![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 30, Issue 7, 788-794, July 2002
Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario, Canada
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results and Discussion
References
|
|---|
Porphyrinogenicity of certain xenobiotics depends upon mechanism-based inactivation of specific cytochrome P450 (P450) enzymes, followed by formation of N-alkylprotoporphyrin IX (N-alkylPP). Examination of the porphyrinogenicity of xenobiotics in animals and extrapolation of the results to humans is associated with ambiguity due, in part, to differences between P450 enzymes. The goal of this study was to develop an in vitro test for the detection of N-alkylPPs, produced in human liver after administration of xenobiotics found to be porphyrinogenic in animals. This goal was achieved using fluorometry to detect N-alkylPP formation following mechanism-based inactivation by porphyrinogenic xenobiotics of single cDNA-expressed human P450 enzymes in microsomes prepared from baculovirus-infected insect cells (Supersomes) and in human liver microsomes. The following combinations of P450 enzymes were major sources of N-alkylPPs in Supersomes: CYP3A4 [3-[(arylthio)-ethyl]sydnone (TTMS)]; CYP1A2 and 2C9 [3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC)]; and CYP2C9, 2D6, and 3A4 [allylisopropylacetamide (AIA)]. Whereas similarities were found between results with human enzymes in Supersomes and their rat orthologs in rat liver microsomes, some differences were found. The results with TTMS and AIA, but not with 4-ethyl DDC, were the same in individual human enzymes expressed in Supersomes and human liver microsomes. We conclude that some differences exist between human liver P450 enzymes and their rat P450 orthologs in liver microsomes. It would therefore be prudent when dealing with xenobiotics in which porphyrinogenicity depends upon N-alkylPP formation to supplement animal data with studies using human P450 enzymes.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
Xenobiotics
which interfere with control of heme biosynthesis and allow porphyrins
to accumulate are referred to as porphyrinogenic. The porphyrinogenic
effects of several xenobiotics (e.g., TTMS1,
4-ethyl DDC, and AIA) depend on their ability to cause mechanism-based inactivation of P450 enzymes (Ortiz de Montellano et al., 1981a
; Ortiz
de Montellano and Grab, 1986; Oritz de Montellano and Mico, 1981c).
These xenobiotics, upon selective P450 enzyme mediated biotransformation, form reactive intermediates at the P450 active site.
The reactive intermediates interact with the heme moiety resulting in
N-alkylation of a pyrrole ring (Marks et al., 1988; Ortiz de
Montellano and Correia, 1995). P450 inactivation and dissociation of
N-alkylheme is followed by loss of iron, yielding N-alkylprotoporphyrin IX (N-alkylPP), some of
which are potent inhibitors of ferrochelatase (EC 4.99.1.1) (De Matteis
et al., 1980; Cole et al., 1981). Ferrochelatase inhibition results in decreased heme production and less control over the rate-limiting enzyme aminolevulinic acid synthase (EC 2.3.1.37; aminolevulinic acid
synthase). Increased aminolevulinic acid synthase activity results in porphyrin accumulation and porphyria (De Matteis and Marks,
1996). The N-alkylPP formed after mechanism-based
inactivation of P450 by AIA, viz.
N-AIAPP2,
does not inhibit ferrochelatase. The porphyrinogenic effect of AIA is
explained as follows: since P450 can be reconstituted after undergoing
mechanism-based inactivation by AIA as a result of the exchange of the
N-alkylated heme moiety (N-AIAPP) for a fresh
heme molecule, AIA functions as a heme-destructive catalyst that
depletes regulatory free heme (Ortiz de Montellano et al., 1985).
The porphyrinogenicity of TTMS, 4-ethyl DDC, and AIA, has been shown in
animals to be dependent on mechanism-based inactivation of specific
P450 enzymes (Marks et al., 1988). Our previous in vivo studies have
elucidated the important rat liver P450 enzymes responsible for
N-alkylPP formation. CYP3A2 is the major source of
N-vinylprotoporphyrin IX (N-vinylPP) after the
administration of TTMS to rats whereas CYP2C11 is the major source of
N-ethylprotoporphyrin IX (N-ethylPP) and
N-AIAprotoporphyrin IX (N-AIAPP) after the administration of 4-ethyl DDC and AIA, respectively (Wong et al., 1998,
1999a).
Currently during toxicity testing, the porphyrinogenicity of
xenobiotics is tested in animals, and it is sometimes a problem to
relate these results to humans. In the case of xenobiotics, which owe
their porphyrinogenicity to mechanism-based inactivation of selective
P450 enzymes with formation of N-alkylPPs, the problem stems, in part, to differences in the P450 enzymes between animals and
humans. This is a particular problem when a xenobiotic is found to be
porphyrinogenic in one species but not in others (Frater et al., 1993).
Therefore, an understanding of the human P450 enzymes targeted by these
xenobiotics is required to establish whether porphyria elicited in
animals is likely to occur in humans. Using microsomes prepared from
human lymphoblastoid cell lines containing single cDNA-expressed human
P450 enzymes, McNamee et al. (1997) used selective enzyme activities to
determine which human P450 enzymes are susceptible to mechanism-based
inactivation after administration of TTMS, 4-ethyl DDC, and AIA. TTMS
and 4-ethyl DDC caused mechanism-based inactivation of human P450
enzymes 1A2 and 3A4, whereas only 4-ethyl DDC caused mechanism-based
inactivation of CYP2C9; neither xenobiotic caused mechanism-based
inactivation of CYP2D6. AIA did not inactivate any of the human P450
enzymes. However, there are several different ways in which xenobiotics can cause mechanism-based inactivation, and hepatic porphyrin accumulation will only occur if N-alkylPP formation
accompanies mechanism-based inactivation (Marks et al., 1988).
Therefore, it is important to determine which human P450 enzymes
targeted for mechanism-based inactivation also elicit
N-alkylPP formation.
In previous studies, we determined that interaction of porphyrinogenic
xenobiotics with in vitro preparations containing hepatic P450 enzymes
in quantities similar to those in commercially available microsomal
products from human lymphobastoid cells (0.5-1.0 nmol total P450) did
not yield N-alkylPPs in sufficient quantity to be detected
by a combination of thin-layer chromatography and UV-visible
spectrophotometry (Wong and Marks, 1999b). Our primary goal is the
development of an in vitro test system for the isolation and detection
of N-alkylPPs, which may be produced in human liver after
administration of xenobiotics, previously found to be porphyrinogenic in animals. We therefore explored a variety of methods to determine which was sufficiently sensitive for the detection of
N-alkylPP formation from in vitro preparations containing
human hepatic P450 enzymes. Our first objective was to use the most
sensitive method to determine which specific P450 enzymes were
responsible for N-alkylPP formation using single
cDNA-expressed human P450 enzymes in microsomes prepared from
baculovirus-infected insect cells (Supersomes). The second objective
was to use the most sensitive method to determine whether
N-alkylPP formation could be detected in human liver
microsomes after interaction with xenobiotics in vitro.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
Source of Chemicals.
TTMS and 4-ethyl DDC were obtained from Color Your Enzyme (Bath,
Ontario, Canada). AIA was obtained as a gift from F. Hoffman-LaRoche (Vaudreuil, Quebec, Canada). Solvents (acetone, dichloromethane, and
methanol) were purchased from VWR Canada (Mississauga, Ontario, Canada). Inducers (
-naphthoflavone, dexamethasone, and phenobarbital sodium) and NADPH were purchased from Sigma-Aldrich (St. Louis, MO). Human liver microsomes and Supersomes were purchased from Gentest
Corp. (Woburn, MA).
Treatment of Animals. Male Sprague-Dawley rats (250 to 300 g) were obtained from Charles River Canada, Inc. (St. Constant, Quebec, Canada). All rats received Purina Laboratory Chow (Purina, St. Louis, MO) and water ad libitum and were housed under controlled conditions (22°C, 14:10 h light/dark cycle) prior to treatment.
Treatment of Male Rats with P450 Inducers.
Rats received either dexamethasone (100 mg/kg, dissolved in 0.5 ml
dimethyl sulfoxide) or phenobarbital sodium (80 mg/kg, dissolved in 0.5 ml deionized water) administered i.p. once daily for 4 days.
-Naphthoflavone (40 mg/kg, dissolved in 0.5 ml dimethyl sulfoxide)
was administered i.p. once daily for 3 days (McNamee et al., 1997).
Preparation of Rat Hepatic Microsomes.
Twenty-four hours after final treatment with specific P450 enzyme
inducer, rats were sacrificed by decapitation, and their livers were
perfused in situ with 150 ml of ice-cold 1.15% (w/v) KCl to remove the
blood. Livers were excised, then minced and homogenized in 3 volumes of
ice-cold phosphate-buffered KCl [1.15% (w/v) KCl, 10 mM
K2HPO4, pH 7.4] using a
Potter-Elvehjem apparatus. The homogenate was centrifuged at
9000g for 20 min. The supernatant was centrifuged at
106,000g for 60 min at 4°C. The resulting eight microsomal
pellets were resuspended in ice-cold 0.1 M
K2HPO4 buffer (pH 7.4)
containing 1.5 mM EDTA and recentrifuged at 106,000g for 60 min at 4°C. Each microsomal pellet was resuspended in 4.0 ml of 0.1 M
K2HPO4 buffer (pH 7.4)
containing 1.5 mM EDTA to yield a protein concentration of 4 to 6 mg/ml
(Wong and Marks, 1999b). Microsomal P450 content was determined on 1 ml
of microsomal suspension prior to mechanism-based inactivation by the
difference spectrophotometric method of Omura and Sato (1964).
Mechanism-Based Inactivation of Rat Hepatic Microsomal P450 with
Porphyrinogenic Xenobiotics.
For studies with TTMS, 4-ethyl DDC, and AIA, liver microsomal
suspensions from dexamethasone-, -naphthoflavone-, or
phenobarbital-pretreated rats were used, respectively. Microsomal
suspensions were incubated with 2.0 mM NADPH and TTMS (0.5 mM), 4-ethyl
DDC (1.0 mM), or AIA (10 mM) for 30 min at 37°C in a shaking water
bath. When one microsomal pellet was used, the final volume was 4 ml.
With consecutive reductions in amount of microsomes used, the final
volume was reduced in a corresponding manner.
Isolation of N-alkylPP from Rat Hepatic
Microsomes.
Following incubation, the reaction mixture (4 ml) was mixed with 5 volumes of ice-cold 5%
H2SO4:methanol (v/v) (20 ml) and shaken in the dark at 4°C for 18 h. The mixture
containing N-alkylPP dimethyl esters was filtered, diluted
with an equal volume of deionized water (24 ml), and then extracted
twice with dichloromethane (2 × 12 ml) in a separatory funnel.
The dichloromethane extract was washed with 5% sodium bicarbonate (24 ml). Zinc acetate (12 µmol) in 1.0 ml methanol was added to the
dichloromethane solution to form the Zn N-alkylPP dimethyl
ester, and the solution was evaporated to dryness. The
N-alkylPP dimethyl ester forms complexes with a variety of
metals leading to the formation of a mixture of metallo-complexes,
particularly during TLC. Preparation of the zinc complex circumvents
this problem. The residue was dissolved in dichloromethane (3 ml), and
an aliquot (1 ml) was taken for UV-visible spectrophotometry, and an
aliquot (2 ml) was taken for fluorometry (see below). The
dichloromethane aliquots were combined and applied to an Analtec silica
gel G TLC plate (2000 µm; Analtech, Newark, DE) and developed in
dichloromethane/methanol (195:30) for 60 min. A single green band
(Rf 0.68-0.74) that fluoresced red
under long wavelength ultraviolet light was eluted from the plate with
acetone and evaporated to dryness under nitrogen, as previously
described by Wong and Marks (1999b). The residue was dissolved in
dichloromethane (1.0 ml) for both UV-visible spectrophotometry and fluorometry.
Quantitation of Zn N-alkylPP Dimethyl Ester by
UV-Visible Spectrophotometry and Fluorometry prior to and following
TLC.
The electronic absorption spectrum of the Zn N-alkylPP
dimethyl ester in dichloromethane (1.0 ml) was determined, and the Zn
N-alkylPP dimethyl ester concentration was calculated using the molar extinction coefficient of 128,000 m
1cm1 at 432 nm (Ortiz
de Montellano et al., 1981b). The dichloromethane solution (1.0 ml) was
diluted with 1.0 ml of dichloromethane, and the fluorescence spectrum
of the Zn N-alkylPP dimethyl ester in dichloromethane was
determined. Using an excitation wavelength of 432 nm (Soret band), the
emission spectrum of the sample was scanned from 600 to 800 nm. The
emission spectrum characteristics of samples containing Zn
N-alkylPP dimethyl esters included peaks at 660 and 720 nm
for N-vinylPP, 655 and 715 nm for N-ethylPP, and
650 and 712 nm for N-AIAPP. The Zn N-alkylPP
dimethyl ester concentration was determined from standard curves
obtained by using known concentrations determined by UV-visible spectrophotometry.
Mechanism-Based Inactivation of Human P450 Enzymes in Supersomes
with Porphyrinogenic Xenobiotics.
Microsomes prepared from baculovirus-infected insect cells
(Supersomes) possessing single cDNA-expressed human P450 enzymes (1A2,
catalogue no. P203; 2C9, catalogue no. P258; 2D6, catalogue no. P217;
or 3A4, catalogue no. P202) were obtained from Gentest Corp. All
Supersomes were prepared from cell lines coexpressing P450 and
oxidoreductase cDNA. Following previously described methods (McNamee et
al., 1997), when required, Supersomes were rapidly thawed in a 37°C
shaking water bath. The Supersomes suspension (1.0 ml for CYP1A2, 2C9,
and 2D6 and 0.5 ml for CYP3A4) was added to 5-ml Erlenmeyer flasks,
containing 2.0 mM NADPH and TTMS (0.5 mM), 4-ethyl DDC (1.0 mM), or AIA
(10 mM). Samples were incubated for 30 min in a 37°C shaking water bath.
Isolation of N-alkylPP from Supersomes Containing CYP1A2, 2C9, 2D6, and 3A4. Following incubation, the reaction mixture was mixed with 5 volumes of ice-cold 5% H2SO4:methanol (v/v) (5 ml) and shaken in the dark at 4°C for 18 h. The mixture containing N-alkylPP dimethyl esters was filtered, diluted with an equal volume of deionized water (6 ml), and then extracted twice with dichloromethane (2 × 3 ml) in a 60-ml separatory funnel. The dichloromethane extract was washed with 5% sodium bicarbonate (6 ml). Zinc acetate (12 µmol) in 1.0 ml of methanol was added to the dichloromethane solution to form the Zn N-alkylPP dimethyl ester. The volume of solution was reduced to 2 ml by a stream of nitrogen prior to fluorometric quantitation. Since the Supersome preparation of CYP3A4 was contained in a volume of 0.5 ml, the above procedure was carried out by reducing volumes of reagents by 50%. Wherever the presence of an N-alkylPP was detected, the experiment was repeated with a second Supersome preparation.
Mechanism-Based Inactivation of Human P450 Enzymes in Human Liver Microsomes and Isolation of N-alkylPP. The procedure described above for Supersomes was used for human liver microsomes, which were also obtained from Gentest Corp. Human liver microsomes possessing elevated CYP3A4 (catalogue no. HG112) contained 6.28 nmol of total P450 in 0.5 ml, of which 53% was CYP3A4, 14% CYP2C9, 7.5% CYP2B6, 11.5% CYP2C19, 1.2% CYP2D6, and 11.2% CYP2E1. The human liver microsomes were obtained from a two-year-old white female who had died of head trauma. During hospitalization, she had received mannitol, phenytoin, ibuprofen, nafcillin, phenobarbital, ranitidine, dobutaine, dopamine, and lorazepam. Human liver microsomes possessing elevated CYP1A2 and CYP2C9 (catalogue no. HG56) contained 4.98 nmol of total P450 of which 25% was CYP1A2, 29% CYP2C9, 12.5% CYP3A4, 11.6% CYP2A6, 5% CYP2C19, 2.2% CYP2D6, and 13.2% CYP2E1. The human liver microsomes were obtained from a 57-year-old white female who had died of an aneurism. Her medical history included hypertension and hepatitis A. Medication given during hospitalization was not available. Whenever an N-alkylPP was detected, the experiment was repeated with a second batch of human liver microsomes with the same catalogue number.
| |
Results and Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
To achieve our primary goal, namely to develop an in vitro test system for the isolation and detection of N-alkylPPs, which may be produced in the human liver after administration of xenobiotics, our first studies were directed to establishing a sensitive technique for detecting N-alkylPP formation.
Isolation and Detection of N-alkylPPs Formed in Rat Liver Microsomes. The lower limit of detection (LLD) of N-alkylPPs by UV-visible spectrophotometry and fluorometry was determined, using known amounts of N-alkylPP in dichloromethane. The LLD was determined by setting the criterion that the peak heights were at least 1.5 times greater than that of the noise level. The fluorometer cuvettes required 2 ml of solution as compared with the spectrophotometer cuvettes, which required 1 ml. The LLD for UV-visible spectrophotometry was found to be 0.16 nmol/ml of N-alkylPP, whereas fluorometry detected as little as 0.04 nmol/2 ml of N-alkylPP. Therefore, it was concluded that fluorometry has greater sensitivity than UV-visible spectrophotometry for detection of N-alkylPP.
To determine which of four methods was the most sensitive, rat liver microsomal suspensions were incubated with NADPH and TTMS for 30 min at 37°C, and N-vinylPP formation was quantified by each of the four methods. The first method explored (UV-visible spectrophotometry without N-alkylPP isolation by TLC) could not be relied upon for N-vinylPP quantitation because the mixture, prior to TLC, still contained impurities with absorbencies that masked the characteristic spectrum of N-vinylPP. On the other hand, fluorometry without TLC could be relied upon to quantitate N-vinylPPs in this mixture, prior to TLC. Using the Soret peak of 432 nm as the excitation wavelength, the N-vinylPP emission spectrum displayed two peaks at approximately 660 and 720 nm, which were not subject to interference by other components in the mixture. While TLC could separate N-alkylPPs from interfering components in the mixture, the procedure results in a 60 to 70% loss of N-alkylPP, and we concluded that since fluorometry could be used without losses incurred by TLC, fluorometry alone was the most sensitive method for the detection of N-alkylPPs formed in vitro. To determine whether the isolation and detection of N-alkylPPs from in vitro preparations containing human hepatic P450 enzymes was feasible for future studies, we determined the minimum amount of P450 required for the detection of N-alkylPP using fluorometry without TLC. We determined the lowest amount of P450 required to produce a detectable amount of N-vinylPP by using the LLD for fluorometry (0.04 nmol/2 ml) and interpolating the value from the graph. TTMS was able to elicit detectable N-vinylPP formation from rat hepatic liver microsomes prepared from dexamethasone-treated liver (0.01 g), containing 0.2 nmol of total P450 (Fig. 1). For N-ethylPP and N-AIAPP detection, after the administration of 4-ethyl DDC and AIA, respectively, we required rat liver microsomes containing approximately 0.5 and 1.0 nmol of total P450. Since, commercially available microsomal products (Gentest Supersomes) contain 0.5 to 1.0 nmol human P450 enzyme, we concluded that these in vitro preparations contained sufficient P450 for use in detecting N-alkylPP formation following mechanism-based inactivation by porphyrinogenic xenobiotics.
|
Isolation and Detection of N-alkylPP from Microsomes
Prepared from Baculovirus-Infected Insect Cells Possessing Single
cDNA-Expressed P450 Enzymes.
The most abundantly expressed P450 enzymes of the human liver are 1A2,
2C9, 2D6, and 3A4, and these enzymes are responsible for the
biotransformation of most drugs that undergo P450-mediated metabolism
in the human liver (Guengerich, 1995). Mechanism-based inactivation of
P450 can involve a number of different pathways. However, with respect
to hepatic porphyria, only the inactivation of those P450 enzymes which
undergo N-alkylation are relevant. The formation of
N-vinylPP, N-ethylPP, and N-AIAPP all
lead to porphyrin accumulation. Therefore, our first objective was to identify the specific human P450 enzymes responsible for
N-alkylPP formation after interaction with TTMS, 4-ethyl
DDC, and AIA.
). Since that time, microsomes from baculovirus-infected insect cells possessing single cDNA-expressed P450 enzymes (Supersomes) became available. We elected to use the microsomes from the
baculovirus-infected insect cells because commercial preparations
contained considerably higher amounts of P450 than the microsomes from
the lymphoblastoid cells, thus enhancing the potential for formation of
N-alkylPP from the heme moiety of P450. We believed that the
change from a lymphoblastoid cell preparation to a baculovirus-infected
insect cell preparation was justifiable since our major goal was to
detect N-alkylPP formation in an in vitro system. We
recognize that differences would be anticipated between these two
systems for a variety of reasons (e.g., a difference in the P450 to
P450 reductase ratio). Thus, microsomes prepared from
baculovirus-infected insect cells (Supersomes) possessing single
cDNA-expressed human P450 enzymes (1A2, 2C9, 2D6, or 3A4) were
incubated with TTMS, 4-ethyl DDC, and AIA, plus NADPH, and
N-alkylPP formation was quantitated using fluorometry.
Controls included both the omission of NADPH or porphyrinogenic xenobiotic from each of the four different human P450s.
When TTMS (0.5 mM), 4-ethyl DDC (1.0 mM), or AIA (1.0 or 10 mM) were
incubated in vitro with Supersomes possessing cDNA-expressed human
CYP1A2, only 4-ethyl DDC elicited N-alkylPP formation in an
NADPH-dependent manner (Figs. 2 and
3). The average ratio of N-ethylPP formation to the amount of CYP1A2 present in
Supersomes (nmol/nmol) was found to be 0.60 (ratio of first trial = 0.94 and ratio of second trial = 0.25). The above results
indicate that N-ethylPP formation occurred concurrently with
mechanism-based inactivation of CYP1A2 in the microsomes from
baculovirus-infected insect cells. The fact that neither TTMS nor AIA
elicited N-alkylPP formation indicates that neither TTMS nor
AIA caused mechanism-based inactivation of CYP1A2, or if they do, then
mechanism-based inactivation takes place by a pathway other than
N-alkylation of one of the pyrrole rings. These results
correspond with results obtained by Wong et al. (1998, 1999a) using rat
liver microsomes from -naphthoflavone-pretreated rats in which
4-ethyl DDC but not TTMS or AIA yielded N-alkylPP formation
from CYP1A1/2 (Table 1).
|
|
|
), that
mechanism-based inactivation of rat CYP3A2 by 4-ethyl DDC was found to
involve fragmentation of the heme moiety to reactive metabolites that irreversibly bind to the P450 apoprotein. It is of interest to compare
the above results with those obtained by Wong et al. (1998, 1999a)
using rat liver microsomes. Thus, TTMS but not 4-ethyl DDC yielded
N-alkylPP formation from rat CYP3A2. On the other hand,
results obtained for AIA with CYP3A4 did not correspond with those of
Wong et al. (1998, 1999a), who did not detect N-alkylPP formation with rat CYP3A2 in liver microsomes (Table 1). This finding
suggests that there are sufficient differences between the active sites
of CYP3A4 and 3A2 to result in different pathways for biotransformation
of AIA. An alternative explanation is the difference in the P450 to
P450 reductase ratio between the two P450 enzymes, one of which is
present in Supersomes and the other in rat microsomes.
A comparison of N-alkylPP formation between Supersomes
containing single cDNA-expressed human P450 enzymes (1A2, 2C9, or 3A4) and rat liver (Wong et al., 1998, 1999a) after treatment with TTMS,
4-ethyl DDC, and AIA can be found in Table 1. The results obtained with
human CYP1A2 correspond with that of rat CYP1A2. The results obtained
with TTMS and 4-ethyl DDC with human CYP3A4 correspond with those
obtained with rat CYP3A2, whereas the results with AIA do not
correspond. The results obtained with TTMS, AIA, and 4-ethyl DDC with
human 2C9 correspond with those observed with rat CYP2C6 for all three
xenobiotics. On the other hand, the results obtained with 4-ethyl DDC
and AIA with human CYP2C9 correspond with those obtained with rat
CYP2C11, but this is not the case with TTMS. The differences observed
between rat and human P450 enzyme orthologs indicate that it would be
useful when dealing with xenobiotics in which porphyrinogenicity
depends upon interaction with P450 enzymes accompanied by
N-alkylPP formation to supplement animal data with studies
using cDNA-expressed individual human P450 enzyme preparations.
The advantage of using expression systems in our studies was that the
effect of TTMS, 4-ethyl DDC, and AIA on individual P450 enzymes could
be determined, without interference by other P450 enzymes.
Nevertheless, results obtained with these expression systems may not
reflect the overall in vivo effects of these compounds, which is
dependent on the specific activity and relative abundance of each P450
enzyme. Moreover, the phospholipid environment, lipid to protein ratio,
and NADPH-P450 reductase to P450 ratio can be markedly different
between native microsomal systems and cDNA-expressed single P450 enzyme
systems (Rodrigues, 1999). Therefore, our second objective was to
determine whether N-alkylPP formation could be detected in
human liver microsomes after interaction with xenobiotics in vitro.
When TTMS (0.5 mM) or AIA (10 mM) were incubated with human liver
microsomes possessing elevated CYP3A4 (HG112), both TTMS and AIA
elicited N-alkylPP formation in an NADPH-dependent manner (Figs. 4 and
5). The amount of N-vinylPP
formation from TTMS and N-AIAPP formation from AIA was found
to be 0.83 nmol (trial one = 0.91 nmol, trial two = 0.84 nmol, and trial three = 0.73 nmol) and 0.38 nmol (trial one = 0.48 nmol and trial two = 0.28 nmol), respectively. In view of the
fact that we obtained both N-vinylPP and N-AIAPP
formation from Supersomes possessing only CYP3A4, these results were
anticipated. When 4-ethyl DDC or AIA was incubated with human liver
microsomes possessing elevated CYP1A2 and 2C9 (HG56), only AIA elicited
N-alkylPP formation in an NADPH-dependent manner (Figs. 4
and 5). The amount of N-AIAPP formation was found to be 0.37 nmol (trial one = 0.42 nmol and trial two = 0.32 nmol). Given
the fact that AIA elicited N-alkylPP formation from
Supersomes possessing CYP2C9, these results were anticipated. However,
the absence of N-ethylPP formation after the interaction of
4-ethyl DDC with human liver microsomes (HG56) was not anticipated,
since N-ethylPP formation was observed with Supersomes
containing either CYP1A2 or 2C9. A possible explanation for this result
is the following: although these human liver microsomes (HG56) contain
elevated CYP1A2 and 2C9, they also contain 0.62 nmol or 12.5% CYP3A4.
Moreover, 4-ethyl DDC caused mechanism-based inactivation of rat
CYP3A2, which is not accompanied by N-alkylPP formation
(Correia et al., 1987). Thus the major portion of 4-ethyl DDC may
undergo biotransformation by CYP3A4 diverting the xenobiotic from
CYP1A2 and CYP2C9.
|
|
).
| |
Footnotes |
|---|
Received September 18, 2001; accepted March 19, 2002.
Supported by the Canadian Institutes of Health Research.
2
The N-alkyl moiety is derived
from AIA by addition of a hydroxyl group to the internal carbon of the
allyl group and a porphyrin nitrogen to the terminal carbon of the
allyl group. The N-alkyl moiety is converted in a
secondary reaction into a lactone by reaction of the hydroxyl with an
amide group (Ortiz de Montellano et al., 1984).
Address correspondence to: Gerald S. Marks, Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario, Canada, K7L 3N6. E-mail: gsm{at}post.queensu.ca
| |
Abbreviations |
|---|
Abbreviations used are: TTMS, 3-[(arylthio)-ethyl]sydnone; 4-ethyl DDC, 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine; AIA, allylisopropylacetamide; P450, cytochrome P450; N-alkylPP, N-alkylprotoporphyrin; N-AIAPP, N-AIA protoporphyrin; N-vinylPP, N-vinylprotoporphyrin; N-ethylPP, N-ethylprotoporphyrin; TLC, thin layer chromatography; LLD, lower limit of detection.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  G. P. Black, K. S. Collins, D. P. Blacquiere, and P.-G. Forkert FORMATION OF N-ALKYLPROTOPORPHYRIN IX FROM METABOLISM OF DIALLYL SULFONE IN LUNG AND LIVER Drug Metab. Dispos., June 1, 2006; 34(6): 895 - 900. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Gamble, K. Nakatsu, and G. S. Marks Comparison of the Formation of N-Alkylprotoporphyrin IX after Interaction of Porphyrinogenic Xenobiotics with Single cDNA-Expressed Human P450 Enzymes in Microsomes Prepared from Baculovirus-Infected Insect Cells and Human Lymphoblastoid Cell Lines+ Drug Metab. Dispos., February 1, 2003; 31(2): 202 - 205. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
