BIOCHEMICAL TOXICOLOGY OF SIMPLE DIORGANYL CHALCOGENIDES

Cristina W. Nogueira¹, Evelise N. Maciel¹, Gilson Zeni¹, Dominguita Graça², João B. T. Rocha¹

1.Departamento de Química – Universidade Federal de Santa Maria – Santa Maria – RS – Brasil – 97105-900
2. Departamento de Patologia - Universidade Federal de Santa Maria – Santa Maria – RS – Brasil – 97105-900
E-mail: jbtrocha@yahoo.com.br

Received: 15 August 2001 / Uploaded 22 August 2001
 

The explosive growth of organochalcogen chemistry, over the last years, can be attributed to the specific properties of organic chalcogenides compounds, which fit the requirements of modern organic synthesis. Most of them are well adapted to chem., regio and stereo-selectivities. In addition, they can be used in mild experimental conditions, which are compatible with the stability end of the substrates and products in the preparation of unsaturated and functional complex molecules, especially in the field of natural products.

The main selenium functional groups have been know for a long time, and some compounds have being described in the last century, but the use of selenium reagents or intermediates is more recent. We can take 1973 as the year modern organo selenium chemistry was born, and it was also the year the first important book dealing with the synthesis and properties of selenium organic compounds was published. Since 1973, several publications have been devoted to increasing our knowledge of the subject.

Dialkyl and diaryl diselenides are useful intermediates in organic synthesis. These compounds can be synthesized by alkylation of the diselenide anion, nucleophilic aromatic substitutions, selenium addition on a Grignard reagent and an organoillithium with subsequent hydrolysis.

As well, vinyl selenides are important reagents and intermediates in organic synthesis^2d and among a large list of described transformations, the ability of these compounds to participate in the formation or carbon-carbon bonds by the Ni-catalyzed cross-coupling reactions with magnesium or zinc reagents is remarkably interesting. Only few synthetic approaches have been reported for 1,1-disubstituted vinylic selenides,^2d, while a large number of methods for 1,2-disubstituted vinyl selenides of E or Z configuration are known. The most common method involves the addition of organo selenols to terminal alkynes under different reaction conditions. It should be noted that the regio- and stereochemistry of the obtained products is very sensitive to the reaction conditions (Scheme 1).

Scheme 1:

The Pd-catalyzed hydroselenation of alkyl mono-substituted acetylenes affords the 1-alkyl-1-phenylseleno ethenes as the main product among the 1,2-disubstituted isomers. The opposite regioselectivity is observed when an aryl group is linked to the triple bond of the starting material. The use of organo selenols as reagents is a remarkable disadvantage that can not be disregarded in view of the difficulties in preparing and handling these very volatile, bad-smelling, toxic and air-sensitive compounds. The use of diphenyl diselenide, a commercially available, stable and odorless solid, could be a more convenient and an alternative reagent to be used to perform the hydroselenation of alkynes by the in situ generation of selenolate anions.

Organo selenium compounds have been described to possess very interesting biological activities, such as 6-Selenopurine 1, selenazoles 2 and p-methoxybenzeneselenol 3 shown to exhibit antitumor activities. Phenyl-2-aminoethylselenide 4 exhibits cardiovascular activities. 2-phenyl-1,2-benziso-selenazol-3(2H)-one (Ebselen) 5 exhibits anti-inflammatory action in a number of experimental models. 5-hydroxy benz-2,1,3-selenadiazole 6 has shown to exhibit antibacterial and anti-fungal activity (Scheme 2).

Scheme 2:

On the other hand, organo tellurium chemistry is an area that has not attracted much attention. Many tellurium compounds possess an intrinsic thermodynamic instability, a property that is characterized by the deposition of the element. It is not unusual for tellurium compounds to exhibit photosensitivity and it is often necessary to perform laboratory procedures in the dark or in subdued light. Another factor that may account for the relative lack of interest in organo tellurium chemistry is that no compound containing tellurium has yet been detected in any living organism. There is no question that the discovery of selenoproteins and selenium-containing enzymes, especially of the mammalian enzyme, glutathione peroxidase, is largely responsible for the great interest in selenium biochemistry, which has developed, in recent years. However, the synthetic potential of this class of organoelemental compounds has only recently received the attention of organic chemists. Actually, vinylic tellurides had been much less studied. However, their promising potential could be anticipated by the combination of the special reactivity of tellurium and particularly the reactivity associated with a carbon-carbon bound. The most interesting synthetic transformation of tellurium compounds is their conversion into reactive organometallic compounds by reaction with organometallic species (Scheme 3).

Scheme 3:

Recently was developed a catalytic coupling process of vinylic tellurides with alkynes giving Z enyne and Z neediness (Scheme 4).

Scheme 4:

The success of these transformations shows that vinylic tellurides can be used for the synthesis of natural products having conjugated unsaturated systems. Another application of these compounds was demonstrated when they were successfully employed in the synthesis of Montiporic Acids A and B (Scheme 5).

Scheme 5:

An additional application of vinylic tellurides was presented when they were used in the synthesis of four polyacetylenic acids isolated from Heisteriaacuminata (Scheme 6).

Scheme 6:

Although tellurium has no known physiological role and is highly toxic, organic compounds of tellurium have been demonstrated to possess aintitumor and antiviral effects, and consequently, the use of organotellurides as a pharmacological agent will probably increase in the near future.

Interaction of Thiols with Diorganyl Chalcogenides

The molecular mechanism underlining selenium toxicity is still not completely understood; however, about 60 years ago Painter proposed that the toxicity of selenium could be related to the oxidation of thiols of biological importance. In fact, selenite (Se⁺⁴) readily oxidizes sulfhydryl groups, producing disulfide and an unstable intermediary now named as selenotrisulfide (or (bis(alkylthio) selenide). Painter²⁹ proposed that the reaction between Se⁺⁴ and –SH groups may take three distinct courses:

However, Painter²⁹ was unable to isolate compounds of the type RS-Se-RS from disulfides, because of the instability of the selenotrisulfide. Different laboratories demonstrated formation of compounds of the type RS-Se-RS of biological significance some years latter. However, Ganther accomplished the isolation in chemical pure form of selenotrisulfide only about 30 years after the original Painter’s proposal.

Tsen and Tapel^30c reported that selenite catalytically oxidized reduced glutathione (GSH) and propose the following sequence of reactions to explain the catalytic effect of Se⁺⁴:

The fact that selenite was a good catalyst for the oxidation of a variety of biologically significant thiols; including, in addition to GSH, cysteine, dihydrolipoic acid and Coenzyme A helped to explain the biochemistry of selenite poisoning, where a significant losses of GSH from the blood and organs of experimental animals were observed . Therefore, these studies confirmed the early proposition of Painter and established that selenite exerts its toxic action by catalyzing the oxidation of biologically significant sulfhydryl-containing molecules.

More recently, Seko and colleagues suggested that selenite reacts with GSH forming reactive superoxide (O_2·^-). This observation was extended by Spallhozl and colleagues, which showed that other thiols reacted with selenite and selenocystine to produce superoxide and hydroperoxide. According to these authors the selenium toxicity will be manifested acutely or chronically only when oxidative damage exceeds antioxidant defenses.

Practically at the same time that the toxicology of selenium was indubitably related to interaction of Se⁺⁴ with sulfhydryl compounds, Schwarz and Foltz concluded that selenium was the essential part of the active organic Factor 3 which prevents liver necrosis in rats fed diets deficient in selenium and vitamin E. Most importantly, they demonstrated that the Factor 3 was not an essential organic dietary constituent, since it could be replaced by selenite. These findings intensified research on the physiological and biochemical role played by selenium in mammals, with special emphasis on its interrelationship with sulfur-containing amino acids. In fact, it has been known for many years that selenium in plants and animals was associated with proteins . Furthermore, exogenous administered Se⁴⁺ became firmly attached to proteins of rats . However, for a long time it was not known whether selenium became bound to proteins because, as pointed out above for selenite, its interacts with reactive –SH groups of a variety of proteins or if it was reduced and incorporated into proteins as a component of amino acids. Now it is well established that incorporation of inorganic Se in proteins is rather complex, involving selenophosphate synthesis from selenide and ATP by a selenophosphate synthetase followed by incorporation of Se into selenocysteine synthesized from seryl-tRNA (Sec) .

Organic selenocompounds such as selenocystine and a variety of diselenides can also react with thiols such as cysteine, dithiothreitol, and reduced glutathione to produce selenols and disulfides. In line with this findings, Günther showed that dithiothreitol (DTT), a compound with extremely low redox potential, reduced a variety of diselenides, forming selenols and trans-4, 5-dihydroxy-1,2-dithiane, the oxidation product of DTT. Some decades ago the reduction of diselenides to selenols by reaction with thiols was considered to be of physiological significance^40, . Indeed, Walter and collaborators hypothesized that selenoamino acids, particularly selenomethionine, could act as reversible catalytically effective biological antioxidants. However, it is now apparent that selenium plays its most fundamental role as a component of the active center of the enzymes glutathione peroxidase and phospholipid hydrogen glutathione peroxidase. In these peroxidases selenium is found as a residue of selenocysteine and during the catalytic cycle the selenol group is oxidized by peroxide to a selenenic acid. In subsequent steps, selenol is regenerated by the reaction of selenenic acid with reduced glutathione. Of particular importance, simple diorganylchalcogenides such as diphenyl diselenide and diphenyl ditelluride and the antioxidant selenocompound, ebselen accelerate the rate of peroxide reduction by a variety of thiols according to the reaction described below:

This reaction is similar to that catalyzed by glutathione peroxidase and is of particular significance for living cells because it decompose hydrogen peroxide, an intermediary that can give origin to the extremely reactive and toxic product OH· . Diorganylcalchogenides and ebselen can also reduce phospholipid hydroperoxides ; thus, protecting biomembranes from peroxidative degradation. The thiol-peroxidase like activity of diorganyl calchogenides can explain, at least in part, the in vitro antioxidant properties of these compounds.

In addition to act as glutathione peroxidase mimic, simple diselenides and ditellurides can accelerate the rate of thiol oxidation even in the absence of peroxide by catalyzing the following reaction:

X = Se, Te

In the presence of oxygen, diorganyl chalcogenides are regenerated:

Although the peroxidase-like activity of diselenides may account for their antioxidant properties, the thiol-diselenide exchange catalyzed by chalcogenides may contribute to their toxicological properties by oxidizing relevant thiol containing metabolites and proteins.

Interaction of Diorganyl Chalcogenides with d -Aminolevulinate Dehydratase

As pointed out above, diorganyl chalcogenides can interact directly with low molecular thiols, oxidizing them to disulfides. Reduced cysteinyl residues from proteins can also react with simple diselenides and ditellurides, which may cause, in the case of enzymes, the loss of catalytic activity. For instance, d -aminolevulinate dehydratase or porphobilinogen synthase is a sulfhydryl-containing enzyme, which is extremely sensitive to oxidizing agents. This enzyme catalyzes the asymmetrical condensation of two molecules of 5-aminolevulinic acids to form porphobilinogen, an intermediary in tetrapyrol biosynthesis. Hence, this enzyme play a fundamental role in most living aerobic and photosynthesizing organisms by participating in heme and chlorophyll biosynthesis. The mechanism of porphobilinogen synthesis is similar in animals and plants; however, the enzyme obtained from these sources exhibits subtle structural diversity.⁴⁹ Accordingly, recent data from our laboratory showed that aminolevulinate dehydratase from plant, in marked contrast to the enzyme from rat, was not inhibited by diphenyl diselenide and diphenyl ditelluride. The divergent response of the plant enzyme to diphenyl diselenide (and possibly to others simple diorganyl chalcogenides) is presumably related to differences in quantity and in the spatial proximity of cysteinyl residues in the three-dimensional structure of plant and mammal enzyme. In fact, plant enzyme has no cysteinyl residues in close spatial proximity as observed in the active site of the mammal enzyme.⁴⁹ In view of this, we have proposed the following scheme to explain why the mammal enzyme is inhibited by diphenyl diselenide and diphenyl ditelluride, while plant enzyme is not affected by these compounds:

The first step in this scheme involves the formation of an unstable intermediary of the type E-Cys-S-SePh and PhSeH. Subsequently, a less reactive cysteinyl residue (represented as Cys**-SH in the scheme above) due to its close spatial proximity to the more reactive residue attacks the sulfur atom of the E-Cys*-S-SePh, producing the oxidized (and inactive) enzyme, and a second molecule of PhSeH. Support for this scheme has also been obtained using low molecular weight thiol-containing molecules. We have observed that DTT (a dithiol) is a better substrate than cysteine or GSH (monothiols) for the oxidation catalyzed by diselenide and ditelluride. The PhSeH molecules formed after reaction with –SH are oxidized back to PhSeSePh by atmospheric O₂. The regeneration of PhSeSePh after oxidation of phenylselenol by oxygen helps to explain the previous observation that the inhibitory effect of PhSeSePh towards rats ALA-D decreases considerably in an anaerobic atmosphere.⁵⁰

The results obtained with mammal ALA-D suggest that proteins containing cysteinyl residues in close spatial proximity in their three-dimensional structure will react more promptly with diorganyl chalcogenides. Consequently, we can suppose that the cellular toxicity of diorganyl chalcogenides may be related, at least in part, to oxidation of sulfhydryl groups of target proteins containing vicinal –SH groups.

Other aspect that must be mentioned here is the presence of a thermolabile factor that catalyzes the thiol/disulfide/selenol/diselenide exchange in rat and fish tissues. Using DTT as an enzyme model, we observed that the rate of oxidation of DTT caused by diselenides (diphenyl diselenide and pCl-diphenyl diselenide) increased considerably when liver supernatants were added to the reaction medium containing DTT and diselenide. Furthermore, heating the tissue fraction for 15 minutes at 100 ºC abolished this effect caused by liver supernatant. Based on this results we postulated that mammal and fish liver posses a thermo labile factor (possibly an enzyme) that catalyses the following reaction:

In analogy to reactions presented above, atmospheric oxygen can regenerate diphenyl diselenide. Furthermore, we also presuppose that thiol-containing enzymes (such as ALA-D) can participate in this exchange reaction between thiol and diselenides.

Neurotoxicity of Organylchalcogenides

Inorganic and organic tellurium compounds are highly toxic to the central nervous system of rodents. Recent histological data from our laboratory have shown that mice exposed to diphenyl ditelluride for one day (0.5 mmol/kg) or 14 days (2.5, 10 and 18.5 m mol/kg) showed accentuated vacuolization of cell bodies and neuropil, and this spongy change was more conspicuous within pontine nuclei in mice brain (Figure 1). Mice exposed to a single (1 mmol/kg) or to 14 doses (0.125 or 0.25 mmol/kg) of diphenyl diselenide showed no neurotoxic histological sign of intoxication. However, a clear inhibition of cerebral ALA-D of mice exposed acutely or chronically to diphenyl ditelluride or diphenyl diselenide was observed. Furthermore, exposure to high doses (250 m mol/kg, s.c) of diphenyl diselenide increased 3 times brain total selenium content, indicating that the brain accumulate a considerable quantity of organochalcogens. These results suggest that brain is a potential target for the toxicity of high lipophilic diorganyl chalcogenides; however, the pathological effect caused in the brain of mice varied considerably depending on the chalcogenide. In fact, exposure to single or multiple doses of diphenyl ditelluride caused a striking neurotoxic effect observed under light optic microscopy; while the analogous selenium compound did not cause any observable histological alteration.

Figure 1: Neurotoxicity of diphenyl ditelluride. Mice were exposed subcutaneously for 14 days to (a) dimethylsulfoxide (2.5 ml/kg); (b) 2.5, (c) 10, or (d) 18.5 m mol/kg of diphenyl ditelluride. Morphologic examination was carried out by light microscopy. Trimmed slices were processed for grade ethanol dehydration, embedded in paraffin blocks, cut into 6 m m sections, stained with hematoxylin and eosin (HE), and examined by light microscopy (x400).
 

The amino acid glutamate, the main excitatory neurotransmitter in the mammalian brain, is believed to play important roles in several physiological and pathological processes. Glutamatergic neurotransmission is achieved through ionotropic (ligand-gated ion channel) and metabotropic receptors (coupled to GTP-binding proteins (G-proteins), modulating second messangers systems). It is reasonable to conceive that modifications in glutamatergic transmission may contribute to initiate or exacerbate neurotoxic effects caused by a variety of agents. In fact, changes in glutamatergic transmission have been implicated in the genesis of a variety of neuronal insults, including cerebral ischemia, hypoxia, trauma, neurodegenerative disorders and acute injuries, i.e. stroke and convulsions.

Similarly, increasing concentrations of the glutamate in the synaptic cleft may produce neurotoxic effects associated with an over stimulation of the glutamatergic system and has been implicated in acute neurological disorders. Guanine nucleotides can antagonize glutamatergic transmission by acting at extracellular sites located at the membrane surface. These extracellular sites are distinct from classical intracellular adenylate cyclase-coupled G-protein site that binds guanosine triphosphate (GTP) , and guanine nucleotide binding to the extracellular site seems to confer neuroprotection against glutamate excitotoxicity.

In order to investigate the participation of glutamatergic system in the neurotoxicity of diorganyl chalcogenides several parameters related to this system were studied in vitro and after in vivo exposure to diphenyl diselenide and diphenyl ditelluride in rats. Nogueira et al (2001) demonstrated that diphenyl diselenide and diphenyl ditelluride inhibit the [³H]glutamate, [³H]MK-801 and total [³H]GMP-PNP binding to rat brain synaptic membrane preparations after both in vitro and ex vivo exposure. These parameters are important indicators of glutamatergic system functionality. Observations suggest that organotellurides are more reactive than structurally related organoselenium compounds, essentially due to their higher electronegativity in relation to carbon, associated with a larger atomic volume.^21a Results from experiments ex vivo⁶⁴ are in agreement with these observations (Table 1). The dose of diphenyl diselenide used was about 8 times higher than that of diphenyl ditelluride and these doses caused similar reductions in [³H]MK-801, [³H]glutamate and [³H]GMP-PNP binding, suggesting that (PhTe)₂ is more toxic than (PhSe)₂ when the dose injected is considered.

An inhibitory effect on [³H]glutamate and [³H]MK-801 binding may indicate that the toxicological properties of organochalcogens may be related, at least in part, to an inhibition of physiological excitatory neurotransmitter systems. Recent evidence has show that a fine tune of glutamatergic system functioning is essential for proper brain functioning. In fact, an unbalanced increase or decrease in glutamatergic systems is highly neurotoxic. However, in vitro the reactivity towards biological systems does not always follow this role, since only [³H]glutamate binding was more sensitive to diphenyl ditelluride than to diphenyl diselenide. [³H]MK-801 and total [³H]GMP-PNP binding were similarly affected by both compounds.

The mechanism(s) underlining the neurotoxicology of organochalcogens is still poorly known, but tentatively involves the oxidation of essential thiols in target proteins^56, (see above in this paper for further discussion). The absence of a protective role of DTT, a reducing agent that protects sulphydryl molecules from oxidants, against the inhibitory effects of (PhSe)₂ and (PhTe)₂ on [³H]glutamate binding to synaptic plasma membranes suggest that the toxicological properties of these organochalcogens are not related to oxidation of –SH groups on glutamate receptors.⁶⁴

Table 1-Effect of diphenyl diselenide and diphenyl ditelluride on [³H]glutamate [³H]MK-801 and total [³H]GMP-PNP binding ex vivo and in vitro to rat brain synaptic membrane preparations. Adapted from Brain Research, 2001, 906, 157.

	Diphenyl diselenide		Diphenyl ditelluride
Ligand	in vitro (100m M)	ex vivo (25m mol/kg)	in vitro (100m M)	ex vivo (25m mol/kg)
[3H]MK-801	30	30	50	30
[3H]Glutamate	50	30	70	30
[3H]GMP-PNP	40	50	40	60

Data represent % binding inhibition in relation to control value (100%).

Our results suggest that organochalcogens affect in a rather complex way the glutamatergic system after acute exposure in rats. The two organochalcogens tested inhibited the binding of ligands to glutamatergic receptors and the binding of [³H]GMP-PNP, presumably to G-proteins that may be linked to these receptors.

Alternatively, organic forms of selenium and tellurium have been pointed out as possible antioxidant agents because they exhibit glutathione peroxidase-like activity and oxidized –SH during the reduction of H₂O₂. In addition, these organochalcogens also retard the lypoperoxidation induced by a variety of oxidants. In fact, recent data from our laboratory suggest that a variety of organochalcogenides [(PhSe)₂, (pCl Se)₂ and (PhTe)₂] are relatively good antioxidants against peroxidation induced by quinolinic acid and sodium nitroprussiate in brain homogenates. In general they are less effective than ebselen; however since no detailed toxicological study have yet been evaluated for diselenide, the therapeutic use of diselenide can not be discarded. Indeed, our unpublished results showed a similar survival dose-response profile to diphenyl diselenide and Ebselen in rats. The LD₅₀ for rats were 750 and 500 m mol/kg diphenyl diselenide and Ebselen, respectively. In contrast diphenyl ditelluride is highly toxic, independent on the route of administration (i.p or s.c) in rats and mice (LD₅₀ values were lower than 1 and 75 m mol/kg respectively).

Can Diorganyl Chalcogenides Be Used Therapeutically?

Recently, it has been demonstrated that Ebselen has a protective effect against brain ischemic insults in humans and in a variety of experimental models of acute excitotoxicity caused by glutamate^47c,. The successful use of Ebselen in clinical trials clearly indicates that seleno-organic compounds, including diorganyl selenides, are promising therapeutic agents. Organotellurium compounds have also been proposed as potential therapeutic antioxidant agents; however, regarding diorganyl tellurides, we have great concern with a possible therapeutic use of such compounds. In fact, diphenyl ditelluride is extremely neurotoxic and even compounds that release the Te atom slowly may represent a neurotoxic time bomb for mammals. Recently, Tiano et al. reported that diaryl tellurides are cito- and genotoxic for trout erythrocytes. In relation to diselenides more detailed chronic and acute toxicological studies are needed in order to ascertain whether they have a good margin of safety. Furthermore, other aspect that deserve investigation is to determine a possible relationship between the thiol-peroxidase activity of diorganyl diselenides with the capacity of these compounds in catalyzing thiol/diselenide exchange, and how these two chemical properties of selenides correlates with their toxicological and pharmacological effects.

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