(Received for publication, April 17, 1995; and in revised form, July 11, 1995)
From the
To determine the basis for unexpected differences in CYP1A1
inducing potencies and efficacies for the diet-derived indole
derivative, indolo[3,2-b]carbazole (ICZ) and
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we conducted a
systematic analysis of events involved in the induced expression of
CYP1A1 in murine hepatoma-derived cell lines (Hepa-1). In contrast to
the effects of TCDD, induction kinetics and CYP1A1 mRNA half-life were
dependent on ICZ concentration, and the response from low doses of
inducer was transient due to rapid clearance of ICZ. TCDD and ICZ
produced the same maximum response (i.e. equal efficacies)
from a TCDD-responsive CAT reporter construct in Hepa-1 cells. When
measured by the immediate responses associated with CYP1A1 expression,
including cellular uptake of inducer, receptor transformation and
binding to DRE (gel mobility shift assay), initiation of transcription
(nuclear run-on assay), and short-term accumulation of mRNA (Northern
blot assay), ICZ also exhibited an efficacy equal to that of TCDD and a
potency that corresponds to its receptor affinity. ICZ is a potent and
selective noncompetitive inhibitor of ethoxyresorufin O-deethylase activity (K = 1.5 nM). Taken together these results
indicate that ICZ is a bifunctional modulator of CYP1A1 expression with
intrinsic efficacy equal to that of TCDD.
Indolo[3,2-b]carbazole (ICZ) ()(Fig. Z1) is a compound of dietary origin present
in the gastrointestinal tract of rodents and humans. ICZ is produced in vivo and in vitro as one of the acid-catalyzed
reaction products of non-nutritive indoles such as indole-3-carbinol
and glucobrassicin that are present in cabbage and Brussels sprouts and
other plants of the Brassica genus(1, 2, 3, 4) . ICZ is also
produced, presumably from the nutritive indole, tryptophan, as a
metabolic product of intestinal bacteria(5) .
Figure Z1: Structure 1. a, indolo[3,2-b]carbazole; b, indole-3-carbinol; c, glucobrassicin.
ICZ is similar in several respects to the potent environmental pollutant, TCDD. Both compounds have immunosuppressive activity in murine fetal thymus organ culture and both substances exhibit potent antiestrogenic activities including inhibition of estrogen-dependent growth of cultured breast tumor cells (6, 7) . Additionally, both ICZ and TCDD induce CYP1A1 activity in animals and in cultured cells(1) . CYP1A1 is a phase I enzyme involved in the metabolism of many drugs and carcinogens. CYP1A1 is also the enzyme thought to be primarily responsible for the inactivation of estradiol in breast tumor cells(8) .
Perhaps key to these similarities in activities is the fact that ICZ and TCDD are nearly isosteric and both compounds are potent Ah receptor agonists(9, 10) . The Ah receptor is a widely occurring, ligand-activated transcription factor that mediates the activation of CYP1A1, CYP1A2, glutathione S-transferase Ya, and quinone reductase genes. Binding to this receptor is thought also to be responsible for most of the toxic effects of TCDD, including tumor promotion, teratogenesis, and lethal anorexia with wasting, that appear to result mechanistically from effects beyond simple induction of xenobiotic metabolizing enzymes (11) .
Current knowledge of the Ah receptor-mediated signaling pathway derives primarily from studies of CYP1A1 induction. The process involves ligand binding to a cytosolic Ah receptor/Hsp90 complex which produces a conformational change that results in translocation of the complex to the nucleus where the receptor combines with the Ah receptor nuclear transporter protein. The heterodimer binds to receptive DNA elements (9, 10, 11) located in the enhancer region of the CYP1A1 gene (12, 13, 14) . By mechanisms yet to be determined, this process leads to an increase in transcription rate.
We are interested in the mechanism of action of
ICZ as a model natural ligand for the Ah receptor. Although ICZ
exhibits high affinity for the Ah receptor, it is halogen-free with low
lipophilicity. ICZ, therefore, is less likely to accumulate and persist
in cells than is TCDD and may exhibit certain properties, including the
long-term effects, that are quite different from those of TCDD. In
previous studies we noted that (a) TCDD is
10-10
times as active as ICZ in the induction
of CYP1A1-dependent enzyme activity in Hepa-1 cells, a difference that
is at least 2 orders of magnitude larger than the difference in binding
affinities for the Ah receptor, and (b) the maximal enzyme
activity induced by ICZ in the cells is only about 60% the maximal
activity induced by TCDD(1) . In the present study we compared
further the CYP1A1 regulatory activities of ICZ and TCDD in the murine
hepatoma cell line in an effort to explain these differences in their
activities and to gain further understanding of the regulation of this
important gene.
Gel retardation analysis of MeSO-, TCDD-,
and ICZ-treated Hepa-1 nuclear extracts was carried out as described
previously(23, 24) . To determine the amount of
protein-DNA complex formed, the specific radiolabeled band was excised
from the dried polyacrylamide gel and quantified by liquid
scintillation. The amount of
P-labeled DRE specifically
bound in the ligand-inducible complex was estimated by measuring the
amount of radioactivity in the inducible protein-DNA complex isolated
from the ligand-treated sample lane, and subtracting the amount of
radioactivity present in the same position in a non-ligand-treated
sample lane. The difference in radioactivity between these samples
represents the ligand-inducible specific binding of
P-DRE
and is expressed as the amount of ligand
AhR
DRE complex
formed.
To determine the EROD inhibitory effect of
ICZ over time, various concentrations of ICZ were preincubated with the
induced microsomes, and the NADPH and the substrate were added at the
end of incubation for enzyme analysis. IC values (ICZ
concentration giving 50% inhibition of EROD activity) were estimated
from the inhibition curves, and the inhibition constant (K
) was determined by Dixon plot(29) .
Figure 1: Effect of ICZ and TCDD on EROD activity in Hepa-1 cells. Cells were treated with different concentrations of inducer for 24 or 48 h. The cells were then harvested for analysis of enzyme activity. Symbols and bars represent mean values and the ranges of two individual determinations.
Fig. 2shows the results of Northern blot analyses of
the CYP1A1 mRNA levels relative to -actin mRNA in Hepa-1 cells
expressed as percent of maximum induction after 4- and 24-h treatment
with a range of concentrations of TCDD and ICZ. Whereas there was no
significant shift in the TCDD curve, there was a clear shift to the
left of the 4-h curve for ICZ compared to the 24-h curve. The
difference in EC
values at 4 h was about 2 orders of
magnitude and the maximum level of induction by ICZ after 4 h
incubation was similar to the maximum level induced by TCDD.
Figure 2:
Concentration dependent effect of ICZ and
TCDD on steady-state levels of CYP1A1 mRNA in Hepa-1 cells. The cells
were incubated with different concentrations of ICZ or TCDD for 4 or 24
h. Total RNA was then isolated and CYP1A1 mRNA was quantified by gel
electrophoresis and Northern hybridization. Following analysis by
PhosphorImager, blots were stripped and reprobed with P-labeled human
-actin cDNA. Experiments were
conducted twice with similar results.
Results
of an experiment to examine the TCDD and ICZ concentration-dependent
activation of chloramphenicol acyltransferase in Hepa-1 cells stably
transfected with a TCDD-responsive bacterial CAT reporter gene are
shown in Fig. 3. The EC values for CAT reporter
induction by 19 h treatment with TCDD and ICZ are similar to the 24-h
EC
values for EROD and CYP1A1 mRNA induction by these
compounds and their potencies differ by over 4 orders of magnitude in
all three assays. In contrast to the results of EROD induction
experiments, however, the maximal levels of CAT activation were similar
for the two inducers.
Figure 3: ICZ and TCDD as inducers of DRE-driven CAT reporter gene activity in Hepa-1 cells. Murine Hepa-1 cells were transfected with the reporter gene construct and treated for 20 h with inducers in the indicated range of concentrations. The experiment was conducted twice with similar results.
Figure 4:
Kinetics of EROD induction by ICZ in
Hepa-1 cells. The cells were treated with either a high (1.35
µM) or a low (67.5 nM) concentration of ICZ, and
were harvested at designated time points for analysis of enzyme
activity. Symbols and bars represent mean values and
the ranges of two individual determinations. Activity induced by
solvent (MeSO) was subtracted for each time point. The
experiment was conducted two times with similar results. RF,
resorufin.
Results of similar studies of steady-state CYP1A1 mRNA levels indicated that the maximal induction of message occurred around 4 h of treatment with the lower concentration of ICZ and around 8 h in response to the higher concentration of ICZ. Consistent with the kinetics for induction of enzyme activity, the level of CYP1A1 mRNA induced by a low concentration of ICZ was also transient and fell to about 20% of the maximal level by 16 h (data not shown).
Figure 5:
Effect of multiple additions of ICZ to the
medium on EROD activity in Hepa-1 cells. The cells were incubated with
270 nM ICZ for 24 h and then ICZ was removed or was added at
the times indicated; group I, no change in medium for 72 h; group II,
additional ICZ was added at 48 h; group III, additional ICZ was added
at 24 h; group IV, the medium was replaced with ICZ-free medium at 24
h. The cells were collected for analysis of EROD activity at the
designated times. Symbols and bars represent mean
values and the ranges of two individual determinations. RF,
resorufin; DMSO, MeSO.
Figure 6: Combined effect of TCDD and ICZ on EROD activity in Hepa-1 cells. The cells were incubated with different concentrations of TCDD together with either 1.35 µM or 67.5 nM ICZ. The cells were harvested after 24 h for analysis of EROD activity. Symbols and bars represent mean values and the ranges of two individual determinations. The experiment was conducted two times with similar results. RF, resorufin.
Figure 7:
Concentration-dependent formation of the
transformed Ah receptor in nuclear extracts of Hepa-1 cells by TCDD and
ICZ. Cells were incubated for 1 h with MeSO (DMSO)
or the indicated concentrations of TCDD or ICZ. Nuclear extracts were
prepared and mixed with
P-labeled DRE3 oligonucleotide,
and the formation of protein-DNA complexes was analyzed by gel
retardation assay (a), and quantified by PhosphorImager (b). The data presented in b are the mean ±
S.D. for three values.
Figure 8: Decay of CYP1A1 mRNA from Hepa-1 cells after ICZ or TCDD treatment. Confluent cells were incubated with ICZ (67.5 nM) or TCDD (10 pM) for 8 h and then treated with actinomycin D (2 µg/ml). Total RNA was isolated at the designated time points, and CYP1A1 mRNA was quantified by PhosphorImager after gel electrophoresis and Northern hybridization.
Figure 9:
Inhibition of microsomal EROD activity by
ICZ. The reaction mixture contained ICZ-induced microsomes, ICZ (10
pM to 10 µM), ethoxyresorufin (0.1-1
µM), and NADPH. Double reciprocal (a) and Dixon (b) plots for the inhibition of microsomal EROD activity by
ICZ are indicated. The experiment was conducted three times with
similar results. DMSO, MeSO; ERF,
ethoxyresorufin; RF, resorufin.
Figure 10: Kinetics of ICZ disappearance. ICZ (4-10 nM) was incubated with ICZ-induced microsomes or TCDD-induced Hepa-1 cells for the times indicated, followed by either addition of the substrate for EROD assay of (a) microsomes and (b) cells, or extraction of aliquots of the microsomal mixture for analyses of ICZ by HPLC (c). Experiments were conducted twice with similar results.
The purpose of this study was to examine the CYP1A1-inducing effects of ICZ as a model natural ligand for the Ah receptor. We have shown that mutant cells deficient in Ah receptor function do not possess detectable EROD activity after ICZ treatment, which confirms the requirement for a competent Ah receptor/AhR nuclear translocator signal transduction system for CYP1A1 induction by ICZ, as for TCDD (31, 32) . Our results, consistent with previous observations(1) , demonstrated that TCDD and ICZ generated parallel concentration-response curves for induction of EROD activity with ICZ producing a lower maximal response than TCDD. Concentration-response curves for induction of the TCDD-responsive CAT reporter and for CYP1A1 mRNA were also parallel for the two inducers, but in these cases maximal responses similar to that of TCDD were produced by ICZ (Fig. 1Fig. 2Fig. 3). Simultaneous treatment of cells with maximally inducing concentrations of both inducers produced no greater response than either inducer alone. This evidence indicates that ICZ and TCDD function by the same mechanism in the induction of CYP1A1 in the Hepa-1 cells and that while ICZ is a less potent inducer than TCDD, it has the same efficacy as an inducer.
In contrast to the effects of TCDD, however, the kinetics of CYP1A1 induction by ICZ were dependent on the dose of inducer. Maximum induction of CYP1A1 mRNA occurred after 8 h of exposure to the higher concentration of ICZ, and after only 4 h incubation with the lower dose of ICZ. Maximum induction of CYP1A1 mRNA occurs after 8 h of exposure to TCDD (37, 38) and in this case the kinetics are reported to be independent of dose of TCDD in the Hepa-1 cells(26) .
Also in contrast to the response from TCDD, CYP1A1 induction by a low concentration of ICZ (67.5 nM) was transient. This transient effect also has been observed in rainbow trout treated with indole-3-carbinol, a precursor of ICZ(39) , and in rodent liver tumor cell lines exposed to aryl hydrocarbons(40, 41) . This transient induction by ICZ in the Hepa-1 cells is most likely due to the clearance of ICZ from the medium. When induced cells were incubated in ICZ-free medium, the EROD activity and CYP1A1 mRNA level dropped by about 85% in 12 and 4 h, respectively. The induction was recovered when ICZ was reintroduced into the medium, indicating that the cells had not become insensitive to the inducer and that the signal transduction system had not been down-regulated following exposure to ICZ. In contrast, following removal of TCDD from the medium, the induced EROD activity remained unchanged for at least another 48 h. The persistent effect of TCDD has been reported by several investigators (38, 40, 42) and is likely due to its high resistance to metabolic degradation.
Since the steady-state level of mRNA is affected by rate of transcription and rate of mRNA degradation, we conducted a series of experiments to compare the effects of ICZ and TCDD on individual components of the Ah receptor-mediated signal transduction pathway. Comparisons of ICZ- and TCDD-induced transformation of the Ah receptor, its translocation to the nucleus, and its binding to DNA, in vitro, indicated that there was an approximate 100-fold difference in potencies for the two compounds and that they produced the same maximum level of transformation and binding to DNA. This difference in potencies, which was observed after 1 h exposure to inducer, corresponds to the difference in Ah receptor binding affinities that we have determined for the compounds. The similarities in maximum levels of Ah receptor transformation, induced mRNA levels, and maximum transcription rate (based on our nuclear run-on experiments), indicate further that while the two compounds have different potencies, they are equally effective as CYP1A1 inducers. This is in contrast to 3-methylcholanthrene, benzanthracene, and certain chlorinated hydrocarbons that have intrinsic inducing efficacies that do not correspond to their high affinities for the Ah receptor(40, 43, 44) .
To compare the
effect of ICZ on the degradation rate of the CYP1A1 mRNA we also
performed actinomycin D chase experiments. Our results showed that
induction with either low (10 pM) or high concentrations (100
pM) of TCDD or a high concentration of ICZ (1.35
µM) produced CYP1A1 mRNA with a half-life of about 4.5 h.
This value is consistent with a 3-h half-life computed previously for
CYP1A1 mRNA in Hepa-1 cells and not directly measured(32) , but
it is considerably shorter than the 14-h half-life previously measured
in -naphthoflavone-induced rabbit hepatocytes(33) .
Treatment of cells with a low concentration of ICZ (67.5 nM)
produced CYP1A1 mRNA with a half-life of only about 1.7 h. This
dependence of the mRNA half-life on inducer concentration has not been
reported previously for Hepa-1 cells. Post-transcriptional regulation
of CYP1A1, however, has been reported for 3-methylcholanthrene- and
TCDD-induced rat hepatocytes, and in TCDD-induced mouse livers based on
the difference between the magnitude of the increase in steady-state
mRNA accumulation and the rate of
transcription(34, 35, 36) .
Post-transcriptional mechanisms also contribute to the regulation of
the CYP1A2 gene as indicated by results of studies of TCDD- and
3-methylcholanthrene-induced rat hepatocytes and
livers(35, 36) , and in the
-naphthoflavone-induced rabbit hepatocytes(33) . Our
results are consistent with a direct role of the inducer in
stabilization of message.
ICZ is not only an inducer of the Cyp1a-1 gene, but also a potent and selective inhibitor of
CYP1A1 enzyme (EROD) activity. With an inhibition constant of 1.5
nM for EROD activity, ICZ is the most potent of the various
synthetic (e.g. -naphthoflavone and 1-ethynylpyrene) and
naturally occurring (e.g. ellipticine, flavonoids, and
coumarins) inhibitors for which inhibition data are
available(45, 46, 47, 48) . Because
ICZ is similar in chemical structure to ellipticine and both compounds
are noncompetitive inhibitors, it is likely that ICZ functions by the
mechanism suggested for ellipticine, that is, by association with heme
and displacement of oxygen from the active site(49) . ICZ,
however, does not exhibit the high degree of cytotoxicity that is
characteristic of the ellipticines. A selectivity in enzyme inhibition
for ICZ is indicated by its lack of effect against
phenobarbital-induced CYP2B1/2B2 (pentoxyresorufin O-dealkylase) activity. The lower maximal induction of EROD
activity induced by ICZ and the suppressive effect of high doses of ICZ
on TCDD-induced EROD activity that we observed can by attributed to the
inhibitory effect of ICZ.
Our observation of the potent inhibitory effect of ICZ against EROD activity provided an indirect method of evaluating ICZ cellular uptake and clearance. The rapid onset and subsequent loss of this inhibition and the similar kinetics in incubations with both microsomes and cells are consistent with a rapid cellular uptake and clearance of ICZ by metabolism to inactive substances. Rapid uptake of ICZ is further indicated by our results showing the potent and rapid effect of ICZ on Ah receptor nuclear translocation and DNA binding and by the rapid induction of transcription indicated by the results of the nuclear run-on assay. Direct evidence for the rapid clearance of ICZ was provided by the results of HPLC analyses of the microsomal incubation mixture. These observations indicate that the decreased potency for CYP1A1 induction by ICZ compared to TCDD is not due to a significantly decreased rate of ICZ uptake but is due to rapid clearance.
In light of our results, a recent report by Kleman et al.(50) suggests that the inducing effects of indolocarbazoles may be highly dependent on variations in inducer structure, cell-type, or enhancer configuration. In contrast to our findings on the response to ICZ in Hepa-1 cells of the chloramphenicol acetyltransferase reporter gene controlled by the full Cyp1a-1 enhancer, the N,N`-dimethylated derivative of ICZ, 5,11-dimethylindolo[3,2-b]carbazole is reported to be equipotent with TCDD in these cells as an inducer of transcription from a CAT reporter gene controlled by a simple, single DRE-containing enhancer. In addition, ICZ and 5,11-dimethylindolo[3,2-b]carbazole are reported to be equipotent with TCDD as inducers of the latter reporter in human hepatoma cells (HepG2). These increased potencies of the indolocarbazoles relative to TCDD could result from decreased metabolic clearance and/or an increased sensitivity to the indolocarbazoles of the Ah receptor-mediated signal transduction pathway.
Taken together our results indicate that ICZ is a bifunctional modulator of CYP1A1 expression in murine Hepa-1 cells. ICZ and TCDD function by the same mechanism and with equal efficacy in the induction of CYP1A1. The decreased potency of ICZ in comparison to its affinity for the Ah receptor may be attributed to rapid clearance of the inducer from these cells. Clearance of inducer also appears to be responsible for the transient ICZ-induced expression of CYP1A1, an effect that is augmented by decreased stability of CYP1A1 mRNA in the absence of the inducer. ICZ is also a selective, noncompetitive inhibitor of CYP1A1 enzyme activity, with a potency for enzyme inhibition that is greater by an order of magnitude than its maximum inducing potency. Thus, at concentrations below those necessary to produce gene activation, ICZ can inhibit CYP1A1-mediated enzyme activity. Whether these quantitative and qualitative effects of ICZ on CYP1A1 expression can be generalized to human systems requires further investigation.