Substituent, structural and positional isomerisation alter anti-oxidant activity of organochalcogen compounds in rats’ brain preparations

1 Introduction

Reactive oxygen species (ROS) are an inevitable by-product of cellular respiration which causes oxidation of lipids, nucleic acids and proteins. ROS induced damage is the underlying cause of various neurodegenerative diseases (Valko et al., 2006; Angel et al., 2005). Cells have sophisticated antioxidant regulatory systems to maintain proper balance of ROS. However, disruption in homeostasis can result in oxidative stress and tissue injury (Perry et al., 2002).

Literature have demonstrated that organoselenium compounds possess very interesting biological activities like, glutathione peroxidase-mimic, lipid peroxidation, radical-scavenging, antiinflammatory, antinociceptive, hepatoprotective, nephroprotective and neuroprotective activity (Nogueira et al., 2004). In this regard our group has shown that selenium-containing molecules are better nucleophiles (and therefore better antioxidants) than classical antioxidants and this has led to the a significant development in synthetic organoselenium compounds with pharmacological potential (Arteel and Sies, 2001; Hassan et al., 2009a,b,c). Moreover, clinical trials in humans revealed beneficial effects of organoselenium compounds such as ebselen in pathological situations (Parnham and Sies, 2000). Similarly, organoselenium compounds are good glutathione peroxidase (GPx) mimics and have shown interesting biological activities in different models of oxidative stress induced damages (Meotti et al., 2004; Hassan et al., 2009a,b). Sulfur compounds are also studied for their antioxidant properties and a recent study indicates that aqueous garlic extract protects against arsenic toxicity (Chowdhury et al., 2008). Biological sulfur-containing compounds, including cysteine, methionine, taurine, glutathione (GSH), N-acetylcysteine (NAC), and other sulfur compounds have been extensively studied for their antioxidant properties (Parcell, 2002; Atmaca, 2004).

This paper describes the synthesis of a series of sulfur and selenium agents (according to the literature methods) and evaluation of their antioxidant potential. In order to understand the biochemical efficacies of these chalcogens, we particularly emphasized on a chemically multidimensional approach towards antioxidant characterization that includes (i) effect of chain length i.e., introduction of CH₂ group (ii) different ring substituent effects i.e., both electron donating and electron withdrawing groups and (iii) not only the effect of ortho, meta and para positioning but also structural changes of the same molecule leading to different isomers and its anti-oxidant effect are described in detail. We have tested the efficacy of these compounds against Fe(II) induced TBARS formation in rat’s brain homogenate. The activities of these compounds are also measured and confirmed by their thiol peroxidase like activity followed by DPPH radical scavenging assay. In vivo work followed the in vitro assays to get a better understanding of its possible toxic profile.

2 Material and method

2.1

2.1 Synthesis of organoselenium and organosulfur compounds

The unsymmetrical diorganyl selenides and sulfides (Fig. 1) were synthesized using the literature procedure (Narayanaperumal et al., 2009; Brindaban et al., 2004). Diphenyl disulphide (CAS No: 882-33-7) was purchased and used for the experiments.

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Figure 1

Structures of organochalcogen compounds.

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Reaction 1. Synthesis of diorganyl selenides. $$\text{RSeSeR}{\displaystyle \underset{(\text{bmim}){\text{BF}}_4/30\text{min}}{\overset{{\displaystyle \genfrac{}{}{0ex}{}{2{\text{R}}^{\prime }\text{X}}{\text{lnl}(1.0\text{eq})}}}{\to }}}2{\text{RSeR}}^{\prime }$$

Reaction 2. Synthesis of diorganyl sulfides. $$\text{RSSR}\underset{{\text{CH}}_2{\text{Cl}}_2/\text{rt}}{\overset{{\displaystyle \genfrac{}{}{0ex}{}{2{\text{R}}^{\prime }\text{X}}{\text{lnl}(1.0\text{eq})}}}{\to }}2{\text{RSR}}^{\prime }$$

3 Results and discussion

The structures of all tested compounds are shown in Fig. 1. The influence of the substituent on organoselenium compounds can be observed through the potential shift in anti-oxidant activities as shown in Fig. 2A. Addition of Methoxy –OCH₃ (mesomerically electron donating group) group at ortho (C-3) and para (C-4) position, significantly improved antioxidant activity. Importantly, the para isomer displayed significantly a higher antioxidant potential than ortho isomer. The results were replicated (C5 and C6) by the addition of another electron donating group (CH_3, inductively electron donating group). Hypothetically, the electron donating group increases the electronic density on selenium atom which may increase its anti-oxidant activity. For this purpose we synthesized three isomers (C7–9), where an electron donating group i.e., (CH₃) was attached to the benzyl ring (this ring does not have a direct bond with selenium). None of the compound showed better anti-oxidant potential than C-6. However, these isomers (C7–9) have better antioxidant activity than parent C1 and C2. These results prove that isomerisation has a significant effect on the anti-oxidant activity of organoselenium compounds. Contrarily, the presence of an electron withdrawing group might destabilize the selenium moiety by withdrawing electronic density and may lead to a decrease in the antioxidant potential. This phenomenon is observed in our results, where C-10, a compound with an electron withdrawing group (chlorine-Cl) did not show any antioxidant potential. In fact, C-10 at the highest tested concentration showed pro-oxidant behavior as apparent from increased TBARS formation as shown in Fig. 2A.

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Figure 2

Effect of organoselenium (A) and organosulfur compounds (B) against Fe(II) induced TBARS formation.

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Like the organoselenium compounds we synthesized structurally related organosulfur compounds and tested their efficacy against Fe(II) Induced TBARS production. The parent compound i.e., diphenyl sulfide (C-11) did not show any activity. It is apparent from Fig. 2B that introduction of the electron donating group did not improve antioxidant potential. Similarly, the electron donating group (CH₃) attached to the benzyl moiety of organosulfur compounds at ortho, meta and para position i.e., C-14, C-15 and C-16 did not show any anti-oxidant activity. Similarly the introduction of the electron donating group (Cl) at C-17 showed pro-oxidant behavior.

Our results indicate that organoselenium compounds are better anti-oxidants than its sulfur analogues. Although selenium shares many chemical properties with its neighboring homologue sulfur, selenium differs from sulfur in a number of ways, the most significant being that selenol is a more powerful nucleophile than thiol. In contrast to thiols, selenols exist mostly in anionic form at neutral pH and represent good reducing groups under normal physiological conditions (Burk, 1994). This possibly could be the main reason for the better anti-oxidant activity of organoselenium compounds than organosulfur analogues.

Organoselenium compounds protected against Fe(II) induced TBARS production at highest tested concentration. Ferryl ion (Bors et al., 1979) perferryl ion (Pederson et al., 1973) and Fe⁺²–O₂^•–Fe⁺³ complexes (Bucher et al., 1983) have been proposed to be involved in the initiation of Fe(II)-dependent lipid peroxidation. A plausible mechanism by which organoselenium compounds are conferring protective action against Fe(II)-induced lipid peroxidation in these homogenate is that organoselenium compounds may be interacting with Fe(II) or its oxidized forms.

Organoselenium compounds showed better thiol peroxidase like activities (Fig. 3A) than their sulfur analogues (Fig. 3B). However, it is important to note that in strong contrast to TBARS inhibitory activity (Fig. 2A) the compounds C7, C8 and C9 did not show any improved thiol peroxidase activity than parent C1 or C2 compound (Fig. 3A). The plausible explanation for this contrasting behavior between thiol peroxidase like activity and TBARS inhibitory activities could be that although GPx measurements are typically used to determine antioxidant activity of selenium compounds, these measurements may not accurately reflect cellular conditions. GPx activity measurements are often determined under conditions that are not physiologically relevant, such as using non-aqueous solutions or non-biological thiols (Sarma and Mugesh, 2005; Mareque et al., 2004; Marnett, 2000).

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Figure 3

Thiol peroxidase activity data of organoselenium (3A) and organosulfur compounds (3B).

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The ability of sulfur compounds to scavenge ROS has been attributed to a possible antioxidant mechanism for these compounds. (Mishra et al., 2008; Kim et al., 2006; Shindo and Brown, 1965; Livingstone and Nolan, 1968; Battin and Brumaghim, 2008). However our results indicated that all sulfur compounds lack anti-oxidant activity. This behavior is apparent from lack of anti-oxidant activity against Fe(II) induced TBARS production (Fig. 2B), thiol peroxidase like activity (Fig. 3B) and inactivity towards DPPH^• radical scavenging assay (Fig. 4). The lack of anti-oxidant activity indicates that these organosulfur compounds neither exhibit metal binding capacity nor act as free radical scavenger which may be produced by fenton reactions.

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Figure 4

DPPH radical scavenging data of organosulfur compounds.

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Based on our in vitro data, C-6 with the highest TBARS inhibition and thiol peroxidase like activity was selected for in vivo work. Our results indicated (Fig. 5A) that C-6 has no effect on TBARS content and confirms the safe position of the tested compound. Organochalcogens can interact directly with low molecular thiols oxidizing them to disulfides (Goeger and Ganther, 1994). Reduced cysteinyl residues from proteins can also react with simple diselenides and ditellurides, which can cause, in the case of enzymes, the loss of catalytic activity. Besides, organoselenium and organotellurium compounds have been reported to inhibit various sulfhydryl group containing enzymes, like 5-lipoxygenase (Björnstedt et al., 1996), protein kinase JNK1 (Park et al., 2000), δ-aminolevulinate dehydratase (Barbosa et al., 1998; Nogueira et al., 2003) and squalene monooxygenase (Gupta and Porter, 2001; Laden and Porter, 2001). However the present study revealed that compound-6 did not inhibit the δ-ALA-D activity (Fig. 5B) rather at the highest tested concentration it increased NPSH content (Fig. 5C). Rats exposed to C-6 presented no hepatic and renal damage apparent from normal enzymatic and biochemical parameters (Fig. 6).

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Figure 5

Effect of compound 6 on TBARS formation (A), α-ALA-D activity (B) and NPSH (C) at different doses in rat’s tissue preparation. Data are reported as mean ± SEM for six rats in each group.

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Figure 6

Effect of compound 6 on toxicological parameters after 3-doses of acute treatment. Data are reported as mean ± SEM for six rats in each group.

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Previously we have shown that acute exposure to high doses of DPDS did not affect plasma transaminase activities or urea and creatinine levels in rodents (Meotti et al., 2003). Furthermore, rabbits chronically exposed to DPDS supplemented diet have hepatic and renal functions within the normal range (de Bem et al., 2007). Taken together, these data suggest that acute exposure to this mono-selenide (C-6) did not induce oxidative stress in rats. Further studies are needed to understand the potential therapeutic and toxicological efficacies of this interesting class of compounds.