Regular ArticleDevelopmental Aspects of Glucocorticoid Regulation of Polycyclic Aromatic Hydrocarbon-Inducible Enzymes in Rat Liver
Abstract
The expression of hepatic cytochrome P4501A1 (P4501A1), glutathione S-transferase Ya subunit (GST), and NAD(P)H:quinone oxidoreductase (QOR) proteins was evaluated in fetal, neonatal, and adolescent rats treated with 3-methylcholanthrene (MC) and the synthetic glucocorticoid dexamethasone (Dex) to elucidate the developmental aspects of glucocorticoid regulation of the induction of drug metabolizing enzymes by polycyclic aromatic hydrocarbons in vivo. These developmental states were chosen to represent either glucocorticoid deplete or replete conditions due to their differences in circulating glucocorticoid levels. Rats were treated with either MC (10 mg/kg body wt) or Dex (10 mg/kg body wt) or a combination of both and sacrificed 24 h later. In neonatal rats, the enzyme activities of P4501A1, GST, and QOR were increased by MC treatment approximately 65-, 1.4-, and 7-fold, respectively. The induction of these enzymes by MC was further potentiated an additional 2-, 1.5-, and 1.4-fold by concomitant Dex treatment. In adolescent male rats, Dex potentiated MC induction of P4501A1 activity (1.7-fold), but repressed MC induction of GST and QOR activities. When the protein contents for the three enzymes were measured by Western blot analyses, a positive correlation was observed with enzyme activities for all conditions except for the adolescent rat, where hepatic protein content of P4501A1 of rats treated with both MC and flex was not significantly increased above the level seen with 3-methylcholanthrene treatment alone. The levels of specific mRNA and transcriptional activity for cytochrome P4501A1, GST Ya isozyme, and QOR closely paralleled the changes seen in their protein content in the livers of neonatal and adolescent rats. Dexamethasone potentiation of P4501A1 expression at the protein and RNA level were clearly statistically significant in the neonatal rat, but not in the adolescent rat, suggesting that the circulating levels of glucocorticoids are sufficiently low during the neonatal period that the full expression of induction of P4501A1 was not attained in the absence of exogenously administered glucocorticoids. These data also demonstrate that glucocorticoids have differential effects on the induction of GST Ya subunit and QOR protein and RNA in the neonatal and adolescent state, possibly related to circulating levels of glucocorticoids.
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The effect of synthetic glucocorticoid, dexamethasone on CYP1A1 inducibility in adult rat and human hepatocytes
2005, FEBS LettersGlucocorticoids act synergistically with polycyclic aromatic hydrocarbons in increasing mRNA and protein levels of CYP1A1 in rat liver. The action of dexamethasone to modify CYP1A1 expression has been investigated in adult human hepatocytes. The effect of dexamethasone on the induction of CYP1A1 by 3-methylcholanthrene is different in rat and human liver cells. Dexamethasone potentiates the induction of CYP1A1 about 3- to 4-fold in rat cells. In human hepatocytes, it reduces CYP1A1 induction by 50–60% at enzyme protein level, while it does not have an effect on CYP1A1 mRNA amount.
Regulation of NAD(P)H:quininone oxidoreductase by glucocorticoids
2004, Toxicology and Applied PharmacologyPrevious studies in neonatal and adolescent rats as well as adrenalectomized rats have demonstrated that glucocorticoids regulate the expression of the rat NAD(P)H:quinone oxidoreductase gene (QOR). We used primary cultures of rat adult hepatocytes to document that added glucorticoids repress both the basal and 1,2-benzanthracene-induced expression of QOR mRNA by 65–70%. QOR enzyme activity and protein were concomitantly suppressed as well. The monotonic concentration response for repression of QOR gene products up to 100 μM DEX concentration demonstrated that the glucocorticoid receptor (GR) was most likely involved in this process. The lack of effect at higher concentration rules out a role for the Pregnane X receptor in this regulation by DEX. In addition, the anti-glucorticoid RU38486 blocked this negative regulation and the protein synthesis inhibitor cycloheximide had no effect on this repression process. Similar results of GR dependence were observed using a luciferase reporter construct containing the 5′-flanking region of the human QOR gene using HepG2 cells. Collectively, these results demonstrate that GR must directly participate in the negative regulation of QOR gene expression by dexamethasone and other glucocorticoids in vivo.
Evaluation of octamethylcyclotetrasiloxane (D<inf>4</inf>) as an inducer of rat hepatic microsomal cytochrome P450, UDP-glucuronosyltransferase, and epoxide hydrolase: A 28-day inhalation study
1998, Toxicological SciencesRepeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufinO-deethylase (EROD) and 7-pentoxyresorufinO-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4groups and more than 20-fold in the 700 ppm D4groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6β-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4can be described as a “PB-like” inducer of hepatic microsomal enzymes in the Fischer 344 rat.
Ligands of four receptors in the nuclear steroid/thyroid hormone superfamily inhibit induction of rat cytosolic aldehyde dehydrogenase-3 (ALDH3c) by 3-methylcholanthrene
1995, Biochemical PharmacologyUsing six ligands that bind to four different receptors in the nuclear steroid/thyroid hormone superfamily, we have examined the effects of these chemicals on induction of the cytosolic aldehyde dehydrogenase (ALDH3c) activity by 3-methylcholanthrene (3MC) in rat liver and uterus. In contrast to negligible activities in the untreated rat, ALDH3c enzyme activities are induced after a single dose of 3MC. Hepatic ALDH3c induction is decreased 60% to 90% when 3MC is administered together with any of the following ligands: estradiol, testosterone, progesterone, hydrocortisol, diethylstilbestrol, or tamoxifen. None of these same doses of chemicals, administered alone, affects ALDH3c enzyme activity. In addition, when these ligands are injected 2 days after 3MC, no changes are observed in liver or uterus ALDH3c induction. These results suggest that ligands that bind to different receptors in the nuclear steroid/thyroid hormone superfamily might inhibit the ALDH3c induction process by polycyclic aromatic hydrocarbons; the molecular mechanism(s) of this inhibitory effect is not yet understood.
Induction in vitro and complete coding region sequence of cytochrome P4501A1 cDNA from cultured whole rat conceptuses during early organogenesis
1994, Biochemical PharmacologyExposures of cultured whole rat conceptuses during organogenesis to 3-methylcholanthrene (MC; 0.025-25 μM), 5,6-benzoflavone (BNF; 5–100 μM) or benz[a]athracene (BA; 5–100 μM) were effected by placement of each of these “MC-type” inducing agents in the culture medium at the time of explantation on day 9.5 of gestation. Conceptuses were then cultured for 48 hr and evaluated on day 11.5 for increased expression of inducible conceptal cytochrome P450 (P450). The three agents each elicited concentration-dependent increases in 7,8-benzoflavone (ANF)-inhibitable ethoxyresorufin O-deethylase (EROD) activities and increased P4501A1 mRNA as detected by primer-specific reverse transcriptase-polymerase chain reaction (RT-PCR) in cell-free preparations of the treated, cultured conceptuses. At effective inducing concentrations, dysmorphogenic or other embryotoxic effects were not detectable. At 20 μM concentrations, the three agents exhibited roughly equal induction that was approximately equivalent in magnitude (6- to 13-fold) to that achieved previously with exposures to MC in utero. Additions to the culture medium of 2.5 to 10 μM concentrations of dexamethasone (DEX) did not alter significantly the magnitude of MC-elicited induction in vitro. Repeated full-length sequencing of an RT-PCR-amplified cDNA revealed a coding region sequence identical to that reported for the P4501A1 sequence from adult rat liver. The results provide a basis for investigations, in the absence of maternal influences, of the regulation of mammalian conceptal P4501A1 in intact tissues during organogenesis, a gestational period critical in terms of the dysmorphogenic and other embryotoxic effects of foreign organic chemicals. The results are also pertinent to studies of embryotoxicity, particularly to the transplacental carcinogenicity, mutagenicity and dysmorphogenicity of P4501A1 substrates.
Mechanisms of NADPH - cytochrome P450 oxidoreductase induction by dexamethasone in the H4IIE rat hepatoma cell line
2020, Canadian Journal of Physiology and Pharmacology