Regulation of Rat Carboxylesterase Expression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD): A Dose-Dependent Decrease in mRNA Levels but a Biphasic Change in Protein Levels and Activity

Dongfang Yang, Yuxin Li, Xiong Yuan, Lynn Matoney and Bingfang Yan1

Department of Biomedical Sciences, University of Rhode Island, Kingston, Rhode Island 02881

Received May 10, 2001; accepted July 18, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carboxylesterases play an important role in the metabolism of endogenous lipids and foreign compounds; therefore, xenobiotic regulation of carboxylesterase gene expression has both physiological and pharmacological significance. We previously reported that ß-naphthoflavone and 3-methylcholanthrene, two potent inducers for cytochrome P4501A enzymes, had opposing effects on the expression of hydrolase S, a secretory carboxylesterase. ß-Naphthoflavone caused suppression, whereas 3-methylcholanthrene caused induction of the expression of this enzyme. The aim of the present study was to determine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), another prototypical cytochrome P4501A inducer, on the expression of this and several other rat carboxylesterases (hydrolases A, B, and C) in liver and extrahepatic tissues. Rats received TCDD treatment at nonlethal (<=10 µg/kg), sublethal (30 µg/kg), or lethal doses (90 µg/kg). The overall hydrolytic activity in the liver microsomes toward both 1-naphthyl- andpara-nitrophenylacetate was markedly increased in the nonlethal dosage groups, but markedly decreased in the lethal dosage group. Consistent with such a biphasic dose-response relationship, the levels of enzyme proteins exhibited an initial increase, followed by a decrease in response to nonlethal and lethal doses, respectively. In contrast, treatment with TCDD caused a dose-dependent decrease on the levels of mRNA encoding these enzymes. All liver carboxylesterases showed a similar pattern of change on activity, protein, and mRNA levels, suggesting that TCDD coregulates the expression of these genes. In the extrahepatic tissues, a similar biphasic change was observed in activity and in protein and mRNA levels. In both liver and kidney, the expression of cytochrome P4501A1 (CYP1A1) was significantly induced in a dose-dependent manner. TCDD is known to upregulate the expression of CYP1A1 gene through the aryl hydrocarbon receptor (AhR). The differential effects on the expression of liver carboxylesterases and CYP1A1 suggest that TCDD regulates the expression of hydrolytic enzymesviaa mechanism(s) other than the AhR-mediated transcription activation, as observed in the CYP1A1 regulation. The different patterns of change on protein and mRNA levels in the nonlethal dosage groups suggest that TCDD regulates the expression of hepatic carboxylesterases by acting on both transcription and translation.

Key Words: carboxylesterase; hydrolase A, B and S; 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carboxylesterases play an important role in the metabolism of endogenous lipids and foreign compounds such as drugs and pesticides (Heymann, 1982Go; Junge and Krisch, 1975Go; Parkinson, 1995Go; Satoh and Hosokawa, 1998Go). In addition to hydrolyzing numerous chemicals, carboxylesterases can catalyze transesterification reaction, which accounts for the conversion of cocaine (a methyl ester) to ethylcocaine (the corresponding ethyl ester) in the presence of ethyl alcohol (Boyer and Petersen, 1992Go). Carboxylesterase activity is widely distributed in mammalian tissues, with the highest levels present in liver microsomes (Morgan et al., 1994aGo; Parkinson, 1995Go; Satoh and Hosokawa, 1998Go). Multiple forms of carboxylesterases are identified in several mammalian species (Parkinson, 1995Go; Satoh and Hosokawa, 1998Go; Yan et al., 1999Go). We previously reported the isolation of cDNAs encoding 4 rat carboxylesterases. They are designated hydrolases A, B, C, and S (Yan et al., 1994Go, 1995aGo,Yan et al., bGo,cGo), respectively. Hydrolase A, B, C, and S are ~70% identical in both the nucleotide and the derived amino acid sequences, with the exception of hydrolases B and C, which are ~95% identical. In addition to liver expression, hydrolase A is abundant in the testis, whereas hydrolase B is abundant in the kidney (Morgan et al., 1994aGo,bGo). Hydrolase S, lacking the retention signal for targeting to the endoplasmic reticulum, is synthesized in the liver but secreted into the blood (Yan et al., 1995cGo).

A wide range of drugs and other xenobiotics is found to alter the expression of carboxylesterases, and xenobiotic regulation of these genes exhibits several characteristics (Morgan et al., 1994aGo,bGo; Parkinson, 1995Go; Satoh and Hosokawa, 1998Go). Some chemicals have similar effects across species, whereas other compounds show a profound species difference (Morgan et al., 1994aGo,bGo; Satoh and Hosokawa, 1998Go; Zhu et al., 2000Go). Phenobarbital, for example, causes induction of carboxylesterases in both rodents and humans, whereas dexamethasone exhibits species-specific effects. In rats, dexamethasone causes marked suppression of the expression of hydrolases A, B, C, and S. In contrast, this compound causes a moderate induction of human carboxylesterases as determined with primary hepatocyte cultures (Zhu et al., 2000Go). Structurally related compounds may also have different effects on carboxylesterase expression. Pregnenolone 16{alpha}-carbonitrile, a synthetic steroid structurally related to dexamethasone, causes either a moderate or a marked induction of several rat carboxylesterases (Hosokawa et al., 1993Go). Finally, chemicals with the same or similar effects on the expression of CYP3 may differentially regulate the expression of carboxylesterases (Hosokawa et al., 1993Go; Morgan et al., 1994aGo,bGo; Parkinson, 1995Go; Satoh and Hosokawa, 1998Go). For example, expression of rat hydrolase S is significantly suppressed by isoniazid but slightly increased by streptozotocin; both of them are CYP2E1 inducers (Yan et al., 1995cGo). The CYP1A enzyme inducers, ß-naphthoflavone and 3-methylcholanthrene, have opposing effects; the former compound suppresses the expression of hydrolase S whereas the latter compound slightly induces it (Yan et al., 1995cGo). The differential response of carboxylesterase genes to the same type of CYP inducers suggests that these chemicals use multiple signaling pathways to exert their biological effects.

The aim of the present study was to determine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), another prototypical CYP4501A inducer, on the expression of hydrolase S and several other rat carboxylesterases (hydrolases A, B, and C) in hepatic and extrahepatic tissues. Rats received TCDD treatment at nonlethal (<=10 µg/kg), sublethal (30 µg/kg), or lethal doses (90 µg/kg). The overall hydrolytic activity in the liver microsomes toward both 1-naphthyl- and p-nitrophenylacetate was markedly increased in the nonlethal dosage groups, but markedly decreased in the lethal dosage group. A similar biphasic change was observed on the levels of enzyme proteins, as determined by Western immunoblotting analyses. In contrast, treatment with TCDD caused a dose-dependent decrease on the levels of mRNA encoding these enzymes. In both liver and kidney, the expression of CYP4501A1 was significantly induced in a strict dose-dependent manner. The differential effects on the expression of liver carboxylesterases and CYP4501A1 suggest that various mechanisms mediate the action of TCDD in regulating the expression of xenobiotic-metabolizing enzymes. The different patterns of change on protein and mRNA levels suggest that TCDD regulates the expression of hepatic carboxylesterases by acting on both transcription and translation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and supplies.
TRI Reagent RNA extraction solution, para-nitrophenylacetate and 1-naphthylacetate, were from Sigma Chemical Co., St. Louis, MO; the kit for primer extension labeling, nitro blue tetrazolium, and 5-bromo-4-chloro-3-indolyl-phosphate were from Promega (Madison, WI). Goat antirabbit-IgG conjugated with alkaline phosphatase was from Pierce (Rockford, IL); and antirat CYP1A antibody was purchased from XenoTech, LLC. (Kansas City, KS). TCDD with a purity of 99% was purchased from Cambridge Laboratory (Woburn, MA). Sprague-Dawley rats (7–8 weeks old) were purchased from Charles River (Wilmington, MA). Anti-hydrolase S and plasmids harboring a cDNA insert encoding hydrolase A, B, or S were described elsewhere (Yan et al., 1994Go, 1995aGo,Yan et al., bGo,cGo). Unless otherwise indicated, all other reagents were purchased from Fisher Scientific (Pittsburgh, PA).

Animal treatment and microsome preparation.
Sprague-Dawley rats (5 per group) were given orally 1, 3, 10, 30, or 90 µg TCDD/kg as described previously (Fan et al., 1997Go). TCDD was dissolved in corn oil, and dosing was conducted by gastric intubation at 5 ml/kg. Control rats received corn oil only. Rats were sacrificed at day 8, internal organs were harvested, and trunk blood was collected. Liver microsomes were prepared by differential centrifugation as described by Lu and Levin (1972) and stored as a suspension in 250 mM sucrose at –80°C. Extrahepatic microsomes were prepared as described by Sonderfan et al. (1989) without addition of the protease inhibitor. The same rats were used for all experiments throughout this study. All rats were housed in an AAALAC-accredited facility and allowed free access to Purina Rodent Chow 5001 and water.

Enzymatic assays.
The enzymatic activity toward para-nitrophenylacetate or 1-naphthylacetate was spectrophotometrically determined with a Beckman DU-520 spectrophotometer, essentially as described previously (Morgan et al., 1994bGo). The hydrolysis of para-nitrophenylacetate to para-nitrophenol was determined from an increase in the absorbance at 400 nm, where the hydrolysis of 1-naphthylacetate to 1-naphthol was determined from an increase in the absorbance at 322 nm. Microsomes were diluted to 25 µg/ml with 100-mM potassium phosphate buffer, pH 7.4. The reactions were conducted in a 1-ml cuvette and initiated by the addition of substrate (10 µl of 100 mM stock in acetonitrile). The extinction of coefficients for para-nitrophenol and 1-naphthol were 13 mM–1cm–1 and 2.2 mM–1cm–1, respectively. All enzymatic determinations were conducted in triplicate and expressed as means ± SD.

Western immunoblotting and nondenaturing gel electrophoresis.
Microsomes (2–20 µg) were subjected to SDS–PAGE according to Laemmli (1970). Samples were transferred electrophoretically to a Trans-Blot nitrocellulose membrane. The immunoblots were blocked in 5% nonfat dry milk and then incubated with the antibody (10 µg/ml) against hydrolase S. Hydrolase A, B, and S were electrophoretically distinct, and anti-hydrolase S cross-reacted with both hydrolases A and B (Yan et al., 1995cGo). The primary antibody was then located by alkaline phosphatase-conjugated goat antirabbit IgG. The blots were stained with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate as described previously (Yan et al., 1995cGo).

Nondenaturing gel electrophoresis for staining esterase activity was conducted as described previously (Yan et al., 1995cGo)). Microsomal protein (5 µg) was solubilized with 0.2% Lubrol and subjected to electrophoresis with a 3% acrylamide stacking gel and a 7.5% acrylamide separating gel. After electrophoresis, the gels were washed for 1 h in 100 mM potassium phosphate buffer (pH6.5), followed by incubating in the same buffer containing 1-naphthylacetate (5 mM) and 4-benzolamino-2,5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt, usually termed Fast Blue RR (0.4 mg/ml). The staining of esterases by this method is based on the formation of a black, insoluble complex between 1-naphthol hydrolyzed from 1-naphthylacetate and Fast Blue RR.

Northern blotting.
Total RNA from control or TCDD-treated Sprague-Dawley adult rats was isolated with a TRI Reagent RNA extraction solution according to the instruction by the manufacturer. RNA samples from each group of rats were pooled. Total RNA (20 µg) was fractionated by electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde. Fractionated RNA was blotted to a Nytran nylon membrane, and mRNA encoding a carboxylesterase was detected with a 32P-labeled cDNA probe as described previously (Yan et al., 1995cGo). The cDNA probe was prepared by radiolabeling a cDNA insert encoding hydrolase A, B, or S with a Prime-a-Gene system as described previously (Yan et al., 1995cGo). To normalize the abundance of 28S rRNA contained in each sample, the same membrane was stripped by boiling 2x for 15 min and reprobed with an oligonucleotide (hybridized with 28S rRNA), 32P-radiolabeled with T4 polynucleotide kinase as described previously (Yan et al., 1995cGo). The intensity (both Northern and Western blots) was determined with a laser scanning densitometer (Biomed Instruments, Inc., Fullerton, CA).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of TCDD treatment on hydrolytic activity in hepatic and extrahepatic tissues.
Rats received TCDD treatment at a nonlethal (<=10 µg/kg), sublethal (30 µg/kg), or lethal (90 µg/kg) dose. Microsomes from both hepatic and extrahepatic tissues were prepared. Hydrolytic activities toward para-nitrophenylacetate and 1-naphthylacetate were determined with individual rat microsomal samples. As shown in Figure 1Go (top), liver microsomal esterase activity toward both substrates was significantly increased (65–100%) in rats treated with nonlethal doses. The maximum increase was observed in the 1-µg/kg group for both substrates. In contrast, treatment of rats with a lethal dose (90 µg/kg) caused a decrease in hydrolytic activity by 50–70%, depending on the substrate used. Kidney microsomal esterase activity was increased among all dosage groups (Fig. 1Go, middle) compared with the control group, although a ~30% decrease was detected from the 30-µg/kg to the 90-µg/kg dosage group. The maximum increase was detected in the 30-µg/kg dosage group with an 85% increase on para-nitrophenylacetate and a 100% increase on 1-naphthylacetate hydrolysis, respectively (middle of Fig. 1Go). Similar to kidney microsomes, testicular microsomes displayed a biphasic change while hydrolyzing both substrates (bottom of Fig. 1Go). The maximum activity in hydrolyzing both para-nitrophenylacetate and 1-naphthylacetate was observed in the 10-µg/kg dosage group (110% and 50%, respectively). Testicular and kidney microsomes exhibited less suppression than liver microsomes in the rats treated with the lethal dose.



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FIG. 1. Effect of TCDD treatment on the hydrolytic activity toward para-nitrophenylacetate or 1-naphthylacetate in both hepatic and extrahepatic tissues. Male rats (5 per group) were treated with corn oil or TCDD at different doses (1–90 µg/kg). Animals were sacrificed 8 days after treatment, and microsomes from liver, kidney and testis were prepared. The microsomal esterase activity of liver, kidney, and testis was spectrophotometrically determined at room temperature with 1.0 mM para-nitrophenylacetate or 1-naphthylacetate. All assays were performed with individual rat samples and repeated 3 times. Hydrolytic rates were expressed as the mean ± SD (µmol/mg protein/min). *Significantly different from control according to Dunnett's test.

 
Effect of TCDD on the hepatic expression of hydrolases A and B.
We previously reported that rat liver microsomes contain 2 major forms of carboxylesterases designated hydrolases A and B (Morgan et al., 1994aGo,bGo). These two enzymes were estimated to account for ~85% of the microsomal esterase activity toward para-nitrophenylacetate. We next examined whether the changes of the hydrolytic activity toward this substrate reflected changes in the expression of hydrolase A and B. Microsomes were pooled from each group and subjected to Western immunoblotting analyses. The immunostaining was quantified by scanning densitometry. As shown in Figure 2Go (left panel), the abundance of both hydrolase A and B in the liver microsomes displayed a biphasic change: the levels of both enzymes were increased in the nonlethal dosage groups but decreased in the lethal dosage group. The maximum increase for hydrolase A (top, left panel) was 20%, which was observed in the 1-µg/kg dosage group. The maximum induction for hydrolase B (bottom, left panel) was slightly higher (30%) than that for hydrolase A, which was observed in the 3-µg/kg dosage group. In contrast, the suppression on the expression of both enzymes was profound. The levels of hydrolase A and B were decreased by 90% and 60%, respectively, in the 90-µg/kg dosage group.



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FIG. 2. Effect of TCDD treatment on the expression of hydrolase A (HA) and B (HB). Male rats (5 per group) were treated with corn oil or TCDD as described in the legend to Figure 1Go. For Western analyses (left panels), liver microsomes (5 µg) pooled from 5 rats were subjected to SDS–PAGE as described in Materials and Methods. Samples were transferred electrophoretically to a Trans-Blot nitrocellulose membrane. The immunoblots were blocked in 5% nonfat dry milk and then incubated with the antibody (10 µg/ml) against hydrolase S. Hydrolase A and B were electrophoretically distinct, and the antibody against hydrolase S cross-reacted with hydrolase A (top left panel) and B (bottom left panel). The primary antibody was then located by alkaline phosphatase-conjugated goat antirabbit IgG. For Northern analyses, total RNA (10 µg) pooled from 5 rats was subjected to 2,2 M formaldehyde agarose electrophoresis and transferred to a Nytran nylon membrane by a vacuum blotting system. The blots were detected with 32P-labeled probes from cDNA encoding hydrolase A (top right panel) or hydrolase B (bottom right panel). To normalize the abundance of 28S rRNA contained in each sample, the same membrane was stripped by boiling 2x for15 min and reprobed with a [32P]ATP-labeled oligonucleotide hybridizing 28 S rRNA (data not shown). The immune staining and the signal on the autoradiographs were quantified by a scanning densitometer and expressed in a bar graph. Figures 2–6GoGoGoGoGo show representative results with pooled samples.

 
We previously showed that xenobiotic regulation of hydrolase S by an array of chemicals primarily affected the accumulated levels of mRNA (Yan et al., 1995cGo). Namely, changes of mRNA levels were correlated well with changes in the protein levels. We next examined whether changes of the mRNA levels of hydrolase A or B showed a similar biphasic pattern to that shown by Western analyses. Total RNA samples were pooled from each group and subjected to Northern blotting analyses. In a striking contrast, the mRNA levels for both enzymes were markedly decreased in a dose-dependent manner (right panel of Fig. 2Go). Treatment of rats with only 1 µg/kg TCDD caused a 30 and 40% decrease in mRNA levels for hydrolase A and B, respectively. Little mRNA encoding hydrolase A or B was detected in the samples from the 90-µg/kg group. The same blots were stripped and reprobed for abundance of 28 S rRNA; little variation was detected (data not shown).

Effect of TCDD on the expression of hydrolase A in the testis and hydrolase B in the kidney.
We previously demonstrated by kinetics and immunoblotting analyses that high levels of hydrolase A were present in the testis, whereas high levels of hydrolase B were present in the kidney (Morgan et al., 1994aGo). We next examined the effect of TCDD on the expression of both enzymes in extrahepatic tissues with samples pooled from each group. As shown in Figure 3Go (left panel), only a slight induction was detected for both hydrolase A and B, notably in the 30-µg/kg TCDD dosage group. In contrast, a 50% decrease of testicular hydrolase A was detected in the 90-µg/kg TCDD group (top, left panel of Fig. 3Go). The suppression of kidney hydrolase B was negligible compared with the control (bottom, left panel of Fig. 3Go). In contrast to the liver, the extrahepatic tissues showed a high degree of correlation between the protein and mRNA levels (Figs. 2, 3GoGo).



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FIG. 3. Effect of TCDD treatment on the expression of hydrolase A (HA) in testis and hydrolase B (HB) in kidney. Rat treatment, sample preparation: Western and Northern analyses were carried out as described in the legend to Figure 2Go, but samples from extrahepatic tissues were used, i.e., testis microsomes (top left panel), kidney microsomes (bottom left panel), testis RNA (top right panel), and kidney RNA (bottom right panel).

 
Effect of TCDD on the abundance of hydrolase S in the liver and serum.
Hydrolase S is a carboxylesterase synthesized in the liver but secreted into the blood (Yan et al., 1995cGo). Hydrolase S, therefore, provided one more step (secretion) that TCDD might regulate. To test this possibility, liver microsomes and sera pooled from each group were subjected to Western blotting analyses. As shown in Figure 4Go, a biphasic change of liver hydrolase S was detected. The maximum induction was ~100% and detected in the 1 µg/kg dosage group (top of Fig. 4Go). In contrast, the abundance of serum hydrolase S displayed only a negligible increase (~10%) (middle of Fig. 4Go). However, the suppressive effects on hydrolase S expression in the 90 µg/kg TCDD group was profound; neither liver nor serum hydrolase S was detected in this group. Similar to the levels of mRNA encoding hydrolase A or B (Fig. 1Go), hydrolase S mRNA levels were decreased in a dose-dependent manner (bottom of Fig. 4Go). In contrast to the biphasic changes on the expression of carboxylesterases, abundance of CYP1A1 in both liver and kidney tissues was increased in a dose-dependent manner (Fig. 5Go). No CYP1A1 was detected in the testis (data not shown).



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FIG. 4. Effect of TCDD treatment on the abundance of hydrolase S (HS) in liver and serum. Rat treatment, sample preparation: Western and Northern analyses were carried out as described in the legend to Figure 2Go, but radiolabeled cDNA encoding hydrolase S was used to detect the RNA blot. To determine the levels of secretary hydrolase S, serum samples (0.5 µl) pooled from 5 rats were analyzed by Western immunoblotting.

 


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FIG. 5. Effect of TCDD treatment on the expression of cytochrome P4501A1. Liver or kidney microsomes pooled from 5 rats were analyzed by Western immunoblotting as described in the legend to Figure 2Go, but the blots were detected with anti-CYP1A1 antibody. Also, different amounts of microsomal proteins were used: liver, 2 µg and kidney, 10 µg.

 
Nondenaturing gel electrophoresis staining for esterase activity in the liver, kidney and testis microsomes.
Hydrolase A and B represent 2 major forms of carboxylesterases in hepatic and extrahepatic tissues, but they are not the only rat carboxylesterases (Morgan et al., 1994aGo,bGo). Nondenaturing gel electrophoresis staining for esterase activity detected several other electrophoretically distinct carboxylesterases toward 1-naphthylacetate (Morgan et al., 1994bGo). In order to determine the effects of TCDD on these enzymes, the same pooled microsomes from the liver, kidney, and testis were subjected to nondenaturing gel electrophoresis, followed by incubation with 1-naphthylacetate and Fast Blue RR. As shown in Figure 6Go, all electrophoretically distinct bands in the liver microsomes showed a biphasic change, namely, an induction was followed by a suppression from the nonlethal to lethal dosage groups. These results suggest that TCDD coregulates the expression of the electrophoretically distinct carboxylesterases and contrasts to dexamethasone, which displays differential effects on these enzymes (Morgan et al., 1994bGo). In the kidney and testis, only the bands corresponding to kidney hydrolase B or testis hydrolase A were apparent, and the patterns of change on these bands were similar to the pattern detected by Western immunoblotting for hydrolase A in the testis or hydrolase B in the kidney (Figs. 3 and 6GoGo).



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FIG. 6. Nondenaturing gel electrophoresis of liver, kidney and testis microsomes from rats treated with corn oil or TCDD. Pooled liver, kidney, and testis microsomes (5 µg) were subjected to nondenaturing gel electrophoresis, as described under Materials and Methods. Staining for esterase activity was based on the formation of a black, insoluble complex between diazotized Fast Blue RR and 1-naphthol, which was released enzymatically from 1-naphthylacetate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carboxylesterases play an important role in the metabolism of endogenous lipids and foreign compounds, therefore, xenobiotic regulation of carboxylesterase gene expression has both physiological and pharmacological significance. TCDD, a widespread environmental contaminant, has been shown to modulate the expression of many drug-metabolizing enzymes (Dragnev et al., 1995Go; Parkinson, 1995Go; Runge-Morris, 1998Go). In most cases, TCDD acts as a positive regulator on these genes. In this report, we describe a biphasic change on the expression of rat carboxylesterases caused by TCDD. Nonlethal TCDD doses cause induction whereas the lethal dose causes suppression. Such a biphasic pattern is limited only to the changes on catalytic activity and enzyme protein levels but not on the accumulated mRNA levels. In the extrahepatic tissues, a similar biphasic change is observed in the expression of hydrolase A or B. However, higher doses are required to exert the maximum induction and the suppression is only moderate or negligible. In both hepatic and extrahepatic tissues, the CYP1A1 gene is abundantly induced in a strict dose-dependent manner.

TCDD-mediated regulation of rat carboxylesterases has several notable characteristics. First, TCDD may exert differential effects on protein and mRNA levels for a carboxylesterase. The abundance of liver hydrolase B protein, for example, is increased by ~30% in the 3-µg/kg TCDD dosage group, whereas the abundance of hydrolase B mRNA is decreased by ~45% in the same group (bottom of Fig. 2Go). Second, TCDD exhibits a tissue-specific effect on the expression of carboxylesterases. In the liver, TCDD causes a dose-dependent decrease on the accumulated levels of mRNA encoding hydrolase A or B (Fig. 2Go). In the extrahepatic tissues, TCDD causes a biphasic change on the accumulated mRNA levels. The nonlethal doses cause a slight increase and the lethal dose causes a moderate decrease (Fig. 3Go). In addition, changes of enzyme protein and mRNA levels are highly correlated in the extrahepatic tissues in all dosage groups but not in the liver (e.g., the nonlethal dosage groups; Figs. 2 and 3GoGo). Third, TCDD coregulates the expression of many carboxylesterases. All electrophoretically distinct carboxylesterases detected by nondenaturing gel electrophoresis show a similar pattern of change in response to TCDD in all dosage groups (Fig. 6Go). Finally, TCDD differs from structurally related chemicals, notably 3-methylcholanthrene and ß-naphthoflavone, in regulating hepatic carboxylesterases. Based on Western analyses, 3-methylcholanthrene has negligible effects on hydrolase A expression, slightly decreases the levels of hydrolase B, and slightly increases hydrolase S (Morgan et al., 1994aGo; Yan et al., 1995cGo). ß-Naphthoflavone decreases the levels of all 3 enzymes, with hydrolase A being suppressed the least and hydrolase B the most (Morgan et al., 1994aGo; Yan et al., 1995cGo). At nonlethal doses (<10 µg/kg), TCDD induces all 3 enzymes, with hydrolase S being increased the most and hydrolase A the least. At the lethal dose, TCDD suppresses the expression of all 3 enzymes, with hydrolase S being suppressed the most and hydrolase B the least (Figs. 2 and 4GoGo). It should be emphasized that effects of 3-methylcholanthrene and ß-naphthoflavone were studied with a single dose known to induce CYP1A1 (Morgan et al., 1994aGo; Yan et al., 1995cGo). Whether they cause a dose-dependent biphasic change on carboxylesterase expression remains to be determined.

TCDD is distributed widely but not evenly among all tissues, which accounts for many differences in tissue-specific responses (Diliberto et al., 1995Go; Hurst et al., 2000Go). Liver has been found to contain 20–100 times as much TCDD as kidney, depending on doses used (Diliberto et al., 1995Go). Such a large difference in disposition may explain, at least in part, the difference of TCDD-mediated regulation on liver and kidney hydrolase B. The maximum induction of liver hydrolase B is detected in the 3-µg/kg dosage group, whereas the maximum induction of kidney hydrolase B is detected in the 30-µg/kg dosage group. Treatment with only 1 µg/kg of TCDD causes a 40% decrease in the accumulated mRNA levels of liver hydrolase B (bottom of Fig. 1Go). In contrast, the mRNA levels of kidney hydrolase B start to show a decrease at 90 µg/kg, the highest dose used in this study. Even in the same organ, different carboxylesterase genes are regulated to a slightly different extent. Based on Western immunoblotting analyses, the gene encoding hydrolase S is the most sensitive, hydrolase A is in the middle, and hydrolase B is relatively more resistant to TCDD treatment, particularly on the suppression phase (sublethal and lethal dosage groups; Figs. 2 and 4GoGo).

TCDD-mediated regulation on the expression of rat carboxylesterase genes is likely to involve multiple mechanisms. Treatment with nonlethal doses, for example, causes an increase in enzyme protein levels, and at the same time, causes a decrease in the accumulated mRNA levels for all carboxylesterases (Figs. 2 and 4GoGo). Such opposing effects suggest that both transcriptional and translational regulations are involved. TCDD-mediated transcriptional regulation of liver carboxylesterases apparently differs from that of liver CYP1A1. The accumulated levels of mRNAs encoding the carboxylesterases studied are markedly decreased in a dose-dependent manner, whereas the CYP1A1 gene is known to be upregulated by transcription activation of TCDD-occupied aryl hydrocarbon receptor (AhR; Figs 2 and 3GoGo; Parkinson, 1995Go; Whitlock, 1993Go). It remains to be determined whether decreased mRNA levels are due to transcriptional repression and/or increased degradation or whether the AhR is involved in such a suppressive regulation. Likewise, it remains to be determined whether the changes on the enzyme protein levels are primarily due to changes in the translation efficiency or degradation rate. In addition, although TCDD is metabolically resistant, secondary and tertiary events resulting from TCDD exposure likely contribute to the overall changes of the expression levels of carboxylesterases.

In summary, we report the regulation of carboxylesterase gene expression by TCDD, one of the most biologically active halogenated aromatic hydrocarbons. Nonlethal doses increase the levels of enzyme expression, but surprisingly, decrease the levels of mRNA. TCDD coregulates the expression of electrophoretically distinct carboxylesterases and exerts tissue-dependent effects. The dose dependence and the magnitude of changes on carboxylesterase differ significantly from those on CYP1A1. The differential effects in the expression of liver carboxylesterases and CYP1A1 suggest that TCDD regulates the expression of xenobiotic-metabolizing enzymes through various mechanisms. The different patterns of change in protein and mRNA levels suggest that TCDD regulates the expression of hepatic carboxylesterases by acting on both transcription and translation. TCDD is a widespread environmental contaminant and produces a plethora of biological effects, including tumor promotion, hepatoxicity, immunosuppression, and development (Birnbaum, 1994Go; Dragnev et al., 1995Go; Roman et al., 1998Go; Hurst et al., 2000Go). Carboxylesterases play an important role in the metabolism of numerous xenobiotics, activation of ester pro-drugs, and detoxication of organophosphates (Ewesuedo and Ratain, 1997Go; Ogasawara et al., 1995Go; Parkinson, 1995Go; Saltz, 1997Go; Satoh and Hosokawa, 1998Go; Stucky-Marshall, 1999Go). Therefore, regulation of carboxylesterases by TCDD has profound pharmacological and toxicological significance.


    ACKNOWLEDGMENTS
 
This work was partially supported by Grant ES07965 from the National Institute of Environmental Health Sciences (NIEHS) and a New Investigator Award from the American Association of Colleges of Pharmacy.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Biomedical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, RI 02881. Fax: (401) 874-5048. E-mail: byan{at}uri.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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