Mutagenic and Analgesic Activities of Aniline Derivatives SUSANA M. SICARDI~, JORGE L. MARTIARENA, AND MARIAT.
IGLESIAS
Received December 7, 1989, from the lnstituto de la Quimica y Metabolismo del Fdrmaco, UBA-CONICET; Facultad de Farmacia y Bioquimica, Accepted for publication October 16, 1990. Universidad de Buenos Ares, Junin 956, 1 1 13-Buenos Alres, Argentma. Abstract 0 Phenacetin (1), acetaminophen (2), acetanilide (3). 4-aminophenol (4), and aniline (5) were tested in S.J.L. Swiss mice for their mutagenic and analgesic activities. The Sanalogues of 1 and 2, 4-mercaptoacetanilide (6) and 4-ethylthioacetanilide (7), respectively,
were synthesized and tested in the same way to define if both activities could be separated by molecular modification. All the compounds tested exhibited analgesic activity with EDsovalues ranging from 12.6 to 158.5 mgikg. The compounds could be arranged in a decreasing order of analgesic activity as follows: 3 > 4 = 5 = 6 > 1 7 > 2. All the compounds, except 6 , were positive mutagens in the micronucleus test (statisticallysignificant).The order of relative mutagenic potencies was 1 = 7 > 4 > 2 = 3 ==5. A nlarrow doseresponse curve relationship was found for 5 and its metabol1:e4, the relative mutagenic potencies of which suggest ring hydroxylation as the major pathway of biotoxification. No parallelism was found between analgesic and mutagenic activities, so they could be separated by pharmacomodulation: 6 was more effective as an analgesic in the acetic acid test than 2, and no mutagenic activity was found at the doses assayed.
-
The detection of longterm risks, such as carcinogenesis and mutagenesis, is one of the goals of the toxicological profile of drugs and chemicals. The control of toxicity or enhanced selectivity in action can be obtained by molecular manipulation, where metabolic considerations or structure-toxicity relationships are taken into account.1.2 On this basis, the purpose of this paper was to compare the analgesic and mutagenic activity of aniline derivatives, with the intention of dissociating both actions by a suitable ring substitution. Phenacetin, acetaminophen (paracetamol), and acetanilide are mild analgesic, aniline derivatives. Phenacetin has been employed worldwide fo:r more than a century. The signs of its chronic toxicity in humans did not become apparent until several decades after the introduction of the drug into medical practice. Diseases such as chronic hemolitic anemia, methemoglobinemia, renal and liver necrosis, hypoglycemic coma, and uroepithelial cancer of the renal pelvis and lower urinary tract are associated with the ingestion of 1g of phenacetin per day for a t least 1 year or smaller daily intakes for many years.3-5 Lethal doses d phenacetin are not associated with hepatic damage, but cyanosis, respiratory depression, and cardiac arrest. Currently, phenacetin is considered a carcinogenic drug, although no mutagenic activity was found in Salmonella typhimurium6 or micronucleus test;7J in the latter test, phenacetin was not assayed at low doses. Carcinogenicity studies were carried out by GranbergOhman et al.,9 who investigated chromosomal aberrations and the frequency of sister-chromatid exchanges in experiments involving Sprague-Dawley rats treated with phenacetin, antipyrine, ,and caffeine, either alone or in different combinations in the diet. Compared with the controls, aberrations, such as gaps, breaks, and structural rearrangements, were increased only in the group treated with phenacetin alone. Although the metabolism of this drug involves multiple pathways,10.11 the possible role of its metabolites in promoting carcinogenicity or renal injury is yet to be unam0022-3549/91/0800-0761$01.00/0 @ 1991, American Pharmaceutical Association
biguously determined. It is agreed that in the major pathway (Scheme I), phenacetin (1) undergoes oxidative deethylation to form acetaminophen.5 It should be noted that acetaminophen (2) is one of the more popular nonprescription analgesic-antipyretic drugs in current use, and it was not mutagenic in the Ames test.12 Chronic toxicity of 2, caused by overdoses, was recently associated with hepatotoxicityla and nephrotoxicity,14J5 both requiring metabolic activation. The covalent binding of ring-labeled 2 to renal and hepatic microsomal protein(s) was identical to the deacetylation product of 2, 4-aminophenol.16 The oxidation of 2 by prostaglandin hydroperoxidase:H,O, led to a transient 4-aminophenoxyl radical 4a, as detected by ESR.17 The 4-amiNH2
VH COCH3
NH2
OH G -0minophenol
aniline
acetanilide
0.
0
LQ
ocetaminoDhen
NHCOCH3
I
0 phenacetin
1
i
I
0. NHCOCH3
r
OC2H5
HO
OH
0'"- 6.1
Scheme CPathways for the conversion of phenacetin to chemically reactive metabolites. Compounds in brackets are proposed intermediates. Journal of Pharmaceutical Sciences I 761 Vol. 80, No. 8, August 1991
nophenoxyl radical resulted, also by in vitro deacetylase action,ls in N-acetyl-4-benzoquinonimine(2a). The major product of aniline oxidative metabolism in all the species tested is 4-aminophenol (4). Also, acetanilide (3) has been identified as a urinary metabolite in rabbits.4 Both chemicals have been found to be negative mutagens when employed in the Ames test.19 Thus, all the compounds described above are chemically and metabolically related to aniline (5), the parent drug. Aniline and its hydrochloride have been studied extensively in the Salmonella typhimurium mutagenicity assay system and, at doses of 2500 pglplate, no mutagenic activity was found.20 However, the presence of an aromatic amino group makes 5 and its derivatives suspected of genotoxic activity. For screening purposes, some tests are used as predictive tools in estimating the carcinogenic or mutagenic potential. In vitro clastogenicity is usually determined by the Ames test, with or without metabolic activation. In vivo activity can be measured by tumorigenic effects for a long-term test or by formation of micronuclei in bone marrow for a short-term test. Substances that are positive in one of these tests are not always positive in the other. Those found positive in both tests, especially if positive in the micronucleus test as well,
are most often tumorigenic in vivo.'Zl The analgesic aromatic amines described in this paper are negative mutagens in the Ames test.. Although some of them deplete glutathione22 and the binding of their metabolites to DNA has been confirmed,l6,23 most. of them have not been studied in vivo over a wide range of doses. Recently, we detected clastogenic activity for 1 and 2 using the micronucleus test.24 In this paper we describe the results of this assay for 1-5, and S-analogues of 1and 2,4-ethylthioacetanilide(71, and 4-mercaptoacetanilide (61, which have been synthesized by conventional methods. Compounds that were considered negative mutagens at 24 h were assayed at 48 and 72 h. Their analgesic activity in the acetic acid-induced writhing test25 was determined and compared.
Experimental Section Animals-Male and female S.J.L. Swiss mice (localbreeding stock) had free access to Purina chow (Nutrimentos,S.A.) and tap water until the timeofexperimentation.They were maintained at 20-25 "C on a 12-h light/lZ-h dark cycle. Chemicals-Commercially available phenacetin (11, acetaminophen (2), acetanilide (3), 4-aminophenol (41, and aniline were purified by recrystallizationor distillation.4-Mercaptoacetanilide(6)
Table I-Mlcronucleated Polychromatic Erythrocyte (MNPCE) Frequency In Bone Marrow Cells of Swiss Mlce Treated intraperltoneally wlth Anlllne and Derivatives" No. 1
2
3
4
5
6
7
MNPCE, Yo t SEM
Compound (CAS-RN) Phenacetin (62-44-2)
Acetaminophen (103-90-2)
Acetanilide (103-84-4)
4-Aminophenol (123-30-8)
Aniline (62-53-3)
4-Mercaptoacetanilide (1126-81-4)
4-Ethylthioacetanilide
(-4
0 2 5 50 100 0 5 50 100 150 200 0 5 50 100 200 400 0 5 50 100 200 0 5 50 100 200 0 5 50 100 150 0 2 5 50 100
2.60 t 0.36 3.30 t 0.40d 3.63 t 0.32d 3.50 t 0.17d 2.50 f 0.33 2.52 f 0.41 2.62 t 0.34 3.00 f 0.53 6.11 f 1.65' 6.13 t 0.62d 3.13 t 0.46 2.75 f 0.41 2.00 t 0.57 5.38 t 0.48d 8.63 f 0.51d 3.75 t 0.28c 4.00 f 0.41d 2.44 f 0.26 2.33 f 0.38 6.40 t 0.36d 21.40 t 1.OBd 7.70 t 0.54d 2.88 f 0.62 2.38 ? 0.45 6.00 2 0.26d 7.50 f 0.34d 7.10 f 0.62d 2.50 t 0.38 3.29 t 0.47 3.13 t 0.26 3.10 t 0.46 2.38 t 0.34 2.50 f 0.44 3.50 f 0.34 4.50 2 0.4F 3.00 f 0.41 2.38 t 0.34
F, obs.'
YO Increase Over Control
MicronucleusInducing Potencye
A
26.9 39.6 34.6
2988.8 1414.6 123.6
142.5 143.3
215.9 144.7
95.6 213.8 36.4 45.5
258.5 292.9 24.9 15.6
162.3 777.0 216.0
352.8 844.6 118.0
108.3 160.4 146.5
200.6 148.5 67.8
80.0
2666.7
6
C
D
E
F
G
n = 10 mice per group; no animal died during the treatment. Fisher's F calculated using one-way ANOVA test; A:F(4,50) = 3.99, p < 0.01; B: F(5,60) = 4.80, p < 0.01; C: F(5,60) = 28.48, p < 0.01; D: F(4,50) = 98.99, p < 0.01; E: F(4.50) = 22.60, p < 0.01; F: F(4,50) = 1.29, p > 0.05; G: F(4.50) = 3.34, p < 0.05. p < 0.01 as compared with the control average and other doses (Newman-Keuls test). p < 0.05 as compared with the control average and other doses (Newman-Keuls test). Calculated by RP = YO increase over controls/dose (mM/kg).
762 I Journal of PharmaceuticalSciences Vol. 80, No. 8, August 7997
and 4-ethylthioacetanilide (7) were synthesized in a conventional manner from 4-chloronitrobenzene.26,27All compounds were examined by TLC for impurities. Micronucleus Test-Swiss mice (20-25g) were divided into groups of 10 (5female, 5 male). Thirty groups received one of the different doses (Table I) of the test drug by ip injection. For each lot of animals, one group was used as control, receiving the vehicle alone as described above. The compounds were dissolved or suspended in an aqueous solution of propyleneglycol (0.15mUmL) to the desired concentration immediately before application. Euthanasia was carried out by cervical dislaication 24 h after the injection. Both femurs were removed from each mouse and bone marrow smears were prepared as described by Schmid.28 The presence of micronuclei was analyzed in 1000 polychromatic erythrocytes (PCE,)on coded slides and the number of micronucleated PCE (MNPCE) in treated and control samples was recorded. The values given in Table I ,are the average SEM of 10 slides of bone marrow cells smears. Statistical analysis between control and treated animals were carried out by the one-way ANOVA test. Post-hoc analysis with the Newman-Keuls test was also completed. In all cases, p values c0.05 were considered significant. Relative potencies were calculated as the ratio of percent increase of MNPCE over controls to the corresponding molar dose of compound. Two groups were treated with 4-mercaptoacetanilide, at an ip dose of 100 mg/kg, followed by euthanasia at 48 and 72 h. Analgesic Activity-The acetic acid-induced writhing test for analgesia was used.25.29 Ten mice per group, weighing 15-25 g, were administered 0.696 acetic acid via ip injection. The test compounds were administered ip as a gum arabic suspension 20 min before acetic acid administration. The response of drug-treated animals measured 5 min after injection of acetic acid was compared with that of those given acetic acid done. The results are exhibited and expressed as potency ratios and median effective dose (]ED,,), as calculated by the Litchfield and Wilcoxon method.30
*
Results and Discussion No sex differences were found in either of the tests (Table I). All the compounds, except 6 , gave significant increases in MNPCE over controls (Table I). In the doses tested, no suppression of bone marrow stem cells was detected. Compounds 3, 4, and 5 displayed highly significant differences between treatments and controls. Furthermore, clastogenic doses showed significant dose-response relationships. The dose-response curves for 3, 4, and 5 show an inverted U-shaped form in the range ofdoses from 50 to 200 mgkg (i.e., these compounds faciilitated a genotoxic action at median doses, but had no effect at low and high doses). In contrast to the above, 1 and its S-analogue 7 showed a moderate but significant increase of micronuclei when treated animals were compai-ed with controls; however, an all-ornone effect seems to occur. When the micronucleus-inducing potency is analyzed, 1 and 7 were the strongest clastogenic drugs, followed by 4. Earlier reports31.32 defined ring hydroxylations as deactivation pathways in aromatic amines. Comparing the relative in vivo potency of annline and its metabolites (3 and 4), the ring hydroxylation seems to be the major pathway of biotoxification; this was also found by Mason and Fischer.18 When 6 was assayed a t different doses and sampling times, it did not cause a significant increase in micronuclei; thus, it is a negative mutagen. Comparing this compound with its analogue 2, the sigma values for 4-SH and 4-OH are 0.15 and -0.37,respectively. The hydroxy group, with a strong electronic effect, activate3 the amino group to N-hydroxylation and the ring to epoxidation. In fact, optimum mutagenic activity in bone marrow cells of aniline-monosubstituted derivatives is associated with the presence of donatingelectron substituents.33 The sigma values above zero define 4-SH as a electron-wit,hdrawinggroup which has the opposite
Table ICAnalgeslc Activities of Aniline and Analogues as Determined by the Acetlc Acid Test in Mice
Compound" 4 5
3 2 6 7 1
Drug
ED,,, mglkgb
4-Aminophenol 28.8 (54.8-1 5.2) Aniline 31.6 (54.6-1 8.4) Acetanilide 12.6 (22.7-7.0) Acetaminophen 158.5 (237.8-105.7) 25.1 (42.7-14.8) 4-Merca~toacetanilide 4-Ethylthio85.1 (136.2-53.2) acetanilide Phenacetin 83.2 (120.6-57.4)
Potency Ratiob 1.1
(2.5-0.5)
2.5 (5.51.1) 12.6 (26.5-6.0) 6.3(12.3-3.2) 3.4 ( 7 . 5 15) 1.O (1.9-0.5)
a The compounds were arranged in order to compare their activities and molecular modification. 95% confidence limits by Litchfield & Wilcoxon method.
effect. On the other hand, it is agreed that thiol groups protect cells from free radicals or electrophylic intermediaries carrying out a repair mechanism.34 All the compounds showed analgesic activity (Table11) with ED,, values ranging from 12.6 to 158.5mgkg. Doseresponse determinations showed a parallelism with dose-percent inhibition (data not shown). The N-acetylation did not alter the clastogenic ability of aniline, but the presence of the N-acetyl group caused a serious drop in activity and toxicity of 2 compared with 4.
Conclusions The micronucleus test was found to be a suitable procedure to detect the clastogenic ability of compounds containing aromatic amino groups, an activity which is not currently detectable by the Ames test. The results obtained in this test for 1 confirm its carcinogenic activity. Compounds 2 and 3 showed a lower micronucleus-inducing potency than 1. As positive mutagens and considering that 2 and 3 are commonly used analgesics, both drugs require further testing by class-A carcinogenicity assays.
References and Notes 1. Drug Design; Ariens, E. J., Ed.; Academic: New York, 1980; Volume 9,pp 1-46. 2. Harvison, P. J.; Forte, A. J. J . Med. Chem. 1986,29,1737-1743. 3. Dacie, J. V. The Haemol tic Anemias: Congenital and Acquired Churchill: London, 196{ p 1041. 4. Radomski, J. L. Ann. Rev. Pharmacol. Toxicol. 1979,19, 129157. 5. Carro-Ciampi, G. Toxicology 1978,10,311-339. 6. Ames, B. N.;McCann, J.; Yamasaky, E. Mutat. Res. 1975,31, 347-364. 7. Hoover, R.; Fraumeni, J. F. Cancer 1981,47,1071-1080. 8. The Collaborative Study Group for the Micronucleus Test Mutat. Res. 1986,172,151-163. 9. Granber -Ohman, I.;Johansson, S.;Hjerpe, A. Mutat. Res. 1980, 70, 13-lf5. 10. Brodie, B. B.; Axelrod, J. J . Pharmacol. Exp. Ther. 1949,97, 5 u-.. 7 -11. Calder, I. C: Creek M. J.; Williams, P. J.; Funder, C. C.; Green, C. R.;Ham,k. N.;?ange, J. D. J.Med.Chem. 1973,16,449-502. 12. King, M. T.; Beikirch,-H.; Eckhardtk, K.; Gocke, E.; Wild, D. Mutat. Res. 1979,66, 33-43. 13. Mitchell, J. R.; Jollow, D. J.; Potter, W. Z.; Davis, D. C.; Gillette, J. R.; Brodie, B. B. J . Pharmacol. Exp. Ther. 1973,187,185-212. 14. McMurtry, R. J.;Snodgrass, W. R.; Mitchell, J. R. Toxicol. Appl. Pharmacol. 1978,46,87-100. 15. Breen, K.;Wandscheer, J. C.; Peignoux, M.; Pessayre, D. Biochem. Pharmacol. 1982,31,115-116. 16. Gram, T.E.; Okine, L. K.; Gram, R. A. Ann. Reu. Pharmacol. Toxicol. 1986,26,259-291. 17. Newton, J. F.; Yoshimoto, M.; Bemstein, J.; Rush, G. F.; Hook, J. B. Toxicol. Appl. Pharmacol. 1983,69,291-306. 18. Mason, R. P.; Fischer, V. Fed. Proc. 1986,45,2493-2499. Journal of Pharmaceutical Sciences I 763 Vol. 80, No. 8, August 1991
19. Zeiger E: Anderson, B.; Haworth S: Lowler, T.; Mortelmans, K. Envirdn. holec. Mutagen. 1988,11 (Suppl. 12),1-158. 20. Kawachi, T.; Yahagi, T.; Kada, T.; Tazima, Y.; Ishidate, M.; Sasaki, M.; Sugiyama, T. ZARC Sci. Publ. 1980,27,323-330. 21. Ishidate, M., Jr.; Harnois, M. C.; Sofuni, T. Mutut. Res. 1988,195, 151-213. 22. Aikawa, K.; Satoh, T.; Kobayashi, K.;Kitagawa, H. Jpn. J. Pharmacol. 1978,285,699-705. 23. Roberts,J. J., Warwick, G. P. Znt. J.Cancer 1966,I, 179-196. 24. Sicardi, S. M.;Martiarena, J. L.; Iglesias, M. T. Actu Farm. Bonaerense 1987,6,71-75. 25. Doherty, N. S.Ann. Rep. Med. Chem. 1987,22,245-252. 26. Gilman, H.; Gainer, G. C. J. Am. Chem. SOC. 1949, 71, 17471749. .
27. Sicardi, S. M.; Lamdan, S.; Gaozza, C. H. J. Heterocycl. Chem. 1973,10,1039-1042. 28. Schmid. W. Mutut. Res. 1975.31.9-15. 29. Koster,'R.; Anderson, M.;de Beer, E. J. Fed. Proc. 1959,18,412.
764 I Journal of Pharmaceutical Sciences Vol. 80, No. 8, August 7997
30. Litchfield, J. T., Jr.; Wilcoxon, F. J.Pharmucol. Exp. Ther. 1949, 113,99-113. 31. Weisberger, J. H.; Weisburger, E. K. Pharmacol. Rev. 1973,25, 1-66. 32. Masson, H. A.; Ioannides, C.; Gorrod, J. W.; Gibson, G. G. Carcinogenesis 1977,4,1583-1586. 33. Sicardi, S.M.;Martiarena, J. L. Universidad de Buenos Aires, 1989,unpublished results. 34. O'Brien, P. J. Free Rad. Biol. Med. 1988,4,169-183.
Acknowledgments This work forms part of a thesis submitted by J.L.M. to the Universidad de Buenos Aires for the de ee of Doctor en Bio uimica y Farmacia. This work was supportedyy grants from the tonsejo Nacional de Investigaciones Cienttficas y 'Tbcnicas, PID no. 30463001 85-88.
IGLESIAS
Received December 7, 1989, from the lnstituto de la Quimica y Metabolismo del Fdrmaco, UBA-CONICET; Facultad de Farmacia y Bioquimica, Accepted for publication October 16, 1990. Universidad de Buenos Ares, Junin 956, 1 1 13-Buenos Alres, Argentma. Abstract 0 Phenacetin (1), acetaminophen (2), acetanilide (3). 4-aminophenol (4), and aniline (5) were tested in S.J.L. Swiss mice for their mutagenic and analgesic activities. The Sanalogues of 1 and 2, 4-mercaptoacetanilide (6) and 4-ethylthioacetanilide (7), respectively,
were synthesized and tested in the same way to define if both activities could be separated by molecular modification. All the compounds tested exhibited analgesic activity with EDsovalues ranging from 12.6 to 158.5 mgikg. The compounds could be arranged in a decreasing order of analgesic activity as follows: 3 > 4 = 5 = 6 > 1 7 > 2. All the compounds, except 6 , were positive mutagens in the micronucleus test (statisticallysignificant).The order of relative mutagenic potencies was 1 = 7 > 4 > 2 = 3 ==5. A nlarrow doseresponse curve relationship was found for 5 and its metabol1:e4, the relative mutagenic potencies of which suggest ring hydroxylation as the major pathway of biotoxification. No parallelism was found between analgesic and mutagenic activities, so they could be separated by pharmacomodulation: 6 was more effective as an analgesic in the acetic acid test than 2, and no mutagenic activity was found at the doses assayed.
-
The detection of longterm risks, such as carcinogenesis and mutagenesis, is one of the goals of the toxicological profile of drugs and chemicals. The control of toxicity or enhanced selectivity in action can be obtained by molecular manipulation, where metabolic considerations or structure-toxicity relationships are taken into account.1.2 On this basis, the purpose of this paper was to compare the analgesic and mutagenic activity of aniline derivatives, with the intention of dissociating both actions by a suitable ring substitution. Phenacetin, acetaminophen (paracetamol), and acetanilide are mild analgesic, aniline derivatives. Phenacetin has been employed worldwide fo:r more than a century. The signs of its chronic toxicity in humans did not become apparent until several decades after the introduction of the drug into medical practice. Diseases such as chronic hemolitic anemia, methemoglobinemia, renal and liver necrosis, hypoglycemic coma, and uroepithelial cancer of the renal pelvis and lower urinary tract are associated with the ingestion of 1g of phenacetin per day for a t least 1 year or smaller daily intakes for many years.3-5 Lethal doses d phenacetin are not associated with hepatic damage, but cyanosis, respiratory depression, and cardiac arrest. Currently, phenacetin is considered a carcinogenic drug, although no mutagenic activity was found in Salmonella typhimurium6 or micronucleus test;7J in the latter test, phenacetin was not assayed at low doses. Carcinogenicity studies were carried out by GranbergOhman et al.,9 who investigated chromosomal aberrations and the frequency of sister-chromatid exchanges in experiments involving Sprague-Dawley rats treated with phenacetin, antipyrine, ,and caffeine, either alone or in different combinations in the diet. Compared with the controls, aberrations, such as gaps, breaks, and structural rearrangements, were increased only in the group treated with phenacetin alone. Although the metabolism of this drug involves multiple pathways,10.11 the possible role of its metabolites in promoting carcinogenicity or renal injury is yet to be unam0022-3549/91/0800-0761$01.00/0 @ 1991, American Pharmaceutical Association
biguously determined. It is agreed that in the major pathway (Scheme I), phenacetin (1) undergoes oxidative deethylation to form acetaminophen.5 It should be noted that acetaminophen (2) is one of the more popular nonprescription analgesic-antipyretic drugs in current use, and it was not mutagenic in the Ames test.12 Chronic toxicity of 2, caused by overdoses, was recently associated with hepatotoxicityla and nephrotoxicity,14J5 both requiring metabolic activation. The covalent binding of ring-labeled 2 to renal and hepatic microsomal protein(s) was identical to the deacetylation product of 2, 4-aminophenol.16 The oxidation of 2 by prostaglandin hydroperoxidase:H,O, led to a transient 4-aminophenoxyl radical 4a, as detected by ESR.17 The 4-amiNH2
VH COCH3
NH2
OH G -0minophenol
aniline
acetanilide
0.
0
LQ
ocetaminoDhen
NHCOCH3
I
0 phenacetin
1
i
I
0. NHCOCH3
r
OC2H5
HO
OH
0'"- 6.1
Scheme CPathways for the conversion of phenacetin to chemically reactive metabolites. Compounds in brackets are proposed intermediates. Journal of Pharmaceutical Sciences I 761 Vol. 80, No. 8, August 1991
nophenoxyl radical resulted, also by in vitro deacetylase action,ls in N-acetyl-4-benzoquinonimine(2a). The major product of aniline oxidative metabolism in all the species tested is 4-aminophenol (4). Also, acetanilide (3) has been identified as a urinary metabolite in rabbits.4 Both chemicals have been found to be negative mutagens when employed in the Ames test.19 Thus, all the compounds described above are chemically and metabolically related to aniline (5), the parent drug. Aniline and its hydrochloride have been studied extensively in the Salmonella typhimurium mutagenicity assay system and, at doses of 2500 pglplate, no mutagenic activity was found.20 However, the presence of an aromatic amino group makes 5 and its derivatives suspected of genotoxic activity. For screening purposes, some tests are used as predictive tools in estimating the carcinogenic or mutagenic potential. In vitro clastogenicity is usually determined by the Ames test, with or without metabolic activation. In vivo activity can be measured by tumorigenic effects for a long-term test or by formation of micronuclei in bone marrow for a short-term test. Substances that are positive in one of these tests are not always positive in the other. Those found positive in both tests, especially if positive in the micronucleus test as well,
are most often tumorigenic in vivo.'Zl The analgesic aromatic amines described in this paper are negative mutagens in the Ames test.. Although some of them deplete glutathione22 and the binding of their metabolites to DNA has been confirmed,l6,23 most. of them have not been studied in vivo over a wide range of doses. Recently, we detected clastogenic activity for 1 and 2 using the micronucleus test.24 In this paper we describe the results of this assay for 1-5, and S-analogues of 1and 2,4-ethylthioacetanilide(71, and 4-mercaptoacetanilide (61, which have been synthesized by conventional methods. Compounds that were considered negative mutagens at 24 h were assayed at 48 and 72 h. Their analgesic activity in the acetic acid-induced writhing test25 was determined and compared.
Experimental Section Animals-Male and female S.J.L. Swiss mice (localbreeding stock) had free access to Purina chow (Nutrimentos,S.A.) and tap water until the timeofexperimentation.They were maintained at 20-25 "C on a 12-h light/lZ-h dark cycle. Chemicals-Commercially available phenacetin (11, acetaminophen (2), acetanilide (3), 4-aminophenol (41, and aniline were purified by recrystallizationor distillation.4-Mercaptoacetanilide(6)
Table I-Mlcronucleated Polychromatic Erythrocyte (MNPCE) Frequency In Bone Marrow Cells of Swiss Mlce Treated intraperltoneally wlth Anlllne and Derivatives" No. 1
2
3
4
5
6
7
MNPCE, Yo t SEM
Compound (CAS-RN) Phenacetin (62-44-2)
Acetaminophen (103-90-2)
Acetanilide (103-84-4)
4-Aminophenol (123-30-8)
Aniline (62-53-3)
4-Mercaptoacetanilide (1126-81-4)
4-Ethylthioacetanilide
(-4
0 2 5 50 100 0 5 50 100 150 200 0 5 50 100 200 400 0 5 50 100 200 0 5 50 100 200 0 5 50 100 150 0 2 5 50 100
2.60 t 0.36 3.30 t 0.40d 3.63 t 0.32d 3.50 t 0.17d 2.50 f 0.33 2.52 f 0.41 2.62 t 0.34 3.00 f 0.53 6.11 f 1.65' 6.13 t 0.62d 3.13 t 0.46 2.75 f 0.41 2.00 t 0.57 5.38 t 0.48d 8.63 f 0.51d 3.75 t 0.28c 4.00 f 0.41d 2.44 f 0.26 2.33 f 0.38 6.40 t 0.36d 21.40 t 1.OBd 7.70 t 0.54d 2.88 f 0.62 2.38 ? 0.45 6.00 2 0.26d 7.50 f 0.34d 7.10 f 0.62d 2.50 t 0.38 3.29 t 0.47 3.13 t 0.26 3.10 t 0.46 2.38 t 0.34 2.50 f 0.44 3.50 f 0.34 4.50 2 0.4F 3.00 f 0.41 2.38 t 0.34
F, obs.'
YO Increase Over Control
MicronucleusInducing Potencye
A
26.9 39.6 34.6
2988.8 1414.6 123.6
142.5 143.3
215.9 144.7
95.6 213.8 36.4 45.5
258.5 292.9 24.9 15.6
162.3 777.0 216.0
352.8 844.6 118.0
108.3 160.4 146.5
200.6 148.5 67.8
80.0
2666.7
6
C
D
E
F
G
n = 10 mice per group; no animal died during the treatment. Fisher's F calculated using one-way ANOVA test; A:F(4,50) = 3.99, p < 0.01; B: F(5,60) = 4.80, p < 0.01; C: F(5,60) = 28.48, p < 0.01; D: F(4,50) = 98.99, p < 0.01; E: F(4.50) = 22.60, p < 0.01; F: F(4,50) = 1.29, p > 0.05; G: F(4.50) = 3.34, p < 0.05. p < 0.01 as compared with the control average and other doses (Newman-Keuls test). p < 0.05 as compared with the control average and other doses (Newman-Keuls test). Calculated by RP = YO increase over controls/dose (mM/kg).
762 I Journal of PharmaceuticalSciences Vol. 80, No. 8, August 7997
and 4-ethylthioacetanilide (7) were synthesized in a conventional manner from 4-chloronitrobenzene.26,27All compounds were examined by TLC for impurities. Micronucleus Test-Swiss mice (20-25g) were divided into groups of 10 (5female, 5 male). Thirty groups received one of the different doses (Table I) of the test drug by ip injection. For each lot of animals, one group was used as control, receiving the vehicle alone as described above. The compounds were dissolved or suspended in an aqueous solution of propyleneglycol (0.15mUmL) to the desired concentration immediately before application. Euthanasia was carried out by cervical dislaication 24 h after the injection. Both femurs were removed from each mouse and bone marrow smears were prepared as described by Schmid.28 The presence of micronuclei was analyzed in 1000 polychromatic erythrocytes (PCE,)on coded slides and the number of micronucleated PCE (MNPCE) in treated and control samples was recorded. The values given in Table I ,are the average SEM of 10 slides of bone marrow cells smears. Statistical analysis between control and treated animals were carried out by the one-way ANOVA test. Post-hoc analysis with the Newman-Keuls test was also completed. In all cases, p values c0.05 were considered significant. Relative potencies were calculated as the ratio of percent increase of MNPCE over controls to the corresponding molar dose of compound. Two groups were treated with 4-mercaptoacetanilide, at an ip dose of 100 mg/kg, followed by euthanasia at 48 and 72 h. Analgesic Activity-The acetic acid-induced writhing test for analgesia was used.25.29 Ten mice per group, weighing 15-25 g, were administered 0.696 acetic acid via ip injection. The test compounds were administered ip as a gum arabic suspension 20 min before acetic acid administration. The response of drug-treated animals measured 5 min after injection of acetic acid was compared with that of those given acetic acid done. The results are exhibited and expressed as potency ratios and median effective dose (]ED,,), as calculated by the Litchfield and Wilcoxon method.30
*
Results and Discussion No sex differences were found in either of the tests (Table I). All the compounds, except 6 , gave significant increases in MNPCE over controls (Table I). In the doses tested, no suppression of bone marrow stem cells was detected. Compounds 3, 4, and 5 displayed highly significant differences between treatments and controls. Furthermore, clastogenic doses showed significant dose-response relationships. The dose-response curves for 3, 4, and 5 show an inverted U-shaped form in the range ofdoses from 50 to 200 mgkg (i.e., these compounds faciilitated a genotoxic action at median doses, but had no effect at low and high doses). In contrast to the above, 1 and its S-analogue 7 showed a moderate but significant increase of micronuclei when treated animals were compai-ed with controls; however, an all-ornone effect seems to occur. When the micronucleus-inducing potency is analyzed, 1 and 7 were the strongest clastogenic drugs, followed by 4. Earlier reports31.32 defined ring hydroxylations as deactivation pathways in aromatic amines. Comparing the relative in vivo potency of annline and its metabolites (3 and 4), the ring hydroxylation seems to be the major pathway of biotoxification; this was also found by Mason and Fischer.18 When 6 was assayed a t different doses and sampling times, it did not cause a significant increase in micronuclei; thus, it is a negative mutagen. Comparing this compound with its analogue 2, the sigma values for 4-SH and 4-OH are 0.15 and -0.37,respectively. The hydroxy group, with a strong electronic effect, activate3 the amino group to N-hydroxylation and the ring to epoxidation. In fact, optimum mutagenic activity in bone marrow cells of aniline-monosubstituted derivatives is associated with the presence of donatingelectron substituents.33 The sigma values above zero define 4-SH as a electron-wit,hdrawinggroup which has the opposite
Table ICAnalgeslc Activities of Aniline and Analogues as Determined by the Acetlc Acid Test in Mice
Compound" 4 5
3 2 6 7 1
Drug
ED,,, mglkgb
4-Aminophenol 28.8 (54.8-1 5.2) Aniline 31.6 (54.6-1 8.4) Acetanilide 12.6 (22.7-7.0) Acetaminophen 158.5 (237.8-105.7) 25.1 (42.7-14.8) 4-Merca~toacetanilide 4-Ethylthio85.1 (136.2-53.2) acetanilide Phenacetin 83.2 (120.6-57.4)
Potency Ratiob 1.1
(2.5-0.5)
2.5 (5.51.1) 12.6 (26.5-6.0) 6.3(12.3-3.2) 3.4 ( 7 . 5 15) 1.O (1.9-0.5)
a The compounds were arranged in order to compare their activities and molecular modification. 95% confidence limits by Litchfield & Wilcoxon method.
effect. On the other hand, it is agreed that thiol groups protect cells from free radicals or electrophylic intermediaries carrying out a repair mechanism.34 All the compounds showed analgesic activity (Table11) with ED,, values ranging from 12.6 to 158.5mgkg. Doseresponse determinations showed a parallelism with dose-percent inhibition (data not shown). The N-acetylation did not alter the clastogenic ability of aniline, but the presence of the N-acetyl group caused a serious drop in activity and toxicity of 2 compared with 4.
Conclusions The micronucleus test was found to be a suitable procedure to detect the clastogenic ability of compounds containing aromatic amino groups, an activity which is not currently detectable by the Ames test. The results obtained in this test for 1 confirm its carcinogenic activity. Compounds 2 and 3 showed a lower micronucleus-inducing potency than 1. As positive mutagens and considering that 2 and 3 are commonly used analgesics, both drugs require further testing by class-A carcinogenicity assays.
References and Notes 1. Drug Design; Ariens, E. J., Ed.; Academic: New York, 1980; Volume 9,pp 1-46. 2. Harvison, P. J.; Forte, A. J. J . Med. Chem. 1986,29,1737-1743. 3. Dacie, J. V. The Haemol tic Anemias: Congenital and Acquired Churchill: London, 196{ p 1041. 4. Radomski, J. L. Ann. Rev. Pharmacol. Toxicol. 1979,19, 129157. 5. Carro-Ciampi, G. Toxicology 1978,10,311-339. 6. Ames, B. N.;McCann, J.; Yamasaky, E. Mutat. Res. 1975,31, 347-364. 7. Hoover, R.; Fraumeni, J. F. Cancer 1981,47,1071-1080. 8. The Collaborative Study Group for the Micronucleus Test Mutat. Res. 1986,172,151-163. 9. Granber -Ohman, I.;Johansson, S.;Hjerpe, A. Mutat. Res. 1980, 70, 13-lf5. 10. Brodie, B. B.; Axelrod, J. J . Pharmacol. Exp. Ther. 1949,97, 5 u-.. 7 -11. Calder, I. C: Creek M. J.; Williams, P. J.; Funder, C. C.; Green, C. R.;Ham,k. N.;?ange, J. D. J.Med.Chem. 1973,16,449-502. 12. King, M. T.; Beikirch,-H.; Eckhardtk, K.; Gocke, E.; Wild, D. Mutat. Res. 1979,66, 33-43. 13. Mitchell, J. R.; Jollow, D. J.; Potter, W. Z.; Davis, D. C.; Gillette, J. R.; Brodie, B. B. J . Pharmacol. Exp. Ther. 1973,187,185-212. 14. McMurtry, R. J.;Snodgrass, W. R.; Mitchell, J. R. Toxicol. Appl. Pharmacol. 1978,46,87-100. 15. Breen, K.;Wandscheer, J. C.; Peignoux, M.; Pessayre, D. Biochem. Pharmacol. 1982,31,115-116. 16. Gram, T.E.; Okine, L. K.; Gram, R. A. Ann. Reu. Pharmacol. Toxicol. 1986,26,259-291. 17. Newton, J. F.; Yoshimoto, M.; Bemstein, J.; Rush, G. F.; Hook, J. B. Toxicol. Appl. Pharmacol. 1983,69,291-306. 18. Mason, R. P.; Fischer, V. Fed. Proc. 1986,45,2493-2499. Journal of Pharmaceutical Sciences I 763 Vol. 80, No. 8, August 1991
19. Zeiger E: Anderson, B.; Haworth S: Lowler, T.; Mortelmans, K. Envirdn. holec. Mutagen. 1988,11 (Suppl. 12),1-158. 20. Kawachi, T.; Yahagi, T.; Kada, T.; Tazima, Y.; Ishidate, M.; Sasaki, M.; Sugiyama, T. ZARC Sci. Publ. 1980,27,323-330. 21. Ishidate, M., Jr.; Harnois, M. C.; Sofuni, T. Mutut. Res. 1988,195, 151-213. 22. Aikawa, K.; Satoh, T.; Kobayashi, K.;Kitagawa, H. Jpn. J. Pharmacol. 1978,285,699-705. 23. Roberts,J. J., Warwick, G. P. Znt. J.Cancer 1966,I, 179-196. 24. Sicardi, S. M.;Martiarena, J. L.; Iglesias, M. T. Actu Farm. Bonaerense 1987,6,71-75. 25. Doherty, N. S.Ann. Rep. Med. Chem. 1987,22,245-252. 26. Gilman, H.; Gainer, G. C. J. Am. Chem. SOC. 1949, 71, 17471749. .
27. Sicardi, S. M.; Lamdan, S.; Gaozza, C. H. J. Heterocycl. Chem. 1973,10,1039-1042. 28. Schmid. W. Mutut. Res. 1975.31.9-15. 29. Koster,'R.; Anderson, M.;de Beer, E. J. Fed. Proc. 1959,18,412.
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30. Litchfield, J. T., Jr.; Wilcoxon, F. J.Pharmucol. Exp. Ther. 1949, 113,99-113. 31. Weisberger, J. H.; Weisburger, E. K. Pharmacol. Rev. 1973,25, 1-66. 32. Masson, H. A.; Ioannides, C.; Gorrod, J. W.; Gibson, G. G. Carcinogenesis 1977,4,1583-1586. 33. Sicardi, S.M.;Martiarena, J. L. Universidad de Buenos Aires, 1989,unpublished results. 34. O'Brien, P. J. Free Rad. Biol. Med. 1988,4,169-183.
Acknowledgments This work forms part of a thesis submitted by J.L.M. to the Universidad de Buenos Aires for the de ee of Doctor en Bio uimica y Farmacia. This work was supportedyy grants from the tonsejo Nacional de Investigaciones Cienttficas y 'Tbcnicas, PID no. 30463001 85-88.
