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Center for Pharmaceutical Research, School of Pharmacy and Medical Sciences, University of South Australia, SA, 5000, Australia
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30%. Androstenedione
6ß-hydroxylase activity showed atypical kinetics. For oleuropein
glycoside, hydroxytyrosol and gallic acid the apparent Ki
values were determined to be 80, 77 and 70 µmol/L, respectively.
Analysis of structural features of inhibitory compounds established
that a 3,4-dihydroxyphenyl ethanol structure was required for
inhibition of androstenedione 6ß-hydroxylase for this group of
compounds.
KEY WORDS: • humans • cytochrome P450 • 17ß-hydroxysteroid dehydrogenase • CYP3A
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); however
concentrations of 2.3–9 mg/L of oleuropein and 1.4–5.6 mg/L of
hydroxytyrosol have been reported. (Montedoro et al. 1992
). The possible beneficial effects of these complex phenols
have been examined in a number of studies. It has been suggested that
they may play a role with respect to the inhibition of platelet
aggregation and arachidonic acid metabolism (Petroni et al. 1994
and 1995
) and also in the prevention of
oxidation of LDL (Visioli et al. 1995
).
Hepatic microsomal enzymes, in particular the cytochromes P450
(CYP)2
, are important in the metabolism of xenobiotic and endogenous
substrates. Most xenobiotics, such as drugs, are metabolized and
generally inactivated along pathways that involve different CYP.
However, if only one CYP is involved in the clearance of a drug, then
inhibition will have a major impact on the therapeutic effects of the
drug (reviewed, Murray 1999
). Inhibition of CYP by nonnutrients has
been demonstrated (Guengerich et al. 1994
) potentially
altering therapeutic effects (Kupferschmidt et al. 1995
). Evidence now indicates that there are three CYP3A
subfamily proteins in humans. Of these CYP3A4 and CYP3A5 are expressed
in adult liver. (Nebert et al. 1991
). Androstenedione
6ß- hydroxylase activity is regarded as being a specific marker for
the CYP3A proteins (Waxman et al. 1991
). Aside from the
CYP, there are many other enzymes that participate in xenobiotic and
endobiotic metabolism. The enzyme 17ß- hydroxysteroid dehydrogenase
(17ß-HSD), also known as 17ß-hydroxysteroid oxidoreductase,
controls the important final step in the biosynthesis of androgens and
estrogens. At least five isoenzymes, that exhibit different patterns of
expression in cells, substrate specificity and regulatory mechanisms,
have been described (Labrie et al. 1997
). Reduction
(e.g., androstenedione to testosterone) utilizes NADPH and NADH as
cofactors whereas NADP and NAD are cofactors for the oxidative
(dehydrogenation) pathway.
The present study was undertaken to examine the effects of some complex phenol compounds on androstenedione 6ß-hydroxylase, and both oxidative and reductive 17ß-HSD activity. Inhibition of androstenedione 6ß-hydroxylase, or oxidative or reductive 17ß-HSD activity, could potentially alter the metabolism of xenobiotics or endobiotics by olive oil phenols.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A sample of pooled human liver microsomes was purchased from Human
Biologics International (Scottsdale, AZ). NADP, NADPH, NADH, isocitrate
dehydrogenase, unlabeled androst-4-ene-3,17- dione (androstenedione),
17ß-hydroxy-4-androsten-3-one (testosterone) and 3ß-hydroxysteroid
dehydrogenase, were purchased from the Sigma Chemical (St. Louis, MO).
[4-14C] Androstenedione (sp. act. 2.0 GBq/mmol) and
[4-14C] testosterone (sp. act. 1.9 GBq/mmol) were
purchased from Amersham (Sydney, Australia).
16ß-Hydroxyandrostenedione was prepared enzymatically by the action
of 3ß-hydroxy steroid dehydrogenase on 3ß,
16ß-dihdroxyandrost-5-ene-17-one (Prof. D. N. Kirk and MRC
steroid Reference Collection, Queen Mary’s College, London, United
Kingdom) as described (Sheets and Estabrook 1985
). Other
hydroxylated testosterone and androstenedione standards were obtained
from the MRC Steroid collection or Steraloids, (Wilton, NH). Oleuropein
was purchased from Indofine Chemicals (Somerville, NJ); gallic acid and
p-coumaric acid were purchased from ICN (Aurora, OH);
3,4- dihydroxybenzoic acid was purchased from BDH (Poole, United
Kingdom); 2-(4-hydroxyphenyl) ethanol was purchased from Fluka (Buchs,
Switzerland); 3,4-dimethoxyphenethyl alcohol and
3,4-dihydroxyphenylacetic acid alcohol were purchased from Aldrich
(Milwaukee, WI). Hydroxytyrosol was synthesized by the LiAlH4 reduction
of 3,4-dihydroxyphenylacetic acid (Baraldi et al. 1983
).
All solvents and other miscellaneous chemicals were at least analytical
reagent grade.
Assays.
Microsomal androstenedione hydroxylase and reductive 17ß-HSD activity
were assayed essentially as previously described (Waxman et al. 1983
). For incubations utilizing NADPH, microsomal fractions
were incubated in a 1 mL reaction mixture containing isocitrate (4
mmol/L), isocitrate dehydrogenase (0.4 I.U.), MgCl2 (8
mmol/L), 1 mmol/L NADP and androstenedione (43.75–87.5 µmol/L, 4.8
MBq/mmol) for 10 min at 37°C, after which the incubation mixtures
were extracted twice with ethyl acetate. Testosterone oxidation was
assayed essentially as described for microsomal androstenedione
hydroxylase and reductive 17ß-HSD activity except that testosterone
(4.8 MBq/mmol) was used as substrate. One mmol/L NAD was used in place
of the NADPH-generating system. Inhibitors, where used, were added
in water, and appropriate control incubations were performed
concurrently. Application to, and development of, TLC plates was
carried out as previously described (Stupans and Sansom 1991
). Zones corresponding to hydroxylated androstenedione
standards and to testosterone were visualized under UV light and
scraped into vials for scintillation spectrometry (ACS; Amersham,
Sydney, Australia).
All assay conditions were optimized with respect to time, protein concentration and substrate concentrations to ensure linearity.
Data analysis.
For each experiment, data are expressed as the mean of triplicate
observations. Kinetic parameters were determined using a range of
inhibitor concentrations and substrate concentrations. The method of
Dixon was used to calculate inhibition constants
(Ki) (Dixon and Webb 1979
). As this
method cannot distinguish the type of inhibition, the
Cornish-Bowden method was used (Cornish-Bowden 1974
). Eadie-Hofstee plots were used to visually detect
deviations from linearity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, were examined as inhibitors of androstenedione 6ß-hydroxylase,
reductive and oxidative 17ß-HSD activities. Data for inhibition of
these activities are shown in Table 1
. There was inhibition of androstenedione 6ß-hydroxylase activity by
three of the selected test compounds, i.e., oleuropein glycoside,
hydroxytyrosol and gallic acid. These same compounds, and also
dihydroxybenzoic acid, also inhibited reductive 17ß-HSD activity,
with little inhibition shown by the other test compounds. Oxidative
17ß-HSD activity was not inhibited by any of the compounds tested;
however, gallic acid stimulated activity by 30%.
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shows the Dixon plot obtained for oleuropein glycoside. The apparent
Ki was determined to be 80 µmol/L.
Similarly for hydroxytyrosol and gallic acid, apparent
Ki values of 77 and 70 µmol/L,
respectively, were determined; however the method cannot distinguish
between competitive and mixed-type inhibition (Dixon and Webb 1979
). The Cornish-Bowden plot did not indicate
typical kinetic inhibition (Cornish-Bowden 1974
) (not
shown). The atypical nature was confirmed by Eadie-Hofstee plots
(Fig. 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This assay was therefore used to determine CYP3A
inhibition. In humans, both oxidative 17ß-HSD
C19 (i.e., testosterone to androstenedione) and
reductive 17ß-HSD C19 activity in hepatic
microsomes are principally mediated by 17ß-HSD type 2 (Labrie et al. 1997
).
Inspection of the molecular structure of the test compounds revealed
several interesting results (Table 1)
. Hydroxytyrosol was found to be a
good inhibitor of androstenedione 6ß-hydroxylase and reductive
17ß-HSD activities, whereas a compound which differs only by the
absence of the 3-hydroxy function [2-(4-hydroxyphenyl) ethanol] was
observed to be a poor inhibitor. The test compound 3,4-dihydroxybenzoic
acid was found to be an inhibitor of reductive 17ß-HSD activity
inhibitor and yet was a poor inhibitor of androstenedione
6ß-hydroxylase activity. It differs from hydroxytyrosol only by the
absence of a two-carbon side chain and the presence of a carboxylic
acid function. The addition of another hydroxy group to
3,4-dihydroxybenzoic acid as in gallic acid markedly increased
inhibitory potency with respect to androstenedione 6ß- hydroxylase
activity. The addition of this hydroxy group also changed the compound
from one that had no effect with respect to oxidative 17ß-HSD
activity to one that stimulated activity. The addition of methyl ether
functions to hydroxytyrosol as in 3,4-dimethoxyphenyl ethanol decreased
inhibitory potency. The test compound p-coumaric acid was a
very poor inhibitor. These observations taken together with the
observation that inhibitory potency with respect to androstenedione
6ß-hydroxylase was not markedly different between oleuropein
glycoside and hydroxytyrosol suggests that the presence of a
3,4-dihydroxyphenyl ethanol structure is required for inhibitory
activity of androstenedione 6ß-hydroxylase. It is interesting to note
that olive oil phenol antioxidant activity is also associated
with the requirement for a 3,4-dihydroxyphenyl 2 carbon side chain
structure (de la Puerta et al. 1999
)
The apparent Ki for the inhibition of
androstenedione 6ß-hydroxylase activity by oleuropein glycoside was
found to be 80 µmol/L. This value is similar to that observed for the
inhibition of CYP3A4 in human liver microsomes by another
food-derived compound, tangeretin
(Ki 72 µmol/L) (Obermeier et al. 1995
) but is higher than that reported for naringenin
(Ki 23 µmol/L) (Ubeaud et al. 1999
) or 6',7'-dihydroxybergamottin (25 µmol/L concentration
required to inhibit 6ß-hydroxytestosterone formation by 50%)
(Edwards et al. 1996
). However it is worth pointing out
that at the present stage the compounds actually responsible for the
effects of grapefruit juice are still unidentified (Bailey et al. 1998
).
The atypical kinetics observed for CYP3A activities have been reported
previously (Schwab et al. 1988
), (Stupans and Sansom 1991
) and recently also for other CYP activities
(Ekins et al. 1998
). Interpretation of inhibition
kinetics in these circumstances is inappropriate.
In view of the important role that steroidal hormones have in the
etiology of hormone-dependent diseases, design of inhibitors to
the 17ß-HSD isoenzymes has received attention
(Tremblay and Poirier 1998
). Flavonoids have been
reported to inhibit reductive 17ß-HSD type 1 (Le Bail et al. 1998
), (Makela et al. 1998
) and oxidative type 2
(Makela et al. 1998
). In this study inhibitors were
identified for reductive microsomal human 17ß-HSD type 2 activity,
i.e., androstenedione to testosterone. Potential inhibitors for this
pathway have not been described to date.
This study is the first report of the potential inhibition of
xenobiotic and endobiotic metabolism by complex phenols derived from
olive oil and other related compounds. We have identified oleuropein
glycoside, hydroxytyrosol and gallic acid as inhibitors of CYP3A and
reductive 17ß-HSD activity. The role of food-derived CYP
inhibitors has been highlighted by studies of 6',7'-
dihydroxybergamottin (Edwards et al. 1996
), and of their
therapeutic importance (Kupferschmidt et al. 1995
). The
physiological role of such inhibition is speculative; however, it
deserves further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received December 8, 1999. Revision accepted April 25, 2000.
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