Department of Biomedical Sciences, University of Rhode Island, Kingston, Rhode Island 02881
Received May 10, 2001; accepted July 18, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: carboxylesterase; hydrolase A, B and S; 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1994a,b
; Parkinson, 1995
; Satoh and Hosokawa, 1998
). Some chemicals have similar effects across species, whereas other compounds show a profound species difference (Morgan et al., 1994a
,b
; Satoh and Hosokawa, 1998
; Zhu et al., 2000
). 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., 2000
). Structurally related compounds may also have different effects on carboxylesterase expression. Pregnenolone 16
-carbonitrile, a synthetic steroid structurally related to dexamethasone, causes either a moderate or a marked induction of several rat carboxylesterases (Hosokawa et al., 1993
). Finally, chemicals with the same or similar effects on the expression of CYP3 may differentially regulate the expression of carboxylesterases (Hosokawa et al., 1993
; Morgan et al., 1994a
,b
; Parkinson, 1995
; Satoh and Hosokawa, 1998
). 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., 1995c
). 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., 1995c
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1997). 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., 1994b). 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 mM1cm1 and 2.2 mM1cm1, respectively. All enzymatic determinations were conducted in triplicate and expressed as means ± SD.
Western immunoblotting and nondenaturing gel electrophoresis.
Microsomes (220 µg) were subjected to SDSPAGE 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., 1995c). 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., 1995c
).
Nondenaturing gel electrophoresis for staining esterase activity was conducted as described previously (Yan et al., 1995c)). 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., 1995c). 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., 1995c
). 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., 1995c
). The intensity (both Northern and Western blots) was determined with a laser scanning densitometer (Biomed Instruments, Inc., Fullerton, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
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., 1994a). 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 3
(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. 3
). The suppression of kidney hydrolase B was negligible compared with the control (bottom, left panel of Fig. 3
). In contrast to the liver, the extrahepatic tissues showed a high degree of correlation between the protein and mRNA levels (Figs. 2, 3
).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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. 2
). 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. 2
). 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. 3
). 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 3
). 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. 6
). 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., 1994a
; Yan et al., 1995c
). ß-Naphthoflavone decreases the levels of all 3 enzymes, with hydrolase A being suppressed the least and hydrolase B the most (Morgan et al., 1994a
; Yan et al., 1995c
). 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 4
). 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., 1994a
; Yan et al., 1995c
). 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., 1995; Hurst et al., 2000
). Liver has been found to contain 20100 times as much TCDD as kidney, depending on doses used (Diliberto et al., 1995
). 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. 1
). 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 4
).
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 4). 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 3
; Parkinson, 1995
; Whitlock, 1993
). 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, 1994; Dragnev et al., 1995
; Roman et al., 1998
; Hurst et al., 2000
). Carboxylesterases play an important role in the metabolism of numerous xenobiotics, activation of ester pro-drugs, and detoxication of organophosphates (Ewesuedo and Ratain, 1997
; Ogasawara et al., 1995
; Parkinson, 1995
; Saltz, 1997
; Satoh and Hosokawa, 1998
; Stucky-Marshall, 1999
). Therefore, regulation of carboxylesterases by TCDD has profound pharmacological and toxicological significance.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boyer, C. S., and Petersen, D. R. (1992). Enzymatic basis for the transesterification of cocaine in the presence of ethanol: Evidence for the participation of microsomal carboxylesterases. J. Pharmacol. Exp. Ther. 260, 939946.[Abstract]
Diliberto, J. J., Akubue, P. I., Luebke, R. W., and Birnbaum, L. S. (1995). Dose-response relationships of tissue distribution and induction of CYP1A1 and CYP1A2 enzymatic activities following acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. Toxicol. Appl. Pharmacol. 130, 197208.[ISI][Medline]
Dragnev, K. H., Nims, R. W., Fox, S. D., Lindahl, R., and Lubet, R. A. (1995). Relative potencies of induction of hepatic-metabolizing enzyme genes by individual PCB congeners. Toxicol. Appl. Pharmacol. 132, 334342.[ISI][Medline]
Ewesuedo, R. B., and Ratain, M. J. (1997).Topoisomerase I inhibitors. Oncologist 2, 359364.
Fan, F., Yan, B., Wood, G., Viluksela, M., and Rozman, K. K. (1997). Cytokines (IL-1ß and TNF-) in relation to biochemical and immunological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats. Toxicology 116, 916.[ISI][Medline]
Heymann, E. (1982). Hydrolysis of carboxylic esters and amides. In Metabolic Basis of Detoxication (W.B. Jakoby, J.R. Bend, and J. Caldwell, Eds.), pp. 291232. Academic Press, New York.
Hosokawa, M., Hattori, K., and Satoh, T. (1993). Differential responses of rat hepatic microsomal carboxylesterase isozymes to glucocorticoids and pregnenolone 16-carbonitrile. Biochem. Pharmacol. 45, 23172322.[ISI][Medline]
Hurst, C. H., DeVito, M. J., Setzer, R. W., and Birnbaum, L. S. (2000). Acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in pregnant Long-Evans rats: Association of measured tissue concentrations with developmental effects. Toxicol. Sci. 53, 411420.
Junge, W., and Krisch, K. (1975). The carboxylesterases/amidases of mammalian liver and their possible significance. CRC Crit. Rev. Toxicol. 3, 371435.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680685.[ISI][Medline]
Lu, A. Y., and Levin, W. (1972). Partial purification of cytochrome P450 and P448 from rat liver microsomes. Biochem. Biophys. Res. Commun. 46, 13341339.[ISI][Medline]
Morgan, E. W., Yan, B., Greenway, D., and Parkinson, A. (1994a). Regulation of two rat liver microsomal carboxylesterase isozymes: Species differences, tissue distribution, and the effects of age, sex, and xenobiotic treatment of rats. Arch. Biochem. Biophys. 315, 513526.[ISI][Medline]
Morgan, E. W., Yan, B., Petersen, D. R., Greenway, D., and Parkinson, A. (1994b). Purification and characterization of two rat liver microsomal carboxylesterases (hydrolases A and B). Arch. Biochem. Biophys. 315, 495512.[ISI][Medline]
Ogasawara, H., Nishio, K., Kanzawa, F., Lee, Y. S., Funayama, Y., Ohira, T., Kuraishi, Y., Isogai, Y., and Saijo, N. (1995). Intracellular carboxyl esterase activity is a determinant of cellular sensitivity to the antineoplastic agent KW-2189 in cell lines resistant to cisplatin and CPT-11. Jpn. J. Cancer Res. 86, 124129.[ISI][Medline]
Parkinson, A. (1995). Biotransformation of xenobiotics. In Casarett & Doull's Toxicology, the Basic Science of Poisons (C.D. Klaassen, Ed.), pp. 139162. McGraw-Hill, New York.
Roman, B. L., Pollenz, R. S., and Peterson, R. E. (1998). Responsiveness of the adult male rat reproductive tract to 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: Ah receptor and Arnt expression, CYP1A1 induction and Ah receptor down-regulation. Toxicol. Appl. Pharmacol. 150, 228239.[ISI][Medline]
Runge-Morris, M. (1998). Regulation of sulfotransferase gene expression by glucocorticoid hormones and xenobiotics in primary rat hepatocyte culture. Chem. Biol. Interact. 109, 315327.[ISI][Medline]
Saltz, L. B. (1997). Clinical use of irinotecan: Current status and future considerations. Oncologist 2, 402409.
Satoh, T., and Hosokawa, M. (1998). The mammalian carboxylesterases: From molecules to functions. Annu. Rev. Pharmacol. Toxicol. 38, 257288.[ISI][Medline]
Sonderfan, A. J., Arlotto, M. P., and Parkinson, A. (1989) Identification of cytochrome P450 isozymes responsible for testosterone oxidation in rat lung, kidney, and testis: Evidence that cytochrome P450a (P450IIAI) is the physiologically important testosterone 7-hydroxylase in rat testis. Endocrinology 125, 857866.[Abstract]
Stucky-Marshall, L. (1999). New agents in gastrointestinal malignancies: 1. Irinotecan in clinical practice. Cancer Nurs. 22, 212219.[ISI][Medline]
Whitlock, J. P. (1993). Mechanistic aspects of dioxin action. Chem. Res. Toxicol. 6, 754763.[ISI][Medline]
Yan, B., Matoney, L., and Yang, D. (1999). Human carboxylesterases in term placenta: Enzymatic characterization, molecular cloning, and evidence for the existence of multiple forms. Placenta 20, 599607.[ISI][Medline]
Yan, B., Yang, D., Brady, M. and Parkinson, A. (1994) Rat kidney carboxylesterase: Cloning, sequencing, cellular localization and relationship to rat liver hydrolase B. J. Biol. Chem. 269, 2968829696.
Yan, B., Yang, D., Brady, M., and Parkinson, A. (1995a). Rat testicular carboxylesterase: Cloning, sequencing, cellular localization, and relationship to liver hydrolase A. Arch. Biochem. Biophys. 316, 899908.[ISI][Medline]
Yan, B., Yang, D., and Parkinson, A. (1995b). Cloning and expression of hydrolase C, a member of the rat carboxylesterase gene family. Arch. Biochem. Biophys. 317, 222234.[ISI][Medline]
Yan, B., Yang, D., and Parkinson, A. (1995c). Rat serum carboxylesterase: Cloning, expression, regulation, and evidence of secretion from liver. J. Biol. Chem. 270, 1912819134.
Zhu, W., Song, L., Matoney, L., LeCluyse, E., and Yan, B. (2000). Dexamethasone differentially regulates the expression of carboxylesterase genes in humans and rats. Drug Metab. Dispos. 28, 186191.