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Bile salt/acid induction of DNA damage in bacterial cells: Effect of taurine conjugation a
Zhi‐Ying Zheng & 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: Zhi‐Ying Zheng & Carol Bernstein (1992) Bile salt/acid induction of DNA damage in bacterial cells: Effect of taurine conjugation, Nutrition and Cancer, 18:2, 157-164, DOI: 10.1080/01635589209514215 To link to this article: http://dx.doi.org/10.1080/01635589209514215
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Bile Salt/Acid Induction of DNA Damage in Bacterial Cells: Effect of Taurine Conjugation
Downloaded by [New York University] at 06:38 29 July 2015
Zhi-Ying Zheng and Carol Bernstein
Abstract Bile salts and acids have been implicated in the etiology of colon cancer, possibly through their ability to cause DNA damage. Taurine-conjugated and nonconjugated forms of three bile salts and one bile acid were tested for DNA repair-inducing potential and for cellular toxicity in a recently developed Escherichia coli chromotest system. The taurine-conjugated forms of sodium deoxycholate and lithocholic acid had reduced ability to induce DNA repair. Also the taurine-conjugated form of lithocholic acid had a reduced lethal effect. These observations suggest that the biotransformation step, whereby bacteria in the intestine remove the taurine added to bile salts in the liver, may be significant in the etiology of colon cancer. (Nutr Cancer 18, 157-164, 1992)
Introduction
Epidemiological evidence indicates that dietary or intracolonic factors are important in causing colon cancer (1-3). Recently, Cheah (4) reviewed 87 studies on bile acids, dietary fats, fecal mutagens, and ketosteroids and concluded that bile acids are the compounds most strongly implicated in the etiology of colorectal cancer. A previous study in our laboratory (5) showed that the primary bile salt sodium chenodeoxycholate (NaCDOC) and the secondary bile salt sodium deoxycholate (NaDOC) caused DNA damage in each of three in vivo test systems. One bacterial system (a modified Escherichia coli chromotest) and two mammalian cell systems were used. Although only these two bile salts were examined and shown to cause DNA damage in living cells, intestinal bacteria of humans can generate 15-20 bile salts from the salts of the two primary bile acids cholic acid and chenodeoxycholic acid (6). Some compounds that cause tumors in the colon have been shown to depend on biotransformation to generate their carcinogenic potential (see summary in Ref. 7). The bile salts secreted into the small intestine are conjugated with glycine or taurine. However, in a major biotransformation carried out by intestinal bacteria, these conjugated forms are The authors are affiliated with the Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724.
Copyright © 1992, Lawrence Erlbaum Associates, Inc.
Downloaded by [New York University] at 06:38 29 July 2015
deconjugated. In healthy humans, unconjugated bile salts constitute about half the luminal bile salts in the lower ileum, and by the time feces are excreted, conjugated bile salts are rarely present (8). If taurine-conjugated bile salts cause less DNA damage than the unconjugated forms, this may explain, to some extent, the lower rates of carcinogenesis in the small intestine than in the colon. Thus we tested four pairs of bile salts or acid, the pairs consisting of nonconjugated and taurine-conjugated forms, for cellular toxicity and DNA repair induction. We used the modified E. coli chromotest developed by Kandell and Bernstein (5), because it is both rapid and quantitative. We tested the bile salts NaDOC, NaCDOC, and sodium cholate (NaC), which are soluble in water, and lithocholic acid (LCA), which is poorly soluble. The water-soluble bile salts come in uniform contact with the test bacteria. However, the poorly soluble bile acid LCA was also tested because, as described by Carey and Cahalane (8), "In the colon, bile salts that escape from the enterohepatic circulation—are deconjugated to form free bile acids [and they] also precipitate to a considerable degree because of their high TpfCa values and in the case of lithocholates because of their high Krafft points." Thus the poorly soluble LCA would be more typical of bile compounds in the colon contents. Of the bile acids in the colon, LCA constitutes about 33%. The test used here is a bacterial assay, which measures damage to DNA in bacterial cells, as reflected in the DNA repair response to that damage. The levels of damage induced by bile salts and acids in DNA of colon cells may not be the same as the levels of damage induced in DNA of bacterial cells. However, damage to bacterial DNA by bile salts or acids is likely to be qualitatively similar to damage to colon cell DNA by these agents, and thus these experiments are relevant to the etiology of colon cancer. Materials and Methods Bacteria and Growth Conditions E. coli strain JL1705 (kindly provided by Dr. J. W. Little, University of Arizona) was constructed from E. coli JL1047 and the plasmid pJWL184 (9). It has a single copy of the structural gene for lacZ 03-galactosidase), which is under the control of sulA, a DNA damage-inducible gene, turned on when SOS DNA repair is induced. Overnight cultures were grown in Luria broth (10). Working stocks were maintained on trypticase agar plates. The medium used in trypticase agar plates consisted of 10 g trypticase peptone (Baxter Healthcare, Tempe, AZ), 5 g NaCl, and 10 g Bacto-agar (Difco Laboratories, Detroit, MI) in 1 liter H2O (pH 7.2). Experiments in which DNA repair induction was measured were carried out on plates containing the above medium to which 80 /ig/ml of Xgal (5-bromo-4chloro-3-indolyl-/3-D-galactoside) and 50 /tg/ml of ampicillin were added. Bacteria to be placed on these plates were suspended in a soft-agar overlay (11), with Xgal added at 80 /tg/ml and ampicillin added at 100 /tg/ml. The agar was cooled to about 45 °C before the addition of Xgal and ampicillin. Chemicals The bile salts used were NaDOC, sodium taurodeoxycholate (NaTDOC), NaC, sodium taurocholate (NaTC), NaCDOC, and sodium taurochenodeoxycholate (NaTCDOC) (Sigma Chemical, St. Louis, MO). Stock solutions of these salts were prepared at 100 mg/ml in sterile deionized water. Stocks of the bile acids LCA and taurolithocholic acid (TLCA) (Sigma Chemical) were prepared in 100% ethanol at 100 mg/ml. Because the LCA only partially dissolved and the TLCA was almost completely insoluble, these were used in the form of suspensions. Mitomycin C (Sigma Chemical) was dissolved at 200 /tg/ml in sterile deionized water.
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Bacterial DNA Repair Induction Test (Modified Chromotest) A 0- to 360-/tl aliquot of bile salt solution or bile acid suspension, or control mitomycin C solution, was mixed with soft-agar overlay medium. Then E. coli JL1705 cells, from an overnight culture, were added at a concentration sufficient to yield 50-200 viable colonies upon plating, and the mixture was plated on Xgal-ampicillin-trypticase plates. Plates were incubated for 19 hours at 37°C. The formation of blue colonies is an indication of a "turned-on" state of SOS DNA repair passed on to cells within the colony (5). As reviewed by Walker (12), the SOS response, which controls turn-on of the sulA:lacZ fusion operon, is buffered against being turned on by small amounts of inducing signal (single-stranded DNA regions caused by DNA damage). Thus there is a barrier that must be overcome before the SOS response is turned on. In addition, Cole and Honore (13) showed that the sulA operon has an unusual arrangement of two turn-off mechanisms, so it can be turned off quickly. This arrangement predicts a completely turned-on or completely turned-off state for sulA:lacZ and, thereby, an on or off state for blue color production. Blue color production should turn on only after a minimum amount of damage accumulates in a cell. This turned-on state, which is due to the cleavage of LexA repressor protein, should be passed on to daughter cells as long as DNA damage continues to be produced. A higher percentage of blue colonies would indicate a higher percentage of colony-forming cells that had "turnedon" DNA repair. There would be few intermediately blue (partly turned-on) colonies on a plate. Blue colonies and total colonies were counted on each plate, with two plates counted for each different dose of the agent tested. Each experiment was carried out three times. Results Previously Established DNA-Damaging Agents As shown in Figure 1A, mitomycin C, an established DNA-damaging agent, caused an increase in the percentage of blue colonies on plates, from 1% to 23%, as its concentration increased from 0 to 3.15 /tM. At the same time, survival of colony-forming ability of cells decreased from 100% to 7%. Two bile salts, NaDOC and NaCDOC, that had previously shown a positive result in this test (5) also caused increases in percentage of blue colonies and a concomitant decrease in survival of colony-forming ability as molar concentration of the bile salts increased. Quantitatively, Figure IB shows that as NaDOC concentration was increased from 0 to 2.7 mM, the percentage of blue colonies increased from 0.7% to 16%, while survival of colony-forming ability fell from 100% to 27%. Figure 1C shows that as NaCDOC concentration increased from 0 to 2.6 mM, the percentage of blue colonies increased from 0.5% to 13%, while survival of colony-forming ability fell from 100% to 32%. Comparison of Nonconjugated With Taurine-Conjugated Bile Salts and Acid Comparisons were then made of the nonconjugated forms of NaC, NaCDOC, NaDOC, and LCA and their taurine-conjugated forms with respect to inactivation of colony-forming ability and increase in blue colony formation. Figure 2A shows that NaC and its taurine conjugate NaTC did not kill cells and only increased blue colony formation slightly when molar concentrations increased from 0 to 2.5 mM. Figure 2B shows that NaCDOC and its taurine conjugate NaTCDOC caused both increased inactivation of colony-forming ability of cells and increased percentage of formation of blue colonies as molar concentrations increased from 0 to 2.5 mM. For these two compounds, taurine conjugation made no difference in lethality or induction of DNA repair. However, the other two bile compounds tested did show differences in the effects of their
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1 £ == LU IOX1
-o-survivors —o—blue colonies
-•-survivors
-o-survivors
—o—blue colonies
—c—blue colonies i i i I i i i [ i i i
.001 0
1.00 2.00 3.00 0
uM mitomycin C
i i i I i i i I i i i
i i i i i i i
1.00 2.00 3.00 0 mM
NaDOC
1.00
i i i
2.00
3.00
mM NaCDOC
Figure 1. Percentage of cells surviving to form colonies on plates (open squares) and percentage of surviving colonies that were blue (open diamonds). Concentrations of mitomycin C (A), sodium deoxycholate (NaDOC) (B), and sodium chenodeoxycholate (NaCDOC) (C) were calculated on assumption that each compound had diffused throughout bottom agar (30 ml) and top agar (3 ml). Each point represents mean from 3 expts. Error bars, SE; bars that are not visible are included in symbols.
nonconjugated and taurine-conjugated forms. Figure 3A shows that both NaDOC and NaTDOC inactivated the colony-forming ability of cells, decreasing from 100% survival of colony-forming units to about 30% when concentrations increased from 0 to about 2.5 mM. However, the nonconjugated NaDOC caused a larger increase in blue colony formation than the taurine-conjugated NaTDOC. As molar concentrations increased for NaDOC, blue colonies rose from 0.7% to 16%, while for NaTDOC blue colonies rose from 1.3% to only 4% at about 2.5 mM. Figure 3B shows that the partially ethanol-soluble LCA caused a small decrease in ability of the cells to form colonies, from 100% to 60% colony-forming units, but a large increase in blue colony formation, from 0.5% to 21 %, when molar concentration of LCA increased from 0 to 24.6 mM. Taurine conjugation of LCA caused a large decrease in its ethanol solubility (see Materials and Methods) along with an inability to inactivate colony formation or induce blue colony formation. A control experiment with ethanol alone was performed in parallel with an experiment with LCA partly dissolved in ethanol to test the possibility that the ethanol solvent, used with LCA, itself had an SOS-inducing effect or an effect on cell survival. Although the LCA caused a reduction in survival and an increase in percentage of blue colonies, as previously found, the cells to which only ethanol was added
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B --o-survivors NaC -o-survivors NaTC -o-blue colonies NaC —»
Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Bile salt/acid induction of DNA damage in bacterial cells: Effect of taurine conjugation a
Zhi‐Ying Zheng & 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: Zhi‐Ying Zheng & Carol Bernstein (1992) Bile salt/acid induction of DNA damage in bacterial cells: Effect of taurine conjugation, Nutrition and Cancer, 18:2, 157-164, DOI: 10.1080/01635589209514215 To link to this article: http://dx.doi.org/10.1080/01635589209514215
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Bile Salt/Acid Induction of DNA Damage in Bacterial Cells: Effect of Taurine Conjugation
Downloaded by [New York University] at 06:38 29 July 2015
Zhi-Ying Zheng and Carol Bernstein
Abstract Bile salts and acids have been implicated in the etiology of colon cancer, possibly through their ability to cause DNA damage. Taurine-conjugated and nonconjugated forms of three bile salts and one bile acid were tested for DNA repair-inducing potential and for cellular toxicity in a recently developed Escherichia coli chromotest system. The taurine-conjugated forms of sodium deoxycholate and lithocholic acid had reduced ability to induce DNA repair. Also the taurine-conjugated form of lithocholic acid had a reduced lethal effect. These observations suggest that the biotransformation step, whereby bacteria in the intestine remove the taurine added to bile salts in the liver, may be significant in the etiology of colon cancer. (Nutr Cancer 18, 157-164, 1992)
Introduction
Epidemiological evidence indicates that dietary or intracolonic factors are important in causing colon cancer (1-3). Recently, Cheah (4) reviewed 87 studies on bile acids, dietary fats, fecal mutagens, and ketosteroids and concluded that bile acids are the compounds most strongly implicated in the etiology of colorectal cancer. A previous study in our laboratory (5) showed that the primary bile salt sodium chenodeoxycholate (NaCDOC) and the secondary bile salt sodium deoxycholate (NaDOC) caused DNA damage in each of three in vivo test systems. One bacterial system (a modified Escherichia coli chromotest) and two mammalian cell systems were used. Although only these two bile salts were examined and shown to cause DNA damage in living cells, intestinal bacteria of humans can generate 15-20 bile salts from the salts of the two primary bile acids cholic acid and chenodeoxycholic acid (6). Some compounds that cause tumors in the colon have been shown to depend on biotransformation to generate their carcinogenic potential (see summary in Ref. 7). The bile salts secreted into the small intestine are conjugated with glycine or taurine. However, in a major biotransformation carried out by intestinal bacteria, these conjugated forms are The authors are affiliated with the Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724.
Copyright © 1992, Lawrence Erlbaum Associates, Inc.
Downloaded by [New York University] at 06:38 29 July 2015
deconjugated. In healthy humans, unconjugated bile salts constitute about half the luminal bile salts in the lower ileum, and by the time feces are excreted, conjugated bile salts are rarely present (8). If taurine-conjugated bile salts cause less DNA damage than the unconjugated forms, this may explain, to some extent, the lower rates of carcinogenesis in the small intestine than in the colon. Thus we tested four pairs of bile salts or acid, the pairs consisting of nonconjugated and taurine-conjugated forms, for cellular toxicity and DNA repair induction. We used the modified E. coli chromotest developed by Kandell and Bernstein (5), because it is both rapid and quantitative. We tested the bile salts NaDOC, NaCDOC, and sodium cholate (NaC), which are soluble in water, and lithocholic acid (LCA), which is poorly soluble. The water-soluble bile salts come in uniform contact with the test bacteria. However, the poorly soluble bile acid LCA was also tested because, as described by Carey and Cahalane (8), "In the colon, bile salts that escape from the enterohepatic circulation—are deconjugated to form free bile acids [and they] also precipitate to a considerable degree because of their high TpfCa values and in the case of lithocholates because of their high Krafft points." Thus the poorly soluble LCA would be more typical of bile compounds in the colon contents. Of the bile acids in the colon, LCA constitutes about 33%. The test used here is a bacterial assay, which measures damage to DNA in bacterial cells, as reflected in the DNA repair response to that damage. The levels of damage induced by bile salts and acids in DNA of colon cells may not be the same as the levels of damage induced in DNA of bacterial cells. However, damage to bacterial DNA by bile salts or acids is likely to be qualitatively similar to damage to colon cell DNA by these agents, and thus these experiments are relevant to the etiology of colon cancer. Materials and Methods Bacteria and Growth Conditions E. coli strain JL1705 (kindly provided by Dr. J. W. Little, University of Arizona) was constructed from E. coli JL1047 and the plasmid pJWL184 (9). It has a single copy of the structural gene for lacZ 03-galactosidase), which is under the control of sulA, a DNA damage-inducible gene, turned on when SOS DNA repair is induced. Overnight cultures were grown in Luria broth (10). Working stocks were maintained on trypticase agar plates. The medium used in trypticase agar plates consisted of 10 g trypticase peptone (Baxter Healthcare, Tempe, AZ), 5 g NaCl, and 10 g Bacto-agar (Difco Laboratories, Detroit, MI) in 1 liter H2O (pH 7.2). Experiments in which DNA repair induction was measured were carried out on plates containing the above medium to which 80 /ig/ml of Xgal (5-bromo-4chloro-3-indolyl-/3-D-galactoside) and 50 /tg/ml of ampicillin were added. Bacteria to be placed on these plates were suspended in a soft-agar overlay (11), with Xgal added at 80 /tg/ml and ampicillin added at 100 /tg/ml. The agar was cooled to about 45 °C before the addition of Xgal and ampicillin. Chemicals The bile salts used were NaDOC, sodium taurodeoxycholate (NaTDOC), NaC, sodium taurocholate (NaTC), NaCDOC, and sodium taurochenodeoxycholate (NaTCDOC) (Sigma Chemical, St. Louis, MO). Stock solutions of these salts were prepared at 100 mg/ml in sterile deionized water. Stocks of the bile acids LCA and taurolithocholic acid (TLCA) (Sigma Chemical) were prepared in 100% ethanol at 100 mg/ml. Because the LCA only partially dissolved and the TLCA was almost completely insoluble, these were used in the form of suspensions. Mitomycin C (Sigma Chemical) was dissolved at 200 /tg/ml in sterile deionized water.
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Bacterial DNA Repair Induction Test (Modified Chromotest) A 0- to 360-/tl aliquot of bile salt solution or bile acid suspension, or control mitomycin C solution, was mixed with soft-agar overlay medium. Then E. coli JL1705 cells, from an overnight culture, were added at a concentration sufficient to yield 50-200 viable colonies upon plating, and the mixture was plated on Xgal-ampicillin-trypticase plates. Plates were incubated for 19 hours at 37°C. The formation of blue colonies is an indication of a "turned-on" state of SOS DNA repair passed on to cells within the colony (5). As reviewed by Walker (12), the SOS response, which controls turn-on of the sulA:lacZ fusion operon, is buffered against being turned on by small amounts of inducing signal (single-stranded DNA regions caused by DNA damage). Thus there is a barrier that must be overcome before the SOS response is turned on. In addition, Cole and Honore (13) showed that the sulA operon has an unusual arrangement of two turn-off mechanisms, so it can be turned off quickly. This arrangement predicts a completely turned-on or completely turned-off state for sulA:lacZ and, thereby, an on or off state for blue color production. Blue color production should turn on only after a minimum amount of damage accumulates in a cell. This turned-on state, which is due to the cleavage of LexA repressor protein, should be passed on to daughter cells as long as DNA damage continues to be produced. A higher percentage of blue colonies would indicate a higher percentage of colony-forming cells that had "turnedon" DNA repair. There would be few intermediately blue (partly turned-on) colonies on a plate. Blue colonies and total colonies were counted on each plate, with two plates counted for each different dose of the agent tested. Each experiment was carried out three times. Results Previously Established DNA-Damaging Agents As shown in Figure 1A, mitomycin C, an established DNA-damaging agent, caused an increase in the percentage of blue colonies on plates, from 1% to 23%, as its concentration increased from 0 to 3.15 /tM. At the same time, survival of colony-forming ability of cells decreased from 100% to 7%. Two bile salts, NaDOC and NaCDOC, that had previously shown a positive result in this test (5) also caused increases in percentage of blue colonies and a concomitant decrease in survival of colony-forming ability as molar concentration of the bile salts increased. Quantitatively, Figure IB shows that as NaDOC concentration was increased from 0 to 2.7 mM, the percentage of blue colonies increased from 0.7% to 16%, while survival of colony-forming ability fell from 100% to 27%. Figure 1C shows that as NaCDOC concentration increased from 0 to 2.6 mM, the percentage of blue colonies increased from 0.5% to 13%, while survival of colony-forming ability fell from 100% to 32%. Comparison of Nonconjugated With Taurine-Conjugated Bile Salts and Acid Comparisons were then made of the nonconjugated forms of NaC, NaCDOC, NaDOC, and LCA and their taurine-conjugated forms with respect to inactivation of colony-forming ability and increase in blue colony formation. Figure 2A shows that NaC and its taurine conjugate NaTC did not kill cells and only increased blue colony formation slightly when molar concentrations increased from 0 to 2.5 mM. Figure 2B shows that NaCDOC and its taurine conjugate NaTCDOC caused both increased inactivation of colony-forming ability of cells and increased percentage of formation of blue colonies as molar concentrations increased from 0 to 2.5 mM. For these two compounds, taurine conjugation made no difference in lethality or induction of DNA repair. However, the other two bile compounds tested did show differences in the effects of their
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-o-survivors —o—blue colonies
-•-survivors
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—c—blue colonies i i i I i i i [ i i i
.001 0
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i i i I i i i I i i i
i i i i i i i
1.00 2.00 3.00 0 mM
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mM NaCDOC
Figure 1. Percentage of cells surviving to form colonies on plates (open squares) and percentage of surviving colonies that were blue (open diamonds). Concentrations of mitomycin C (A), sodium deoxycholate (NaDOC) (B), and sodium chenodeoxycholate (NaCDOC) (C) were calculated on assumption that each compound had diffused throughout bottom agar (30 ml) and top agar (3 ml). Each point represents mean from 3 expts. Error bars, SE; bars that are not visible are included in symbols.
nonconjugated and taurine-conjugated forms. Figure 3A shows that both NaDOC and NaTDOC inactivated the colony-forming ability of cells, decreasing from 100% survival of colony-forming units to about 30% when concentrations increased from 0 to about 2.5 mM. However, the nonconjugated NaDOC caused a larger increase in blue colony formation than the taurine-conjugated NaTDOC. As molar concentrations increased for NaDOC, blue colonies rose from 0.7% to 16%, while for NaTDOC blue colonies rose from 1.3% to only 4% at about 2.5 mM. Figure 3B shows that the partially ethanol-soluble LCA caused a small decrease in ability of the cells to form colonies, from 100% to 60% colony-forming units, but a large increase in blue colony formation, from 0.5% to 21 %, when molar concentration of LCA increased from 0 to 24.6 mM. Taurine conjugation of LCA caused a large decrease in its ethanol solubility (see Materials and Methods) along with an inability to inactivate colony formation or induce blue colony formation. A control experiment with ethanol alone was performed in parallel with an experiment with LCA partly dissolved in ethanol to test the possibility that the ethanol solvent, used with LCA, itself had an SOS-inducing effect or an effect on cell survival. Although the LCA caused a reduction in survival and an increase in percentage of blue colonies, as previously found, the cells to which only ethanol was added
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B --o-survivors NaC -o-survivors NaTC -o-blue colonies NaC —»
