http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.928725

RESEARCH ARTICLE

Evidence for the contribution of non-covalent steroid interactions between DNA and topoisomerase in the genotoxicity of steroids

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Ronald D. Snyder1, Patrick A. Holt2, Jon M. Maguire2, and John O. Trent2 1

RDS Consulting Services, 3335 Grand Falls Blvd, Maineville, OH, United States and 2University of Louisville, James Graham Brown Cancer Center, 505 South Hancock St, Louisville, KY, United States Abstract

Keywords

Fifty two steroids and 9 Vitamin D analogs were docked into ten crystallographically-defined DNA dinucleotide sites and two human topoisomerase II ATP binding sites using two computational programs, Autodock and Surflex. It is shown that both steroids and Vitamin D analogs exhibit a propensity for non-covalent intercalative binding to DNA. A higher predicted binding affinity was found, however, for steroids and the ATP binding site of topoisomerase; in fact these drugs exhibited among the highest topo II binding observed in over 1370 docked drugs. These findings along with genotoxicity data from 26 additional steroids not subjected to docking analysis, support a mechanism wherein the long known, but poorly understood, clastogenicity of steroids may be attributable to inhibition of topoisomerase. A ‘‘proof of principle’’ experiment with dexamethasone demonstrated this to be the likely mechanism of clastogenicity of, at least, this steroid. The generality of this proposed mechanism of genotoxicity across the steroids and vitamin-D analogs is discussed.

Aqueous solubility, DNA intercalation, docking studies

Introduction Many hormonal steroids and structurally related chemicals exhibit genotoxicity when tested in in vitro and in vivo models. A review of the steroidal genotoxicity literature reveals that, for the most part, the effects are at the chromosomal rather than the gene (mutation) level (Joosten et al., 2004). Steroids and related molecules, in general, possess none of the classic ‘‘structural alerts’’ known to be associated with covalent DNA modification. Instead, genotoxicity has been conjectured to arise from formation of reactive oxygen species (ROS) (resulting from redox cycling, DNA base oxidation, malondialdehyde DNA adducts arising from lipid peroxidation), or bulky adduct formation (Bolton et al., 2000; Joosten et al., 2004; Kasper, 2001; Kulling et al., 1999; Liehr, 2000; Russo & Russo, 2004). All of these most likely contribute to steroidal genotoxicity on a case-by-case basis, but the early observations that steroids and other related small molecules can intercalate into DNA and that they likely have evolved this capability for gene regulatory purposes (Hendry et al., 2007), opens the possibility that these noncovalent DNA interactions of steroids may contribute more than covalent interactions to the observed genotoxicity.

Address for correspondence: Ronald D. Snyder, RDS Consulting Services, 3335 Grand Falls Blvd, Maineville, OH 45039, United States. E-mail: [email protected]

History Received 22 May 2014 Accepted 23 May 2014 Published online 27 June 2014

The use of computational models to predict non-covalent DNA binding has been recently described (Holt et al., 2008; Ricci & Netz, 2009) and we have recently reported the results of such studies with more than 1350 drug structures (Snyder et al., 2013). In the present communication, we examine the docking results from 61 steroids and vitamin D analogs reported in this earlier paper but not further analyzed. Cell-based data is also presented suggesting that, while physico-chemical properties confound the issue somewhat, the genotoxicity of these molecules is likely to be due, at least in part, to DNA intercalation and perturbation of topoisomerase function.

Materials and methods DNA and topoisomerase docking studies The computational programs, Autodock and Surflex, as well as the crystallographic structures from Protein Databank used for docking in these studies, have been described in detail earlier (Snyder et al., 2013). Briefly, ten distinct crystal structures derived from the intercalative binding of different drugs into specific DNA sequences were used. This use of crystal structures of stably-intercalated drugs, provides a real comparator for determining intercalation capability as opposed to using variously arbitrarily unwound DNA sequences. The chosen sites provide a reasonable representation of the most common intercalation sites. Because little is known about the sequence preferences of the drugs docked in this study, analysis of all individual sites and computation of

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R. D. Snyder et al.

the mean binding across all sites was deemed to be a reasonable approximation of overall binding ability. The structures represent the standard CG intercalation sites (CG1-3) and a reverse CG site (CG4) and atypical intercalation sites, TG (TG1 and extended sequence TG2), CA, CT, GT, and AG sites. The Surflex and Autodock simulations included the complementary interactions within the expanded base step of the intercalation site. Two human type IIA topoisomerase binding sites, PDB entries 1ZXN and 1ZXM were also used as docking sites. Surflex, which uses structural and shape determinants, and Autodock, which uses electrostatic determinants, were run using standard previously published parameters (Holt et al., 2008) of Multi 5 and ‘‘Random’’ and 5 docks of 2E7 energy evaluations, respectively. These parameters have been shown to reproduce the crystal structures of intercalated daunorubicin and ellipticine using the standard scoring methods of Surflex and Autodock. Data handling After the generation of data from the docking studies, Autodock and Surflex spreadsheets were established containing the drug name, docking values for each site (and mean docking values). These data are provided in the appendix of our earlier paper (Snyder et al., 2013). Surflex values are estimates of the binding affinity (log(Kd)). Autodock values, since based on the free energy of binding using a modified AMBER force field, were generated as negative values and were converted to positive values for ease of depiction. Higher negative values indicate stronger binding. For DNA docking, the mean of all 10 sites was calculated and this mean was used to rank order the drugs with respect to binding affinity. Also calculated were the mean values for each sequence across the total number of compounds. Covalent binding was not examined per se, as these docking programs do not have that capability under the conditions employed. V79 cell-based micronucleus studies Dexamethasone-induced micronucleus formation in Chinese Hamster V79 cells, and its modulation by chloroquine was assessed as described previously (Snyder & Arnone, 2002 and references therein).

Results For simplicity, the term ‘‘steroid’’ used throughout this paper will refer to both steroids and glucocorticoids although the two are not necessarily considered structurally or functionally equivalent. Table 1 provides the structures of those steroids included in this study along with an indication of clastogenicity derived primarily from a recent review by Joosten et al. (2004). For ease of presentation, the steroids are presented in groups defined by the presence and position of ring double bonds. Also for simplicity, complex substitution at position 17 of some steroids is described in abbreviated form. Table 2 provides the structures of the Vitamin D analogs used in the study. These tables demonstrate the structural complexity

Drug Chem Toxicol, Early Online: 1–8

which has contributed to the difficulty in identifying an SAR around genotoxicity. Table 3 contains data from 52 steroidal molecules subjected to computational docking and 26 additional molecules, which were not initially considered for the docking studies since they were not included in the MDDR database employed. Existing data with respect to genotoxicity, and aqueous solubility is presented. Of those steroids with available genotoxicity data, 47% (27/58) were positive in in vitro chromosome aberration assays. Additionally, 50% (20/40) exhibited in vivo genotoxicity. Twenty eight of 36 (77%) exhibited consistent findings, positive or negative, for the in vitro and in vivo assays. It is noteworthy that the HPBL system appears to be somewhat more responsive to steroid-induced aberration induction than are the mammalian cell lines (CHO, V79, SHE) with 56% (22/29) and 38% (10/26) positives, respectively. However, of 13 steroids which were tested in both systems 11/13 (85%) showed concordant results. Having a complete dataset from one or the other assay system would facilitate determination of an SAR for clastogenicity. Figure 1 indicates that of those non-clastogenic molecules for which solubility data was available, 96% (23/24) had aqueous solubility less than 60 mg/mL. By contrast, among genotoxic steroids, 45% (9/20), showed aqueous solubility of 60 mg/mL and higher. Across the entire dataset, there is only one obvious SAR linking structure to genotoxicity. All nine of those molecules with all 3 double bonds in the A-ring, and, thus, carrying the 3-OH rather than the keto group, (estradiols, estriols, estrones) were positive clastogens. Absence of 1 or 2 of those double bonds and presence of the 3-keto reduced the positivity to, in both cases, 37% (6/16). This would not seem to be explainable solely on the basis of aqueous solubility. Attempts at elucidating relationships between other structural features and genotoxicity were unsuccessful. DNA and topoisomerase binding are also shown in Table 3. For those compounds that were docked, a mean overall docking value across all 10 DNA sites is shown using both Surflex and Autodock. The average of this mean across all steroids tested was 3.68 for Surflex and 6.75 for Autodock. These values compare to an overall mean for all 1370 docked molecules of 6.2 and 5.7, respectively (see appendix ref 1). In other words, the steroids as a class, bound DNA more poorly than the global MDDR population of molecules, when docked using Surflex (which is more dependent on shape and structure), but appreciably better than the global population when using Autodock (which is more dependent on electrostatics). Looking at topoisomerase binding, the average of means for steroids was 6.6 and 8.9 compared to the global values of 7.5 and 6.0 for Surflex and Autodock, respectively. Again, while steroids generally bind topoisomerase poorly in a structure-dependent model (exceptions are evident in Table 3), they bind very strongly in a charge-dependent model. In fact, the steroids (and Vitamin D analogs, see below), are the highest binding molecules of the entire set of 1370 docked drugs examined (Snyder et al., 2013). Unfortunately, however, there is no apparent simple relationship between the strength of topoisomerase binding and steroid genotoxicity even when aqueous solubility is also

Mechanisms of steroid clastogenicity

DOI: 10.3109/01480545.2014.928725

3

Table 1. Steroid structures evaluated in this study employing conventional ring-numbering system.

11 1 2

R2

16

14

8 5

4

17

9

10

3

18

13

R

19

R1

R

12

15

7 6

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. Double Bonds None Androstane Androstanolone Oxandrolone Oxymetholone Ursodiol 1–2;3–4;5–10 Estrone Estriol Estradiol Ethynyl estradiol Mestranol Cyclodiol Cyclotriol 2 4 OH estradiol 1–2;4–5 Atamestane Beclometasone Budesonide Ciclesonide Clobetasol Flunizolide Deflazacort Alclometasone Betamethasone Dexamethasone Depredone Exemestane Fluocinonide Fluticasone Halometasone Methylprednisolone Mometasone Prednicarbate Prednisolone Prednisone Halobetasol Triamcinolone 1–2 only Finasteride Dutasteride 4–5 only Drospirenone Desogestrel Dimethisterone Fluoxymesterone Formestane Hydrocortisone Levonorgestrel Medroxyprogest

3

6

9

11

16

keto keto keto OH OH OH OH OH methoxy

OH

OH OH keto keto keto keto keto keto keto keto keto keto keto keto keto keto keto keto keto keto keto keto keto keto

17

18

19

OH OH/C C/OH Pentenoate

C C C C C

C C C C C

keto OH OH OH/Ethynyl OH/Ethynyl Cyclopentane Cyclopentane OH

Other

Clastogenicity

2-O 2-C¼COH 7-OH

? ? Pos Neg/Neg Neg/Neg

C C C

Pos/pos C Pos Pos/Pos Pos/Pos H Pos H Pos H Pos H Pos

C C

keto C(O)CO complex complex C(O)CCl complex complex OH/C(O)COH OH/C(O)COH OH/C(O)COH OH/C(O)C Keto complex OH/C(O)SCF OH/C(O)COH C(O)COH OH/C(O)CCl OH/C(O)COH OH/C(O)COH OH/C(O)COC OH/C(O)CCl OH/C(O)COH

C C C C C C C C C C C C C C C C C C C C C C

C C C C C C C C C C C C C C C C C C C C C C

keto keto

C(O)N-tertbu C(O)N-3FCphen

C C

C C

4-N 4-N

C-Pos C-Neg

keto

carbolactone

C

C

6,7 and 15, 16 cycloprop

H-Neg

OH/Ethynyl OH/Ethinyl OH/C keto-ethynyl OH/C(O)COH OH/Ethynyl OH/C(O)COH

CC C C C C CC C

Cl F F F F C¼C F F F C

F F F Cl

F

F F

C OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH keto OH OH

keto keto keto keto keto keto keto

C F

OH OH

C

C C C C

C C C

C OH

C C C C C

1-C

7-Cl

2-Cl

4-OH

H Neg C-Neg H Neg H Neg C-Neg C-Neg Pos ? H-Pos H-Pos ? H¼Pos H-Neg Neg/Neg ? ? C-Neg ? Pos/Pos Pos Neg C-Neg

C-Neg H-Neg H-Pos ? H-Pos C-Neg Pos/Pos (continued )

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R. D. Snyder et al.

Drug Chem Toxicol, Early Online: 1–8

Table 1. Continued

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Double Bonds Methyl testost Nandrolone Nesterone Norelgestromin Norethisterone Norgestimate Norgestrel Progesterone Testosterone Spironolactone Eplerenone 4–5;9–11 Anecortave 4–5;9–10 or 11–12 Dienogest Mifepristone Gestrinone Trimegestone Trenbolone Lilopristone Onapristone 4–5;6–7 Canrenone Chlormadinone Cyproterone Megestrol Nomogestrol Dydrogesterone 2–3 only Trilostane 5–10 only Norethynodrel Tibolone

3

6

9

11

17

18

19

OH/C OH OC(O)C/C(O)C OH/Ethynyl OH/Ethynyl Ethynyl OH/Ethinyl Acetyl OH carbolactone oxylactone

C C

C

CC C CC CC C C C C

C C C C

OH/C(O)COC(O)C

C

C

OH/cyanomethyl OH/Methylethynyl OH/ethynyl C(O)C(O)C OH OH/C¼CCOH OH/CCCOH

C C CC C C C C

spiro-lactone OH/C(O)C OH/Acetyl OH/Acetyl OH/C(O)C C(O)C

C C C C C C

C C C C

OH

OH

C

C

keto keto

OH/Ethinyl OH/Ethinyl

C C

keto keto keto oxime keto oxime keto keto keto keto keto

16

C¼C

keto keto keto keto keto keto keto keto keto keto keto keto keto keto

NDMP*

NDMP NDMP

Cl Cl Cl

Other

Clastogenicity

7-acetylthio 9–11 Epoxy

? Pos/Pos H-Neg H-Neg H-Pos ? H-Pos H-Neg H-Neg H-Neg C-Neg ? H-Neg C-Neg ? Pos/Pos Neg/Neg H-Neg H-neg H-Neg/C-Pos H¼Pos H-Pos/C-Neg H-Neg ? ?

C 4–5 epoxy 2-carbonitrile 7-C

? H-Pos Neg

*N-dimethylphenyl. Clastogenicity data is also included: ? ¼ no data available; H is HPBL chromosome aberration assay; C is CHO, V79, or SHE chromosome aberration assay.

taken into account. Steroids also display no strong sequence preference for any of the DNA intercalation sites when assessed in Surflex or Autodock (not shown). The Vitamin D analogs are B ring-open analogs of steroids (Table 2). Vitamin D analogs are classified as fat- soluble vitamins and all but doxercalciferol have aqueous solubility below 10 mg/mL. As shown in Table 4, of the nine analogs evaluated for DNA and topoisomerase docking, only doxercalciferol is known to be genotoxic. However, an incomplete dataset is available with only four of nine compounds having chromosome aberration data. As with the steroids, DNA binding scores were higher using Autodock than with Surflex, but strong topoisomerase binding was apparent in both models. Again, no DNA sequence selectivity was observed with either docking model (not shown). We have previously demonstrated that, in some cases, chemical treatment-induced formation of micronuclei in in vitro systems can be antagonized by catalytic inhibitors of topoisomerase II (Snyder & Arnone, 2002; Snyder, 2000). This is interpreted as indicating the likelihood that the original clastogenicity observed required a catalytically active topoisomerase. As shown in Table 5, the clastogenicity of dexamethasone was strongly antagonized by the presence of chloroquine, a catalytic inhibitor of topoisomerase II in V79

cells at concentrations of both agents which did not appreciably reduce cell proliferation (as evidenced by lack of reduction in binucleated cells; not shown). While not definitive, these findings are consistent with the hypothesis that dexamethasone exerts its clastogenicity via topoisomerase inhibition. The use of additional in vitro cell-based studies is clearly required to confirm and expand these findings.

Discussion It is very likely that the genotoxicity of steroids occurs through more than a single mechanism as there is a great deal of structural complexity off of the steroid scaffold. Clearly, active oxygen would be expected to be involved in the genotoxicity of some molecules and DNA adduct formation via the ethynyl and other constituents is also expected. Hydrophobicity, as previously suggested (Dorn et al., 2007) and as confirmed herein, must certainly also be a factor. This would seem to be the case with the Vitamin D analogs as well, which, while equaling the steroids in both computational DNA and topoisomerase binding, lack any genotoxicity, with the exception of doxercalciferol. Consistently, doxercalciferol has some aqueous solubility (about 50 mg/mL) whereas all the others have aqueous solubility below 10 mg/mL. It must be

Mechanisms of steroid clastogenicity

DOI: 10.3109/01480545.2014.928725

Table 2. Vitamin D analog structures evaluated in this study employing conventional ring-numbering system.

CH3

22

28

H3C

21 20

CH3

12

18

11

8

CH3 26

CH3 27

16

14

15

7

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25

17

13

9

10

4 3

HO

23

24

CH2 19

1 2

. 1 Doxercalciferol Calcitriol Calcipotriol Alfacalcidol Tacalcitol Maxacalcitol Paracalcitol Falecalcetriol Secalciferol

22

OH OH OH OH OH OH OH

O

22–23 db YES NO YES NO NO NO YES NO NO

25

28 C

OH OH/CYCLPR OH OH OH OH/di-TFM* OH

C OH

*Di-trifluoromethyl.

tentatively concluded that, in the absence of information to the contrary, the low aqueous solubility of these fat-soluble vitamins may be the primary contributing factor to their lack of genotoxicity. The results of the docking studies reported herein, provide another possible mechanism of steroid genotoxicity: DNA intercalation with consequent topoisomerase inhibition. The speculation that steroids can intercalate into DNA has been around for some time (Hendry et al., 2007, for review). Cohen et al., had shown in 1969 that steroids had favorable shape and thermodynamics to bind DNA. By 1977, it was appreciated by Hendry et al. (1977), that a stereochemical complementarity existed between DNA and small molecules such as estrogens and subsequent studies by these investigators demonstrated conclusively that this stereocomplementarity (1) allowed for intercalation, (2) correlated with biological activity, (3) most likely had an evolutionary gene regulatory role, and (4) could be put to advantage in the design of novel nuclear receptortargeted drugs (Hendry et al., 1986, 1994; Hendry & Mahesh, 1995; Hendry, 1988). We have previously evaluated three-dimensional computational DNA docking as a model for non-covalent DNA interaction, primarily intercalation (Snyder et al., 2004, 2006). The present studies expand this assessment, indicating that steroids, and vitamin D analogs, in fact, computationally bind both DNA and topoisomerase to a much greater extent than

5

do most other structural classes of molecules. The observation that the DNA binding was stronger in Autodock than in Surflex, suggests that perhaps it is the distribution of charge rather than the general space-filling shape of the steroids that facilitates binding. However, extrapolation of this conclusion to the actual biological situation is hampered by the fact that the intercalation templates chosen for these studies were not optimized for steroids. For the same reason, the lack of a sequence dependency for steroids as might be predicted from modeling studies which demonstrated a preference of steroids for a 50 -TG-30 -50 -CA-30 site (Chandrasekhar et al., 1987; Sidell et al., 2005) is also not unexpected. Thus, while the pocket in the crystal structure of the anthracycline (for example)- DNA complex may allow for the binding of other molecules, such as steroids, this pocket is not optimized for those structures. Thus, the data presented herein show only that steroids and Vitamin D analogs can intercalate into some unwound sequences in which other molecules have been shown to intercalate; no conclusions can be made with respect to optimal sequences for such intercalation. A relative estimation of the strength of this intercalative binding is made in comparison to planar acridines (3 fused rings), anthracyclines (four fused rings), and phenanthrenes (the closest structural similarity to steroids bearing a scaffold consisting of the A, B and C steroid rings), which have Autodock DNA binding means of approximately 6.8, 5.7 and 8.1, respectively (Snyder et al., 2013). As described above the mean autodock binding of the steroids was 7.75, very close to that of the phenanthrenes and well above the global mean of 5.7. In vitro micronucleus studies in V79 cells (Table 5) indicate that the clastogenicity of dexamethasone is effectively ablated in the presence of a catalytic topoisomerase inhibitor, chloroquine. We have previously concluded that ablation of the clastogenicity of other structurally diverse molecules likely reflects a requirement that an active topoisomerase be present in order to generate a clastogenic response (Snyder, 2000, 2007). So, how can the docking data generated herein help explain the genotoxicity of steroids? Several non-mutually exclusive mechanisms can be envisioned: First, steroids might intercalate directly into DNA and, depending on the strength of that association, inhibit the normal functioning of the topoisomerase in much the same way as do the clastogenic anthracyclines, bioflavonoids and fluoroquinolones. Docking and earlier modeling data indicate that intercalation is possible, and in fact, likely. Interference with topoisomerase action by steroids has also been previously postulated (Joosten et al., 2004). That no apparent association occurs between computational DNA binding affinity and genotoxicity, precludes a simple explanation, but this might relate to physico-chemical properties such as aqueous solubility. Second is the possibility that the steroid binds to a receptor protein which is then carried to an estrogen response element in the DNA. The complex might then drop off of the DNA after inserting the steroid into the DNA. This would have the same consequence as in the first scenario but would predict that clastogenic breakpoints would be localized to ER sites. This would be testable by fluorescent probing of micronuclei or metaphase spreads for the presence of ER elements.

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R. D. Snyder et al.

Drug Chem Toxicol, Early Online: 1–8

Table 3. Summary of steroid docking and genotoxicity findings.

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Steroid 16-alpha oh estrone 2-methoxy estrone Alclometasone Androstane Androstanolone Anecortave Atamestane Beclomethasone Betamethasone Budesonide Canrenone Chlormadinone Ciclesonide Clobetasol Cyclodiol Cyclotriol Cyproterone Deflazacort Depredone Desogestrel Dexamethasone Dienogest Dimethisterone Drospirenone Dutasteride Dydrogesterone Eplerenone Estradiol Estradiol 2-OH Estradiol 4OH Estriol Estrone Ethinyl estradiol Exemestane Finasteride Flunisolide Fluocinolide Fluoxymesterone Fluticasone Formestane Fulvestrant Gestodene Halobetasol Halometasone Hydrocortisone Levonorgestrel Lilopristone Medroxyprogesterone Megestrol Mestranol Methylprednisolone Methyltestosterone Mifepristone Mometasone Nandrolone Nesterone Nomegestrol Norelgestromin Norethindrone Norethynodrel Norgestimate Norgestrel Onapristone Oxandrolone Oxymetholone Polyestradiol Prednicarbate Prednisolone

Surflex mean Mean

Auto Mean

Sur Topo

Auto Topo

In vitro genotox

In vivo positive genotox

H20 sol (mg/mL)

SHE neg SHE-Neg 4.2

5.6

3.3

10.9

neg Ames-Neg

2.4 2.9

6.5 6.6

6.41 10

8.3 10.3

4.3 4.1 4

6 6.6 7.6

5.9 7.9 10.2

9 9.3 9.9

4.3

8.1

6.3

3.7 3.2 3.2 3.3 5.7 4.5

7.1 6.3 6.4 8.2 5.4 7

4 6 7.8 6.7 8.6 6.2

8.7 9.2 8.4 8.8 6.2 8.4

2.8 3.7

7.3 7

4.6 5.4

9.1 9

3.1 5.3

7 6.8

2.9 7.6

8.5 7.5

4.9

6.4

6.8

7.3

10

2.8 0.9 2.8 3.2

6.7 6.6 5.8 5.8

6.5 4.6 5.8 6.7

8.5 8.2 8.8 8.8

2.6 3.12 6.6

6.5 6.9 6.7

5.4 7.5 9.4

8.8 8.6 8.1

2.4 2 4.7 3.8

5.8 4.9 6.7 7.8

4.9 3.5 8.3 6.2

7.4 6.2 9.7 8.7

3.2 3.5

7.1 7.5

4.6 5.8

9.2 9.3

3.5 2.5 5.6 3.4 6.1

6.3 6.8 9.1 6.5 8.1

5.6 5.6 8.1 2.3 10.5

10.6 8.5 9.7 8.4 11.4

5.2 4.2 3.7

8.5 7.7 7.8

7.7 6.9 5.6

10.2 8.5 8.5

4.4

7.9

7.2

10.1

2.5 5.3 3.9 2.2

6.3 6.8 6.1 5.4

6.2 6.6 7.6 9.4

8.6 7.5 9.1 9.6

9.00 MLA-Neg HPBl-Neg CHO-Neg HPBL-Pos HPBL-Neg HPBLNeg; CHO-Pos HPBL-Pos HPBL-Neg CHO-Neg HPBL Pos HPBL Pos HPBL-Pos, CHO-Neg HPBL-Pos CHO-Neg HPBL-Pos HPBL-Neg HPBL-Neg HPBL-Neg CHO-Neg CHO-Neg HPBL, CHO-Pos, V79-Pos SHE-Pos SHE-Pos CHO-Pos HPBL, CHO-Pos HPBL, CHO-Pos HPBL-Pos CHO-Pos CHO-Neg HPBL-Neg HPBL-Pos HPBL, CHO-Neg

Neg Neg Pos Pos Pos Pos Neg pos pos Pos

6.00

Pos Neg

0.2–3 89.00 0.60

Neg Neg Equivocal Neg Pos

Pos Pos Neg Pos Pos Neg

HPBL-Neg Neg Abs HPBL-Pos CHO-Neg HPBL-Neg HPBL, CHO-Pos HPBL-Neg HPBL-Pos CHO-Neg CHO-Neg HPBL-Pos HPBL-Neg HPBL-Neg HPBL-Pos HPBL-Pos

49.00 66.00 10.70 1.50 3.80

2.00 1.00 15.00 3.60 400.00 30.00 11.30 6.00 11.70 10.00 9.70 67.00 0.50 6.00

Neg Pos Pos

280.00 2.00

Neg Pos Pos

3.00 3.00 3.70 120.00

Neg

3.00 5.00 60.00

Neg Pos Neg Neg

6.00 7.00 40.00

HPBL-Pos HPBL-Neg HPBL-Pos HPBL, CHO-Neg

5.00

Neg

16.00 5.00

HPBL, CHL-Pos

Neg Pos

5.00 220.00 (continued )

Mechanisms of steroid clastogenicity

DOI: 10.3109/01480545.2014.928725

Steroid Prednisone Progesterone Propylmesterolone Spironolactone Testosterone Tibolone Trenbolone Triamcinolone Trilostane Trimegestone Ursodiol

Surflex mean Mean

Auto Mean

Sur Topo

Auto Topo

2.8 2.5

2.5 7

7.7 6.6

9.1 9.3

2.4 5.8 3.1

7 8 7

4.5 8.9 6.5

9.7 10.9 9.3

3.7 2.8 4.6

5.8 6.3 7.7

7.4 6.9 7.6

7.9 8.1 8.8

7

In vitro genotox

In vivo positive genotox

H20 sol (mg/mL)

CHL-Pos HPBL-Neg HPBL-Neg HPBL-Neg HPBL-Neg CHO-Neg HPBL, CHO-Neg CHO-Neg

Neg Pos Neg

312.00 5.00 22.00 23.00

Neg Neg Neg Neg

20.00 80.00 4.70

HPBL, CHO-Pos Neg Abs, Ames

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Original data for docking is found in the Appendix to reference one. In vitro and in vivo genotoxicity data is taken from Joosten et al. (2004) as well as the Physicians’ Desk Reference data on aqueous solubility was from Drug Bank and other miscellaneous online sources.

14

Table 5. Antagonism of Dexamethasone-induced micronuclei in V 79 cells by chloroquine.

12 10

Treatment

8

Control Dexamethasone 100 mM 50 mM 25 mM 10 mM Chloroquine 34 mM

6 4 2 0 0 to 5 µg/mL

5.1 to 60 µg/mL

>60 µg/mL

Figure 1. Relationship of steroid aqueous solubility to genotoxicity. The distribution of genotoxicity- positive (black bars) and negative (gray bars) steroids at aqueous solubilities of 0–5 mg/mL, 5.1 to 60 mg/mL and 460 mg/mL is presented. Data taken from sources noted in Table 3.

Table 4. Summary of Vitamin D analog binding data and genotoxicity information. Surflex Auto Vitamin D analog Alfacalcidol Calcipotriol Calcitriol Doxercalciferol Falecalcitriol Maxacalcitol Paricalcitol Secalciferol Tacalcitol

Mean 3.6 4.2 4.2 3.8 3.6 4 3.3 3.6 4.5

Sur

Auto

Mean Topo Topo 7.2 7.2 6.8 7.8 7 8.8 10.1 6.7 6.8

9.2 9.3 8.6 7.8 9.6 9.6 7.6 9 9.8

Genotoxicity

9.2 10.2 Neg Chrom Abs 8.9 9.8 Pos Chrom abs 7 8.8 10.1 Neg chrom abs 9.2 9.1 Neg Chrom Abs

Vivo Neg Neg Neg Neg Neg Neg Neg Neg

It is interesting in this regard, however, that the most potent anti-estrogen yet discovered, para-hydroxy-3-phenylacetylamino-2,6-piperidinedione was synthesized for its ability to bind to the consensus estrogen DNA binding sequence rather than to the receptor (Sidell et al., 2005). Third is the possibility of interactions between the steroid and topoisomerase or topoisomerase/DNA complex. It must be kept in mind here that the data generated in this paper relate to specific binding of molecules to the ATP binding site, of human topoisomerase. One must be cautioned against over interpretation of these data as indicating a functional effect of such binding on topoisomerase activity.

Chloroquine + 100 mM Dex Chloroquine + 50 mM Dex Chloroquine + 25 mM Dex

Percent binucleated cells with MN (N ¼ 3)

Fold control MN cells

1.2 ± 0.6 17.6 ± 4.8 12.8 ± 4.9 6.4 ± 2.6 3.3 ± 1.2 2.2 ± 0.7

100 14.6 10.6 5.3 2.8 1.8

N¼3

Percent Dex alone MN

4.1 ± 2.2 3.3 ± 1.7 2.5 ± 1.1

23 26 40

V79 cells were treated as indicated for 3 h. Fresh medium containing cytochalasin B was added and the cells were harvested after approximately 18 h.

Further important information would most certainly be obtained from docking of steroids into the DNA/topoisomerase binary complex to more closely represent the actual site of interaction. That having been said, there are some intriguing observations that may be germaine. The piperidinedione mentioned above carries the same dione terminus as the bisdioxopiperazines (e.g. ICRF-187), maleimide (Jensen et al., 2002), mitindomide (Hasinoff et al., 1997), rebeccamycin (Long et al., 2002), and merbarone (Fortune & Osheroff, 1998), all of which are catalytic topoisomerase inhibitors known to bind to the ATP binding site of that enzyme (Classen et al., 2003; Hu et al., 2002). Several chemically-related pyrazole and tetrahydroindazole N-aryl ketones are also topoisomerase inhibitors (Gomez et al., 2007; Wiener et al., 2007). Thus, this dione terminus may play a crucial role in blocking ATP hydrolysis and may, therefore be a genotoxic determinant. As a matter of further interest, clastogenic cytidine, guanosine and thymidine analogs also possessing similar Northo ketone structures, also bind at the topoisomerase ATP binding site as evidenced by high docking scores (Snyder et al., 2013) and, while their clastogenicity has rightfully been ascribed to alterations in DNA synthesis and repair, it is reasonable to now ask if there may also be ATP-mimetic

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R. D. Snyder et al.

competition at the topoisomerase ATPase site. In fact, some substituted purines have been shown to be topoisomerase inhibitors (Jensen et al., 2005). Additional direct evidence for steroid-topoisomerase interaction is weak but this has not been examined to any great extent. Bufalin, a steroid constituent of a traditional Chinese herbal medicine, is a catalytic topoisomerase inhibitor, although it is not clear if this relates to ATPase activity as this was not specifically evaluated (Pastor et al., 2002). Other cardiotonic steroids such as ouabain and digoxigenin are known to bind to and inactivate Na,K-ATPase (Cornelius & Mahmoud, 2008; Herman et al., 2010). A crystal structure is available for the ouabain/shark Na-K-ATPase complex but the structural relatedness of these two ATP binding sites is not known. In summary, both steroids and vitamin D analogs appear to be able to bind non-covalently to DNA and human topoisomerase. While the former finding is no surprise, predicted binding to topoisomerase was unexpected. The lack of a strong relationship between binding to either target and genotoxicity diminishes confidence that such non-covalent interactions are important in steroid genotoxicity. However, additional cell-based micronucleus studies using catalytic inhibitors and non-cell based studies with purified topoisomerase in the presence of steroids would be important next steps in understanding if the in silico docking experiments are biologically relevant.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Evidence for the contribution of non-covalent steroid interactions between DNA and topoisomerase in the genotoxicity of steroids.

Fifty two steroids and 9 Vitamin D analogs were docked into ten crystallographically-defined DNA dinucleotide sites and two human topoisomerase II ATP...
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