Release of reactive selenium species from phthalic selenoanhydride in the presence of hydrogen sulfide and glutathione with implications for cancer research

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Release of Reactive Selenium Species from phthalic

selenoanhydride in the presence of hydrogen sulfide and

glutathione with implications for cancer research

Received 00th January 20xx,

Accepted 00th January 20xx

Ammar Kharma^a,b, Anton Misak^a, Marian Grman^a, Vlasta Brezova^c, Lucia Kurakova^d, Peter Baráth^e,

Claus Jacob^b, Miroslav Chovanec^f, Karol Ondrias^aand Enrique Domínguez-Álvarez*^g

DOI: 10.1039/x0xx00000x

The last decade has witnessed a renewed interest in selenium (Se) as an element able to prevent a range of illnesses in

humans, mainly through supplementation. However, such supplementation relies on species such as sodium selenite or

selenomethionine that proved to have limited solubility and bioavailability, thus leading to a limited activity. To overcome

this limitation, other selenium species need to be explored, as the phthalic selenoanhydride (R-Se), which is soluble in

physiological media. R-Se releases various Reactive Selenium Species (RSeS), including hydrogen selenide (H₂Se), that can

interact with cellular components, such as glutathione (GSH) and hydrogen sulfide (H₂S). This interplay between R-Se and

the intracellular components provides a sophisticated biochemical release mechanism that could be behind the noteworthy

biological activities observed for this compound. In order to investigate the interactions of phthalic chalcogen anhydrides

with H₂S or GSH, we have employed UV-VIS spectrophotometry, electron spin resonance spectroscopy (ESR) and plasmid

DNA (pDNA) cleavage assay. We found that apart from R-Se, the other analogues do not have ability to scavenge the ^•cPTIO

radical or to cleave pDNA on their own. In contrast, the scavenging potency of ^•cPTIO radical and of O₂^•–radical exerted by

R-Se and of its sulfur analogue (R-S) significantly increased when they were evaluated in presence of H₂S. However, GSH only

changed the radical scavenging activity of R-Se. These new discoveries may explain some of the biological activities

associated with this class of compounds and opens a new approach to ascertain the possible mechanisms underlying their

biological actions.

diseases.^3-8However, the molecular mechanism of action is not

fully understood yet for some of these selenoproteins.

Introduction

Selenium (Se) is an essential element for human health and its

deficiency causes severe disorders, such as Keshan or Kashin-

Beck diseases, which are endemic of farming self-sufficient

regions with low levels of Se in the soil.^1,2Both Se-containing

compounds and the Se atom present in the active sites of 25

mammalian selenoproteins identified so far, participate in key

cellular and physiological processes, as well as diseases such as

cancer, inflammation, immunity, type 2 diabetes or liver

During recent years, Se-compounds have gained substantial

interest as H₂Se donors, as potential anti-cancer agents or as

selenocompounds with potential use in selenium

supplementation.^8-11Among other effects of Se-compounds

related to cancer, they could induce apoptosis in cancer cells

through the production of Reactive Oxygen Species (ROS) and

thus inducing oxidative stress (OS).^12,13Furthermore, certain Se-

compounds can damage DNA. Then, they may not protect

against cancer and other chronic diseases, and can even cause

or enhance some types of cancer. These facts indicate that Se

14,15

may exert a broad pattern of toxic effects.

The exact

^a.Institute of Clinical and Translational Research, Biomedical Research Centre,

University Science Park for Medicine, Slovak Academy of Sciences, Dúbravská

cesta 9, 845 05 Bratislava, Slovak Republic.

mechanisms underlying the beneficial and toxic effects of the

Se-compounds are not fully understood yet and they are still

under intensive investigation, due to the interest of the

potential applications of this dual behaviour: these pro-

oxidant/antioxidant Se-compounds could act as novel cellular

redox modulators.

^b.Division of Bioorganic Chemistry, School of Pharmacy, Saarland University,

Campus B2 1, D-66123 Saarbruecken, Germany.

^c.Faculty of Chemical and Food Technology, Slovak University of Technology,

Radlinskeho 9, 812 37 Bratislava, Slovak Republic.

^d.Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius

University, Ulica Odbojárov 10, 832 32 Bratislava, Slovak Republic.

^e.Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38

Bratislava, Slovakia.

^f.Cancer Research Institute, Biomedical Research Centre, University Science Park

for Medicine, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava,

Slovak Republic.

^g.Instituto de Química Orgánica General, Consejo Superior de Investigaciones

Científicas (IQOG, CSIC), Juan de la Cierva 3, 28006 Madrid, Spain.

Electronic Supplementary Information (ESI) available: [details of any supplementary

information available should be included here]. See DOI: 10.1039/x0xx00000x

In this context, the accessibility and reactivity of the selenols

(mainly deprotonated at biological pH values) and selenol-

derived compounds, such as Se-methyl selenocysteine and

diselenides,^16-19may be behind the reported chemopreventive

activity of different Se-containing compounds, which has been

reviewed extensively by numerous authors.^11,20-25Several

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mechanisms have been proposed to explain the inside the cells, and at the same time, it would enable its release

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chemopreventive activity, such as the direct scavenging of free in an “activated” form as one of the Reac^Dti^Ov^Ie^{: 1}S⁰e^.1l⁰e³n⁹i^/u^Cm^9NS^Jp⁰e²²c⁴ie⁵s^G,

radicals,^26,27the amelioration of the toxic effects of anticancer ready to exert immediately a wide variety of biological effects.

drugs,²⁸the glutathione peroxidase (GPx)-like activity,^29,30toxic Besides, these compounds have proved in previous studies that

elements such as arsenic by protecting PC12 cells from arsenic can exert a selective action for being less toxic in non-tumour

induced oxidative stress ³¹or radiotherapy;³²the modulation of cells than in cancer cells.^49,51Thus, they could be used as safer

the intracellular redox homeostasis³³and of the protein redox modulators.

kinases.³⁴

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Herein, we have studied further the possible mechanism(s)

In the last twenty years, H₂S (H₂S/HS^–/S^2–) has been emerging as underlying these initial promising biological activities of R-Se,

a new gaseous signalling molecule besides nitric oxide (^●NO) and we will ascertain if these hypothesized interactions with H₂S

and carbon monoxide (CO). H₂S is produced endogenously in and GSH take effectively place. Activities of the R-O and phthalic

almost all mammalian cells and affects many physiological and thioanhydride (R-S) were examined in parallel for comparison.

35-37

pathological processes.

H₂S has mostly beneficial effects Since H₂S and GSH interact with several biologically active

under conditions of oxidative stress by reacting with reactive molecules modulating their activities.^42,46-48,53Interactions of

oxygen and nitrogen species. ^38-42It has both the pro- and anti- H₂S and GSH with these three phthalic anhydride derivatives

cancer effects depending on the cell type, concentration and and their molecular consequences were also analysed;

43-45

interaction with other cellular molecules.

Glutathione expecting that in these instances, the R-Se would be the most

(GSH) is another intracellular natural antioxidant, which has reactive one thanks to its higher expected reactivity. The

many biological roles including modulation of cellular redox reduction of the ^•cPTIO and superoxide (O^2•–) radicals, and

homeostasis and protection against reactive oxygen and plasmid DNA (pDNA) cleavage assay were employed to monitor

nitrogen species.^46,47As it has been determined in a previous these consequences. We show that only R-Se displays any

work, sodium selenite (Na₂SeO₃) and selenium tetrachloride significant biological activity on its own. This activity augmented

(SeCl₄) have ability to interact separately with GSH and H₂S. This considerably when tested in combination with H₂S or GSH. In

fact could be behind their biological effects.⁴⁸However, as contrast, GSH showed no impact over the activities of R-S or of

mentioned above, sodium selenite can have reduced R-O, whilst H₂S was efficient to some extent in this context.

bioavailability and exert toxic effects. ^14,15Thus, it is desirable to Hence, products of the H₂S or GSH interaction with R-Se and R-

move towards novel Se-containing compounds that retain this S show free radical scavenging properties and appear to cleave

observed ability to interact with relevant sulfur compounds pDNA, potentially explaining some of their biological activities.

(herein, GSH and H₂S), and, at the same time, show a reduced

toxicity in comparison with sodium selenite. This would improve

Results and discussion

the applicability of these new selenium-based redox

modulators, and simultaneously, open a new approach in Se-

To unveil the possible mechanisms underlying the reported

supplementation, by finding of redox-active derivatives with

activities of R-Se against cancer, we have designed different

less toxicity.

experiments with R-Se, R-S and R-O, as mass spectrometry (ESI-

MS), spectrophotometrically-monitored radical scavenging and

In this context, our previous data showed promising

electron paramagnetic resonance (EPR); in an attempt to

chemopreventive, antiproliferative, cytotoxic, free radical

ascertain how this promising Se-containing compound can

scavenging, pro-apoptotic and multidrug-resistance (MDR)

interact with different cellular redox targets.

reversing activity of phthalic selenoanhydride (R-Se, Figure 1),

the Se-analogue of phthalic-anhydride.^49-52Probably, these

Chemistry

reported biological activities are directly related to the Se atom,

In the present work, we have evaluated three chalcogen

as no relevant biological effects were displayed by its oxygen

analogue, the phthalic anhydride (R-O).⁵¹A hypothesis which

may explain the amazing biological properties of R-Se draws the

attention to the lability of the CO-Se chemical bond.⁴⁹This

lability suggests the interesting possibility that R-Se behaves in

the organism as a prodrug: It enables the internalization of the

compound in the cells, and once inside, it can release selenium

slowly inside the cell, in form of H₂Se, of other uncharged Se-

species as nanoparticles of selenium or of charged Se-anions

able to interact swiftly with cellular components.^49,51Due to

their particular chemical affinity towards thiol-containing

agents, such as hydrogen sulfide (H₂S) or glutathione (GSH), and

with the enzymes and proteins which are components of the

cell thiolstat, such reactivity may initiate pronounced biological

responses. In this way, R-Se could result to be a very simple,

elegant and straightforward method to transport selenium

phthalic anhydrides, as shown in Figure 1: the phthalic

selenoanhydride (X=Se R-Se), the phthalic thioanhydride

(X=S R-S) and the phthalic anhydride (X=O R-O), as well as



phthalic acid (R-OH). R-O and R-OH were commercially

available, whereas R-Se and R-S were synthesized following an

adaption of the procedure previously described for the

synthesis of R-Se (Figure 1). ⁴⁹

In brief words, elemental selenium or sulfur are reduced with

lithium aluminium hydride, and the in situ formed hydrogen

chalcogenide attacks the phthaloyl chloride to form a reactive

intermediate. Sulphuric acid is added to form the desired final

R-Se or R-S. Compounds were isolated in the form of stable

solids, whose purity was assessed through NMR and LC-MS.

2 | J. Name., 2012, 00, 1-3

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of water to later generate the protonated R-O (m/z =

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DOI: 10.1039/C9NJ02245G

181.04956). The majority of these peaks can be seen when R-Se

is analysed together with Na₂S (Figure 4). The remaining peaks,

especially those with m/z>245, are the result of complex

couplings with other R-Se molecules/fragments, and with water

or with the solvent (methanol). The low abundance of the

protonated molecular peak in ESI-MS spectrum may be an

indicator of the readiness of the R-Se compound to react with

different compounds, and this reactivity can explain the

biological activities found so far for this bioactive compound.

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When Na₂S is added to the R-Se solution in 50% methanol/H₂O

(Figure 4), the protonated molecular peak of the R-Se (m/z =

212.94487) disappears, although the m/z = 244.97110 (result of

the methanol addition to this peak and also with Se isotopic

pattern) can be observed, but with a significant lower

abundance than in the spectrum of R-Se alone. It is also possible

to observe the peaks of lower m/z that were hypothesized

above in Figure 2: m/z = 135.04414, m/z = 149.02, m/z =

163.03908 and m/z = 181.04958. Besides, new peaks appear.

The most relevant of them is the m/z = 197.02664, which is the

equivalent of the m/z = 244.97110 but replacing the Se atom by

sulfur (Figure 4 inset). This peak can be formed through the

coupling of H₂S (generated in situ from Na₂S) with the m/z =

163.03908. Finally, two peaks (m/z = 266.95303 and m/z =

219.00859) with a difference of 48 Da are found, difference that

can be attributed to Se-S change, taking into account that the

first peak presents the characteristic isotopic pattern of Se.

Tentative structures that could explain these two peaks are the

polyhydroxy-containing compounds drawn in Figure 4 inset. In

any case, the absence of the R-Se protonated molecular peak

and the two sulfur-containing peaks (m/z = 197.02664, m/z =

219.00859) together can be an indicative of an interaction

between R-Se and Na₂S.

Figure 1. A). Chemical structure of the chalcogen derivatives of the

phthalic anhydride. X= Se R-Se; S R-S; O R-O. Synthetic

procedure for R-Se and R-S. B) Hypothesized reactions that would

lead to the release of hydrogen selenide (H₂Se) from R-Se in the

physiological media.

ESI-MS

R-Se showed a complex pattern of fragmentation (Figure 2) in

ESI-MS (electrospray ionization mass spectrometry) spectrum

taken in a 50% methanol/H₂O solution (Figure 3). The possible

fragments corresponding with the main peaks observed in the

ESI are suggested in Figure 2.

According to the data obtained, ESI-MS experiments show how

this Se-compound, in the electrospray ionization conditions,

suffers specific fragmentations (Figure 2) and how it forms

complex couplings with the solvent and other R-Se

molecules/fragments, indicating that this compound has a high

reactivity. This fact could be an indicative of potential

interactions of R-Se with the reactive species present in the cell,

such as ROS, Reactive Nitrogen Species (RNS) and the sulfur

compounds of the redox thiolstat. To prove this hypothetic

interaction between R-Se and sulfur species present in cells

such as H₂S, we also applied the ESI-MS methodology to a

mixture of R-Se and Na₂S. In this second ESI-MS spectrum, it is

observed how the peaks of the initial R-Se are practically

irrelevant, whereas its fragments peaks are the main peaks.

Additionally, new peaks related to couplings of its fragments

with the added sulfur atoms start to be observed, proving also

the reaction between the two chalcogen compounds.

Figure 2: Hypothesized fragmentation pattern for protonated R-Se

in ESI-MS.

The M⁺H molecular peak of the R-Se is easily recognisable

thanks to the characteristic isotopic pattern of the Se-

containing fragments, and the remaining peaks of low m/z can

be assigned to specific fragments (Figure 2). Briefly, the

protonated molecular peak (m/z = 212.94487) is attacked by

methanol to generate the fragment with m/z = 244.97104 (also

with the characteristic isotopic pattern of Se). This fragment

loses a molecule of hydrogen selenide, leading to the m/z

=163.03906, which can suffer an additional fragmentation

(release of CO, m/z = 135.04408) or can incorporate a molecule

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 3

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180626 CTRL 50%MeOH_180626 #5-45 RT: 0.09-0.89 AV: 41 NL: 7.01E5

T: FTMS + c ESIFull ms [110.00-500.00]

135.04408

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z=1

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z=?

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z=1

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z=?

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z=?

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0

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Figure 3: Control R-Se ESI spectrum in 50% methanol/H₂O. Inset: zoom of the protonated molecular peak showing the characteristic

isotopic pattern of Se.

180626 CTRL 50%MeOH Na2S_180626 #56 RT: 1.18 AV: 1 NL: 7.98E4

T: FTMS + c ESIFull ms [110.00-500.00]

163.03908

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OH

O

HO

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m/z = 197.03

m/z = 219.01

m/z = 266.95

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Figure 4: Experimental ESI-MS spectrum of R-Se + Na₂S in 50% methanol/H₂O. Inset: Structures suggested to explain the differential peaks

in respect to R-Se ESI-MS spectrum.

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Results of the Reduction of ^•cPTIO by Phthalic-Anhydride

H₂S/R-Se and H₂S/R-S is different. cPTIO reduction potency of the

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H₂S/R-Se mixture was several folds higher than (≥ 5 x)_Vt_ih_ewat_Aro_tif_clH_{e O}S_n/_liR_ne-

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DOI: 10.1039/C9NJ02245G

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S (Figure 6E). Reduction of cPTIO strongly depended on H₂S/R-Se

molar ratio: it was low at 0.5 and 1 H₂S/R-Se molar ratio but

increased significantly at 2 and 4 molar ratios (Figure 6B,F). On the

Since H₂S is endogenously produced in living organisms, we studied

the interaction of H₂S with R-Se. We observed that H₂S interacts with

R-Se. Since H₂S and Se derivatives were reported to interact with

radicals,⁵⁴it was of high interest to reveal whether products of the

H₂S/R-Se reaction interact with radicals and how this interaction is

unique with comparison to other phthalic-anhydride derivatives.

Therefore, we studied the potency of H₂S, R-Se, R-S, R-O and R-OH

and the interaction products of H₂S/R-Se, H₂S/R-S, H₂S/R-O and

H₂S/R-OH to reduce the ^•cPTIO radical.

•

other hand, reduction of cPTIO gradually increased with H₂S/R-Se

molar ratio (Figure 6D,F). The results show that H₂S interacting with

R-Se and R-S (but not with R-O or R-OH) forms reactive products,

which reduce ^•cPTIO radical. The order of potency is as follows:

H₂S/R-Se > H₂S/R-S >> H₂S/R-O ~ R-OH = 0.

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H₂S Potentiates R-Se and R-S (But Not R-O or R-OH) in Reducing

^•cPTIO. Since H₂S is endogenously produced in organisms and

exogenous H₂S donors are being considered to be used in medicine,

we have studied interaction of H₂S with anhydride derivatives and

the ability of products of this interaction to reduce the ^•cPTIO radical.

H₂S in the presence of R-Se or R-S, but not R-O or R-OH, significantly

increased rate and potency of the compounds to reduce ^•cPTIO

(Figures 5A-inset, 5B and 6).

•

Figure 5. Reduction of cPTIO by H₂S, R-Se, R-S, RO and R-Se/H₂S

•

mixture. (A) Time resolved UV-VIS spectra of 100 µM cPTIO after

addition of 100 µM R-S. Spectra were recorded every 30 s for 20 min.

The first spectrum was recorded 15 s after R-S addition. Arrows

indicate decrease of ABS at 356 and 560 nm. Inset: Kinetics of

•

changes in absorbance at 560 nm of 100 µM cPTIO after addition

Figure 6. The effect of R-Se and R-S on the kinetics of ^•cPTIO

reduction in the absence and presence of H₂S. (A) The effect of R-Se

(indicated by arrow) of: 100 µM H₂S (black); 50 and 100 µM R-Se (red

and pink); 50 and 100 µM R-S (blue and cyan); 50 and 100 µM R-O

(green and dark green) and mixture of 100 µM H₂S with 100 µM R-O

(dark yellow). Means ± SEM, n = 2 - 4. (B) Time resolved UV-VIS

spectra of the interaction of 100 µM ^•cPTIO with 100 µM H₂S (3 times

repeated every 30 s, black) and subsequent addition of 12.5 µM R-

Se. Spectra were recorded every 30 s for 20 min, the first spectrum,

indicated by the red line, was measured 15 s after addition of R-Se.

Inset: details of the time resolved spectra of the ^•cPTIO/H₂S (100/100

µM/µM) interaction before (black) and after addition of R-Se (12.5

µM, the first spectrum is indicated by the solid red line, which is

followed each 30 s by: long dashed red, medium dashed red, short

dashed red, dotted red, solid blue line, long dashed blue, medium

dashed blue, etc.).

•

and R-OH on the time-dependent reduction of cPTIO/H₂S. Kinetics

of changes in absorbance at 560 nm of 100 µM ^•cPTIO after addition

(indicated by arrow) of 100 µM R-Se alone (dark green) and

compared it to the addition of 0 (dark yellow), 6.25 (red), 12.5 (blue),

25 (pink), 50 (cyan) and 100 µM (green) R-Se to ^•cPTIO/H₂S (100/100

µM/µM). 50 µM R-OH added to ^•cPTIO/H₂S (black; 100/100 µM/µM).

Data were collected from UV-VIS spectra every 30 s for 20min. (B)

•

The effect of H₂S on kinetics of reduction of cPTIO in the presence

of R-Se. Kinetics of changes in absorbance at 560 nm of 100 µM

^•cPTIO after addition (indicated by arrow) of 100 µM H₂S alone (dark

yellow) and after addition of 50 µM R-Se to 0 µM (dark green), 25 µM

(red), 50 µM (blue), 100 µM (pink) and 200 µM H₂S (cyan). (C) The

•

effect of R-S on the kinetics of cPTIO reduction in the presence of

•

Notably, 6.25, 12.5 and 25 µM of R-Se in the presence of 100 µM H₂S

had two time-dependent phases of the decreased concentration of

100 µM ^•cPTIO. The first, it was fast decrease (in ≤ 2 min) followed by

the second gradual decrease (Figure 6A). On the other hand, 6.25,

12.5 and 25 µM of R-S decreased ^•cPTIO in the first phase slower than

what was observed when R-Se was employed (≤ 5 min), but later

^•cPTIO concentration did not decrease significantly (Figure 6C). This

indicates that the molecular mechanism of ^•cPTIO reduction by

H₂S. Kinetics of changes in absorbance at 560 nm of 100 µM cPTIO

after addition (indicated by arrow) of 100 µM R-S alone (dark green)

and comparison to the addition of 0 (dark yellow), 6.25 (red), 12.5

•

(blue), 25 (pink), 50 (cyan) and 100 µM (green) R-S to cPTIO/H₂S

•

(100/100 µM/µM). (D) The effect of H₂S on the kinetics of cPTIO

reduction in the presence of R-S. Kinetics of changes in absorbance

•

at 560 nm of 100 µM cPTIO after addition (indicated by arrow) of

100 µM H₂S alone (dark yellow) and after addition of 50 µM R-S to 0

µM (dark green), 25 µM H₂S (red), 50 µM (blue), 100 µM (pink) and

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200 µM H₂S (cyan). (E) Comparison of the potency of R-Se and R-S to

reduce ^•cPTIO in the presence of H₂S. Reduction of ^•cPTIO (100 µM)

by R-Se and R-S (100 µM) and reduction of ^•cPTIO in the presence of

H₂S (100 µM) after addition of 0, 6.12, 12.5, 25, 50 and 100 µM R-Se

or R-S. The changes in absorbance at 560 nm of 100 µM ^•cPTIO were

taken from (A,C) at the 5^thmin after the addition of compound. (F)

H₂S and GSH Interacting with Na₂Se-derivat_Viv_iee_ws_ArtiR_cle_ed_Ou_nlc_ine_e

^•cPTIO. ESI-MS experiments (Figures 3, 4) show that H₂S

DOI: 10.1039/C9NJ02245G

interacts with R-Se (and its derivatives). We observed that the

H₂S/R-Se mixture, but not the H₂S/R-O or H₂S/phthalic acid

•

mixtures, reduced cPTIO (Figure 6). Based on these data, we

suppose that intermediates and/or products of H₂S interaction

with Se (released from R-Se) and/or with R-Se derivatives, are

•

Comparison of the potency of R-Se and R-S to reduce cPTIO in the

presence of different concentration of H₂S. Reduction of ^•cPTIO (100

µM) by R-Se and R-S (50 µM) in the presence of 0, 25, 50, 100, 200

µM H₂S. The changes in absorbance at 560 nm of 100 µM ^•cPTIO were

taken from (B,D) at the 5^thmin after the addition of the compound.

Means ± SEM, n = 2 - 4.

•

responsible for cPTIO reduction. To confirm this, we studied

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the interaction of H₂S and GSH with Se derivatives using Na₂Se.

UV-VIS spectra of freshly prepared 100 µM Na₂Se changed

gradually for 20 min (Figure 8A), which could be an indication of

a potential slow unspecific interaction with any of the

compounds present in the solution, as the diethylene-

triaminepentaacetic acid (DTPA), sodium phosphate, the

solvent (water) or most probably with oxygen, which acts as an

oxidant. UV-VIS spectra of H₂S/Na₂Se (100/100 µM/µM) also

changed gradually for 20 min (Figure 8B). Since the time

dependence of the UV-VIS spectra of Na₂Se (Figure 8A) and

Na₂Se/H₂S mixture (Figure 8B) were different (marked by

arrows), we confirm the interaction of H₂S with Na₂Se

derivatives.

GSH Potentiates R-Se and R-S (But Not R-O or R-OH) in Reducing

^•cPTIO. GSH is a tri peptic (glutamate-cysteine-glycine) natural

antioxidant, whose intracellular concentrations are in the range of

0.5 to 10 mM.^46,47Therefore, we studied the effect of GSH on the

reducing potency of the phthalic-anhydride derivatives. GSH (100

and 500 µM) did not reduce the ^•cPTIO (100 µM) radical alone (Figure

7), as observed in our previous study.⁴²However, the GSH/R-Se

(200/50 and 500/50 µM/ µM) and GSH/R-S (200/50 and 500/50

µM/µM) mixtures significantly reduced ^•cPTIO (Figure 7). Kinetics of

^•cPTIO reduction by the mixtures were different for R-Se and R-S. In

case of R-S, it was an exponential decay, but in case of R-Se an

induction period was observed. The addition of R-O to the

The time resolved UV-VIS spectra of ^•cPTIO/Na₂Se

•

(100/100 µM/µM) shows no cPTIO reduction by Na₂Se alone,

since ABS at 356 nm (marked by arrow) did not decrease over

the time (Figure 8C,F), nor ABS at 560 nm (Figure 8C-inset). H₂S

(100 µM) or GSH (500 µM) had only minor effects (≤ 7%) on their

•

^•cPTIO/GSH mixture did not cause cPTIO reduction (Figure 7). The

results indicate that the reducing potency of GSH is significantly

enhanced upon its interaction with R-Se and R-S. In control

experiments, R-O and phthalic acid did not reduce ^•cPTIO

themselves, neither in the presence of 500 µM GSH (Figure 7). This

indicates that the presence of Se and S in phthalic anhydride

derivatives after interaction with H₂S and GSH is responsible for the

reduction of ^•cPTIO.

•

own in terms of cPTIO (100 µM) reduction within 20 min.

However, addition of freshly prepared Na₂Se (100 µM) to

H₂S/^•cPTIO (100/100 µM/µM) or GSH/^•cPTIO (500/100 µM/µM)

mixture reduced ^•cPTIO (decreased ABS at 356 and 560 nm) in

< 1 min (Figure 8D,E,F), indicating a formation of reducing

species during the interaction of Na₂Se-derivatives with H₂S and

•

GSH, which reduce the cPTIO radical. The results support the

suggestion that H₂S and GSH significantly potentiated the

reducing properties of Se derivatives, which are released from

R-Se.

Discussion of the Reduction of ^•cPTIO by Phthalic-Anhydride

Derivatives and H₂S

•

Regarding the reduction of the cPTIO radical, R-S and R-Se

showed a higher capacity to reduce this radical in comparison

with H₂S. But H₂S was most effective in the reduction of the

^•cPTIO free radical than the phthalic acid and phthalic

anhydride. This fact suggests that the mechanism that explains

this reduction must be related to a characteristic reaction of R-

S and R-Se that is not demonstrated by the R-O or by the R-OH.

A potential candidate for the reaction led to this observation

could be then the release of sulfide or selenide anions,

respectively as the release of O^2-is non-existent.H₂S is produced

endogenously and exert relevant biological effects and

functions. It can be found in tissue cells in non-negligible

physiological concentrations that reach even higher than

1 µM.⁵⁵Besides, its local space-time concentration in

microenvironments can be even several folds higher. This fact

indicates that the H₂S/R-Se interaction may be involved also in

Figure 7. Effect of GSH on ^•cPTIO reduction kinetics in the presence

of R-Se, R-S, R-O and R-OH. Kinetics of the changes in absorbance at

560 nm of 100 µM ^•cPTIO after addition (indicated by arrow) of 100

(dashed dark-yellow) and 500 µM GSH (solid dark yellow); after

•

addition of 50 µM R-Se to the cPTIO/GSH (100/200 µM/µM; pink),

to the ^•cPTIO/GSH (100/500 µM/µM; red); after addition of 50 µM R-

S to the ^•cPTIO/GSH (100/200 µM/µM; cyan), to the ^•cPTIO/GSH

(100/500 µM/µM; blue); after addition of 50 µM R-O to the

^•cPTIO/GSH (100/200 µM/µM; dashed green), to the ^•cPTIO/GSH

(100/500 µM/µM; solid green); after addition of 50 µM R-OH to the

^•cPTIO/GSH (100/500 µM/µM; black). Means ± SEM, n = 2 - 3.

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the biological activities of R-Se. Thus, we have evaluated how it of H₂S with R-Se is dependent on the molar ratio_Vb_iee_wtw_Arte_ice_len_Ont_lh_ine_e

DOI: 10.1039/C9NJ02245G

interacts with these chalcogen phthalic derivatives. Results Se analogue and H₂S: when R-Se is predominant or when both

were in line with the previous observations and supported our are equimolar, a low reduction is observed. However, when the

hypothesis of the S^2-and Se^2-release: The addition of H₂S to R-S molar ratio H₂S/R-Se is 2 to 4, the detected reduction increased

and R-Se potentiated the above mentioned reduction of ^•cPTIO significantly. This fact could suggest that a reaction between H₂S

radical. It is noteworthy the fact that the addition of H₂S and R-Se takes place and that the concentration of the first

promoted the reduction exerted by R-Se more than the one potentiates this reaction; although the R-Se is also crucial as its

induced by R-S. On the other hand, the addition of H₂S to R-O replacement by R-S reduced the observed potentiation effect.

and R-OH did not exert any effect. Interestingly, the interaction

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Figure 8. UV-VIS spectra of interaction of Na₂Se with ^•cPTIO/H₂S and ^•cPTIO/GSH. (A) Time resolved UV-VIS spectra of 100 µM Na₂Se in 100

mM sodium phosphate, 100 µM DTPA, pH 7.4 buffer at 37 °C. Spectra were collected every 30 s for 20 min, the first spectrum was measured

15 s after addition of Na₂Se. Inset are details. The first spectrum is indicated by the solid red line, which is followed each 30 s by: long dashed

red, medium dashed red, short dashed red, dotted red, solid blue line, long dashed blue, medium dashed blue, etc.; marked by arrows. (B)

Time resolved UV-VIS spectra of the interaction of 100 µM Na₂Se with 100 µM H₂S. Spectra were collected every 30 s for 20 min, the first

spectrum, indicated by the solid red line, was measured 15 s after addition of Na₂Se to H₂S. Inset: details of the time resolved spectra of HS^–

, peak at 230 nm. (C) Time resolved UV-VIS spectra of the interaction of 100 µM ^•cPTIO with 100 µM Na₂Se (^•cPTIO - 3 times repeated every

30 s, black) and subsequent addition of 100 µM Na₂Se. Spectra were collected every 30 s for 20 min, the first spectrum, indicated by the solid

red line, was measured 15 s after addition of Na₂Se. The arrow indicates ABS at 356 nm. Inset: details of the time resolved spectra of the

^•cPTIO/Na₂Se (100/100 µM/µM) interaction before (black) and after addition of Na₂Se (100 µM). (D) Time resolved UV-VIS spectra of the

interaction of Na₂Se with ^•cPTIO/H₂S. Control ^•cPTIO/H₂S (100/100 µM/µM; 3 times repeated every 30 s, black), and subsequent addition of

100 µM Na₂Se. Spectra were collected every 30 s for 20 min, the first spectrum, indicated by the red line, was measured 15 s after addition

•

of Na₂Se. The arrows indicate decrease of ABS at 356 and 560 nm. Inset: details of the time resolved spectra of the cPTIO/H₂S (100/100

µM/µM) interaction before (black) and after addition of Na₂Se (100 µM). (E) Time resolved UV-VIS spectra of the interaction of Na₂Se with

^•cPTIO/GSH. Control ^•cPTIO/GSH (100/500 µM/µM; 3 times repeated every 30 s, black), and subsequent addition of 100 µM Na₂Se. Spectra

were collected every 30 s for 20 min, the first spectrum, indicated by the solid red line, was measured 15 s after addition of Na₂Se. The arrows

•

indicate decrease of ABS at 356 and 560 nm. Inset: details of the time resolved spectra of the cPTIO/GSH (100/500 µM/µM) interaction

before (black) and after addition of Na₂Se (100 µM). (F) Kinetics of the interaction of Na₂Se (100 µM, marked by arrow) with ^•cPTIO (100 µM,

black), ^•cPTIO/H₂S (100/100 µM/µM, blue) and ^•cPTIO/GSH (100/500 µM/µM, red) monitored as changes of ABS at 356 nm with correction

to 420 nm; means ± SEM, n = 2 - 3.

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View Article Online

DOI: 10.1039/C9NJ02245G

Another sulfur-containing biogenic compound with crucial R-OH, the increasing concentrations of GSH had no effects on

functions is GSH, which can reach intracellular concentrations in pDNA cleavage (Figure 10B).

the range 0.5 – 10 mM. Thus, we have also evaluated its

interaction with the phthalic anhydride derivatives, as this

interaction may be involved in the R-Se biological effects. And

effectively, similar results of potentiation of R-Se and R-S ability

•

to reduce the cPTIO radical were obtained, being then again

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more significant for R-Se than for R-S. The phthalic anhydride

derivatives could interact also with other components of the

cellular thiolstat, or even with other different enzymes. For

example, they could be activated through different cellular

enzymes, such as disulfide reductases and esterases. However,

further research needs to be done in future works to confirm

this extent. In this study we have selected GSH and H₂S as

representative compounds of the sulfur containing components

involved in the redox thiolstat. The enhancement of the R-Se

activity in respect to the potentiation observed for R-S by the

Figure 9. The effect of anhydride-derivatives on pDNA integrity. The

two thiols evaluated (H₂S and GSH) was observed therefore, we

increasing concentrations of R-Se, R-S, R-O or phthalic acid were

tested how H₂S and GSH reduced the ^•cPTIO radical in the

incubated with pDNA for 30 min at 37°C and the resulting pDNA

absence of R-Se, finding that in this case there was no

forms were resolved using agarose gel electrophoresis. Mean ± SEM,

interaction. This confirms the key role of R-Se in this interaction,

n = 3.

and the requirement of having both Se and S species to have a

more effective interaction.

As we detected relatively similar pDNA cleavage efficiencies at

1:1 and 1:3 molar ratios of R-Se:H₂S, we checked whether there

was a difference in kinetics between the two reactions.

However, the two reactions displayed the same (linear) time-

dependent pDNA cleavage efficiency, suggesting that within a

1:1-1:3 (R-Se:H₂S) molar ratio window, R-Se is a rate-limiting

factor in the reaction (Figure 11).

Cleavage of pDNA

We wanted to ascertain whether the products of the H2S/R-Se

and/or GSH/R-Se interaction can directly attack pDNA without

contribution of other (unknown) biologically important

molecules and/or pathways. Briefly, the pDNA cleavage assay

can detect any activity that attacks and disrupts the sugar-

phosphate backbone of DNA (e.g. reactive oxygen species, free

radicals etc.).

Summing up, we have observed that R-S, R-O and R-OH only

exerted a limited pDNA cleavage activity; whereas this activity

increased significantly when R-Se was employed in

a

concentration-dependent manner. Interestingly, the addition of

H₂S and GSH modulated strongly this pDNA cleavage action, and

showed a bell-shaped effect, suggesting that kinetics may be a

rate-limiting factor in this action. In this case, the pDNA

cleavage exerted by the remaining phthalic derivatives tested in

the presence of H₂S increased in a concentration dependent

manner of H₂S, which may be caused by the H₂S-related pDNA

cleavage effect. Thus, it is noteworthy that the pDNA cleavage

in the absence of H₂S and the bell-shaped modulation in the

presence of H₂S and GSH are only observed when R-Se is

employed, which underlines the unique redox-modulating

properties of R-Se; and which indicates the possibility of the

formation of a S-Se intermediate when H₂S interacts with R-Se.

H₂S and GSH Interacting with Phthalic-Anhydride Derivatives

Cleave pDNA. To compare the pDNA cleavage activity caused or

mediated by the phthalic-anhydride derivatives, increasing

concentrations of these compounds were incubated with pDNA

in vitro and the resulting reaction mixtures were subjected to

electrophoretic separation to resolve the individual pDNA

forms. R-S, R-O or R-OH had only minor effects on pDNA

cleavage. In contrast, R-Se cleaved pDNA in a concentration-

dependent manner at concentrations ≥ 50 µM (Figure 9).

Notably, H₂S modulated pDNA cleavage activity of the

anhydride derivatives depending on the H₂S/anhydride

derivative molar ratio. In the presence of 50 µM of R-Se, the

increasing concentrations of H₂S caused the bell-shaped effects

with a maximum level being reached at 100/50 and 200/50

µM/µM H₂S/R-Se molar ratios. The effect of 50 µM of R-S, R-O

and R-OH was increased with the increasing H₂S concentrations

(Figure 10A). In the presence of 100 µM H₂S, pDNA cleavage

activity of R-Se was several folds higher with comparison to R-S,

R-O or R-OH. Also GSH modulated the pDNA cleavage activity of

the anhydride derivatives. In the presence of 50 µM R-Se,

increasing concentrations of GSH mediated the bell-shaped

effects with a maximum level being seen at 100/50 µM/µM

GSH/R-Se molar ratio. In the presence of 50 µM R-S, R-O or

The effect of R-Se, R-S and R-O without and with H₂S to Scavenge

•

the O₂^-Radical or Its Derivatives.

Since we observed that the mixture of H₂S and R-Se or R-S

significantly potentiated ^•cPTIO reduction, it was of interest to

•

–

study if the mixture can scavenge O₂radicals. The EPR spin

•

trap method based on the reaction of O₂^–with BMPO to form

the ^●BMPO-OOH adduct was employed.⁵⁶The O₂^●–radical anion

solution (prepared by dissolving KO₂in DMSO) was diluted in

phosphate buffer (pH 7.4; 37°C) and trapped by BMPO.

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OOH/^•BMPO-adducts (Figure 12C,D), a superposition of at least

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•

DOI: 10.1039/C9NJ02245G

two radicals, BMPO-OOH and BMPO-OH, was recognized. H₂S

(50 µM) had similar effects as R-S, however its potency to

decrease the quantity of the ^•BMPO-adducts and ratio of

^•BMPO-OOH/^•BMPO-OH was lower in comparison to R-S (Figure

12E1-E3; Figure 13).

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Figure 10. The effect of increasing concentrations of H₂S (A) and GSH

(B) on pDNA integrity in the presence of the anhydride-derivatives R-

Se, R-S, R-O and R-OH at 50 µM concentrations. Mean ± SEM, n = 3-

5.

•

Figure 12. EPR spectra of ^•BMPO in the presence of O₂and

−

modulated by R-Se, R-S and R-O without and with H₂S.

•

Representative EPR spectra of the BMPO-adducts were monitored

in 10% v/v saturated KO₂/DMSO solution in 50 mM sodium

phosphate buffer, 0.1 mM DTPA, pH 7.4, 37^oC in the presence of the

studied species investigated and 20 mM BMPO. Sets of individual EPR

spectra of the ^•BMPO-adducts monitored upon 15 sequential scans,

s (A1-H1), starting acquisition 2 min after sample

preparation in: control 10% v/v KO₂/DMSO in the buffer (A1); the

KO₂/DMSO in the presence of 25 µM R-Se (B1); 25 µM R-S (C1); 25

each 42

Figure 11. Comparison of time-dependent pDNA cleavage by H₂S/R- µM R-O (D1); 50 µM H₂S (E1); mixture of 25/50 µM/µM R-Se/H₂S

Se molar ratios 50/50 and 150/50 µM/µM. The H₂S/R-Se mixture was (F1); mixture of 25/50 µM/µM R-S/H₂S (G1) and the mixture of 25/50

incubated with pDNA for the given time. I_Rof ncDNA was normalized µM/µM R-O/H₂S (H1). The spectra A2-H2 show details of the

relative to 50 µM R-Se. Mean ± SEM, n = 3.

accumulated first ten A1-H1 spectra. The spectra A3-H3 show details

of the accumulated last five A1-H1 spectra. The intensities of the

time-dependent EPR spectra (A1-H1) and detailed spectra (A2-H2

and A3-H3) are comparable, as they were measured under identical

EPR settings.

•

Under these conditions, the relative intensity of the BMPO-

OOH adduct decreased slowly over the time and was

comparable to the values reported under physiological

conditions (Figure 12A1-A3).⁵⁶The addition of R-Se or R-O

(25 µM) had minor effect on the ^•BMPO-OOH adduct formation,

its concentration or rate of decay (Figure 12B1-B3,D1-D3 and

Figure 13A,B). In contrast, R-S (25 µM) significantly decreased

the quantity of the ^•BMPO-adducts (Figure 12C1-C3; Figure

13A,B) and from the decreased ratio of the ^•BMPO-

The presence of H₂S (50 µM) in R-Se or R-S (25 µM) solution

significantly decreased the quantity of the ^•BMPO-adducts

(Figure 12F1-F3,G1-G3; Figure 13A,B) and significantly

decreased the ratio of the ^•BMPO-OOH/^•BMPO-adducts (Figure

•

13C,D), where a superposition of at least two radicals, BMPO-

•

OOH and BMPO-OH, were recognized. On the other hand, a

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mixture of H₂S (50 µM) with R-O (25 µM) caused similar effect Final discussion

View Article Online

At the sight of the results presented here^Din^O,^Iw^{: 1}e^0.s¹⁰u³g⁹g^/e^Cs⁹t^Nt^Jh⁰a²t²⁴th^5Ge

products of H₂S or GSH interaction with R-Se, having free radical

scavenging and pDNA cleavage activities, can also affect

intracellular molecules other than DNA. Based on the well-

known consequences of oxidative stress, protein oxidation is

also highly expected. We are aware of the fact that much more

effects should be examined to obtain a more complete picture

on action of products of H₂S or GSH interaction with R-Se and

our intention is to present these promising initial results.

as H₂S alone (Figure 12H1-H3; Figure 13). Based on the

•

decreasing quantity of the BMPO-adducts, we suggest that R-

S, H₂S/R-Se and H₂S/R-S scavenge the ^•BMPO-OOH/OH adducts,

•

which may include a direct scavenging of O₂^–or its derivatives.

The decreasing ratio of the ^•BMPO-OOH/^•BMPO-adducts

indicates that the compounds cause the decomposition of

^•BMPO-OOH to ^•BMPO-OH and scavenge both ^•BMPO-adducts.

However, we cannot exclude the possibility of trapping an

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•

unknown radical by BMPO, which decomposed to BMPO-OH

before measurement of the sample. Our data suggest that R-S,

H₂S/R-Se and H₂S/R-S have high potency to scavenge different

radicals.

In summary, the results confirm our initial hypothesis: the

selenoanhydride (R-Se) can act as a H₂Se donor, serving as a

prodrug that enables the internalisation of the selenium in the

cells, and the subsequent release of H₂Se and ionic species of Se

inside the cell. Besides, we have proved that H₂S and GSH

interact with R-Se, and that the intermediates and/or products

of this interaction have significant properties to reduce

(scavenge) the ^•cPTIO and superoxide (O₂^•–) radicals or its

derivatives and to cleave pDNA. The observed antioxidant

(reducing) properties of intermediates and/or products of the

•

H₂S/R-Se and GSH/R-Se interaction to reduce cPTIO, scavenge

•

−

●

O₂and decompose BMPO-OOH to BMPO-OH, indicate that

they may modulate redox properties and free radical signalling.

However, qualifying the significance of these observations is a

challenge for future research

Experimental

Chemical Synthesis of the Anhydride-derivatives

Figure 13. The effects of compounds on the ^•BMPO-adducts radicals.

The effects of compounds (25 µM) and their mixture with H₂S (50

µM) on quantity of the •BMPO-adducts radicals (double integral of

EPR spectra from Figure 12) in the presence of 10% v/v saturated

KO₂/DMSO solution. Average radical quantity during 2-9 (A, from

Figure 12A2-H2) and 10-13 (B, from Figure 12A3-H3) min after

sample preparation. The effects of compounds (25 µM) and their

mixture with H₂S (50 µM) on EPR intensity of the ration of the first

over the second line spectra of the •BMPO-adducts radicals (data

from Figure 12). Average ration during 2-9 (C, from Figure 12A2-H2)

and 10-13 (D, from Figure 12A3-H3) min after sample preparation.

R-Se and R-S (Figure 1) were synthetized according to a

procedure based in the one previously described in literature⁴⁹,

with minor modifications, whereas R-O was commercially

available. Briefly, a suspension of grey selenium (for R-Se) or

elemental sulfur (for R-S) in water-free tetrahydrofuran is

reduced by a drop wise addition of lithium aluminium hydride.

Once completed the reaction (visible by the ceasing of the

generation of molecular hydrogen), phthaloyl chloride is added

to the reaction and left reacting till the end of the reaction

(usually 1 h). Then, solution is filtered to eliminate the metallic

Buffer: 50 mM sodium phosphate, 0.1 mM DTPA, pH 7.4, 37^oC. salts generated during the process, and over the filtrate, a 10

Means ± SEM, n=2

mL of concentrated sulfuric acid are added drop wise. Mixture

is left reacting and the solid formed is filtered and washed with

chloroform. The product isolated from the organic fraction is

recrystallized in hexane.

To summarize this section, taking into account the ability of the

phthalic derivatives to reduce the ^•cPTIO radical, we have

studied also how these derivatives interact with O₂^–observing

•

The structure of the compounds R-Se and R-S was confirmed by

NMR-¹H, and their purity, by LC-MS. Spectra are provided in

Supplementary Material and they are in accordance with

bibliography. The purity of both compounds was 100%,

according LC-MS (see data in Supplementary), so both

derivatives were suitable for biological evaluation as they

accomplish the 95% of purity considered as the minimum

threshold purity value required for biological assays. The

chemical reactions of this synthetic procedure were shown in

Figure 1. It is quite interesting to see how the reaction normally

used to get the oxygen anhydrides (dehydration of the phthalic

how they interfere with the formation of the ^●BMPO-OOH

adduct in the presence of KO₂and BMPO. In this experiment, R-

•

–

Se and R-O showed a minor interaction with O₂radical,

whereas R-S significantly decreased the formation of the

^●BMPO-OOH adduct. Interestingly, the addition of H₂S to R-Se

and to R-S significantly enhances their capacity to decrease the

formation of the ^●BMPO-OOH adduct, again highlighting the

enhanced activity of the products of the interaction between

H₂S and R-Se: The presence of both Se and S atoms seems to be

crucial for all these activities.

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acid) also serves to synthesize the sulfur and selenium argon) H₂O was added, and in 10 s aliquot of the stock Na₂Se

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anhydrides analogues. What is more, it is noteworthy to point solution (10 mM) was added to UV-V^DIS^OIc^{: 1}u⁰v^.1e⁰t³t⁹e^/Cc⁹o^Nn^Jt⁰a²i²n⁴in^5Gg

the selectivity of the formation of the respective thio- and studied compounds. 100 mM sodium phosphate buffer

selenoanhydride when an oxygen atom is bound to the second supplemented with 100 µM DTPA, pH 7.4, 37°C, was employed

carbonyl of the intermediate chalcogen phthalate.

for UV-VIS experiments. 50 and 25 mM sodium phosphate

buffer, supplemented with 100 and 50 µM DTPA

(diethylenetriaminepentaacetic acid), pH 7.4, 37°C was used for

electron paramagnetic resonance (EPR) studies.

In a previous work in a PhD dissertation⁵⁷, to learn more about

the reactivity of different phthalic derivatives to form the

phthalic selenoanhydride (R-Se) following this procedure, a

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synthetic study was performed. R-Se was synthesized according UV-VIS of ^•cPTIO

to the procedure mentioned above departing from phthaloyl

To basic 900 - 990 µl solution of 100 mM sodium phosphate,

chloride and using lithium aluminium hydride for the reaction.

The yield before recrystallization in this case was a 94%. When

the reaction was carried using water as solvent and employing

sodium borohydride as reducing agent, yield before

recrystallization was 47%. Reaction could also use different

phthalic derivatives as substrates (always employing lithium

aluminium hydride), in this case with different yields. We

explored phthalic anhydride and N-hydroxyphthalimide,

achieving yields of 16% and 62%, respectively. Interestingly

(unpublished results), phthalimide did not render the R-Se after

the dehydration with sulfuric acid. It seems that a selenazine is

formed instead of the selenoanhydride, obtaining the 1H-

benzo[d][1,2]selenazine-1,4(3H)-dione. Unfortunately, this

compound could not be obtained with a satisfactory purity and

more research needs to be done to isolate and characterize this

compound.

100 µM DTPA buffer (pH 7.4, 37°C) the final concentrations of

100 µM ^•cPTIO and Na₂S was added and the UV-VIS spectra (900

– 190 nm) were recorded 3 x 30 s. Studied compounds, R-Se, R-

S and R-O (50 mM in DMSO) firstly dissolved in 50 µl buffer and

vortexed for 3 s were added and the spectra were recorded

every 30 s for 20 min using a Shimadzu 1800 (Kyoto, Japan)

spectrometer at 37°C (blank was H₂O). For our study, the ^•cPTIO

extinction coefficient at 560 nm of 920 M^-1cm^–1was used.

•

Scavenging of the cPTIO radical by Na₂S (H₂S) or GSH and its

mixture with studied compounds R-Se, R-S and R-O was

determined as a decrease of absorbance at 356 and 560 nm

•

(absorption maximum of cPTIO) after subtraction of baseline

absorbance, which were determined at 730 or 420 nm,

respectively.⁴²

Plasmid DNA Cleavage

The pDNA cleavage assay, which detects a disruption of the

sugar-phosphate backbone of DNA, was used to study if the

products of the H₂S/R-Se and/or GSH/R-Se interaction can

directly attack pDNA. In this assay, even a single hit is trapped,

as it converts the circular supercoiled DNA molecule into its

nicked relaxed circular form. These two forms display distinct

mobility in agarose gels, and therefore they can easily be

distinguished and quantified.

For pDNA cleavage assay, the anhydride-derivatives were

dissolved in ultrapure deionized water at

1 mM final

concentration by vortexing and 1 min water bath sonication,

subsequently aliquoted and stored at -80°C before their use. For

UV-VIS, EPR and ESI studies, the anhydride-derivatives were

dissolved in anhydrous DMSO at 50 mM concentration,

aliquoted and stored at -80°C before being used.

ESI-MS Measurement

The pBR322 vector (4.361 kb, New England Biolabs, N3033L)

was used in pDNA cleavage assay. In this assay, all samples

contained 200 ng of pDNA in a final volume of 20 µl of buffer

composed of 25 mM sodium phosphate and 50 µM DTPA

(pH 7.4). Three different assay conditions were used: i) pDNA

per se (control), ii) pDNA + phthalic-anhydride derivatives, and

iii) pDNA + anhydride-derivative + Na₂S or GSH. The resulting

mixtures were incubated for 30 min at 37°C. Afterwards, the

reaction mixtures were subjected to 0.6% agarose gel

electrophoresis. The samples were electrophoresed in TBE

buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) at

5.5 V/cm for 2 h. The gel was stained with Gel Red™Nucleic Acid

Gel Stain. Finally, the gels were photographed using a UV

transilluminator. To quantify the pDNA cleavage efficiency, the

integrated densities of two identified pBR322 forms (a

supercoiled and a nicked circular form) in each lane were

quantified using Total Lab TL100 image analysis software

(Nonlinear Dynamic Ltd., USA).

Saturated R-Se was prepared in 50% methanol/H₂O, vortexed

for 2-3 min, sonicated in water bath for 1-2 min and centrifuged

for 2 min. The sample without and with 7 mM Na₂S (~ 9 pH) was

incubated for 1 min at 37⁰C and 55 µl of the supernatant was

used to measure ESI-MS spectra (Orbitrap Elite,

ThermoScientific).

Chemicals for UV-VIS and EPR measurements

The studied compounds, R-Se, R-S and R-O in DMSO (50 mM)

were used after thawing. The spin trap 5-tert-butoxycarbonyl-

5-methyl-1-pyrroline-N-oxide (BMPO, 100 mM, ENZO Life

Sciences AG, Switzerland) was prepared in deionized H₂O,

stored at -80^oC and used after thawing. The radical 2-(4-

carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

(^•cPTIO, 10 mM, Cayman 81540 or Sigma C221) in deionized H₂O

was stored at -20^oC for several weeks. Na₂S as a source of H₂S

(100 mM; SB01, DoJindo, Japan) was prepared in deionized H₂O,

stored at -80⁰C and used after thawing. Na₂S dissociates in

solution and reacts with H⁺to yield H₂S, HS⁻and a trace of S²⁻. EPR of ^•BMPO-adducts

We use the term H₂S to encompass the total mixture of H₂S, HS⁻

and S²⁻. To Na₂Se powder (Alfa Aesar, 36187, stored under

To study the ability of R-Se, R-S and R-O without and with H₂S

•

-

to scavenge the O₂radical produced in DMSO/KO₂solution,

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sample preparation and EPR measurements were conducted in interact with GSH and H₂S. This is of crucial importance, as it

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accordance with previously reported protocols.⁴²The solution would enable detecting new compounds that could behave as

DOI: 10.1039/C9NJ02245G

(final concentrations) of BMPO (20 mM), DTPA (100 µM) in Se-based redox modulators in a potential Se supplementation.

sodium phosphate buffer (50 mM, pH 7.4) was incubated for 1 An example would be the phthalic selenoanhydride (R-Se)

min at 37°C. An aliquot of the compounds studied was added, reported in this work, which maintains the capacity of sodium

followed by the addition of Na₂S in 3 s and saturated KO₂/DMSO selenite to react with key components of the redox thiolstat (as

solution (10% v/v DMSO/final buffer) 3 s later. The sample was GSH and H₂S) and simultaneously, according to previous works,

mixed for 5 s and the first EPR spectrum was recorded 2 min shows a lower toxicity against non-tumour cells.

after the addition of KO₂/DMSO solution at 37°C. The sets of

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individual EPR spectra of the ^•BMPO spin-adducts were

Conflicts of interest

recorded as 15 sequential scans, each 42 s, with a total time of

11 min. Each experiment was repeated at least twice. EPR

spectra of the ^•BMPO spin-adducts were measured on a Bruker

EMX spectrometer, X-band ~9.4 GHz, 335.15 mT central field, 8

mT scan range, 10 mW microwave power, 0.1 mT modulation

amplitude, 42 s sweep time, 20.48 ms time constant, and

20.48 ms conversion time at 37°C.

There are no conflicts to declare.

Author Contributions: Conceptualization, E.D-A., C.J., M.C., and

K.O.; Methodology, K.O., M.C., and V.B.; Validation, K.O., M.C.,

V.B., A.M., M.G., and E.D.-A.; Formal Analysis, A.M., and K.O.,

Investigation, A.M., M.G., A.K., V.B., L.K., P.B., M.C., K.O., and E.

D.-A.; Resources, A.K., C.J., and E.D.-A.; Writing-Original Draft

Preparation, K.O., M.C., and E.D.-A.: Visualization, K.O., A.M.,

M.G., and E.D.-A.; Supervision, K.O., C.J., M.C., and E.D.-A.;

Project Administration, K.O., C.J., M.C., and E.D.-A.; Funding

acquisition, K.O.

The relative quantity of the ^•BMPO-adducts radicals was

calculated as double integral of EPR spectra. Since EPR spectra

were mostly low intensity, which did not permit spectral

simulation, to quantify relative ratio of the ^•BMPO-

OOH/^•BMPO-adducts, the ratio of EPR intensity of the first line

over the second line was used. The ratio is ~ 1 at ~ 100 % of

^•BMPO-OOH (Figure 12A2) and ~ 0.5 at ~ 0 % of ^•BMPO-OOH.⁴²

The lower ratio (lower than 1) indicates higher concentration of

Acknowledgements

•

This research was funded by the Slovak Research

&

other BMPO-adducts, in which mostly BMPO-OH radicals are

present.

Development Agency, grant numbers APVV-15-0371, 14-0783,

15-0565 and 17-0384 and the Scientific Grant Agency of the

Slovak Republic, grant numbers VEGA 1/0041/15, 1/0026/18,

2/0146/16, and 2/0014/17. The authors would also like to

acknowledge the financial support of the Agencia Estatal

Consejo Superior de Investigaciones Científicas (Spain, project

201780I027) and of the University of Saarland. We also thank,

Prof. Dr. Anna K. H. Hirsch and Dr. Eleonora Diamanti from

Helmholtz Institute for Pharmaceutical Research in Saarland

(HIPS) for helping with MS spectra measurements.

Conclusions

Understanding of the molecular mechanism of biological effects

of phthalic-anhydride derivatives could lead to development of

more efficient drugs for treatment of cancer and ROS related

diseases. To achieve this, we found, that phthalic-anhydride

derivatives, R-Se, R-S, R-O and R-OH (≤ 50 µM) on their own

have minor potency to reduce/scavenge radicals or cleave

pDNA. However, the potency of R-Se and R-S, but not R-O or R-

OH, significantly increased after interacting with H₂S and GSH.

Notes and references

Our in-vitro data revealed unique properties of the H₂S/R-Se,

GSH/R-Se and H₂S/R-S mixtures to reduce the ^•cPTIO and

superoxide radicals. The unique potency of the H₂S/R-Se

mixture to cleave pDNA has the bell-shaped dependence on H₂S

and GSH concentrations, whereas potency of H₂S/R-S increased

linearly with H₂S, but did not increase with GSH concentration.

The results underline that the interactions of R-Se and R-S with

H₂S and GSH enhanced significantly the different activities

monitored, thus indicating that the intermediates and/or the

products of the interaction of R-Se and R-S with endogenous H₂S

and GSH have significant antioxidant properties and that they

can damage DNA. These findings may contribute to understand

more-in-depth the unique biological effects reported so far for

R-Se and R-S. Besides, these findings open a new so far

unexplored approach to study the action of the Se-containing

compounds. These experiments, for example, can be applied to

the different selenium species that have been used until now in

supplementation, to ascertain which ones have more ability to

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Article Doi

Release of reactive selenium species from phthalic selenoanhydride in the presence of hydrogen sulfide and glutathione with implications for cancer research

DOI: 10.1039/c9nj02245g

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Authors:

Article abstract of DOI:10.1039/c9nj02245g

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