We have shown that PA metabolism catalyzed by MPO resulted in the formation of a protein free radical on MPO, which was then trapped by DMPO and subsequently identified by the anti-DMPO antibody. In general, aromatic amines are well-known substrates for peroxidases. Examination of the Soret region of peroxidases is a common method for analyzing their catalytic cycle. We found that PA caused the accumulation of MPO Compound II. Our previous study with aminoglutethimide, another aromatic amine drug, showed that it kept MPO in its Compound II state for at least 60 min (entire scan duration), whereas with PA, Compound II reduction back to the resting enzyme began after 28 min (). Kettle and Winterbourn (19
) have previously shown that other aromatic amine drugs inhibited hypochlorous acid formation, which has been attributed to the accumulation of Compound II. It is likely that PA also fits into this category of poor peroxidase substrates, since good substrates would efficiently reduce Compound II back to the resting state.
PA has previously been shown to form an N
-hydroxylamine and N
-chloro-PA when oxidzed by activated neutrophils or MPO/H2
, and MPO/H2
, respectively (40
). It was shown previously that approximately 20% of PA results in N
-chloro-PA in the presence of MPO/H2
and PA at pH 6 (41
). We did not observe any attenuation from chloride, however, either because our experiments were carried out at pH 7.4, where no N
-chloro-PA was formed, or because PA inhibited HOCl formation as described above. In this study, we have shown the peroxidase metabolism of PA to a substituted-phenyl radical demonstrated by ESR using spin trapping. The phenyl structure of the MNP adduct is unambiguous (32
). Previously, an almost identical spectrum was found with sulfanilamide after UV photolysis (33
). This spectrum was assigned to 4-sulfamoylbenzene-t
-butylnitroxide. We recently showed that a very similar spectrum was observed in a similar system using aminoglutethimide; its metabolism also resulted in the formation of a phenyl radical (21
). Therefore, based on the ESR spectrum obtained and its analogy to sulfanilamide as well as aminoglutethimide, we conclude that the PA free radical adduct with DMPO or MNP results from the cleavage of the N-C bond of the aniline moiety, with subsequent trapping of the phenyl radical (). This phenyl radical metabolite may be responsible for the formation of an MPO protein free radical by hydrogen abstraction or, possibly, its addition across an amino acid double bond (). However, we also detected a nitrogen-centered cation radical with DMPO which was not observed at lower DMPO concentrations. The trapping of a nitrogen-centered radical with MNP could form a nitrogen-nitrogen bond of lower stability than the nitrogen-carbon bond of the DMPO-phenyl radical adduct. This suggests that the cation radical is the first radical metabolite formed, as expected. In addition, the PA cation radical will be a strong oxidant with an oxidation potential of about 1.03 V (42
). As such, it may oxidize one or more aromatic amino acid residues of MPO.
PA free radical metabolites formed and trapped by DMPO and MNP
In this study, MPO-catalyzed PA metabolism was immunochemically found to produce a positive response to the anti-DMPO antibody. We recently showed that aminoglutethimide also exhibited this activity (21
). PA is another example of drug oxidation by an MPO/H2
system that resulted in a DMPO-protein adduct, () which we propose occurs by the following reactions (equations 1
Interestingly, MPO, which itself catalyzed the oxidation of the drug to a free radical, was also the target of the resulting free radical reactive drug metabolite. As mechanism-based inhibitors of peroxidases have been proposed to form reactive free radical intermediates that bind to the heme active site and inhibit enzyme activity (43
), we assayed the remaining peroxidase activity after incubation with PA. However, we found no inhibition of MPO peroxidase activity after treatment with PA (data not shown). It is unlikely, therefore, that a heme adduct results; a free radical on the protein chain (amino acid radical) is more likely. The affinity purified MPO was deglycosylated and the DMPO was still attached (data not shown), ruling out the possibility of a glycosyl radical. The specific site of radical formation, however, still remains to be identified.
The inhibition of anti-DMPO detection when MPO inhibitors were included in the reaction suggests that MPO catalytic activity is necessary for MPO protein free radical formation. ABAH (45
) and azide (46
) are both inhibitors of MPO, and both attenuated DMPO-MPO adduct formation. Ascorbate is a substrate in that MPO/H2
can oxidize ascorbate, but perhaps more importantly, ascorbate can rapidly reduce many radical intermediates formed by peroxidases (47
). However, ascorbate is not taken up by HL-60 cells (38
) even though we previously showed that ascorbate attenuated anti-DMPO detection when incubated before the addition of drug (21
). Dehydroascorbate is taken up by cells and reduced intracellularly to ascorbate. It is certain that ascorbate oxidation in the cell media resulted in sufficient amounts of dehydroascorbate, which when to be taken up by the cells, was then reduced to ascorbate. It is probable that ascorbate inhibited DMPO-MPO nitrone formation due to its reduction of the PA free radical metabolites, which prevented a radical attack on MPO.
The involvement of PA radical metabolites in MPO radical formation was also proven with the use of NAPA, an N
-acetylated metabolite of PA. Others have shown that activated neutrophils produced significantly fewer cytotoxic NAPA metabolites compared to PA (48
). Also, it has previously been shown that N
-acetylbenzidine is a poorer peroxidase substrate than benzidine (49
). A free amine should thus have a lower oxidation potential than its corresponding N
-acetylated derivative. Consistent with this trend, we found that treating HL-60 cells with NAPA resulted in less DMPO bound to protein than in cells treated with PA.
We have shown above that NAPA has a greater ionization potential than PA. Ionization potential has been shown to have a linear relationship with energy of the highest occupied molecular orbital (50
), and Koopman’s theorem states that the latter is equal to the first ionization potential. Ionization potential is also related linearly to the oxidation potential (51
). It is likely that less protein-DMPO resulted from NAPA metabolism because of its unfavorable oxidation potential. The importance of oxidation potential was shown in a study that investigated horseradish peroxidase/H2
-catalyzed binding of aromatic hydrocarbons to DNA, where above a certain ionization potential, no DNA binding was observed (52
The biphasic nature of DMPO-MPO formation (, inset) could be a result of prooxidant behavior of PA at low concentrations, after which PA reaches a threshold concentration where it becomes an antioxidant (53
). This biphasic effect was also found with aminoglutethimide, suggesting that it could be a general phenomenon.
A better characterized side effect of PA than agranulocytosis is lupus, since there are clear serological biomarkers (anti-nuclear antibodies) that are associated with this autoimmune disease. When the role of PA acetylation was evaluated in patients, those patients receiving NAPA did not develop lupus, nor were anti-nuclear antibodies detected in their serum (55
). Hydralazine is a hydrazine-based, anti-hypertensive drug that has a high risk of lupus. A study showed that hydralazine-induced lupus occurred almost exclusively in slow acetylator phenotype individuals (56
), suggesting that the free amine of the drug was responsible for induction of the side-effect. Unfortunately, PA-induced agranulocytosis has a serum profile resembling the asymptomatic patient, as opposed to PA induced lupus (57
). This fact, combined with the lower incidence of agranulcytosis compared with lupus, makes it difficult to reach a conclusion regarding the effect of acetylation polymorphisms on PA-induced agranulocytosis.
HL-60 cells were used to investigate whether DMPO-protein adducts could be detected in intact cells because, similar to human neutrophils, HL-60 cells contain 47.5 μg MPO / 107
). Neutrophils can be primed by many different agents, all which result in the association of the NADPH oxidase complex which produces H2
). We used glucose/glucose oxidase to generate H2
, which simulates the respiratory burst in neutrophils or macrophages. The formation of H2
as a co-substrate fueled the oxidation of PA by MPO. The model we have used may illustrate a possible outcome of PA use under a condition of inflammation since neutrophils and macrophages both produce H2
during infection. Our study showed that both enzymatically and with HL-60 cells, NAPA resulted in less protein free radical formation than PA.
In addition to the immunological detection of protein-DMPO in HL-60 cells formed from PA metabolism, the ESR spectra of HL-60 cell cytosol unequivocally showed the presence of a protein free radical that was PA-dependent and attenuated in both systems with MPO inhibitors, further validating its immunological detection. However, these ESR experiments could not provide information regarding the identity of the protein target. The Western blot from HL-60 cell cytosol indicated a DMPO-containing band of between 50 and 75 kDa, which led us to consider that the target may be the heavy chain of MPO whose molecular weight has been reported from 55 to 62 kDa (16
). Through the use of concanavalin A, an affinity purification resin for glycoproteins, we were able to partially purify MPO from the HL-60 cells and detect DMPO binding induced by PA. Mass spectrometry of the corresponding Coomassie blue stained gel showed that this band was MPO.
We propose that the free radical metabolism of PA may be implicated in agranulocytosis. A case report of two patients under PA treatment showed that their serum IgG was immunoreactive against leukemia cell lines (HL-60 or K-652 cells), resulting in T-cell mediated cytotoxicity (61
). There was approximately 2- to 4-fold more complement-induced lysis against HL-60 cells than K-562 cells. Interestingly, K-562 cells do not constitutively express MPO, in contrast to HL-60 cells (62
We propose that free radical formation on MPO by PA free radical metabolites may be implicated in agranulocytosis. We propose that free radical modification of MPO or other neutrophil proteins in vivo
may lead to anti-neutrophil antibody formation and granulocyte death via immune-mediated mechanisms. We believe that MPO free radicals are formed either by oxidation of the protein by a PA-derived free radical, or through covalent binding by a PA-derived free radical across a double bond. It should be pointed out that PA-derived electrophiles covalently bind to MPO, resulting in an immunogen. Uetrecht’s group has shown, through the use of an anti-PA antibody, that PA itself became bound to MPO after metabolism by MPO/H2
. The neutrophils from a patient undergoing PA therapy exhibited a 58 kDa protein that was reactive towards anti-PA (63
). This protein is believed to be covalently bound to PA through an electrophilic metabolite of PA (e.g., N
-chloro-PA), or possibly via a free radical metabolite. It would be of great interest to identify this protein, which could possibly be MPO. Although we have determined that MPO forms a free radical in HL-60 cells, it is certainly not the only target for free radical formation () and may or may not be the most relevant target in relation to agranulocytosis. In this regard, a recent study in dogs treated with sulfonamide, an aromatic amine drug, showed that anti-MPO and anti-cathepsin G antibodies were detected in sera (64
In summary, we have shown that PA, a drug associated with agranulocytosis, induced the formation of an MPO free radical in a pure enzymatic system and in HL-60 cells. These findings warrant further study into the toxicological relevance of an MPO free radical in vivo and the possible role it may play in the pathogenesis of PA-induced agranulocytosis. Future studies will be conducted with a larger set of MPO substrates to ascertain whether or not there is a correlation between drug-induced MPO protein free radical formation and agranulocytosis.