Quantitative structure activity relationships for the biotransformation and toxicity of halogenated benzene - derivatives : implications for enzyme catalysis and reaction mechanisms

Organisms are frequently exposed to low molecular weight xenobiotic compounds. An advanced enzymatic machinery modifies these compounds into more hydrophilic metabolites which are subsequently excreted from the body. This process of biotransformation aims to detoxify bodyforeign compounds. Ironically, reactive intermediates may also be formed during the biotransformation process and can interact with macromolecules or receptors, with possible toxicological consequences (bioactivation). Toxicological testing of all new and existing compounds is a moneyand time-consuming problem and therefore alternatives are urgently needed. The main objective of the studies decribed in this thesis, which is outlined in chapter 1 , was to describe QSARs (quantitative structure-activity relationships) for the biotransformation and toxicity of halogenated aminoand nitrobenzene derivatives, Special attention was focussed on the most important phase I biotransformation enzyme involved, the cytochrome P450 system. In addition, in the course of the investigations some attention was paid to the glutathione/glutathione S-transferase dependent phase 2 biotransformation pathway.Chapter 2 gives an overview of the biotransformation enzymes primarily involved in the metabolism of halogenated amino- and nitrobenzene derivatives with respect to their function, regulation, occurrence, molecular/biochemical mechanisms and role in bioactivation and detoxication of xenobiotics. Biotransformation of amino- and nitrobenzene derivatives plays a crucial role in the generation of reactive intermediates assumed to contribute to the toxicity of this class of compounds. Since biotransformation of nitro- and aminobenzene derivatives is known to include pathways leading to both detoxication and bioactivation, insight into factors that direct the rates and regioselectivities of biotransformation processes or interactions with nucleophiles, like macromolecules or the tripeptide glutathione, will help to gain insight in factors that direct processes of, and chances on, bioactivation or detoxication of these compounds. Regioselective enzymatic conversion of relatively small aromatic substrates in the relatively large and aspecific active sites of cytochromes P450 is assumed to depend predominantly on chemical characteristics of the substrates, whereas for relatively large substrates stereoselective positioning through steric constraints of the protein core in the active site becomes more important (see also chapter 2 ). Therefore, the relative differences in reactivity of various sites in a small aromatic compound can be expected to affect the possibilities for enzymatic conversion of the molecule. These considerations prompted us to investigate whether computer calculated molecular orbital characteristics of halogenated benzene derivatives, in combination with insight into the molecular mechanisms of their enzymatic conversion, could provide a basis for the prediction of their metabolic fate.Chapter 3 presents a study on the regioselectivity and underlying mechanisms for the cytochrome P450-catalyzed aromatic hydroxylation of monofluoroanilines. This study provides insight into the molecular mechanism of the cytochrome P450-catalyzed aromatic hydroxylation of molecules containing a heteroatom as well as factors that influence the regioselectivity of hydroxylation. Three mechanisms for aromatic hydroxylation can be proposed; hydrogen abstraction (I), or electron abstraction followed by proton release (II) both leading to formation of a NH . radical. Upon rearrangement of the radical and OH- rebound from the (FeOH) 3+species, the aminophenol product is formed. Aromatic hydroxylation might also proceed by a direct interaction of the high-valent iron-oxo cytochrome (FeO) 3+intermediate with the π-electrons of the aromatic ring resulting in a so-called σ-adduct (III), which rearranges to the aminophenol, either directly or through formation of epoxides and/or ketones as intermediates. First, it was demonstrated that the regioselectivity of the aromatic hydroxylation was influenced by the position of fluoro-substituents at the aniline-ring and that the observed regioselectivity for hydroxylation of these small aromatics was not influenced by the relatively aspecific and large hydrophobic active sites of the cytochromes P450. As expected, a fluorine-substituent induces an effect on the electronic characteristics of the aniline-molecule and this effect was quantified using molecular orbital calculations. Considering the possible molecular mechanisms for aromatic hydroxylation, it appeared that the observed in vitro and in vivo regioselectivity correlated best with the frontier orbitals of importance for a direct interaction of the (FeO) 3+species with the π-electrons of the aromatic molecule e g. the density distribution of the HOMO/HOMO-1. The spin density distribution of the NH . radicals -a parameter of importance for the hydrogen abstraction as well as electron abstraction plus proton release mechanisms- could not explain the observed regioselectivities, indicating that the regarding mechanisms are less likely. In a later study it was demonstrated that for a series of fluorobenzenes the regioselectivity for aromatic hydroxylation could even be predicted on the basis of the frontier orbital density distribution for electrophilic attack within 6% accuracy (r=0.96) [Rietjens et al., 1993]. For the fluoroanilines the C4 position is hydroxylated to higher extent, whereas the C2/C6 positions are hydroxylated to an extent lower than their chemical reactivity predicts. This deviation might result from steric hindrance of the amino moiety for electrophilic attack, a stereoselective positioning of the substrate through an interaction of the amino moiety with amino acid residues in the active site as has been described for P450 debrisoquine 4-hydroxylase, or a dipole-dipole or electronic interaction between the substrate and the activated cytochrome P450 (FeO) 3+species. It is stressed here that the juxtaposition of these small substrates in the active site with respect to the Fe 3+resting state as determined on the basis of 1H-NMR T1 relaxation or cristallography studies, might not represent the actual orientation with respect to the reactive (FeO) 3+intermediate actually performing the hydroxylation step [Koerts et al., 1995].In chapter 4 the aromatic hydroxylation of anilines was further investigated with special emphasis on possible relationships between kinetic parameters and both the physicochemical and electronic substrate characteristics. This was done in order to provide a basis for molecular orbital based quantitative structure activity relationships (MO-QSARs) for kinetic characteristics of the cytochrome P450-mediated aromatic hydroxylation of a homologous series of aniline-derivatives. It was demonstrated that the k cat for C4-hydroxylation in a series of substituted anilines strongly correlates with the HOMO energy of the anilines for the iodosobenzene supported P450 reaction in isosafrole induced microsomes. This observation is in accordance with a mechanism that proceeds by an initial electrophilic interaction of the (FeO) 3+intermediate with the frontier ic electron of the aniline-substrate. In a NADPH/oxygen-supported P450 system, however, it was demonstrated that the interaction of the (FeO) 3+species on the aniline-substrate is no longer rate-limiting, and therefore cannot be described by this QSAR. However, when the electrophilic reactivity of the substrates becomes too low, as is the case for 2,3,5,6-tetrafluoroaniline (E HOMO of -9.24 eV) the initial attack of the cytochrome (FeO) 3+on the substrate might become the rate-limiting step in the overall catalysis. The relatively low conversion rate observed for aromatic hydroxylation of fluorobenzenes for example [Rietjens et al., 1993], might be explained by the relatively low electrophilic reactivity of these compounds. If the resulting metabolite is less toxic than its parent compound, a decreased conversion might have implications for the toxicity and the other way around.To fully describe the cytochrome P450-mediated biotransformation of these aniline- derivatives, a sensitive and efficient analytical technique was developed for the detection and quantification of 2-aminophenols ( chapter 5 ). The principle of the method was based on a dimerization reaction of 2-aminophenols to an intensively colored 2- hydroxy-isophenoxazin-3-one in an acidic environment using ferric ions as the catalyst. It was demonstrated that this method was also applicable for the detection and quantification of halogenated 2-aminophenol derivatives.Besides aromatic hydroxylation reactions, also oxidative dehalogenation reactions mediated by cytochromes P450 were investigated. Halogen substituents are often introduced into molecules to block positions at the aromatic ring of drugs or agrochemicals for bioactivation or biodegradation. Fluorine substituents are frequently used for this purpose, due to the strong C-F bond, and a Van der Waals radius that almost equals that of a hydrogen. In order to study the molecular mechanism of oxidative aromatic dehalogenation as well as the consequences of halogen substitution for regioselective hydroxylation and the formation of reactive intermediates, the study described in chapter 6 was undertaken. Using halogenated anilines as the model compound, the effect of a varying halogen substituent patterns on the cytochrome P450-catalyzed dehalogenation of 4- halogenated anilines to 4-aminophenols was investigated. In the case of C4-fluorinated aniline derivatives, the cytochrome P450-mediated metabolic pathway has been unequivocally demonstrated to result in direct formation of a reactive 1,4- benzoquinoneimine and fluoride anions the primary reaction products [Rietjens et al., 1993]. These reactive benzoquinoneimine metabolites may interact with cellular macromolecules and hence can lead to destruction of molecules essential to living cells.The study described in chapter 6 clearly demonstrated that upon the cytochrome P450-mediated oxidative dehalogenation to the primary reactive 1,4-benzoquinoneimine a fluorine substituent at C4 position of the aromatic aniline-ring was more easily eliminated than a chloro-, bromo- or iodo-substituent. A similar decrease in dehalogenation was observed in a NADPH/0 2 supported microsomal P450 system, as well as in a tBuOOH supported microsomal P450 system, or a system with purified reconstituted P4502B1, or in a system with the heme-based mini-enzyme microperoxidase 8. This indicates that the decrease in dehalogenation was not a result of a change in rate-limiting steps in the P450 catalysis or a change in the contribution of P450 enzymes with a change in the halogen substituent. Structureactivity relationship principles were applied to investigate the reaction mechanism of dehalogenation. The results obtained strongly indicate that the possibilities for the cytochrome P450-mediated dehalogenation of 4-halogenated anilines to 4-aminophenol metabolites are dependent on i) the characteristics of the halogen that has to be eliminated, the most electronegative and smallest halogen (fluorine) being the one most easily eliminated and ii) the electron-withdrawing capacities of the other substituents in the aromatic ring, electron-withdrawing substituents decreasing the relative rate of the reaction. The conclusion that not only a fluoro-, but also a chloro-, bromo- and iodo- substituent, is eliminated as a halogen anion serves as the best explanation for the observations in this study. In addition, it was demonstrated that blocking the C4-position for aromatic hydroxylation by cytochromes P450 resulted in a metabolic switch from dehalogenation and 4-aminophenol formation, to formation of 2-aminophenol- and N- hydroxyaniline-derivatives. Although halogen substitution at the C4-position of an aniline indeed leads to a decreased metabolism at that site, the formation of the reactive 1,4- benzoquinoneimine instead of 4-aminophenol as the primary metabolite, as well as the switch to N-hydroxylation, giving rise to reactive hydroxylamino- and nitroso-derivatives may have considerable toxicological implications.The biological activity of numerous aniline derivatives has been shown to be closely related to the cytochrome P450 mediated oxidative attack at their nitrogen center, yielding products with increasing toxic properties, namely N-hydroxyaniline and nitrosobenzene derivatives. Reactive N-hydroxyanilines cause for example ferrihemoglobin formation (methemoglobinemia) with concomittant co-oxidation to nitrosobenzenes. The resulting nitrosobenzenes in turn are able to interact with cysteine residues of hemoglobin or with the tripeptide glutathione [Eyer, 1988]. On the other hand, the cytochrome P450 mediated aromatic hydroxylation of anilines may represent a detoxification pathway due to the efficient conjugation of the resulting phenolic metabolites and subsequent excretion from the body. However, the cytochrome P450 mediated formation of aminophenol metabolites has been proposed to play a role in the nephrotoxicity of halogenated anilines, occuring predominantly at the proximal tubule and to a lesser extent at the distal tubule [Lo et al., 1990 & 1991; Rankin et al., 1986a & b; Valentovic et al., 1992]. Chapter 7 presents a clear example of a metabolism-toxicity relationship study. Since the cytochrome P450 mediated regioselective hydroxylation seems to direct the toxicity of aniline compounds, a study was performed on the relationships between the regioselectivity of the hydroxylation of C4-substituted 2-fluoroaniline derivatives and their toxic endpoints nephrotoxicity and/or methemoglobinernia. Depending on the derivative, nephrotoxicity at the tubular site and/or methemoglobinernia was shown to occur. The extent of nephrotoxicity induced by C4-substituted 2-fluoroanilines was shown to be related to the extent of C4-hydroxylation, and the extent of methemoglobinernia was shown to be related to the extent of N-hydroxylation. Consequently, a change in the regioselective hydroxylation from the cytochrome P450-mediated C4-hydroxylation to N-hydroxylation resulted in a change of toxic endpoint from nephrotoxicity to methemoglobinemia.In chapter 8 a study was directed at understanding the marked differences in biotransformation pathways of anilines and their chemically oxidized analogues nitrobenzenes. In vivo and in vitro, 2,5-difluoroaniline was demonstrated to become predominantly metabolized through a cytochrome P450-mediated pathway, whereas 2,5- dihuoronitrobenzene is predominantly converted through glutathione conjugation, and the a minor extent through nitroreduction and cytochrome P450-mediated aromatic hydroxylation. On the basis of computer calculations a hypothesis was presented that might explain the differences in metabolic pathways on the basis of their molecular orbital substrate characteristics. It was suggested that the HOMO of the thiolate anion of glutathione will interact more efficiently with the LUMO of a nitro compound (E LUMO = -1.57 eV) than with the LUMO of a less electrophilic amino compound (E LUMO = -0.08 eV). Concerning the aromatic hydroxylation on the other hand, it was suggested that the SOMO of the cytochrome P450 (FeO) 3+intermediate can interact more efficiently with the amino compound (E HOMO = -8.83 eV) than with the nitro compound (E HOMO = -10.29 eV). These considerations are in accordance with the observation in chapter 4, that the reaction rate of the cytochrome P450-mediated C4-hydroxylation decreases with decreasing electrophilic reactivity of an aniline substrate. Substrates with an E HOMO value below -9.2 eV were converted at a relatively low rate by the cytochromes P450. The considerations are also in accordance with the recent observation that the rate of both the chemical and glutathione S-transferase catalyzed glutathione conjugation of fluoronitrobenzenes increases with decreasing E LUMO values [Rietjens et al., 1995].Halogenated nitrobenzenes are known to become metabolized by nitroreduction, glutathione conjugation and aromatic hydroxylation. For some of the toxic effects of nitrobenzenes, the formation of reactive metabolites is a prerequisite. Reduction of nitrobenzenes to the aminobenzene analogues, via the formation of methemoglobinemia-inducing intermediates (nitrosobenzene, N-hydroxyaniline or radical intermediates), is a clear example of bioactivation of nitrobenzenes. The different susceptibility for nitroreduction, and also the presence of competing biotransformation pathways, like glutathione conjugation and aromatic hydroxylation, have been proposed to set the chances for methemoglobinernia exerted by nitrobenzene-derivatives. Applying QSAR approaches, a study was performed in order to investigate whether a metabolism-toxicity relationship exists for a series of fluoronitrobenzenes providing insight in the relative importance of these two suggestions. Chapter 9 describes a combined study concerning the influence of the substituent pattern of fluoronitrobenzenes on their biotransformation and their capacity to induce methemoglobinemia. It was demonstrated that increased possibilities for the conjugation of fluoronitrobenzenes to cellular nucleophiles was accompanied by decreased contributions of aromatic hydroxylation and nitroreduction. A QSAR could be described for the rate of conjugation with the tripeptide glutathione or with bovine serum alburnine (a model for cellular nucleophiles) and calculated parameters for electrophilicity of the fluoronitrobenzenes, showing that with increasing number of fluoro-substituents the conjugation pathway will become more important. In addition, the intrinsic reactivity of the fluoronitrobenzenes for nitroreduction by cecal microflora and rat liver preparations was not related to the in vivo methemoglobin-forming capacity. This observation, in combination with the QSAR for conjugation, led to the conclusion that the different methemoglobinemic capacity must rather result from differences in the inherent direct methemoglobinemic reactivity of the various toxic metabolites, and/or from the difference in reactivity of the fluoronitrobenzenes with glutathione or other cellular nucleophiles. As a result of an increased electrophilic reactivity of fluoronitrobenzenes, another toxic endpoint than methemoglobinemia can be expected.In the final study described in chapter 10 , comparative MO-QSAR studies clearly demonstrated that outcomes on both the regioselectivity and the rate of the cytochrome P450-catalyzed aromatic hydroxylation could be described on the basis of frontier orbital characteristics of the substrates. The MO-QSARs obtained for rats, were also valid for other species, including man. These results strongly support the conclusion that the conversion of the relatively small halogenated benzene derivatives in the relatively large and aspecific active sites of the mammalian cytochromes P450 -even when derived from various species- are mainly dependent on chemical reactivity parameters of the substrates. This importance of the substrate characteristics also dominates over the influence of the specific P450 enzymes in determining the regioselectivity of biotransformation both in vitro and in vivo.In conclusion, the studies described in this thesis cleary demonstrate that molecular orbital calculations in combination with the frontier orbital theory are a usefull additional tool to study the mechanism of cytochrome P450 enzyme catalysis. In addition to the theoretical considerations on the molecular reaction mechanisms and enzyme catalysis, empirical data on the metabolism are essential to construct valid rules or even quantitative structure activity relationships for the biotransformation and toxicity of series of related compounds. Insight into the possibilities for biotransformation have been demonstrated to provide insight into the formation of toxicity determining reactive intermediates. Defining QSARs for biotransformation have been shown to offer an approach to explain the type and extent of the toxic effects exerted by the series of halogenated amino- and nitrobenzene derivatives.Refined models, which do not only focus on the chemical reactivity parameters of the substrates, but also take into account possible stereoselective positioning or interactions with specific amino acid residues in the active site, will contribute to a better prediction of metabolic profiles of existing and new drugs, agrochemicals and other industrially rele