Metabolism of 1-fluoropyrene and pyrene in marine flatfish and terrestrial isopods

Abstract

Monofluorinated polycyclic aromatic hydrocarbons (F-PAHs) are useful reference compounds for a broad spectrum of PAH studies. The pyrene metabolite 1-hydroxypyrene is often used as a biomarker of PAH exposure. Two species, isopod (Porcellio scaber) and flatfish (Platichthys flesus), that produce 1-hydroxypyrene as the major intermediary metabolite and have distinct phase-II conjugates, were selected to investigate the cytochrome P450 catalyzed metabolism of 1-fluoropyrene. The fluorine atom blocks one of the four most favored carbon atoms, yielding different metabolite patterns and the results obtained in the selected species were compared with metabolite profiles obtained for unsubstituted pyrene. Charge densities in 1-fluoropyrene measured with ¹³C-NMR were used to predict possible positions of hydroxylation, which were confirmed by ¹⁹F-NMR. Additionally, the retention behaviour of conjugated 1-fluoropyrene metabolite isomers in reversed-phase liquid chromatography on a polymeric alkyl-bonded phase was interpreted based on the slot model. Whereas three phase-I metabolites were found in isopod hepatopancreas, only two were observed in flatfish bile. Phase-II metabolism appeared unaffected by the fluorine substituent. It was concluded that the phase-I enzyme cytochrome P450 is non-regioselective in the isopod: the activation is mostly influenced by the electron density distribution. In contrast, the enzymatic oxidation in the flounder is more selective. These differences will affect to what extent pyrene metabolite measurements can be used to assess the impact of PAHs to different species.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed throughout the environment, because they are emitted by many sources such as industrial combustion and discharge of fossil fuels, residential heating and motor vehicle exhausts (Bjorseth and Ramdahl, 1985). Because of their mutagenic and carcinogenic properties (Harvey, 1991) PAHs are routinely analysed in a variety of matrices including air, water, solids and tissue samples (Wise and Schantz, 1995). Often, their metabolites, which are the result of biotransformation, and not the parent PAHs, are responsible for the main toxic effects. PAH metabolites have therefore received much attention in the literature (Guengerich, 2000). In the field of monitoring, the use of PAH metabolites as biomarkers of PAH exposure has a number of advantages over straightforward PAH analysis (Levin, 1995). The presence of PAH metabolites indicates the environmental occurrence of PAHs, their availability for uptake by an organism and the capability of the organism to biotransform these PAHs. The analysis of pyrene metabolites as a means to assess bioavailability and biotransformation capacity has proven successful in both occupational (DeCaprio, 1997) and environmental (Myers et al., 1991; Escartin and Porte, 1999a, Escartin and Porte, 1999b) monitoring studies. Recently, Stroomberg et al. (1999) reported on the formation of pyrene conjugates by the terrestrial isopods, Porcellio scaber and Oniscus asellus (Crustacea). The presence of pyrene metabolites in the bile of environmentally exposed flatfish was demonstrated by Ariese et al. (1993) for flounder, Platichthys flesus, and for English sole (Parophrys vetulus) by Krahn et al. (1987). The major enzyme class responsible for the initial biotransformation of PAHs is the cytochrome P450 enzyme superfamily (Snyder, 2000). Within one species, a large number of genes can be found, expressing a range of different cytochrome P450 enzymes with varying activities towards numerous substrates. The net result of the accumulated enzyme activities will influence the formation of reactive intermediates and hence influence the risk for genotoxic effects. It has been stressed that knowledge of similarities and dissimilarities of biotransformation pathways is necessary for the development of biomarkers and for the modelling of chemical fate in ecosystems (Livingstone, 1998).

In the present study, we use 1-fluoropyrene as a model compound to study the in vivo metabolism of pyrene in P. scaber and P. flesus. Both species form 1-hydroxypyrene (phase-I) as the major intermediate metabolite, which is then conjugated (phase-II) with a variety of functional groups (Stroomberg et al., 1999; Ariese et al., 1993). The conjugates are subsequently excreted. In addition, both animal species show a clear correlation between PAH contamination and the level of pyrene metabolites in their bile (P. flesus) (Myers et al., 1991) or hepatopancreas (P. scaber) (Stroomberg et al., 2002). Although the two species produce the same intermediate phase-I product, there are indications that the responsible enzyme systems are different: In flounder the cytochrome P450 1A1 gene has been described (Williams et al., 2000). For terrestrial isopods no such detailed information is available. However, in the terrestrial isopod O. asellus the presence of a cytochrome P450-like protein has been reported by Zanger et al. (1997), using cytochrome P450 1A1 antibodies. In vitro enzymatic oxidation of pyrene into 1-hydroxypyrene with microsomes of O. asellus and P. scaber has been determined as a measure of cytochrome P450 activity (De Knecht et al., 2001). It was, however, shown that a typical cytochrome P450 1A inhibitor, α-naphthoflavone, only partly inhibits the in vitro enzymatic oxidation of pyrene into 1-hydroxypyrene with microsomes of O. asellus (De Knecht et al., 2001). In crustaceans other PAH metabolising cytochrome P450 isoforms (Cyp 2 and 3) are more abundant than cytochrome P450 1A (James and Boyle, 1998).

Recently, Luthe and Brinkman (2000) and Luthe et al. (2002a) synthesised and tested monofluorinated PAHs (F-PAHs) for use as internal standards in PAH analysis. Their behaviour in sample pre-treatment (e.g. solid phase extraction), chromatography (GC-MS, LC-UV), and low-temperature Shpol'skii fluorescence spectroscopy were studied and showed that the physico-chemical properties of F-PAHs are surprisingly similar to those of the corresponding parent PAHs (Luthe et al., 2001a, Luthe et al., 2001b, Luthe et al., 2002b). This is caused by two antagonistic effects of fluorine substitution on the molecule's polarity: on the one hand the creation of a permanent dipole moment and on the other hand the reduction of the London forces (Luthe et al., 2002a). Once incorporated at a specific carbon atom, the fluorine is not easily removed; this is the result of the exceptional strength of the C–F bond in electron-rich aromatic systems, as was for instance found in high-temperature studies (Luthe and Wiersum, 2002). Another advantage, important for the molecular fit in enzyme receptors is the similar size of fluorine and hydrogen in aromatic systems caused by C–F secondary binding of the π-system with the p-orbitals of fluorine (Brooke, 2000).

In the highly symmetrical pyrene molecule, hydroxylation takes place preferentially at one of four equivalent carbon atoms: C-1, C-3, C-6, and C-8. According to standard nomenclature the product would be called 1-hydroxypyrene in all cases. In 1-fluoropyrene the C-1 position is effectively blocked, leaving the C-3, C-6, and C-8 positions available for hydroxylation (see Fig. 1 for chemical structures). However, these positions are no longer equivalent, and different product isomers may be formed, depending on the electronic influence of fluorine and steric effects in the enzyme receptor.

In this study, the phase-II metabolites of 1-fluoropyrene found in isopod hepatopancreas and flounder bile were separated by LC with diode-array UV and fluorescence detection in series. Several phase-I metabolite isomers were formed and for each a cluster of conjugates was observed. We interpreted the formation of different isomers on the basis of the model of electrophilic substitution. Preferential sites for hydroxylation are characterized by a high electron density. Commonly used calculation methods such as CNDO/2 and AM1 are not particularly suitable for our purpose, because many factors (e.g. mesomeric, π-inductive, steric and/or direct electric field effects) can play a role. These methods only distinguish between positions with a high electron density and positions with a low electron density. Therefore, we rather relied on substituent-induced chemical shifts in ¹³C-NMR to determine the actual distribution of electronic charges in the molecule, and this was verified by carrying out a nitration reaction, a typical electrophilic substitution reaction. ¹⁹F-NMR was used to identify the synthetic product isomers. A modified slot model (Wise and Bonnett, 1981) was used to explain the LC retention behaviour in order to help identify different isomers.