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

Article abstract of DOI:10.1080/0049825021000017894

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 nmolmin^-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 μM), 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 nmolmin^-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 μM). 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 6β-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 Ndemethylase 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.

Full text of DOI:10.1080/0049825021000017894

xenobiotica, 2003, vol. 33, no. 1, 1–11
4-Biphenylaldehyde and 9-anthraldehyde: two
fluorescent substrates for determining P450
enzyme activities in rat and human
S. MARINI{, E. GRASSO{, V. LONGO{, P. PUCCINI{,
B. RICCARDI{ and P. GIOVANNI GERVASI{*
{ Istituto di Fisiologia Clinica, Area della Ricerca CNR, via G. Moruzzi 1, I-56100 Pisa,
Italy
{ Chiesi Farmaceutici spa, via Palermo 26/A, I-43100 Parma, Italy
Received 25 March 2002
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 ðVmaxÞ of both aldehydes above the control values, which
were 6.7 Æ 1.1 and 3.3 Æ 0.6 nmol minÀ1 mgÀ1 protein for 4-BA and 9-AA, respectively.
On the otherhand, the Km’s, which were similar for both aldehydes (about 25 mm), 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 Æ 0.2 and
0.42 Æ 0.1 nmol minÀ1 mgÀ1 protein for 4-BA and 9-AA, respectively) were lower than
those of rat but with similar Km’s (20–26 mm).
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 6ꢀ-testosterone hydroxylase and the oxidation
of 4-BA ðr ¼ 0:74Þ or9-AA ðr ¼ 0:80Þ and between the oxidation of 4-BA versus 9-AA
ðr ¼ 0:74Þ. Weak correlations were also found between the 2B6-linked S-mephenytoin N-
demethylase and the oxidation of 4-BA ðr ¼ 0:58Þ or9-AA ðr ¼ 0:65Þ.
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 livermicorsomes 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.
Introduction
Aldehydes are commonly considered to be converted to acids in biological
systems by the action of aldehyde dehydrogenase (Lindahl and Peterson 1991).
More recently, some examples have demonstrated that hepatic microsomes from
* Author for correspondence; e-mail: p.gervasi@imd.pi.cnr.it
Xenobiotica ISSN 0049–8254 print/ISSN 1366–5928 online # 2003 Taylor& Francis Ltd
http://www.tandf.co.uk/journals
DOI: 10.1080/0049825021000017894

2
S. Marini et al.
various animals have the ability to oxidize xenobiotic aldehydes to carboxylic acids
by the cytochrome P450 system (Watanabe et al. 1990, 1992). It has been reported
that the oxidation of aromatic aldehydes such as tolualdehydes and tetrahydro-
cannabinol to the corresponding acids is primarily catalysed in rat and mouse by
hepatic CYPs belonging to the 2C subfamily (Watanabe et al. 1993, 1995).
Aliphatic aldehydes such as acetaldehyde and 2,2,2-trifluoroacetaldehyde are
oxidized mainly by CYP2E1 (Kaminsky et al. 1992, Kunitoh et al. 1997). On
the other hand, retinals have been reported to undergo oxidative conversion to the
corresponding retinoic acids primarily by the CYP1A2 and CYP3A4 forms,
although with different affinities (Zhang et al. 2000). It has also been reported
that 9-anthraldehyde (9-AA) is a good substrate for CYPs and may be assayed by
an easy fluorimetric method (Watanabe et al. 1991), although the CYPs involved in
this reaction are not known. Many studies have been devoted to identifying probe
substrates for specific CYPs. Fluorimetric assays, for example, the O-dealkylation
of coumarins (Mayer et al. 1990) and resorufins (Burke and Mayer 1983), are
particularly useful because of their sensitivity and ability to select fluorophores in a
wavelength range that obviates artefacts deriving from endogenous compounds.
In the current study, we have investigated the metabolic specificity of 9-AA
and of anothernovel aormatic aldehyde, 4-biphenylaldehyde (4-BA), against
microsomes from control and induced (male and female) rats, and some purified
rat CYP1A1, 2E1, 2B1 and 2C11. In addition, we have studied the metabolic
activity and specificity of these two aldehydes in man by using a well-characterized
bank of human liver microsomes, a series of cDNA-expressed human P450
enzymes and selective CYP inhibitors.
Materials and methods
Chemicals
9-Anthraldehyde, 9-anthracene carboxylic acid (9-ACA), 4-biphenylaldehyde,
4-biphenyl carboxylic acid (4-BCA) and cytochrome b5 were obtained from Sigma
Chemical Co. (St Louis, MO, USA). CYP2B6 was purchased from Gentest Corp.
(Woburn, MA, USA). All other chemicals and solvents used were obtained from
common commercial sources.
Animals and treatments
Male and female Sprague–Dawley rats (Charles River, Como, Italy), weighing
200–250 g, were used. They were fed with food pellets (Altromin1, Rieper,
Bolzano, Italy) and water ad libitum. Inducers administered intraperitoneally
(i.p.) in water were: sodium phenobarbital (PB) and pyrazole (PYR) at 80 and
200 mg kgÀ1, respectively, for 3 days. ꢀ-Naphthoflavone (ꢀNF) at 40 mg kgÀ1
,
dexamethasone (DEX) at 50 mg kgÀ1 and clofibrate (CLO) at 250 mg kgÀ1 were
given in corn oil for 3 days. Animals were killed by CO2 asphyxia; the livers were
removed and microsomes prepared as described by Longo et al. (1986). The
washed microsomal pellets were resuspended in 100 mm phosphate buffer, 1 mm
EDTA, pH 7.4, and stored at À808C. Protein content was determined according to
Lowry et al. using bovine albumin as standard.

4-Biphenylaldehyde and 9-anthraldehyde oxidation by rat and human P450s
Human liver microsomes
3
Three pooled liver microsomes (HL-1, HL-2, HL-3) and 14 individual
samples (10 male, fourfemales, aged 2–68 years), negative forHIV and hepatitis,
were obtained from Xeno Tech’s reaction phenotyping kit (Kansas City, KS,
USA). Each human sample was supplied with the CYP content and majorCYP
marker activities such as 7-ethoxyresorufin O-deethylase, coumarin 7-hydroxylase,
7-ethoxy-4-trifluoromethylcoumarin, tolbutamide methyl-hydroxylase, testoster-
one 6ꢀ-hydroxylase, chlorzoxazone 6-hydroxylase, lauric acid 12-hydroxylase,
paclitaxel (taxol)-6ꢁ-hydroxylase, dextromethorphan O-demethylase, mepheny-
toin N-demethylase and S-mephenytoin 40-hydroxylase.
cDNA-expressed enzymes
Human recombinant CYP1A1, 1A2, 2A6, 2C9, 2D6, 2E1 and 3A4 were
expressed from E. coli using plasmids containing cDNA of various forms kindly
supplied by F. P. Guengerich (Vanderbilt University, Nashville, TN, USA).
4-BA and 9-AA oxidation assay
4-BA and 9-AA oxidation activity was determined by measuring the formation
of 4-BCA or9-ACA, as descirbed by Watanabe
et al. (1991). In brief, the
incubation mixture contained (in 1 ml), hepatic microsomes (0.1–0.5 mg protein
mlÀ1), 0.5 mm NADPþ, 10 mm glucose 6-phosphate, 10 mm MgCl2; 1 unit glucose
6-phosphate deydrogenase, and 4-BA or 9-AA as substrate in 100 mm phosphate
buffer, pH 7.4. The reaction was carried out at 378C generally for 15 min and
terminated with 1 ml 0.5 N NaOH and extracted with 4 ml ethyl acetate. Aqueous
phase (1 ml) was transferred to another tube and 4-BCA or 9-ACA extracted with
4 ml ethyl acetate afterthe addition of 1 ml 0.5 N HCl. The ogranic phase was
scanned for fluorescence spectra with a Perkin-Elmer spectrofluorimeter. The
formation of 4-BCA was determined at 290 nm excitation and 330 nm emission,
whereas 9-ACA was determined at 255 nm (excitation) and 458 nm (emission).
Both 4-BCA and 9-ACA carried through the extraction procedure under identical
conditions were used as standards and were linear at least up 50 nmol. The
extraction phase is necessary to remove completely the fluorescence of substrate
(4-BA or9-AA).
Reconstituted system
CYP1A1, 2B1, 2C11, 2E1 and NADPH-cytochrome P450 reductase were
purified from microsomes of male Sprague–Dawley rats (Amato et al. 1996).
The reconstituted system contained 100 mm phosphate buffer, pH 7.4, 0.1 mm
P450, 0.3 mm P450 reductase, 30 mg dilauroyl phosphatidylcholine (DLPC) and
substrate (9-AA or 4-BA) in 0.5 ml of the incubation mixture. DLPC was prepared
in waterand sonicated immediately before use. Aftera 30-min pre-incubation at
room temperature, the reaction was carried out at 378C for30 min afteraddition of
an NADPH-regenerating system and analysed for the formation 4-BCA or 9-ACA
as already described.
Membranes from E. coli expressing human recombinant CYPs (50 pmol) were
pre-incubated with 50 pmol purified rat P450 reductase, 15 mg dilauroylphospha-

4
S. Marini et al.
tidylcholine and 50 pmol cytochrome b5 (in the case of incubation with CYP3A4)
for 60 min at room temperature. Potassium phosphate buffer (100 mm, pH 7.4),
MgCl2 (3 mm) and substrate (4-BA or 9-AA) were added at the final concentrations
indicated. The reactions were started by addition of an NADPH-generating
system. After60 min of incubation at 37 8C, reactions were terminated with 1 ml
0.5 N NaOH and analysed as described above.
Inhibition studies
This approach involves an evaluation of the effects of chemical inhibitors of
selected CYPs on the metabolism of 4-BA or9-AA by human livermicrosomes. In
the chemical inhibition experiments, ꢁ-naphthoflavone (ꢁ-NF), 8-methoxypsor-
alene (8-MOP), sulphaphenazole (SULF), quinidine (QUIN), 4-methylpyrazole
(4-MP), S-mephenytoin (MEF) and ketoconazole (KET) ortorleandomycin
(TAO), were used as inhibitors of CYP1A2, 2A6, 2C9, 2D6, 2E1, 2C19 and
3A4, respectively (Parkinson 1996, Koenigs et al. 1997, Sai et al. 2000). The
inhibitors were dissolved in methanol and added to the incubation mixture before
the reaction was initiated. The incubations were carried out with 0.2 mg pooled
samples of human liver microsomes in a final volume of 0.5 ml and at a concentra-
tion of 4-BA or9-AA 100 mm.
Results
Fluorescence characteristics of 4-BCA and 4-BA
The fluorescence spectrum of 4-BCA in ethyl acetate has excitation and
emission maxima at 290 and 330 nm, respectively. No fluorescence was detected
with 4-BA at a concentration up 0.01 mm. However, the 4-BA may be completely
separated by the extraction method described above.
These properties make 4-BA a suitable substrate for a direct assay of micro-
somal monoxygenase activities.
Oxidation of 4-BA and 9-AA by rat liver microsomes
As previously reported for 9-AA (Watanabe et al. 1991), 4-BA is also oxidized
by CYPs in rat liver microsomes to the corresponding carboxylic acid 4-BCA.
This oxidation was dependent on NADPH and molecularoxygen and was
inhibited by CO, typical fora CYP-dependent reaction; NADH was much less
þ
effective as the cofactorand both NADP and NADþ were ineffective (data not
shown).
To establish which CYPs were most active in the oxidation of 4-BA and 9-AA,
incubations were carried out using liver microsomes from control rats and rats
treated with classical CYP inducers; ꢀNF, PB, PYR, DEX and CLO were used to
induce CYP1A1/2, 2B1/2, 2E1, 3A1/2 and 4A1/2, respectively (Ryan and Levin
1990). With all types of microsomes, the oxidation of the two aldehydes followed
simple Michaelis–Menten kinetics and were linear up to 30 min and for 0.3–0.5 mg
microsomal protein mlÀ1. From Lineweaver–Burk plots of 4-BA and 9-AA
oxidation, the apparent kinetic constants were determined (table 1).

4-Biphenylaldehyde and 9-anthraldehyde oxidation by rat and human P450s
5
Table 1. Apparent kinetic constants for the oxidation of 4-BA and 9-AA by CYP2B1 and 2C11 and by
liver microsomes from control and male pretreated rats.
9-AA
4-BA
Microsomes
orpurified
CYP
Vmax
Vmax
Km
(mm)
(nmol minÀ1 mgÀ1
protein)
Km
(mm)
(nmol minÀ1 mgÀ1
protein)
MajorCYP
Control male
Control female
ꢀNF
PB
PYR
DEX
CLO
P4502B1
P4502C11
2C11
2C12
1A
2B
2E1
3A
25 Æ 7
8 Æ 3*
24 Æ 5
38 Æ 8
21 Æ 5
19 Æ 7
23 Æ 6
3.3 Æ 0.6
0.9 Æ 0.3*
0.7 Æ 0.2*
11 Æ 2.5*
1.6 Æ 0.5*
5.4 Æ 1.3
3.7 Æ 0.5
7.2
26 Æ 9
30 Æ 6
18 Æ 5
47 Æ 15
43 Æ 12
55 Æ 18
27 Æ 7
6.7 Æ 1.1
3.8 Æ 0.8*
4.3 Æ 0.5*
14 Æ 2.7*
5.6 Æ 0.6
8.5 Æ 2.1
4.8 Æ 0.8
5.2
4A
9.6
7.4
Values are means Æ SD of three experiments performed with different microsomal preparations.
Each preparation contained pooled livers from two to three rats. The incubations were performedÀa1t
378C with a substrate concentration ranging from 1 to 100 mm and a protein concentration of 1 mg ml
.
The incubation of the reconstituted system containing CYP2B1 or 2C11 was carried out as described in
the Materials and methods.
Vmax and Km are expressed as nmol minÀ1 mgÀ1 protein and mm, respectively; in the case of
purified P450, the activity was expressed as nmol minÀ1 nmolÀ1 P450.
* Significantly different from male control microsomes, p < 0:5, Student’s t-test.
The formation rates of both 4-BCA and 9-ACA by hepatic microsomes from
PB-treated rats were increased about threefold with respect to those of microsomes
from control male rats indicating that CYP2B1, highly induced in PB microsomes,
may play an important role in these reactions. On the other hand, with microsomes
from ꢀNF-, PYR-, DEX- and CLO-treated rats, the kinetic parameters were
similar or lower than those of microsomes of male control rats implying that the
CYPs induced in these microsomes CYP1A1/2, 2E1, 3A1/2, 4A1/2 are not
involved in the oxidation of these aldehydes.
The oxidation rates of 4-BA and 9-AA were also significantly higher in the
male than in female microsomes suggesting a catalytic role of the male-
specific CYP2C11, the major CYP constitutively expressed in rat liver (Fujita
et al. 1987). The apparent involvement of CYP2C11 and 2B1 in the oxidation
of these aldehydes was also supported by the finding that these enzymes in
a reconstituted system could catalyse these oxidations with a consistent
turnover rate. On the contrary, CYP1A1 and 2E1 were unable to perform these
reactions.
Oxidation of 4-BA and 9-AA by human liver microsomes
Both 4-BA and 9-AA are readily oxidized by human liver microsomes
following simple Michaelis–Menten kinetics and were linear with time up to
20 min and with protein up 0.5 mg mlÀ1 (data not shown). The apparent kinetic
parameters estimated by Eadie–Hofstee plots in three pooled human liver
microsomes are shown in table 2. The mean apparent Km’s forthe two
substrates were similar whereas the mean apparent Vmax for4-BA was lagrer
than that for9-AA.

6
S. Marini et al.
Table 2. Kinetic parameters for the oxidation of 4-BA and 9-AA in three pooled human liver
microsome samples.
4-BA
9-AA
Liver
microsomes
Km
Vmax
Km
Vmax
HL-1
HL-2
15.4
17
1.8
1.7
21
11
0.3
0.45
HL-3
Mean (ÆSD)
26
19.4 Æ 5.7
1.4
1.6 Æ 0.2
35
22.3 Æ 12
0.51
0.42 Æ 0.1
Vmax is expressed as nmol minÀ1 mgÀ1 protein Km as mm.
Correlation studies
The rates of 4-BA and 9-AA oxidation were determined in microsomes from 14
individual human livers at a substrate concentration of 200 mm (figure 1). All the
human liver microsomal samples examined demonstrated the ability to oxidize
both aldehydes. The rates of 4-BCA formation exhibited a 2.8-fold range from
1.1 to 3.05 nmol minÀ1 mgÀ1 protein, and the rates of the 9-ACA formation showed
a 3.2-fold range from 0.25 to 0.81 nmol minÀ1 mgÀ1 protein. These rates were
then correlated against marker catalytic activities for the major human CYPs,
determined in these samples. The marker activities were 7-ethoxyresorufin
O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), S-mephenytoin
N-demethylase (CYP2B6), 7-ethoxy-4-trifluoromethylcoumarin deethylase
(CYP2B6), paclitaxel 6ꢁ-hydroxylase (CYP2C8), tolbutamide methyl-hydroxylase
(CYP2C9), S-mephenytoin 40-hydroxylase (CYP2C19), dextromethorphan
O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), testosterone
6ꢀ-hydroxylase (CYP3A4), lauric acid 12 hydroxylase (CYP4A9/11). The
correlation coefficients obtained are detailed in table 3. For 4-BA and 9-AA, a
high correlation was obtained with CYP3A4 (testosterone 6ꢀ-hydroxylase)
(r ¼ 0:76 and 0.8, p < 0:001) and a weak correlation with the catalytic activities
Figure 1. Oxidation rates of 4-BA (&) and 9-AA (&) by 14 human livermicroscome samples. Each
incubation performed for 20 min at 378C contained, in a volume of 0.5 ml, 0.2 mg of microsomal
protein, NADPH-generating system and 200 mm of each substrate; the corresponding acids were
analysed as described in Materials and Methods. Data are the average of two determinations.

4-Biphenylaldehyde and 9-anthraldehyde oxidation by rat and human P450s
7
Table 3. Correlation of the oxidation rates of 4-BA and 9-AA with specific CYP activities in 14 human
livermicrosomes.
Correlation
coefficient ðrÞ
Activities
4-BA
9-AA
7-Ethoxyresorufin O-deethylase (1A2)
Coumarin 7-hydroxylase (2A6)
7-Ethoxy-trifluoromethylcoumarin deethylase (2B6)
S-mephenytoin N-demethylase (2B6)
Paclitaxel 6ꢁ-hydroxylase (2C8)
Tolbutamide methyl-hydroxylase (2C9)
S-mephenytoin 40-hydroxylase (2C19)
Dextromethorphan O-demethylase (2D6)
Chlorzoxazone 6-hydroxylase (2E1)
Testosterone 6ꢀ-hydroxylase (3A4)
Lauric acid 12-hydroxylase (4A9/11)
4-Biophenyl aldehyde oxidase
0.30
0.32
0.62
0.58
0.14
0.38
0.19
70.17
0.55
0.76
70.30
7
0.04
0.63
0.60
0.65
0.46
0.48
0.34
70.08
0.48
0.80
0.07
0.74
Correlation coefficients were calculated from the data of figure 1. Marker activites were supplied
by Xenotch. Bold numbers mean a good or weak correlation among the various activies.
7-ethoxy-4-trifluoromethyl coumarin O-deethylase (r ¼ 0:62 and r ¼ 0:60,
p < 0:01) and S-mephenytoin N-demethylase (r ¼ 0:58 and 0.65, p < 0:01), both
markers for CYP2B6. In addition, a good correlation was also observed between
the oxidation rates of 4-BA versus 9-AA ðr ¼ 0:74Þ.
Oxidation of 4-BA and 9-AA by cDNA expressed human CYPs
Rates of 9-AA and 4-BA oxidation by the cDNA expressed human CYP1A1,
1A2, 2A6, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A4 þ b5 are shown in figure 2.
Conversion of 9-AA and 4-BA to the corresponding acids was only detected
with CYP2A6, 2B6, 2C9, and 3A4; with 9-AA as substrate, turnover numbers
Figure 2. Oxidation rates of 4-BA (&) and 9-AA (&) to the corresponding carboxylic acids by various
cDNA expressed human CYPs. Activities were measured with 200 mm aryl aldehydes under
conditions described in Materials and Methods. Data are the average of two determinations.

8
S. Marini et al.
100
90
80
70
60
50
40
30
20
10
0
INHIBITORS
Figure 3. Effect of specific CYP inhibitors on the oxidation rate of 4-BA (&) and 9-AA (&), both at
100 mm. Values are the average of two determinations performed with two pooled human liver
microsomes (HL-1 and HL-2) as described in Materials and Methods.
were 1.6, 6.4, 0.3 and 1.6 nmol minÀ1 nmolÀ1 CYP, whereas with 4-BA as
substrate, turnover numbers were 4, 6, 1.5 and 2.1 nmol minÀ1 nmolÀ1 CYP,
respectively. With both substrates, CYP2B6 exhibited the highest activity and
CYP3A showed an increase in activity in the presence of cytochrome b5.
Chemical inhibition
Two pooled human liver microsomes (HL1 and HL2) were incubated with 4-
BA and 9-AA (at 100 mm) in the presence of selective chemical CYP inhibitors and
their conversion rates to the corresponding acids were determined. The inhibitors
used were ꢁ-NF (2 mm), 8-MOP (10 mm), SULF (2 mm), MEF (100 mm), QUIN
(1 mm), 4-MP (50 mm), KET (0.5 mm) and TAO (100 mm), selective forthe CYP1A2,
2A6, 2C9, 2C19, 2D6, 2E1 and 3A4 (Parkinson 1996, Koenigs et al. 1997, Sai et al.
2000), respectively.
As shown in figure 3, the oxidations of 4-BA and 9-AA were markedly
inhibited by KET, TAO and 8-MOP and, at a lesserextent, by SULF. These
data suggest that CYP3A4, 2A6 and 2C9 are likely involved in the oxidation of the
two aldehydes.
Discussion
A previous study reported that mouse liver microsomes can catalyse the
oxidation of 9-AA to 9-ACA and that this reaction may be assayed by a simple
fluorimetric method (Watanabe et al. 1991). In the present paper, we describe that
this fluorometric assay may also be used to determine the microsomal oxidation of
4-BA to the corresponding carboxylic acid 4-BCA. The assay takes advantage of

4-Biphenylaldehyde and 9-anthraldehyde oxidation by rat and human P450s
9
the fluorescence of 4-BCA, which is easily separated from the interference of 4-BA
by using the same extraction method described for 9-AA metabolism. As found for
9-AA and otheraldehydes (Watanabe et al. 1991, Kunitoh et al. 1997), it was
demonstrated that 4-BA, which has not been investigated previously, may also be
substrate for the CYP system. The CYP forms responsible for 9-AA metabolism
and, of course, those ones for 4-BA are unknown. We obtained evidence that more
than one form metabolized both 9-AA and 4-BA in the rat. The results indicated
that in the untreated male rats the oxidation of these aldehydes is mediated
primarily by the sex-specific CYP2C11, the major CYP constitutively expressed
in male rat liver (Fujita et al. 1989). The experiments with microsomes from pre-
induced rats and purified enzymes provided evidence that CYP2B1 and 2C11 but
not CYP1A1/2, 2E1, 3A1/2 and 4A1/2 could oxidize these aldehydes with a
consistent turnover.
The 4-BA and 9-AA metabolism to the corresponding acids was also studied in
three preparations of pooled liver microsomes. Investigations into the oxidation
kinetics of these compounds revealed a single phase in Eadie–Hofstee plots with
similarmean Km’s (19.4 Æ 5.7 and 22.3 Æ 12 mm for4-BA and 9-AA, respectively),
supporting the hypothesis that a major CYP(s) with similar affinities (Km) may be
responsible for their oxidation. Despite the similarity in Km, in a bank of 14 human
liver microsomes the mean oxidation rate of 9-AA (0.51 Æ 0.16 nmol minÀ1 mgÀ1
protein) was about 4-fold lower than that of 4-BA (1.8 Æ 0.54 nmol minÀ1 mgÀ1
protein).
A comparison of the kinetic parameters for the oxidation of 4-BA and 9-AA in
rat microsomes versus human microsomes shows similar Km’s, but a 4–10-fold
higher Vmax for both 4-BA and 9-AA in rat microsomes (tables 1 and 2). This
difference may be explained by considering the high constitutive expression of the
CYP2C11 (about 40% of total CYP) in rat (Lewis 1998), its catalytic turnover
observed in the oxidation of these aldehydes (table 1) and the lack of a similar
expression of homologous CYPs in human liver.
To evaluate the contribution of CYPs in the oxidation of 4-BA and 9-AA in
human, studies were conducted using cDNA-expressed CYPs, correlation analy-
sis, and selective chemical inhibitors. When the oxidation of 4-BA and 9-AA was
examined by cDNA-expressed CYPs, it was found that both were extensively
metabolized by CYP2A6, 2B6 and, at lowerextent, 2C9 and 3A4, the latterin the
presence of b5. Only trace amounts of aldehyde metabolism were observed with
othercDNA-expressed CYPs.
Using a bank of 14 human liver microsomes, good correlations were obtained
between the oxidation rate of 4-BA and 9-AA ðr ¼ 0:74Þ and between the oxidation
rates of both aldehydes (r ¼ 0:76 for4-BA, r ¼ 0:80 for9-AA) with testosterone
6ꢀ-hydroxylase (table 3). This enzymatic activity, which is considered to be
catalysed by CYP3A4 (Parkinson 1996, Wrighton et al. 1993), is the most
abundant form in human liver microsomes, accounting for up to 40% of total
expressed CYP (Shimada et al. 1994). A weak correlation was also observed with
S-mephenytoin N-demethylase (r ¼ 0:65 for9-AA, r ¼ 0:58 for4-BA) and 7-
ethoxy-4-trifluoromethylcoumarin (r ¼ 0:60 for9-AA, r ¼ 0:62 for4-BA), both
dependent on CYP2B6 (Ekins et al. 1997, Jo et al. 1998). Since this enzyme is
expressed at low levels (about 1%) in human liver microsomes (Lewis 1998), its
contribution to the oxidation of these aldehydes is likely rather small.

10
S. Marini et al.
A weak correlation was also found between coumarin-7-hydroxylase, a marker
forCYP2A6 (Lewis 1998), and 9-AA oxidation ( r ¼ 0:63). However, no correla-
tion was found between the 4-BA oxidation rate and coumarin 7-hydroxylase
(r ¼ 0:32); on the basis of the finding obtained with the cDNA-expressed
CYP2A6, a correlation with the oxidation of 4-BA would be expected. Possibly,
this discrepancy may be due to the low expression (about 4%) of this enzyme in
human liver(Shimada et al. 1994) and the limited numberof human samples used.
Experiments were also performed with chemical inhibitors selective for CYPs.
The results indicated that the oxidation of 4-BA and 9-AA was extensively
inhibited by TAO and ketoconazole (at low concentration), both considered
inhibitors of CYP3A4 (Say et al. 2000). An inhibition of metabolism of both
aldehydes was also observed with 8-MOP, a mechanism-based inhibitor of
CYP2A6 (Koenigs et al. 1997) and, with sulfaphenazole although at a lower
extent, selective forCYP2C9 (Jo et al. 1998). In the light of the data of the
correlation analysis, cDNA-expressed CYP catalysis and chemical inhibition
presented, it is apparent that, in human liver, CYP3A4 makes a major contribution
to the biotransformation of both 4-BA and 9-AA.
However, as the oxidation of these aldehydes was also demonstrated with
CYP2B6, 2A6 and 2C9 then, under certain conditions, these forms may play an
important role particularly in extrahepatic tissues, where the expression of
CYP3A4 is low.
Overall, the results of the present study demonstrate that both 9-AA and 4-BA
can be substrates for a numbers of different CYPs in rat and in human liver. Owing
to sequence homology of CYPs between species (Smith 1991, Soucek and Gut
1992), we would expect similarenzymes to be responsible forthe 4-BA and 9-AA
oxidation. Indeed, for the orthologous rat CYP2B1 and human CYP2B6, the
substrate specificity towards these aldehydes appears to be maintained. However,
between human CYP3A4 and rat CYP3A1/2 a different catalytic behaviour was
apparent: whereas CYP3A4 appeared the most important catalyst, the highly
expressed CYP3A1/2 in microsomes from DEX-treated rats did not enhance the
oxidation rates of these aldehydes above their control values, suggesting a minor
contribution, if any, of these rat forms. As it is well known that the members of
CYP2A and 2C subfamilies have a very different substrate specificity (Lewis
1998), no comparison can be made between rat CYP2A1/2 and human CYP2A6
and between CYP2C11- and CYP2C9-mediated catalysis.
In conclusion, 4-BA and 9-AA are not CYP-specific substrates whose oxidation
is mediated by CYP2C11 and CYP2B1 in rat and by CYP3A4, 2B6, 2A6 and 2C9
in human. Consequently, these fluorescent probes should be used with caution
when comparing metabolism across species. Furthermore, our results obtained
show that 9-AA and particularly 4-BA undergo oxidation with a high Vmax and
may offera distinct advantage overotherfluoerscent substartes in testing CYP
activities in a certain species when only a small amount of tissue is available or
when extrahepatic tissues have to be examined.
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