Deciphering the Nongenomic, Mitochondrial Toxicity of Tamoxifens As

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Deciphering the non-genomic, mitochondrial toxicity of tamoxifens as determined by cell metabolism and redox activity. Theodossis Athanassios Theodossiou, Sebastien Wälchli, Cathrine Elisabeth Olsen, Ellen Skarpen, and Kristian Berg ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00734 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 24, 2015

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ACS Chemical Biology

Deciphering the non-genomic, mitochondrial toxicity of tamoxifens as determined by cell metabolism and redox activity.

Theodossis Athanassios Theodossiou1*, Sebastien Wälchli2, Cathrine Elisabeth Olsen1, Ellen Skarpen3 and Kristian Berg1. 1

Department of Radiation Biology, Institute for Cancer research, The Radium

Hospital, Oslo University Hospital, Montebello, Oslo, 0379, Norway 2

Department of Immunology and Department for Cellular Therapy, Institute for

Cancer research, The Radium Hospital, Oslo University Hospital, Montebello, Oslo, 0379, Norway 3

Department of Biochemistry, Institute for Cancer research, The Radium Hospital,

Oslo University Hospital, Montebello, Oslo, 0379, Norway *Correspondence: [email protected]

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Abstract Tamoxifen is not only considered a very potent chemotherapeutic adjuvant for estrogen receptor positive breast cancers, but also a very good chemo-preventive drug. Recently there has been a rising amount of evidence for a non-genomic cytotoxicity of tamoxifen, even in estrogen receptor negative cells, which has greatly confounded researchers. Clinically the side-effects of tamoxifen can be very serious, ranging from liver steatosis to cirrhosis, tumorigenesis or onset of porphyrias. Herein, we deciphered the non-genomic, mitochondrial cytotoxicity of tamoxifen in estrogen receptor positive MCF7 versus triple-negative MDA-MB-231 cells, employing

the

mitochondrial

complex

III

quinoloxidising-centre

inhibitor

myxothiazol. We showed a role for hydroxyl-radical-mediated lipid peroxidation, catalyzed by iron, stemming from the redox interactions of tamoxifen quinoid metabolites with complex III, resulting in Fenton-capable reduced quinones. The role of tamoxifen semiquinone species in mitochondrial toxicity was also shown together with evidence of mitochondrial DNA damage. Tamoxifen caused an overall metabolic (respiratory and glycolytic) rate decrease in the Pasteur type MCF cells while in the Warburg type MDA-MB-231 cells the respiratory rate was not significantly affected and the glycolytiv rate was significantly boosted. The non-genomic cytotoxicity of tamoxifens was hence associated to the metabolic phenotype and redox activity of the cells, as in the present paradigm of Pasteur MCF7s versus Warburg MDA-MB-231 cells. Our present findings call for caution in the use of the drug, especially as a chemopreventive and /or in cases of iron overload diseases.


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Introduction Tamoxifen (TAM) is a triphenylethylene nonsteroidal selective estrogen receptor modulator (SERM) used both in the treatment of primary

1

and metastatic breast

cancer 2. TAM was also approved in the US as a chemopreventive agent for the decrease of breast cancer onset in high-risk groups 3, although this is an issue of controversy due to the drug’s serious side effects, most of which are related to hepatic dysfunction and disease 4-12. SERMs like TAM are estrogen antagonists and have a high affinity for estrogen receptors (ER), comparable to those of 17β-estradiol (E2)

13

. ER are transcription

factors that regulate the expression of estrogen responsive genes; in this sense when TAM and its metabolites bind to them they trigger genomic effects like cytostasis due to induction of a G1 cell cycle block

14

, or cell apoptosis e.g. due to transcriptional

regulation of Bcl-2 family proteins 15. TAM induced cell death has also been observed in ER negative cell lines as well however, and with various modes of death

16-20

. Ιn

MCF7 cells the mode of death seems to be associated with the dose administered 21; an acute cytotoxicity not inhibitable by estradiol (E2) leading to necrotic death was observed for doses over 10 µM whilst a gradual death with onset at day 3 was registered for doses ≤ 1 µM. It has been reported that, TAM related cell death in MCF7 cells is associated with the induction of autophagy causes, like high concentrations of Zn2+

22

21-23

, attributed to various

or mutant KRAS degradation 23. The role

of autophagy observed following TAM administration is not clear; even though aberrant autophagy stimulation can eventually promote cell death mechanisms 24, the primary function of autophagy is that of a pro-survival cellular mechanism. Several recent studies show TAM-related toxic effects in cell mitochondria 20, 25, 26. In addition, TAM has been postulated to cause collapse of the mitochondrial membrane



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potential and inhibit electron transfer at the electron transport chain (ETC) complex III in isolated cell mitochondria

27

. Furthermore, structural changes to the

mitochondrial membranes have been suggested to explain the adverse effects of TAM on oxidative phosphorylation (OXPHOS)

28

. In fact, in a recent review

29

, the

mitochondria have been proposed as the gateway for tamoxifen-induced liver injury. TAM and its metabolite 4-hydroxytamoxifen (4-OHT) were found to incorporate strongly into membranes, like in isolated sarcoplasmic reticulum vesicles 30, as well as biomimetic model membranes structural properties

31

, inducing modifications in their physical and

32

. TAM appears to specifically concentrate in the hydrophobic

membrane core, perturbing the lipid bilayer structure and modulating the membrane fluidity

31, 32

. This membrane disruption has been elsewhere reported to lead to

hemolysis of human erythrocytes 33. In our recent publication

25

, we showed that TAM localizes in cell mitochondria in

both MCF7 and DU145 ER positive cells. When administered following inhibition of mitochondrial respiration mainly at the quinoloxidizing centre (qo) of complex III by myxothiazol (MYXO), TAM induced severe and rapid cytotoxicity to MCF7 but not DU145 cells. The observed MCF7 cytotoxicity could not be of a genomic nature because of its rapid onset and severity, and although both cell lines were ER positive there was no analogous effect in the DU145 cells. In addition, the synergistic cytotoxicity seen in the MCF7 cells was not responsive to treatment with E2. In general the overall frame of TAM cytotoxic and cytostatic effects is somewhat hazy. There seem to be two pharmacological effects of TAM interweaved in its action, one genomic maybe antiproliferative or even apoptogenic and one non genomic and cytotoxic also relevant to ER negative cells.


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In the present work we undertook to study the TAM and MYXO cytotoxic synergism in MCF7 cells using the triple negative breast cancer cell line MDA-MB-231 in an ancillary fashion, in an attempt to elucidate the mechanism behind the observed rapid, non-genomic cytotoxicity. In addition the two cell lines were selected because of their different metabolic phenotypes: Pasteur (MCF7) versus Warburg (MDA-MB231) 34, 35. MATERIALS AND METHODS The detailed methods and experimental procedures can be found in the supporting information. RESULTS AND DISCUSSION The impact of MYXO on TAM cytotoxicity and its intracellular localization. In view of our recent results 25, we set out to further explore the observed synergistic action of TAM with ETC complex III quinoloxidizing centre (Qo) inhibitor MYXO in MCF7 cells. Initially we examined the dose response of both TAM and MYXO with respect to their synergistic cytotoxicity (Fig. S1 A, B) and concurred to an optimal strategy of 15 µM TAM and 1.4 µM MYXO, administered simultaneously. We also verified the lack of synergy in MDA-MB-231 cells as is shown in Fig. S1 C. In order to explore the divergent effects of TAM and MYXO in MCF7 and MDAMB-231 cells the subcellular localization of TAM was investigated in the two cell lines using NDMTAM-FITC (see supporting info. Fig. S2) in the presence and absence of MYXO. Representative confocal images in Fig. S2 A, B, revealed a profound co-localization of NDMTAM-FITC with MitoTracker® Deep Red FM in both cell lines. Nevertheless, it could also be seen that NDMTAM-FITC exhibited a residual extra-mitochondrial cytoplasmic distribution, possibly engaging cytoplasmic 5 ACS Paragon Plus Environment


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antiestrogen targets. In both MCF7 and MDA-MB-231 cells the co-treatment with MYXO made no clear difference in the TAM localization. Although we registered a strong mitochondrial NDMTAM-FITC localization in the ERβ-negative MDA-MB231 we additionally investigated whether the observed mitochondrial TAM accumulation in MCF7, which proved to be unaffected by co-incubation with MYXO, might have been linked to a mitochondrial localization of ERβ as previously suggested

36-38

. To this end we performed anti-ERβ immunofluorescence on MCF7

cells (supplementary data; Fig. S3). The representative confocal microscopy images showed a primarily nuclear along with a secondary, much smaller cytoplasmic localization of ERβ. No apparent mitochondrial colocalization was observed either in controls, or following treatment with TAM (15 µM), MYXO (1.4 µM) or TAM+MYXO, overnight. Cell treatment with leptomycin B, an inhibitor of nuclear export, notably increased the nuclear build-up of ERβ. These results together with the prominent mitochondrial localization of TAM in MDA-MB-231 which lack estrogen receptors, suggested that TAM is mitotropic because of its physicochemical properties (lipophilic, cationic and a triphenylethylene moiety bearing resemblance to the mitotropic triphenylphosphonium species) rather than due to its ERβ affinity. Cytotoxic evaluation of strategies combining various TAM metabolites with MYXO. It is well documented that TAM is only active as a Z isomer and further that it is primarily metabolized by cytochrome P450 to 4-Hydroxytamoxifen (4-OHT) and NDMTAM 39. This first level of TAM metabolism is shown in Fig. 1 A. We undertook to study the cytotoxic synergy of TAM primary metabolites and their isomers with MYXO on MCF7 cells. The results are presented in Fig. 1 B; The most efficient metabolite of TAM with respect to cytotoxic synergy with MYXO was found to be (Z)


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4-OHT as compared to TAM, NDMTAM and the least active racemic, (E,Z) 4-OHT. On this basis and unless otherwise specified, the combination of 15 µM (Z) 4-OHT (here forth 4-OHT) and 1.4 µM MYXO will be mainly used and will be refered to as “strategy”. We also subjected the two cell lines to isobologram analysis40 with respect to our strategy (Fig S4, A, B). From this figure it can be seen that in the case of MCF7 (Fig. S4, A) the strategy acts in a synergistic mode, where as in the case of MDA-MB231 (Fig. S4, B) the simultaneous application of 15 µM 4-OHT and 1.4 µM MYXO barely had additive effects. The effect of modulators on the viability of cells treated with a 4-OHT and MYXO strategy. As described above, it appeared likely that the non-genomic, rapid cytotoxic component of TAM action and more so the synergy of TAM and MYXO, was primarily due to a mitochondrial chain of events. Thus, a number of relevant modulators were applied to our 4-OHT+MYXO strategy in MCF7 cells (Fig. 1 C). Modulators inhibiting the quinone-reducing centres of complexes I and III (ROT and ANTI-A respectively) were used one at a time, without completely inhibiting the respiratory activity as well as modulators relating to the respiratory metabolism and ATP production and trafficking (FCCP, OLIGO and ATR, which we will revisit later in the text). In all cases except for co-treatment with OLIGO, the modulators acted as antagonists to the 4-OHT/MYXO strategy. In addition to the previous non-effective application of 17β-Estradiol with MYXO 25, we evaluated FULV (a potent antiestrogen, ICI-182780) both as a modulator of our strategy (Fig. 1 C), but also in lieu of 4-OHT with MYXO (Fig. S5, supplementary information) with null effect in both cases. This further precluded estrogen receptor related i.e. genomic action of our strategy, within our regime timeframe, since



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fulvestrant was previously found to be more genomically efficient than TAM and hence more proapoptotic 15. The abrogation of cytotoxicity by application of the modulators ROT and ANTI-A to our strategy, was immediately suggestive of quinone species implication. 4-OHT may be further metabolized in cells to 3.4-dihydroxytamoxifen to an o-hydroquinone) as well as quinone methide

42

41

(3-4-diOHT, equivalent

and o-quinone species

43

- the

latter two as products of oxidation of 4-OHT and 3,4-diOHT (Fig. 1D) . In our previous work

25

GSH monoethyl-ester application had a clear cytoprotective

effect against TAM+MYXO cytotoxicity. In the present work, the cysteine antioxidant NAC was found to confer a null effect even though employed at a high concentration (4 mM, Fig. 1 C); This points to GSH binding quinone methide and/or o-quinone as suggested elsewhere

42, 43

, for consequent exocytosis via γ-GT, rather than primarily

exerting antioxidant action.

The implication of TAM quinoid metabolites in the MYXO enhanced 4-OHT cytotoxicity in MCF7 cells; 4-OHT is a reductive substrate of complex III The possible implication of quinoid moieties in the cytotoxic action of our strategy led us hypothesize that these species might function as substrates of the quinoloxidizing and quinonereducing centres of complex III, much alike the natural substrate, coenzyme Q10. The possibility of such an implication is graphically portrayed in Fig. 1E and the easier to visualise Fig. S6; the quinoid species would in such an eventuality accept one electron at Qi thus initially becoming semiquinones or semiquinone radicals, due to the enhanced stability of semiquinones at this site

44

.

This electron transfer would be inhibitable by ANTI-A, while a similar electron transfer could possibly take place at the quinonereducing site of complex I, in this


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case inhibitable by ROT. Conversely, the respective reduced quinoid species docked at Qo could lose one (o-semiquinones) or two electrons (o-hydroquinones), which would facilitate cytochrome c1 reduction; this electron stripping would be inhibited by MYXO. We next set out to explore this possibility by employing a methodology we had previously applied to the quinone photosensitizer hypericin 45. Cytochrome c was added to submitochondrial particles (SMPs), to evaluate its heme reduction from ferric to ferrous, by the quinoloxidising centre of complex III, which can be spectrophotometrically recorded (see supplementary information).

In the present

case, since the o-hydroquinone 3-4-diOHT was not readily available, we employed 4OHT itself since it has the possibility to redox-cycle to its respective quinone methide. The results of this study are presented in Fig. 1 F, where also NDMTAM was employed (as a non –OH containing TAM metabolite) and where the initial linear phases of enzyme (i.e. complex III) activity are shown; the slopes by linear fits to these graphs represent the enzyme maximal activity values (Table S1). From the graphs in Fig. 1 F as well as from the extrapolated maximal activity values of Table S1, we could make several useful deductions. Firstly 4-OHT functioned as a reductive substrate of complex III and this enzymatic activity was entirely inhibited by MYXO. Surprisingly, 4-OHT was also able to directly reduce cyt C, nonenzymatically, at ¼ the respective enzymatic rate, presumably due to its OH moiety. This activity could not be inhibited by MYXO (data not shown). These results indicate a close affinity for 4-OHT and/or cyt c to the SMPs, since in the presence of SMPs all cyt c reduction was inhibitable by MYXO. The non –OH containing TAM metabolite NDMTAM exhibited some enzymatic activity (< ½ that of 4-OHT). This was only partly inhibited by MYXO, probably indicating the involvement of complex III independent activity. Nonetheless, NDMTAM was found to be totally incapable of



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non-enzymatic cyt c reduction (Fig. 1 F). On this basis it appears that TAM metabolites directly interact with the quinoloxidizing centre of complex III.

Implication of the Fenton reaction: iron chelators, mannitol and hydroxyl radicals Previous results implicated Zn (II) in Tamoxifen induced autophagy documented for its antioxidant capacity

46

species as suggested by Hwang et. al.

22

22

. Zinc is well

and does not fit the remit of a pro-oxidant

. One well-established property of Zn as a

diamagnetic metal ion however, is the stabilization of semiquinone radicals e.g. in EPR

47

for their easier detection. On a different note, both quinones

semiquinone anion radicals

49

48

and catechol

form metal ion complexes especially with Fenton

reactive metal ions such as Fe (II). The above led us to investigate the influence of iron chelators on our strategy in MCF7 cells. In particular, we employed DFO (predominantly a ferric ion chelator), oPhen (primarily a ferrous ion chelator), their combination and also EDTA for the chelation of extracellular metal ions. The results are shown in Fig. 2 A; both DFO, ophen and their combination yielded a remarkable abrogation of the strategy induced cytotoxicity in relation to the respective chelator controls, while EDTA had no effect. This suggested a cytotoxic role of intracellular Fe ions, quite possibly acting as Fenton reaction catalysts, since the Fenton Reaction results in the formation of OH•. We consequently proceeded with the evaluation of the effect of mannitol, a specific hydroxyl radical scavenger 50, 51. The results are presented in Fig. 2 B; in this graph, it is of great interest that although mannitol abrogated the 4-OHT cytotoxicity in both cell lines, the strategy related cytotoxicity was only abrogated in MCF7 cells and not MDA-MB-231 cells. These results suggested a harmful effect of hydroxyl radicals in


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MCF7 cells treated with 4-OHT (±MYXO). We next strived to record the intracellular localization of hydroxyl radicals in MCF7 cells employing a novel specific hydroxyl radical probe 52. Representative confocal microscopy images are displayed in Fig. 3A; these images reveal a basal hydroxyl-radical signal in control cells which is significantly enhanced in 4-OHT treated cells and even more so in strategy treated cells. In Fig.3B the Pearson’s correlation coefficients between the mitochondrial stain and the hydroxyl radical probe are plotted for all treatment groups. The correlation coefficient significantly increases from the control (~0.1) to the 4-OHT treated cells (~0.55) and even more in the 4-OHT and MYXO group (~0.78). This unequivocally demonstrates the implication of hydroxyl radicals in 4-OHT (± MYXO) mitochondrial damage.

Investigation of lipid peroxidation in strategy treated MCF7 cells Following these results we labelled MCF7 cells for lipid peroxidation using linoleamide alkyne as an oxidation target and utilizing click chemistry for labelling of peroxidation products. The results of incubation for 6 h with 4-OHT ± MYXO appear in the representative images of Fig. 4. A profound increase of lipid peroxidation could be seen for the 4-OHT and strategy treated groups. In both cases the lipid peroxidation

associated

green

fluorescence

was

punctate,

perinuclear and

substantially higher than the basal levels in control cells. Although in both 4-OHT and strategy treated groups the lipid peroxidation associated green fluorescence exhibited only partial co-localization with that of Mitotracker® Deep Red FM it was clearly localized in the mitochondrial vicinity and was interweaved with mitochondrial fluorescence. The lack of complete co-localization could be attributed to the fact that mitochondria with advanced lipid peroxidation (i.e. dissipating their ∆Ψm or losing



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membrane integrity) may not have been able to efficiently retain Mitotracker® Deep Red FM, since functional/intact inner mitochondrial membranes are required for optimal staining. The 4-OHT and strategy associated lipid peroxidation exhibits, however, a completely different subcellular pattern from that following administration of cumene hydroperoxide which seems to primarily affect the plasma membrane (Fig. 4). These results indicate that the observed lipid peroxidation pattern is treatment specific.

The role of ATP related modulators, respiration and cell metabolism. In MCF7 cells uncoupling of mitochondrial respiration from ATP production by use of FCCP, and inhibition of adenine nucleotide transport from the mitochondrial matrix by ATR afforded notable protection from the applied strategy, whilst direct inhibition of the F1F0-ATPase by OLIGO exacerbated the cytotoxicity (Fig 1C). These results can be better understood in light of the metabolic phenotypes of the two cell lines employed in the present study. For this purpose we probed the oxygen consumption rates (OCR) and media acidification rates (MAR) of both MCF7 and MDA-MB-231 cells treated with 4-OHT (15 µM), MYXO (1.4 µM) or strategy for 0, 7 and 24h using the XFe96 Seahorse Analyzer (Seahorse BioScience Copenhagen, Denmark) (Fig. 5). From these data the different metabolic profiles of the two cell lines were evident: In normal conditions the OCR of MCF7 cells was 4-5 times higher than that of MDAMB-231 cells, while their media acidification rates corresponding to their glycolytic rates were comparable. In both cell lines the introduction of MYXO almost immediately collapsed the respiratory activity. This caused an elevation in media acidification rates in both cell lines, albeit MDA-MB-231 seemed to have a higher “distress” glycolytic capacity. Upon 4-OHT administration a time dependent


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inhibition of the respiratory capacity was observed in MCF7 cells reaching their 1/3 capacity at 24h, while a small decline in the glycolytic activity was also observed, down to their ¾ capacity at 24h. MDA-MB-231 cells seemed not to respond metabolically to the 4-OHT insult and in general terms they maintained their metabolic rates even slightly increased their glycolytic activity. Upon strategy administration in both cell lines the respiratory rates collapsed while with regard to the glycolytic rates these remained higher than the basal ones for MDA-MB-231 and receded substantially for MCF7 at 24 h following strategy application. The oxygen consumption drop in MCF7 was from ~225 pmole/min down to practically 0 pmole/min, while in MDA-MB-231 the corresponding drop was from ~60 pmole/min to ~5 pmole/min. This metabolic collapse of MCF7 (Fig. S9, circle) could also be one of the reasons of the synergistic cytotoxicity of 4-OHT and MYXO. MCF7 cells perform respiratory ATP production at normoxic conditions and also switch to glycolysis under hypoxia, whilst MDA-MB-231 rely on glycolysis for ATP production in both normoxic and hypoxic circumstances

34, 35

, presumably due to the

considerably lower respiration rates of MDA-MB-231 (Fig. 5). In this context MCF7 cells experienced a significant bioenergetic stress by the combination of MYXO and 4-OHT as this strategy both “chocked” their respiration and suppressed their glycolytic activity. The metabolic charts of MCF7 and MDA-MB-231 following the administration of 4-OHT, MYXO or strategy are shown in Fig. S9 A and B as oxygen consumption rates versus media acidification rates. If we take into account the ATP measurements (Fig. S10 A,B), it seems that MDA-MB-231 cells maintained constant ATP levels, except for their elevation upon MYXO treatment which is in good accordance with the data in Fig. 5 B. With regard to MCF7 cells there was an ATP elevation both after treatment with MYXO and 4-OHT+MYXO at 7h, while the ATP



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levels dropped profoundly in the 4-OHT (± MYXO) groups 24h post-treatment (consistent with the metabolic drops shown in Fig. 5 A). Accordingly, we believe that in the case of strategy application to MCF7 cells and while the cell ATP needs are (to an extend) covered by glycolysis, a ∆Ψm collapse 27 possibly triggers the F1F0-ATPase to function in reverse mode, pumping H+ out of the mitochondrial matrix. At the same time ATR an adenine nucleotide translocase (ANT) inhibitor

53, 54

, traps ATP in the

matrix for the F1F0-ATPase to hydrolize and further fuel its reverse function; it is even possible, since ATR locks the ANT in the cytoplasm side open conformation

54

, that

ANT can also allow glycolysis derived ATP to enter the matrix and be hydrolised by the F1F0-ATPase functioning in reverse, to overcome the energy crisis

55

. The

protonophore FCCP uncouples the respiration from ATP production, at the same time markedly increasing respiration levels, in an effort to pump more H+ out of the matrix. Also, in both MCF7 and MDA-MB-231 cells, FCCP uncoupling pushes the metabolism to anaerobic glycolysis (data not shown). Finally the FCCP protective effect from strategy cytotoxicity, may stem from H+ flooding the mitochondrial matrix, possibly protonating the tamoxifen semiquinone and quinone methide species. There is also of course the eventuality of Fe-ATP complex formation which can be very harmful to mitochondrial membranes 56.

Mode of cell death following 4-OHT and MYXO strategy treatment. We further evaluated the mode of death conferred to MCF7 cells by our synergy. We employed a second viability assay side by side with MTT at 24h following strategy administration. This assay quantified the total lysosomal β-hexosaminidase (HEXO-β) content in cells which directly relates to the cell number. The HEXO-β assay was found to be in good agreement with the back-to-back MTT assays in both cell lines


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showing a 4-OHT MYXO synergy in MCF7 cells but not in MDA-MB-231 cells (data not shown). We subsequently looked at the LDH release from both cell lines (MCF7 and MDA-MB-231) immediately following incubation (24h) to explore the possibility of necrotic death. The results appear in Fig. S7 A and clearly delineate necrosis as a possible mode of death for 4-OHT (± MYXO). However in the case of MCF7 (but not MDA-MB-231 cells) the 4-OHT+MYXO group cell death was higher than that of the 4-OHT group. These results were also in good agreement, as to the lack of apoptosis, with the results from a flow-cytometric TUNEL (TdT/DNA) assay we performed on the TAM/MYXO strategy at 6, 12 and 24 h post incubation, and which showed only a basal level of apoptosis, and no notable differences between control and treatment groups (Fig. S13). Clonogenicity assays were performed on both cell lines treated for 24h with our strategy (Fig. S7B). In the case of MDA-MB-231, approx. 1/3 of the cells survived the 4-OHT treatment while less than 5% of the MCF7 cells survived the same treatment. This is in good accordance with the lack of ER in the MDA-MB-231 cell line and hence lack of a genomic cytostasis, whilst the profound suppression of clonogenicity in the 4-OHT treated MCF7 cells is presumably due to the genomic interaction with the estrogen receptors. Furthermore, MYXO exerted a strong and total suppression of clonogenicity in both cell lines conversely to its mild and subtoxic effects at 24h following treatment (Fig. 6B). The striking conclusion that can be drawn from the data as described above is that although MYXO prevented the oxidation of quinoloid species at Qo and conversely ROT and ANTI-A inhibited the reduction of quinones at Qi which are expected to be deleterious to cells

57

, the treatment responses obtained from the two cell lines

indicated a cytotoxic role for reduced quinoid species. MYXO promoted cytotoxicity in MCF7 cells when combined with 4-OHT, but not MDA-MB-231, whilst ROT and



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ANTI-A, protected from this cytotoxicity although they promoted the intracellular accumulation of (oxidized) quinones. So we set forth to investigate how reduced TAM quinoid species contributed to TAM related cytotoxicity. TAM induced autophagy 23

22,

was also studied by western blots of LC3B (Light Chain 3 protein cleavage, Fig. S8

A). In these blots we observed analogous enhanced autophagy in the TAM and TAM+MYXO groups in contrast to basal autophagy signals in the media control and MYXO-only groups. both in MCF7 and MDA-MB-231 cells. Thus the role of autophagy seems to be limited to recycling damaged material and not regulating the cytotoxicity. That is why application of WORT had no effect on the strategy (Fig. 1 C), notwithstanding the fact that in western blots WORT was found to alleviate 4OHT (± MYXO) related autophagy (Fig. S8 B). This is was somewhat expected since mitochondria were undergoing fatal impairment anyway, so irrespective of autophagy (recycling) they ended up having lost their vital functions. In order to find whether autophagy and/or mitochondrial damage depleted the number of mitochondria in the treated cells, we isolated and amplified the mtDNA from these cells, quantified it with Quant-iT™ PicoGreen® dsDNA Reagent, and compared it to the controls. The results in Fig. S11 A, show that treatment with 4-OHT (15 µM) for 7h consistently produced cells with depleted mtDNA, suggesting mitochondria number depletion. However when applying agarose gel electrophoresis to the isolated mtDNA, we observed laddering in the strategy treated (15 µM 4-OHT and 1.2 µM MYXO for 7h) cells’ mtDNA which suggested direct damage to the mtDNA (Fig. S11 B) most probably due to the TAM quinoid species. In contrast MYXO and 4-OHT single treatments were not found to cause any mtDNA laddering.

Hypothesis testing: Inducing strategy cytotoxicity in MDA-MB-231 by mitochondrial uncoupling and upregulating respiration. 16 ACS Paragon Plus Environment

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We subsequently assumed as a rational extrapolation of all the above, that application of the protonophore FCCP to MDA-MB-231 cells should (i) have no immediate deleterious effect on its own since these cells do not rely on OXPHOS for ATP generation but (ii) since uncoupling highly increases the respiration rates (Fig. 6 A), FCCP should sensitize MDA-MB-231s to our strategy. These were our exact findings indeed, summarized in Fig. 6 B: An additional ca. 35% viability reduction upon FCCP application, compared to the 4-OHT/MYXO group without FCCP, signifying sensitization to the applied strategy. In the present study we have investigated the non-genomic component of TAM action which has for several years puzzled researchers. TAM was expected to act primarily as E2 agonist/antagonist affecting cells at the genomic level, e.g. by exerting 14

cytostasis due to an induction of a G1 cell cycle block apoptosis by transcriptional regulation of BCl-2 proteins

, or the propagation of

15

. Herein we have shown

that the non-genomic component of TAM cytotoxicity is determined by a combination of factors related to cell metabolism and intracellular redox activity which may incur irreversible damage to cell mitochondria. Our findings suggest that cell metabolism regulates TAM cytotoxicity in different ways ranging from the chemical modification of TAM into its active metabolites intracellularly, to the bioenergetic phenotype of the cells (Pasteur or Warbourg) which also determines the extent of the intracellular TAM redox reactions according to the level of respiration. These redox reactions may either lead to the formation of deleterious tamoxifen semiquinone species and their radicals, or to the reduction of Fe via tamoxifen quinoid species redox cycling to Fenton active Fe (II) which can facilitate lipid peroxidation of mitochondrial membranes. In order to verify our hypothesis we applied the strategy three other cell types (Fig. S12). In Fig. S12 A, the difference



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between the additive effect (Fig. S4) calculated by the individual effects of 4-OHT and MYXO and the combined effect of the application of strategy is shown for MDAMB-231. MDA-MB-468, T-47D, MCF7 and BT-474 cells, in ascending order of OCR. In Fig. S12 B the ratio of calculated (additive) effect over the experimental strategy effect are presented for the same cell lines. It is evident from the graphs in Fig. S12 that the synergistic effect between 4-OHT and MYXO increased with increasing level of basal respiration, which further verifies our hypothesis. In a nutshell, based on the present study, an overview of what is happening upon strategy administration is proposed in the cartoon of Fig. 7: Following the two levels of TAM metabolism, as reviewed above (Fig. 1 A,D) its various metabolites can interact with complex III, undergoing reduction at Qi (blocked by ANTI-A) and oxidation at Qo (inhibited by MYXO). In consequence, upon MYXO application the tamoxifen matabolites can still use Qi for the production of hydroquinones, semiquinones and the associated semiquinone radicals which are deleterious to the cell, but also 4-OHT is itself preserved for further metabolism. The resulting semiquinone radicals can be further stabilized by diamagnetic metal ions such as Zn (II) and thus have their lifetime prolonged inducing further damage. This is, in all likelihood, why elsewhere

22

, zinc modulation and/or chelation reduced TAM

cytotoxicity. Apart from the toxic effects of the semiquinone species, the hydroquinones are capable of reducing ferric ions to their more active and toxic ferrous form which can then catalyze mitochondrial H2O2 to form hydroxyl radicals and cause lipid peroxidation to mitochondrial membranes thus causing substantial and, in cases, irreversible damage to the mitochondria. Apart from their redox-cycling at complex III, there might be a second redox cycle between the TAM metabolites and iron (either free or complexed e.g. with ATP) and a 18 ACS Paragon Plus Environment

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concomitant third one between iron and mitochondrial H2O2 generating highly active hydroxyl radicals which cause lipid peroxidation to mitochondrial membranes and consequently irreversible mitochondrial damage. This can prove fatal to cells above a critical threshold irrespective of the onset of any pro-survival mechanisms (e.g. autophagy). Furthermore intracellular Zn (I) can reduce Fe (III) exacerbating the cytotoxicity as it would fuel the mitochondria with more Fenton-active ferrous iron. All the above constitute the non-genomic action of TAM which is not at all dependent on the existence of estrogen receptors and can be further exacerbated in Pasteur type cells by blocking the quinoloxidising centre of mitochondrial complex III. Iron is very essential in the mitochondria for heme biosynthesis and Fe-S cluster production or repair, where it is tightly regulated 58, especially due to the high basal mitochondrial ROS production 59, so it is not clear how TAM metabolites manage to interact with mitochondrial iron. Apart from direct interaction with free iron, one other possibility is the direct formation of complexes between the quinones and iron which can be implicated in Fenton type reactions 48. Several other iron complexes can induce mitochondrial membrane damage by catalyzing the formation of hydroxyl radicals and thus promoting lipid peroxidation; amongst these, Fe(II)-ATP complex was shown to be very effective to that end 56. In fact Fe(II)-ATP complex was found to have a biphasic effect on mitochondrial damage: One component dependent and one independent of lipid peroxidation

56

. In a very interesting study

60

, Minotti

showed that Adriamycin (redox active quinone, aka doxorubicin) was able to reduce membrane bound iron thus facilitating its release from microsomes and consequently its participation in lipid peroxidation. This is in line with observations of TAM and 4OHT interacting with both model and native mebranes resulting in structural modifications and strong fluidization of the membrane hydrophobic regions

31, 32

. In



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Page 20 of 40

fact, this could be the mechanism of membrane- or protein- bound iron displacement, resulting in harmful interactions with the released iron. This is corroborated further, by the fact that in our experiments, administration of iron in the form of the cellpermeable FeSO4 salt (50-500 µM) had a null effect on the 4-OHT MYXO strategy either in MCF7 or MDA-MB-231 cells (data not shown). It has been reported that TAM and 4-OHT may exert antioxidant activity

61, 62

and

even protect against lipid peroxidation 63. However all these studies were conducted in either biomimetic models or isolated membranes. These systems are probably unable to generate TAM and 4-OHT metabolites and include enzymatic processes (vide supra). Conversely, in a nude mouse MCF7 tumour model 64, lipid peroxidation was found to be substantially higher in TAM-sensitive tumours than in TAM-resistant ones which exhibited baseline lipid peroxidation levels. Elsewhere, In ER-negative cells, TAM at lower doses than our regime, induced apoptosis through caspase-3 and c-jun NH2-terminal kinase 1 pathways, probably initiated by oxidation at the cell plasma membrane

65

, since lipid soluble vitamin E treatment blocked the pathway

activation and the TAM induced apoptosis. The TAM and 4-OHT concentrations chosen in the present study are pharmacologically relevant; current therapeutic doses of TAM involve a daily dose of 20 mg for several weeks 13. A clinical study by Kisanga et al. 66, on 120 breast cancer patients showed that after 28 days of administration at 20 mg per day the TAM concentration in the cancer tissue ranged from 0.6-7 µM; however due to its highly lipophilic nature, its concentration within membranes can reach values 10 to 60-fold those in serum

67

. Moreover current clinical doses of tamoxifen (20 mg/day) were

designed only to facilitate the genomic action of tamoxifen. We however believe, that the administration of one or more high-dose boluses of tamoxifen, could activate the 20 ACS Paragon Plus Environment

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non-genomic action of tamoxifen in estrogen dependent or independent lesions. This regimen is clinically relevant as in a parallel study we currently administer one or two 5 mg tamoxifen boluses to ~20g mice without any toxic effects (data not shown). Relating the current work to previous studies, the rapid cell death component seen by Kallio et. al

20

who used TAM in doses similar to ours, could be due to serum-

starvation which could exacerbate the TAM-related autophagy since TAM was administered in non-FBS supplemented media

68

. Elswhere

21

TAM and 4-OHT

administered to MCF7 cells at concentrations of 10-5 M caused (non-genomic) necrotic death within 24 h, similar to our findings, whereas submicromolar doses led to a more gradual death by day 3 (mainly genomic component). Apparently, the genomic and no genomic effects of TAM run concurrently in cells. The non-genomic effect causes escalated necrosis through the mechanism involving quinoid species and/or lipid peroxidation as shown herein; above 20 µM 4-OHT or TAM we found this effect to be acutely cytotoxic to the cells within hours from administration. The genomic effect takes longer to act and can be both cytostatic apoptotic

20, 26

14

as well as pro-

to cells with estrogen receptors. In low, submicromolar doses, the

genomic effect is predominant, whilst as the dose increases, the non-genomic effect gradually becomes dominating, quite possibly as an apoptotic insult 16-18, 65, 69 at doses lower than used in the present work. The mode of death switches to necrotic as the dose increases 20, 25. The implication of quinoid species in TAM cytotoxicity strongly suggests that TAM should be administered with caution. The levels of TAM reached following four weeks of therapy in normal tissue (1.1-4 µM)

66

could generate enough quinoid

species to promote new, unwanted carcinogenesis in healthy cells

57, 70

. Electrophilic

quinones may alkylate nucleophilic sites in macromolecules (e.g. DNA) while 21 ACS Paragon Plus Environment


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hydroquinones may also undergo rearrangements that could produce reactive alkylating species

71

; one such bioreductive alkylation agent is mitomycin

kind of alkylation and possibly downstream crosslinking

72

. This

73

, in the present case of

TAM would be relevant to mitochondrial DNA (mtDNA) damage, observed herein (vide supra). It can be deduced from the present study that TAM should be avoided in cases of iron overload diseases such as β-thalassemia, hemochromatosis and porphyria cutanea tarda

74

. In fact TAM administration in rats resulted in liver iron overload (liver

siderosis)6, while tamoxifen treatment in patients has been linked with the onset of non-alcoholic steatohepatitis (NASH)

7

Elsewhere

8, 9

, several TAM-related NASH

cases were reported to have progressed to liver cirrhosis. TAM has also been implicated in causing porphyria cutanea tarda

75, 76

; these rare cases have been

associated with TAM-related NASH and hepatotoxicity, leading to inactivation of uroporphyrinogen decarboxylase. The porphyria symptoms subsided and liver toxicity tests normalized within a few weeks following TAM treatment termination

75

. In

addition to the above, cholestatic jaundice has been sporadically reported in patients under tamoxifen treatment

10, 11

. However the most serious side effect of long-term

tamoxifen treatment seems to be the development of hepatocellular carcinomas

12, 77,

78

.

ACKNOWLEDGEMENTS K.B. and T.A.T. would like to gratefully acknowledge the European Community for financially supporting this work through the Marie Curie IntraEuropean Fellowship HYPERTAM (327075). We are thankful to Dr. Konstantina Yannakopoulou and Mr. A. Ricardo Gonçalves of Institute for Nanoscience and Nanotechnology, NCSR 22 ACS Paragon Plus Environment

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Demokritos, for their kind provision of N-desmethyltamoxifen-FITC. Finally we would like to thank Seahorse Biosciences for the generous loan of a Seahorse XFe96 analyser, which made the metabolic studies possible. REFERENCES 1. 2.

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FIGURE LEGENDS Figure 1. Tamoxifen metabolites and interaction with mitochondria and ETC complex III. A) First level of TAM intracellular metabolism by cytochrome P450 to 4-hydroxytamoxifen

(4-OHT)

and

N-desmethyltamoxifen

(NDMTAM).

B)

Synergistic cytotoxicity of TAM and its metabolites (15 µM) (including stereoisomers E -inactive, Z) with 1.4 µM MYXO (overnight incubation) in MCF7 cells. The cytotoxicity was determined by standard MTT assays 24h post-incubation. C) Effects of strategy (15 mM 4-OHT + 1.4 µM MYXO) modulators on the viability of MCF7 cells. Cells were pre-incubated with the modulators for 2h and then coincubated with the strategy overnight. The cytotoxicity was determined by standard MTT assays 24h post-incubation. The red dashed line represents survival following application of strategy alone (blue bar). D) Second level of TAM intracellular metabolism into quinoid species. RED: reduction, OX: oxidation E) Schematic representation of the


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possible redox interactions of 4-OHT and TAM quinoid metabolites with ETC complex III quinone reducing (Qi) and quinol oxidizing centres (Qo) (see also Fig. 5). F) Reduction of cytochrome c (30 µM) by complex III (0.285 mg protein submitochondrial particles, SMP) with 4-OHT or NDMTAM (200 µM) as the substrate. The kinetics are shown as difference in absorbance ratio (550/540 nm) : — ⚫— 4-OHT + SMP + cyt c; —⚫— NDMTAM+ SMP + cyt c; —⚫— 4-OHT + SMP + cyt c with 10 µM MYXO; —⚫— NDMTAM + SMP + cyt c with 10 µM MYXO; —⚫— 4-OHT+cyt c, no SMP; —⚫— NDMTAM +cyt c, no SMP and — ⚫— just SMP with cyt c. Figure 2. Application of iron chelators and mannitol. A) Effects of iron chelators desferoxamine (DFO, 15 µM), 1,10 phenanthroline (o-phen 10 µM), EDTA (2 mM) and their combination on the cytotoxicity of strategy application (15 µM 4-OHT, 1.4 µM MYXO) in MCF7 cells. Cells were pre-incubated with the chelators for 2 h and then co-incubated with chelators and strategy for 24 h. The cytotoxicity was assessed with MTT assays immediately following the 24 h co-incubation. B) Effects of the hydroxyl radical scavenger mannitol (5 mM) on the cytotoxicity of either 20 µM 4OHT or strategy (15 µM 4-OHT, 1.4 µM MYXO) application, in MCF7 cells. Cells were pre-incubated with mannitol for 2 h and then co-incubated with mannitol and 4OHT or strategy for 24 h. The cytotoxicity was assessed with MTT assays immediately following the 24 h co-incubation. Figure 3. Live cell confocal microscopy of hydroxyl radical formation in MCF7 cells following 4-OHT and strategy treatment. A) Intracellular imaging of HPF, a hydroxyl radical probe; the cells were incubated with 15µM 4-OHT± 1.4 µM MYXO for 1h and consequently 10 µM HPF (Life Technologies Inc.) for 45 min. During the



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last 15 minutes of HPF incubation 200 nM MitoTracker® Deep Red FM was added to the cells. HPF fluorescence is depicted in green and MitoTracker® Deep Red FM fluorescence in red; yellow (right column) shows green and red co-localization. B) Pearson’s correlation coefficients between the two colour channels in A (HPF and MitoTracker® Deep Red FM) for the three treatment groups, media control, 4-OHT (15 µM) and strategy (4-OHT 15 µM and MYXO 1.4 µM). Figure 4. Confocal microscopy of lipid peroxidation in fixed MCF7 cells using Click-iT® lipid peroxidation detection. Cells were co-incubated with 50 µM linoleamide alkyne (LAA) on the one hand and 4-OHT or strategy (15µM 4-OHT± 1.4 µM

MYXO) for 6h or 100 µM cumene hydroperoxide (positive control) for 2h. LAA oxidation led to alkyne modifications at the nucleophilic side chains of proteins. These proteins could then be labelled by Alexa Fluor 488 azide, employing copper-catalyzed click chemistry upon cell fixation (4% formaldehyde 15 min) and permeabilization (Triton-X-100 0.5% 10 min). The cells were loaded with 150 nM MitoTracker® Deep Red FM for 20 min just prior to fixation. Alexa Fluor 488 fluorescence is shown in green and depicts lipid peroxidation while the MitoTracker® Deep Red FM fluorescence is shown red. Yellow (-orange) denotes collocalization where the mitochondrial membrane potential has not yet been lost due to advanced lipid peroxidation membrane damage.

Figure 5. Metabolic Studies. Oxygen consumption

and media acidification

rate measurements in A) MCF7 and B) MDA-MB-231 cells using the Seahorse XFe96 analyzer. Cells were treated with media only, 15 µM 4-OHT, 1.4 µM MYXO or 15 µM 4-OHT & 1.4 µM MYXO (strategy) for 0, 8 or 24 h. Figure 6. Application of FCCP induces the 4-OHT & MYXO cytotoxic synergy in MDA-MB-231 cells A) Effects of a mitostress test on MDA-MB-231 cells. The first 4 points represent the basal respiration OCR, while 1µM OLIGO, 1µM FCCP 30 ACS Paragon Plus Environment

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and 1µM ANTI-A & ROT were sequentially injected to the cells allowing for 3 OCR measurements after each injection. It is clear that upon addition of 1µM FCCP the maximal respiratory capacity is reached which is ~1.3-fold the basal respiration. B) Effect of FCCP application on the cytotoxicity of strategy (15µM 4-OHT± 1.4 µM MYXO) in MDA-MB-231 cells. After FCCP application cells become sensitized to strategy and respond similarly to the Pasteur type MCF7 cells. Figure 7. Mechanistic hypothesis. Upon TAM (illustrated by the main metabolite 4OHT) application the quinoid metabolites undergo redox cycling in ETC complex III quinonereducing and quinoloxidizing (Qo) centres. This redox coupling could be coupled with free (or complexated) Fe, producing Fenton reactive Fe2+ which could redox cycle with intramitochondrial H2O2 producing highly reactive hydroxyl radicals leading to lipid peroxidation. Moreover, upon addition of strategy, inhibition of ETC complex III quinoloxidizing (Qo) centre by MYXO promotes the production and accumulation of deleterious semiquinone species (including semiquinone radicals) at the quinonereducing (Qi) centre of complex III. Oxidised iron (Fe3+) could further oxidize Zn to Zn2+ which is notorious for stabilizing (prolonging the life of) the semiquinone species. The semiquinone species and in particular semiquinone radicals, which are also produced at (Qi) i.e. close to the mitochondrial matrix could be responsible for the mtDNA damage only observed upon strategy application, i.e. inhibition of (Qo) with MYXO (Fig. S11B).



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Figure 1.



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Figure 2.


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Figure 3.



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Figure 4.


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Figure 5.



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Figure 6.


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Figure 7.



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Graphical abstract: Herein, we offer a mechanistic insight into the non-genomic, mitochondrial cytotoxicity of tamoxifen. Upon tamoxifen application (represented here by its primary metabolite 4-OHT) its secondary quinoid metabolites undergo redox cycling in ETC complex III quinonereducing and quinoloxidizing (Qo) centres. More importantly, inhibition of ETC complex III quinoloxidizing (Qo) centre by myxothiazol promotes the production and accumulation of deleterious semiquinone tamoxifen species (including semiquinone radicals) at the quinonereducing (Qi) centre of complex III. Oxidised iron (Fe3+) could further oxidize Zn to Zn2+ which is notorious for stabilizing (prolonging the life of) the semiquinone species. The semiquinone species and in particular semiquinone radicals, which are also produced at (Qi) i.e. close to the mitochondrial matrix could be responsible for the mtDNA damage observed upon inhibition of (Qo) with myxothiazol and parallel tamoxifen application. Secondly, the above described redox coupling could lead to the generation of Fenton reactive Fe2+ which further could redox cycle with intramitochondrial H2O2 producing highly reactive hydroxyl radicals leading to the herein documented lipid peroxidation. 338x190mm (300 x 300 DPI)

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Deciphering the Nongenomic, Mitochondrial Toxicity of Tamoxifens As

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