Eur J Clin Pharmacol (2001) 57: 377386

DOI 10.1007/s002280100330

PHARMACOKINETICS AND DISPOSITION

Sherwin K.B. Sy Bing-Kou Tang

Aleksandra Pastrakuljic Eve A. Roberts Werner Kalow

Detailed characterization of experimentally derived human hepatic

CYP1A1 activity and expression using differential inhibition
of ethoxyresorun O-deethylation by uvoxamine
Received: 20 February 2001 / Accepted in revised form: 23 May 2001 / Published online: 13 July 2001
Springer-Verlag 2001

Abstract Objective: To characterize the distribution of

mathematically derived human hepatic CYP1A1 activity
using dierential inhibition of ethoxyresorun O-deethylation (EROD) by uvoxamine.
Methods: Quantitative CYP1A1- and CYP1A2-mediated EROD activities were determined in 42 human livers
using dierential inhibition of EROD by uvoxamine.
CYP1A2-specic activity was also measured by phenacetin O-deethylation and caeine 3-demethylation.
Distributions of CYP1A1-mediated EROD and CYP1A2 probe activities were analyzed using cumulative
distribution (probit) plots and the Kolgomorov-Smirnov
test. Age eect on CYP1A1- and CYP1A2-mediated
EROD activities was evaluated using descriptive statistics and analysis of variance.
Results: The derived CYP1A1 protein concentration of
0.58 1.04 pmol/mg was only 4% of the derived
CYP1A2. Since CYP1A1 is intrinsically far more active
than CYP1A2 in mediating EROD, contribution of
CYP1A1 to EROD represented approximately 2540%
of CYP1A2 contribution. Three of the 42 livers exhibited no CYP1A1-mediated EROD. Approximately 8%

of the individuals showed high CYP1A1 activity phenotype based on cumulative distribution curve analysis.
Hepatic CYP1A1 activity was more variable than that of
CYP1A2. The variance of CYP1A1-mediated EROD
was signicantly dierent from that of CYP1A2, using
the Kolgomorov-Smirnov statistical test. Even though
not statistically signicant, an age-related pattern in
CYP1A1-mediated activity was identied: activity was
high in the pre-puberty group, then decreased in the
young/mature adult group and, nally, a slight increase
was observed in old age.
Conclusions: Distribution pattern in CYP1A1-mediated
EROD suggests that the low derived CYP1A1 expression is most likely induced rather than constitutive.
CYP1A1 activity deviates from log-normal distribution;
the variations in hepatic CYP1A1 activity may aect the
conversion of procarcinogens to carcinogens. The agerelated trend in CYP1A1-mediated EROD activity hints
that CYP1A1 responsiveness to inducers may change
with age as well as with exposure to environmental inducers. These ndings prompt (1) future genotyping
studies to determine whether increased CYP1A1 inducibility is a result of genetic factors and (2) studies to
address whether CYP1A1 inducibility changes with age.

S.K.B. Sy (&)
DuPont Pharmaceuticals Company, Stine-Haskell Research
Center, Bldg. 112, P.O. Box 30 (1090 Elkton Rd),
Newark, DE, 19714 USA
E-mail: Sherwin.K.Sy@dupontpharma.com
Tel.: +1-302-3665161
Fax: +1-302-4510054

Keywords CYP1A1 CYP1A2 Dierential

inhibition EROD Fluvoxamine

S.K.B. Sy B.-K. Tang W. Kalow

Department of Pharmacology, University of Toronto,
Toronto, Ontario, Canada
A. Pastrakuljic
Department of Clinical Pharmacology and Toxicology,
Hospital for Sick Children Toronto, Ontario, Canada
E.A. Roberts
Division of Gastroenterology and Nutrition, Hospital
for Sick Children, Toronto, Department of Pharmacology,
University of Toronto, Toronto, Ontario, Canada

Introduction
The microsomal cytochrome P450 (CYP) system metabolizes numerous exogenous and endogenous compounds. The liver is the main organ for xenobiotic
metabolism and contains major CYPs. The CYP1A
subfamily, namely CYP1A1 and CYP1A2, represents
CYPs that catalyze the activation of procarcinogens and
promutagens to active metabolites, which cause DNA
damage (Shimada et al. 1996). CYP1A1 is of additional
interest because it is the most active cytochrome in

378

metabolizing polycyclic aromatic hydrocarbon (PAH)

into active species (Roberts-Thomson et al. 1993; Shimada et al. 1996). Formation of DNA adducts in these
active species is thought to be the initial step in carcinogenesis. Moreover, CYP1A1 expression is highly inducible by environmental xenobiotics through the aryl
hydrocarbon (Ah) receptor (Swanson and Bradeld
1993). Activation of the Ah receptor by PAHs, polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated
dibenzofurans and polychlorinated biphenyls increases
the transcription and translation of CYP1A1, resulting
in increased CYP1A1 expression (Okey et al. 1994). The
relationship between CYP1A1 expression and increased
susceptibility to chemical-induced carcinogenesis remains an area of debate. Researches have been undertaken to explain how genetic variation in metabolic
activation of procarcinogens may increase the risk of
various forms of cancers (Shields et al. 1993; Le Marchand et al. 1998).
Certain genetic variation of CYP1A1 has been shown
to be highly inducible. Previous epidemiological studies
have shown that the highly inducible phenotype of
CYP1A1, MspI polymorphism, is more commonly
found in cancer patients. The MspI mutation in the 3anking region of CYP1A1 gene is associated with lung
cancer in Japan, Hawaii, and Korea (Kawajiri et al.
1990; Xu et al. 1996; Le Marchand et al. 1998). The
MspI polymorphism combined with lack of glutathione
S-transferase-l1 is linked to higher incidence of lung
cancer in the Asian population (Hayashi et al. 1992;
Nakachi et al. 1993; Kihara et al. 1995). Another mutation linked to the MspI polymorphism has been
identied in exon 7 of the CYP1A1 gene. The combined
MspI and exon 7 polymorphism has been associated
with an elevated risk of lung and endometrial cancers
(Nakachi et al. 1995; Sugimura et al. 1995; Goto et al.
1996; Esteller et al. 1997; Mooney et al. 1997; Spivack
et al. 1997). Another study found evidence of interaction
between these two polymorphisms and cigarette consumption (Ishibe et al. 1997). A recent study reported
the link of occurrence of hepatocellular carcinoma to
MspI polymorphism of the CYP1A1 gene (Yu et al.
1999). Increased CYP1A1 expression is thought to
increase the risk of carcinogenesis in mice. A direct relationship between CYP1A1 levels and oxidative DNA
damage was recently demonstrated (Quan et al. 1994).
In direct contrast to previous claims of association of
highly inducible CYP1A1 polymorphism and carcinogenesis, the Finnish, Norwegian, German, North
American, and Taiwanese population have not established a correlation between MspI mutation and lung
cancer (Tefre et al. 1991; Hivonen et al. 1992; Shields
et al. 1993; Drakoulis et al. 1994; Lin et al. 2000). Other
contradicting evidence shows that an increase in number
of DNA adducts is linked to the lack of MspI polymorphism in individuals engaged in the chimneysweeping occupation. Studies have shown that CYP1A1
mediates inactivation of carcinogens (Koser et al. 1988).
In light of this, it is important to determine CYP1A1

activity in humans, especially in the liver which is the

major site of rst-pass metabolism.
The nding that the highly inducible CYP1A1 polymorphism is linked to hepatocellular carcinoma (Yu
et al. 1999) is of interest since CYP1A1 was thought to
be only expressed in extrahepatic organs such as the lung
(Ioannides and Parke 1990; Shimada et al. 1992, 1994).
Numerous studies have examined hepatic CYP1A1 expression, and the majority of them reported no detectable CYP1A1 protein. Immunoblotting technique using
polyclonal antibodies against rat CYP1A1 could not
detect human hepatic CYP1A1 protein (Schweikl et al.
1993). However, hepatic CYP1A1 mRNA was detected
in numerous studies (Omiecinski et al. 1990; McKinnon
et al. 1991; Schweikl et al. 1993; Hakkola et al. 1994).
Recently, two separate studies have shown low levels of
CYP1A1 expression. Using uvoxamine and isosafrole
as inhibitors of ethoxyresorun O-deethylation (EROD)
activity, the concentration of CYP1A1 protein in four
uninduced human liver samples was estimated to range
from 0.4 pmol to 2.7 pmol CYP1A1/mg protein (Pastrakuljic et al. 1997). The result was conrmed independently by Drahushuk et al. (1998) using a
monoclonal antibody directed against a marine scup
P450E. CYP1A1 was detected in all 20 samples they
studied. While there has been evidence of CYP1A1 expression in the human liver in vivo, interindividual
variation in hepatic CYP1A1 activity has not been
determined as yet.
The present study was undertaken to determine the
proportion of highly active CYP1A1 in the population.
The distribution of CYP1A1 activity was analyzed and
compared with probe activities of CYP1A2 using a
detailed statistical model. Another objective of the study
was to investigate the eect of age on CYP1A1-mediated
EROD.

Materials and methods

Chemicals and drugs
Caeine and paraxanthine were purchased from Sigma Chemical
Co. (St. Louis, Mo.); phenacetin, paracetamol, methanol, MgCl2,
KH2PO4, and Na2HPO4 were acquired from BDH Inc. (Toronto,
Ont.); isopropanol, dichloromethane and ethanol were obtained
from Caledon Laboratories Ltd. (Georgetown, Ont.). NADPH was
bought from Boehringer Mannheim GmbH (Germany). Ethoxyresorun was from Molecular Probes Inc. (Junction City, Ore.);
dimethylsulfoxide (DMSO) was from Caledon (Georgetown, Ont.);
and uvoxamine was from Solvay Kingswood Inc. (Scarborough,
Ont.).
Enzyme sources
The human hepatic microsomes used in this study were prepared
from frozen livers obtained from Dr. Eve Roberts at the Hospital
for Sick Children (Toronto, Ont.). The human hepatic tissues were
obtained from surplus portions of normal livers used for transplant. Hepatic microsomes were prepared by the method of Tyndale et al. (1989). Protein concentrations were measured using a
BCA protein kit (Pierce Chemical Co., Rockford, Ill.) with bovine

379
serum albumin as standard. All microsomes were stored at 70C.
Microsomes from cell lines expressing either CYP1A1 or CYP1A2
were purchased from the Gentest Corp. (Cambridge, Mass.). The
concentrations of CYP1A1 and CYP1A2 were 44 pmol/mg and
106 pmol/mg microsomal protein, respectively, as stated by Gentest Corp.
Analysis of CYP1A1- and CYP1A2-mediated EROD
Inhibition studies were carried out with either microsomes from
human livers or expressed CYP1A isozymes, as described in Pastrakuljic et al. (1997), with modications. A 1.45-ml mixture containing 0.2 mg/ml hepatic microsome, 0.42 lM 7-ethoxyresorun,
sodium phosphate buer (0.1 M, pH 7.6) and uvoxamine (0, 20 or
100 lM nal concentration) were pre-incubated at 37C for 5 min
followed by the addition of 50 ll 0.05 g/ml reduced nicotinamide
adenine dinucleotide phosphate (NADPH) to start the incubation.
The incubation was stopped after 10 min by adding 2 ml methanol.
As a control, a mixture containing all of the above components
except uvoxamine and NADPH was used as a blank. EROD was
measured spectrophotometrically as previously described by Roberts et al. (1993). The uorescence of the product was determined
using a Perkin-Elmer LS-5 uorescence spectrophotometer (Perkin-Elmer, Norwalk, Conn.) with an excitation wavelength of
580 nm and emission wavelength of 600 nm. The response was
quantied by comparison with the uorescence of the resorun
standards. Each EROD measurement was subtracted from the
blank.
For kinetic studies, 0.2 mg/ml microsomal protein from two
selected livers (L27 and L31) were mixed with 0.13.4 lM 7-ethoxyresorun. Similar conditions for inhibition studies were performed as stated above. The estimation of Michaelis-Menten
parameters was obtained using the program ENZFITTER (Elsevier-Biosoft Co. 1987).
The hepatic concentrations of CYP1A1 and CYP1A2 and
EROD activity of each isozyme were determined as described by
Pastrakuljic et al. (1997). CYP1A1- and CYP1A2-mediated EROD
activities were calculated using the following equations:
V1A1 =Vtotal 1=0:76Vi =Vtotal
V1A2 =Vtotal 1

Vremaining =Vtotal ;

1=0:76Vi =Vtotal 0:32Vremaining =Vtotal

was added to the incubation mixture, which was then extracted

with dichloromethane (3 ml, vortex for 30 s) to remove unreacted
phenacetin. The aqueous layer was separated by centrifugation for
5 min, and the organic phase was discarded. HCl (0.18 ml 1 N) and
200 mg of ammonium sulfate were added to decrease the pH to
approximately 33.5. Under acidic conditions, paracetamol was
extracted twice with 3 ml dichloromethane/isopropanol (85:15),
vortexed for 30 s, and centrifuged for 5 min. The combined extract
was evaporated under a stream N2 gas and reconstituted in 0.2 ml
HPLC mobile phase. The mobile phase of 0.1 ml was injected onto
the Ultrasphere column (Beckman, 5 lm, 25 mm4.6 cm). The
mobile phase consisted of 1.5% isopropanol, 0.1% acetonitrile,
0.05% acetic acid, and 98.35% water. The ow rate was 1 ml/min.
Retention times of acetaminophen and the internal standard were
14 min and 16 min, respectively, as monitored by ultraviolet detection at 250 nm. The detection limit of paracetamol formation
was 0.2 ng.
Analysis of caeine 3-demethylation
This assay was performed based on the method of Gu et al. (1992).
The incubation mixture of 0.4 ml contained 0.1 mg caeine or
dimethylxanthine as substrate, 0.1 ml phosphate buer solution
(0.2 M, pH 7.4), 0.1 ml 1.15% KCl, 0.1 ml 0.95% MgCl2, and
0.5 mg NADPH. The mixture was incubated at 37C for 60 min,
the reaction was terminated by the addition of 0.05 ml 1.5 M HCl.
Then, 0.02 ml N-acetyl-4-aminophenol (1 mg/100 ml H20) as internal standard and 150 mg ammonium sulfate were added. The
mixture was extracted with 8 ml chloroform:isopropanol (85:15,
v:v), and 0.1 ml was injected onto an Ultrasphere ODS column
(Beckman, 5 lm, 25 mm4.6 cm). The xanthines and urates were
monitored using an ultraviolet spectrophotometer at 280 nm.
Limits were set at 0.3 lM.
HPLC system
The HPLC used in this study was the Shimadzu HPLC System
(Shimadzu Corp., Kyoto, Japan) with LC-6A liquid chromatograph, SCL-10A system controller, SPD-10A UV-VIS detector,
SIL-10A auto injector, and C-R4A chromatopac.

where V1A1 and V1A2 are CYP1A1- and CYP1A2-mediated EROD

activities, respectively, Vtotal is the uninhibited EROD activity, Vi
is the activity of EROD at 20 lM uvoxamine inhibition, and
Vremaining is the remaining uninhibited EROD activity at 100 lM
uvoxamine inhibition.
CYP1A1 and CYP1A2 concentrations in the hepatic microsome were extrapolated from V1A1 and V1A2 based on the equations below:
CYP1A1 concentration 1=7:7V1A1
CYP1A2 concentration 1=0:73V1A2

Data analysis
Probit plots were constructed using the NTV statistical program
(NTV, copyright 1992 by M. Patel and L. Endrenyi) to illustrate
distribution patterns. The slope of the probit plots was estimated
using least-square regression analysis. The Kolgomorov-Smirnov
test was used to compare the variance of the normalized data of
CYP1A1 and CYP1A2 probes. Data were normalized by dividing
individual values by the mean of the probe activity. Descriptive
statistics, analysis of variance (ANOVA) and regression analyses
were determined using Excel 97 software. Kolgomorov-Smirnov
test was performed using Statistica software.

Results
Analysis of phenacetin O-deethylation
The phenacetin O-deethylase activity was determined by measuring
the product acetaminophen. A high-performance liquid chromatography (HPLC) procedure was used for the quantication of
acetaminophen. The 1-ml mixture containing 0.2 mg microsomal
protein, 0.2 mg NADPH, 0.2 ml 1.15% KCl, 0.2 ml 0.95% MgCl2
and 0.2 mM phosphate buer (pH 7.4) was pre-incubated at 37C
in a shaking water bath for 5 min. Then, the substrate phenacetin
to give a nal concentration of 0.2 mM was added to initiate the
reaction. After 30 min of incubation, the reaction was terminated
by addition of 0.08 ml NaOH (to pH 10), and cooled on ice. The
assay internal standard (1,3-dimethyluric acid; 0.06 ml 1 lg/ml)

The activity of CYP1A1 was determined using a novel

dierential inhibition of EROD activity by uvoxamine
(Pastrakuljic et al. 1997). Ethoxyresorun is a specic
substrate for the CYP1A subfamily (Burke et al. 1994).
cDNA-expressed CYP1A1 and CYP1A2 exhibit manyfold higher EROD activities than do other expressed
human CYPs (Lee et al. 1991). Previous studies (Berthou et al. 1991; Forrester et al. 1992) showed that
EROD activity correlated with the immunoquantied
CYP1A2 concentration in the liver. To address the

380

question of specicity of CYP1A1 and CYP1A2 for

EROD activity at low concentrations of 7-ethoxyresorun, we searched the literature for indications of
which other enzymes exhibited EROD activity. The
recently discovered CYP1B1 was shown to catalyze the
O-deethylation of 7-ethoxyresorun at low micromolar
range in cDNA-expressed systems. However, the presence of CYP1B1 in the human liver was not detected by
Western blot at all, neither was the presence of its
mRNA by polymerase chain reaction (PCR) amplication conclusively shown (Sutter et al. 1994; Shimada
et al. 1996, 1997; Crespi et al. 1997; Murray et al. 1997).
At the ethoxyresorun concentration of 0.42 lM used in
this study, EROD activity is overwhelmingly due to
CYP1A1 and CYP1A2, though CYP3A, 2B and 2C may
contribute to a small extent at higher substrate concentrations (Burke et al. 1994). As Pastrakuljic et al. (1997)
have shown, there must be a small third component in
our assay that is not inhibited by uvoxamine in the
human liver microsomes. We speculate that it represents
the summation of those small contributions to EROD
activity by other enzymes. This result is not surprising
because residual activity of EROD was present in all
livers even after inhibition by large amounts of CYP1A
antibodies (Burke et al. 1994).
The rates of EROD activity at the concentration of
0.42 lM 7-ethoxyresorun varied considerably from
sample to sample, but the reaction rates were proportional to protein concentration up to 0.25 mg/ml and
an incubation time up to 10 min. Protein concentration
and incubation time for our reactions were chosen to be
within the linear range, 0.2 mg/ml and 10 min, respectively. The dierential inhibition of EROD activity using the uvoxamine method was simplied to only three
uvoxamine concentrations namely, 0, 20, and 100 lM.
Without uvoxamine inhibition, EROD activity would
be resulting primarily from both CYP1A1 and
CYP1A2. With 2 lM uvoxamine concentration, the
EROD would be mainly due to CYP1A1. At 100 lM
uvoxamine concentration, both CYP1A1 and
CYP1A2 were inhibited, and the remaining activity was
due to other enzymes not inhibited by uvoxamine.
Equations V1A1 and V1A2 accounted for the partial

Table 1 Validation of the

dierential uvoxamine inhibition of ethoxyresorun O-deethylation (EROD) assay using
articial mixtures of cDNAexpressed enzymes. Percentage
recovery as an assessment of
accuracy is close to 100%

Added amount

inhibition of CYP1A isozymes at 2 lM uvoxamine

concentration.
Since the molar concentrations of CYP1A1 and
CYP1A2 in the commercially available cDNA preparations were provided by Gentest, the concentrations of
the CYP1A isozymes could be calculated from their
activities. The assay was tested for validation in articial
mixtures of expressed CYP1A1 and CYP1A2, in order
to examine the accuracy and precision of the inhibition
assay. As shown in Table 1, a series of articial mixtures
of the cDNA-expressed CYP1A1 and CYP1A2 was
tested at ranges close to their observed concentrations in
the liver. The measured amounts of CYP1A1 and
CYP1A2 in the articial mixture, determined from the
dierential inhibition assay, were close estimates of
the added amount. The coecients of variation (CV) of
the mean recovery were 16% for CYP1A1 and 8% for
CYP1A2 (Table 1).
A total of 42 livers were collected and microsomes
were prepared. Eighteen donors were male; twenty-one
donors were female and three were undetermined. The
ages of the donor at the time of harvest ranged from
0.7 years to 55 years.
Individual liver microsomes were screened for their
CYP1A1 and CYP1A2 EROD activities. Caeine 3demethylation (Ca3D) and phenacetin O-deethylation
(POD) were also determined for CYP1A2 activity.
CYP1A1 and CYP1A2 protein concentrations were derived from CYP1A1- and CYP1A2-mediated EROD
activities, respectively, by proportion to cDNA-expressed isozyme specic activities. Table 2 presents the
summary statistics of CYP1A1 and CYP1A2 protein
and activities in the collection of human livers. Of the 42
livers, 3 exhibited no CYP1A1 activity. The average
CYP1A1-mediated EROD was 4.58.0 pmol/mg/min
corresponding to 0.581.04 pmol/mg derived CYP1A1
protein concentration. Estimated CYP1A1 protein
concentration represented 4% of CYP1A2 concentration. However, activity of CYP1A1-mediated EROD
was 2530% that of CYP1A2, since CYP1A1 has higher
activity for EROD than CYP1A2.
Activities of CYP1A1 and CYP1A2 exhibited large
intersubject variability. The CVs, calculated as standard

Measured amount

Recovery*

CYP1A1
(pmol/mg)

CYP1A2
(pmol/mg)

CYP1A1
(pmol/mg)

CYP1A2
(pmol/mg)

CYP1A1
(%)

CYP1A2
(%)

0
0.4
2.6
2.6
2.6
2.6
2.6
15.8

60
60
0
3.7
60
60
156
60

0
0.45
2.84
1.89
2.52
2.7
2.25
14.4

60.8
67
0
3.4
55
56
168
65

125
109
73
97
104
87
91

101
112

92
92
93
108
108

*The mean recoveries are 9716 and 1008 for CYP1A1 and CYP1A2, respectively. The coecients
of variation are 16% for CYP1A1 and 8% for CYP1A2

381
Table 2 Summary of derived
CYP1A isozyme concentrations, probe activities of CYP1A1, and CYP1A2 in human
hepatic microsomes. CYP1A2
activites were measured by caffeine 3-demethylation (Ca3D),
phenacetin O-deethylation
(POD) and CYP1A2-mediated
7-ethoxyresorun O-deethylation (EROD). CYP1A1 activity
was measured by means of
CYP1A1-mediated EROD

CYP1A protein
(pmol/mg)

MeanSD

Coecient of
variation (%)

Range

CYP1A1
CYP1A2
CYP1A activities
(pmol/mg/min)
CYP1A1-mediated EROD
CYP1A2-mediated EROD*
POD**
Ca3D**

0.581.04
15.715.9

180
101

(04.9)
(0.569.6)

42
42

4.58.0
11.411.6
120100
4834

180
101
83
71

(038.3)
(0.3650.8)
(174010
(5.893)

42
42
34
34

*Signicant dierences (P<0.05) for comparison with CYP1A1-mediated EROD, using KolgomorovSmirnov test
**Signicant dierences (P<0.01) for comparison with CYP1A1-mediated EROD, using Kolgomorov-Smirnov test

deviation divided by the mean, were comparable for the

three CYP1A2 probes. The CVs ranged from 71% to
101% for CYP1A2 activities. CYP1A1-mediated EROD
exhibited a higher CV.
To determine whether there were any signicant differences in the distribution pattern among the measurements, the Kolgomorov-Smirnov test that compares
variances was used to analyze the normalized data. The
normalized data was based on the ratio of the individual
value divided by the mean value of the group measured
by the same probe. The data indicated no dierence in
the distribution of CYP1A2-probe activities. CYP1A1mediated EROD was signicantly dierent from other
probes (P<0.05).
Distributions of CYP1A1 and CYP1A2 activities
were also evaluated using probit plots with logarithmic
abscissa (Fig. 1). Based on visual inspection, probit
plots of CYP1A2 probe activities showed log-normal
distribution. However, probit plots of CYP1A1-mediated EROD exhibited deviation from normality above
the third quartile. Three (8%) of the 42 livers expressed
high CYP1A1 activity. Of the three high CYP1A1
activity livers, two were from children ages 24 years
(one male and one female), and one was from a female
donor 42 years of age. Deviation from normality in the
lower estimates may be due to assay sensitivity or
limitation of the deconvolution technique in the probit
regression algorithm. Since the slopes of probit plots
would indicate the magnitude of standard deviations,
the slopes of the probit were compared using linear
regression. Due to the non-linearity in the tails of the
plots, the points from the rst to the third quartiles
were regressed to allow comparison. Three sets of
CYP1A2 data obtained via EROD, POD and Ca3D
showed very similar slopes (slopes=2.6, 2.78, and 2.97,
respectively). The slope for CYP1A1 was much shallower with a value of 1.8.
Studies were conducted to determine which kinetic
parameters exhibited the greatest variability. Kinetic
parameters for EROD due to CYP1A1 and CYP1A2
were determined in two human hepatic microsomes
exhibiting high (L27 hepatic microsomes) and intermediate (L31 hepatic microsomes) capacities to O-deethylate
7-ethoxyresorun. Hepatic microsomes were incubated

Fig. 1 Probit plots of the distribution of CYP1A1- and CYP1A2mediated ethoxyresorun O-deethylation (EROD), caeine 3demethylation (Ca3D), and phenacetin O-deethylation (POD) in
a collection of human livers. Approximate slopes between the rst
and third quartiles for CYP1A1-mediated EROD, CYP1A2mediated EROD, Ca3D, and POD are 1.8, 2.6, 2.97, and 2.78,
respectively

with 0.13.4 lM 7-ethoxyresorun. The relationship

between 7-ethoxyresorun concentration and EROD
due to CYP1A1 or that due to CYP1A2 adhered to
Michaelis-Menten kinetics. The kinetic parameters are
summarized in Table 3. Despite variable Vmax, the apparent Km values of EROD due to CYP1A1 and
CYP1A2 were approximately 0.1 lM and 0.2 lM,
respectively, for both microsomal samples. The apparent
Km of EROD due to CYP1A1 was consistent for
both microsomal samples (L27 and L31). A similar
phenomenon was observed for the apparent Km of
EROD due to CYP1A2. These values were close to that
reported for cDNA expressed enzymes (Penman
et al. 1994).
Given that the majority of the high CYP1A1 activity
was in the young age group, the eect of age on derived
CYP1A1- and CYP1A2-mediated EROD activity was
assessed (Fig. 2). One liver sample without documented
age of donor was excluded from analysis. CYP1A1- and

382
Table 3 Estimated kinetic parameters (SEM) of CYP1A1- and
CYP1A2-mediated ethoxyresorun O-deethylation (EROD) through dierential inhibition by uvoxamine were close estimates to

that reported for EROD in cDNA-expressed CYP1A1 and CYP1A2 microsomes. Two human livers (L27 and L31) were used to characterize the kinetics. NA data not available

Identication
code

Vmax
pmol/mg/min

Vmax/CYP1A
pmol/min/pmol CYP1A

Km
lM

Reference

CYP1A1

L27
L31
cDNA-expressed

19.51
8.60.8
NA

4.8
10.5
NA

0.10.02
0.10.04
0.087

Present study
Present study
Penman et al. 1994

CYP1A2

L27

593

0.85

0.210.04

Present study

252
NA

1.2
NA

0.180.05
0.24

Present study
Penman et al. 1994

L31
cDNA-expressed

order of average CYP1A1-mediated EROD activity was:

age less than 13 years, 40 years or more, 1340 years. As
for CYP1A2, EROD activities due to this isozyme were
very similar for the groups with age less than 13 years
and 1340 years, while the group of age 40 years or
more exhibited slightly lower CYP1A2 activity.
There is a concern that CYP1A1 activity may be related to that of CYP1A2 due to the methodological aspect of the study rather than a functional relationship.
Should high CYP1A2 activity automatically result in a
tendency toward high CYP1A1 activity as a consequence of the method of evaluation, CYP1A1-mediated
activity would correlate very strongly with that of
CYP1A2 and CYP1A2-mediated EROD. To address
this issue, correlation analysis of estimated CYP1A1mediated activity versus that of CYP1A2 was performed. Figure 3 shows the relationship between
CYP1A1-mediated EROD versus CYP1A2-mediated
EROD for both linear and logarithmic scales. In contrast to what was suspected to be a methodological
tendency of high CYP1A2-mediated EROD to drive
CYP1A1-mediated activity in the same direction, we
found a low but borderline-signicant correlation coefcient of 0.32 (P<0.05) between CYP1A1-mediated
EROD and CYP1A2-mediated EROD.
Multiple correlation in Table 4 shows that CYP1A1mediated EROD correlation with POD and Ca3D is low
but signicant. As expected, all CYP1A2 probes were
strongly correlated to each other.
Fig. 2 Age eect on estimated CYP1A1- (A) and CYP1A2- (B)
mediated ethoxyresorun O-deethylation (EROD) activities. Ages
of donor at time of liver harvest were separated into groups of age
<13 years, 1340 years, and 40 years or more. Open circles
represent individual estimates of CYP1A-mediated EROD activity
and horizontal line represents the mean of the group. Since the
logarithm of zero is undened, the three subjects with no CYP1A1
activity, one belonged to the group 1340 years and two to the
group 40 years or more, were not included in the rst gure but
were included in the mean estimate

CYP1A2-mediated EROD activities for this liver sample

were 1.39 pmol/mg/min and 30.3 pmol/mg/min,
respectively. The ages of donors at the time of liver
harvest were separated into groups of less than 13 years,
1340 years, and 40 years or more. Rank in descending

Discussion
Though the association of CYP1A1 activity to carcinogenesis is still a subject of debate, its role in activation of
xenobiotics to carcinogenic agents is an active area of
research interest. In animals and humans, CYP1A1 is
thought to be constitutively expressed only in extrahepatic organs (Ioannides and Parke 1990; Shimada et al.
1992, 1994) but readily inducible in the liver through the
aryl hydrocarbon receptor. Even though CYP1A1
transcription occurs in the liver (Schweikl et al. 1993),
detection of CYP1A1 protein in uninduced liver is debatable. A previous immunoblotting technique using
antibodies against rat CYP1A1 failed to detect CYP1A1

383

Fig. 3 Correlation between CYP1A2-mediated ethoxyresorun Odeethylation (EROD) and CYP1A1-mediated EROD in linear
scale (A) and logarithmic scale (B)
Table 4 Multiple correlation between phenacetin O-deethylation
(POD), caeine 3-demethylation (Ca3D), CYP1A2-mediated ethoxyresorun O-deethylation (CYP1A2-EROD) and CYP1A1-mediated ethoxyresorun O-deethylation (CYP1A1-EROD). The
three CYP1A2 probe activities, namely POD, Ca3D and CYP1A2EROD correlate strongly with each other. CYP1A1-EROD correlation coecients with CYP1A2 probes are low but borderline
signicant

Ca3D
CYP1A2 EROD
CYP1A1 EROD

POD

Ca3D

0.96*
0.83*
0.31**

0.81*
0.33**

CYP1A2
EROD

0.32**

* P<0.001; **P<0.05

in human liver (Schweikl et al. 1993). However, monoclonal antibody against marine scup P450E shows human hepatic CYP1A1 signal in the uninduced state
(Drahushuk et al. 1998). It is not clear why the two
antibodies would produce such dierent results.
Our laboratory has previously separated hepatic
CYP1A1 activity from that of CYP1A2 using dierential
inhibition of EROD by uvoxamine (Pastrakuljic et al.
1997). The underlying principle of the assay is based on
the fact that the Ki value for CYP1A2 is 800 times lower

than that for CYP1A1. At low uvoxamine concentration, CYP1A2-mediated EROD activity is inhibited,
while CYP1A1-mediated EROD activity is spared. At
high uvoxamine concentration, both CYP1A1 and
CYP1A2 are inhibited. Fluvoxamine concentrations
have been chosen to provide optimized inhibition of each
enzyme. CYP1A1- and CYP1A2-mediated EROD activities were measured using derived mathematical
equations (Pastrakuljic et al. 1997). It is noted here that
the current method used to assess CYP1A1 and CYP1A2
activities is indirect but operational. Until a specic
substrate of CYP1A1 is discovered, the current method
provides a way to separate CYP1A1 contribution to
EROD activity from that of CYP1A2.
In the present study, we demonstrated that the method
accurately predicts the amount of CYP1A1 and CYP1A2
in articial mixtures of cDNA-expressed enzymes. The
added amounts of the two cDNA-expressed isozymes
were selected to mimic their actual concentrations in the
liver. The mathematical equations previously developed
by Pastrakuljic et al. (1997) assumed no structural mutation in either of the isozymes that would aect their
enzyme kinetics. In support of this assumption, we
showed that the estimated Michaelis-Menten kinetics,
specically Km in the two livers with intermediate and
high CYP1A1 activities, is similar to that reported in the
literature for cDNA-expressed enzymes. Km is very
similar for CYP1A1 and CYP1A2 in both livers. Even
though the only known CYP1A1 structural polymorphism has been found in exon 7 of its gene, CYP1A1
kinetics was not altered following expression of the mutant allele in Escherichia coli and yeast (Zhang et al. 1996;
Persson et al. 1997). As for CYP1A2, phenotyping
showed no evidence of variability that could potentially
aect its kinetics (Nakajima et al. 1994). Another support
for the validity of the assay is high correlation of
CYP1A2-mediated EROD activity to POD and Ca3D,
both of which are specic probes for CYP1A2.
In this study, CYP1A1-mediated EROD activity was
quantied in 42 human livers. Three of the livers did not
exhibit CYP1A1 activity. CYP1A1 contribution to
EROD activity was approximately 2540% of CYP1A2mediated EROD. However, estimated CYP1A1 protein
concentration, ranging from 0 pmol/mg to 4.9 pmol/mg,
is approximately 4% of CYP1A2 concentration. Since it
is not possible to rule out background exposure to
environmental xenobiotics that may induce CYP1A1
expression through the aryl hydrocarbon receptor, the
low estimated expression of CYP1A1 and the fact that
three livers did not exhibit CYP1A1 activity suggest that
CYP1A1 expression may be induced rather than constitutive. Consistent with our observation, low CYP1A1
protein concentrations were detected in all 20 livers using a specic CYP1A1 monoclonal antibody, as reported by Drahushuk et al. (1998). Interestingly, the
highest CYP1A1 protein concentration using an immunoblotting technique (Drahushuk et al. 1998) was identical to our highest estimated CYP1A1 concentration
derived from dierential enzyme inhibition study.

384

The CV of CYP1A1 was about twice as large as that

of CYP1A2. The striking interindividual variability of
CYP1A1 might be due to the fact that some livers did
not exhibit CYP1A1 activity, while approximately 8%
of the livers exhibited high CYP1A1 activity. No historical data, such as smoking or drinking habits, were
provided except for the age of the donor at the time of
liver harvest. Since these liver tissues were from surplus
portions of those used in pediatric liver transplant, the
livers were from deceased healthy individuals who were
tested negative for human immunodeciency virus and
hepatitis C. In the three high CYP1A1 activity livers,
two livers came from children aged 24 years and one
was from a female donor 42 years of age.
Given that the majority of the high CYP1A1 activity
livers was from children, the trend of CYP1A1-mediated
EROD activity in relation to age was evaluated in this
study. A pattern in CYP1A1-mediated EROD activity
was identied: activity was high in the pre-puberty
group, then decreased in the young/mature adult group
and, nally, there was a slight increase in old age.
However, none of these changes was signicant. Overall,
a decreasing trend of CYP1A1 activity from pre-puberty
to old age was observed. The signicance of the nding
in vivo is still debatable, since the observed trend might
be due to random chance resulting from small sample
size. Of particular interest is the slight increase in
CYP1A1 activity from adult to elderly. Since exposure
of the elderly donors to environmental pollutant was not
documented, we could only speculate that the slight increase in CYP1A1 activity in the elderly might be related
to accumulation of environmental inducers over time.
As for CYP1A2, our observation that old age has a
tendency toward lower CYP1A2 activity is conrmed by
decreased clearance of a substrate of CYP1A2, brofaromine, in the elderly (Zeeh et al. 1996). Tanaka (1998)
also reported the same trend for CYP1A2-mediated
drug metabolism.
The present study also investigated the distribution of
CYP1A1-mediated activity along with CYP1A2 activity
using three CYP1A2 probes. Distribution of CYP1A1mediated EROD activity is clearly dierent from that of
CYP1A2. The probit plot of CYP1A1-mediated EROD
activity exhibited a shallower slope, corresponding to
greater interindividual variation than that of CYP1A2.
In addition, the variance of CYP1A1 activity was signicantly dierent from the three probe activities of
CYP1A2 based on a statistical distribution (Kolgomorov-Smirnov) test, suggesting that distinct regulatory
pathways for the two isozymes may be involved. Several
studies have shown separate induction pathways of
CYP1A1 and CYP1A2. Exposure of human liver slices
to PCDD, which is a prototypic ligand of the Ah receptor, resulted in signicant induction of CYP1A1
protein but no change in CYP1A2 induction (Drahushuk et al. 1998). In the Ah-receptor knock-out mouse,
both transcription and translation of CYP1A2 genes
were increased after treatment with piperonyl butoxide
and acenaphthylene, while induction of CYP1A1 was

not detected (Ryu et al. 1996). The probable presence of

divergent pathways in the regulation of CYP1A1 and
CYP1A2 may explain, at least in part, why the distributions of the two isozymes are dierent.
However, the extent of variability among CYP1A2
probe activities was not signicantly dierent based on
the Kolgomorov-Smirnov test and a comparison of the
CV of the three CYP1A2 probe activities. Considerable
interindividual variability in CYP1A2 activity was consistent with previous ndings showing 5- to 40-fold
variation in CYP1A2 mRNA (Farin et al. 1993) and 67fold interindividual variation in CYP1A2 protein in
human liver (Sesardic et al. 1998). In vivo caeine metabolism varied 16-fold in Chinese population (Ou-Yang
et al. 2000). Furthermore, 6- to 200-fold variations of
CYP1A2 activity have been reported in in vivo studies
with dierent CYP1A2 markers, derived mainly from
urinary metabolites of caeine (Butler et al. 1992;
Nakajima et al. 1994; Tang et al. 1994; Le Marchand
et al. 1997). A possible explanation for inconsistent
variability of CYP1A2 activity is the mixed population
in the study, such as some smokers. Methodological issues such as the quality of samples in in vitro studies,
confounding factors and assumptions when using urinary metabolic ratios may also aect variability in
CYP1A2 phenotype results. Detailed modeling of caffeine metabolism and examination of the CYP1A2 gene
shows lack of a polymorphism in CYP1A2 in Caucasians (Welfare et al. 1999). The study also concluded
that the wide interindividual variation in activity might
be due to environmental factors.
The variability observed in CYP1A1-mediated
EROD activity suggests a possible polymorphic response. Our study indicates that CYP1A1-mediated activity in the liver deviates from the log-normal
distribution with 8% of the individuals showing high
activity. At present, there does not appear to be an explanation for the observed high CYP1A1 activity individuals. Future studies are directed at characterizing
CYP1A1 polymorphism using a genotype study of this
liver bank. Furthermore, an age-related eect on
CYP1A1 and CYP1A2 induction will be studied to understand the mechanisms involved. A combination of
the dierential inhibition of EROD by uvoxamine,
molecular biology techniques and induction of CYP1A
isozymes in precision-cut human liver slices would provide a powerful tool to address these issues.
Acknowledgements This work was supported by the Medical Research Council of Canada grant MT13760. We would like to thank
Mr. Jared Shockcor, Ms. Marie Hildebrandt and Mr. John Mondick for expert editorial assistance.

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