Mutation Research, 298 (1992)53-60
53
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
MUTGEN 01826
Mutagenic activity of oxathiolane steroids: structural requirement for the genotoxic activity in Salmonella and E. coli Shafat A. Qadri, Shabana Islam and Masood Ahmad Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University,Aligarh 202 002, India
(Received 9 October 1991) (Revision received31 March 1992) (Accepted 12 June 1992)
Keywords: Mutagenesis; Steroid-induced damage and repair; Salmonella typhimurium; Escherich& coli; Colony-formingability;
Ames testing Summary Oxathiolanes and disulfonyl derivatives of steroids were tested for mutagenic activity in the Ames tester strains. The test compounds exhibited mutagenic activity without metabolic activation although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA, lexA and rer of E. coli was observed as compared to their wild-type counterpart in the presence of the test steroid. Structural features which appear to be crucial for the mutagenic activity in these steroidal drugs are: (i) an electron-donating group at position 3, and (ii) a bulky group anchored at the 5th and 6th positions. The test steroids appear to damage DNA which in turn initiates the SOS repair with the concomitant induction of mutation.
Several steroids have wide pharmacological applications which include antitumor (Moreau et al., 1979) antiinflammatory (Verma, 1981) and anti-AIDS (Barnes, 1987) activities. Steroidal dithioketals may have a future role in cancer chemotherapy (Chang et al., 1955; Blickenstaff and Foster, 1961). Besides there are reports on the enhancement of mutagenic activity by contraceptive steroids (Rao et al., 1983). Recently, we have reported mutagenic activity and the mechanism of action of certain synthetic steroids including aziridines, dithiolanes, sulfines and sulfones
Correspondence: Dr. M. Ahmad, International Centre for Genetic Engineering and Biotechnology,Nil Campus, Shaheed Jit Singh Marg, New Delhi-ll0 067, India.
(Islam and Ahmad, 1991; Islam et al., 1991). We considered it worthwhile to investigate the mutagenicity and structure-function relationship of some other steroidal-oxathiolane, -thiones and -disulfone which were synthesized recently for pharmacological purposes (Shafiullah et al., 1990). Further studies regarding their mechanism of action are in progress. Materials and methods Steroids
The structure and symbols of the steroids are listed in Table 1. The steroids were synthesized by Shafiullah et al. (1990) according to the method of Shopee et al. (1956) using triethylamine as a catalyst (Durdan et al., 1960). After repeated
54 TABLE 1 THE STEROIDS USED IN THIS STUDY Steroid
Symbol
Melting point (°C)
3/3-Acetoxy-5a-cholestano[6a,5-d]',3'-oxathiolane-2'-thione
1
181
Structure
~ HsC--~--O
1
~
OS
\/
C
II
S 3/3-Chloro-5a-cholestano[6a,5-d]',3'-oxathiolane-2'-thione
~
163
Hl7
_-- _:_ OS
\/
C
II S 3/3-Hydroxy-5a-cholestano[6a,5-d]',3'-oxathiolane-2'-thione
III
132
18 C8H17
HO
- _ OS
\/
C
II
5a-Cholestano[6a,5-d]-l',3'oxathiolane-2 '-thione
IV
87-88
~
S Ht7
OS
\/
C
II
S 5a-Cholestan-3,6-dione
V
165
18 c8nI7
bis ethylene dithiolane
02
802 02S~_~SO2
HI7
55 Table 1 (continued) Steroid
Symbol
Melting point (°C)
Cholest-5-ene
VI
94-95
crystallization the purity and identity of each compound were confirmed by elementary analysis and IR, N M R and mass spectra (Shafiullah et al., 1990). Thin-layer chromatography (TLC) indicated that the test steroids were chromatographically homogeneous. All the steroids were dissolved in 3 0 - 4 0 % acetone plus 70-60% dimethyl sulfoxide (DMSO) mixture. This solvent system is recommended for Ames testing (Maron et al., 1981). Since little is known about the stability of the steroids in solution, the doses were prepared immediately before use. The potency of the mutagenic behavior was determined on the basis of the number of revertants at a particular dose exhibiting a linear d o s e - r e s p o n s e relationship (de Serres and Ashby, 1981; Levin et al., 1984). The background counts obtained in the presence of solvent as control were always subtracted.
18 CsHI7
the livers of Aroclor-1254-induced BalbC mice. $9 mix was freshly prepared for each experiment according to Maron and Ames (1983). A m e s mutagenicity test
The preincubation test was performed as described by Maron and Ames (1983). To 0.3 ml phosphate buffer, p H 7.4, 0.2 ml of bacterial culture and the required dose of the test steroid were added. The mixture was vortexed and incubated for 20 min at 37°C. Then 3.0 ml of molten TABLE 2 CHARACTERISTICS OF E. coli K-12 AND Salmonella typhimurium STRAINS Strain Relevant genetic markers designation
Ames tester strains as well as E. coli K-12 strains were used for the detection of genotoxicity of the steroids. The characteristics of the strains used are described in Table 2. The Ames strains were maintained as frozen stock and grown as described by Maron and Ames (i983). Each strain was tested on the basis of associated genetic markers raising it from a single colony from the master plate. Media
The media for the A m e s tester strains were essentially the same as described by Maron and Ames (1983). Nutrient broth contained (per liter): peptone, 5 g, b e e f extract, 1.5 g, yeast extract, 1.5 g, sodium chloride, 5 g (all from Hi-Media, India). The bacterial strains of E. coli K-12 were always plated on nutrient broth containing 1.5% ( w / v ) agar (Hi-Media). $9 fraction was prepared from
Source
Ames strains
TA97a Bacteria
Structure
uvrB, hisD6610, bio, rfa,
B.N. Ames
R-factor plasmid- pKM101 TA98
uvrB, hisD3052, bio, rfa,
B.N. Ames
R-factor plasmid- pKM101 TA100 TA102
TA104
B.N. Ames R-factor plasmid- pKM101 rfa, R-factor plasmid- pKM101, B.N.Ames multicopy plasmid pAQ1 containing hisG428 auxotrophic marker and tet r B.N. Ames uvrB, hisG428, rfa, R-factor plasmid pKM101
uvrB, hisG46, bio, rfa,
E. coli K-12 strains ABl157 thi-1, argE3, thr-1, leuB6, proA2, hisG4, lacY1, F , Str r, As AB2463 recA13, thi-1, argE3, thr-1, leuB6, proA2, hisG4, F-, Str r, As AB2494 lexA, thi-1, thr-1, leuB6, proA2, hisG4, metB, lacyl, F-, Str r, As BS39 rer, arg, thr-1, leu, pro, his, F-, str r, As
P. Howard Flanders P. Howard Flanders P. Howard Flanders B.S. Srivastava
178.0_+6.0 32.0+_ 3.5 198.0-+ 9.0 269.0-+ 7.5 427.0+25.0
151.0_+ 6.5 51.0+ 2.5 128.0± 4.5 240.0_+ 6.0 442.0± 7.5
180.0-+ 8.0 37.0-+ 2.5 212.0-+ 4.5 245.0_+12.5 399.0+14.0
163.0-+ 2.0 39.0+ 2.0 174.0_+ 4.5 252.0_+ 4.5 412.0-+ 5.0
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
I
II
III
IV
V
VI c (parent steroid) 307.0_+ 5.0 48.0-+ 6.0 265.0_+10.0 255.0_+10.5 260.0_+ 7.5
228.0_+ 8.5 52.0_+ 7.0 282.0_+33.0 383.0_+14.0 1093.0_+27.3
188.0_+ 5.5 11.0_+ 3.0 255.0_+ 5.0 360.0_+ 4.7 501.0-+10.3
186.0+ 4.5 14.0-+ 1.5 405.0_+ 3.7 319.0_+ 5.0 700.0_+36.5
120.0_+ 4.0 21.0_+ 2.5 138.0-+ 3.5 345.0+12.3 1037.0+_10.5
151.0+ 5.0 27.0+ 4.3 446.0-+21.5 601.0-+ 8.5 943.0-+29.0
0.25
4.5 3.0 6.3 4.5 5.0
328.0_+ 7.0 51.0-+ 5.5 268.5_+ 4.3 239.0_+20.5 214.5+ 6.5
282.0+ 6.4 84.0_+ 6.5 284.0_+ 9.0 401.0_+ 7.3 1162.0_+21.0
193.0+ 7.8 18.0_+ 2.5 274.0_+ 7.5 505.0_+16.0 652.0_+ 6.5
212.0-+ 25.0_+ 428.0± 353.0_+ 807.0+
92.0_+ 3.5 49.0+ 2.3 178.0-+ 9.0 580.0_+ 6.0 819.0-+12.5
168.0± 6.0 30.0-+ 4.5 511.0-+ 5.5 670.0_+ 6.0 1126.0-+ 8.5
0.5
3.0 7.5 5.5 7.3 2.5
6.3 2.0 5.8 3.0 7.5
340.0_+ 10.0 57.5-+ 7.5 260.5_+ 6.0 241.0_+ 1 2 . 0 240.0_+ 1 0 . 0
296.0-+ 3.5 53.0_+ 11.2 300.5_+ 7.5 506.0_+ 4.0 1200.5+ 1 0 . 0
201.0-+ 31.0_+ 294.0+ 530.0_+ 809.0_+
236.0_+ 2 2 . 0 18.0_+ 4.5 441.0+ 6.3 928.0_+ 5.5 1277.0-+ 3 5 . 3
118.0_+ 33.0+ 352.0-+ 472.0± 677.0+
188.0-+ 6.3 33.0-+ 4.5 382.5-+ 2 1 . 2 754.0_+ 7.5 1216.0_+120.3
1.0
6.0 4.5 2.3 3.5 6.0
350.0_+ 3.0 53.0_+ 4.0 251.0_+ 9.0 221.5_+17.5 283.5_+53.5
280.0_+ 6.5 46.0+ 4.8 226.0_+14.0 727.0_+18.0 1098.0_+4.5
255.0-+ 1.5 55.0+ 3.5 296.0_+35.0 510.0+ 8.5 782.0_+ 7.5
196.0_+14.4 31.0_+ 4.0 355.0_+ 3.5 1238.0_+ 7.5 1060.0-+21.2
146.0_+ 54.0-+ 258.0-+ 506.5+ 657.0-+
305.0+ 5.7 54.0-+ 3.5 326.0_+11.0 640.5+ 4.0 877.5_+94.3
5.0
339.5_+ 0.0 31.0_+ 3.5 256.5_+ 3.5 231.0_+28.0 100.0_+ 5.0
272.0_+ 5.3 36.0_+ 2.5 306.0_+ 4.5 388.0_+13.2 992.0_+ 3.5
217.0+ 2.5 47.0_+ 4.5 238.0+ 9.0 394.0_+ 5.5 772.0_+ 8.0
42.0_+ 2.6 19.0± 6.0 330.0_+ 4.5 583.0_+ 7.3 751.0-+ 8.0
133.0+ 5.0 71.0-+ 3.5 243.0_+ 3.5 570.0± 8.5 650.0+ 4.5
258.0+ 5.5 51.0-+ 1.5 308.0+ 4.3 528.0+ 8.2 871.0_+ 8.8
10.0
347.5+ 7.5 10.0-+ 3.5 164.0+ 5.0 282.5_+ 9.5 48.0_+ 7.5
254.0_+ 3.9 36.0+ 2.5 386.0+_ 8.0 311.0+ 7.5 763.0_+11.5
196.0_+ 4.5 10.0+ 3.0 253.0_+ 1.5 310.0_+ 8.0 552.0-+ 5.5
20.0± 7.0 16.0_+ 7.5 170.0_+10.0 423.0_+15.0 441.0_+ 8.5
53.0± 7.5 46.0_+10.0 158.0± 4.5 340.0-+ 5.5 527.0+ 6.0
262.0-+ 3.5 20.0+ 2.0 258.0-+ 4.3 369.0+_ 5.0 456.0_+ 6.5
15.0
a The number represents actual histidine revertants in terms of mean-+ SD; 0.0, not a single colony. b The molar concentration of the steroid for 1 /xg per plate was equivalent to 3.7, 3.8, 4.0, 4.2, 3.4 and 5.1 nM for steroids I-VI respectively. c The data of the parent compound (steroid VI) have been adopted from Islam and Ahmad (1991).
314.5_+27.5 33.0_+ 4.5 263.0_+15.0 254.0_+21.0 218.0_+ 8.0
175.0_+ 2.5 27.0_+10.0 220.0_+ 0.5 263.0+ 7.0 382.0_+ 7.0
Control
Histidine + revertants/plate a Steroid (/zg/plate) b
Ames strains
Steroid
REVERSION OF AMES TESTER STRAINS IN THE PRESENCE OF STEROIDS WITHOUT METABOLIC ACTIVATION
TABLE 3A
319.0+ 0.0± 64.0 ± 230.0+ 0.0 ±
98.0 + 30.0± 191.5+ 273.0 ± 410.0±
47.0 ± 0.0 ± 113.0± 208.0 ± 310.0±
6.0 0.0 6.0 9.5 0.0
2.0 2.5 3.5 8.0 9.0
5.0 0.0 7.0 3.0 9.5
0.0± 0.0 13.0i 4.3 58.0+ 9.0 195.0_+ 10.0 200.5 ± 12.0
0.0_+ 0.0 0.0 _+ 0.0 98.5 + 4.0 135.0_+ 9.0 296.0 + 10.0
105.0 _+ 4.5 0.0_+ 0.0 161.0_+ 5.0 -210.0 ± 9.5 387.0 + 15.5
20.0
57 TABLE 3B REVERSION OF AMES TESTER STRAINS IN PRESENCE OF STEROID III AFTER METABOLICACTIVATION BY POSTMITOCHONDRIALSUPERNATANT($9 FRACTION) Ames Strain
Histidine ÷ revertants/plate a Steroid(p.g/plate) Control
TA97a 205.0+3.5 TA98 65.8+3.0 TA100 193.0+6.0 TA102 283.5-+2.5 TA104 461.5+4.5
0.25
0.5
267.0+_ 5.0 357.5+ 4.5 290.0+ 7.5 176.0+ 6.0 517.5+15.0 562.0_+14.0 370.0-+ 4.0 315.0-+ 5.5 921.5+ 8.6 1035.0+ 8.0
1.0
5.0
10.0
15.0
20.0
492.0+_3.0 427.5+ 7.5 320.0+ 6.3 53.0+2.5 0.0+ 0.0 236.5+ 7.5 71.5_+10.0 49.0+ 8.6 67.0+4.5 21.0+_3.0 509.0+ 7.5 427.5+11.5 381.0+ 8.0 272.0+9.5 116.0+ 6.5 1133.5_+10.0 1421.0_+12.0 759.5+20.0 527.0+6.3 215.0+ 7.0 1440.0-+15.0 1276.0+ 6.3 955.0_+ 7.5 502.0+3.5 240.0_+ 7.5
a The number represents actual histidine revertants in terms of mean + SD. 0.0, not a single colony. soft agar was added and the mixture was plated on glucose-supplemented minimal media. The plates were incubated at 37°C for 48 h. For the metabolic activation of comparatively more potent steroids, 20 /,d of $9 liver homogenate mix per plate was added. All the experiments were performed in triplicate. At each dose point duplicate plates were used.
Steroid I, II, III and I V treatment of E. coli K-12 The SOS-defective recA, lexA and rer mutants of E. coli K-12 as well as the isogenic wild-type strain were harvested by centrifugation from exponentially growing cultures (1-3 × l0 s viable counts/ml). The pellets so obtained were suspended directly in 0.01 M MgSO 4 solution and treated with doses of 0.5/x g / m l culture of steroids separately. Samples were drawn at regular intervals, suitably diluted and plated to assay the colony-forming ability. Plates were incubated overnight at 37°C. Solvent as control was also run simultaneously. Results
Reversion o f A m e s tester strains in the presence of steroids I - V I The reversion of Ames tester strains by the test steroids I - V I at the indicated doses in the absence of liver microsomal fraction is shown in Table 3A. The steroids displayed the maximum mutagenic activity with strains TA104 and TA102. The tester strains TA98 and TA97a usually did
not respond to steroids I - I V while TA100 responded comparatively weakly to the test steroids. The steroids exhibited mutagenicity at lower doses (0.25-5.0 /zg/plate equivalent to 0.94-1.9 nM final concentration approx.) but became lethal after attaining a peak value. The strains could usually be listed in the order of their sensitivity to steroids as follows: TA104 > TA102 > TA100 > TA97a > TA98. The mutagenicity exhibited by the steroids of the same series on the basis of structure was as follows: III > I > II > IV > VI. The mutagenic activity of steroid III was found to be highest even in the absence of liver microsomal ($9) fraction. Moreover, metabolic activation markedly enhanced the mutagenic activity of steroid III (Table 3B).
Survival of E. coli K-12 treated with steroids I - I V The survival patterns of recA, lexA and rer mutants of E. coli in the presence of steroids I - I V are shown in Fig. 1 ( A - D ) . The mutants exhibited a significant decline in colony-forming units (CFU) compared with their isogenic wildtype counterpart. The lexA mutant was found to be more sensitive than the recA and rer mutants. The recA and rer mutants exhibited almost equal sensitivity towards the test steroids. Moreover, it was observed that upon 2 h incubation of recA and rer mutants in the presence of test steroids there was no significant damage but after 6 h of incubation a significant decline in CFU was observed compared with the wild-type strain. The
5~
100
5O
D U_
g
1oo ~
]
v 0_
50
o'2, 0
2
4
60 Time ( h )
2
4
Fig. 1. Survivalof E. coli K-12 strains exposed to test steroids: e, wild type; o, recA; @, lexA; @, rer. (A) Steroid III; (B) steroid I; (C) steroid II; (D) steroid IV. Dose of steroid, 0.5 p,g/ml.
survival of the test E. coil strains in the presence of steroid III after 6 h treatment was recA, 45.0%; lexA, 1.0%; rer, 25.0%; wild-type, 94.2%. The steroids could be listed in order of their potency towards the mutants as follows: Ill > I > 1I > IV. Discussion
Although several short-term mutagenicity testing systems have been developed during the past 15 years, the Ames Salmonella system is recognized as the valid indicator of mutagenicity even today (Maron and Ames, 1983). However, it is always desirable to carry out a range of tests for a better understanding of the actual behavior of the test compounds (Maron and Ames, 1983). We have, therefore, employed two test systems of the potentially mutagenic behavior of the test steroids: (i) the Ames test, and (ii) the survival of D N A repair-defective E. coli strains. Our results indicate a remarkable degree of mutagenicity of the test steroids (Table 3A,B). The increase in the number of revertant colonies in the presence of the test steroid in comparison to that of the
control (Table 3A,B) indicates that the test compounds are mutagenic while the number of reverrant colonies provides an index of the mutagenic activity of the samples (de Serres and Ashby, 1981; Levin et al., 1984). The maximum degree of mutagenicity of the test steroids was generally observed with Ames mutants TAI02 and TA104. These strains contain A : T base pairs at the critical site of mutation with the difference of carrying the mutation in the multicopy plasmid or on the chromosome (Levin et al., 1982; Table 2). This suggests that the test steroids preferentially act upon A : T base pairs to bring about transition mutation involving an A T base pair (Table 3A,B). Moreover, it is noteworthy that even in the absence of $9 microsomal fraction, usually the tester strains responded significantly (Table 3A). The addition of microsomal fraction, however, further enhanced the mutagenic activity of test steroid IlI (Table 3B). These results are in agreement with our earlier findings (Islam and Ahmad, 1991; Islam et al., 1991). It was found that among the closely related steroids, those which contained substituted groups at position 3 were more mutagenic than the one devoid of any group at that position (Table 3A). This finding further substantiates our idea that the moiety after getting attached to the steroidal nucleus becomes more mutagenic compared with the parent steroid and individual moiety (Islam and Ahmad, 1991). Moreover, an increase in the number of moieties attached to the steroid appears to further enhance the mutagenic activity. If the disulfonyl moiety is attached to the 3rd position in addition to the 6th position the activity was further enhanced (Table 3A; Islam et al., 1991). We recently reported that dithiolanes and sulfonyl derivatives of steroids displayed mutagenic activity in proportion to the electronegativity of the halogen atom at the 3rd position of the steroidal nucleus. In addition we also reported that as the number of oxygen atoms increases in the substituted groups the mutagenic activity of the steroid also increases (Islam et al., 1991). Our results obtained with steroid V (Table 3A) are consistent with our previous finding. It is postulated that the inducible error-prone repair pathway presumably involving the recA and lexA genes could potentially operate on sev-
59
eral types of lesions in D N A whether produced by radiation or by other agents (Walker, 1985; Strauss, 1989). The recA, lexA and rer mutants of E. coli were found to be highly sensitive to the test steroids (Fig. 1), suggesting damage to the DNA of the exposed cells as well as the role of recA, lexA and rer genes to cope with the hazardous effect. These genes are believed to initiate the error-prone repair process in cells exposed to radiation and hazardous chemicals as well as other non-physiological stresses (Witkin, 1976; Walker 1985; Musarrat and Ahmad, 1990). The rer gene is also an inducible gene and regulates the coordination between the repair and replication of damaged DNA (Srivastava, 1976, 1978). This idea gains further support from Ames testing studies (Table 3A) with the Salmonella strains triggering the error-prone SOS response (Little et al., 1989). In fact, the Salmonella strains per se lack error-prone repair (Walker, 1984) due to the absence of a functional umuD gene (Herrera et al., 1988). However, this error-pron e repair in Salmonella is regained in the presence of plasmid pKM101 (McCann et al., 1975) which contains analogs of u m u C and umuD genes (Walker and Dobson, 1979; Walker, 1984). Similar findings of recA and lexA sensitivity have also been obtained with other synthetic steroids (Islam and Ahmad, 1991; Islam et al., 1991). The Salmonella tester strain TA102 detects a variety of oxidants and other agents as mutagens (Levin et al., 1982): Our test steroids exhibited a marked degree of mutagenicity with strains TA102 and TA104 (Table 3A). The substituted group present at position 3 is thought to be responsible for the interaction with any reactive species in the system to form oxygen radical species (Islam et al., 1991; Islam and Ahmad, 1991). Our findings gain further support for this idea since the increase in the number of potentially mutagenic oxygen radical loci (e.g., oxygen atoms) increases the mutagenic activity of the steroid. Our findings are consistent with the idea that the test steroids bring about the DNA damage and thus the treated ceils initiate the SOS response with the induction of mutation. Moreover, our studies further support the idea that oxygen radical formation in living systems increases the risk of chemical carcinogenesis (Hochstein and
Atallah, 1988) and certain steroids might be instrumental in the causation of cancer.
Acknowledgements Financial support from UGC, New Delhi (to S.A.Q.) and ICMR, New Delhi (to S.I.) is gratefully acknowledged.
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60 McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens: bacterial tester strains with R-factor plasmids, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Moreau, R.C., J.P. Fournier and G. Brouly-Boye (1979) Synthesis and in vitro cytotoxic properties of chloro-alkyl phenylsulfones, Ann. Pharm., 37, 429-432. Musarrat, J., and M. Ahmad (1991) Damage and mutagenesis of bacteriophage lambda induced by high pH, Mutagenesis, 6, 207-211. Rao, T.K., B.E. Allen, J.T. Cox and J.L. Epler (1983) Enhancement of mutagenic activity in Salmonella by contraceptive steroids, Mutation Res., 69, 48-54. Shafiullah, Shamsuzzaman and R.K. Pathak (1990) Synthesis of steroidal oxathiolane thiones, Acta Chem. Hung., 125, 701-704. Shopee, C.W., R.J. Bridgewater, D.N. Jones and G.H.R. Summers (1956) Steroid and Walden inversion, part XXXIII. The configuration of the coprostanyl halides, J. Chem. Soc., Part If, 2492-2499.
Srivastava, B.S. (1976) Radiation sensitivity of a mutant of Escherichia coli K-12 associated with DNA replication: Evidence for a new repair function, Mol. Gen. Genet., 143, 327-332. Srivastava, B.S. (1978) The role of inducible gene rer of Escherichia coli K-12 in DNA repair and mutagenesis, Mutation Res., 52, 1-9. Strauss, B.S. (1989) In vitro mutagenesis and DNA-repair, Ann., Ist. Super. Sanita, 25, 177-189. Verma, R.K. (1981) Topical antiinflamatory (Squib, E.R., and Sons, Inc.), U.S. Patent 4252733 (cl. 26-397.45; CO7JI/00), 4 pp. Walker, G.C. (1984) Mutagenesis and inducible response to DNA damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Walker, G.C. (1985) Inducible DNA-repair systems, Annu. Rev. Biochem, 54, 425-457.
53
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
MUTGEN 01826
Mutagenic activity of oxathiolane steroids: structural requirement for the genotoxic activity in Salmonella and E. coli Shafat A. Qadri, Shabana Islam and Masood Ahmad Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University,Aligarh 202 002, India
(Received 9 October 1991) (Revision received31 March 1992) (Accepted 12 June 1992)
Keywords: Mutagenesis; Steroid-induced damage and repair; Salmonella typhimurium; Escherich& coli; Colony-formingability;
Ames testing Summary Oxathiolanes and disulfonyl derivatives of steroids were tested for mutagenic activity in the Ames tester strains. The test compounds exhibited mutagenic activity without metabolic activation although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA, lexA and rer of E. coli was observed as compared to their wild-type counterpart in the presence of the test steroid. Structural features which appear to be crucial for the mutagenic activity in these steroidal drugs are: (i) an electron-donating group at position 3, and (ii) a bulky group anchored at the 5th and 6th positions. The test steroids appear to damage DNA which in turn initiates the SOS repair with the concomitant induction of mutation.
Several steroids have wide pharmacological applications which include antitumor (Moreau et al., 1979) antiinflammatory (Verma, 1981) and anti-AIDS (Barnes, 1987) activities. Steroidal dithioketals may have a future role in cancer chemotherapy (Chang et al., 1955; Blickenstaff and Foster, 1961). Besides there are reports on the enhancement of mutagenic activity by contraceptive steroids (Rao et al., 1983). Recently, we have reported mutagenic activity and the mechanism of action of certain synthetic steroids including aziridines, dithiolanes, sulfines and sulfones
Correspondence: Dr. M. Ahmad, International Centre for Genetic Engineering and Biotechnology,Nil Campus, Shaheed Jit Singh Marg, New Delhi-ll0 067, India.
(Islam and Ahmad, 1991; Islam et al., 1991). We considered it worthwhile to investigate the mutagenicity and structure-function relationship of some other steroidal-oxathiolane, -thiones and -disulfone which were synthesized recently for pharmacological purposes (Shafiullah et al., 1990). Further studies regarding their mechanism of action are in progress. Materials and methods Steroids
The structure and symbols of the steroids are listed in Table 1. The steroids were synthesized by Shafiullah et al. (1990) according to the method of Shopee et al. (1956) using triethylamine as a catalyst (Durdan et al., 1960). After repeated
54 TABLE 1 THE STEROIDS USED IN THIS STUDY Steroid
Symbol
Melting point (°C)
3/3-Acetoxy-5a-cholestano[6a,5-d]',3'-oxathiolane-2'-thione
1
181
Structure
~ HsC--~--O
1
~
OS
\/
C
II
S 3/3-Chloro-5a-cholestano[6a,5-d]',3'-oxathiolane-2'-thione
~
163
Hl7
_-- _:_ OS
\/
C
II S 3/3-Hydroxy-5a-cholestano[6a,5-d]',3'-oxathiolane-2'-thione
III
132
18 C8H17
HO
- _ OS
\/
C
II
5a-Cholestano[6a,5-d]-l',3'oxathiolane-2 '-thione
IV
87-88
~
S Ht7
OS
\/
C
II
S 5a-Cholestan-3,6-dione
V
165
18 c8nI7
bis ethylene dithiolane
02
802 02S~_~SO2
HI7
55 Table 1 (continued) Steroid
Symbol
Melting point (°C)
Cholest-5-ene
VI
94-95
crystallization the purity and identity of each compound were confirmed by elementary analysis and IR, N M R and mass spectra (Shafiullah et al., 1990). Thin-layer chromatography (TLC) indicated that the test steroids were chromatographically homogeneous. All the steroids were dissolved in 3 0 - 4 0 % acetone plus 70-60% dimethyl sulfoxide (DMSO) mixture. This solvent system is recommended for Ames testing (Maron et al., 1981). Since little is known about the stability of the steroids in solution, the doses were prepared immediately before use. The potency of the mutagenic behavior was determined on the basis of the number of revertants at a particular dose exhibiting a linear d o s e - r e s p o n s e relationship (de Serres and Ashby, 1981; Levin et al., 1984). The background counts obtained in the presence of solvent as control were always subtracted.
18 CsHI7
the livers of Aroclor-1254-induced BalbC mice. $9 mix was freshly prepared for each experiment according to Maron and Ames (1983). A m e s mutagenicity test
The preincubation test was performed as described by Maron and Ames (1983). To 0.3 ml phosphate buffer, p H 7.4, 0.2 ml of bacterial culture and the required dose of the test steroid were added. The mixture was vortexed and incubated for 20 min at 37°C. Then 3.0 ml of molten TABLE 2 CHARACTERISTICS OF E. coli K-12 AND Salmonella typhimurium STRAINS Strain Relevant genetic markers designation
Ames tester strains as well as E. coli K-12 strains were used for the detection of genotoxicity of the steroids. The characteristics of the strains used are described in Table 2. The Ames strains were maintained as frozen stock and grown as described by Maron and Ames (i983). Each strain was tested on the basis of associated genetic markers raising it from a single colony from the master plate. Media
The media for the A m e s tester strains were essentially the same as described by Maron and Ames (1983). Nutrient broth contained (per liter): peptone, 5 g, b e e f extract, 1.5 g, yeast extract, 1.5 g, sodium chloride, 5 g (all from Hi-Media, India). The bacterial strains of E. coli K-12 were always plated on nutrient broth containing 1.5% ( w / v ) agar (Hi-Media). $9 fraction was prepared from
Source
Ames strains
TA97a Bacteria
Structure
uvrB, hisD6610, bio, rfa,
B.N. Ames
R-factor plasmid- pKM101 TA98
uvrB, hisD3052, bio, rfa,
B.N. Ames
R-factor plasmid- pKM101 TA100 TA102
TA104
B.N. Ames R-factor plasmid- pKM101 rfa, R-factor plasmid- pKM101, B.N.Ames multicopy plasmid pAQ1 containing hisG428 auxotrophic marker and tet r B.N. Ames uvrB, hisG428, rfa, R-factor plasmid pKM101
uvrB, hisG46, bio, rfa,
E. coli K-12 strains ABl157 thi-1, argE3, thr-1, leuB6, proA2, hisG4, lacY1, F , Str r, As AB2463 recA13, thi-1, argE3, thr-1, leuB6, proA2, hisG4, F-, Str r, As AB2494 lexA, thi-1, thr-1, leuB6, proA2, hisG4, metB, lacyl, F-, Str r, As BS39 rer, arg, thr-1, leu, pro, his, F-, str r, As
P. Howard Flanders P. Howard Flanders P. Howard Flanders B.S. Srivastava
178.0_+6.0 32.0+_ 3.5 198.0-+ 9.0 269.0-+ 7.5 427.0+25.0
151.0_+ 6.5 51.0+ 2.5 128.0± 4.5 240.0_+ 6.0 442.0± 7.5
180.0-+ 8.0 37.0-+ 2.5 212.0-+ 4.5 245.0_+12.5 399.0+14.0
163.0-+ 2.0 39.0+ 2.0 174.0_+ 4.5 252.0_+ 4.5 412.0-+ 5.0
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
TA97a TA98 TA100 TA102 TA104
I
II
III
IV
V
VI c (parent steroid) 307.0_+ 5.0 48.0-+ 6.0 265.0_+10.0 255.0_+10.5 260.0_+ 7.5
228.0_+ 8.5 52.0_+ 7.0 282.0_+33.0 383.0_+14.0 1093.0_+27.3
188.0_+ 5.5 11.0_+ 3.0 255.0_+ 5.0 360.0_+ 4.7 501.0-+10.3
186.0+ 4.5 14.0-+ 1.5 405.0_+ 3.7 319.0_+ 5.0 700.0_+36.5
120.0_+ 4.0 21.0_+ 2.5 138.0-+ 3.5 345.0+12.3 1037.0+_10.5
151.0+ 5.0 27.0+ 4.3 446.0-+21.5 601.0-+ 8.5 943.0-+29.0
0.25
4.5 3.0 6.3 4.5 5.0
328.0_+ 7.0 51.0-+ 5.5 268.5_+ 4.3 239.0_+20.5 214.5+ 6.5
282.0+ 6.4 84.0_+ 6.5 284.0_+ 9.0 401.0_+ 7.3 1162.0_+21.0
193.0+ 7.8 18.0_+ 2.5 274.0_+ 7.5 505.0_+16.0 652.0_+ 6.5
212.0-+ 25.0_+ 428.0± 353.0_+ 807.0+
92.0_+ 3.5 49.0+ 2.3 178.0-+ 9.0 580.0_+ 6.0 819.0-+12.5
168.0± 6.0 30.0-+ 4.5 511.0-+ 5.5 670.0_+ 6.0 1126.0-+ 8.5
0.5
3.0 7.5 5.5 7.3 2.5
6.3 2.0 5.8 3.0 7.5
340.0_+ 10.0 57.5-+ 7.5 260.5_+ 6.0 241.0_+ 1 2 . 0 240.0_+ 1 0 . 0
296.0-+ 3.5 53.0_+ 11.2 300.5_+ 7.5 506.0_+ 4.0 1200.5+ 1 0 . 0
201.0-+ 31.0_+ 294.0+ 530.0_+ 809.0_+
236.0_+ 2 2 . 0 18.0_+ 4.5 441.0+ 6.3 928.0_+ 5.5 1277.0-+ 3 5 . 3
118.0_+ 33.0+ 352.0-+ 472.0± 677.0+
188.0-+ 6.3 33.0-+ 4.5 382.5-+ 2 1 . 2 754.0_+ 7.5 1216.0_+120.3
1.0
6.0 4.5 2.3 3.5 6.0
350.0_+ 3.0 53.0_+ 4.0 251.0_+ 9.0 221.5_+17.5 283.5_+53.5
280.0_+ 6.5 46.0+ 4.8 226.0_+14.0 727.0_+18.0 1098.0_+4.5
255.0-+ 1.5 55.0+ 3.5 296.0_+35.0 510.0+ 8.5 782.0_+ 7.5
196.0_+14.4 31.0_+ 4.0 355.0_+ 3.5 1238.0_+ 7.5 1060.0-+21.2
146.0_+ 54.0-+ 258.0-+ 506.5+ 657.0-+
305.0+ 5.7 54.0-+ 3.5 326.0_+11.0 640.5+ 4.0 877.5_+94.3
5.0
339.5_+ 0.0 31.0_+ 3.5 256.5_+ 3.5 231.0_+28.0 100.0_+ 5.0
272.0_+ 5.3 36.0_+ 2.5 306.0_+ 4.5 388.0_+13.2 992.0_+ 3.5
217.0+ 2.5 47.0_+ 4.5 238.0+ 9.0 394.0_+ 5.5 772.0_+ 8.0
42.0_+ 2.6 19.0± 6.0 330.0_+ 4.5 583.0_+ 7.3 751.0-+ 8.0
133.0+ 5.0 71.0-+ 3.5 243.0_+ 3.5 570.0± 8.5 650.0+ 4.5
258.0+ 5.5 51.0-+ 1.5 308.0+ 4.3 528.0+ 8.2 871.0_+ 8.8
10.0
347.5+ 7.5 10.0-+ 3.5 164.0+ 5.0 282.5_+ 9.5 48.0_+ 7.5
254.0_+ 3.9 36.0+ 2.5 386.0+_ 8.0 311.0+ 7.5 763.0_+11.5
196.0_+ 4.5 10.0+ 3.0 253.0_+ 1.5 310.0_+ 8.0 552.0-+ 5.5
20.0± 7.0 16.0_+ 7.5 170.0_+10.0 423.0_+15.0 441.0_+ 8.5
53.0± 7.5 46.0_+10.0 158.0± 4.5 340.0-+ 5.5 527.0+ 6.0
262.0-+ 3.5 20.0+ 2.0 258.0-+ 4.3 369.0+_ 5.0 456.0_+ 6.5
15.0
a The number represents actual histidine revertants in terms of mean-+ SD; 0.0, not a single colony. b The molar concentration of the steroid for 1 /xg per plate was equivalent to 3.7, 3.8, 4.0, 4.2, 3.4 and 5.1 nM for steroids I-VI respectively. c The data of the parent compound (steroid VI) have been adopted from Islam and Ahmad (1991).
314.5_+27.5 33.0_+ 4.5 263.0_+15.0 254.0_+21.0 218.0_+ 8.0
175.0_+ 2.5 27.0_+10.0 220.0_+ 0.5 263.0+ 7.0 382.0_+ 7.0
Control
Histidine + revertants/plate a Steroid (/zg/plate) b
Ames strains
Steroid
REVERSION OF AMES TESTER STRAINS IN THE PRESENCE OF STEROIDS WITHOUT METABOLIC ACTIVATION
TABLE 3A
319.0+ 0.0± 64.0 ± 230.0+ 0.0 ±
98.0 + 30.0± 191.5+ 273.0 ± 410.0±
47.0 ± 0.0 ± 113.0± 208.0 ± 310.0±
6.0 0.0 6.0 9.5 0.0
2.0 2.5 3.5 8.0 9.0
5.0 0.0 7.0 3.0 9.5
0.0± 0.0 13.0i 4.3 58.0+ 9.0 195.0_+ 10.0 200.5 ± 12.0
0.0_+ 0.0 0.0 _+ 0.0 98.5 + 4.0 135.0_+ 9.0 296.0 + 10.0
105.0 _+ 4.5 0.0_+ 0.0 161.0_+ 5.0 -210.0 ± 9.5 387.0 + 15.5
20.0
57 TABLE 3B REVERSION OF AMES TESTER STRAINS IN PRESENCE OF STEROID III AFTER METABOLICACTIVATION BY POSTMITOCHONDRIALSUPERNATANT($9 FRACTION) Ames Strain
Histidine ÷ revertants/plate a Steroid(p.g/plate) Control
TA97a 205.0+3.5 TA98 65.8+3.0 TA100 193.0+6.0 TA102 283.5-+2.5 TA104 461.5+4.5
0.25
0.5
267.0+_ 5.0 357.5+ 4.5 290.0+ 7.5 176.0+ 6.0 517.5+15.0 562.0_+14.0 370.0-+ 4.0 315.0-+ 5.5 921.5+ 8.6 1035.0+ 8.0
1.0
5.0
10.0
15.0
20.0
492.0+_3.0 427.5+ 7.5 320.0+ 6.3 53.0+2.5 0.0+ 0.0 236.5+ 7.5 71.5_+10.0 49.0+ 8.6 67.0+4.5 21.0+_3.0 509.0+ 7.5 427.5+11.5 381.0+ 8.0 272.0+9.5 116.0+ 6.5 1133.5_+10.0 1421.0_+12.0 759.5+20.0 527.0+6.3 215.0+ 7.0 1440.0-+15.0 1276.0+ 6.3 955.0_+ 7.5 502.0+3.5 240.0_+ 7.5
a The number represents actual histidine revertants in terms of mean + SD. 0.0, not a single colony. soft agar was added and the mixture was plated on glucose-supplemented minimal media. The plates were incubated at 37°C for 48 h. For the metabolic activation of comparatively more potent steroids, 20 /,d of $9 liver homogenate mix per plate was added. All the experiments were performed in triplicate. At each dose point duplicate plates were used.
Steroid I, II, III and I V treatment of E. coli K-12 The SOS-defective recA, lexA and rer mutants of E. coli K-12 as well as the isogenic wild-type strain were harvested by centrifugation from exponentially growing cultures (1-3 × l0 s viable counts/ml). The pellets so obtained were suspended directly in 0.01 M MgSO 4 solution and treated with doses of 0.5/x g / m l culture of steroids separately. Samples were drawn at regular intervals, suitably diluted and plated to assay the colony-forming ability. Plates were incubated overnight at 37°C. Solvent as control was also run simultaneously. Results
Reversion o f A m e s tester strains in the presence of steroids I - V I The reversion of Ames tester strains by the test steroids I - V I at the indicated doses in the absence of liver microsomal fraction is shown in Table 3A. The steroids displayed the maximum mutagenic activity with strains TA104 and TA102. The tester strains TA98 and TA97a usually did
not respond to steroids I - I V while TA100 responded comparatively weakly to the test steroids. The steroids exhibited mutagenicity at lower doses (0.25-5.0 /zg/plate equivalent to 0.94-1.9 nM final concentration approx.) but became lethal after attaining a peak value. The strains could usually be listed in the order of their sensitivity to steroids as follows: TA104 > TA102 > TA100 > TA97a > TA98. The mutagenicity exhibited by the steroids of the same series on the basis of structure was as follows: III > I > II > IV > VI. The mutagenic activity of steroid III was found to be highest even in the absence of liver microsomal ($9) fraction. Moreover, metabolic activation markedly enhanced the mutagenic activity of steroid III (Table 3B).
Survival of E. coli K-12 treated with steroids I - I V The survival patterns of recA, lexA and rer mutants of E. coli in the presence of steroids I - I V are shown in Fig. 1 ( A - D ) . The mutants exhibited a significant decline in colony-forming units (CFU) compared with their isogenic wildtype counterpart. The lexA mutant was found to be more sensitive than the recA and rer mutants. The recA and rer mutants exhibited almost equal sensitivity towards the test steroids. Moreover, it was observed that upon 2 h incubation of recA and rer mutants in the presence of test steroids there was no significant damage but after 6 h of incubation a significant decline in CFU was observed compared with the wild-type strain. The
5~
100
5O
D U_
g
1oo ~
]
v 0_
50
o'2, 0
2
4
60 Time ( h )
2
4
Fig. 1. Survivalof E. coli K-12 strains exposed to test steroids: e, wild type; o, recA; @, lexA; @, rer. (A) Steroid III; (B) steroid I; (C) steroid II; (D) steroid IV. Dose of steroid, 0.5 p,g/ml.
survival of the test E. coil strains in the presence of steroid III after 6 h treatment was recA, 45.0%; lexA, 1.0%; rer, 25.0%; wild-type, 94.2%. The steroids could be listed in order of their potency towards the mutants as follows: Ill > I > 1I > IV. Discussion
Although several short-term mutagenicity testing systems have been developed during the past 15 years, the Ames Salmonella system is recognized as the valid indicator of mutagenicity even today (Maron and Ames, 1983). However, it is always desirable to carry out a range of tests for a better understanding of the actual behavior of the test compounds (Maron and Ames, 1983). We have, therefore, employed two test systems of the potentially mutagenic behavior of the test steroids: (i) the Ames test, and (ii) the survival of D N A repair-defective E. coli strains. Our results indicate a remarkable degree of mutagenicity of the test steroids (Table 3A,B). The increase in the number of revertant colonies in the presence of the test steroid in comparison to that of the
control (Table 3A,B) indicates that the test compounds are mutagenic while the number of reverrant colonies provides an index of the mutagenic activity of the samples (de Serres and Ashby, 1981; Levin et al., 1984). The maximum degree of mutagenicity of the test steroids was generally observed with Ames mutants TAI02 and TA104. These strains contain A : T base pairs at the critical site of mutation with the difference of carrying the mutation in the multicopy plasmid or on the chromosome (Levin et al., 1982; Table 2). This suggests that the test steroids preferentially act upon A : T base pairs to bring about transition mutation involving an A T base pair (Table 3A,B). Moreover, it is noteworthy that even in the absence of $9 microsomal fraction, usually the tester strains responded significantly (Table 3A). The addition of microsomal fraction, however, further enhanced the mutagenic activity of test steroid IlI (Table 3B). These results are in agreement with our earlier findings (Islam and Ahmad, 1991; Islam et al., 1991). It was found that among the closely related steroids, those which contained substituted groups at position 3 were more mutagenic than the one devoid of any group at that position (Table 3A). This finding further substantiates our idea that the moiety after getting attached to the steroidal nucleus becomes more mutagenic compared with the parent steroid and individual moiety (Islam and Ahmad, 1991). Moreover, an increase in the number of moieties attached to the steroid appears to further enhance the mutagenic activity. If the disulfonyl moiety is attached to the 3rd position in addition to the 6th position the activity was further enhanced (Table 3A; Islam et al., 1991). We recently reported that dithiolanes and sulfonyl derivatives of steroids displayed mutagenic activity in proportion to the electronegativity of the halogen atom at the 3rd position of the steroidal nucleus. In addition we also reported that as the number of oxygen atoms increases in the substituted groups the mutagenic activity of the steroid also increases (Islam et al., 1991). Our results obtained with steroid V (Table 3A) are consistent with our previous finding. It is postulated that the inducible error-prone repair pathway presumably involving the recA and lexA genes could potentially operate on sev-
59
eral types of lesions in D N A whether produced by radiation or by other agents (Walker, 1985; Strauss, 1989). The recA, lexA and rer mutants of E. coli were found to be highly sensitive to the test steroids (Fig. 1), suggesting damage to the DNA of the exposed cells as well as the role of recA, lexA and rer genes to cope with the hazardous effect. These genes are believed to initiate the error-prone repair process in cells exposed to radiation and hazardous chemicals as well as other non-physiological stresses (Witkin, 1976; Walker 1985; Musarrat and Ahmad, 1990). The rer gene is also an inducible gene and regulates the coordination between the repair and replication of damaged DNA (Srivastava, 1976, 1978). This idea gains further support from Ames testing studies (Table 3A) with the Salmonella strains triggering the error-prone SOS response (Little et al., 1989). In fact, the Salmonella strains per se lack error-prone repair (Walker, 1984) due to the absence of a functional umuD gene (Herrera et al., 1988). However, this error-pron e repair in Salmonella is regained in the presence of plasmid pKM101 (McCann et al., 1975) which contains analogs of u m u C and umuD genes (Walker and Dobson, 1979; Walker, 1984). Similar findings of recA and lexA sensitivity have also been obtained with other synthetic steroids (Islam and Ahmad, 1991; Islam et al., 1991). The Salmonella tester strain TA102 detects a variety of oxidants and other agents as mutagens (Levin et al., 1982): Our test steroids exhibited a marked degree of mutagenicity with strains TA102 and TA104 (Table 3A). The substituted group present at position 3 is thought to be responsible for the interaction with any reactive species in the system to form oxygen radical species (Islam et al., 1991; Islam and Ahmad, 1991). Our findings gain further support for this idea since the increase in the number of potentially mutagenic oxygen radical loci (e.g., oxygen atoms) increases the mutagenic activity of the steroid. Our findings are consistent with the idea that the test steroids bring about the DNA damage and thus the treated ceils initiate the SOS response with the induction of mutation. Moreover, our studies further support the idea that oxygen radical formation in living systems increases the risk of chemical carcinogenesis (Hochstein and
Atallah, 1988) and certain steroids might be instrumental in the causation of cancer.
Acknowledgements Financial support from UGC, New Delhi (to S.A.Q.) and ICMR, New Delhi (to S.I.) is gratefully acknowledged.
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