Regulation of sulfotransferase gene expression by glucocorticoid hormones and xenobiotics in primary rat hepatocyte culture

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Abstract

In the rat liver, hydroxysteroid sulfotransferase-a (HST-a) and aryl sulfotransferase IV (ASTIV) represent two major rat hepatic sulfotransferases that are important to xenobiotic metabolism. Prototypic CYP1A1 and CYP2B/3A inducers regulate rat hepatic sulfotransferase gene expression although not necessarily in a coordinate direction. It has been previously reported that in vivo treatment with CYP1A1 inducer 3-methylcholanthrene (3-MC) suppresses rat hepatic HST-a mRNA expression in a dose-dependent manner. Similarly, HST-a and ASTIV mRNA levels become suppressed or induced, respectively, following in vivo treatment with phenobarbital (PB)-like CYP2B/3A inducers or prototypic CYP3A inducers such as glucocorticoid hormones. In the whole animal, sulfotransferase gene expression is modulated by members of the hypothalamic/pituitary–adrenal–gonadal hormone axis. However, studies in primary rat hepatocyte culture suggest that prototypic P450 inducers regulate HST-a and ASTIV gene expression directly at the level of the hepatocyte. Glucocorticoid-mediated sulfotransferase expression was compared with the regulation of tyrosine amino transferase (TAT), a gene that is transcriptionally regulated by ligand bound glucocorticoid receptor. It was found that lower doses of dexamethasone (DEX, 10−7 M) produced concomitant increases in ASTIV and TAT mRNA expression, whereas HST-a mRNA expression continued to rise as the DEX dose was increased through 10−5 M. When hepatocytes were co-incubated with DEX and antiglucocorticoid/antiprogestin RU-486, DEX-stimulated HST-a mRNA expression was not significantly inhibited by RU-486, but ASTIV and TAT mRNA expression were inhibited to a similar extent. The results suggested that ASTIV, like TAT, is likely regulated by a classical glucocorticoid receptor mediated mechanism, whereas HST-a is probably regulated by glucocorticoids via an alternative mechanism. In contrast to the positive effects of glucorticoid hormones, HST-a and ASTIV mRNA expression was negatively regulated by xenobiotics such as PB-like CYP2B/3A inducers or aryl hydrocarbon receptor (AhR) agonist CYP1A1 inducers. Incubation of primary cultured rat hepatocytes with PB or structurally dissimilar PB-like inducers clotrimazole, diphenylhydantoin, heptachlor, γ-hexachlorocyclohexane or 2,2′,4,4′,5,5′-hexachlorobiphenyl suppressed HST-a and ASTIV mRNA levels. Also, incubation of primary cultured rat hepatocytes with CYP1A1 inducer β-naphthoflavone or with archetypic AhR agonist, 2,3,7,8-tetrachloro-p-dioxin (TCDD) markedly suppressed HST-a and ASTIV mRNA expression. These data suggest that the rat HST-a and ASTIV genes are positively regulated by glucocorticoid hormones and negatively regulated by xenobiotics as a result of molecular and cellular mechanisms that act directly on the hepatocyte.

Introduction

HST-a and ASTIV represent the two major classes of sulfotransferase enzyme in the rat liver cytosol that are especially important to xenobiotic metabolism. The rat is a convenient mammalian model for studying the intricate processes that control sulfotransferase gene expression. Well designed studies to investigate the molecular and cellular mechanisms that regulate rat hepatic sulfotransferase gene expression may ultimately serve as a useful paradigm for understanding endocrine or xenobiotic determinants that modulate hepatic sulfotransferase gene expression in humans. Progress in molecular cloning will greatly facilitate our efforts to discern isoform-specific sulfotransferase-mediated effects within the aryl sulfotransferase (AST or ST1) and hydroxysteroid sulfotransferase (HST or ST2) gene subfamilies. Of the ST2 cDNAs that have been characterized thus far, the ST2 isoform (or isoforms) traditionally referred to as ‘HST-a’ is considered to be more abundant in the female relative to the male rat liver, readily catalyzes the sulfation of hydroxysteroids such as dehydroepiandrosterone (DHEA) and bioactivates hydroxy methyl polycyclic aromatic hydrocarbon carcinogens to highly reactive sulfate esters 1, 2. The ST1 subfamily members with ‘ASTIV’ characteristics include ST1A1 which catalyzes the sulfation of a variety of phenols and ST1C1 which, by comparison, more readily bioactivates the carcinogenic intermediate N-OH-2-acetylaminofluorene 3, 4. The ASTIV and HST-a cDNA probes that have been employed in the studies presented in this manuscript and in foundation studies 5, 6, most likely detect a number of individual ST1 and ST2 isoforms, respectively. A major challenge of future work in this field will be to characterize the biological significance of isoform-specific sulfotransferase-mediated events. In this report, the trivial names such as ‘ASTIV’ and ‘HST-a’ will be used to refer to the members of the ST1 and ST2 gene subfamilies that are recognized by the class-specific ASTIV and HST-a cDNA probes.

The HST-a and ASTIV genes are differentially regulated with respect to age- and gender-related signals 7, 8. In particular, hormones of the hypothalamic/pituitary–adrenal–gonadal hormone axis have been implicated in the molecular regulation of these genes. Rat hepatic HST-a mRNA expression is suppressible by androgens at the level of gene transcription [9]and gender-specific fluctuations in growth hormone levels may be partly responsible for gender-associated differences in hepatic HST-a gene expression 10, 11. Endocrinopathies such as diabetes also affect hepatic sulfotransferase expression. Hepatic estrogen and hydroxysteroid sulfotransferase gene expression become altered in genetically obese mice with non-insulin-dependent diabetes 12, 13, 14and it has been shown that rat hepatic HST-a mRNA and protein expression is significantly suppressed in streptozotocin-induced insulin-dependent diabetes [6].

Although the sulfotransferases are not generally recognized to be ‘xenobiotic inducible’, prototypic P450 inducers do produce biologically significant effects on rat hepatic sulfotransferase mRNA expression, protein levels and enzyme activity. For example, as typical CYP3A1 inducers, glucocorticoid hormones induce aryl sulfotransferase gene expression in cultured rat tracheobronchial cells 15, 16, alter the enzyme activity and mRNA expression of individual rat hepatic ST1 and ST2 isozymes following in vivo treatment [17]and induce HST-a and ASTIV mRNA expression in primary cultured rat hepatocytes [5]. In addition, prototypic phenobarbital (PB)-like CYP2B/3A (unpublished data) and AhR agonist CYP1A1 inducers1 suppress rat hepatic HST-a and ASTIV gene expression in vivo and in primary rat hepatocyte culture. The in vivo administration of glucocorticoid or xenobiotic test agents is complicated by the fact that these agents produce pleiotropic systemic effects. Therefore, more incisive studies in primary rat hepatocyte culture were designed to focus on defining the critical cellular factors and pathways that trans-activate sulfotransferase gene expression. This manuscript summarizes the most recent findings on the effects of glucocorticoid hormones and xenobiotic agents on rat hepatic HST-a and ASTIV gene expression in primary rat hepatocyte culture.

Section snippets

Reagents

Molecular biology reagents, steroid chemicals and PB-like CYP2B/3A inducing agents were purchased from Sigma (St. Louis, MO). TCDD and other AhR agonists were purchased from ChemSyn (Lenexa, KS). Radionuclide [α-32P]dATP (>3000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). cDNA probes were random prime labeled according to established methods. Collagenase (type I) was purchased from Worthington Biochemical (Freehold, NJ). Matrigel substratum for hepatocyte culture was purchased

Results and discussion

In vivo treatment with pharmacologic doses of glucocorticoid hormones results in significant increases in rat hepatic HST-a (mRNA and protein) and ASTIV mRNA expression [5]and stimulates HST-a enzyme activity (Table 1). These results are in agreement with those recently published by Liu and Klaassen which show that the mRNA and enzyme activity expression of many of the individual ST1 and ST2 enzymes in rat liver are increased following in vivo administration of glucocorticoid hormones [17].

The

Acknowledgements

The author would like to thank her research assistant Ms Kelly Rose for her technical contribution to this work and also her friend and colleague Dr Thomas A. Kocarek for his intellectual guidance and assistance with primary rat hepatocyte culture procedures. This investigation was supported by NIH grant ES05823 and NIEHS Center Grant ES06639.

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