-DMSO +DMSO -ACN +ACN
Metyrapone DCPO DCPT 4-DCTD
Effect of Cyclic Imides on Cytochrome P450 (CYP) Activity in Rat Liver Microsomes.
Kathryn J. McGonegal, Christine M. Crincoli, Niti N. Patel, Peter J. Harvison*. Department of Pharmaceutical Sciences.
Cyclic imide-containing compounds may be inhibitors of cytochrome P450s (CYPs). To
examine this possibility, the following compounds were evaluated for their ability to inhibit
the CYP-mediated ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin (PROD)
activities: 3-(3,5-dichlorophenyl)-2,4 thiazolidinedione (DCPT), 3-(3,5-dichlorophenyl)-2,4
oxazolidinedione (DCPO) and 3-(3,5-dichlorophenyl)-4-thiazolidinone (4-DCTD). Each
compound was incubated (37 °C, pH 7.4) at a concentration of 0.2 mM with rat liver
microsomes (final protein concentration 0.1 mg/ml), and the cofactor NADPH. Reactions
were initiated by the addition of either ethoxyresorufin or pentoxyresorufin (final
concentration 0.005 mM). Resorufin (product) formation was monitored by measuring
fluorescence. Separate incubations were also done using the known CYP inhibitor,
metyrapone, and the vehicle, dimethyl sulfoxide (DMSO). Percent inhibition of EROD
activity was as follows: 31.8% (metyrapone), 55.0% (DCPT), 28.4% (DCPO), and 74.5%
(4-DCTD). The extent of PROD inhibition was 26.7% (metyrapone), 81.1% (DCPT), 31.1%
(DCPO), and 49.2% (4-DCTD). DMSO had little effect on either activity. Our results show
that metyrapone and DCPO inhibited EROD and PROD activity to the same extent. 4-
DCTD was a more potent inhibitor of EROD activity than DCPT. In contrast, DCPT more
effectively inhibited PROD activity than did 4-DCTD. Since EROD and PROD activities may
be catalyzed by different CYP isozymes, we conclude that 4-DCTD and DCPT exhibit
some isozyme selectivity for inhibition.
DCPT DCPO 4-DCTD
Figure 1. Structures of compounds evaluated for cytochrome P450 (CYP) inhibitory activity.
Abbreviations: DCPT, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione; DCPO, 3-(3,5-dichlorophenyl)-
2,4-oxazolidinedione; 4-DCTD, 3-(3,5-dichlorophenyl)-4-thiazolidinone.
Figure 2. Ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD)
Figure 5. Inhibition of EROD and PROD by cyclic
imide-containing compounds and metyrapone.
An asterisk (*) indicates that inhibition of PROD is
significantly different (p < 0.05) from EROD for the
compound. A dagger (†) indicates that inhibition of
EROD by the compound is significantly different (p <
0.05) from metyrapone. A double dagger (‡)
indicates that inhibition of PROD by the compound
is significantly different (p < 0.05) from metyrapone
Ethoxyresorufin O-deethylase (EROD)/Pentoxyresorufin O-depentylase (PROD)
EROD and PROD were measured in microsomes as indices of CYP activities. Incubations were
conducted in 48-well cell culture plates (Costar 3548, Corning, NY). In short, male rat liver
microsomes (2.0 mg/mL, Xenotech, Lenexa, KS) were incubated for 30 minutes at 37°C with a
known (metyrapone) or potential (DCPT, DCPO or 4-DCTD) inhibitor, an NADPH-regenerating
system (120 mM MgCl2
; 200 mM glucose-6-phosphate; 40 mM NADP+
; 2U/50µL glucose-6-
phosphate dehydrogenase) in a 111.1 mM potassium phosphate-containing 1.1 mM EDTA
buffer solution. Inhibitors were added in acetonitrile (ACN) or dimethylsulfoxide (DMSO). Control
experiments were conducted with the solvents to determine if they were producing any
inhibition. After the preincubation period, either 7-ethoxyresorufin (0.5 mM) or 7-
pentoxyresorufin (0.5 mM) were added. The plates were incubated at 37°C with shaking for 30
minutes with a fluorescence reading taken every two minutes. The resorufin concentration was
measured against a standard curve generated using concentrations ranging from 0-1000
pmol/mL. Specific activity was calculated from the amount of resorufin formed over the
incubation period multiplied by the final protein concentration. Fluorescence readings were
taken on a Perkin-Elmer (Wellesley, MA) plate reader (Model # N450-1001) set at excitation
wavelength of 530 nm and emission wavelength of 590nm using HTS 7000 series software.
• The organic solvent acetonitrile inhibited EROD activity by approximately 22%, but had no effect on PROD
activity (Figs. 3 and 4)
• Neither enzyme activity was inhibited by DMSO; therefore this solvent was used in all experiments (Figs. 3
• DCPO, which contains a 2,4-oxazolidinedione ring, inhibited EROD and PROD activity to the same extent
(ca. 30%, Fig 5). Similar results were obtained for the known CYP inhibitor, metyrapone (Fig. 5).
• The sulfur containing compounds, DCPT and 4-DCTD, were effective inhibitors (≥ 50%) of both enzyme
activities (Fig. 5). DCPT, which contains a 2,4-thiazolidinedione ring, inhibited PROD to a greater extent
than EROD (81% vs. 55%, respectively). In contrast, the 4-thiazolidinone ring-containing compound, 4-
DCTD, was a more potent inhibitor of EROD than PROD (74.5% vs. 49.2%, respectively).
• We previously found that 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT, Fig. 1)
produced liver damage in rats (Kennedy et al., 2003). In contrast, 3-(3,5-dichlorophenyl)-
2,4-oxazolidinedione (DCPO, Fig. 1) and 3-(3,5-dichlorophenyl)-4-thiazolidinone (4-DCTD,
Fig. 1) were not hepatotoxic (Kennedy et al., 2003; Patel et al., 2005).
• Further studies suggested that DCPT must undergo metabolism by hepatic cytochromes
P450 (CYPs) to exert its hepatotoxic effects (Crincoli et al., 2004, 2005, 2006). Since this
implies that DCPT may be a CYP substrate, we were interested in determining if this
compound could inhibit metabolism of other CYP substrates. To study this possibility, the
effect of DCPT on two well-known CYP-mediated reactions, ethoxyresorufin-O-deethylase
(EROD) and pentoxyresorufin-O-deethylase (PROD), was evaluated. In these reactions
the substrates are converted to resorufin (Fig. 2), which can be measured fluorometrically.
For comparison purposes, DCPO, 4-DCTD and the known CYP inhibitor metyrapone (Fig.
1) were also evaluated for their ability to inhibit EROD and PROD activities.
Brouwer WG and Blem AR, US Patent # 4,664,694 (1987).
Crincoli C, Patel N and Harvison PJ, The Toxicologist 78 (2004). [2004 Society of Toxicology meeting, abstract #499]
Crincoli CM, Patel NN, Tchao R and Harvison PJ, The Toxicologist 84 (2005). [2005 Society of Toxicology meeting,
Crincoli CM, Patel NN, Tchao R and Harvison PJ, The Toxicologist 90 (2006). [2006 Society of Toxicology meeting,
Fujinami A, Ozaki T and Yamato S, Agric. Biol. Chem. 35: 1707-1719 (1971).
Kennedy EL, Tchao R and Harvison PJ, Toxicology 186: 79-91 (2003).
Patel NN, Crincoli CM, Tchao R and Harvison PJ, The Toxicologist 84 (2005). [2005 Society of Toxicology meeting,
DCPT and DCPO were synthesized by a modification of the method of Fujinami et al.
(1971). The procedure reported by Brouwer et al. (1987) was followed for the preparation
The authors would like to thank Dr. Anil D’mello and Mr. Ganesh Charela, USP Dept. of Pharmaceutical Sciences,
for the EROD and PROD assay procedures.
Supported by NIH grant # ES012499 to P.J.H.
• These experiments have shown that cyclic imide-containing compounds, such as DCPO, DCPT and 4-
DCTD, can inhibit two “standard” CYP-mediated reactions.
• Compared to DCPO, the greater inhibitory activity of DCPT and 4-DCTD could be due to the presence of a
sulfur atom in their cyclic imide rings. This may represent a “soft spot” in the compounds for metabolism by
• EROD and PROD may be catalyzed by different CYP isozymes in rat liver microsomes. Since we observed
differential effects on these enzymes activities with DCPT and 4-DCTD, this could indicate that these
compounds exert some selectivity for inhibition of CYP isozymes.
• Further experiments will be necessary to determine if the inhibition is reversible or irreversible.
-DMSO +DMSO -ACN +ACN
Figure 3. Effect of organic solvents on
ethoxyresorufin-O-deethylase (EROD) activity. An
asterisk (*) indicates that the reaction rate is significantly
different (p < 0.05) from incubations conducted in the
absence of the solvent. Abbreviations: DMSO,
dimethylsulfoxide; ACN, acetonitrile.
Figure 4. Effect of organic solvents on
pentoxyresorufin-O-deethylase (PROD) activity.
Abbreviations: DMSO, dimethylsulfoxide; ACN,
Results are expressed as mean ± SE (n=3). The data were analyzed by a one-way analysis of
variance (ANOVA) followed by a Student-Newman-Keuls post hoc test or t-test when necessary.
Differences between the means were considered significant when p< 0.05.
Effect of Treatments on CYP Activity
Effect of Solvents on CYP Activity