Mutagenicity of Metronidazole: Presence of Several Active Metabolites in Human Urine William T. Speck,3,
4
Aaron B. Stein,5 and Herbert S. Rosenkranz ,5,~,
SUMMARY-Mutagenic activity was found in the 'Urine of 10 patients given therapeutic dosages of metronidazole orally or per vagina. Paper chromatographic separation revealed that mutagenicity in the urine was associated with unmodified metronidazole and at least four of its known urinary metabolites. Activity was also recovered in a region of the chromato· gram heretofore not assigned to a metronidazole metabolite.J Natl Cancer Inst 56: 283-284, 1976.
1-(2-Hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) is commonly used in the treatment of amebiasis, T,rich?monas vaginitis, and infections caused by anaerobI.C mICrobes. Recently, the reported mutagenicity of this wIdely used agent has received considerable attention (1-3), especially because such mutagenic activity was present in the urine of patients receiving therapeutic dosages of metronidazole (2). We now confirm the presence of mutagenic activity in the urine of patients given this drug either orally or per vagina (pv) and show that this activity is due to unmodified metronidazole and at least four of its metabolites. MATERIALS AND METHODS
Urine.- The specimens were obtained from patients who had received metronidazole (Flagyl; G. D. Searle & Co., Chicago, Ill.) longer than 24 hours. First-voided morning specimens were sterile-filtered and stored at - 20° C. Clinical details of the patients are in table 1. Mutagenesis assay.-The technique devised and perfected by Ames and associates (4) was used to detect mutagenic activity. In this procedure, the tester strain (a histidine-requiring Salmonella typhimurium) plus the mutagen are incorporated into an agar overlay; after incubation at 37° C for 2 days, revertants to histidine independence (i.e., mutants) are counted. The tester strain was S. typhimurium T AIOO (5). A mutant of TAIOO deficient in nitro reductase was obtained by selecting colonies capable of growth in the presence of 20 p.g 5-nitro-2-furaldehyde semicarbazone (nitrofurazone) /ml medium (6). One derivative, T AIOOFRI, was used in this study. Colorimetric analysis (6) revealed that it contained 4% of the nitrofurazone reductase activity of its parent strain. Furthermore, TAIOO-FRI was resistant to a variety of organic nitro compounds (e.g., picrolonic acid, nitrofluorene, nitronaphthalene, metronidazole), which indicates that it is deficient in a nonspecific nitro reductase activity. Paper chromatography.-One hundred-,p.l quantities of urine were deposited across 15 cm Whatman 3MM paper, and descending chromatography was carried out with n-butanol saturated with water (86: 14). Development proceded for a minimum of 45 cm. The chromatogram was dried and then inspected with UV light, and strips (2 X 2 cm) were excised. These strips were numbered and placed on top of agar plates containing the tester strain in the overlay. The plates were incubated at 37° C for 48 hours in the dark (7); the paper strips were removed and the number of mutants in the area previously covered by the paper was determined. We enumerated mutants in this way and did not count the mutants around
1, 2
7
t~e paper disc because the tester strain TAIOO gives a hIgh background (spontaneous revertants), which makes the usual enumeration more difficult.
RESULTS AND DISCUSSION Mut~genic a~tivity in the urine of patients given me!romdazole eI.ther orally or through vaginal suppositones was readIly demonstrable with unconcentrated urine samples (table I). Prolonged storage at - 20° Chad no effect on mutagenic activity. This activity depended on the amount of urine incorporated into the agar over~ay..The dose-response obtained with pure metronidazole IS gIven for comparison. It can be concluded that the ?bse:ved activ~t~ is due to metronidazole and/or one of Its nitro-contaInIng metabolites, since mutagenic activity could not be demonstrated with S. typhimurium TAIOOFRI, a nitro-reductase-deficient mutant as the tester strain (table I). The mutagenic activity is probably due to a hydroxylamino derivative, and TAIOO-FRI cannot metabolize the parent compounds to this activated form. Subjecting the urine samples to paper chromatography revealed that mutagenic activity could be recovered consistently in several specific ~reas of the chromatogram (table 2). Most speCImens yIelded at least four active areas; their rates of migration and those of known metabolites (8) of metronidazole are listed in table 2. One of the gen~tically active metabolites (Rf 0.31) had not been descnbed heretofore. Because this material is not mutagenic for TAIOO-FRl, we can assume that it too contains the nitro function. Studies of its chemical structure must await its isolation in sufficient quantities. ~he fin.d~ngs confi~m the previously reported (2) mutagenI~ actIVity of unne from patients receiving therapeutic dosages of metronidazole. In addition, we demonstrated that such activity is not restricted to patients taking the medication orally. Furthermore, we have shwn that the observed mutagenic activity is not restncted ~o met.ronidazole, but that it is possessed by several of ItS unnary metabolites. This finding indicates that, .although man can metabolize metronidazole, he does not detoxify it to substances with no genetic activity. These :esults, observed in a human population on t?erapeutIc. dosages. of a commonly prescribed medication, are dIsconcertIng when one considers the relationship be!ween mutagenic and carcinogenic activity and the earlIer report of the carcinogenicity of metronidazole for rodents (9).
Received July 28, 1975; accepted September 19, 1975. Supported by Public Health Service research contract NOICP33395 from the Division of Cancer Cause and Prevention National Cancer Institute. ' 3 Department of Pediatrics, College of Physicians and Surgeons Columbia University, New York, N. Y. 10032. ' 4 Vivian Allen Fellow in Clinical Medicine. 5 Department of Microbiology, College of Physicians and Surgeons Columbia University. ' 6 ~ecipient .of a Research Career Development Award from the NatIonal Institute of General Medical Sciences. 7 ~e thank Dr. Bruce N. Ames, University of California, Berkeley, CalIf. for the Salmonella typhimurium T AIOO. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 56, NO.2, FEBRUARY 1976
283
284
SPECK, STEIN, AND ROSENKRANZ TABLE
Patient or compound
Dosage
b
l.-Mutagenicity of urine samples for S. typhimurium Clinical indication
OJ
Revertan ts /plate Amount/plate TA100
1003
250 mg po
Trichomonas vaginitis
1015
250 mg po
Trichomonas vaginitis
1016 1017 1018
250 mg po 250 mg po 750 mg po
Trichomonas vaginitis Trichomonas vaginitis Amebic liver abscesses
1019
250 mg po
Trichomonas vaginitis
1020
500 mg pv
Trichomonas vaginitis
1021
250 mg po
Trichomonas vaginitis
1022
250 mg po
Trichomonas vaginitis
1023
250 mg po
Trichomonas vaginitis
0.1 ml 0.2 ml 0.5 ml 0.1 ml 0.2 ml 0.1 ml 0.1 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml
Metronidazole
Ethyl methanesulfonate
1,134 1,924 4,785 456 971 581 434 829 1,534 400 933 229 423 333 630
103 171 357 455
TA100-FR1
80 33 28 25
61 116 287 529 556 766
19
5,000
5,000
" Mutagenicity is expressed as the average of the number of revertants/replicate plates from which the spontaneous background values were subtracted (""200 and 120 colonies/plate for TA100 and TA100-FRI, respectively). Urine specimens from untreated adult controls had no mutagenic activity and did not influence background values. b Dosages were given three times a day; po = orally. TABLE
2.-Rates of paper chromatographic migration of mutagenic activity OJ Urinary metabolites
Mutagenic activity Specimen Urine
Rf
Rf
0.10-0.18
0.15 0.17
0.31 0.69-0.74 0.77-0.86 Metronidazole
therapeutic regimen does not exist or an unsatisfactory clinical response has been demonstrated. A substance with mutagenic and carcinogenic potentials should not be used in trivial clinical situations.
Chemical structure I-Acetic acid-2-methyl-5nitroimidazole 1-(2-Hydroxyethyl)-2carboxylic acid-5-nitroimidazole
Unknown Not reported 1-(2-Hydroxyethyl)-20.71 hydroxymethyl-5-nitroimidazole Metronidazole 0.79
0.77-0.84
" Determined by descending paper chromatography in n-butanol saturated with water. Since mutagenic activity was determined with excised paper strips (2 X2 cm), the values for rates of migration (Rf) are expressed as ranges in which genetic activity was recovered. The RI values for known urinary metabolites are taken from (8).
Because the recommended therapeutic regimens are doses of metronidazole between 250 and 750 mg thrice daily for 10 days, and because metronidazole has a long serum half-life (10), humans seem especially at risk. We are convinced that this form of antibacterial and antiprotozoal therapy should be restricted to life-threatening clinical situations in which a satisfactory alternate
REFERENCES (1) VOOGD CE, VAN DER STEL JJ, JACOBS JJ: The mutagenic action of nitroimidazoles. I. Metronidazole, nimorazole, dimetridazole and ronidazole. Mutat Res 26:483-490, 1974 (2) LEGATOR MS, CONNOR TH, STOECKEL M: Detection of mutagenic activity of metronidazole and niridazole in body fluids of humans and mice. Science 188:1118-1119, 1975 (J) Med Lett Drugs Ther 17:53-54, 1975 (4) AMES BN, LEE FD, DURSTON WE: An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Nat! Acad Sci USA 70:782-786, 1973 (5) MCCANN J, SPINGARN NE, KOBORI J, et al: Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc Nat! Acad Sci USA 72:979-983, 1975 (6) MCCALLA DR, REUVER A, KAISER C: Mode of action of nitrofurazone. J Bacteriol 104:1126-1134, 1970 (7) SPECK WT, ROSENKRANZ HS: Base substitution mutations induced in Salmonella strains by visible light (450 nm). Photochem Photobiol 21 :369-371, 1975 (8) STAMBAUGH JE, FEO LG, MANTHEI RW: The isolation and identification of the urinary oxidative metabolites of metronidazole in man. J Pharmacol Exp Ther 161:373-381, 1968 (9) RUSTIA M, SHUBIK P: Induction of lung tumors and malignant lymphomas in mice by metronidazole. J Nat! Cancer Inst 48:721-729, 1972 (10) RALPH ED, CLARKE JT, LIBKE RD, et al: Pharmacokinetics of metronidazole as determined by bioassay. Antimicrob Agents Chemother 6:691-696, 1974
4
Aaron B. Stein,5 and Herbert S. Rosenkranz ,5,~,
SUMMARY-Mutagenic activity was found in the 'Urine of 10 patients given therapeutic dosages of metronidazole orally or per vagina. Paper chromatographic separation revealed that mutagenicity in the urine was associated with unmodified metronidazole and at least four of its known urinary metabolites. Activity was also recovered in a region of the chromato· gram heretofore not assigned to a metronidazole metabolite.J Natl Cancer Inst 56: 283-284, 1976.
1-(2-Hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) is commonly used in the treatment of amebiasis, T,rich?monas vaginitis, and infections caused by anaerobI.C mICrobes. Recently, the reported mutagenicity of this wIdely used agent has received considerable attention (1-3), especially because such mutagenic activity was present in the urine of patients receiving therapeutic dosages of metronidazole (2). We now confirm the presence of mutagenic activity in the urine of patients given this drug either orally or per vagina (pv) and show that this activity is due to unmodified metronidazole and at least four of its metabolites. MATERIALS AND METHODS
Urine.- The specimens were obtained from patients who had received metronidazole (Flagyl; G. D. Searle & Co., Chicago, Ill.) longer than 24 hours. First-voided morning specimens were sterile-filtered and stored at - 20° C. Clinical details of the patients are in table 1. Mutagenesis assay.-The technique devised and perfected by Ames and associates (4) was used to detect mutagenic activity. In this procedure, the tester strain (a histidine-requiring Salmonella typhimurium) plus the mutagen are incorporated into an agar overlay; after incubation at 37° C for 2 days, revertants to histidine independence (i.e., mutants) are counted. The tester strain was S. typhimurium T AIOO (5). A mutant of TAIOO deficient in nitro reductase was obtained by selecting colonies capable of growth in the presence of 20 p.g 5-nitro-2-furaldehyde semicarbazone (nitrofurazone) /ml medium (6). One derivative, T AIOOFRI, was used in this study. Colorimetric analysis (6) revealed that it contained 4% of the nitrofurazone reductase activity of its parent strain. Furthermore, TAIOO-FRI was resistant to a variety of organic nitro compounds (e.g., picrolonic acid, nitrofluorene, nitronaphthalene, metronidazole), which indicates that it is deficient in a nonspecific nitro reductase activity. Paper chromatography.-One hundred-,p.l quantities of urine were deposited across 15 cm Whatman 3MM paper, and descending chromatography was carried out with n-butanol saturated with water (86: 14). Development proceded for a minimum of 45 cm. The chromatogram was dried and then inspected with UV light, and strips (2 X 2 cm) were excised. These strips were numbered and placed on top of agar plates containing the tester strain in the overlay. The plates were incubated at 37° C for 48 hours in the dark (7); the paper strips were removed and the number of mutants in the area previously covered by the paper was determined. We enumerated mutants in this way and did not count the mutants around
1, 2
7
t~e paper disc because the tester strain TAIOO gives a hIgh background (spontaneous revertants), which makes the usual enumeration more difficult.
RESULTS AND DISCUSSION Mut~genic a~tivity in the urine of patients given me!romdazole eI.ther orally or through vaginal suppositones was readIly demonstrable with unconcentrated urine samples (table I). Prolonged storage at - 20° Chad no effect on mutagenic activity. This activity depended on the amount of urine incorporated into the agar over~ay..The dose-response obtained with pure metronidazole IS gIven for comparison. It can be concluded that the ?bse:ved activ~t~ is due to metronidazole and/or one of Its nitro-contaInIng metabolites, since mutagenic activity could not be demonstrated with S. typhimurium TAIOOFRI, a nitro-reductase-deficient mutant as the tester strain (table I). The mutagenic activity is probably due to a hydroxylamino derivative, and TAIOO-FRI cannot metabolize the parent compounds to this activated form. Subjecting the urine samples to paper chromatography revealed that mutagenic activity could be recovered consistently in several specific ~reas of the chromatogram (table 2). Most speCImens yIelded at least four active areas; their rates of migration and those of known metabolites (8) of metronidazole are listed in table 2. One of the gen~tically active metabolites (Rf 0.31) had not been descnbed heretofore. Because this material is not mutagenic for TAIOO-FRl, we can assume that it too contains the nitro function. Studies of its chemical structure must await its isolation in sufficient quantities. ~he fin.d~ngs confi~m the previously reported (2) mutagenI~ actIVity of unne from patients receiving therapeutic dosages of metronidazole. In addition, we demonstrated that such activity is not restricted to patients taking the medication orally. Furthermore, we have shwn that the observed mutagenic activity is not restncted ~o met.ronidazole, but that it is possessed by several of ItS unnary metabolites. This finding indicates that, .although man can metabolize metronidazole, he does not detoxify it to substances with no genetic activity. These :esults, observed in a human population on t?erapeutIc. dosages. of a commonly prescribed medication, are dIsconcertIng when one considers the relationship be!ween mutagenic and carcinogenic activity and the earlIer report of the carcinogenicity of metronidazole for rodents (9).
Received July 28, 1975; accepted September 19, 1975. Supported by Public Health Service research contract NOICP33395 from the Division of Cancer Cause and Prevention National Cancer Institute. ' 3 Department of Pediatrics, College of Physicians and Surgeons Columbia University, New York, N. Y. 10032. ' 4 Vivian Allen Fellow in Clinical Medicine. 5 Department of Microbiology, College of Physicians and Surgeons Columbia University. ' 6 ~ecipient .of a Research Career Development Award from the NatIonal Institute of General Medical Sciences. 7 ~e thank Dr. Bruce N. Ames, University of California, Berkeley, CalIf. for the Salmonella typhimurium T AIOO. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 56, NO.2, FEBRUARY 1976
283
284
SPECK, STEIN, AND ROSENKRANZ TABLE
Patient or compound
Dosage
b
l.-Mutagenicity of urine samples for S. typhimurium Clinical indication
OJ
Revertan ts /plate Amount/plate TA100
1003
250 mg po
Trichomonas vaginitis
1015
250 mg po
Trichomonas vaginitis
1016 1017 1018
250 mg po 250 mg po 750 mg po
Trichomonas vaginitis Trichomonas vaginitis Amebic liver abscesses
1019
250 mg po
Trichomonas vaginitis
1020
500 mg pv
Trichomonas vaginitis
1021
250 mg po
Trichomonas vaginitis
1022
250 mg po
Trichomonas vaginitis
1023
250 mg po
Trichomonas vaginitis
0.1 ml 0.2 ml 0.5 ml 0.1 ml 0.2 ml 0.1 ml 0.1 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml
Metronidazole
Ethyl methanesulfonate
1,134 1,924 4,785 456 971 581 434 829 1,534 400 933 229 423 333 630
103 171 357 455
TA100-FR1
80 33 28 25
61 116 287 529 556 766
19
5,000
5,000
" Mutagenicity is expressed as the average of the number of revertants/replicate plates from which the spontaneous background values were subtracted (""200 and 120 colonies/plate for TA100 and TA100-FRI, respectively). Urine specimens from untreated adult controls had no mutagenic activity and did not influence background values. b Dosages were given three times a day; po = orally. TABLE
2.-Rates of paper chromatographic migration of mutagenic activity OJ Urinary metabolites
Mutagenic activity Specimen Urine
Rf
Rf
0.10-0.18
0.15 0.17
0.31 0.69-0.74 0.77-0.86 Metronidazole
therapeutic regimen does not exist or an unsatisfactory clinical response has been demonstrated. A substance with mutagenic and carcinogenic potentials should not be used in trivial clinical situations.
Chemical structure I-Acetic acid-2-methyl-5nitroimidazole 1-(2-Hydroxyethyl)-2carboxylic acid-5-nitroimidazole
Unknown Not reported 1-(2-Hydroxyethyl)-20.71 hydroxymethyl-5-nitroimidazole Metronidazole 0.79
0.77-0.84
" Determined by descending paper chromatography in n-butanol saturated with water. Since mutagenic activity was determined with excised paper strips (2 X2 cm), the values for rates of migration (Rf) are expressed as ranges in which genetic activity was recovered. The RI values for known urinary metabolites are taken from (8).
Because the recommended therapeutic regimens are doses of metronidazole between 250 and 750 mg thrice daily for 10 days, and because metronidazole has a long serum half-life (10), humans seem especially at risk. We are convinced that this form of antibacterial and antiprotozoal therapy should be restricted to life-threatening clinical situations in which a satisfactory alternate
REFERENCES (1) VOOGD CE, VAN DER STEL JJ, JACOBS JJ: The mutagenic action of nitroimidazoles. I. Metronidazole, nimorazole, dimetridazole and ronidazole. Mutat Res 26:483-490, 1974 (2) LEGATOR MS, CONNOR TH, STOECKEL M: Detection of mutagenic activity of metronidazole and niridazole in body fluids of humans and mice. Science 188:1118-1119, 1975 (J) Med Lett Drugs Ther 17:53-54, 1975 (4) AMES BN, LEE FD, DURSTON WE: An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Nat! Acad Sci USA 70:782-786, 1973 (5) MCCANN J, SPINGARN NE, KOBORI J, et al: Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc Nat! Acad Sci USA 72:979-983, 1975 (6) MCCALLA DR, REUVER A, KAISER C: Mode of action of nitrofurazone. J Bacteriol 104:1126-1134, 1970 (7) SPECK WT, ROSENKRANZ HS: Base substitution mutations induced in Salmonella strains by visible light (450 nm). Photochem Photobiol 21 :369-371, 1975 (8) STAMBAUGH JE, FEO LG, MANTHEI RW: The isolation and identification of the urinary oxidative metabolites of metronidazole in man. J Pharmacol Exp Ther 161:373-381, 1968 (9) RUSTIA M, SHUBIK P: Induction of lung tumors and malignant lymphomas in mice by metronidazole. J Nat! Cancer Inst 48:721-729, 1972 (10) RALPH ED, CLARKE JT, LIBKE RD, et al: Pharmacokinetics of metronidazole as determined by bioassay. Antimicrob Agents Chemother 6:691-696, 1974
