Chapter Two - Free Radical Metabolites in Arylamine Toxicity
Introduction
Aromatic amines (arylamines) are a ubiquitous class of chemical compounds, which are found in many xenobiotics, ranging from therapeutic agents (drugs) to environmental pollutants produced from industrial by-products. Although there are some instances where the aromatic amine compound itself has been suggested as the direct toxicant, there is an overwhelmingly large body of evidence which indicates metabolism of aromatic amines is a requirement for them to exhibit toxicity. There are at least three requirements for the association of aromatic amine metabolism with toxicity: (1) the particular type of toxicity determines whether a causal effect of the aromatic amine metabolism can be established. This is based on deductive, mechanistic, or anecdotal evidence; (2) the particular metabolite(s) that is/are identified within the biological system under study; and (3) the identification and function of the target that the arylamine metabolite interacts with. For example, chemical carcinogenesis is a relatively well-established paradigm of a particular toxic endpoint that is associated with the necessary metabolism of an aromatic amine to a reactive metabolite that binds to DNA bases. The interaction of aromatic amines with a biological system (isolated enzymes, cells, isolated organs, or the intact organism) will typically produce metabolites which will have varying degrees of reactivity. The type of metabolites produced will dictate their subsequent reactivity, and the metabolic pathways are usually good predictors of what to expect in terms of reactive metabolites. Before discussing metabolic pathways and the specific case of free radicals, it is worthwhile to refer to some historic examples of fundamentally important studies regarding the definition of a reactive metabolite, which is something that is taken for granted today.
One of the first (if not the first) description of a reactive metabolite, using this term precisely, was used in the context of sulfonamide activity on bacterial growth. In an early report, a reactive metabolite was used to describe the enediol glucoreductone (triose reductone or 2,3-dihydroxyacrolein) as an important source of bacterial energy. Sulfonamides reacted with this metabolite forming an adduct that would prevent the cell from using this metabolite [1]. However, in a lengthy publication describing the physicochemical basis of energy metabolism, electron acceptors (containing double bonds) were described as reactive structures [2]. The fundamental contributions of Elizabeth and James Miller dealt with studies on aromatic amine dyes induced carcinogenesis. In one of their foundational studies on p-dimethylaminoazobenzene, the equivalent to a reactive metabolite is denoted as the primary carcinogen:
Furthermore,…the parent carcinogen [compound administered] might be converted in the body to a derivative which could be properly considered as the primary carcinogen.
After a decade into the late 1950s, the term “reactive metabolite” was used to describe the intermediate through which 2-aminofluorene and related arylamines (including aniline) reacted with isolated liver proteins (Fig. 2.1) [4]: “…the working hypothesis has previously been advanced … that reactive metabolites of carcinogens may act as cytoplasmic mutagens…” What is interesting to point out from the Millers’ studies and Hultin’s was the observation that reactive metabolites were compounds that reacted with liver proteins which was considered important for their toxic (carcinogenic) effects. The concept of protein reactions is important, since it is the topic that will be discussed toward the end of this chapter. Metabolism was very important as Cramer et al. later identified and characterized N-hydroxy-N-acetylaminofluorene as an important metabolite of 2-aminofluorene [5]. This finding was significant, as 2-aminofluorene produces many other metabolites but identifying the reactive metabolite(s) was critical for underpinning of the carcinogenic mechanism of action.As a working hypothesis, we have considered that at least one of the primary carcinogens is either p-dimethylaminoazobenzene itself or its metabolite, p-monomethylaminoazobenzene, a mixture of these dyes, or a compound very closely related to them [3].
The large body of significant work demonstrating DNA adduct formation and the association with carcinogenesis will not be the focus of this chapter (readers are referred to http://monographs.iarc.fr/ENG/Monographs/vol99/mono99-6.pdf for a comprehensive summary). Rather, the focus will be on the reactive metabolites—specifically free radical metabolites—which are produced from arylamine xenobiotics, and their association and role in mechanistic toxicology.
Section snippets
Arylamine Toxicity
If there was a toxic xenobiotic hall of fame, the arylamines would be among the most appropriate inductees. Although these molecules form the basis of some very useful therapeutic agents, the arylamine substructure also appears in many different toxic reactions. It was shown that aniline and anilide substructures of drugs have the highest frequency of idiosyncratic adverse drug reactions in a comparison of 200 drugs that have been withdrawn from the market [6]. From this study, the
Arylamines to Nitroaromatics—Six Degrees of Redox Separation
It is very important to understand that arylamines are species that have a fully reduced, electron-rich (therefore oxidizable) amino group. The oxidation of this group fully leads to nitro compounds, but the reactive metabolites—be they free radicals or electrophiles—all occur in between these fully reduced or fully oxidized states. In Fig. 2.3, the fully reduced arylamine (1) can undergo one-electron oxidation to form the N-centered cation radical (2) or neutral arylamino radical (3). The
Sources and Systems that Generate Free Radical Metabolites from Arylamine Xenobiotics
Taking into account the various redox states of arylamines, there are a large number of metabolic pathways by which they can undergo activation. As illustrated in Fig. 2.2 and described above, the reduced arylamine (ArNH2) species requires oxidation in order to form reactive metabolites. As such, any conditions, enzymatic or otherwise, which favor oxidation can potentially activate the arylamine. Thus, a highly oxidizing system will not surprisingly produce the fully oxidized nitroaryl species.
Reactions of Arylamine Radicals with Cellular Antioxidants
There have been many in vitro studies demonstrating the potential of arylamine radicals to oxidize cellular thiols, reduced flavin mononucleotides, and ascorbic acid. We demonstrated that the carcinogenic arylamines benzidine and 2-aminofluorene were most potent in oxidizing reduced nicotinamide adenine dinucleotide (NADH) or reduced glutathione (GSH) in a horseradish peroxidase/H2O2 system, and this was followed closely by 1-naphthylamine (not carcinogenic), o-anisidine, 4-aminobiphenyl,
Reactions Between Arylamine Radicals and DNA
The early work that investigated free radical metabolites of arylamine carcinogens is attributed to Bartsch and Hecker, and Floyd et al., who demonstrated peroxidase-mediated activation of 2-aminofluorene metabolites (i.e., N-hydroxyl-2-aminofluorene and N-acetoxy-hydroxylaminofluorene). It was demonstrated that N-hydroxy-N-2-acetylaminofluorene was metabolized to 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene by horseradish peroxidase [81]. As this reaction was inhibited by
Arylamine Drugs and Blood Dyscrasias
Blood dyscrasias cover all aspects of hematotoxicity, which perturb the normal levels of any blood cell types or alters their function. The most well-known blood dyscrasia induced by arylamine drugs is methemoglobinemia. This reaction is caused by the oxidation of oxyhemoglobin with concurrent production of superoxide from oxygen, which is catalyzed by the hydroxylamine metabolite of an arylamine; the latter ends up forming a nitroso metabolite. The key metabolic concept is the production of
Diverting Toxic Arylamine Free Radical Generation to Detoxification: Importance of Free Radical Targets
Although this chapter is focused on toxicity mechanisms, there is a flipside to the arylamine free radical reactions described thus far. Although the formation of a free radical that is diffusible, that is, not “caged” as in enzyme active sites, is usually considered in this chapter as a toxification process, there is precedent for industrial utility for such reactions. One example is the detoxification of pollutants, which includes arylamines. As discussed above, soil (more precisely,
Mixtures Toxicology—Application to Arylamine Free Radicals
Mixtures toxicology is an area of toxicology that engages researchers in determining the toxicity mechanism of multiple components. The toxic effects of mixtures according to one textbook have been listed to produce: (a) enhanced toxic effects, (b) reactions at low concentrations, and (c) multiple targets of attack (modified from [178]). Although mixtures can involve hundreds of substances, when considering arylamine radicals it is relevant to consider bimolecular reactions that produce free
Protein Radicals and Toxicity—Future Prospects
Studies from our group have demonstrated that monocyclic arylamines (including arylamine drugs) are capable of producing protein radicals, particularly on myeloperoxidase. These studies were carried out in neutrophil precursor-like cells (human promyelocytic leukemia, HL-60 cells), which normally synthesize myeloperoxidase. The detection of protein-free radical was performed using immuno-spin trapping which requires the addition of a spin trap to cells in order to react with the protein (or
Closing Remarks
It appears as though arylamine xenobiotics will continue to come up on the radar of health of disease. A recent study on the transient receptor potential of vanilloid receptor antagonists evaluated a lead compound that, when hydrolyzed, produced a mutagenic arylamine derivative [195]. Similarly, anticancer compounds were recently synthesized utilizing aniline substructures which demonstrated toxicity against two different cancer cell lines [196]. Landfills and Superfund sites (in the United
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