ipso-Substitution by the Oxygen Atom of the Active Species
Abstract
When various p-substituted phenols (substituent = NO2, CN, CH2OH, COCH3, COPh, COOH, F, Cl, and Br) were incubated with rat liver microsomes, the substituent was eliminated to produce hydroquinone, and the reaction was inhibited by CO and a cytochrome P450-specific inhibitor. In the case of p-cresol (substituent = CH3),p-toluquinol was formed instead of hydroquinone. Experiments using 18O2 proved that the elimination is accompanied with ipso-substitution by the oxygen atom of the active species in cytochrome P450. These results are similar to those in a cytochrome P450 chemical model system (Ohe, T.,et al., Tetrahedron Lett. 42, 7681–7684, 1995), implying that the model is a good mimic of cytochrome P450. Substrates that lack a hydroxy group, namely p-substituted toluenes, did not undergo the reaction, thus indicating that a hydroxy group at the p-position to the eliminated substituent is necessary for this pathway. This is the same as the result obtained with the cytochrome P450 model. Finally, to elucidate how the substituent is eliminated, we attempted to detect the product derived from the eliminated group with several substrates. Results indicated that the mechanism of the substituent elimination can be divided into two types: the substituent is eliminated as an anion or as a cation.
Cytochrome P450 monooxygenases catalyze diverse oxidations, such as hydroxylation of aliphatic and aromatic carbons, epoxidation of olefins, N-dealkylation of amines, and O-dealkylation of ethers by activation of molecular oxygen (1). These enzymes play major roles in the metabolism of a wide variety of drugs and organic compounds (2-5). In addition, endogenous substances—such as fatty acids, steroids, fat-soluble vitamins, and prostaglandins—are transformed by the actions of cytochrome P450 (6).
Many metalloporphyrins have been synthesized in attempts to develop practical catalysts for oxidative reactions and used to elucidate the molecular mechanisms of biological oxygen atom activation and oxidation of substrates (7). We have used various metalloporphyrins as cytochrome P450 chemical models for studying drug metabolism1 (8-17).
Metabolism studies are crucial to evaluate the safety and effects of drugs, food additives, pesticides, and other industrial chemicals. Metabolites are usually obtained from biological samples—such as urine, bile, and liver microsomal reaction mixtures—but the amounts are small, and new metabolites and metabolic pathways can be difficult to identify. In addition, the toxicity of many chemicals is due to the initial formation and subsequent reactions of highly reactive metabolites that cause tissue injury (18, 19). However, it is difficult to detect such reactive metabolites directly in biological samples and to determine the origin of the toxicity, because most of them form adducts with cellular constituents, leading to cellular damage. For these reasons, we have applied a variety of cytochrome P450 models to drug metabolism studies (8-17). Cytochrome P450 models are able to provide relatively large amounts of metabolites and reactive metabolites, and are also useful to analyze the metabolic mechanisms.
The present study was focused on the cytochrome P450-catalyzed conversion of p-substituted phenols. The metabolism of phenol derivatives is interesting, because most aromatic compounds are hydroxylated to phenol derivatives by cytochrome P450. We have already reported that p-phenoxyphenol and p-methoxyphenol suffer “cleavage of the oxygen-aromatic ring bond” in cytochrome P450 models and rat liver microsomes (14). It was also proved that this cleavage reaction is accompanied with ipso-substitution by the oxygen atom of the active species, and a hydroxy group of the substrate is necessary for this pathway to operate. On the basis of these results, we supposed that similar reactions might occur generally in various p-substituted phenols other thanp-hydroxyarylethers (fig. 1). Various substituents might be replaced by the cytochrome P450 active species. Catechol derivatives formation and conjugation by UDP-glucuronosyltransferase or aryl sulfotransferase are well-known metabolic pathways of p-substituted phenols.ipso-Substitution might be the third metabolic pathway ofp-substituted phenols.
We have recently shown that such a reaction proceeds in a cytochrome P450 model system that consists ofmeso-tetrakis(2,6-difluorophenyl)porphinatoiron (III) chloride and mCPBA2 (16). These results encouraged us to examine whether this kind of oxidative reaction occurs with cytochrome P450 itself. Dehalogenation of halogenated phenols is known to be a cytochrome P450-mediated metabolic process (20-24) that also supports our hypothesis. Elimination of substituents other than halogen groups is a completely new type of metabolic pathway. It would be especially interesting if this type of reaction occurs with substrates whose substituents are attached to the aromatic ring through a carbon—carbon bond, such as p-acetylphenol,p-hydroxybenzyl alcohol, p-hydroxybenzoic acid,etc., because the carbon—carbon bond is very stable and has generally been thought to resist metabolism. We also report the results of the elimination of halogen groups for comparison.
We conducted this study to characterize the above metabolic pathway in rat liver microsomes and to study its mechanism in detail.
Materials and Methods
Chemicals.
p-Toluquinol was prepared by the photooxygenation ofp-cresol in the presence of Rose Bengal according to the method of Endo et al. (25). Similarly, 1-hydroxy-cis-7-oxabicyclo[4.3.0]nona-2-en-4-one was synthesized by the photooxygenation of 2-(4-hydroxyphenyl)ethyl alcohol (p-hydroxyphenethyl alcohol) in the presence of Rose Bengal (25). p-Hydroxyphenyl benzoate was synthesized by reaction of hydroquinone and benzoyl chloride in the presence of sodium carbonate (26). Phenyl p-hydroxybenzoate was obtained by treatment of sodium carbonate after reaction ofp-hydroxybenzoic acid, phenol, and phosphorus oxychloride (27).
These four compounds were purified by silica gel column chromatography and identified on the basis of 1H-NMR and mass spectra.
NADP+ and G-6-P were purchased from Boehringer Mannheim GmbH (Mannheim, Germany), and were stored at 4°C. G-6-P DHase (E.C. 1.1.1.49) from Baker’s yeast was purchased from Sigma Chemical Co. (St. Louis, MO) and was stored at −20°C.18O2 gas was from ISOTEC (Miamisburg, OH). All other chemicals were of the purest grade commercially available.
Preparation of Microsomes.
Male Wistar rats (6 weeks old, 150–200 g each) were treated by intraperitoneal injection of phenobarbital (60 mg/kg in saline) for 3 days and killed 24 hr after the last injection. Hepatic microsomes were prepared as previously described (8). Microsomal protein concentration was determined by the Lowry et al. (28) method using bovine serum albumin as a standard, and the cytochrome P450 content was measured as described by Omura and Sato (29).
Microsomal Incubations.
Detection of Hydroquinone and Quinols. The incubation mixture containing liver microsomes (4 mg protein), substrate (1 mM), KCl (60 mM), MgCl2 (4 mM), G-6-P (4 mM), and G-6-P DHase (5 units) in 2.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) was preincubated for 3 min at 37°C. The reaction was initiated by adding NADP+ (final 0.4 mM). After incubation for 20 min at 37°C, the mixture was treated with 2 ml of ice-cold ethyl acetate to stop the reaction and extract the products, and the organic phase was separated and concentrated by argon flushing. Products formed were trimethylsilylated with BSTFA and pyridine. After removal of excess BSTFA by argon flushing, the residue was dissolved in a small amount of acetone and analyzed by GC/MS (Shimadzu QP5000, capillary column DB-5 30 m; J & W Scientific, Folsom, CA). Injection temperature was 220°C. Initial column temperature was 100°C for 3 min; then, it was raised at 10°C/min to 250°C, followed by an isothermal hold at this temperature.
Detection of Formaldehyde (Substrate = p-Hydroxybenzyl Alcohol).
This incubation was conducted under the same conditions as described. After incubation for 20 min at 37°C, the mixture was treated with 1 ml of 20% trichloroacetic acid aqueous solution to stop the reaction. Amounts of formaldehyde formed were determined by Nash’s method (30,31).
Detection of Benzoic Acid, Etc. (Substrate = p-Benzoylphenol).
This incubation was conducted under the same conditions as described. After incubation for 20 min at 37°C, the mixture was treated with 1 ml of 2 N HCl aqueous solution to stop the reaction. Products were extracted with 2 ml of ethyl acetate, and the organic phase was separated and concentrated by argon flushing. Products formed were trimethylsilylated with BSTFA and pyridine. After removal of excess BSTFA by argon flushing, the residue was dissolved in a small amount of acetone and analyzed by GC/MS (Shimadzu QP5000, capillary column DB-5 30 m; J & W Scientific). Injection temperature was 220°C. Initial column temperature was 80°C for 3 min; then, it was raised at 10°C/min to 250°C, followed by an isothermal hold at this temperature.
Detection of Nitrate Ion (Substrate = p-Nitrophenol).
This incubation was conducted under the same conditions as described. After incubation for 20 min at 37°C, the mixture was treated with 0.5 ml of 20% trichloroacetic acid aqueous solution to stop the reaction. The Griess assay (32, 33), a spectrophotometric determination for nitrite, was used to quantify the nitrate levels in the microsomal incubation mixtures. Briefly, the incubation mixture was centrifuged, and 2 ml of the supernatant was reduced with cadmium powder to convert nitrate to nitrite. The resulting sample was filtered. To 1 ml of the filtrate, an equal volume of Griess reagent (1% sulfanilic acid, 0.1%N-1-naphthylethylenediamine dihydrochloride in 5% H3PO4) was added and immediately mixed. After 30 min, the amount of the product was measured in terms of the absorbance at 550 nm.
Inhibition of Hydroquinone Formation by CO and Metyrapone.Inhibition by CO.
This incubation was performed under the same conditions as described, except that it was done under an atmosphere of 20% O2/80% CO.
Inhibition by Metyrapone.
This incubation was performed under the same conditions as described, except it contained metyrapone (2 mM).
Microsomal Incubation Under 18O2.
This incubation was conducted under the same conditions as used for the microsomes-NADPH/O2 system, except that it was done under an atmosphere of 18O2/argon. The components, except NADP+, were contained in a two-necked flask, and NADP+ was separately placed in a glass vessel connected to the flask. The system was also connected by a glass tube to a vial containing 100 ml of 18O2 gas (89 atom%), a balloon-containing argon gas, and a vacuum pump. Although immersed in an ice bath, the incubation mixture was evacuated 10 times, and the atmosphere was replaced with argon gas each time. After a final evacuation, the seal to the 18O2 gas reservoir was broken, and the gas was allowed to distribute through the system, followed by the introduction of argon gas to equalize the pressure. The reaction was subsequently initiated by addition and mixing of the NADP+. The 18O content in hydroquinone was calculated from the 256/254 (M+ + 2/M+) peak ratio in the mass spectrum of the trimethylsilylated derivative, then corrected to take account of the original 18O content of18O2 (14).
Results and Discussion
Oxidation of p-Substituted Phenols by Liver Microsomal Cytochrome P450.
Based on the previous results of oxidation by the cytochrome P450 model (fig. 2A), various p-substituted phenols (X = F, Cl, Br, NO2, CN, CH3, CH2OH, COCH3, COPh, and COOH) were incubated with rat liver microsomes (NADPH/O2 system). Figure2B shows the results on substituent elimination from variousp-substituted phenols to afford hydroquinone, which was identified as the trimethylsilylated derivative by GC/MS. However, the primary product, in some cases, may be p-benzoquinone, not hydroquinone, because p-benzoquinone is easily transformed to hydroquinone by reduction dependent on rat liver microsomes-NADPH (14).
All of the substituents, except X = CH3, were eliminated, and relatively large amounts of hydroquinone were formed when p-halogenophenols (especially X = F),p-nitrophenol, p-hydroxybenzyl alcohol, andp-benzoylphenol were used as substrates. Results obtained herein were similar to the results in the cytochrome P450 model system (fig. 2, A and B), implying that the cytochrome P450 model is a good mimic of cytochrome P450. It is interesting that this type of reaction occurred even in the case of some substrates whose substituents are attached to the aromatic ring through a carbon—carbon bond, (such as p-acetylphenol,p-hydroxybenzyl alcohol, p-hydroxybenzoic acid,etc.), because the carbon—carbon bond, which is very stable and has generally been thought to resist metabolism, was cleaved. Carbon—carbon bond cleavage is known to be catalyzed by some cytochrome P450 forms involved in the synthesis of steroid hormones from cholesterol in steroidogenic tissues (i.e. the adrenal cortex, testis, and ovary) (33). However, the cleavage reaction in the present study is entirely different from such cases.
When microsomes or NADP+ were omitted from the complete system, hydroquinone was not detected.
CO inhibition of hydroquinone formation was measured. Data shown in table 1 demonstrate that the formation of hydroquinone was significantly inhibited in the presence of 20% O2/80% CO. Further, a cytochrome P450-specific inhibitor, metyrapone, also suppressed the formation of hydroquinone (table 1). These results indicate that the substituent eliminations were catalyzed by cytochrome P450.
In the case of p-cresol, p-toluquinol was formed (3.56 ± 0.08 nmol/nmol P450) instead of hydroquinone (fig.3). The product was identified as the trimethylsilylated derivative by GC/MS on the basis of its retention time and mass fragment pattern, compared with those of the synthesized authentic compound. This indicates that the methyl group is difficult to eliminate, and the reaction stopped before this step. This finding is significant for metabolism studies, because p-toluquinol is a toxic metabolite that binds covalently to DNA, RNA, or other cellular macromolecules. To apply this metabolic reaction to a different structure, p-hydroxyphenethyl alcohol was used as a substrate. Interestingly, we detected a bicyclic product (i.e.1-hydroxy-cis-7-oxabicyclo[4.3.0]nona-2-ene-4-one) as the trimethylsilylated derivative by GC/MS (fig. 4). It was identified on the ground of its retention time and mass fragment pattern, compared with those of the synthesized authentic compound. This result shows that p-hydroxyphenethyl alcohol was converted into the corresponding quinol by cytochrome P450, followed by undergoing intramolecular cyclization (fig. 4). Further studies of the applicability of this metabolic cyclization to a variety of compounds are in progress.
To elucidate the mechanism of these unusual substituent eliminations catalyzed by cytochrome P450, microsomal incubations were performed under 18O2/argon mixtures (molecular oxygen is the origin of oxygen in the iron-oxenoid active species of cytochrome P450), and the reaction products were analyzed by GC/MS. The18O content in hydroquinone was calculated from the 256/254 (M+ + 2/M+) peak ratio in the mass spectrum of the trimethylsilylated derivative, then corrected to take account of the original 18O content of 18O2. The 18O contents in hydroquinone were >80% in every case (table 2), showing clearly that one oxygen of hydroquinone was mostly derived from the oxygen atom of the active species on cytochrome P450. The substituent elimination occurred, accompanied with the replacement of the substituent by the oxygen atom of the active species, as in the case of the cytochrome P450 model (16). This type of reaction is often called “ipso-substitution.” When p-cresol was used as a substrate under 18O2/argon mixtures, the18O content in p-toluquinol formed was almost 100%. This indicates that the oxygen atom of the active species attacked the root of the methyl group, and the methyl group was not eliminated but remained.
Requirement of Hydroxy Group on Substrate.
p-Substituted phenols, which have a phenolic hydroxy group at the p-position to the substituent, were used as substrates (as described herein), because we assumed that a hydroxy group at the p-position to the eliminated substituent might be required for the ipso-substitution, by analogy with the result obtained in the cytochrome P450 model (16). To confirm the requirement of a hydroxy group, we investigated the microsomal reactions of p-substituted phenol analogs that lack a hydroxy group, namely p-substituted toluenes. Littlep-cresol was detected when any p-substituted toluene was used as the substrate (data not shown). These results show that a hydroxy group at the p-position is indispensable for elimination. This finding is the same as the result obtained in the cytochrome P450 model system (16).
Studies on the Mechanism of ipso-Substitution.
On the basis of the data presented herein, we suggest in fig.5 a possible mechanism for metabolicipso-substitution. First, p-substituted phenols are hydroxylated at ipso-position with the same mechanism that we proposed previously (14, 16). This intermediate (i.e. quinol) breaks down to affordp-benzoquinone or hydroquinone, resulting in elimination of the substituent. We have a good basis for thinking thatipso-substitution proceeds via a quinol intermediate, in that we detected p-toluquinol instead of hydroquinone when p-cresol was used as a substrate. This finding indicates that ipso-substitution probably occurs through a quinol in the case of other p-substituted phenols.
There are two types of substituent elimination from the quinol intermediate. When the substituent is eliminated with acceptance of the C—X bond electron pair, p-benzoquinone is formed and the eliminated group is an anion. We will hereafter use the term “type I elimination” to refer to this kind of reaction (fig. 5). When the substituent is eliminated with donation of the C—X bond electron pair to the aromatic ring, hydroquinone is formed and the eliminated group is a cation. Hereafter, we will use the term “type II elimination” to refer to this kind of reaction (fig. 5).
It is a very interesting problem to determine which type of elimination (type I or type II) occurs with each ipso-substitution. This might depend on the nature of the substrate. We have already reported that the substituent is, in the case of p-alkoxyphenol andp-phenoxyphenol, eliminated as an alcohol form or a phenol form during ipso-substitution (14). Therefore, it was concluded that the ipso-substitution of these phenols proceeds via type I elimination. To elucidate the type of elimination for the other substrates, we further investigated the leaving groups for some p-substituted phenols that yielded a relatively large amount of hydroquinone (p-hydroxybenzyl alcohol,p-benzoylphenol, and p-nitrophenol).
p-Hydroxybenzyl Alcohols.
To quantify HCHO, the Nash assay (a colorimetric determination for HCHO) was used when p-hydroxybenzyl alcohol was used as a substrate. Results are shown in table 3. The yield roughly corresponds to the amount of hydroquinone formation fromp-hydroxybenzyl alcohol. This result demonstrates that the leaving group is HCHO. The substituent is eliminated with donation of the C—CH2OH bond electron pair to the aromatic ring in this case (fig. 6). Accordingly, we conclude that theipso-substitution of p-hydroxybenzyl alcohol proceeds via type II elimination.
p-Benzoylphenols.
Detection of PhCHO and PhCOOH was attempted by GC/MS, whenp-benzoylphenol was used as a substrate. PhCHO was not obtained at all,3 whereas PhCOOH was formed (table 3) in a yield roughly corresponding to the amount of hydroquinone formation from p-benzoylphenol. In this case, the substituent is eliminated with donation of the C—COPh bond electron pair to the aromatic ring and, at the same time, is transformed to PhCOOH owing to the aqueous condition, in which H2O attacks the carbonyl group of the substituent (fig.7). Accordingly, it may be concluded that theipso-substitution of p-benzoylphenol proceedsvia type II elimination.
Another possible mechanism can be considered for this elimination reaction. p-Benzoylphenol may be converted to the ester form (p-hydroxyphenyl benzoate), accompanied with Baeyer-Villiger rearrangement catalyzed by cytochrome P450, followed by enzyme-catalyzed hydrolytic cleavage to give hydroquinone and PhCOOH. The ester was not detected by GC/MS in the reaction mixtures, because it is unstable and hydrolyzed easily in this system.
If the ipso-substitution of p-benzoylphenol proceeds via Baeyer-Villiger rearrangement, another possible ester form (phenyl p-hydroxybenzoate) should also be formed. However, no products of hydrolysis of this ester, namely phenol andp-hydroxybenzoic acid, were formed. In addition, Baeyer-Villiger rearrangement does not require a hydroxy group at thep-position; but, the cleavage reaction did not occur whenp-benzoyltoluene was used as the substrate. We conclude that the mechanism of the ipso-substitution ofp-benzoylphenol is not via Baeyer-Villiger rearrangement but via the direct replacement of the substituent with the oxygen of the active species.
p-Nitrophenols.
Detection of NO3− was attempted, whenp-nitrophenol was used as the substrate. To convert NO3− into NO2−, the reaction mixtures were reduced by cadmium after the incubation, and Griess assay was used. As a result, NO2− was detected (as shown in table 3). When the Griess assay was used without reduction, no NO2− was detected in the microsomal reaction mixtures. This indicates that NO3−, and not NO2−, exists originally in incubation mixtures. This yield roughly corresponds to the amount of hydroquinone formation fromp-nitrophenol.
If NO3− is eliminated fromp-nitrophenol directly, the elimination seems to proceedvia the type II process (fig. 8B). In this case, the substituent is eliminated with donation of the C—NO2 bond electron pair to the aromatic ring and is transformed to NO3− through attack of H2O on the nitro group.
However, it is known that hemoproteins in aerobic aqueous solution convert NO2− to NO3−(35), so that NO2− formed fromp-nitrophenol can be oxidized and changed into NO3− in the microsomal reaction mixtures. Therefore, we consider that the nitro group is eliminated as an anion (NO2−) via the type I mechanism, and NO2− is oxidized to NO3− by a hemoprotein (i.e. P450) in the microsomal system (fig. 8A).
It is not yet clear which type of elimination accompanies theipso-substitution of p-nitrophenol.
Studies of Dehalogenation.
Dehalogenation of halogenated phenols and anilines is known to be a cytochrome P450-mediated metabolic process (20-24, 36-40). Rietjenset al. (37) proposed that the reaction proceeds by formation of the halogen anion and chemically reactive benzoquinone(imine) as the primary product. They recently studied the conversion of various halogenated anilines and reported (41) that elimination of a fluorine substituent from the aromatic ring is easier than that of other halogen substituents.
Our results are similar to theirs. We found a decrease in hydroquinone formation in the order p-fluorophenol >p-chlorophenol > p-bromophenol. To examine whether the observed order in dehalogenation might be the result of a decrease in the overall metabolism in the aforementioned order, formation of metabolites resulting from hydroxylation at theo-position to the phenolic hydroxy group, namely catechol derivatives, was also determined.
Table 4 presents catechol derivatives formation and the total amounts of products, calculated as the sum of the amounts of products formed via ipso-substitution ando-hydroxylation. Formation of m-hydroxylated metabolites and NIH-shifted metabolites was not observed. Results demonstrate an increase in catechol derivatives formation in the orderp-fluorophenol < p-chlorophenol <p-bromophenol. Thus, the total amounts ofipso-substitution and o-hydroxylation are not influenced by the type of halogen at the p-position. These results clearly indicate that the observed order of dehalogenation is not due to the difference of overall ability of cytochrome P450 to oxidize each p-halogenophenol.
We showed in our previous paper a decrease in dehalogenation product formation in the order p-fluorophenol >p-chlorophenol > p-bromophenol in the cytochrome P450 chemical model [Fe(III)porphyrin-mCPBA system] (16). The change in the extent of ipso-substitution with change in the halogen substituent is thus not due to a change in the ability of the substrates to bind the cytochrome P450 enzyme, because the same tendency was seen in our cytochrome P450 chemical model system without a substrate binding site, and the overall conversion ofp-halogenophenols remained unaltered in the microsomal system.
Cnubben et al. (41) also reported a similar tendency in the metabolism of p-halogenoanilines by the microsomal system, cytochrome P450IIB1 and MP-8 (MP-8, consisting of a protoporphyrin IX heme covalently bound to an oligopeptide of eight amino acids, is prepared by the enzymatic hydrolysis of cytochrome c and catalyzes cytochrome P450-like reactions). If the mechanism forp-halogenophenols is the same as that forp-halogenoanilines, these results might be consistently explained as follows.
Two possibilities may be considered to explain the change in the extent of ipso-substitution with a change in the halogen substituent. The first possibility is based on the consideration that the order of yields in p-halogenophenols (F > Cl > Br) inversely reflects the order of size of the halogen substituents (F < Cl < Br). The cytochrome P450 active species is more hindered in its approach to the ipso-position by a larger substituent such as bromine. The second possibility was suggested by Cnubben et al. that the change in dehalogenation with change in the halogen substituent is due to an intrinsic electronic parameter of various p-halogenophenols dependent on the halogen substituent. The order of yields in p-halogenophenols (F > Cl > Br) corresponds to the order of electronegativity of halogen substituents (F > Cl > Br).
It is not clear what causes the observed change in dehalogenation, but these findings are extremely important for the following reason. A C—F bond is generally thought to be inert and difficult to break, and incorporation of a fluorine substituent into drugs has been used as a means of blocking biodegradation and bioactivation of the compounds. However, our findings show that a fluorine substituent attached to an aromatic ring can be metabolically eliminated to substantial extent.
In conclusion, we have shown that the substituent elimination of various p-substituted phenols, which is a novel metabolic pathway catalyzed by cytochrome P450, occurs in a rat liver microsomal system in accordance with results obtained in a cytochrome P450 chemical model system. Thus, the use of cytochrome P450 models as an approach to metabolism research seems to be effective for discovering novel metabolic pathways and analyzing their metabolic mechanisms.
It is interesting to note that this reaction occurs even in the case of some substrates whose substituents are attached to the aromatic ring through a carbon—carbon bond, because the carbon—carbon bond had been thought to be very stable and resistant to metabolism. It was proved that the substituent elimination is accompanied withipso-substitution by the oxygen atom of the active species, and a hydroxy group at the p-position to the substituent to be eliminated is necessary for this pathway. The elimination can be divided into two types: type I (anion) or type II (cation) elimination.
Finally, there are two points that we should emphasize. The first is that hydroquinone or p-benzoquinone, a highly toxic metabolite, is formed in this metabolic pathway. Therefore, these findings may have important implications for drug metabolism studies and could help to explain some of the side effects of certain drugs. The second is that this metabolic pathway is applicable to a wide range of drugs and environmental chemicals, because most aromatic compounds are hydroxylated to phenol derivatives by cytochrome P450. Further studies of the occurrence of this metabolic pathway with a variety of compounds are in progress.
Footnotes
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Send reprint requests to: Dr. Tadahiko Mashino, Kyoritsu College of Pharmacy, 1-5-30 Shibakoen Minato-Ku, Tokyo 105, Japan.
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↵1 This work is Part XI in the series, “Application of Chemical Cytochrome P450 Model Systems to Studies on Drug Metabolism.”
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↵3 It was confirmed that benzaldehyde was converted into benzyl alcohol, as well as benzoic acid in the microsomes-NADPH/O2 system. However, benzyl alcohol was not obtained at all when p-benzoylphenol was used as a substrate. Therefore, we consider that benzaldehyde was not formed primarily.
- Abbreviations used are::
- mCPBA
- m-chloroperoxybenzoic acid
- G-6-P
- glucose-6-phosphate
- G-6-P DHase
- glucose-6-phosphate dehydrogenase
- BSTFA
- N,O-bis(trimethylsilyl)trifluoroacetamide
- CO
- carbon monoxide
- Received July 8, 1996.
- Accepted September 27, 1996.
- The American Society for Pharmacology and Experimental Therapeutics