Free Radical Metabolites in Arylamine Toxicity

Abstract

Aromatic amines (also known as arylamines) form a very important class of xenobiotics. The arylamine substructure is found in pesticides, carcinogens, and drugs. It is therefore unsurprising that arylamine drugs possess varying degrees of toxicity which ultimately depend on dose, exposure, and the particular genetic makeup of the individual. Arylamines have been shown to undergo oxidation reactions to produce reactive metabolites. This chapter will focus on a subset of reactive metabolites which are the arylamine-free radical metabolites. A detailed discussion on how these free radical metabolites form is presented, and association between the latter and toxicity reactions are discussed. Particular emphasis is devoted to the subject of arylamine-induced blood dyscrasias and recent advances as well as future prospects in this area.

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:

…the parent carcinogen [compound administered] might be converted in the body to a derivative which could be properly considered as the primary carcinogen.

Furthermore,

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].

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.

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.