution of penicillin (100,000 U/ml) plus streptomycin (10,000 /tg/ml, Sigma Chemical) and 10% fetal bovine serum (GIBCO, Grand Island, NY) before use. For maintenance of growing cells, 2.8 x 105 cells were seeded into a 250-ml tissue culture flask containing 10 ml growth medium and placed in a 37°C 5% CO2 incubator. Cells for use in UDS experiments were grown on coverslips placed in the bottom of a petri dish. Growth was allowed until a confluent monolayer formed. CHO cells were grown as monolayer cultures in 250-ml tissue culture flasks until needed for an experiment. Alpha medium (GIBCO) supplemented with 10% fetal bovine serum and 0.5% of a solution of penicillin (10,000 U/ml) plus streptomycin (10,000 /tg/ml) was used as growth medium. Bacterial SOS DNA Repair Induction Test This test is a modified version of the standard SOS chromotest (10). An aliquot of 0-150 /tl of the agent being tested was added to melted top (overlay) agar kept at 42°C and vigorously mixed. E. coli JL1705 cells, grown as an overnight culture in Luria broth, were added to the top agar in sufficient number to yield 50-200 viable colonies, and the mixture was plated on BBL trypticase plates. The plates were then incubated for 19 hours at 37°C, colonies were scored as either blue or white, and the total surviving fraction, S/So, was calculated. Each experiment was repeated three times. CHO Cytotoxicity Assay Differential cytotoxicity of CHO cells defective in DNA repair was tested using the method described by Hoy and co-workers (20). UDS in Human Foreskin Fibroblast Cells Coverslips carrying a confluent monolayer of freshly grown human foreskin fibroblast cells were rinsed with phosphate-buffered saline (PBS, 8.0 g NaCl, 0.2 g KC1, 0.12 g KH2PO4, 0.91 g Na2HPO4 in 1 liter, adjusted to pH 7.5 with NaOH). Then 5.0 ml PBS containing 10 raM hydroxyurea were used to cover cells for 15 minutes at room temperature to inhibit replicative DNA synthesis (22). Medium covering the cells was then replaced by treatment medium consisting of 5.0 ml PBS, 10 mM hydroxyurea, 1% dimethyl sulfoxide (DMSO), and the agent to be tested for 20 minutes at room temperature, or slides were exposed to 30 J/m 2 of ultraviolet (UV) light and then returned to 5.0 ml PBS containing 10 mM hydroxyurea for 20 minutes. All further work was carried out under yellow light to prevent possible photoreactivation of DNA damage. The cells were then rinsed twice with PBS and placed in petri dishes containing growth medium plus 1.0 ftCi/ml [3H]thymidine (sp act 60-68 Ci/mmol) and incubated in light-tight containers at 37°C in a 5% CO2 incubator for 21 hours. The petri dishes were rinsed three times with PBS, and a 1 % solution of sodium
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citrate was added and left on the coverslips for 10 minutes to swell the nuclei of the cells. The cells were then fixed with three 10-minute treatments of 3:1 ethanol-acetic acid. The fixer was poured off, and the coverslips were allowed to dry. Coverslips were mounted on slides and covered with Kodak NTB-3 emulsion and exposed at 4°C for seven days. After the slides were developed, cells were strained with hematoxylin. Grains over nuclei were counted, and grains in an equal area, as judged by eye, in the upper right-hand area adjacent to the nucleus, were also counted and subtracted from the grains over the nucleus to give a net grain count for each nucleus. If the upper right-hand area was obscured by debris or blemish, then the equal area, as judged by eye, in the lower left-hand area adjacent to the nucleus was used for background subtraction. Infrequent nuclei with net grain counts over 50 were considered to be undergoing replicative synthesis and were not included in the study. All slides were coded before evaluation, so that they were scored without knowledge of their treatments.
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Tests of Genotype o/E. coli and CHO Mutant Strains E. coli JL1705 cells were plated on Bacto MacConkey agar (Difco Laboratories) before they were used in each test for DNA damage. Bacteria capable of fermenting lactose on this agar produce a localized pH drop when forming a colony, which, followed by absorption of the neutral red in the agar, imparts a deep red color to the colony. E. coli JL1705 cells, in our tests, formed white to light pink colonies, indicating that the operon containing lacZ was either repressed or expressed at low levels, as expected in the absence of externally caused DNA damage. The standard spot test part of the SOS chromotest (10), using mitomycin C as a known DNA-damaging agent, was also performed as a control before each experiment. The expected blue ring consistently formed with this strain of E. coli, indicating that the operon containing lacZ is expressed in response to DNA damage. CHO cells were tested for sensitivity to DNA damage by irradiation with UV light, a known DNA-damaging agent. Wild-type strain AA8 showed a "least effective amount" of UV irradiation (with which diminished cell growth was detected) of 50 seconds, EM9 had a least effective amount of 40 seconds, and UV4 had at least effective amount of 10 seconds, when each strain was exposed to a UV lamp delivering 1 J/m2/second for 10, 20, 30, 40, and 50 seconds. Chemicals Sodium deoxycholate (ICN Biomedicals, Costa Mesa, CA) and sodium chenodeoxycholate (Sigma Chemical) were dissolved in water. Chenodeoxycholic acid (Sigma Chemical) was dissolved in DMSO. The [3H]thymidine (ICN Biomedicals) had a specific activity of 60-68 /tCi/mM. Results Bile Salt Induction of DNA Damage in Bacteria Measured by a Modified SOS Chromotest We were not able to use the standard SOS chromotest (10), which is performed using E. coli PQ37. This strain carries a mutation in gene uvrA, which causes a defect in excision repair and thus higher sensitivity to some DNA-damaging agents. It also has a mutation in rfa, which makes the strain lipopolysaccharide deficient. This allows better diffusion of certain molecules into the cells but also makes them very sensitive to bile salts. In fact, the recommended test for the proper phenotype of E. coli PQ37 is assay for sensitivity to sodium deoxycholate or inability to grow on agar plates that contain deoxycholate (10). In the standard assay, growth is usually allowed both in liquid media and on solid agar for a spot
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test. The blue color produced in the liquid media or around the spot test on solid agar is then measured. However, even when an E. coli strain with wild-type membrane (rfa+) was used, sodium deoxycholate proved to be too toxic for use during liquid growth. In addition, the spot test was difficult to quantitate because E. coli grows more slowly in the presence of sodium deoxycholate on plates. Thus, we used both a different strain of E. coli and a different quantitative plate assay. The strain we used, E. coli JL1705, was similar to strain PQ37 in that lacZ was under the control of sulA, but JL1705 did not carry a mutation in rfa (or a mutation in uvrA). Then we assayed the frequency with which colonies on a plate containing the test compound turned blue at 19 hours of incubation as well as survival of colony-forming ability on the plates. As shown in Figure 1, the established DNA-damaging agent mitomycin C caused an increase in percentage of blue colonies produced on a plate, from 12% to 80%, as the concentration of mitomycin C increased from 0 to 700 ng/ml. At the same time, survival of colony-forming ability of cells on the plate decreased from 100% to 44%. The increase in percentage of blue colony formation and the decrease in survival followed regular trends. The larger error bars observed, for two of the seven points, indicating percent occurrence of blue colonies, reflect the occasional counting of plates with about 50 colonies per plate. At this lower number of total colonies, a 30% or 40% blue colony frequency refers to 15 or 20 blue colonies on the plate. Some fluctuation in such small numbers is expected. During treatment of E. coli cells with a DNA-damaging agent, there are a number of consequences. Some cells turn on the SOS response. A small fraction of those with the SOS response turned on and a larger fraction of those that had not turned on this response are killed by the increasing concentration of the DNA-damaging agent. As long as the SOS response offers protection from the DNA-damaging agent, an increasing fraction of surviving colonies will be blue, as shown in Figure 1. If the SOS response is turned on in cells with a higher frequency than "turned-on" cells are killed, then there will also be an absolute increase (rather than just a relative increase among survivors) in the percentage of cells that form blue colonies in a population. Figure 2 shows that, after mitomycin C treatment, there is an absolute increase in percentage of cells that went on to form blue colonies. Because the data shown in Figure 2 can be obtained from the data shown in Figure 1 by a simple calculation, the error bars were left out of Figure 2 to promote clarity. The modified SOS chromotest was then used with the two bile salts, sodium chenodeoxycholate and sodium deoxycholate. With each bile salt the results obtained were very similar to those obtained with mitomycin C. As shown in Figure 3, survival of 100
Figure 1. Percentage of colonies that were blue and percentage of cells surviving and able to form colonies (blue or white) on plates. Indicated concentration of mitomycin C used to treat cells was calculated assuming that mitomycin C diffused throughout 3 ml of top agar and 30 ml of bottom agar on the plate. Each point represents mean from 3 experiments; bars represent SE.
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blue colonies
200
400
Mitomycin-C
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ng/ml
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80 (A
O
o
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E2 Killed • White • Blue
o
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o
IUUMUMI 0
141
235
353
471
70S
Mitomycin-C ng/ml Figure 2. Fate of cells placed on plates with indicated concentrations of mitomycin C in agar. Cells can grow to form blue colonies, grow to form white colonies, or fail to grow (be killed).
B
100
200
300
Chenodeoxycholate
400
pg/tnl
500
—
60-
O.
10
100
200
Deoxycholate
300
400
500
pg/ml
Figure 3. Percentage of colonies that were blue and percentage of cells surviving and able to form colonies (blue or white) on plates with indicated concentration of bile salts. Each point represents mean from 3 experiments; bars represent SE. Colonies were grown in the presence of sodium chenodeoxycholate (A) or sodium deoxycholate (B).
colony-forming ability went from 100% to about 20%, while the percentage of blue colonies among the survivors rose from about 8% to about 80%. In addition, as shown in Figure 4, the absolute percentage of cells going on to form blue colonies rose after treatment with chenodeoxycholate from 6% to a maximum of 20% of the population and for deoxycholate from 8% to a maximum of 18% of the population. In these two cases of bile salt treatment, the absolute percentage of surviving blue colonies at the highest concentration of bile salt was less than that with an intermediate concentration of bile salt. At the highest concentration there was less than 20% survival of cells able to form colonies. At this high level of lethality, cells with SOS repair "turned on" were being killed at a higher frequency than new cells were being turned on to SOS repair, because there were actually very few cells left that could be newly turned on. Overall, the data displayed in Figures 3 and 4 indicate that the bile salts tested behave like the known DNA-damaging agent mitomycin C.
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0 • I]
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Chenodeoxycholate
353
fig/ml
441
0
132
176
Deoxycholate
265
Killed White Blue
397
|jg/ml
Figure 4. Fate of cells placed on plates with indicated concentrations of bile salt in agar. Cells can grow to form blue colonies, grow to form white colonies, or fail to grow (be killed). Colonies were grown in the presence of sodium chenodeoxycholate (A) or sodium deoxycholate (B).
Bile Compound Induction ofDNA Damage in Human Foreskin Fibroblasts Measured by UDS When UDS was measured with newborn human foreskin fibroblast cells, the DNAdamaging agent UV light was used as a positive control. UV light is known to cause a strong UDS response in mammalian cells (23). As shown in Table 1, treatment of foreskin fibroblast cells with increasing concentrations of sodium deoxycholate or chenodeoxycholic acid caused a highly significant turn-on of DNA repair, reflected by UDS. The estimated standard error of the mean for grains per cell was calculated by using the standard formula (24) A
a where x is the number of grains in a nucleus and N is the number of nuclei counted. The increase in UDS was not as great with the bile salt or acid as with UV light, but different DNA-damaging agents are known to turn on DNA repair, as reflected by UDS, to different extents (23). Differential Cytotoxicity of Bile Salts to DNA Repair-Defective CHO Cells Three strains of CHO cells were used in these experiments: the wild-type strain AA8 and mutant strains UV4 and EM9. UV4 is defective in excision repair and cross-link removal, and EM9 is defective in strand rejoining. Table 2 summarizes the results obtained. UV4 was significantly more sensitive to killing by sodium chenodeoxycholate than the wild-type strain AA8 but not more sensitive to sodium deoxycholate. EM9 was significantly more sensitive to killing by sodium deoxycholate than the wild-type strain but not more sensitive to killing by sodium chenodeoxycholate. The results were reproduced in three separate experiments, and scoring of the results was the same whether performed by the investigators or by other individuals who had not been told which compounds or which cells were in each well.
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Table 1. Unscheduled DNA Synthesis in Human Foreskin Fibroblasts Expt No. 1
Treatment Deoxycholate
UV
2
Deoxycholate
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UV
3
Chenodeoxycholic acid
UV
4
Chenodeoxycholic acid
UV
Dose" 0.0
12.5 25.0 50.0 100.0 250.0 30.0 0.0
12.5 25.0 50.0 75.0 100.0 30.0 0.0
25.5 50.0 100.0 250.0 30.0 0.0
50.0 75.0 100.0 150.0 200.0 30.0
No. of Slides
No. of Cells
Grains/Cell6
Fold Increase0
3 3 5 4 4 4 3 6 6 6 5 5 5 3 3 3 3 3 3 3 4 3 3 3 3 3 3
450 450 750 600 600 600 450 900 900 900 750 750 750 450 450 450 450 450 450 450 600 450 450 450 450 450 450
3.93 ± 0.43 5.17 ± 0.52 8.36 ± 0.47 7.36 ± 0.51 7.81 ± 0.49 6.92 ± 0.48 12.54 ± 0.40 4.38 ± 0.32 6.63 ± 0.40 8.74 ± 0.40 6.94 ± 0.46 5.51 ± 0.36 7.31 ± 0.42 21.87 ± 0.42 4.25 ± 0.43 3.63 ± 0.30 4.72 ± 0.42 7.58 ± 0.57 6.00 ± 0.45 16.57 ± 0.49 3.72 ± 0.34 3.36 ± 0.32 4.89 ± 0.40 5.14 ± 0.44 6.19 ± 0.45 5.45 ± 0.38 16.15 ± 0.27
1.00 1.31 2.13* 1.87* 1.99* 1.75* 3.22* 1.00 1.51* 1.53* 1.58* 1.26* 1.57* 4.99* 1.00 0.85 1.10 1.78* 1.30* 3.22* 1.00 0.90 1.31 1.38* 1.66* 1.47* 4.34*
a: Expressed as jig/ml or as seconds of ultraviolet irradiation (UV). b: Expressed as means ± estimated SE c: Statistical significance is as follows: *,p £ 0.005; t, P £ 0.01.
Table 2. Bile Salt Cytotoxicity Toward CHO Wild-Type and Mutant Strains Strain AA8 (wild type) UV4 (excision repair mutant) EM9 (defective in strand rejoining)
Compound Chenodeoxycholate Deoxycholate Chenodeoxycholate Deoxycholate Chenodeoxycholate Deoxycholate
Least Effective Concentration" 1,000 1,000 500
1,000 1,000 500
a: Concentration (/ig/ml) that results in clear decrease in cell viability by test of Hoy et al. (20). When established procedure is followed, a 2-fold difference is considered significant (20).
Discussion The results obtained with the modified bacterial SOS chromotest showed that increasing concentrations of either of two bile salts, sodium deoxycholate or sodium chenodeoxycholate, increased the frequency of blue colony formation in a strain carrying a sulA:lacZ fusion as a result of a turning-on of expression of the sulA operon. Turn-on of this operon occurs as one of a spectrum of responses to DNA damage in E. colt. The sulA
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promoter has been found to be very powerful and to allow production of a large number of transcripts when repression is removed (25). The turn-on of this operon in our experiments, as measured by the frequency with which colonies become blue, correlated with the degree to which the colony-forming ability of cells was inactivated by the bile salts tested. These results are expected if the progressive inactivation of cells in a population is caused by intracellular DNA damages, some of which trigger (and are repaired by) a DNA repair pathway induced as part of the SOS response. The results obtained with bile salt treatment (Figures 3 and 4) were similar to the results obtained with the established DNA-damaging agent mitomycin C (Figures 1 and 2). Thus our findings indicate that bile salts are likely to be DNA-damaging agents in vivo in E. coli. The standard SOS chromotest has been validated by Quillardet and co-workers (26), who used this assay to test 83 compounds and found that 90% of the compounds mutagenic in the Ames test were also SOS inducers. Our modified chromotest is, in principle, the same as the standard test and gave a positive result with an established DNA-damaging agent. It should be a valid indicator of DNA damage as well. One factor that should be considered when interpreting these results is an effect found by Schmellik-Sandage and Tessman (27). Their work indicated that mutations that alter cell membrane permeability can cause blue colony formation in a sulA:lacZ fusion strain by allowing extra Xgal to enter and be made blue, even without DNA damage. It may be possible that increasing amounts of bile salts may cause lethality by damaging the membrane, thereby increasing permeability to Xgal. An effect on cell membranes might be expected to affect all the colonies on a plate. For some colonies to be blue and some not blue, one must postulate a complicated explanation of a lineage of blue-producing cells with an epigenetic alteration leading to vulnerable membranes. There is no basis for postulating such an epigenetic effect. Such a cell lineage would presumably give rise to small sick colonies, if the cause of bile salt lethality was to cause membrane deterioration and Xgal permeability. The blue colonies, however, had the same approximate range of sizes and vigor as the nonblue colonies, so a membrane effect does not seem likely. On the other hand, as reviewed by Walker (9), the SOS response, which controls turn-on of the sulA:lacZ fusion, is buffered against being substantially induced by small amounts of inducing signal (where the signal is single-stranded DNA regions caused by damage). Thus there is a barrier, which must be overcome, before the system is turned on. Furthermore, Cole and Honore (25) showed that the sulA operon had an unusual arrangement of two "shut-off mechanisms, so it can be turned off quickly. This arrangement predicts an on-or-off state for sulA:lacZ and, thus, an on-or-off state for blue color production. Blue color production would only turn on after a certain amount of damage accumulated in a cell. Furthermore, this turned-on state, due to cleavage of the LexA repressor protein, could be passed on to progeny cells as long as DNA damage signals continue to be present. The blue colonies, with SOS repair turned on, should have normal size and vigor, because repair of DNA damage in these cells is elevated. Such a normal spectrum of blue colony size and vigor was observed. Thus a DNA repair response is a more likely explanation for our results than a deterioration of membrane permeability. Our test of UDS, as a response to treatment of human newborn foreskin fibroblasts with a bile acid or bile salt, showed a highly significant increase in UDS upon treatment with bile salt/acid (Table 1). Because UDS reflects excision repair in mammalian cells (11), these results indicate that bile salt/acid causes DNA damage in foreskin cells and that excision repair is induced to deal with the damage. Although UDS was induced, it appeared to reach a plateau (Table 1). This may indicate that higher levels of DNA damage inhibit expression of DNA repair enzymes. In fact, at higher doses of the bile compounds than those shown in Table 1, toxicity was shown by cell detachment. Harbach and co-workers (23) reported a similar nonincrease of UDS with increased exposure to a known genotoxic compound, benzo[cr]pyrene. Their result was obtained in a validation study of UDS in response to known
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genotoxic agents applied to rat hepatocytes. The highly significant levels of UDS induced by the bile compounds in our study indicate that the compounds cause DNA damage, whereas the plateau of UDS in response to higher levels of compounds and the detachment of cells at still higher levels indicate that bile compounds are toxic to cells at high levels. Recent work by Suter and Romagna (28) indicates that 10 mM hydroxyurea, used in our experiments to suppress replicative DNA synthesis, can cause a small increase in UDS in rat hepatocytes. In fact, the somewhat high background levels of counts, in the absence of bile salt or acid, are similar to those reported by them (28). However, known genotoxic agents were also tested by them in the presence of 10 mM hydroxyurea in similar liquid scintillation tests of induced DNA repair, and the small amount of repair induced by hydroxyurea did not obscure the positive DNA repair responses induced by these agents. In our case, hydroxyurea was present both in the media for the negative control cells, without bile salt or acid, and in the media for the test of UDS in response to bile salt or acid. The positive increase in UDS in the presence of sodium deoxycholate plus hydroxyurea and chenodeoxycholic acid plus hydroxyurea was highly significantly above the UDS shown in the presence of hydroxyurea alone. The third kind of repair we tested, CHO mutant strain sensitivity to bile salts, assessed excision repair or cross-link removal with one strain and strand break repair with another strain (20). We found (Table 2) that mutant CHO strain UV4, defective in excision repair and cross-link removal, was more sensitive to killing by sodium chenodeoxycholate than the wild-type strain. This indicates that sodium chenodeoxycholate causes a type of DNA damage that can normally be repaired by excision repair or cross-link removal. Mutant CHO strain EM9, on the other hand, was more sensitive to killing by sodium deoxycholate than the wild-type strain. This indicates that sodium deoxycholate causes a DNA damage that can be repaired by a mechanism involving strand break repair. UV4 was not sensitive to killing by sodium deoxycholate, and EM9 was not sensitive to killing by sodium chenodeoxycholate. This implies that the two bile salts caused different kinds of DNA damage. Colon cancer is the second most frequent cause of cancer mortality in the United States, occurring at a rate of about 120,000 cases a year. The probability of developing colorectal cancer, from birth to 70 years, is about 4% (29). Individuals from low-risk populations, such as those of Japan and Poland, have 10-40 new cases of colon cancer per 100,000 per year among 65- to 75-year-old individuals. Upon migration to the United States, individuals from these low-risk populations experience the almost 10-fold higher rates (i.e., 150-300 new cases per 100,000 per year among 65- to 75-year-old individuals) of the United States (30,31). Thus it is likely that some life-style factor is important in the etiology of colon cancer. Epidemiological studies have shown that populations at high risk for colon cancer have increased concentrations of bile acids and related compounds in the feces (32-35). Dietary factors have a strong influence on levels of bile acids in the feces. In healthy humans a change to a diet of 25% more fat changes the concentration of fecal bile acids from 9.6 ± 0.5 to 14.3 ± 0.5 mg/g dry feces (36). In particular, deoxycholate is present as about 3 mg/g of dry feces (6), and the damage it causes is likely to result in DNA strand breaks, as discussed above. These studies, and other studies reviewed by Cheah (5), indicate a likely role for bile acids in the etiology of colon cancer. The results reported here indicate that bile salt/acid may have a direct and central role by causing DNA strand breaks in colon cells. There are two likely alternative outcomes of DNA damage caused by raised levels of bile salts or acids in the colon. One is that the DNA damage causes death of cells in the colon mucosa. This, in turn, would lead to a compensatory proliferation of colon cells, causing an elevated incidence of mutated cells, as a result of errors during the extra replications. An alternative outcome is that the DNA damage causes increased mutations directly by inducing errors of DNA replication or of DNA repair synthesis. Mutation, resulting either from extra
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cell proliferation or from induced errors of replication or repair, could lead to carcinogenesis. One can ask, if colon cells, as we postulate, are subject to increased carcinogenesis by raised levels of bile salts or acids, why are the liver and gallbladder, which produce and store bile salts, not subject to carcinogenesis. The answer to this may lie in the different levels of biotransformation capability of the colon compared with the liver and gallbladder. In a xenobiotic biotransformation study, McMahon and others (37) investigated the possible relationship of colon-specific carcinogenesis by azoxymethane and methylazoxymethanol in rats to enzyme changes in the colon and liver with age. They summarized the findings of others, indicating that the presence of high levels of alcohol dehydrogenase and other enzymes in the colon served to toxify methylazoxymethanol and give it carcinogenic potential in the colon. On the other hand, enzymes present at high levels in the liver activate, transform, or decompose azoxymethane and methylazoxymethanol in a different way. Similarly, bile salts or acids may be detoxified after entering liver or gallbladder cells by enzymes present there, but such enzymes may not be available in colon cells. Acknowledgments and Notes The authors thank Dr. Harris Bernstein for critical reading of the manuscript. This investigation was supported by Grant No. 82-2679 from the Arizona Disease Control Research Commission (Phoeniz, AZ) and a grant from the University of Arizona Cancer Center Institutional Research Grant Committee. Address reprint requests to Dr. C. Bernstein, Dept. of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724. Submitted 21 November 1990; accepted in final form 19 June 1991.
References 1. Hill, MJ: "Bile Acids and Colorectal Cancer in Humans." In Dietary Fiber, Basic and Clinical Aspects, GV Vahouny and D Kritchevsky (eds). New York: Plenum, 1986, pp 497-513. 2. Wynder, EL: "Amount and Type of Fat/Fiber in Nutritional Carcinogenesis." Prev Med 16, 451-459, 1987. 3. Bruce, WR: "Recent Hypotheses for the Origin of Colon Cancer." Cancer Res 47, 4237-4242, 1987. 4. Willet, W: "The Search for the Causes of Breast and Colon Cancer." Nature Lond 338, 389-394, 1989. 5. Cheah, PY: "Hypotheses for the Etiology of Colorectal Cancer-An Overview." Nutr Cancer 14, 5-13, 1990. 6. Nagengast, FM, Hectors, MPC, Buys, WAM, and van Tongeren, JHM: "Inhibition of Secondary Bile Acid Formation in the Large Intestine by Lactulose in Healthy Subjects of Two Different Age Groups." Eur J Clin Invest 18, 56-61, 1988. 7. Carey, MC: "Bile Acids and Bile Salts: Ionization and Solubility Properties." Hepatology 4, 66S-71S, 1984. 8. Carey, MC, and Cahalane, MJ: "Enterohepatic Circulation." In The Liver: Biology and Pathobiology, IM Arias, WB Jakoby, H Popper, D Schachter, and DA Shafritz (eds). New York: Raven, 1988, pp 573-616. 9. Walker, GC: "Mutagenesis and Inducible Responses to Deoxyribonucleic Acid Damage in Escherichia coli." Microbiol Rev 48, 60-93, 1984. 10. Quillardet, P, and Hofnung, M: "The SOS Chromotest, a Colorimetric Bacterial Assay for Genotoxins: Procedures." Mutat Res 147, 65-78, 1985. 11. Friedberg, EC: DNA Repair. New York: Freeman, 1985. 12. Watabe, J, and Bernstein, H: "The Mutagenicity of Bile Acids Using a Fluctuation Test." Mutat Res 158, 45-51, 1985. 13. Bernstein, H: "The Proper Evaluation of Bile Acid Mutagenicity by a Fluctuation Test." Mutat Res 191, 125-128, 1987. 14. Suzuki, K, and Bruce, WR: "Increase by Deoxycholic Acid of the Colonic Nuclear Damage Induced by Known Carcinogens in C57BL/6J Mice." JNCI 76, 1129-1132, 1986. 15. Kawasumi, H, Kaibara, N, and Shigemasa, K: "Cocarcinogenic Activity of Bile Acids in the Chemical Transformation of C3H/10 T1/2 Fibroblasts In Vitro." Oncology 45, 192-196, 1988. 16. Reddy, BS, Watanabe, K, Weisburger, JH, and Wynder, EL: "Promoting Effect of Bile Acids in Colon Carcinogenesis in Germ-Free and Conventional F344 Rats." Cancer Res 37, 3238-3242, 1977. 17. Reddy, BS, and Watanabe, K: "Effect of Cholesterol Metabolites and Promoting Effect of Lithocholic Acid in
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Colon Carcinogenesis in Germ-Free and Conventional F344 Rats." Cancer Res 39, 1521-1524, 1979. 18. Cheah, PY, and Bernstein, H: "Modification of DNA by Bile Acids: A Possible Factor in the Etiology of Colon Cancer." Cancer Lett 49, 207-210, 1990. 19. Lin, LL, and Little, JW: "Isolation and Characterization of Noncleavable (Ind - ) Mutants of the LexA Repressor of Escherichia coli K-12." J Bacteriol 170, 2163-2173, 1988. 20. Hoy, CA, Salazar, EP, and Thompson, LH: "Rapid Detection of DNA-Damaging Agents Using RepairDeficient CHO Cells." Mutat Res 130, 321-332, 1984. 21. Steinberg, CM, and Edgar, RS: "A Critical Test of a Current Theory of Genetic Recombination in Bacteriophage." Genetics 47, 187-208, 1962. 22. Williams, GM: "Detection of Chemical Carcinogens by Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures." Cancer Res 37, 1845-1851, 1977. 23. Harbach, PR, Aaron, CS, Wiser, SK, Grzegorczyk, CR, and Smith, AL: "The In Vitro Unscheduled DNA Synthesis (UDS) Assay in Rat Primary Hepatocytes. Validation of Improved Methods for Primary Culture Including Data on the Lack of Effect of Ionizing Radiation." Mutat Res 216, 101-110, 1989. 24. Croxton, FE: Elementary Statistics. New York: Dover, 1953, p 228. 25. Cole, ST, and Honore, N: "Transcription of the sulA-ompA Region of Escherichia coli During the SOS Response and the Role of an Antisense RNA Molecule." Mol Microbiol 3, 715-722, 1989. 26. Quillardet, P, de Bellcome, C, and Hofnung, M: "The SOS Chromotest, a Colorimetric Bacterial Assay for Genotoxins: Validation Study With 83 Compounds." Mutat Res 147, 79-95, 1985. 27. Schmellik-Sandage, CS, and Tessman, ES: "Signal Strains That Can Detect Certain DNA Replication and Membrane Mutants of Escherichia coli: Isolation of a New ssb Allele, ssb-3. "J Bacteriol 172, 4378-4385, 1990. 28. Suter, W, and Romagna, F: "DNA Repair Induced by Various Mutagens in Rat Hepatocyte Primary Cultures Measured in the Presence of Hydroxyurea, Guanazole or Aphidicolin." Mutat Res 231, 251-264, 1990. 29. Li, FP: "Patients at High Risk of Cancer of the Large Bowel." In Colorectal Cancer, G Steele, Jr, and RT Osteen (eds). New York: Dekker, 1986, pp 27-40. 30. Staszewski, J, and Haenszel, W: "Cancer Mortality Among the Polish-Born in the United States." JNCI 35, 291-297, 1965. 31. Haenszel, W, and Kurihara, M: "Studies of Japanese Migrants. I. Morbidity from Cancer and Other Diseases Among Japanese in the United States." JNCI 40, 43-68, 1968. 32. Reddy, BS, and Wynder, EL: "Metabolic Epidemiology of Colon Cancer: Fecal Bile Acids and Neutral Steroids in Colon Cancer Patients and Patients With Adenomatous Polyps." Cancer 39, 2533-2539, 1977. 33. Hill, MJ: "The Role of Colon Anaerobes in the Metabolism of Bile Acids and Steroids, and Its Relation to Colon Cancer." Cancer 36, 2387-2400, 1975. 34. Reddy, BS, Hedges, AR, Laakso, K, and Wynder, EL: "Metabolic Epidemiology of Large Bowel Cancer." Cancer 42, 2832-2838, 1978. 35. Domellof, L, Darby, L, Hanson, D, Mathews, L, Simi, B, et al.: "Fecal Sterols and Bacterial BetaGlucuronidase Activity: A Preliminary Metabolic Epidemiology Study of Healthy Volunteers from Umea, Sweden, and Metropolitan New York." Nutr Cancer 4, 120-127, 1982. 36. Reddy, BS: "Diet and Excretion of Bile Acids." Cancer Res 41, 3766-3768, 1981. 37. McMahon, TF, Peggins, JO, Centra, MM, and Weiner, M: "Age-Related Changes in Biotransformation of Azoxymethane and Methylazoxymethanol In Vitro." Xenobiotica 20, 501-513, 1990.
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Bile salt/acid induction of DNA damage in bacterial and mammalian cells: Implications for colon cancer a
Risa L. Kandell & Carol Bernstein
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Department of Microbiology and Immunology, College of Medicine , University of Arizona , Tucson, AZ, 85724 Published online: 04 Aug 2009.
To cite this article: Risa L. Kandell & Carol Bernstein (1991) Bile salt/acid induction of DNA damage in bacterial and mammalian cells: Implications for colon cancer, Nutrition and Cancer, 16:3-4, 227-238, DOI: 10.1080/01635589109514161 To link to this article: http://dx.doi.org/10.1080/01635589109514161
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Bile Salt/Acid Induction of DNA Damage in Bacterial and Mammalian Cells: Implications for Colon Cancer Downloaded by [New York University] at 21:27 22 May 2015
Risa L. Kandell and Carol Bernstein
Abstract Two bile salts, sodium chenodeoxycholate and sodium deoxycholate, induced a DNA repair response in the bacterium Escherichia coli. Similarly, a bile acid and a bile salt, chenodeoxycholic acid and sodium deoxycholate, induced DNA repair (indicated by unscheduled DNA synthesis) in human foreskin fibroblasts. Also, DNA repair-deficient Chinese hamster ovary (CHO) cells were found to be more sensitive than normal cells to killing by bile salts. In particular, mutant UV4 CHO cells, defective in DNA excision repair and DNA cross-link removal, were more sensitive to sodium chenodeoxycholate, and mutant EM9 CHO cells, defective in strand-break rejoining, were more sensitive to sodium deoxycholate than wild-type cells. These results indicate that bile salts/acid damage DNA of both bacterial and mammalian cells in vivo. Previous epidemiological studies have shown that colon cancer incidence correlates with fecal bile acid levels. The findings reported here support the hypothesis that bile salts/acids have an etiologic role in colon cancer by causing DNA damage. (Nutr Cancer 16, 227-238, 1991)
Introduction
Epidemiological evidence has long pointed to dietary factors or intracolonic factors as important in colon cancer (1-4). As reviewed by Cheah (5), bile acids, dietary (oxidized) fat, fecal mutagens, and ketosteroids have been the four most widely studied potential etiologic agents in colorectal cancer. Cheah (5) concluded that bile acids are the most strongly implicated on the basis of evidence in 87 studies relating to six different areas: 1) population comparisons, 2) case control comparisons, 3) animal experiments, 4) bacterial experiments, 5) mammalian cell line experiments, and 6) DNA damage experiments. The primary bile acid chenodeoxycholic acid constitutes about 35% of the bile acids in the gallbladder (6). It is usually modified by bacteria of the intestine, so that it constitutes only 0.5-1.0% of the bile acids in the feces. An important secondary bile acid is deoxycholic acid, The authors are affiliated with the Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724.
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which constitutes about 52% of the bile acids in the feces (6). Because it is recirculated through the enterohepatic shunt, it is also present as about 20% of the bile acids in the gallbladder. These two bile acids were chosen for study because one is eliminated from the colon by bacterial modification and the other is introduced in the colon because of bacterial action. The two bile acids might have pointed to a role of bacterial toxification or detoxification of the bile acids in genotoxic activity. However, no great difference in their level of DNA-damaging effects was found in our study. Although a distinction between bile acids and bile salts should be made, organic chemists have traditionally employed the term bile acid as a generic name for this cholanic class of biological compounds (7). However, bile acids have an aqueous solubility of 10~8 to 10~3 M, and bile salts have an aqueous solubility of 1-2 M (7). In the intestinal content, bile salts are the major form present. However, as noted by Carey and Cahalane (8), "whenever the gut luminal pH lies below the precipitation values of bile salts, the latter may precipitate from solution as the sparingly soluble undissociated bile acids. This occurs under normal physiological conditions in both stomach and colon." Thus, both bile acids and bile salts could be important in the etiology of colon cancer. In describing our work, we specifically indicate whether a bile acid or salt was used. A number of tests are widely employed to assess DNA-damaging abilities of compounds. In Escherichia coli, DNA-damaging agents induce a set of gene functions known collectively as the SOS response (9). The SOS chromotest was devised with E. coli to measure DNA repair (10) by use of a strain in which there is an operon fusion placing lacZ, the structural gene for /3-galactosidase, under the control of the SOS gene sulA or sfiA. In this strain, DNA damage results in turn-on of sulA and also lacZ. The /3-galactosidase produced can cleave 5-bromo-4-chloro-3-indoIyl-/3-D-galactoside (Xgal) to form a blue product. We used a modification of the standard SOS chromotest to detect the DNA repair response to DNA damage caused by bile salts in E. coli. One method for investigating DNA repair in mammalian cells is to measure unscheduled DNA synthesis (UDS). This method assays the limited DNA synthesis that occurs during excision repair (for review, see Ref. 11). DNA damage in mammalian cells can also be detected by differential cytotoxicity of DNA repair-deficient lines of Chinese hamster ovary (CHO) cells, compared with wild-type CHO cells. We used both UDS and differential cytotoxicity to detect DNA damage caused by bile acids or salts in mammalian cells. Although bile acids have been shown to be mutagenic to bacterial cells in a modified Ames Salmonella test (12,13), indicating that bile acids cause DNA damage in bacteria, the results were not dramatic and should be confirmed. Other results showing that bile acids can act as mammalian tumor promoters and cocarcinogens (14-17) or can damage bacteriophage DNA in vitro (18) did not address the question of whether bile acids can cause DNA damage in mammalian cells. Our results indicate that bile salts/acids can directly cause DNA damage in vivo both in bacteria and mammalian cells. This is consistent with the hypothesis that bile salts/acid are important in the etiology of colon cancer, perhaps as carcinogenic agents. Materials and Methods
Bacterial and Mammalian Strains E. coli strain JL1705 (kindly provided by Dr. J. W. Little, University of Arizona) has the structural gene for lacZ, /3-galactosidase, under the control of sulA (sfiA) and was constructed from E. coli JL1047 and plasmid pJWL184 (19). Human foreskin fibroblasts were kindly provided by Dr. M. R. Haussler (University of Arizona). The CHO wild-type strain AA8 and mutant strains EM9 and UV4 were kindly provided by Dr. C. A. Hoy (20).
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Growth and Maintenance of Cells E. coli JL1705 cells were grown as overnight cultures in Luria broth, and working stocks of the strain were maintained on trypticase plates, where the trypticase agar consisted of 10 g trypticase peptone (Baxter Healthcare, Tempe, AZ), 5 g NaCl, and 10 g Bacto-agar (Difco, Detroit, MI) in 1 liter of H2O, adjusted to pH 7.2. The test for SOS induction was carried out on trypticase plates to which Xgal (80 /tg/ml) and ampicillin (50 jtg/ml) were added; bacteria were suspended in a soft agar overlay (21) with ampicillin (100 fig/ml) added. Melted agar was cooled to 42 °C before the addition of Xgal or ampicillin. Xgal was diluted in A7,Ar-dimethylformamide at a concentration of 20 mg/ml and stored at —20°C. Human foreskin fibroblasts were grown as a monolayer culture in filter-sterilized McCoy's 5A medium (modified, Sigma Chemical, St. Louis, MO) at pH 7.5 and supplemented with 0.5% solution of penicillin (100,000 U/ml) plus streptomycin (10,000 /tg/ml, Sigma Chemical) and 10% fetal bovine serum (GIBCO, Grand Island, NY) before use. For maintenance of growing cells, 2.8 x 105 cells were seeded into a 250-ml tissue culture flask containing 10 ml growth medium and placed in a 37°C 5% CO2 incubator. Cells for use in UDS experiments were grown on coverslips placed in the bottom of a petri dish. Growth was allowed until a confluent monolayer formed. CHO cells were grown as monolayer cultures in 250-ml tissue culture flasks until needed for an experiment. Alpha medium (GIBCO) supplemented with 10% fetal bovine serum and 0.5% of a solution of penicillin (10,000 U/ml) plus streptomycin (10,000 /tg/ml) was used as growth medium. Bacterial SOS DNA Repair Induction Test This test is a modified version of the standard SOS chromotest (10). An aliquot of 0-150 /tl of the agent being tested was added to melted top (overlay) agar kept at 42°C and vigorously mixed. E. coli JL1705 cells, grown as an overnight culture in Luria broth, were added to the top agar in sufficient number to yield 50-200 viable colonies, and the mixture was plated on BBL trypticase plates. The plates were then incubated for 19 hours at 37°C, colonies were scored as either blue or white, and the total surviving fraction, S/So, was calculated. Each experiment was repeated three times. CHO Cytotoxicity Assay Differential cytotoxicity of CHO cells defective in DNA repair was tested using the method described by Hoy and co-workers (20). UDS in Human Foreskin Fibroblast Cells Coverslips carrying a confluent monolayer of freshly grown human foreskin fibroblast cells were rinsed with phosphate-buffered saline (PBS, 8.0 g NaCl, 0.2 g KC1, 0.12 g KH2PO4, 0.91 g Na2HPO4 in 1 liter, adjusted to pH 7.5 with NaOH). Then 5.0 ml PBS containing 10 raM hydroxyurea were used to cover cells for 15 minutes at room temperature to inhibit replicative DNA synthesis (22). Medium covering the cells was then replaced by treatment medium consisting of 5.0 ml PBS, 10 mM hydroxyurea, 1% dimethyl sulfoxide (DMSO), and the agent to be tested for 20 minutes at room temperature, or slides were exposed to 30 J/m 2 of ultraviolet (UV) light and then returned to 5.0 ml PBS containing 10 mM hydroxyurea for 20 minutes. All further work was carried out under yellow light to prevent possible photoreactivation of DNA damage. The cells were then rinsed twice with PBS and placed in petri dishes containing growth medium plus 1.0 ftCi/ml [3H]thymidine (sp act 60-68 Ci/mmol) and incubated in light-tight containers at 37°C in a 5% CO2 incubator for 21 hours. The petri dishes were rinsed three times with PBS, and a 1 % solution of sodium
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citrate was added and left on the coverslips for 10 minutes to swell the nuclei of the cells. The cells were then fixed with three 10-minute treatments of 3:1 ethanol-acetic acid. The fixer was poured off, and the coverslips were allowed to dry. Coverslips were mounted on slides and covered with Kodak NTB-3 emulsion and exposed at 4°C for seven days. After the slides were developed, cells were strained with hematoxylin. Grains over nuclei were counted, and grains in an equal area, as judged by eye, in the upper right-hand area adjacent to the nucleus, were also counted and subtracted from the grains over the nucleus to give a net grain count for each nucleus. If the upper right-hand area was obscured by debris or blemish, then the equal area, as judged by eye, in the lower left-hand area adjacent to the nucleus was used for background subtraction. Infrequent nuclei with net grain counts over 50 were considered to be undergoing replicative synthesis and were not included in the study. All slides were coded before evaluation, so that they were scored without knowledge of their treatments.
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Tests of Genotype o/E. coli and CHO Mutant Strains E. coli JL1705 cells were plated on Bacto MacConkey agar (Difco Laboratories) before they were used in each test for DNA damage. Bacteria capable of fermenting lactose on this agar produce a localized pH drop when forming a colony, which, followed by absorption of the neutral red in the agar, imparts a deep red color to the colony. E. coli JL1705 cells, in our tests, formed white to light pink colonies, indicating that the operon containing lacZ was either repressed or expressed at low levels, as expected in the absence of externally caused DNA damage. The standard spot test part of the SOS chromotest (10), using mitomycin C as a known DNA-damaging agent, was also performed as a control before each experiment. The expected blue ring consistently formed with this strain of E. coli, indicating that the operon containing lacZ is expressed in response to DNA damage. CHO cells were tested for sensitivity to DNA damage by irradiation with UV light, a known DNA-damaging agent. Wild-type strain AA8 showed a "least effective amount" of UV irradiation (with which diminished cell growth was detected) of 50 seconds, EM9 had a least effective amount of 40 seconds, and UV4 had at least effective amount of 10 seconds, when each strain was exposed to a UV lamp delivering 1 J/m2/second for 10, 20, 30, 40, and 50 seconds. Chemicals Sodium deoxycholate (ICN Biomedicals, Costa Mesa, CA) and sodium chenodeoxycholate (Sigma Chemical) were dissolved in water. Chenodeoxycholic acid (Sigma Chemical) was dissolved in DMSO. The [3H]thymidine (ICN Biomedicals) had a specific activity of 60-68 /tCi/mM. Results Bile Salt Induction of DNA Damage in Bacteria Measured by a Modified SOS Chromotest We were not able to use the standard SOS chromotest (10), which is performed using E. coli PQ37. This strain carries a mutation in gene uvrA, which causes a defect in excision repair and thus higher sensitivity to some DNA-damaging agents. It also has a mutation in rfa, which makes the strain lipopolysaccharide deficient. This allows better diffusion of certain molecules into the cells but also makes them very sensitive to bile salts. In fact, the recommended test for the proper phenotype of E. coli PQ37 is assay for sensitivity to sodium deoxycholate or inability to grow on agar plates that contain deoxycholate (10). In the standard assay, growth is usually allowed both in liquid media and on solid agar for a spot
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test. The blue color produced in the liquid media or around the spot test on solid agar is then measured. However, even when an E. coli strain with wild-type membrane (rfa+) was used, sodium deoxycholate proved to be too toxic for use during liquid growth. In addition, the spot test was difficult to quantitate because E. coli grows more slowly in the presence of sodium deoxycholate on plates. Thus, we used both a different strain of E. coli and a different quantitative plate assay. The strain we used, E. coli JL1705, was similar to strain PQ37 in that lacZ was under the control of sulA, but JL1705 did not carry a mutation in rfa (or a mutation in uvrA). Then we assayed the frequency with which colonies on a plate containing the test compound turned blue at 19 hours of incubation as well as survival of colony-forming ability on the plates. As shown in Figure 1, the established DNA-damaging agent mitomycin C caused an increase in percentage of blue colonies produced on a plate, from 12% to 80%, as the concentration of mitomycin C increased from 0 to 700 ng/ml. At the same time, survival of colony-forming ability of cells on the plate decreased from 100% to 44%. The increase in percentage of blue colony formation and the decrease in survival followed regular trends. The larger error bars observed, for two of the seven points, indicating percent occurrence of blue colonies, reflect the occasional counting of plates with about 50 colonies per plate. At this lower number of total colonies, a 30% or 40% blue colony frequency refers to 15 or 20 blue colonies on the plate. Some fluctuation in such small numbers is expected. During treatment of E. coli cells with a DNA-damaging agent, there are a number of consequences. Some cells turn on the SOS response. A small fraction of those with the SOS response turned on and a larger fraction of those that had not turned on this response are killed by the increasing concentration of the DNA-damaging agent. As long as the SOS response offers protection from the DNA-damaging agent, an increasing fraction of surviving colonies will be blue, as shown in Figure 1. If the SOS response is turned on in cells with a higher frequency than "turned-on" cells are killed, then there will also be an absolute increase (rather than just a relative increase among survivors) in the percentage of cells that form blue colonies in a population. Figure 2 shows that, after mitomycin C treatment, there is an absolute increase in percentage of cells that went on to form blue colonies. Because the data shown in Figure 2 can be obtained from the data shown in Figure 1 by a simple calculation, the error bars were left out of Figure 2 to promote clarity. The modified SOS chromotest was then used with the two bile salts, sodium chenodeoxycholate and sodium deoxycholate. With each bile salt the results obtained were very similar to those obtained with mitomycin C. As shown in Figure 3, survival of 100
Figure 1. Percentage of colonies that were blue and percentage of cells surviving and able to form colonies (blue or white) on plates. Indicated concentration of mitomycin C used to treat cells was calculated assuming that mitomycin C diffused throughout 3 ml of top agar and 30 ml of bottom agar on the plate. Each point represents mean from 3 experiments; bars represent SE.
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Figure 3. Percentage of colonies that were blue and percentage of cells surviving and able to form colonies (blue or white) on plates with indicated concentration of bile salts. Each point represents mean from 3 experiments; bars represent SE. Colonies were grown in the presence of sodium chenodeoxycholate (A) or sodium deoxycholate (B).
colony-forming ability went from 100% to about 20%, while the percentage of blue colonies among the survivors rose from about 8% to about 80%. In addition, as shown in Figure 4, the absolute percentage of cells going on to form blue colonies rose after treatment with chenodeoxycholate from 6% to a maximum of 20% of the population and for deoxycholate from 8% to a maximum of 18% of the population. In these two cases of bile salt treatment, the absolute percentage of surviving blue colonies at the highest concentration of bile salt was less than that with an intermediate concentration of bile salt. At the highest concentration there was less than 20% survival of cells able to form colonies. At this high level of lethality, cells with SOS repair "turned on" were being killed at a higher frequency than new cells were being turned on to SOS repair, because there were actually very few cells left that could be newly turned on. Overall, the data displayed in Figures 3 and 4 indicate that the bile salts tested behave like the known DNA-damaging agent mitomycin C.
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0 • I]
0
132
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Chenodeoxycholate
353
fig/ml
441
0
132
176
Deoxycholate
265
Killed White Blue
397
|jg/ml
Figure 4. Fate of cells placed on plates with indicated concentrations of bile salt in agar. Cells can grow to form blue colonies, grow to form white colonies, or fail to grow (be killed). Colonies were grown in the presence of sodium chenodeoxycholate (A) or sodium deoxycholate (B).
Bile Compound Induction ofDNA Damage in Human Foreskin Fibroblasts Measured by UDS When UDS was measured with newborn human foreskin fibroblast cells, the DNAdamaging agent UV light was used as a positive control. UV light is known to cause a strong UDS response in mammalian cells (23). As shown in Table 1, treatment of foreskin fibroblast cells with increasing concentrations of sodium deoxycholate or chenodeoxycholic acid caused a highly significant turn-on of DNA repair, reflected by UDS. The estimated standard error of the mean for grains per cell was calculated by using the standard formula (24) A
a where x is the number of grains in a nucleus and N is the number of nuclei counted. The increase in UDS was not as great with the bile salt or acid as with UV light, but different DNA-damaging agents are known to turn on DNA repair, as reflected by UDS, to different extents (23). Differential Cytotoxicity of Bile Salts to DNA Repair-Defective CHO Cells Three strains of CHO cells were used in these experiments: the wild-type strain AA8 and mutant strains UV4 and EM9. UV4 is defective in excision repair and cross-link removal, and EM9 is defective in strand rejoining. Table 2 summarizes the results obtained. UV4 was significantly more sensitive to killing by sodium chenodeoxycholate than the wild-type strain AA8 but not more sensitive to sodium deoxycholate. EM9 was significantly more sensitive to killing by sodium deoxycholate than the wild-type strain but not more sensitive to killing by sodium chenodeoxycholate. The results were reproduced in three separate experiments, and scoring of the results was the same whether performed by the investigators or by other individuals who had not been told which compounds or which cells were in each well.
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Table 1. Unscheduled DNA Synthesis in Human Foreskin Fibroblasts Expt No. 1
Treatment Deoxycholate
UV
2
Deoxycholate
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UV
3
Chenodeoxycholic acid
UV
4
Chenodeoxycholic acid
UV
Dose" 0.0
12.5 25.0 50.0 100.0 250.0 30.0 0.0
12.5 25.0 50.0 75.0 100.0 30.0 0.0
25.5 50.0 100.0 250.0 30.0 0.0
50.0 75.0 100.0 150.0 200.0 30.0
No. of Slides
No. of Cells
Grains/Cell6
Fold Increase0
3 3 5 4 4 4 3 6 6 6 5 5 5 3 3 3 3 3 3 3 4 3 3 3 3 3 3
450 450 750 600 600 600 450 900 900 900 750 750 750 450 450 450 450 450 450 450 600 450 450 450 450 450 450
3.93 ± 0.43 5.17 ± 0.52 8.36 ± 0.47 7.36 ± 0.51 7.81 ± 0.49 6.92 ± 0.48 12.54 ± 0.40 4.38 ± 0.32 6.63 ± 0.40 8.74 ± 0.40 6.94 ± 0.46 5.51 ± 0.36 7.31 ± 0.42 21.87 ± 0.42 4.25 ± 0.43 3.63 ± 0.30 4.72 ± 0.42 7.58 ± 0.57 6.00 ± 0.45 16.57 ± 0.49 3.72 ± 0.34 3.36 ± 0.32 4.89 ± 0.40 5.14 ± 0.44 6.19 ± 0.45 5.45 ± 0.38 16.15 ± 0.27
1.00 1.31 2.13* 1.87* 1.99* 1.75* 3.22* 1.00 1.51* 1.53* 1.58* 1.26* 1.57* 4.99* 1.00 0.85 1.10 1.78* 1.30* 3.22* 1.00 0.90 1.31 1.38* 1.66* 1.47* 4.34*
a: Expressed as jig/ml or as seconds of ultraviolet irradiation (UV). b: Expressed as means ± estimated SE c: Statistical significance is as follows: *,p £ 0.005; t, P £ 0.01.
Table 2. Bile Salt Cytotoxicity Toward CHO Wild-Type and Mutant Strains Strain AA8 (wild type) UV4 (excision repair mutant) EM9 (defective in strand rejoining)
Compound Chenodeoxycholate Deoxycholate Chenodeoxycholate Deoxycholate Chenodeoxycholate Deoxycholate
Least Effective Concentration" 1,000 1,000 500
1,000 1,000 500
a: Concentration (/ig/ml) that results in clear decrease in cell viability by test of Hoy et al. (20). When established procedure is followed, a 2-fold difference is considered significant (20).
Discussion The results obtained with the modified bacterial SOS chromotest showed that increasing concentrations of either of two bile salts, sodium deoxycholate or sodium chenodeoxycholate, increased the frequency of blue colony formation in a strain carrying a sulA:lacZ fusion as a result of a turning-on of expression of the sulA operon. Turn-on of this operon occurs as one of a spectrum of responses to DNA damage in E. colt. The sulA
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promoter has been found to be very powerful and to allow production of a large number of transcripts when repression is removed (25). The turn-on of this operon in our experiments, as measured by the frequency with which colonies become blue, correlated with the degree to which the colony-forming ability of cells was inactivated by the bile salts tested. These results are expected if the progressive inactivation of cells in a population is caused by intracellular DNA damages, some of which trigger (and are repaired by) a DNA repair pathway induced as part of the SOS response. The results obtained with bile salt treatment (Figures 3 and 4) were similar to the results obtained with the established DNA-damaging agent mitomycin C (Figures 1 and 2). Thus our findings indicate that bile salts are likely to be DNA-damaging agents in vivo in E. coli. The standard SOS chromotest has been validated by Quillardet and co-workers (26), who used this assay to test 83 compounds and found that 90% of the compounds mutagenic in the Ames test were also SOS inducers. Our modified chromotest is, in principle, the same as the standard test and gave a positive result with an established DNA-damaging agent. It should be a valid indicator of DNA damage as well. One factor that should be considered when interpreting these results is an effect found by Schmellik-Sandage and Tessman (27). Their work indicated that mutations that alter cell membrane permeability can cause blue colony formation in a sulA:lacZ fusion strain by allowing extra Xgal to enter and be made blue, even without DNA damage. It may be possible that increasing amounts of bile salts may cause lethality by damaging the membrane, thereby increasing permeability to Xgal. An effect on cell membranes might be expected to affect all the colonies on a plate. For some colonies to be blue and some not blue, one must postulate a complicated explanation of a lineage of blue-producing cells with an epigenetic alteration leading to vulnerable membranes. There is no basis for postulating such an epigenetic effect. Such a cell lineage would presumably give rise to small sick colonies, if the cause of bile salt lethality was to cause membrane deterioration and Xgal permeability. The blue colonies, however, had the same approximate range of sizes and vigor as the nonblue colonies, so a membrane effect does not seem likely. On the other hand, as reviewed by Walker (9), the SOS response, which controls turn-on of the sulA:lacZ fusion, is buffered against being substantially induced by small amounts of inducing signal (where the signal is single-stranded DNA regions caused by damage). Thus there is a barrier, which must be overcome, before the system is turned on. Furthermore, Cole and Honore (25) showed that the sulA operon had an unusual arrangement of two "shut-off mechanisms, so it can be turned off quickly. This arrangement predicts an on-or-off state for sulA:lacZ and, thus, an on-or-off state for blue color production. Blue color production would only turn on after a certain amount of damage accumulated in a cell. Furthermore, this turned-on state, due to cleavage of the LexA repressor protein, could be passed on to progeny cells as long as DNA damage signals continue to be present. The blue colonies, with SOS repair turned on, should have normal size and vigor, because repair of DNA damage in these cells is elevated. Such a normal spectrum of blue colony size and vigor was observed. Thus a DNA repair response is a more likely explanation for our results than a deterioration of membrane permeability. Our test of UDS, as a response to treatment of human newborn foreskin fibroblasts with a bile acid or bile salt, showed a highly significant increase in UDS upon treatment with bile salt/acid (Table 1). Because UDS reflects excision repair in mammalian cells (11), these results indicate that bile salt/acid causes DNA damage in foreskin cells and that excision repair is induced to deal with the damage. Although UDS was induced, it appeared to reach a plateau (Table 1). This may indicate that higher levels of DNA damage inhibit expression of DNA repair enzymes. In fact, at higher doses of the bile compounds than those shown in Table 1, toxicity was shown by cell detachment. Harbach and co-workers (23) reported a similar nonincrease of UDS with increased exposure to a known genotoxic compound, benzo[cr]pyrene. Their result was obtained in a validation study of UDS in response to known
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genotoxic agents applied to rat hepatocytes. The highly significant levels of UDS induced by the bile compounds in our study indicate that the compounds cause DNA damage, whereas the plateau of UDS in response to higher levels of compounds and the detachment of cells at still higher levels indicate that bile compounds are toxic to cells at high levels. Recent work by Suter and Romagna (28) indicates that 10 mM hydroxyurea, used in our experiments to suppress replicative DNA synthesis, can cause a small increase in UDS in rat hepatocytes. In fact, the somewhat high background levels of counts, in the absence of bile salt or acid, are similar to those reported by them (28). However, known genotoxic agents were also tested by them in the presence of 10 mM hydroxyurea in similar liquid scintillation tests of induced DNA repair, and the small amount of repair induced by hydroxyurea did not obscure the positive DNA repair responses induced by these agents. In our case, hydroxyurea was present both in the media for the negative control cells, without bile salt or acid, and in the media for the test of UDS in response to bile salt or acid. The positive increase in UDS in the presence of sodium deoxycholate plus hydroxyurea and chenodeoxycholic acid plus hydroxyurea was highly significantly above the UDS shown in the presence of hydroxyurea alone. The third kind of repair we tested, CHO mutant strain sensitivity to bile salts, assessed excision repair or cross-link removal with one strain and strand break repair with another strain (20). We found (Table 2) that mutant CHO strain UV4, defective in excision repair and cross-link removal, was more sensitive to killing by sodium chenodeoxycholate than the wild-type strain. This indicates that sodium chenodeoxycholate causes a type of DNA damage that can normally be repaired by excision repair or cross-link removal. Mutant CHO strain EM9, on the other hand, was more sensitive to killing by sodium deoxycholate than the wild-type strain. This indicates that sodium deoxycholate causes a DNA damage that can be repaired by a mechanism involving strand break repair. UV4 was not sensitive to killing by sodium deoxycholate, and EM9 was not sensitive to killing by sodium chenodeoxycholate. This implies that the two bile salts caused different kinds of DNA damage. Colon cancer is the second most frequent cause of cancer mortality in the United States, occurring at a rate of about 120,000 cases a year. The probability of developing colorectal cancer, from birth to 70 years, is about 4% (29). Individuals from low-risk populations, such as those of Japan and Poland, have 10-40 new cases of colon cancer per 100,000 per year among 65- to 75-year-old individuals. Upon migration to the United States, individuals from these low-risk populations experience the almost 10-fold higher rates (i.e., 150-300 new cases per 100,000 per year among 65- to 75-year-old individuals) of the United States (30,31). Thus it is likely that some life-style factor is important in the etiology of colon cancer. Epidemiological studies have shown that populations at high risk for colon cancer have increased concentrations of bile acids and related compounds in the feces (32-35). Dietary factors have a strong influence on levels of bile acids in the feces. In healthy humans a change to a diet of 25% more fat changes the concentration of fecal bile acids from 9.6 ± 0.5 to 14.3 ± 0.5 mg/g dry feces (36). In particular, deoxycholate is present as about 3 mg/g of dry feces (6), and the damage it causes is likely to result in DNA strand breaks, as discussed above. These studies, and other studies reviewed by Cheah (5), indicate a likely role for bile acids in the etiology of colon cancer. The results reported here indicate that bile salt/acid may have a direct and central role by causing DNA strand breaks in colon cells. There are two likely alternative outcomes of DNA damage caused by raised levels of bile salts or acids in the colon. One is that the DNA damage causes death of cells in the colon mucosa. This, in turn, would lead to a compensatory proliferation of colon cells, causing an elevated incidence of mutated cells, as a result of errors during the extra replications. An alternative outcome is that the DNA damage causes increased mutations directly by inducing errors of DNA replication or of DNA repair synthesis. Mutation, resulting either from extra
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cell proliferation or from induced errors of replication or repair, could lead to carcinogenesis. One can ask, if colon cells, as we postulate, are subject to increased carcinogenesis by raised levels of bile salts or acids, why are the liver and gallbladder, which produce and store bile salts, not subject to carcinogenesis. The answer to this may lie in the different levels of biotransformation capability of the colon compared with the liver and gallbladder. In a xenobiotic biotransformation study, McMahon and others (37) investigated the possible relationship of colon-specific carcinogenesis by azoxymethane and methylazoxymethanol in rats to enzyme changes in the colon and liver with age. They summarized the findings of others, indicating that the presence of high levels of alcohol dehydrogenase and other enzymes in the colon served to toxify methylazoxymethanol and give it carcinogenic potential in the colon. On the other hand, enzymes present at high levels in the liver activate, transform, or decompose azoxymethane and methylazoxymethanol in a different way. Similarly, bile salts or acids may be detoxified after entering liver or gallbladder cells by enzymes present there, but such enzymes may not be available in colon cells. Acknowledgments and Notes The authors thank Dr. Harris Bernstein for critical reading of the manuscript. This investigation was supported by Grant No. 82-2679 from the Arizona Disease Control Research Commission (Phoeniz, AZ) and a grant from the University of Arizona Cancer Center Institutional Research Grant Committee. Address reprint requests to Dr. C. Bernstein, Dept. of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724. Submitted 21 November 1990; accepted in final form 19 June 1991.
References 1. Hill, MJ: "Bile Acids and Colorectal Cancer in Humans." In Dietary Fiber, Basic and Clinical Aspects, GV Vahouny and D Kritchevsky (eds). New York: Plenum, 1986, pp 497-513. 2. Wynder, EL: "Amount and Type of Fat/Fiber in Nutritional Carcinogenesis." Prev Med 16, 451-459, 1987. 3. Bruce, WR: "Recent Hypotheses for the Origin of Colon Cancer." Cancer Res 47, 4237-4242, 1987. 4. Willet, W: "The Search for the Causes of Breast and Colon Cancer." Nature Lond 338, 389-394, 1989. 5. Cheah, PY: "Hypotheses for the Etiology of Colorectal Cancer-An Overview." Nutr Cancer 14, 5-13, 1990. 6. Nagengast, FM, Hectors, MPC, Buys, WAM, and van Tongeren, JHM: "Inhibition of Secondary Bile Acid Formation in the Large Intestine by Lactulose in Healthy Subjects of Two Different Age Groups." Eur J Clin Invest 18, 56-61, 1988. 7. Carey, MC: "Bile Acids and Bile Salts: Ionization and Solubility Properties." Hepatology 4, 66S-71S, 1984. 8. Carey, MC, and Cahalane, MJ: "Enterohepatic Circulation." In The Liver: Biology and Pathobiology, IM Arias, WB Jakoby, H Popper, D Schachter, and DA Shafritz (eds). New York: Raven, 1988, pp 573-616. 9. Walker, GC: "Mutagenesis and Inducible Responses to Deoxyribonucleic Acid Damage in Escherichia coli." Microbiol Rev 48, 60-93, 1984. 10. Quillardet, P, and Hofnung, M: "The SOS Chromotest, a Colorimetric Bacterial Assay for Genotoxins: Procedures." Mutat Res 147, 65-78, 1985. 11. Friedberg, EC: DNA Repair. New York: Freeman, 1985. 12. Watabe, J, and Bernstein, H: "The Mutagenicity of Bile Acids Using a Fluctuation Test." Mutat Res 158, 45-51, 1985. 13. Bernstein, H: "The Proper Evaluation of Bile Acid Mutagenicity by a Fluctuation Test." Mutat Res 191, 125-128, 1987. 14. Suzuki, K, and Bruce, WR: "Increase by Deoxycholic Acid of the Colonic Nuclear Damage Induced by Known Carcinogens in C57BL/6J Mice." JNCI 76, 1129-1132, 1986. 15. Kawasumi, H, Kaibara, N, and Shigemasa, K: "Cocarcinogenic Activity of Bile Acids in the Chemical Transformation of C3H/10 T1/2 Fibroblasts In Vitro." Oncology 45, 192-196, 1988. 16. Reddy, BS, Watanabe, K, Weisburger, JH, and Wynder, EL: "Promoting Effect of Bile Acids in Colon Carcinogenesis in Germ-Free and Conventional F344 Rats." Cancer Res 37, 3238-3242, 1977. 17. Reddy, BS, and Watanabe, K: "Effect of Cholesterol Metabolites and Promoting Effect of Lithocholic Acid in
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Colon Carcinogenesis in Germ-Free and Conventional F344 Rats." Cancer Res 39, 1521-1524, 1979. 18. Cheah, PY, and Bernstein, H: "Modification of DNA by Bile Acids: A Possible Factor in the Etiology of Colon Cancer." Cancer Lett 49, 207-210, 1990. 19. Lin, LL, and Little, JW: "Isolation and Characterization of Noncleavable (Ind - ) Mutants of the LexA Repressor of Escherichia coli K-12." J Bacteriol 170, 2163-2173, 1988. 20. Hoy, CA, Salazar, EP, and Thompson, LH: "Rapid Detection of DNA-Damaging Agents Using RepairDeficient CHO Cells." Mutat Res 130, 321-332, 1984. 21. Steinberg, CM, and Edgar, RS: "A Critical Test of a Current Theory of Genetic Recombination in Bacteriophage." Genetics 47, 187-208, 1962. 22. Williams, GM: "Detection of Chemical Carcinogens by Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures." Cancer Res 37, 1845-1851, 1977. 23. Harbach, PR, Aaron, CS, Wiser, SK, Grzegorczyk, CR, and Smith, AL: "The In Vitro Unscheduled DNA Synthesis (UDS) Assay in Rat Primary Hepatocytes. Validation of Improved Methods for Primary Culture Including Data on the Lack of Effect of Ionizing Radiation." Mutat Res 216, 101-110, 1989. 24. Croxton, FE: Elementary Statistics. New York: Dover, 1953, p 228. 25. Cole, ST, and Honore, N: "Transcription of the sulA-ompA Region of Escherichia coli During the SOS Response and the Role of an Antisense RNA Molecule." Mol Microbiol 3, 715-722, 1989. 26. Quillardet, P, de Bellcome, C, and Hofnung, M: "The SOS Chromotest, a Colorimetric Bacterial Assay for Genotoxins: Validation Study With 83 Compounds." Mutat Res 147, 79-95, 1985. 27. Schmellik-Sandage, CS, and Tessman, ES: "Signal Strains That Can Detect Certain DNA Replication and Membrane Mutants of Escherichia coli: Isolation of a New ssb Allele, ssb-3. "J Bacteriol 172, 4378-4385, 1990. 28. Suter, W, and Romagna, F: "DNA Repair Induced by Various Mutagens in Rat Hepatocyte Primary Cultures Measured in the Presence of Hydroxyurea, Guanazole or Aphidicolin." Mutat Res 231, 251-264, 1990. 29. Li, FP: "Patients at High Risk of Cancer of the Large Bowel." In Colorectal Cancer, G Steele, Jr, and RT Osteen (eds). New York: Dekker, 1986, pp 27-40. 30. Staszewski, J, and Haenszel, W: "Cancer Mortality Among the Polish-Born in the United States." JNCI 35, 291-297, 1965. 31. Haenszel, W, and Kurihara, M: "Studies of Japanese Migrants. I. Morbidity from Cancer and Other Diseases Among Japanese in the United States." JNCI 40, 43-68, 1968. 32. Reddy, BS, and Wynder, EL: "Metabolic Epidemiology of Colon Cancer: Fecal Bile Acids and Neutral Steroids in Colon Cancer Patients and Patients With Adenomatous Polyps." Cancer 39, 2533-2539, 1977. 33. Hill, MJ: "The Role of Colon Anaerobes in the Metabolism of Bile Acids and Steroids, and Its Relation to Colon Cancer." Cancer 36, 2387-2400, 1975. 34. Reddy, BS, Hedges, AR, Laakso, K, and Wynder, EL: "Metabolic Epidemiology of Large Bowel Cancer." Cancer 42, 2832-2838, 1978. 35. Domellof, L, Darby, L, Hanson, D, Mathews, L, Simi, B, et al.: "Fecal Sterols and Bacterial BetaGlucuronidase Activity: A Preliminary Metabolic Epidemiology Study of Healthy Volunteers from Umea, Sweden, and Metropolitan New York." Nutr Cancer 4, 120-127, 1982. 36. Reddy, BS: "Diet and Excretion of Bile Acids." Cancer Res 41, 3766-3768, 1981. 37. McMahon, TF, Peggins, JO, Centra, MM, and Weiner, M: "Age-Related Changes in Biotransformation of Azoxymethane and Methylazoxymethanol In Vitro." Xenobiotica 20, 501-513, 1990.
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