Vol.3, No.6, 795-798 (2012) Agricultural Sciences
Interaction of ergotamine with liver Cytochrome P450
3A in rats
Ali S. Moubarak*, Hehai Wang, Zelpha B. Johnson, Charles F. Rosenkrans
Department of Animal Science, University of Arkansas, Fayetteville, USA; *Corresponding Author: moubarak@uark.edu
Received 12 July 2012; revised 27 August 2012; accepted 15 September 2012
This study was conducted to investigate the
effect of the ergot alkaloid, ergotamine (ET), on
the induction of CYP3A and the interaction in
vivo and in vitro with ET. Sprague-Dawley rats
were treated intraperitoneally for 4 days as fol-
lows: control (injecting with 0.5 ml of only corn
oil); dexamethasone treatment (injecting with
100 mg/kg of dexamethasone in corn oil); and
ergotamine treatment (injecting with 100 mg/kg
of ergotamine in corn oil). Liver tissues were
collected from each group (n = 5, total of 30 rats)
and liver microsomes were prepared. Cyto-
chrome CYP3A activity was evaluated using ET
and its isomer as substrates in medium con-
taining liver microsomes and NADPH at 37˚C for
30 min. HPLC was used to measure the disap-
pearance of the substrate and the appearance of
the metabolites. Liver microsomes from rats
pretreated with dexamethasone were five times
more (P < 0.01) active than microsomes from the
control animals in the biotransformation of ET
(32.1 and 7.0 nM/min/mg protein, respectively;
SE = 4.83) or ET-isomer (21.6 and 4.7 nM/min/mg
protein, respectively; SE = 1.7079) into its cor-
responding ET metabolites. The ergotamine treat-
ment produced no increase (P > 0.05) in activity
of CYP3A when compared to the control group
(5.2 vs 7.0 nM ET/min/mg protein; SE = 4.83) or
ET isomer (1.5 vs 4.7 nM ET isomer/min/mg
protein; SE = 1.70). When ketoconazole was
used as specific inhibitor of CYP3A, ergotamine
metabolisms were inhibited in a dose dependent
fashion reaching a maximum at an inhibitor to
substrate ratio of greater than one and LD50 at
0.5 nM of ketoconazole/mg protein. The data
presented in this study suggest that although
the ergot alkaloids ergotamine and its isomer
are ideal substrates for the isozyme CYP3A,
these compounds have no effect on the induc-
tion of CYP3A after 4 days of treatment.
Keywords: Microsomes; Liver; Rat
Cytochrome P450 (CYP) enzyme systems play a key
role in the biotransformation of many endogenous and
exogenous compounds including both toxins and drugs
[1-4]. The CYP enzyme family consists of a large num-
ber of proteins with different substrate specificities and
catalytic properties which are membrane-bound, mostly
localized to the endoplasmic reticulum and in mitochon-
drial inner membranes. CYP1-3 families are active in the
metabolism of xenobiotics with CYP3A subfamilies be-
ing the most important in drug metabolism [5]. The me-
tabolism of ergot alkaloids, such as bromocriptine, ergo-
tamine and other structurally similar ergot derivatives is
mediated mainly by CYP3A4 [6]. Moochhala et al. [7]
reported that bromocriptine interferes with P450-de-
pendent oxidative metabolism of xenobiotics. Later it
was demonstrated that cytochrome P450 3A exhibits a
particularly high affinity for ergopeptides. The CYP3A
family expression patterns differ based on maturity.
CYP3A4 is mainly expressed in adult liver, CYP3A5 in
extrahepatic tissues, and CYP3A7 in fetal liver [8]. Ac-
tivation or inhibition of the induction process of such
enzyme systems can have severe consequences. Witkamp
et al. [9] reported that tiamulin, a semi-synthetic antibi-
otic frequently used in agricultural animals, strongly in-
hibited the hydroxylation rate of testosterone at the 6
beta-position via the formation of a cytochrome P450
3A4 metabolic intermediate complex in both microsomes
and hepatocytes.
The induction of CYP3A4 is mainly regulated by the
novel orphan receptor or pregnane X receptor (PXR) [10,
11], but other receptors including the constitutively ac-
tive receptor (CAR) and, indirectly, the glucocorticoid
receptor (GR) are involved [12,13]. Dexamethasone, one
of the glucocorticoids, influences several aspects of
CYP3A induction. However, most of these effects are not
dependent on GR binding to CYP genes, but rather on
complex protein-protein interplay between GR and vari-
ous other receptors [14]. Information on how or if ET fits
Copyright © 2012 SciRes. OPEN ACCESS
A. S. Moubarak et al. / Agricultural Sciences 3 (2012) 795-798
into the induction processes of rat CYP3A is limited or
not available. Therefore, this report was designed to
study the effects of administering ET in vivo on the in-
duction of CYP3A family using dexamethasone as a spe-
cific CYP3A inducer for similarity and to evaluate the in
vitro interaction of ET with CYP3A using ketoconazole
as specific inhibitor. Ergotamine and similar ergot alka-
loids are extremely susceptible to photolytic and air oxi-
dation, hydration and isomer formation at the C-8 posi-
tion of the ergolene ring. Therefore, ergotamine isomer
was included in this study.
All the chemicals and reagents used in these experi-
ments were of the highest quality available and were
purchased from Sigma Chemical Co. (St. Louis. Mo)
unless stated otherwise.
Sprague-Dawley rats (n = 30 rats; BW ~250 g) were
allowed ab libitum access to water and chow. Rats were
randomly assigned to treatment and treated intraperito-
neally for 4 consecutive days with one of the following
treatments: 1) Control treatment (n = 5 rats, injections of
0.5 ml of corn oil); 2) DXM treatment (n = 20 rats, injec-
tions of 100 mg/kg dexamethasone in 0.5 ml of corn oil);
and 3) ET treatment (n = 5 rats, injections of 100 mg/kg
Ergotamine in 0.5 ml of corn oil). Approximately 24 h
after the last injection (except in DXM treatment), each
rat was anesthetized with chloroform, decapitated, and
liver harvested. Livers were stored at –20˚C until micro-
somes were prepared. In the DXM treatment, five rats
were removed each day for time dependence evaluation
and livers were harvested in the same manner.
Michaelis-Menten kinetics was used to evaluate the
interaction of CYP3A with ET and the linearity of the
Lineweaver-Burk plot of 1/V versus 1/S was further ex-
amined by measuring enzyme activity at various concen-
trations (2.0 to 20.0 μM) of the enzyme substrate, ET, in
the presence and absence of kentozole (3.0 and 5.0 μM).
Liver microsomes were prepared as reported by Mou-
barak and Rosenkrans [15]. Briefly, liver tissue (3 - 5 g)
were diced with scissors and then washed with 150 mM
sodium chloride buffer. The diced tissue was ground (1 g
tissue/10 ml of buffer (250 mM sucrose, 100 mM Tris-
HCl, 1 mM EDTA, pH 7.4)) with ice-cold medium using
a precooled blender for 10 to 20 sec and further homoge-
nized using a Polter-Elvehjem (5×). The homogenate was
sequentially centrifuged at 800 g for 10 min, at 13,500 g
for 20 min collecting the supernatant and then at 105,000
g for 60 min collecting the pellet which contained the
microsomal fraction. The pellet (microsomal fraction)
was resuspended in buffer containing 100 mM sodium
phosphate and 20% v/v glycerol. The protein concentra-
tion after resuspension was approximately 40 mg/ml.
Aliquots of microsomal suspensions were stored at
–20˚C and were used within 20 to 30 days. Protein con-
centration was determined by either the method of
Lowry [16] using serum albumin as standard or by BCA
Protein Assay (Pierce Chemical kit no. 23225).
Ergotamine (ET) and its isomer (ET_iso) were used as
substrates to assay presumptive CYP3A4 activity in vitro.
Our standard assay methods for the metabolism of ET
were analyzed in 330 μl of assay medium, 100 μl of co-
factor generating system (NADPH), 20 μl of ET (final
concentration was 4 µg/ml of fully isomerized ET), and
50 μl microsomal protein (final concentration was 0.4
mg/ml). The assay medium (pH 7.4) consisted of 100
mM potassium phosphate, 0.1 mM EDTA, and 5.0 mM
MgCl2. The NADPH generating system consisted of
assay medium with 10 mM NADP+, 10 mM D-glucose-
6-phosphate, and 2.0 U/ml of Glucose-6-phosphate de-
hydrogenase. The reactions were initiated by adding the
NADPH generating system and were terminated after 30
min by adding 100 μl of 94% acetonitrile and 6% glacial
acetic acid. After the stop solution was added the mixture
was centrifuged at 12,000 g for 4 min. Supernatant (150
μl) from each reaction was examined for the disappear-
ance of ET and ET_iso and the appearance of their me-
tabolites (M1, M2 from ET and M1_iso and M2_iso
from ET_iso) using the HPLC method described by
Moubarak and Rosenkrans [15].
Induction data for the dexamethasone treated rats were
analyzed using one-way ANOVA with day as the inde-
pendent variables and concentration of remaining sub-
strate or metabolite as the dependent variables. Ergo-
tamine and dexamethasone induction data after 4 days of
treatment were analyzed using one-way ANOVA with
treatment as the independent variable and concentration
of remaining substrate or metabolites as the dependent
variables. The kinetics data of kentoconazole inhibition
was interpreted using the double reciprocal plot method
of 1/S vs 1/V.
Data in Figure 1 represents the disappearance of ET
and its isomer after incubation with liver microsomes
from rats that had been treated sequentially for 1, 2, 3, or
4 days with 100 mg/kg of dexamethasone, a specific in-
ducer of CYP3A. Dexamethasone induced CYP3A activ-
ity in rats to a significant (P < 0.01) level only after the
third and the fourth days of treatments. These results
indicate that the mechanism of DXM induction of rat
liver microsomal CYP3A was time dependent, and it
took at least three to four successive days of treatments
to reach the maximum level of activity. Liver micro-
somes from rats pretreated with dexamethasone were
five times more (P < 0.01) active than microsomes from
Copyright © 2012 SciRes. OPEN ACCESS
A. S. Moubarak et al. / Agricultural Sciences 3 (2012) 795-798 797
Days of treatment
nM converted
ET isomer
Figure 1. The effects of intraperitoneal injections of dexa-
methasone (DXM) on the induction of rat liver CYP3A activity
over four consecutive days of injection. Comparisons were
made within substrate (ETor ET isomer). Bars containing the
same letter were not different significantly (P > 0.05).
the control animals in the biotransformation of ET (32.1
and 7.0 nM/min/mg protein, respectively; SE = 4.83) or
ET-isomer (21.6 and 4.7 nM/min/mg protein, respec-
tively SE = 1.7079) into its corresponding ET metabo-
Ergotamine treatment for 4 consecutive days produced
no significant (P > 0.05; 5.2 vs 7.0 nM ET/min/mg pro-
tein; SE = 4.83 and 1.5 vs 4.7 nM ET isomer/min/mg
protein; SE = 1.70) increase in the CYP3A activity over
that of the control animals (Figure 2). Moubarak et al.
[17] showed that treatment of rats with similar ergot al-
kaloids dihydroergotamine (DHET) or ergonovine (EN)
at a concentration of 100 mM did not produce any sig-
nificant increase in the CYP3A activity over that of con-
trol rats. Although it is generally accepted that most
compounds that are metabolized by CYP’s are to some
degree inducers of that CYP’s enzyme system [10], data
from this study and from a previous study in our labora-
tory have indicated that all of the ergot alkaloids studied
(ergotamine, dihydroergotamine and ergonovine) have
very small or no induction effects on CYP3A in rats, yet
those alkaloids have been shown to be metabolized by rat
CYP3A. Previous studies have demonstrated that both
DXM and ET can also inhibit the in vitro metabolism of
tacrolimus mediated by the CYP3A subfamily [18,19].
Another study showed that dihydroergocryptine, a do-
pamine agonist for the treatment of Parkinson’s disease,
has an inhibitory effect on CYP3A4-mediated testoster-
one metabolism and additionally could induce CYP3A4
and CYP2E1 mRNA when added at 10 μM to cultured
human hepatocytes [20]. Moubarak et al. [17] reported
that both ergonovine and dihydroergotamine inhibited in
vitro CYP3A4 activity in a dose dependent manner when
ET was used as a substrate, producing quadratic inhibi-
tion curves. One can ask, is it possible for a group of
compounds such as the ergot alkaloid group (ET, DHET,
EN, etc.) to play a double role? One role is to be involved
at the upstream induction of CYP3A and another is to
directly interact with the structure of the enzyme inhibit-
ing its catalytic activity. Ergot alkaloids have been dem-
onstrated to affect the cytochrome p450 system, espe-
cially isoenzyme CYP3A4, by binding to the isoenzyme
as a substrate [15]. The linearity of the Lineweaver-Burk
plot of 1/V(Vmax), versus 1/S (ET and ET isomer ) shows
that the interaction between the substrate (ET) and
CYP3A in both the presence and absence of ketocona-
zole (Figure 3) follows Michaelis-Menten kinetics. Fur-
thermore, the fact that the slope did not change with the
addition of ketoconazole, and the decrease in the velocity
of the reaction (Vmax), indicated an uncompetitive inhibi-
tion. Thus ketoconazole interacts with the enzyme-sub-
strate complex.
In this study we have demonstrated that although both
ET and ET isomer are ideal substrates for CYP3A, they
appear to have no induction effect on rat hepatic CYP3A
during the 4 days of treatment when compared to the
classic dexamethasone induction. The data from the in
vitro interaction among ET, CYP3A and its specific in-
hibitor (ketoconazole) showed that the binding site for
Control DXMET
Treatmen t
nM converted
ET isomer
Figure 2. The amount of substrate (ET or ET isomer) remain-
ing after incubation with rat hepatic CYP3A preparation from
control (CON), Dexamethsone (DXM) and Ergotamine (ET)
treated rats. Comparisons between treatments were made within
substrate. Bars containing the same letter were not different
significantly (P > 0.05).
-0.15-0.1 -0.0500.050.10.15
0 uM
3.0 uM
5.0 uM
Figure 3. Lineweaver-Burk plots of rat hepatic CYP 3A4 activ-
ity in 0, 3, and 5 μM of ke-toconazole.
Copyright © 2012 SciRes. OPEN ACCESS
A. S. Moubarak et al. / Agricultural Sciences 3 (2012) 795-798
Copyright © 2012 SciRes.
ET was different from that for ketoconazole in rat hepatic
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