Investigation of genotoxic effects of the anti-asthmatic and anti-inflammatory drugs Apocynin and Acetosyringenin in the Salmonella typhimurium mutagenicity assay and the SCE-test with human lymphocytes

Phytomedicine Vol. 4/1995, pp. 319-322 © 1995 by Gustav Fischer Verlag, Stuttgart : Jena . New York

Investigation of genotoxic effects of the anti-asthmatic and anti-inflammatory drugs Apocynin and Acetosyringenin in the Salmonella typhimurium mutagenicity assay and the SeE-test with human lymphocytes S. PFUHLER1, P. STEHRER-SCHMID1, W. DORSCH2, H. WAGNER3 and H. U. Wolf1 1

2

3

Abteilung Pharmakologie und Toxikologie, Universitar Ulm, Albert-Einstein-Allee 11, D-89081 Ulm (FRG) Aidenbachstr. 118, D-81397 Miinchen (FRG) Institut fur Pharmazeutische Biologie, Ludwig-Maximilians-Universirar, Karlsstr. 29, D-80033 Miinchen (FRG)

Summary

We examined the possible genotoxicity of the recently isolated or developed anti-as thmatic and anti-inflammatory substa nces Apocynin (CAS 498-02-2) and Acetosyringeni n (CAS 2478-38-8) using short term test systems. Apocynin is a constituent of Picrorhiza kurroa, a plant from the Himalaya region. Acetosyringenin is its methoxylated synthetic derivative. The test systems used were the Salmonella typhimurium mutagenicity assay (Ames test) with the tester strains TA 97, TA 98, TA 100, and TA 102, and the sister chromatid exchange assay (SCE-Test) performed with human peripheral lymphocytes. The genetic endpoints covered by these test systems are the induction of gene mutations and the indu ction of SCEs, which is interpreted as an indicator for DNA-damaging properties of the test comp ound. The results obtained with Apocynin and Acetosyringenin do not demonstrate genotoxic activities of these compounds regarding the investigated endpoints. Key words: Apocynin, Acetosyringenin, genotoxicity, Salmo nella typhim urium mutagenicity assay, sister chromatid exchange test, human peripheral lymphocyt es. Introduction

The anti-asthmatic and anti-inflammatory drugs that were investigated in this study were isolated from a plant which is app lied in the traditional medicine of its home region. The pheno l derivative 4-Hydroxy-3-methoxy-acetophenone (trivial name Apocynin, [1] former name AcetovaniIlone) is a constituent of root extracts of the medical herb Picrorhiza kurroa Royle ex Benth. - a small plant with tuberous roots that occurs naturally in the Himalaya region in heights above 3 000 m. Picrorhiza kurroa has been extensively used in oriental medicine for a variety of conditions like lung and liver disease, fever, rheumatic disease etc. Picrorhiza kurroa roo t powder was found to inhibit histamine

release in vitro (Dorsch and Wagner, 1991). The anti-inflammatory activity of Apocynin is based on the inhibition of the release of mediators of inflammation such as thromboxane A2 (Engels, Renirie, t'Hart et aI., 1992), and also the inhibition of neutrophil superoxide production (t'Hart, Simons, Knaan -Shanzer et aI., 1990). 3,5-Dimethoxy-4-hydroxy-acetophenone (Acetosyringenin, [2] former name Acetosyringone), a methoxylated semisynthetic derivative of Apocynin, has a high anti-asthmatic activity (Stuppner and Wagner, 1989; Stuppner, Dorsch, Gropp et aI., 1991). Acetosyringenin was shown to be the most effective inhibitor of bronchial constriction in an in vivo comparison of 25 acetophenones with the plethysmographic guinea pig mod el (Dorsch, Muller, Christoffel et aI., 1994).

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O~CH3

on and Ames, the S9-mix contained 10 % liver homogenate corresponding to 1 mg Protein per plate. For SCE anal ysis the S9-fraction was mixed with the cofactors NADP (5 mol/I) and Glc 6-P (10 mol/I) and used in an end concentration of 3,3 % (0,6 mg Protein/ml).

7

~

1 OCH3

O-Glucosyl

1 Androsin

2 Acetosyringen in

For the basic screening of genotoxic properties we decided to use the Ames test and the in vitro sister chromatid exchange (SCE) test with human peripheral lymphocytes because these test systems are generally well accepted and a large data base for validation exists. The Salmonella typh imurium strains that were applied are recommended by Maron and Ames (Maron and Ames, 1983) for basic screening. Th ey detect base substitution (TA 100, TA 102 ) as well as frameshift mutations (loss or addition of base pairs, TA 97 and TA 98) and deletion (TA 100) in the histidine operon. Strain TA 102 is especially effective in the detection of mutations produced by oxidi zing substances. The induction of SCEs by chemical substances is regarded as a sensitive indicator for their inherent DNA-damaging activity (Speit, 1993; Tucker, Auletta, Cimbo et al., 1993). Furthermore, the changes of the pr oliferation index give information about the cytotoxicity of the test compounds.

Salmon ella mutagen icity assay The mutagenicity assay with Salmonella typhimurium was performed as described by Maron and Ames (Maron and Ames, 1983 ) with the Salmonella typh imurium stra ins TA97, TA9 8, TA 100 , and TA 102. We decided to perform the pre incub ation assay, a more sensitive test modification (Yahagi, Degawa, Seino et aI., 1975; M aron and Ames, 1983) with preincubation of bacteria and test substances for 30 min at 37 °C in the presence or absence of S9-mix containing 10 % of S9 rat liver homogenate . All samples were dissolved in 25 ul of DM SO. Three plates per sample were incubated at 37°C for 48 h. Revertant colonies were counted by means of a colony counter (Artek autocount). The follow ing positive controls were used: with S9: all strains 2-aminoanthracene: 10 ug/plate, without S9: TA 97: ICR 1911 ug/plate, TA 98: daunomycin 3 ug/plate, TA 100: sodium azide 50 ug/plate, and TA 102: mitomycin C 2 flg/ plate.

SCE assay with huma n peripheral lymph ocytes

Material and Methods Chemicals Cyclophosphamide, daunomycin hydrochloride, and sodium azide were purchased from Sigma (St. Louis, USA); Colcemid, 3',5'-bromodeoxyuridine and nicotineamide adenine dinucleotide phosphate (NADP) from Boehringer GmbH (Mannheim, FRG). RPMI 1640 medium and phytohemagglutinin M were obtained from Biochrom KG (Berlin, FRG). Acridine mutagen ICR 191 was a product from Fluka AG (Buchs, CH ), mitom ycin C from Serva Feinbi ochemica (He idelberg, FRG ) and Arochlor 1254 from Analabs Inc. (N ort h Haven, CT, USA - no mor e available ).

Substances tested Apocynin (CAS 2478-38-8) were FRG). The purity (Apocynin ) and 98

498-02-2) and Acetosyringenin (CAS purchased from Aldrich (Steinheim, was repo rted by Aldrich to be 97 % % (Acetosyringenin) .

Metab olic activation Arochlor 1254-induced rat liver S9 fract ion was prepared from male Wistar rats as described by M aron and Ames (Maron and Ames, 1983 ). The composition of the S9-mix for use in the Ames test was the same as described by M ar-

Five dr op s of freshly extracted hep ar inized blood were pipetted into a sterile culture tube containing 3 ml of RPMI 1640 culture medium compl eted with 15 % fetal calf seru m, 2.5 ug/rnl Phytohaemagglutinin M and 10 ug/ ml BrdUrd. After careful mixing the tubes were placed in an incubator (37 °C, normal air with 5 % CO 2 ), The test substances, solvent, and positive controls, resp., were added 48 hr after initiation of th e whole blood cultures. Cultures were treated in duplicate with each concentration of the test or control substance for 2 hr (with S9) or for the final 24 hr. Th e cultures for treatm ent in the presence of liver homogenate were centri fuged (l 50xg, 10min) and th e culture medium was drawn and 3 ml of serum-free RPMI medium was added. To each culture 100 ul of S9 fraction were added, revealing an end conc entration of 3.3 % of S9 in the cultures. Then the test substances and control substances , resp., were added and th e cultures were incubated for 2 hr in a 3 rc water bath. After the 2 hr tr eatment, the cells were wash ed with Hank's solution and reincubated with fresh serum- substituted medium. Colcemid (2 umol/l) was added 2 hr pr ior to the harvest of the cultures. After 72 hr the cultures were centrifuged, resuspended in 0.4 % KCI (20 min ) and fixed three tim es in MeOH/glacial acetic acid 3: 1. The cell solution was th en dropped on cold (4°C) slides. For SCE visualisation, the dry slides were irradiated with an UV-Iamp (254 nrn, distance 12 em) for 20 min. Subsequently, the slides were incu-

Investigation of the genotoxic effects

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Table 1. Number of revertants per plate with test chemicals Apocynin and Acetosyringenin in Salmonella typh imurium strains TA 97, TA 98, TA 100 and TA 102 in the presence (+59) and absence (-59) of S9 mix. TA9 7 +59

TA98 +59

TA100 +59

TA102 +59

Apocynin [ug/plate]

- 59

Buffer 0 10 33 100 330 1000 4000 Positive control

200 ± 14 190± 7 178 ± 11 179 ± 12 174± 6 179± 9 167± 2 tox 26 95 ± 16

212±11 189 ± 11 198 ± 5 204± 2 184± 2 191 ± 8 188 ± 3 tox 1258 ± 64

30 ± 3 28 ± 1 28 ± 1 27± 1 27± 5 26± 0 26 ± 2 tox 1267 ±4 9

39± 3 38 ± 3 32± 4 37± 1 40 ± 3 38 ± 1 39± 7 37± 7 1198 ± 120

137± 8 131 ± 3 131 ± 10 148± 6 133± 9 129 ± 17 131 ± 2 tox 767 ±46

151 ± 13 146 ± 6 137 ± 11 139 ± 5 123± 5 130± 12 117± 1 110±12 1394 ±58

261± 8 245 ± 8 161 ± 11 249± 13 253±19 254± 7 215 ± 15 tox 1033 ± 37

317 ± 8 322± 2 353± 4 333 ± 18 341 ± 8 327 ±20 298 ±21 tox 1566 ± 64

200± 14 190± 7 178 ± 4 169 ± 5 169 ± 18 166 ± 12 164± 8 157 ± 3 2695 ± 16

212 ±11 189 ± 11 200 ± 9 190± 5 203 ± 17 192±11 192 ± 19 tox 1258 ± 64

30 ± 3 28 ± 1 26± 3 24± 2 26 ± 6 24± 4 23 ± 2 20± 1 1276 ±49

39± 3 38 ± 3 33 ± 3 34± 3 36± 4 35 ± 4 34± 4 41± 7 1198 ± 120

137 ± 8 131 ± 3 132± 7 134 ± 11 146± 3 135 ± 9 130± 2 tox 767 ± 46

151 ± 13 146 ± 6 138 ± 4 127± 19 143± 4 126 ± 11 133± 3 118±10 1394 ±58

262 ± 8 251 ± 4 254± 17 241±10 242± 6 225± 4 212 ± 8 tox 1210 ±20

361 ± 20 375 ±23 355 ±21 360 ± 15 363 ±20 340±30 317±12 tox 1867 ± 39

- 59

- 59

-59

Acetosyringenin [ug/pla re] Buffer 0 10 33 100 330 1000 4000 Positive control

ta x: thin or no background lawn Table 2. Induct ion of SCEs and effects on lymphocyte growth in the presence (+59) and absence (-59) of 59-mix. Apocynin [ug/ml] 0 3,3 10 33 100 Positive contro l

Mean values [SCFlmitosis] - 59 +59

Standard deviation -59

+59

Proliferat ion index -S9 +59

6.36 6.42 6.18 6.50 6.92 25. 60 *

7.72 7.70 7.28 7.44 7.66 60.36 *

1.65 1.82 1.80 1.83 1.65 4.75

1.82 1.77 1.80 1.53 1.85 7.99

2.20 2.08 2.01 1.81 1.62 2.06

1.96 1.97 1.96 2.0 1 1.81 1.66

6.18 6.08 6.10 6.00 5.94 54.60 "

8.30 7.84 7.82 8.08 8.14 63.42 "

1.59 1.59 1.52 1.73 1.42 8.04

1.94 1.75 1.76 2.02 2.08 9.01

2.13 2.27 2.22 2.11 1.93 2.25

2.17 2.18 2.11 2.11 2.04 1.85

Acetosyringenin [ug/rnl] 0 3,3 10 33 100 Positive control

* Significantly different from control (P < 0.0 1)

bated in 2x SSC for 30 min at 60°C and then stained with 5 % Giemsa in Soerensen buffer (ph 6.8) . SCEs were scored in 25 cells per treatm ent (50 cells per concentration) and the data were tested for significance by the one-ta iled Student's t-test. Proliferation of the lymphocytes was evaluated by scoring the amount of first (M1), second (M2 ) and third or more (M3) mitotic divisions. The proliferat ion index (PI) was calculated using the equation PI = (LM 1 xl + LM2 x2 + LM3 x3)/L(M1 + M2 + M 3). The positive con-

troIs used were: cyclophosp hamide 2.5 ug/ ml with 59 and mitomycin C 0.25 ug/ rnl without 59.

Results and Discussion Table 1 shows the effects of the test substa nces with S.

typhimurium TA97, TA98, TA 100 and TA 102 in the presence or abscence of rat liver homogenate. Apocynin

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and Acetosyringenin did not show any mutagenic response either with or without metabolic activation. At high concentrations (4 mg per plate) both test chemicals are toxic in several tester strains, the background lawn of the histidine prototroph bacteria was very thin or all bacteria died. The results of the whole blood/SCE assay are shown in Table 2. Apocynin and Acetosyringenin do not increase the number of SCEs per mitosis significantly. Both substances are tested up to a dose where they reduce the proliferation index clearly (100 ug/rnl). At higher doses they avoid the growth of the lymphocytes and lead to cell death. None of the tested acetophenones increases the number of revertants in the Ames test or the number of sister chromatid exchanges in the SCE-test over spontaneous level. Our results agree with earlier studies. Nestmann et al. (1980) reported negative results in the Ames test with Apocynin and Acetosyringenin in strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 using the standard plate incorporation assay and S9 without cofactors. Apocynin was investigated in an in vitro SCE study with isolated lymphocytes performed only without S9 by Jansson (Jansson, Curvall, Hedin et aI., 1988) and found to give negative results. Both acetophenones did not increase the frequency of gene conversion in the yeast Saccharomyces cerevisiae strain D7, but Apocynin gave a positive result in S. cerevisiae strain XV185-14C (Nestmann and Lee, 1983) a very rarely used and not well validated test system. These investigations were performed only without adding metabolizing mammalian liver enzymes. Consequently, the results obtained up to now do not give any evidence for a genotoxic activity of Apocynin and Acetosyringenin.

tional Medicine? Int. Arch. Allergy Appl. Immunol. 94: 262-264, 1991. Engels, E, Renirie, B. E, t'Hart, B. A., Labadie, R. P., Nijkamp, E P.: Effects of apocynin, a drug isolated from the roots of Picrorhiza kurroa, on arachidonic acid metabolism. FEBS Lett. (Netherlands) 305 (3): 254-256, 1992. Jansson, T., Curvall, M., Hedin, A., Enzell, C. R.: In vitro studies of the biological effects of cigarette smoke condensate. III. Induction of SCE by some phenolic and related constituents derived from cigarette smoke. A study of structure-activity relationships. Mutation Research 206: 17-24, 1988. Maron, D. M., Ames, B. N.: Revised methods for the Salmonella mutagenicity test. Mutation Research 113: 173-215, 1983. Nestmann, E. R., Lee, E. G.-H.: Mutagenicity of constituents of pulp and paper mill effluent in growing cells of Saccharomyces cerevisiae. Mutation Research 119: 273-280,1983. Nestmann, E. R., Lee, E. G.-H., Matula, T. I., Douglas, G. R., Muller, J. c.: Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/Mammalian - Microsome Assay. Mutation Research 79: 203-212,1980. Speit, G. in: Mutationsforschung und genetische Toxikologie, (Fahrig, R., ed) Wissenschaftliche Buchgesellschaft, Darmstadt 263-273,1993. Stuppner, H., Dorsch, W., Gropp, M., Keppler, P., Wagner, H.: Antiasthmatische Aktivitat von Picrorhiza kurroa - Identifizierung von Wirksubstanzen. Sci. Pharm. 59: 70, 1991. Stuppner, H., Wagner, H.: Minor Iridoid and Phenol Glycosides of Picrorhiza kurroa. Planta medica 55: 467 -469, 1989. t'Hart, B. A., Simons, J. M., Knaan-Shanzer, S., Bakker, N. P., Labadie, L. P.: Antiarthritic activity of the newly developed neutrophil oxidative burst antagonist apocynin. Free Radic. Biol. Med. (United States) 9 (2): 127-131, 1990. Tucker, J. D., Auletta, A., Cimbo, M. c., Dearfield, K. L., Jacobsen-Kram, D., Tice, R. R., Carrano, A. V.: Sister-chromatid exchange: second report of the Gene-Tox program. Mutation Res. 297: 101-180, 1993. Yahagi, T., Degawa, M., Seino, Y., Matsushima, T., Nagao, M., Sugimura, T., Hashimoto, Y.: Mutagenicity of carcinogenic Azo Dyes and their derivatives. Cancer Letters 1: 91-96, 1975.

References Dorsch, W., Muller, A., Christoffel, V., Stuppner, H., Antus, S., Gottsegen, A., Wagner, H.: Antiasthmatic acetophenones - a in vivo study on structure activity relationship. Phytomedicine 1: 47-54,1994. Dorsch, W., Wagner, H.: New Antiasthmatic Drugs from Tradi-

Address

S. Pfuhler, Abteilung Pharmakologie und Toxikologie, Universitat DIm, Albert-Einstein-Allee 11, D-89081 DIm (FRG)