Effects of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by human cytochrome P450 enzymes

Article abstract of DOI:10.1080/004982599238632

1. Effects of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by 12 recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes have been investigated. 2. Arachidonic acid (50 μM) significantly inhibited CYP1A1- and 1A2-dependent 7-ethoxycoumarin O-deethylations, CYP2C8-dependent taxol 6α-hydroxylation and CYP2C19-dependent R-warfarin 7-hydroxylation. This chemical also inhibited slightly the xenobiotic oxidations catalysed by CYP1B1, 2B6, 2C9, 2D6, 2E1 and 3A4 in recombinant enzyme systems. 3. Retinol, retinoic acid and cholecalciferol were strong inhibitors for xenobiotic oxidations catalysed by recombinant CYP1A1, 2C8 and 2C19. 4. Dixon plots of inhibitions of CYP1A1-, 1A2-, 2C8- and 2C19-dependent xenobiotic oxidations by arachidonic acid, of CYP1A1-, 2B6- and 2C19-dependent activities by retinol, and of CYP1A1- and 2C19-dependent activities by cholecalciferol indicated that these chemicals inhibit P450 activities mainly through a competitive mechanism. 5. In human liver microsomes, arachidonic acid inhibited CYP1A2-dependent theophylline hydroxylation, CYP2C8-dependent taxol 6α-hydroxylation and CYP2C19-dependent omeprazole 5-hydroxylation. Taxol 6α-hydroxylation was also inhibited by retinol and retinoic acid, and omeprazole 5-hydroxylation was inhibited by retinol in human liver microsomes. 6. These results suggest that xenobiotic oxidations by P450 enzymes are affected by endobiotic chemicals and that the endobiotic-xenobiotic interactions as well as drug-drug interactions may be of great importance when understanding the basis for pharmacological and toxicological actions of a number of xenobiotic chemicals.

Full text of DOI:10.1080/004982599238632

xenobiotica , 1999, vol . 29, no. 3, 231±241
Eåects of arachidonic acid, prostaglandins, retinol,
retinoic acid and cholecalciferol on xenobiotic
oxidations catalysed by hum an cytochrom e P450
enzymes
H. YAMAZAKI and T. SHIMADA*
Osaka Prefectural Institute of Public Health, 3±69 Nakamichi 1-chome, Higashinari-ku,
Osaka 537, Japan
Received 25 August 1998
1. Eåects of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol
on xenobiotic oxidations catalysed by 12 recombinant human cytochrome P450 (P450 or
CYP) enzymes and by human liver microsomes have been investigated.
lm
2. Arachidonic acid (50
) signi®cantly inhibited CYP1A1- and 1A2-dependent
a
7-ethoxycoumarin O-deethylations, CYP2C8-dependent taxol 6 -hydroxylation and
CYP2C19-dependent R-warfarin 7-hydroxylation. This chemical also inhibited slightly
the xenobiotic oxidations catalysed by CYP1B1, 2B6, 2C9, 2D6, 2E1 and 3A4 in
recombinant enzyme systems.
3. Retinol, retinoic acid and cholecalciferol were strong inhibitors for xenobiotic
oxidations catalysed by recombinant CYP1A1, 2C8 and 2C19.
4. Dixon plots of inhibitions of CYP1A1-, 1A2-, 2C8- and 2C19-dependent xenobiotic
oxidations by arachidonic acid, of CYP1A1-, 2B6- and 2C19-dependent activities by
retinol, and of CYP1A1- and 2C19-dependent activities by cholecalciferol indicated that
these chemicals inhibit P450 activities mainly through a competitive mechanism.
5. In human liver microsomes, arachidonic acid inhibited CYP1A2-dependent
a
theophylline hydroxylation, CYP2C8-dependent taxol 6 -hydroxylation and CYP2C19-
a
dependent omeprazole 5-hydroxylation. Taxol 6 -hydroxylation was also inhibited by
retinol and retinoic acid, and omeprazole 5-hydroxylation was inhibited by retinol in
human liver microsomes.
6. These results suggest that xenobiotic oxidations by P450 enzymes are aåected by
endobiotic chemicals and that the endobiotic-xenobiotic interactions as well as drug-drug
interactions may be of great importance when understanding the basis for pharmacological
and toxicological actions of a number of xenobiotic chemicals.
Introduction
Cytochrome P450 (P450 or CYP) consists of a superfamily of enzymes that
catalyse the oxidation of many xenobiotic chemicals such as drugs, toxic chemicals
and carcinogens as well as endobiotic chemicals including steroids, fatty acids,
prostaglandins and lipid-soluble vitamins (Guengerich and Shimada 1991, Guen-
gerich 1995, Nelson et al. 1996). Xenobiotic oxidations have been shown to be
catalysed mainly by P450 enzymes belonging to the families of CYP1, 2 and 3 in
mammals (Nebert et al. 1989) and these P450 enzymes have also been reported as
having a role in the metabolism of endobiotic chemicals (Guengerich and Shimada
1991). Endobiotics reported to be catalysed by these P450 forms in man include
testosterone and progesterone (6b- and 2b-hydroxylations) by CYP3A4, oestradiol
and oestrone (2-hydroxylations) by CYP1A2 and CYP3A4, oestradiol (4-hy-
droxylation) by CYP1B1, retinol and arachidonic acid (oxidations) by CYP2C8 and
2C9 (Daikh et al. 1994, Zeldin et al. 1995, 1996b), arachidonic acid (epoxidation) by
* Author for correspondence.
Xenobiotica ISSN 0049±8254 print}ISSN 1366±5928 online ’ 1999 Taylor & Francis Ltd
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232
H. Yamazaki and T. Shimada
CYP2J (Zeldin et al. 1996a, 1997), arachidonic acid (oxidations) by CYP1A2, 2C8,
2C9 and 2E1 (Rifkind et al. 1995), and dehydroepiandrosterone 3-sulphate (16a-
hydroxylation) by CYP3A7 (Kitada et al. 1987).
We have recently shown that CYP2C19 (and also CYP2C9 at slower rates)
catalyses progesterone to form 21-, 16a- and 17a-hydroxyprogesterones and
testosterone to form androstenedione in human liver microsomes (Yamazaki and
Shimada 1997b). The conclusions reached are based on the initial ®ndings that
progesterone and other steroid chemicals strongly inhibit the R-warfarin 7-
hydroxylation by CYP2C19 and S-warfarin 7-hydroxylation by CYP2C9 (Yamazaki
and Shimada 1997b).
The present study was undertaken to determine if arachidonic acid, prostag-
landins, retinol, retinoic acid and cholecalciferol inhibit P450-dependent xenobiotic
oxidations in man because these chemicals are oxidized by several forms of human
P450 enzymes (Daikh et al. 1994, Rifkind et al. 1995, Zeldin et al. 1995, 1996a, b,
1997). We used recombinant human P450 enzymes including CYP1A1, 1A2, 1B1,
2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 4A11 and also human liver
microsomes. All of the recombinant P450s were obtained from the baculovirus
expression systems that co-express NADPH-P450 reductase (Yamazaki et al. 1998).
Substrates used for the determination of P450 catalytic activities included
theophylline, 7-ethoxycoumarin, coumarin, taxol, S- and R-warfarin, omeprazole,
bufuralol, nifedipine, and lauric acid.
M aterials and m ethods
Chemicals
Arachidonic acid, retinol, retinoic acid, cholecalciferol and lauric acid were purchased from Sigma
Chemical Co. (St Louis, MO, USA). Prostaglandin E1, E2, A1, A2, B1 and B2 and 20-hydroxy-
prostaglandin E1 and E2 were kindly donated by Ono Pharmaceutical Co. (Osaka, Japan). Other
chemicals and reagents used in this study were obtained from sources described previously or were of
highest quality commercially available (Shimada et al. 1997, Yamazaki and Shimada 1997b).
Enzymes preparations
Recombinant CYP2C9, 2C19, 2D6 and 3A4 expressed in microsomes of Trichoplusia ni cells infected
with a baculovirus containing human P450 and rabbit NADPH-P450 reductase cDNA inserts were
prepared as previously described (Shaw et al. 1997, Yamazaki et al. 1997). Other recombinant P450
enzymes including CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2D6 and 2E1 in baculovirus system co-
expressing human NADPH-P450 reductase were obtained from Gentest Co. (Woburn, MA, USA); the
P450 contents in these latter systems were used as described in the data sheets provided by the
manufacturer.
Human liver samples were obtained from organ donors or patients undergoing liver resection as
m
described previously (Shimada et al. 1994). Liver microsomes were suspended in 10 m Tris-Cl buåer
m
(pH 7.4) containing 1.0 m EDTA and 20 % glycerol (v v) (Guengerich 1994).
}
Enzyme assays
P450-dependent drug oxidation activities were determined by the HPLC methods as described
previously (Shimada et al. 1994, 1997, Yamazaki and Shimada 1997a, b, Yamazaki et al. 1998). Brie¯y,
standard incubation mixtures (®nal volume of 0.20 ml) for theophylline 8-hydroxylation by CYP1A2 and
m
bufuralol 1´-hydroxylation by CYP2D6 contained P450 (5 pmol) in 100 m potassium phosphate buåer
m
(pH 7.4) (25 m when CYP2D6 was used) containing an NADPH-generating system (0.10 mol of
mol glucose 6-phosphate and 0.05 units glucose 6-phosphate dehydrogenase) and
l
NADP+,
1
l
theophylline (100 nmol) or bufuralol (40 nmol). Product formation was determined by HPLC with a
l
5
m C analytical column (4.63 150 mm, Kanto Chemical, Tokyo, Japan). 7-Hydroxylation of R- and
S-warfarin (both ®nal incubation volumes of 0.20 ml) were determined in systems containing CYP2C9
")
m
or 2C19 and in some instances CYP1A1 and 1A2 (5 pmol) in 50 m potassium phosphate buåer (pH 7.4)
containing the NADPH-generating system and R- or S-warfarin (20 nmol). The separation of
l
hydroxylated warfarin metabolites by HPLC was carried out with a 5- m C analytical column.
")
The incubation mixture (®nal volume of 0.20 ml) for 7-ethoxycoumarin O-deethylation by CYP1A1,

Eåects of endobiotics on xenobiotic oxidations by human P450s
233
m
1A2, 2B6 and 2E1 and for coumarin 7-hydroxylation by CYP2A6 consisted of P450 (5 pmol) in 100 m
potassium phosphate buåer (pH 7.4) containing the NADPH-generating system and 7-ethoxycoumarin
l
(20 nmol) or coumarin (20 nmol). Product formation was determined by HPLC using a 5 m C
")
analytical column (unpublished data).
Nifedipine oxidation by CYP3A4, taxol 6 -hydroxylation by CYP2C8 and lauric acid
hydroxylation by CYP4A11 were determined as described (Tanaka et al. 1990, Dirven et al. 1991).
a
x
-
Kinetic parameters for substrate oxidation by recombinant P450 enzymes and human liver
microsomes were estimated using a computer program (KaleidaGraph program, Synergy Software,
Reading, PA, USA) designed for nonlinear regression analysis.
Other assays
Concentrations of P450 and protein were estimated spectrally by the methods of Omura and Sato
(1964) and Lowry et al. (1951) respectively.
Results
Eåects of several endobiotic chemicals on xenobiotic oxidations by recombinant human
P450 enzymes
Eåects of 50 lm concentration of each of arachidonic acid, prostaglandin E2,
retinol, retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by 12
forms of cDNA-expressed human P450 enzymes were investigated (®gure 1).
Arachidonic acid signi®cantly inhibited the CYP1A1- and 1A2-dependent
7-ethoxycoumarin O-deethylations, 2C8-dependent taxol 6a-hydroxylation and
2C19-dependent R-warfarin 7-hydroxylation. This chemical also moderately
suppressed the CYP2B6-dependent 7-ethoxycoumarin O-deethylation, CYP2C9-
dependent S-warfarin 7-hydroxylation, CYP2D6-dependent bufuralol 1 -hydrox-
´
ylation, CYP2E1-dependent 7-ethoxycoumarin O-deethylation and CYP3A4-
dependent nifedipine oxidation.
Prostaglandin E2 did not strongly inhibit the xenobiotic oxidations catalysed by
recombinant P450 enzymes except that it aåected slightly the activities of CYP2B6-
dependent 7-ethoxycoumarin O-deethylation and CYP2C9-dependent S-warfarin
7-hydroxylation. We also determined the eåects of several prostaglandin derivatives
including 20-hydroxyprostaglandin E2, prostaglandin E1 and its 20-hydroxy-
derivative, prostaglandins A1 and A2, and prostaglandins B1 and B2 as well as
prostaglandin E2 on CYP2C9-dependent S-warfarin 7-hydroxylation and found
that these derivatives did not inhibit signi®cantly the CYP2C9-catalytic activities
(data not shown).
Retinol strongly inhibited xenobiotic oxidations catalysed by CYP1A1, 2B6,
2C8, 2C9 and 2C19. Retinoic acid was also observed to be an inhibitor of CYP1A1-,
2B6-, 2C8- and 2C19-dependent xenobiotic oxidations. Cholecalciferol was a
strong inhibitor for taxol 6a-hydroxylation by CYP2C8 and also inhibited the
activities catalysed by CYP1A1 and 2C19.
Nature of inhibition of CYP1A1- and 1A2-dependent xenobiotic oxidations by
arachidonic acid, retinol and cholecalciferol
Analysis (Dixon plots) of the inhibition of CYP1A1-dependent R-warfarin
7-hydroxylation by arachidonic acid, retinol and cholecalciferol, and of CYP1A2-
dependent R-warfarin 7-hydroxylation by arachidonic acid were carried out in
recombinant enzyme systems (®gure 2). CYP1A1-dependent R-warfarin 7-hy-
droxylation was competitively inhibited by arachidonic acid, retinol and chole-
C
calciferol having Ki 10±20 lm .
CYP1A2-dependent R-warfarin 7-hydroxylation was also competitively inhi-

234
H. Yamazaki and T. Shimada
lm
Figure 1. Eåects of several chemicals (50 ) on xenobiotic oxidation activities catalysed by
recombinant CYP1A1 (A), 1A2 (B), 1B1 (C), 2A6 (D), 2B6 (E), 2C8 (F), 2C9 (G), 2C19 (H), 2D6
(I), 2E1 (J), 3A4 (K) and 4A11 (L). Activities determined were 7-ethoxycoumarin O-deethylation
a
(A±C, E, J), coumarin 7-hydroxylation (D), taxol 6 -hydroxylation (F), S- and R-warfarin
7-hydroxylation (G and H respectively), bufuralol 1´-hydroxylation (I), nifedipine oxidation (K)
x
and lauric acid -hydroxylation (L). Substrate concentrations used were 100
P450 concentrations used were 1 pmol 200 l incubation mixtures except that 5 pmol 250
lm
in all cases and
l
l
l
}
}
incubations were used for CYP1B1, 2C8 and 3A4. Control activities (nmol products formed
min nmol P450) without chemicals were 29.7 for CYP1A1, 2.1 for CYP1A2, 0.02 for CYP1B1,
}
}
6.2 for CYP2A6, 1.2 for CYP2B6, 0.91 for CYP2C8, 0.22 for CYP2C9, 0.06 for CYP2C19, 7.5
for CYP2D6, 2.9 for CYP2E1, 29.4 for CYP3A4 and 35.3 for CYP4A11. Data are the average of
duplicate determinations.
Figure 2. Dixon plots of the eåects of arachidonic acid (A), retinol (B) and cholecalciferal (C) on R-
warfarin 7-hydroxylation catalysed by recombinant CYP1A1 and of the eåects of arachidonic acid
(D) on R-warfarin 7-hydroxylation catalysed by recombinant CYP1A2. R-warfarin was added at
lm
lm
(E ) in all cases. Data are the average of duplicate determinations.
20
(D ) and 100

Eåects of endobiotics on xenobiotic oxidations by human P450s
235
Figure 3. HPLC pro®les of the eåects of arachidonic acid on theophylline hydroxylation by human
liver microsomes (HL-19). Theophylline hydroxylation was determined in the absence (A) or
lm
presence (B) of arachidonic acid (at 10
absence of arachidonic acid were 3.3 for N1-demethylation (a), 4.7 for N3-demethylation (b) and
). Control activities (pmol min nmol P450) in the
}
}
l
6.0 for 8-hydroxylation (c). Liver microsomal P450 concentration used was 350 pmol 250 l and
}
incubations were carried out at 37ÊC for 30 min. None of the matabolites was formed when the
NADPH-generated system was omitted from the incubation mixture.
a
Figure 4. Taxol 6 -hydroxylation activities by recombinant human P450 enzymes. Data are average of
duplicate determinations.
a
Figure 5. Dixon plots of the eåects of arachidonic acid on taxol 6 -hydroxylation catalysed by
lm
lm
(E ). Data are
recombinant CYP2C8. Substrate concentrations used were 20
the average of duplicate determinations.
(D ) and 100

236
H. Yamazaki and T. Shimada
a
Figure 6. Taxol 6 -hydroxylation by recombinant CYP2C8 (A) and human liver microsomes (HL-19)
(B) and the eåects of arachidonic acid (C), retinol (D), retinoic acid (E) and cholecalciferol (F) on
a
liver microsomal taxol 6 -hydroxylation activities. These chemicals and substrate were added at
l
concentration to the reaction mixture. P450 concentrations were 5 pmol 250 l for
lm
CYP2C8 and 125 pmol ml for liver microsomes. Control activities for taxol 6 -hydroxylation
10
}
a
}
(nmol min nmol P450) in the absence of chemicals were 0.91 for CYP2C8 and 0.034 for liver
a
microsomes. 6 -, 6 -Hydroxytaxol. None of the metabolites was formed when the NADPH-
}
}
a
generating system was omitted from the incubation mixture.
bited by arachidonic acid in recombinant enzyme system (®gure 2D). To determine
if liver microsomal CYP1A2-catalytic activities are aåected by arachidonic acid, the
eåects of this chemical on the hydroxylation of theophylline, one of the marker
activities for CYP1A2 (Tjia et al. 1996, Shimada et al. 1997), were examined (®gure
3). Arachidonic acid extensively inhibited the N1- and N3-demethylation and
8-hydroxylation of theophylline in human liver microsomes.
Inhibition of CYP2C8-dependent taxol 6a-hydroxylation by endobiotic chemicals
It has previously been reported that CYP2C8 is the principal enzyme catalysing
taxol 6a-hydroxylation in man (Rahman et al. 1994). Other studies have, however,
shown that CYP3A4 5 and other P450 enzymes as well as CYP2C8 catalyse taxol
}
hydroxylation by human liver microsomes (Cresteil et al. 1994, Kumar et al. 1994,
Sonnichsen et al. 1995, Royer et al. 1996). Of the 12 forms of recombinant human
P450 enzymes examined in this study, only CYP2C8 was found to catalyse taxol 6a-
hydroxylation (®gure 4). CYP2C19 only hydroxylated taxol at a very low rate and
other forms including CYP1A1, 1A2, 1B1, 2A6, 2C9, 2C18, 2D6, 2E1, 3A4, 3A5
and 4A11 did not catalyse taxol hydroxylation at detectable rates.
Inhibition of CYP2C8-dependent taxol 6a-hydroxylation was of a competitive
C
nature and the Ki determined was
7 lm in the recombinant enzyme system
(®gure 5).
Human liver microsomal taxol 6a-hydroxylation activities were inhibited by
arachidonic acid, retinol and retinoic acid (®gure 6).
Nature of inhibition of CYP2C9- and CYP2C19-dependent xenobiotic oxidations in
recombinant and human liver microsomal P450 systems
R-warfarin 7-hydroxylation by recombinant CYP2C19 was inhibited by ara-
chidonic acid and retinol mainly by a competitive mechanism (®gure 7). The Ki
!
determined in these cases were 10 lm.

Eåects of endobiotics on xenobiotic oxidations by human P450s
237
Figure 7. Dixon plots of the eåects of arachidonic acid (A) and retinol (B) on R-warfarin
7-hydroxylation catalysed by recombinant CYP2C19. Substrate concentrations used were
lm
lm
(E ). Data are the average of duplicate determinations.
20
(D ) and 100
Table 1. Eåects of arachidonic acid and retinol on S-warfarin 7-hydroxylation and omeprazole
5-hydroxylation catalysed by human liver microsomes (HL-19).
S-warfarin 7-hydroxylation
Omeprazole 5-hydroxylation
(pmol min mg protein) (%)
lm
Œ
Œ
Chemical
(pmol min mg protein)
}
(%)
}
}
}
None
Arachidonic acid
Retinol
±
2.8‰ 0.3
2.2‰ 0.4
2.4‰ 0.3
(100)
(79)
(86)
220‰ 25
112‰ 18*
105‰ 25*
(100)
(51)
(48)
10
10
!
Values are means ‰ SD of triplicate determinations. *p 0.01.
Œ
Bracket indicates the % of control activity (in the absence of arachidonic acid and retinol).
Two reactions in human liver microsomes, S-warfarin 7-hydroxylation and
omeprazole 5-hydroxylation, were chosen for the determinations of activities of
CYP2C9 and 2C19 respectively (table 1) (Yamazaki and Shimada 1997a, Yamazaki
et al. 1997). Both arachidonic acid and retinol inhibited signi®cantly the CYP2C19-
dependent omeprazole 5-hydroxylation by human liver microsomes. The eåects of
these chemicals on S-warfarin 7-hydroxylation by human liver microsomes were
less marked.
Discussion
Drug-drug interactions associated with P450 enzymes have been shown to be
one of the important factors in causing serious side eåects in man when two or more
drugs are dosed to patients simultaneously (Guengerich 1997). At least two
mechanisms underlying P450-dependent drug-drug interactions have been sug-
gested (Periti et al. 1992, Li et al. 1997). One is induction of particular form(s) of
P450 by a drug causing an increase in the metabolism of co-administered drug(s)
(Li et al. 1997). In this case, the pharmacological and toxicological actions of
co-administered drugs may be altered, depending on the reactivities of the
metabolites that are more inactive or reactive than the parent drugs (Li et al. 1997).
The other mechanism, which is suggested to be more important than the
induction mechanism in P450-associated drug interactions, is selective inhibition of
speci®c form(s) of P450 by a drug, resulting in decreases in the metabolism of co-

238
H. Yamazaki and T. Shimada
administered drugs (Guengerich 1997). There are at least four important mecha-
nisms underlying P450 inhibition by drugs, namely (1) competitive inhibition, (2)
reversible inhibition by nitrogen heterocycles, (3) inhibition through formation of
metabolite intermediate complexes and (4) mechanism-based inhibition (Gibaldi
1993, Halpert 1995). Among these, competitive inhibition has been considered as
the most common in drug-drug interactions in man (Halpert et al. 1994, Halpert
1995). Since some of the P450 enzymes involved in xenobiotic oxidations in
mammals have also been reported to catalyse oxidations of endobiotic chemicals
such as arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol
(Theoharides and Kupfer 1981, Hayashi et al. 1988a, b, Gonzalez 1990, Ryan and
Levin 1990, Tanaka et al. 1990, Guengerich and Shimada 1991, Laethem and Koop
1992, Daikh et al. 1994, Rifkind et al. 1995, Zeldin et al. 1995, 1996a, b, 1997), the
possibility exists that these endobiotic chemicals aåect drug oxidations by acting as
competing substrate for the binding sites of P450 enzymes.
Our current results have shown that arachidonic acid, retinol, retinoic acid and
cholecalciferol inhibited the xenobiotic oxidations catalysed by particular forms of
P450 based on the results obtained from recombinant human P450 enzymes.
Reactions inhibited by these endobiotic chemicals (at 50 lm concentration) in
recombinant P450 systems included the CYP1A1-dependent 7-ethoxycoumarin O-
deethylation by arachidonic acid, retinol, retinoic acid and cholecalciferol, the
CYP1A2-dependent 7-ethoxycoumarin O-deethylations by arachidonic acid, the
CYP2C8-dependent taxol 6a-hydroxylation by arachidonic acid, retinol, retinoic
acid and cholecalciferol, and the CYP2C19-dependent R-warfarin 7-hydroxylation
by arachidonic acid, retinol, retinoic acid and cholecalciferol. Dixon plots of the
inhibition of CYP1A1-, 1A2-, 2C8- and 2C19-dependent xenobiotic oxidations by
arachidonic acid, of CYP1A1-, 2B6- and 2C19-dependent activities by retinol and
of CYP1A1- and 2C19-dependent activities by cholecalciferol support the view that
these chemicals inhibit P450 activities mainly through a competitive mechanism. It
is also found that arachidonic acid, retinol and cholecalciferol inhibited CYP1A1-
C
this connection to note our previous ®ndings that S-mephenytoin 4 -hydroxylation
dependent R-warfarin 7-hydroxylation having Ki
10±20 lm. It is interesting in
´
(by CYP2C19) and S-warfarin 7-hydroxylation (by CYP2C9) are competitively
=
inhibited by progesterone with Ki
9 and 17 lm respectively in human liver
microsomes (Yamazaki and Shimada 1997b).
Human liver microsomal studies support the view that arachidonic acid inhibited
theophylline hydroxylation catalysed by CYP1A2, taxol 6a-hydroxylation catalysed
by CYP2C8 and omeprazole 5-hydroxylation catalysed by CYP2C19. We also
observed that retinol inhibited CYP2C19-supported omeprazole 5-hydroxylation
and arachidonic acid, retinol and retinoic acid inhibited CYP2C8-supported taxol
6a-hydroxylation in human liver microsomes.
It is not known at present whether these endobiotic chemicals really aåect the
biotransformation of xenobiotic chemicals when the latter chemicals are admini-
stered to man in vivo. The levels of the endobiotics in the body, particularly in the
microsomal fractions of several organs including liver, lung, kidney, intestine and
others, may be important in understanding the basis for endobiotic-xenobiotic
interactions in man. However, detailed information on the exact levels of these
endobiotic chemicals in the microsomal membranes are lacking, whereas the plasma
levels of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol
are reported to be in the range 0.1±10 lm (Japanese Biochemical Society 1979).

Eåects of endobiotics on xenobiotic oxidations by human P450s
239
Since the levels of endobiotic chemicals in the microsomal membranes may be
higher than those in the blood, the possibility exists that these endobiotic chemicals
aåect the biotransformation of drugs by inhibiting P450 catalytic activities.
It has previously been reported that CYP2C8 is the principal enzyme in
catalysing taxol 6a-hydroxylation in human liver microsomes (Rahman et al. 1994).
Other studies have, however, shown that CYP3A4 5 and some other P450 enzymes
}
also catalyse taxol hydroxylation by human liver microsomes (Cresteil et al. 1994,
Kumar et al. 1994, Sonnichsen et al. 1995, Royer et al. 1996). Of the 12 forms of
recombinant human P450 enzymes examined in the current study, CYP2C8 had the
highest catalytic activity for taxol 6a-hydroxylation. CYP2C19 had a very low rate
of taxol hydroxylation and other forms including CYP1A1, 1A2, 1B1, 2A6, 2C9,
2C18, 2D6, 2E1, 3A4, 3A5 and 4A11 did not catalyse taxol at signi®cant levels.
These results support the importance of CYP2C8 in the 6a-hydroxylation of taxol
by human liver microsomes, consistent with the results reported recently by
Rahman et al. (1994).
Prostaglandins were not very good inhibitors for xenobiotic oxidations catalysed
by recombinant P450 enzymes, as compared with arachidonic acid, retinol, retinoic
acid and cholecalciferol. Since prostaglandin E2 slightly inhibited the activities of
CYP2B6-dependent 7-ethoxycoumarin O-deethylation and CYP2C9-dependent S-
warfarin 7-hydroxylation, we also determined the eåects of several prostaglandin
derivatives (including 20-hydroxyprostaglandin E2, prostaglandin E1 and its 20-
hydroxy-derivative, prostaglandins A1 and A2, and prostaglandins B1 and B2) on
CYP2C9-dependent S-warfarin 7-hydroxylation. The results suggested again that
these prostaglandin derivatives were not very good inhibitors of CYP2C9-dependent
S-warfarin 7-hydroxylation activity. It should, however, be mentioned that
prostaglandins are rapidly metabolized by several forms of P450 in microsomal
membranes of the liver, lung, kidney and intestine (Vatsis et al. 1982, Kaku et al.
1984, Miyata et al. 1994, Yamamoto et al. 1984, Laethem and Koop 1992, Karara et
al. 1993).
In conclusion, the present results suggest that xenobiotic oxidations by P450
enzymes are aåected by endobiotic chemicals and that endobiotic-xenobiotic
interactions as well as drug-drug interactions may be of great importance in the
understanding of the basis for pharmacological and toxicological actions of a
number of xenobiotic chemicals.
Acknow ledgem ents
This work was supported in part by grants from the Ministry of Education,
Science and Culture of Japan, the Ministry of Health and Welfare of Japan and the
Developmental and Creative Studies from Osaka Prefectural Government.
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