Patai S., Rappoport Z. 1996 The chemistry of functional groups. The chemistry of amino, nitroso, nitro and related groups. Part 2 / 0999 1039

diimine, respectively. In fact, during the reaction of 4-ethoxy-40-nitrosodiphenylamine and pentafluoro-40 -nitrosodiphenylamine with equivalent amounts of GSH the quinone diimines 10a68 and 10b56 were the main products. However, in the reaction of 4- nitrosophenol with varying proportions of GSH, the quinonimine 9a was formed only as a minor product45. Most probably, the quinoid resonance structure of the parent nitrosoarene decelerates the rate of the nitroso/thiol interaction36 (see Section II.B.2) and gives rise to further nucleophilic reaction centers besides the nitroso group. These pathways, however, remain to be elucidated in detail.

E. Formation of N-Hydroxysulfonamide

Recently, a new product was discovered during reaction of the polycyclic 2-nitroso-6- methyldipyrido[1,2-a:30,20 -d]imidazole with GSH and cysteine, respectively43. Structural elucidation by UV/Vis, 1H-NMR, IR and FAB-MS and the stability towards hydrolysis71 (see Table 1) revealed an N-hydroxysulfonamide. The authors suggested it to be formed by addition of the nitrosoarene to sulfinic acid, emerging by hydrolysis of the metastable sulfinamide. In fact, nitrosoarenes are known to react with aryl-3,4,7,71,89 as well as with alkylsulfinic acids7,90 to form stable N-hydroxysulfonamides. However, the glutathione sulfinamide decay in neutral solution probably proceeded too slowly to deliver the large amounts of glutathione sulfinic acid required within a few minutes. In addition, a sulfinamide was not observed during the reaction with cysteine. Both thiols, however, formed large amounts of the corresponding arylamine. Its precursor sulfenamide was detected immediately after the reaction start by FAB-MS37, but it was not observed 10 min later by HPLC, despite the low thiol proportions employed43. Therefore, the sulfenamide is probably the unstable ancestor of the sulfinic acid (see equations 6 and 7). Transferred to other nitrosoarenes, formation of an N-hydroxysulfonamide is only to be expected from electron-rich nitrosoarenes forming the sulfenamide, provided the sulfenamide exhibits a particularly marked instability towards hydrolysis (see Section II.D.2).

F. Reaction Pathways Involving Radical Intermediates

Formation of hydronitroxide radicals during nonenzymic reduction of nitrosobenzene, 2-nitroso-1-naphthol and 2-nitroso-1-naphthol-4-sulfonic acid with reducing agents such as NADH, GSH, cysteine, N-acetylcysteine and other thiols has been observed by ESR spectroscopy5,91,92. The reaction was carried out with 5 mM nitrosoarene and 5 mM thiol in 0.1 M phosphate buffer, pH 7.4, under either air or nitrogen. Radical formation did not depend on atmospheric oxygen. Interestingly, in the presence of higher concentrations of the reducing agents, e.g. 10 mM thiol, no ESR signal was detected. These data have been interpreted as being indicative of a one-electron reduction of nitrosoarenes leading to the hydronitroxide radical as the initial reaction product, followed by reduction of the radical to the hydroxylamine with excess thiols91 93.

Formation of phenylhydronitroxide radicals, DMPO (5,5-dimethyl-1-pyrroline- N-oxide)/glutathiyl and DMPO/hemoglobin thiyl free radical adducts has been detected in erythrocytes of rats in vivo after administration of nitrosobenzene and phenylhydroxylamine, respectively92,94. The data, however, could also be interpreted in a different way:

In fact, neutral, partially aqueous solutions containing nitrosobenzene and phenylhydroxylamine yielded phenylnitroxide radicals95. However, excess thiol will reduce the nitrosoarene completely, thereby excluding the comproportionation reaction 11. Reaction 9 is thermodynamically highly unfavored, and formation of thiyl radicals could be only demonstrated in the presence of high concentrations of the spin trapping agent DMPO, e.g. at 100 mM92. Since the yields of spin adducts of thiyl radicals were much higher when phenylhydroxylamine instead of nitrosobenzene was reacted with GSH in buffer or with red cells, it was proposed that the species responsible for the oxidation of the thiols to produce the thiyl free radicals in vivo and in vitro was the phenylhydronitroxide radical generated in the reaction of phenylhydroxylamine with oxyhemoglobin92. Hence, the radical pathway of reaction 8 appears less likely, and the radicals detected stem probably from the reaction chain 10, 11, 9.

III. BIOLOGICAL SIGNIFICANCE

A. Introduction

Overt external exposure of living organisms to nitrosoarenes is a rare event, and chemists are usually aware of the hazard potential of C-nitroso compounds. Nonetheless, we are daily exposed to nitrosoarenes that are generated within our cells.

To maintain a proper milieu interieur,´ our body is faced with eliminating efficiently the useless or harmful ballast of foreign compounds incorporated daily. Since lipophilic xenobiotics are usually not excreted by the kidneys, higher organisms have evolved a variety of metabolic reactions to produce more hydrophilic derivatives that are easily eliminated by the renal route. The liver is the central organ to fulfill this task, but other organs may share it, too. In doing so, the organism runs some risk because reactive intermediates can be formed which may injure the cells where they arise, or if sufficiently stable some distant sensitive organs they reach while travelling through the body.

Aromatic amines and nitroaromatics are typical representatives of lipophilic compounds undergoing extensive metabolism. These substances are widely used in industrial manufacturing of dyes, pesticides, plastics and ammunition, constitute significant environmental pollutants, which are also produced in cigarette smoke, and are constituent moieties of various drugs. Finally, a variety of heterocyclic aromatic amines with a remarkably mutagenic potential are prepared daily in our kitchen while cooking or frying food. Irrespective of their origin, all these compounds have to be considered as being potentially responsible for allergic, toxic, mutagenic and carcinogenic effects. Of the various metabolic pathways involved, N-oxygenation and nitroreduction are generally accepted as being the most important toxication reactions that occur in the body of mammals (for reviews see References 16 and 96 99).

The reactive intermediate oxidation states of this class of compounds, namely, the N-hydroxyarylamines and nitrosoarenes, are in rapid metabolic equilibrium. The nitrosoarenes are readily reduced to the corresponding N-hydroxyarylamines, both enzymically and nonenzymically, and the N-hydroxyarylamines are quickly re-oxidized by autoxidation and, particularly effective, by oxyhemoglobin17,19,34,100.

Scheme 10 depicts the most important reactions of N-oxygenated arylamines as detected in red blood cells27. The most important elimination reaction of N-hydroxyarylamines in blood is the co-oxidation with oxyhemoglobin under formation of the nitroso compound. Depending on substituents, the nitrosoarenes can (A) reversibly bind to the hemoglobin iron like gaseous ligands40, are (B) enzymically reduced to the parent N-hydroxyarylamines by methemoglobin reductase (NADPH) thereby sustaining the

HO SG

N

SCHEME 10. Reaction pathways of N-oxygenated arylamines in erythrocytes

catalytic cycle (Kiese cycle17,101) of methemoglobin formation, or (C, D) undergo addition reactions with thiols. Of these, the reactive cysteine residues in hemoglobin (in human hemoglobin only cys 93 of the ˇ-chain) (C) and the abundant reduced glutathione (GSH)

(D) are of primary importance.

For the last years, covalent binding of nitrosoarenes to hemoglobin SH groups with formation of a sulfinamide that can be hydrolyzed in vitro has attracted particular interest102,103. Biomonitoring of hemoglobin-bound residues has been proposed as a suitable approach to control exposure to, and toxication of, potential carcinogenic arylamines and nitroarenes in persons at risk104 108.

Although hemoglobin adducts of aromatic amines and nitroarenes appear to be good dosimeters for the biologically effective dose of a carcinogen delivered as N-hydroxy or nitroso compound and to correlate with target tissue DNA adduct formation109, several factors probably intervene to limit the quality of such a correlation108. In this context also nongenotoxic actions on tissue-specific tumor formation by arylamines or nitroarenes

have to be considered110. As recently shown in isolated liver mitochondria, the initiating and promoting hepatocarcinogen 2-acetylaminofluorene starts, after transformation into 2-nitrosofluorene, a redox cycle, thereby disturbing the intramitochondrial thiol status. The mitochondrial insult due to thiol depletion and calcium release may lead to cell death, entailing stimulation of cell proliferation that may amplify the altered clone with the genotoxic hit111. Hence, cellular actions of N-hydroxyarylamines other than on DNA should gain more interest in carcinogenicity studies.

This overture highlights only some aspects which, in the opinion of the authors, shed light on the biological, mostly toxicological implications related to the reactions of nitrosoarenes with thiols. The following part presents more in-depth information on selected topics that may exemplify some general principles of reaction pathways occurring under physiological conditions. It covers by no means all of the pertinent literature. Particular emphasis has been put on substituent effects that govern distinct reaction pathways with cellular thiols.

B. Monocyclic Nitrosoaromatics

1. Nitrosobenzene generated from nitrobenzene and aniline

The capacity of aniline and, to a less extent, nitrobenzene to produce hemolysis, methemoglobin and denaturated hemoglobin (Heinz bodies) following poisoning is well known and has been linked to the hepatic biotransformation of these substances into proximate toxic compounds such as phenylhydroxylamine14,16,101,112. This derivative, entering red blood cells within the liver, reacts very fast (k D 2 ð 103 M 1 s 1) with oxyhemoglobin to yield ferrihemoglobin and nitrosobenzene (Scheme 10, reaction B)100. The latter, being a better ligand for the ferrous iron of hemoglobin than dissolved molecular oxygen40, is rapidly sequestered from further reactions (Scheme 10, reaction A) and thus can escape the liver via red blood cells34,113.

Nitrosobenzene itself hardly produces any ferrihemoglobin in solutions of purified oxyhemoglobin but gives rise to many equivalents of ferrihemoglobin in red cells under normal metabolic conditions. In doing so, nitrosobenzene has to be reduced to phenylhydroxylamine, which in turn is co-oxidized with oxyhemoglobin to yield ultimately nitrosobenzene and ferrihemoglobin (Scheme 10, reaction B). Since reactive reduced oxygen species, expectedly superoxide and hydrogen peroxide derived thereof, did not influence ferrihemoglobin formation100, the co-oxidation process was suggested to be more complex, including formation of phenylhydronitroxide radicals and compound I- and II-type hemoglobin intermediates92,114,115.

Formation of free phenylhydronitroxide radicals was observed in live mice immediately after injection of nitrosobenzene116 and in blood in vitro upon addition of nitrosobenzene117. The intermediate phenylhydronitroxide radical arising from the cooxidation of phenylhydroxylamine and oxyhemoglobin92 is reduced by thiols, yielding thiyl radicals that were detected as DMPO (5,5-dimethyl-1-pyrroline-N-oxide)/glutathiyl free radical adducts and DMPO/hemoglobin thiyl free radical adducts (for reaction details see Section II.F). These adducts were observed in rat and human blood in vitro, and in rats in vivo after administration of aniline, phenylhydroxylamine, nitrosobenzene and nitrobenzene, respectively92. Conceivably, these thiyl radicals may react with oxygen under formation of reactive oxygen species or with adjacent amino acid residues, thus explaining hemoglobin denaturation and membrane damage that underly Heinz body formation, hemolysis and reduced life span112. Of course, impaired antioxidative capacity such as in glucose-6-phosphate dehydrogenase deficiency will enhance the susceptibility of red cells towards nitrosoaromatics, whether formed from arylamines or nitroarenes118.

When nitrosobenzene was incubated with human red cells, GSSG was formed together with glutathione adducts which, upon acid treatment, liberated aniline and glutathione sulfinic acid. In addition, nitrosobenzene was bound to the globin moiety (Scheme 10, reaction C). Acid hydrolysis liberated aniline (about 70%) and stoichiometric amounts of cysteic acid (after total hydrolysis of the globin), indicating formation of a sulfinamide19. It should be mentioned, however, that some radioactive material from [U-14C]-labelled nitrosobenzene remained bound to the globin moiety even after extensive acid hydrolysis10,19. The nature of these adducts remains still to be elucidated. Conceivably, a sulfenamide cation may have added to nucleophilic sites under formation of stable adducts (see Section II.D.3).

Collectively, the data from in vitro and in vivo experiments confirm the reactions of nitrosobenzene with thiols as observed in mere chemical systems.

2. Nitroso-procainamide from procainamide

Procainamide [19; 4-amino-N-(2-diethylaminoethyl)benzamide], used as an antiarrhythmic drug, is associated with the highest incidence of drug-induced Lupus erythematodes and with agranulocytosis (4% incidence)119. Procainamide is metabolized by rat and human microsomes and by leucocytes to yield N-hydroxy-procainamide120. In the presence of oxygen the [14C]-label of N-hydroxy-procainamide was found to be bound to proteins, which was prevented by ascorbic acid, NADPH and GSH. In the reaction with GSH a compound was isolated that liberated parent procainamide and glutathione sulfinic acid and exhibited a molecular mass consistent with a glutathione sulfinamide. Besides, a labile compound was detected that regenerated procainamide in the presence of GSH and, therefore, was tentatively assigned as a sulfenamide60. In addition, nitroso-procainamide reacted with cysteine and GSH under physiological conditions with formation of N- hydroxy-procainamide and stoichiometric amounts of the disulfides. Nitroso-procainamide also was found to bind irreversibly to mouse hemoglobin121.

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Binding of N-hydroxy-procainamide (in the presence of oxygen) to histone proteins of white blood cells was markedly reduced by ascorbic acid, but the material bound was not liberated by acid treatment120,122. Hence, it was suggested by the authors that nitrosoprocainamide may have reacted with something other than sulfhydryl groups, particularly since histone proteins contain very few sulfhydryl groups60,120,122.

Binding of nitroso-procainamide to histone proteins may perturb chromatin structure or catabolism, resulting in immunogenic forms of DNA-free histones. In fact, all sera of patients (n D 24) with procainamide-induced Lupus showed IgG and IgM antibody activity against various histone components of chromatin (chromosome subunits)122. The nature of the procainamide adduct to histone proteins still awaits elucidation.

3. 3-Nitrosobenzamide

3-Nitrosobenzamide and 6-nitroso-1,2-benzopyrone are among the most active C-nitroso compounds that inactivate the eukariotic nuclear protein poly(ADP-ribose) polymerase at

one zinc finger site, thereby completely suppressing the proliferation of leukemic and other malignant human cells. The cellular event elicited by these C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesium-dependent endonuclease123. The most probable mechanism underlying the destabilization of zinc coordination to poly(ADP-ribose) polymerase was related to the oxidation of the cysteine ligands in the zinc finger peptide by the C-nitroso compounds124.

3-Nitrosobenzamide gained even more interest when it was found that the compound inhibited acute infection of cultured human lymphocytes by human immunodeficiency virus type 1. The retroviral nucleocapsid protein of HIV-1 contains two domains, each of which binds zinc stoichiometrically with three cysteine thiols and one histidine imidazole group. These zinc complexes and the protein folding that they stabilize are essential for viral genome recognition during budding, genomic RNA packaging, and early events in viral infection. Since these zinc finger sequences are completely conserved and essential for viral replication they appeared as a prime target for antiviral chemotherapy. Treatment of HIV-1 with 3-nitrosobenzamide resulted in a loss of zinc from the virion, as well as from synthetic HIV-1 zinc finger polypeptide, coincidental with viral inactivation125,126.

The mechanism of zinc deprivation by 3-nitrosobenzamide was elucidated most recently. When the reconstituted nucleocapsid protein p7 of HIV-1 (15 mM) was incubated with 3-nitrosobenzamide (300 mM) at pH 7.5, three disulfide bonds per protein molecule were formed while 3-nitrosobenzamide was reduced to the hydroxylamine. Molecular masses of p7 adducts augmented by one or two 3-nitrosobenzamide residues were observed by electrospray ionization MS, consistent with covalent bond formation between cysteine sulfur and the nitroso nitrogen atom127.

These findings point to intermediate semimercaptal formation of 3-nitrosobenzamide with one of the three cysteine residues per domain followed by intramolecular thiolytic cleavage by an adjacent cysteine residue of the triad that normally forms the ligand for the zinc atom. The carboxamide substituent meta to the nitroso group ( m D C0.2855) should favor hydroxylamine formation by thiolytic cleavage at the expense of the sulfinamide pathway. In addition, intramolecular reactions with a properly orientated second thiol group will greatly facilitate the observed reaction. Nevertheless, sulfinamide formation should equally result in loss of zinc binding capacity, and the observed masses would be consistent with a sulfinamide structure, too. Possibly, quantitative data will be provided in future to foster the proposed127 mechanism.

4. Nitroso derivatives of chloramphenicol

Chloramphenicol [20; CAP; D-( )-threo-1-(p-nitrophenyl)-2-(dichloroacetamido)-1,3- propanediol] is an important antibiotic due to its broad activity against a number of clinically relevant microbial pathogens and its ability to penetrate easily the blood brain barrier. Besides human application, CAP became widely and routinely used in veterinary practice and is used in Europe in most animal productions including fish128.

However, the use of CAP was soon restricted after its association with bone marrow depression and aplastic anemia. The underlying biochemical lesion is still obscure, and adequate animal models are lacking. Since thiamphenicol, a CAP analogue where the nitro function has been replaced by a MeSO2-group, has never been associated with aplastic anemia, Yunis and coworkers suggested that the p-nitro group of CAP may be involved in the development of aplastic anemia129,130.

When the nitroso analogue became available131 this hypothesis was tested experimentally. Nitroso-CAP has proved to be considerably more toxic to cultured human bone marrow cells than CAP and to irreversibly inhibit DNA synthesis as well as the growth of

pluripotential hematopoetic stem cells. Moreover, 500-times more [14C]-labelled nitrosoCAP was irreversibly bound to viable bone marrow cells than [14C]-labelled CAP132. From all these data it seemed reasonable to suspect nitroso-CAP as the most probable candidate responsible for the bone marrow cell injury.

Although nitroso-CAP was not detected in vivo, there is ample evidence for nitro reduction of CAP133,134. Nitroso-CAP reacts very rapidly with GSH in chemical systems with formation of a semimercaptal-type intermediate (k D 5.5 ð 103 M 1 s 1, pH 7.4, 37 °C) which gives rise to a sulfinamide22. Nitroso-CAP is rapidly eliminated from human blood in vitro, 90% in less than 15 s. Only 5% is covalently bound to plasma proteins, mainly albumin, the remainder being metabolized in red cells by the very rapid adduct formation with GSH. Preferentially, the sulfinamide but also N-hydroxy-CAP rapidly give rise to amino-CAP in the blood23. It is thus conceivable that part of amino-CAP observed in the blood after CAP administration may have originated from intermediate nitroso-CAP as suggested most recently134. In any event, nitroso-CAP, whether formed by microorganisms in the intestine or produced in the liver, will be degraded in blood before reaching the bone marrow23,130,135.

Interestingly, another CAP metabolite emerged as a favorite proximate toxic candidate, dehydroCAP (21)135. This compound is fairly stable in blood and can readily reach the bone marrow cells. DehydroCAP itself inhibits myeloid colony growth136,137. Perhaps the most important aspect of dehydroCAP is that, in contrast to CAP, it is readily reduced by human bone marrow homogenate even under aerobic conditions130,138. One can assume that the 4-nitrosopropiophenone derivative will react with GSH in a similar way as 4-nitrosoacetophenone to give quite exclusively the hydroxylamine and GSSG25,36, thereby inducing a marked oxidative stress. Further investigations will show whether this metabolic pathway is indeed responsible for the CAP-induced aplastic anemia.

New aspects of CAP exposure and toxicity arose when residues of CAP metabolites were detected in kidney, liver and muscles of chickens that had received oral doses of CAP 12 days before being slaughtered139. Of these, nitroso-CAP, dehydroCAP and dehydroCAP base [1-(p-nitrophenyl)-2-amino-3-hydroxypropanone] appear of particular toxicological importance. The results, however, should be confirmed since it is quite unexpected that a reactive compound such as nitroso-CAP can be detected in organs 12 days after dosing with CAP.

Finally, a toxicological impact is given by the fact that CAP readily decomposes in aqueous solution when exposed to sunlight UV or tungsten light140. Decomposition of CAP has also been observed in vivo when CAP-dosed rats were exposed to UV-A light. Of the degradation products detected, p-nitrosobenzoic acid appears to be most relevant because it induces methemoglobinemia and inhibits DNA synthesis in rat bone marrow

cells. Moreover, covalent binding of [ring-3H]-CAP to tissue of ears and skin of the back of rats was several times higher when the animals were exposed to light compared with controls kept in the dark141. The influence of UV-A on covalent binding of xenobiotics to endogenous material in the skin is considered an important aspect of the occurrence of photoallergic effects141.

Since p-nitrosobenzoic acid has been shown to have a half-life of some 4 min in rat blood, this intermediate, once formed in the capillaries of the irradiated skin, may meet the requirement of sufficient stability to reach sensitive targets as the bone marrow while travelling through the blood141. The reactivity of p-nitrosobenzoic acid with thiols appears not to have been tested hitherto. From the known Hammett constant ( p D C0.4555) one may deduce that the compound will show a reactivity in between the reactivities of nitrosobenzene and p-nitrosoacetophenone.

Taken together, CAP may yield different nitroso derivatives through widely varying pathways. Depending on the substituent, sulfinamides may be formed that deplete cellular thiols and give rise to haptenization of macromolecules, thereby inducing antigenicity. Thiol-mediated reduction of the nitroso compounds, yielding autoxidizable hydroxylamines, may induce oxidative stress with formation of reactive oxygen species that could well be responsible for DNA toxicity.

5. Nitroso derivatives of chloroanilines

Propanil (3,4-dichloropropionanilide) is an important arylamide herbicide that is used in rice, barley, oat and wheat fields. The 3,4-dichloroaniline moiety is also found in the N-substituted phenylureas linuron, diuron and neburon. Hence, exposure to 3,4-dichloroaniline derivatives will be common and has been associated with methemoglobinemia in humans142.

N-Hydroxy-3,4-dichloroaniline has been identified as microsomal metabolite and detected in the blood of propanil-treated rats. Its amount appeared sufficient to account for the hemolytic activity of the parent compound and to be largely responsible for the methemoglobinemia observed143. Interestingly, 3,4-dichloroaniline itself has been shown to produce ferrihemoglobin in bovine red cells in vitro with formation of 3,4-dichloronitrosobenzene144, confirming the peroxidative activity of oxyhemoglobin leading to N-oxygenation145. Moreover, intraperitoneal administration of 3,4-dichloronitrosobenzene to rats produced ferrihemoglobin over much longer periods than an equivalent dose of nitrosobenzene146. This finding is consistent with the behavior of 3,4-dichloronitrosobenzene in the reaction with GSH. At the usual GSH concentration found in red cells, i.e. 2 mM, about 90% of the nitroso compound forms the hydroxylamine that can re-enter the Kiese cycle while side reactions leading to the sulfinamide are small25. Nevertheless, binding of 3,4-dichloroaniline to hemoglobin of rats was observed143,147.

6. Nitroso derivatives of sulfonamide drugs

Dapsone (22; 4,40 -diaminodiphenyl sulfone) is an established antileprotic and antiinflammatory drug that is also effective in the therapy of Pneumocystis carinii pneumonia

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and against chloroquine-resistant Plasmodium falciparum148. Its use is often limited by its dose-dependent toxicity, such as methemoglobinemia and hemolysis. It is also responsible for occasional life-threatening disorders such as agranulocytosis149.

The toxicity of dapsone is due to the cytochrome P-450-catalyzed oxygenation leading to N-hydroxydapsone. This major metabolite enters red cells and is co-oxidized with oxyhemoglobin to generate a nitroso derivative and methemoglobin148.

GSH levels of red cells were diminished upon incubation with N-hydroxydapsone only when glucose was absent, or in glucose-6-phosphate dehydrogenase-deficient red cells150. On the other hand, oxidation of purified hemoglobin was greatly enhanced by GSH151. Prior depletion of GSH by diethylmaleate led to a fall in both methemoglobin and dapsone formation compared with untreated cells148. Hence, it was suggested by the authors that GSH, rather than NADPH methemoglobin reductase, was chiefly responsible for the process of methemoglobin generation and parent amine formation from N-hydroxydapsone in human red cells.

Two GSH-dependent pathways were proposed: reduction of the nitroso derivative by GSH to yield the hydroxylamine together with GSSG, and adduct formation to yield a labile sulfenamide that ultimately gives rise to dapsone152. The latter pathway appears less likely, considering the electron-withdrawing sulfonamide substituent with a Hammett p constant of C0.5855. Rather, the nitroso derivative of dapsone is suggested to react in a similar way as 4-nitrosoacetophenone ( p D C0.5555) which has been shown

to yield nearly exclusively the N-hydroxy derivative and GSSG25,36. Two other routes for the GSH-mediated amine formation are conceivable. The enhanced formation of N- hydroxydapsone facilitates its enzymic reduction to the amine16,17. In addition, the amine could be formed by enzymic cleavage of the sulfinamide as detected for nitrosobenzene19.

Sulfamethoxazole [23; N1-(5-methylisoxazol-3-yl)sulfanilamide] is a clinically important sulfonamide that is mainly used together with trimethoprime in fixed combination (cotrimoxazole). The use of this sulfonamide has been associated with a variety of idiosyncratic reactions, including fever, lymphadenopathy, skin rash, hepatitis, nephritis and blood dyscrasias153. The incidence of these reactions is in the range of 1:5000 in the normal population but is much more common in patients with AIDS154. Sulfamethoxazole is metabolized to the N-hydroxy derivative by cytochrome P-450 and peroxidases, e.g. of white blood cells. Under physiological conditions the N-hydroxy derivative appears to be rapidly and spontaneously oxidized to yield the nitroso compound that is more reactive and more toxic than the N-hydroxy precursor155. When 1 mM GSH was added to 50 mM nitroso-sulfamethoxazole quantitative formation of N-hydroxy-sulfamethoxazole was observed. At much lower concentrations of GSH, e.g. 100 mM, significant quantities of a sulfinamide were formed together with a short-lived intermediate which was tentatively assigned as a semimercaptal28.

O

These data, e.g. predominant thiolytic cleavage of the semimercaptal at physiological GSH concentrations, is in line with the expected behavior in considering the positive Hammett constant of the sulfonamide substituent ( p D C0.5855). It is to be expected that cells with lowered GSH content or impaired enzymic capacity to reduce GSSG will be

more susceptible towards conjugate formation and thus depletion of GSH. Such a vitious cycle may underly cellular toxicity of HIVIII-B-infected lymphocytes and hypersensitivity reactions154.

Sulfasalazine [24; 4-hydroxy-40-(2-pyridylsulfamoyl)azobenzene-3-carboxylic acid] is commonly used for the treatment of inflammatory bowel disease. The drug consists of two moieties, sulfapyridine [N1-(2-pyridyl)sulfanilamide] and 5-aminosalicylic acid, which are linked by an azo bond. Sulfasalazine is hardly absorbed in the intestine and reaches the colon unchanged where bacteria cleave the azo link, liberating sulfapyridine which is absorbed, while 5-aminosalicylic acid is thought to exert its antiinflammatory effects locally. The moiety responsible for its toxicity is considered to be sulfapyridine which can be metabolized to yield N-hydroxy-sulfapyridine (for review see Reference 156). A wide range of adverse effects of sulfapyridine has been reported, including leucopenia, intravascular hemolysis, particularly in glucose-6-phosphate dehydrogenase deficient patients, and methemoglobinemia in up to 40% of the patients.

COOH

HO

O

N N S N

H

O N

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N-Hydroxy-sulfapyridine was shown in vitro to produce ferrihemoglobin and to be cytotoxic to mononuclear leucocytes. Co-incubation with ascorbic acid, GSH, or N- acetylcysteine did abolish cytotoxicity but did not inhibit ferrihemoglobin formation156, indicating that the nitroso derivative may be the ultimate cytotoxic agent for leucocytes while in red cells the Kiese cycle16,101 may be still operating. These data are suggestive that nitroso-sulfapyridine is mainly reduced to the N-hydroxy derivative at high concentrations of GSH as found in human red cells.

7. Nitroso derivatives of dinitrobenzenes

1,3-Dinitrobenzene is an intermediate employed in chemical syntheses of a large number of compounds used in the dye, explosives and plastics industry. The compound is known to induce methemoglobinemia and to cause testicular toxicity with the Sertoli cell being the major target. Nitro reduction was observed in erythrocytes, in rat Sertoligerm cell cocultures and in rat testicular subcellular fractions, and it was shown that 3-nitrosonitrobenzene was formed that was considerably more toxic. Testicular toxicity was enhanced when the intracellular thiol levels were reduced by pretreatment with diethylmaleate. In turn, pretreatment with cysteamine or ascorbate reduced the toxicity of 1,3-dinitrobenzene and 3-nitrosonitrobenzene.

These findings suggest that formation of 3-nitrosonitrobenzene and the corresponding hydroxylamine may elicit a futile redox cycle, using up reduced cofactors such as GSH and NADPH in the Sertoli cell (for literature see Reference 157). The strong electronwithdrawing properties of the nitro group ( m D C0.7; p D C0.855) are in line with

this view. Accordingly, the semimercaptal will be stabilized29,157 and the hydroxylamine pathway will be favored.

From this it becomes clear that low molecular thiols will not lead to eventual detoxication of these nitrosonitroaromatics but will sustain a redox cycle that may be