4-Biphenylaldehyde and 9-anthraldehyde: two fluorescent substrates for determining P450 enzyme activities in rat and human

Abstract

1. 4-Biphenylaldehyde (4-BA) and 9-anthraldehyde (9-AA) were examined as substrates for cytochrome P450 (CYPs) enzymes in rat and human. Both aldehydes were oxidized by CYPs to fluorescent carboxylic acids, which can be assayed with a high sensitivity by an easy fluorimetric method. 2. With liver microsomes from control and induced rats, the oxidation of both 9-AA and 4-BA followed simple Michaelis-Menten kinetics. Only microsomes from rats pretreated with phenobarbital (a strong inducer of P4502B1/2) could increase (about threefold) the oxidation rates (V(max)) of both aldehydes above the control values, which were 6.7+/-1.1 and 3.3+/-0.6 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively. On the other hand, the (K)(m)'s, which were similar for both aldehydes (about 25 micro M), did not change significantly with any inducer. The use of purified rat CYP1A1, 2E1, 2B1 and 2C11 in a reconstituted system showed that only 2B1 and 2C11 could oxidize both substrates with a high turnover. 3. In human liver microsomes, the oxidation rates of these aldehydes (1.6+/-0.2 and 0.42+/-0.1 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively) were lower than those of rat but with similar K(m)'s(20-26 microm). 4. The oxidation of these aldehydes was also determined with cDNA-expressed CYP1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1 and 3A4 and with a characterized bank of 14 human liver microsomes. In a reconstituted system, only CYP2B6, 2A6, 3A4 and with a lower turnover 2C9 oxidized both substrates. 5. Among the CYP marker activities of the 14 human samples, good correlations were only observed between CYP3A-dependent 6 beta-testosterone hydroxylase and the oxidation of 4-BA (r = 0.74) or 9-AA (r = 0.80) and between the oxidation of 4-BA versus 9-AA (r = 0.74). Weak correlations were also found between the 2B6-linked S-mephenytoin N- demethylase and the oxidation of 4-BA (r = 0.58) or 9-AA (r = 0.65). 6. Inhibition experiments revealed that the oxidation of these aldehydes was inhibited by ketoconazole, 8-methoxypsoralene and sulphophenazole, selective inhibitors for P4503A6, 2A6 and 2C9, respectively. 7. In summary, based on the use of cDNA-expressed CYPs, correlation analysis and chemical inhibition, the metabolism in human liver microsomes of these aldehydes appears primarily catalysed by CYP3A, although CYP2A6, 2B6 and 2C9 may play a role. 9-AA and particularly 4-BA, owing to the high rate of its metabolism, may be two novel useful fluorescent probe substrates for assaying CYP activities in various species.

Cytochromes P450 catalyze oxidation of α,β-unsaturated aldehydes

“…Also, hepatic microsomes from various animals possess the ability to oxidize xenobiotic aldehydes to carboxylic acids (Figure 1) by the P450 system [27]. More recently, model substrates, 9-AA and 4-biphenylaldehyde, have been used to measure the activity of several P450s [28,29]. These fluorogenic substrates provide simple methods to measure the acid products formed from the P450-dependent oxidation of aromatic aldehydes.…”

“…The oxidation of anthracene-9-carboxaldehyde (9-AA) by P450 enzymes (Figure 1) was determined by measuring the formation of anthracene-9-carboxylic acid (9-ACA) as described by Watanabe et al [27] and Marini et al [28]. In brief, the incubation mixture included recombinant P450s (50 nM) or mouse liver microsomes (0.25 mg/mL, ∼50 nM P450), an NADPH-regenerating system consisting of 0.25 mM NADPH, 4.25 mM isocitric acid, 50 mM MgCl 2 , and 1.3 Units/mL isocitrate dehydrogenase, 0.025 mM 9-AA, and 0.1 M potassium phosphate buffer, pH 7.4.…”

“…Watanabe et al [31] showed that P4502c29 is an aldehyde oxygenase utilizing a cannabinoid aldehyde at low μM concentrations as a substrate. Therefore, we sought to evaluate the ability of a number of P450s for their ability to oxidize a synthetic aldehyde substrate, 9-AA [27,28]. Several P450s rapidly metabolized the aldehyde compound, while others apparently did not oxidize this aldehyde to its carboxylic acid (Table 1).…”

High-Affinity Binding of [3H]Cimetidine to a Heme-Containing Protein in Rat Brain

“…Activities of CYP2B6, CYP2C19, and CYP2C11 were quantified by measurement of 9-anthraldehyde oxidation, similar to the methods of Marini et al (2003). Briefly, recombinant CYP2B6 (rCYP2B6) (2 pmol, 26 g of protein; BD Biosciences) was preincubated for 15 min at 37°C with 9-anthraldehyde (5 M final) and inhibitors in 100 mM potassium phosphate buffer, pH 7.4, at a volume of 766 l. The reaction was initiated by the addition of an NADPH-regenerating system (NADPH-RS) (0.4 mM NADP ϩ , 0.85 mM glucose 6-phosphate, 0.1 U/ml glucose-6-phosphate dehydrogenase, and 0.85 mM MgCl 2 ; BD Biosciences) and incubated for 60 min at 37°C in a final volume of 1 ml.…”

Aldehydes and Cardiovascular Disease

“…CYPs can catalyze the oxidation of aldehydes to acids with the incorporation of molecular oxygen into the products (Amunom et al, 2005). More recently the commercial substrate, 9-anthaldehyde (9-AA), has been used to measure the activity of several CYPs (Amunom et al, 2005;Marini et al, 2003). In mammals, the oxidation of aromatic aldehydes, such as tetrahydrocannabinal, to its acid is primarily catalyzed in mouse by hepatic CYPs belonging to the 2E family Watanabe et al, 1991).…”