Formation of Potentially Toxic Metabolites of Polycyclic Aromatic Compounds (PAHs) in Reactions Catalyzed by Human Metabolizing Enzymes

2. Metabolic Toxication of PAHs

Depending on the structural properties of the compounds and the enzymes involved, the PAHs participate predominantly as substrates in oxidations (participating at 67% of the reactions) involving C and N atoms (Figure 1).

Table 2 includes suggested or proposed toxic species such as electrophilic nitrenium ion formation following O-sulfation or O-acetylation reactions. The oxidation reactions involve the formation of dihydrodiols, epoxides, and dihydrodiol epoxides (all catalyzed by P450 enzymes), and quinones (catalyzed by AKR and P450 enzymes), with a minor contribution of COX enzymes. Reactions of N-oxidations are catalyzed by P450, LPO, MPO, and PGHS enzymes. In some examples, e.g., the formation of quinones, the reaction is selective to the substrate configuration. For instance, oxidation of 7-methylbenz[a]anthracene-3,4-diol is preferential for (-)-3S,4S-, and that of (+)-benzo[a]pyrene-7,8-dihydrodiol is preferential for (-)-7R,8R-oxidation (Table 2). The significance of the formation of o-quinones and other reactive oxygen species for the metabolism and toxicity of different chemicals (e.g., carcinogens and drugs) has been discussed previously [6,8]. Reductive reactions participate at 12% in the toxication of the PAHs with prevailing nitro-reductions to amino groups. Of other reactions, O-sulfations participated at 14%, and O-acetylations at 7% (Figure 1). The data also show that depending on the toxicant structure, acetylation, and sulfation reactions in some examples occur after previous reduction by NPR enzyme followed by oxidation. Examples are the O-acetylation (catalyzed by NAT enzymes) and O-sulfation (catalyzed by SULT enzymes) of 2-nitrofluorene, 2- and 3-nitrobenzanthrone (3-NBA), and other nitro-PAHs. The 3-nitrobenzanthrone is, for instance, toxified by reduction of the nitro to the amino group and consequent N-oxidation, or by N-sulfation and N-acetylation which resulted in the formation of the DNA adducts. It was suggested that P450, PO, NAT, and SULT enzymes may play an important role in the metabolism of 3-NABA and its metabolites to reactive species forming DNA adducts, participating in the genotoxicity of the compounds (Figure 2, Table 2 and references therein).

1-Nitropyrene, as well as dinitropyrene derivatives, were suggested to be activated by the catalytic activity of P450 1B1 and 1A2 enzymes by nitroreduction to aminopyrene and subsequent N-hydroxylation, which after acetylation would yield a nitrenium ion forming DNA adducts (Table 2 and references therein). Thus, human P450 enzymes were suggested to have activities for both ring oxidations and reductions of nitropyrenes followed by O-acetylation/O-sulfation and binding to DNA [9,10] (Table 2). Additional examples of toxication by conjugation reactions are reactions of hydroxylated metabolites of methyl- and ethylpyrene, which exert toxic activity after sulfation by SULT enzymes’ catalytic activity (Table 2 and references therein).

3. Enzymes

The calculated participation of human metabolizing enzymes in the toxication of the PAHs and their metabolites (Table 2) shows the dominant involvement of P450 enzymes (58%), followed by AKR (16%), SULT (15%), NPR (3%), NAT (6%) and a group of minor participating enzymes composed of LPO, MPO, PGHS, NQO1, XOR, and COX, which participate to the extent of 3% (Figure 3, Table 2).

For comparison, considering the toxication of drugs, the dominant role of P450 enzymes was also recorded, but to an extent at 72%. The differences between the toxication of drugs and PAHs are also recorded in the composition of a group of minor participating enzymes which is in the case of drug toxication composed of AADAC, ADH, CAT, CES, COX, NPR, LPO, HB + H₂O₂) [8].

4. P450 Enzymes

The analysis showed the dominant role of P450 Family 1 (P450 1A1, 1A2, and 1B1) in the toxication of PAHs participating collectively at 75% of the reactions with domination of P450 1A1 enzyme (at 32%). The contribution of other P450 enzymes was 3A4 (8%) and 2W1 (4%). The group of minor participating enzymes which is composed of P450s 2B6, 2C9, 2C10, 2D6, 2E1, 2F1, 3A5, 3A7, and 4B1, participated altogether at 13% (Figure 4).

In the previous analysis on the toxication of general carcinogenic chemicals the dominant role of P450 enzymes was also shown to participate in 66% of the reactions, as well as the dominant participation of cytochrome P450 Family 1 (P450 1A1, 1A2, and 1B1) participating with 58%. P450 3A4 enzyme participated in the toxication of general carcinogens with 10% [4] comparably to the present analysis with participation at 8% (Figure 4). These results show the dominant role of P450 enzymes in the toxication of both PAHs and general carcinogens for the compounds taken into analysis.

5. Effect of the Structure of PAHs on the Toxication Reactions

To analyze the effects of the structure composition of the PAHs analyzed (Table 2) on the toxification reactions, participating enzymes, and metabolites formed, the data were divided into those that relate to the compounds with C-atoms (C-PAHs, 64 compounds), and to those with C- and N-atoms (N-PAHs, 34 compounds) in the structure. An example of the C-PAH compound toxication presented is the role of P450, AKR, COX, SULT, and EH enzymes in the metabolic toxication of benzo[a]pyrene (Figure 5).

The reactions include oxidative reactions e.g., hydroxylation, epoxidation, hydrolysis to dihydrodiols, o-quinone formation, and sulfoconjugation. The properties of benzo[a]pyrene as a substrate and its metabolites in the reactions catalyzed by human P450 enzymes, the factors influencing the reactions, and the kinetic data have been reported before implying that the metabolic pathway of B[a]P and its metabolites, is very complex and has been the subject of extensive research by different research teams over time [6], Table 2 and references therein.

The studies published on the mutagenicity of PAHs showed dependence on the presence and position of the N-atom in the structure of the compound. For example, P450 1A2 contributed to the mutagenicity of 10-azaBaP more than the recombinant human P450 1A1 enzyme. It was suggested that the presence of the nitrogen atom in the structure led to P450 1A2 as the major enzyme and not P450 1A1. The P450 1A1 is the major enzyme for oxidation/toxication of B[a]P possessing C- atoms and no N-atoms in the structure [6,11] (Table 2 and references therein). Furthermore, the change of the position of the N- atom in the structure of the 1,4- and 1,10-diazachrysene resulted in the change in the enzymes responsible for their mutagenicity, e.g., 1,10-diazachrysene was toxified solely by P450 1A2, while 1,4-diazachrysene by 1A2 and 2A6 enzymes [11,12] (Table 2). In addition, studies on the metabolism of 1-, 2-, and 4-nitropyrene derivatives by human cytochrome P450 enzymes catalyzing oxidative and reductive pathways showed dependence upon the position of the nitro group [13]. The present analysis shows clear distinctions between C-PAH and N-PAH groups of compounds in both the extent of toxication and in the enzymes that catalyze the reactions. The present results show that the N-PAH group of compounds participated in the toxication reactions at 66% (Figure 6) compared to the C-PAH group of compounds participating at 51%) (Figure 7).

It is also shown that the AKR enzymes participated in the activation of C-PAHs (at 28%) (Figure 7) and did not participate in the activation of N-PAHs (Figure 6). In addition, NAT enzymes participated in the activation of N-PAHs (at 14%) (Figure 6), but did not participate in the activation of C-PAHs (Figure 7). The differences in the structure of the two groups of compounds have also been noticed in the toxication reactions catalyzed by P450 enzymes and the participation of minor participating enzymes. The P450 1A1 enzyme participated at a lower extent in the toxication N-PAHs (at 23%) (Figure 8) compared to C-PAHs (at 41%) (Figure 9).

Minor differences are noticed regarding the P4501A2 participation which was at 27% in the group of NPAHs (Figure 8) vs. 22% in the group of C-PAHs (Figure 9). The P450 3A4 enzyme is considered a minor participating enzyme in the toxication of the C-PAH group of compounds, which participated in the toxication of the N-PAH group at 19% (Figure 8). In the C-PAH group minor participating enzymes comprise 7 enzymes (P450s 2B6, 2C9, 2C19, 2D6, 2F1, 2W1, and 3A4) participating at 16% (Figure 9). In the N-PAH group of compounds the group of minor participating enzymes participated in the toxication reactions with 15% and comprised four enzymes (P450s 2A6, 2B6, 2D6, and 2W1 (Figure 8).

6. Concluding Remarks

Previous analyses of published data on the participation of human metabolizing enzymes in the toxication of drugs showed preferential participation of P450 enzymes with minor involvement of AOX, SULT, FMO, and MPO enzymes [8]. Data analysis on the toxication of carcinogenic chemicals also showed P450 enzymes as the most prominent participating enzymes examined and the contribution of SULT, NAT, FMO, AKR, and COX enzymes [4]. The predominant contribution of P450 enzymes in catalyzing toxication reactions of chemicals of diverse structures is suggested to be the result of extensive research and the development of affordable methods to be used in research with this group of enzymes [8].

The present paper analyzes data on the toxication of PAHs, constituents of environmental pollutants, by human metabolizing enzymes. Analysed are the effects of the structural characteristics such as the presence of the nitrogen atom in the structure on the participating enzymes and products formed. The results show that oxidation reactions prevail over reductions, sulfation, and acetylation. Of the enzymes, toxications by P450s are revealed as the major enzymes. Within the group of P450 enzymes, the major participation goes to P450 Family 1 (P450s 1A1, 1A2, and 1B1), and of these P450 1A1 participated to the major extent. The analysis of the effect of the chemical structure on the toxications of C-PAHs (the compounds that contain C-atoms and no N-atoms in the structure, vs. N-PAH (compounds containing both C- and N-atoms in the structure) revealed differences between these two groups of compounds in both the extent and the enzymes that catalyze the reactions.