Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of cytochrome P450 1A1, the aryl hydrocarbon receptor, and the aryl hydrocarbon receptor nuclear translocator in rat brain and pituitary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants causing a wide variety of pathological alterations, with the most severe being progressive anorexia and body weight loss. These features suggest a possible involvement of the nervous system and neuroendocrine-related organs including the pituitary gland. However, so far there is little evidence for direct effects of TCDD on these areas. In the present study, male Sprague-Dawley rats were treated with a single oral dose of TCDD (10 microg/kg) and euthanized 1, 3, or 28 days after treatment. The expression of cytochrome P450 1A1 (CYP1A1), the aryl hydrocarbon receptor (AHR), and the aryl hydrocarbon receptor nuclear translocator (ARNT) were analyzed in different brain regions and pituitaries using semiquantitative RT-PCR and Western blotting. Relative levels of CYP1A1 mRNA and protein were dramatically increased in the pituitary. A significant increase in CYP1A1 mRNA was also detected in all the brain regions examined including olfactory bulb, striatum-caudate, hypothalamus, hippocampus, cortex, cerebellum, and substantia nigra. The increase in the expression was time-dependent with the highest level observed 1 day after TCDD treatment. The AHR and ARNT mRNAs were detected in the same areas but in contrast to CYP1A1 the changes in AHR and ARNT mRNA expression were limited to the 28-day time point. The present results provide evidence for the presence of CYP1A1, AHR, and ARNT in the central nervous system and in the pituitary, suggesting that TCDD may exert a direct effect on these regions.     

Activation of CYP1A1 gene expression during primary culture of mouse hepatocytes

Expression of CYP1A1 mRNA in mouse hepatocytes in primary culture was investigated. The expression was obvious on day 3 of culture without addition of any known ligands of the aryl hydrocarbon receptor and increased with culture period. Removal of insulin from and addition of hydrogen peroxide to the medium enhanced and suppressed the expression, respectively. The CYP1A1 mRNA expression was also enhanced in the presence of anti-oxidant, t-butylhydroquinone, in the medium. Several kinds of kinase inhibitors markedly increased the CYP1A1 mRNA expression. In contrast, the inhibitory expression was prolonged in the presence of okadaic acid, a potent inhibitor of serine/threonine phosphatase PP1 and PP2. These observations suggest that there might be a repressive pathway in the regulation of CYP1A1 mRNA expression and that the presently observed expression pathway differs at several points from those previously reported, such as ligand-activated aryl hydrocarbon receptor- or omeprazole-mediated expression. Modulation of CYP1A2 mRNA expression after exposing hepatocytes to agents affecting phosphorylation pathways differed from that of CYP1A1 mRNA. This implies that regulatory pathways for CYP1A1 and CYP1A2 expression may differ.     

Developmental and tissue-specific expression of AHR1, AHR2, and ARNT2 in dioxin-sensitive and -resistant populations of the marine fish Fundulus heteroclitus.

Fundulus heteroclitus is a well-characterized marine fish model for studying aryl hydrocarbon toxicity. The F. heteroclitus population in New Bedford Harbor (NBH), a Superfund site in southeastern Massachusetts, exhibits heritable resistance to the toxic effects of planar halogenated aromatic hydrocarbons (PHAHs), including 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs). To investigate the role of the aryl hydrocarbon receptor (AHR) signal transduction pathway in PHAH resistance, we measured the relative levels of AHR1, AHR2, and ARNT2 mRNA in whole embryos at different developmental stages and in dissected tissues of adults, comparing expression of these genes in NBH fish with fish from a reference site (Scorton Creek, MA [SC]). Expression of both AHR1 and AHR2 mRNA increased during development, achieving maximum levels prior to hatching. Maximal embryonic expression of AHR1 was delayed relative to AHR2. Whole NBH and SC embryos exhibited no discernable differences in expression of these genes. As we have previously observed, adult SC fish expressed AHR2 and ARNT2 mRNA in all tissues examined, while AHR1 was expressed predominantly in brain, heart, and gonads. In contrast, AHR1 mRNA was widely expressed in NBH fish, appearing with unusual abundance in gill, gut, kidney, liver, and spleen. This AHR1 expression pattern was not observed in the lab-reared progeny of NBH fish, demonstrating that constitutive AHR1 expression in gill, gut, kidney, liver, and spleen is not a heritable phenotype. Furthermore, widespread AHR1 expression was not induced in reference-site fish by TCDD or PCB mixtures, suggesting that aberrant AHR1 expression is not simply a normal physiological response of contaminant exposure. These results identify ubiquitous AHR1 expression as an attribute unique to feral NBH F. heteroclitus, and they represent a first step in determining the regulatory mechanisms underlying this expression pattern and its possible role in TCDD resistance.     

Variation in induced CYP1A1 levels: relationship to CYP1A1, Ah receptor and GSTM1 polymorphisms

The genotypic basis of interindividual variation in levels of induced CYP1A1 activity has been investigated by screening both the CYP1A1 gene and the Ah receptor gene (AhR) for both previously described and novel polymorphisms. A 103-fold level of interindividual variation in induced CYPlA1 activity [ethoxyresorufin O-deethylase (EROD)] was observed in lymphocytes from a group of 30 Caucasian volunteers. High levels of induced EROD activity did not correlate with the presence of CYP1A1*2 or CYP1A1*4 alleles or with the GSTM1 null genotype. Novel CYP1A1 alleles with the base substitutions C4151T, G-469A and C-459T respectively, were detected by screening the coding exons and approximately 1 kb of upstream sequence in 20 individuals by single-strand conformational polymorphism (SSCP) analysis but none of the three novel alleles appeared to be associated with high induced CYP1A1 activity in the study group. Screening of the 11 exons of the AhR gene by SSCP analysis confirmed the existence of the previously described G1721A polymorphism in a Caucasian population and a novel allele (G1768A which results in the amino acid substitution V5701) was also detected. The novel allele was very rare in Caucasians though more common in African-Americans. Individuals with at least one copy of the G1721A AhR variant allele showed a significantly higher level of induced CYP1A1 activity compared with individuals negative for the polymorphism (P = 0.0001). A similar finding was obtained for induced CYP1A1 protein levels determined by immunoblotting. Levels of induced CYP1A1 activity were also found to show a sex difference with women showing a significantly lower induced activity compared with men. We conclude that genotypes for the G1721A AhR polymorphism and gender appear to be determinants of levels of induced CYP1A1 activity and that interindividual variation in levels of induced CYP1A1 activity appears to be associated more with regulatory factors than polymorphism in the CYP1A1 gene.     

Co-exposure to metals modulates CYP1A mRNA inducibility in Atlantic tomcod Microgadus tomcod from two populations

Populations from urbanized and industrialized sites are often exposed to mixtures of chemical contaminants including aromatic hydrocarbons (AHs) and heavy metals. The effects of mixtures of these contaminants on these populations are largely unknown. The Hudson River Estuary is highly contaminated with a variety of AHs including, PCBs and PAHs, and metals, and its population of Atlantic tomcod Microgadus tomcod bioaccumulates those which are persistent. The Hudson River's tomcod population exhibits resistance to persistent AHs as exemplified by significantly decreased inducibility of hepatic cytochrome P4501A (CYP1A) mRNA. We used hepatic CYP1A mRNA inducibility in tomcod from the Hudson River and a sensitive population to investigate the effects of acute co-exposure to metals on aryl hydrocarbon receptor (AHR)-mediated gene expression. Adult tomcod from the Hudson River and the cleaner Miramichi River were i.p. injected with one dose of benzo[a]pyrene (B[a]P) or coplanar PCB77 and graded doses of four metals, As, Cd, Cr, and Ni, and levels of hepatic CYP1A mRNA and protein were assayed. We observed no effects of metals treatment on basal levels of hepatic CYP1A mRNA expression, but all four metals significantly reduced CYP1A mRNA inducibility in tomcod from one or both populations. The magnitude of the inhibition of CYP1A mRNA inducibility differed among the metals and fish from the two populations. Also, the profile of the metals modulation of induced CYP1A mRNA showed differences that depended on the time after treatment of sacrifice. Our results demonstrate that co-exposure to several metals can impact inducible, but not basal levels of CYP1A expression and perhaps other toxicities mediated by the AHR.     

Regulation of cytochrome P-450 (CYP) 1B1 in mouse Hepa-1 variant cell lines: A possible role for aryl hydrocarbon receptor nuclear translocator (ARNT) as a suppressor of CYP1B1 gene expression

Cytochrome P-450 (CYP) 1B1 expression in mouse hepatoma (Hepa-1) wild-type (WT) cells was compared with responses in Hepa-1 variants LA1 and LA2, which, respectively, exhibit low aryl hydrocarbon receptor (AhR) level and defective AhR nuclear translocator (ARNT) protein. 10T1/2 mouse embryo fibroblasts express predominantly CYP1B1 and at a 100 times higher level than in Hepa-1 cells, whereas they express about 300-fold lower CYP1A1 than Hepa-1 cells. The expression of CYP1B1 in WT and LA1 variant, although at a much lower level, follows that of CYP1A1, reflecting their common regulation through the AhR. The LA2 (ARNT-defective) cells showed a major difference between CYP1B1 and CYP1A1 expression. Although CYP1A1 mRNA levels in LA2 were extremely low and unresponsive to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), basal CYP1B1 mRNA and protein were expressed at levels similar to those seen in TCDD-induced WT. The elevated basal CYP1B1 mRNA in LA2 cells decreased by 50% after transient transfection of ARNT cDNA, in parallel with substantial restoration of CYP1A1 induction. This implicates ARNT as a suppressor of CYP1B1 basal expression in Hepa cells. In transient CYP1B1-luciferase constructs in LA2 cells, ARNT shows stimulatory effects in the enhancer region but an inhibitory effect on the proximal promoter. Two CYP1B1 enhancer elements [xenobiotic-responsive element (XRE) 1/2 and XRE4] formed TCDD-unresponsive complexes of similar mobility to TCDD-stimulated AhR-ARNT complex with XRE5. However, because these two complexes were formed to the same extent in LA2 as in WT cells, they cannot be due to ARNT or contribute to ARNT-regulated suppression.     

CYP1A1 levels in lung tissue of tobacco smokers and polymorphisms of CYP1A1 and aromatic hydrocarbon receptor

Induction of a polycyclic aromatic hydrocarbon-metabolizing cytochrome P450 isoform CYP1A1 is regulated by aromatic hydrocarbon receptor (AHR). High inducibility of CYP1A1, possibly due to genetic polymorphisms, has been considered to be a risk factor for lung cancer in tobacco smokers. The relationship between low or high pulmonary expression of CYP1A1 and polymorphic genotypes of CYP1A1 and AHR was investigated in 73 active smokers. CYP1A1 expression was determined in surgical lung samples by measuring ethoxyresorufin O-deethylase (EROD) activity and by immunostaining for CYP1A1 protein. The most common allelic variants of CYP1A1 and AHR in Finns, i.e. the MspI variant (CYP1A1*2A), I462V variant (CYP1A1*2B), and -459C to T variant of CYP1A1 and the R554K variant (AHR*2) of AHR were studied using polymerase chain reaction based methods. EROD activity correlated positively with the daily cigarette consumption (r = 0.45). There was additional variation in EROD activity independent of the amount of smoking e.g. among those who smoked one pack per day until the day of operation, EROD activity ranged from 4-142 (median 48) pmol/min/mg. The frequencies of the MspI, 462V, and -459T variant alleles of CYP1A1 and 554K variant allele of AHR were 0.158, 0.055, 0.055 and 0.075, respectively. No differences were observed in the frequencies of polymorphic genotypes between the smokers with low and those with high expression, when the relationship was studied using a regression analysis adjusted for cigarette consumption. Our results thus indicate that the interindividual variation of CYP1A1 levels in smokers' lung tissue is not attributable to genetic polymorphisms of CYP1A1 or AHR tested in this study.     

Analysis of Ah receptor-ARNT and Ah receptor-ARNT2 complexes in vitro and in cell culture.

ARNT and ARNT2 proteins are expressed in mammalian and aquatic species and exhibit a high level of amino acid identity in the basic-helix loop-helix PER/ARNT/SIM domains involved in protein interactions and DNA binding. Since the analysis of ARNT2 function at the protein level has been limited, ARNT2 function in aryl hydrocarbon receptor (AHR)-mediated signaling was evaluated and compared to ARNT. In vitro, ARNT and ARNT2 dimerized equally with the AHR in the presence of 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) and ARNT2 outcompeted ARNT for binding to the AHR when expressed in excess. In contrast, activation of the AHR with 3-methylcholanthrene or benzo[a]pyrene resulted in predominant formation of AHR*ARNT complexes. ARNT2 expressed in Hepa-1 cell culture lines with reduced ARNT protein resulted in minimal induction of endogenous CYP1A1 protein compared to cells expressing ARNT, and mutation of the putative proline residue at amino acid 352 to histidine failed to produce an ARNT2 that could function in AHR-mediated signaling. However, the expression of ARNT2 in wild-type Hepa-1 cells reduced TCDD-mediated induction of endogenous CYP1A1 protein by 30%, even though AHR*ARNT2 complexes could not be detected in nuclear extracts. Western blot analysis of numerous mouse tissues and various cell culture lines showed that both endogenous ARNT and ARNT2 could be detected in cells derived from kidney, central nervous system, and retinal epithelium. Thus, ARNT2 has the ability to dimerize with the liganded AHR in vitro and is influenced by the activating ligand yet appears to be limited in its ability to influence AHR-mediated signaling in cell culture.     

Benzyl butyl phthalate induces necrosis by AhR mediation of CYP1B1 expression in human granulosa cells

We investigated the signaling pathway of the arylhydrocarbon receptor (AhR) on HO23 cells (immortalized human granulosa cells (hGC)) mediated by benzyl butyl-phthalate (BBP). BBP (1 μM) significantly increased the mRNA and protein levels of AhR, aryl hydrocarbon receptor nuclear translocator (ARNT) and cytochrome-P450 (CYP)1B1 in HO23 cells. Treatment with 3',4'-dimethoxyflavone (3',4'-DMF) or AhR siRNA significantly reduced AhR and CYP1B1, but CYP1A1 was not affected by 3',4'-DMF or AhR siRNA, suggesting that increases in CYP1A1 may not regulated by AhR. BBP induced the AhR fusion protein to localize and accumulate around the nucleus, and AhR heterodimerization with ARNT was observed in the nucleus by immunoprecipitation. Chromatin immunoprecipitation and reporter assays revealed the effect of BBP on CYP1B1, but not CYP1A1. Necrosis was significantly increased in HO23 cells after BBP treatment, and 3',4'-DMF, AhR siRNA or CYP1B1 siRNA knockdown blocked this phenomenon. These data suggest that BBP-induced HO23 cell necrosis is AhR and CYP1B1 dependent.