The Ah receptor, cytochrome P450IA1 mRNA induction, and aryl hydrocarbon hydroxylase in a human lymphoblastoid cell line.

The immunosuppressive and carcinogenic effects of aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (MC) on B lymphocytes of adult rodents and the induction of cytochrome P450IA1 and aryl hydrocarbon hydroxylase (AHH) in human mitogen-activated lymphocytes and B-lymphoblastoid cell lines are believed to be mediated by the Ah receptor. However, there has not been a direct demonstration or characterization of the Ah receptor in defined populations of any of these cells. We report here the detection and characterization of an abundant, high-affinity B lymphocyte Ah receptor in the AHH-inducible human B lymphoblastoid cell line BCR-5. Our results represent the first characterization of a human lymphocyte receptor in a well-defined lymphocyte population. Sucrose density gradient analysis of BCR-5 cytosols incubated with [3H]TCDD revealed a characteristic 9 S specific binding peak. The maximum concentration of Ah receptor was about 200 fmol/mg protein. Specific binding to the Ah receptor was also detected with [3H]MC and, to a lesser extent, with [3H]benzo[alpha]pyrene. The apparent binding affinity (Kd) for [3H]TCDD (determined by saturation analyses) was about 5 nM. A specific [3H]TCDD-Ah receptor complex which sedimented at 5 S was extracted from nuclei of BCR-5 cells incubated at 37 degrees with [3H]TCDD. The Ah receptor of BCR-5 cells is thus similar in characteristics to that identified in other cell lines. When BCR-5 cells were exposed in culture for 24 hr to increasing concentrations of benz[alpha]anthracene there was a concentration-dependent increase in induction and a good correlation (r = 0.98) between the level of induced AHH activity and the relative abundance of cytochrome P450IA1 mRNA. The human B lymphoblastoid cell line BCR-5, therefore, has a complete regulatory mechanism for Ah receptor-mediated induction of cytochrome P450IA1 that is essentially the same as that which has been well established in many rodent species. The accessibility of human blood lymphocytes and the ease of establishment of B lymphoblastoid cell lines from any donor provide a source of pure cultures of human B lymphocytes which can be grown continuously in vitro for the study of mechanisms related to Ah receptor-mediated cytochrome P450IA1 induction, immunosuppression and carcinogenesis.     

The cytosolic receptor binding affinities and AHH induction potencies of 29 polynuclear aromatic hydrocarbons

The dose-response rat hepatic cytosolic receptor-binding avidities, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction potencies in rat hepatoma H-4-II E cells in culture were determined for 29 polynuclear aromatic hydrocarbons. It was apparent that the magnitude of the EC50 values for these in vitro responses were strongly dependent on structure. Dibenz[a,h]anthracene (1.6 X 10(-8) M), 7-methylbenz[a]anthracene (1.6 X 10(-8) M), 3-methylcholanthrene (2.8 X 10(-8) M) and picene (4.5 X 10(-8) m) exhibited the highest affinity for the receptor protein and these compounds were only 5-fold less active the 2,3,7,8-tetrachlorodibenzo-p-dioxin (1 X 10(-8) M). All of the compounds which were active in the receptor-binding and monooxygenase enzyme-induction assays possessed one common structural feature, namely the presence of a phenanthrene structure fused with at least 1 benzo ring. The results also demonstrated that there was not any apparent correlation between the receptor-binding avidities and in vitro monooxygenase enzyme-induction potencies for the most active polynuclear aromatic hydrocarbons.     

Detection and characterization of a low affinity form of cytosolic Ah receptor in livers of mice nonresponsive to induction of cytochrome P1-450 by 3-methylcholanthrene

Ah "nonresponsive" mice (prototype, DBA/2) show no significant increase in hepatic P1-450 (P450IA1) when treated with 3-methylcholanthrene or other nonhalogenated polycyclic aromatic hydrocarbons. Potent halogenated aromatics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce P1-450 in liver of nonresponsive mice, but the dose required is approximately 15-fold higher than in "responsive" mice (prototype, C57BL/6). It was postulated several years ago that the genetic basis of nonresponsiveness was a "defect" in the Ah receptor, which normally binds TCDD and other inducers and mediates the induction process. Cytosolic Ah receptor hitherto had not been detectable in hepatic cytosol from nonresponsive mice. Using a modified sucrose gradient assay that we developed in studies on human tissue [Cancer Res. 47:4861-4868 (1987)], we now have detected cytosolic Ah receptor in nonresponsive mice. By saturation analysis, the concentration of specific binding sites for [3H]TCDD in hepatic cytosol from DBA/2J mice was (mean +/- SE) 55 +/- 6.6 fmol/mg of cytosolic protein (n = 21) compared with 133 +/- 7.1 fmol/mg (n = 21) in responsive C57BL/6J mice. Ah receptor also was detected in significant concentrations in other nonresponsive strains; SWR/J, AKR/J, RF/J, and DBA/2N. The sedimentation coefficient on sucrose gradients was the same (approximately 9 S) in nonresponsive as in responsive strains. The major difference in nonresponsive mice is that hepatic cytosolic Ah receptor has an apparent affinity for [3H]TCDD that is about 10-fold lower than in responsive strains; Kd in DBA/2J mice = 16 +/- 2.5 nM (n = 21) and Kd in C57BL/6J mice = 1.8 +/- 0.2 nM (n = 21). Thus, nonresponsive mice do possess the cytosolic Ah receptor in liver. However, the receptor is present in reduced concentration and appears to be a low affinity form, possibly as the result of a mutation in the gene(s) coding for the receptor protein(s).     

Comparison of hepatic and extra hepatic induction of cytochrome P4501A by graded doses of aryl hydrocarbon receptor agonists in Atlantic tomcod from two populations

Atlantic tomcod Microgadus tomcod from the Hudson River, New York, are exposed to high levels of polycyclic aromatic hydrocarbons (PAHs) and bioaccumulate mixtures of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and polychlorinatedfurans (PCDD/Fs). Previous studies demonstrated that hepatic cytochrome P4501A (CYP1A) mRNA was not inducible in tomcod from the Hudson River treated with single doses of PCB77 or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but was inducible with PAHs. In this study, we sought to determine if CYP1A mRNA was inducible with higher doses of these and other halogenated aromatic hydrocarbons (HAHs) in Hudson River tomcod and if decreased sensitivity to gene inducibility occurs across all tissues. Tomcod from the Hudson River and the cleaner Miramichi River, New Brunswick, were treated individually with graded doses of TCDD and coplanar PCBs (PCB77, PCB81, PCB126, PCB169) and profiles of hepatic CYP1A mRNA expression were compared between the two populations. CYP1A mRNA inducibility was also compared in multiple tissues of tomcod from the two rivers that were treated with PCB77. Additionally, hepatic CYP1A mRNA was characterized in Miramichi River tomcod treated with pairs of PCB congeners that included aryl hydrocarbon receptor (AHR) agonists and antagonists. Hepatic CYP1A mRNA was significantly inducible by all agonists in tomcod from the Miramichi River and TCDD and two of four PCBs in tomcod from the Hudson River. CYP1A mRNA was also significantly inducible in four of five tissues of tomcod from the Miramichi River but only in liver of Hudson River tomcod. In summary, CYP1A mRNA inducibility was approximately two orders of magnitude less sensitive in tomcod from the Hudson River than in those from the Miramichi River. But when achieved, maximum levels of CYP1A expression were similar in tomcod from the two populations. Co-administration of PCB126 and PCB77 did not produce significantly greater CYP1A mRNA induction than administration of PCB126 alone and co-administration of mono-ortho-substituted PCB105 significantly decreased CYP1A mRNA inducibility by PCB77. These results indicate that CYP1A mRNA expression is significantly inducible by HAHs in tomcod from the Hudson River and suggest that all components of the AHR pathway are present and functional, but that the pathway is less sensitive to activation than in tomcod from the Miramichi River. Our results also indicate that CYP1A mRNA levels in environmentally exposed fish may not reflect additive tissue burdens of PCB congeners.     

Serum withdrawal leads to reduced aryl hydrocarbon receptor expression and loss of cytochrome P4501A inducibility in PLHC-1 cells

Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several systems and in response to a variety of treatments. The significance of these findings is unclear, because the effects of such changes on subsequent responses to AHR ligands seldom have been measured. We tested the ability of changes in serum used in cell culture medium to alter expression of the AHR and induction of cytochrome P4501A (CYP1A) in PLHC-1 teleost hepatoma cells. Culture of early-passage cells in serum-free medium for 2 days led to a loss of CYP1A inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, culture in 10% delipidated calf serum increased the TCDD-induced levels of both CYP1A protein and enzymatic activity relative to levels in cells cultured in 10% complete calf serum. These effects were consistent between 8 and 24hr post-treatment, indicating that the kinetics of induction were unaffected. In cells cultured in serum-free medium for 1 and 2 days there was a progressive loss of CYP1A inducibility. This loss of response paralleled a time-dependent decline in AHR protein, as measured by specific binding of [3H]TCDD. Using an operational model for AHR action in PLHC-1 cells, the measured reduction in AHR could be shown to predict the loss of CYP1A induction. Expression of AHR protein was unaffected by culture in 10% delipidated serum. The effects of serum-free medium and delipidated serum were found only in early-passage cells; inducibility of CYP1A and expression of AHR protein in late-passage cells were unaffected by serum withdrawal. Comparison of early- and late-passage cells revealed a 2-fold greater rate of proliferation in the latter, suggesting that a growth advantage is coincident with loss of the serum-dependency of AHR expression. These results provide a quantitative link between changes in receptor expression and a downstream response, establishing a foundation for future studies of receptor expression and sensitivity to toxic responses in vitro and in vivo.     

Induction of cytochrome P450 1A in the American Eel by model halogenated and non-halogenated aryl hydrocarbon receptor agonists

Eels (Anguilla sp.) have phylogenetic, life history, and morphological characteristics which distinguish them from many other species that have been examined for cytochrome P450 1A (CYP1A) induction. Members of the family Anguillidae often occur in regions of the coastal environment that are heavily impacted by chemical contamination. Although eels have been suggested to be a useful species for biomonitoring, the sensitivity with which eel CYP1A is induced by aryl hydrocarbon receptor (AHR) agonists is not known. We investigated the dose-dependent induction of hepatic CYP1A in the American eel (Anguilla rostrata). Eels from an uncontaminated site were injected intra-peritoneally with the model AHR agonists ß-naphthoflavone (BNF), benzo[a]pyrene (B[a]P) or 3,3′,4,4-tetrachlorobiphenyl (TCB) at increasing doses (BNF at 0.1, 1, 10 and 100 mg/kg, B[a]P at 0.1, 1 and 10 mg/kg, and TCB at 0.1, 1, 10 and 20 mg/kg). All three compounds produced dose-dependent induction of CYP1A content and catalytic activity. An estimated ED₅₀ for induction of liver microsomal EROD activity by TCB was approximately 5 mg/kg, indicating only moderate sensitivity. At comparable doses of 1 and 10 mg/kg (or 3–4 and 30–40 μmol/kg), BNF and B[a]P had 2–3-fold greater effect than TCB in eliciting hepatic CYP1A induction. Injection of radiolabeled B[a]P and TCB resulted in similar dose-dependent concentrations of these compounds in eel liver, and the hepatic inducer concentrations and CYP1A levels were correlated positively. Eels collected from New Bedford Harbor (NBH), a Superfund site highly contaminated by polycyclic aromatic hydrocarbons and polychlorinated biphenyls, had levels of microsomal CYP1A protein and EROD activity that were equivalent to the highest levels induced experimentally. Eels from less contaminated sites had correspondingly less CYP1A expression. The responses to B[a]P or BNF as compared to TCB suggest a lower efficacy and/or potency for CYP1A induction by TCB which could involve differences in the mechanisms of responses to these compounds in eels. However, the moderate sensitivity and the CYP1A induction in NBH eels support suggestions that eels may be useful in monitoring more contaminated regions.     

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.     

Ah receptor mediating induction of cytochrome P450IA1 in a novel continuous human liver cell line (Mz-Hep-1). Detection by binding with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin and relationship to the activity of aryl hydrocarbon hydroxylase.

The Ah receptor regulates induction of cytochrome P450IA1 and mediates certain toxicities of polyhalogenated aromatics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It has been characterized previously in continuous cell lines, notably the mouse hepatoma line Hepa 1, the human squamous cell carcinoma line A431, and the human liver cell line Hep G2. The present work extends our knowledge of the Ah receptor in continuous human liver cell lines. Ah receptor can be detected in Mz-Hep-1, a hepatitis B virus-negative cell line derived from a Thorotrast-induced hepatocellular carcinoma. The mean concentration of Ah receptor in Mz-Hep-1 cells was 341 +/- 22 fmol/mg cytosol protein (mean +/- SEM, nine separate determinations). This is equivalent to approximately 30,000 sites per cell. The concentration of Ah receptor in Mz-Hep-1 cells is similar to that in Hepa 1 cells and approximately three times higher than that in Hep G2 cells. The Mz-Hep-1 Ah receptor sedimented in continuous sucrose gradients at approximately 9 S. Specificity of binding by [3H]TCDD was demonstrated by competitive binding of non-radiolabeled 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene (MC), and dibenz[a,h]anthracene in 50-fold molar excess. Phenobarbital, which is not a substrate for P450IA1, did not compete with [3H]TCDD for binding to Mz-Hep-1 Ah receptor. Dexamethasone and estradiol also did not compete with [3H]TCDD for binding, suggesting non-identity of Ah receptor with glucocorticoid or estrogen receptor. In separate experiments, glucocorticoid receptor was identified in Mz-Hep-1 cells. By Scatchard plot analysis, the apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Mz-Hep-1 Ah receptor was estimated to be 4.4 nM, compared to 0.8 nM in Hepa 1 cells. By Woolf plot analysis the Kd was 5.4 nM, compared to 1.2 nM in Hepa 1 cells. The [3H]TCDD.Ah receptor complex extracted from nuclei of Mz-Hep-1 cells incubated with [3H]TCDD in culture at 37 degrees sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was detectable in Mz-Hep-1 cells after pretreatment with inducing chemicals. Mz-Hep-1 cells have the highest concentrations of Ah receptor in any continuous human liver cell line thus far investigated. The Mz-Hep-1 Ah receptor is similar physicochemically to that described in murine systems. AHH activity is inducible in Mz-Hep-1 cells.     

Developmental toxicity of 4-ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH receptor isoforms and hepatic cytochrome P4501A metabolism

Polycyclic aromatic hydrocarbons (PAHs) derived from fossil fuels are ubiquitous contaminants and occur in aquatic habitats as highly variable and complex mixtures of compounds containing 2 to 6 rings. For aquatic species, PAHs are generally accepted as acting through either of two modes of action: (1) "dioxin-like" toxicity mediated by activation of the aryl hydrocarbon receptor (AHR), which controls a battery of genes involved in PAH metabolism, such as cytochrome P4501A (CYP1A) and (2) "nonpolar narcosis", in which tissue uptake is dependent solely on hydrophobicity and toxicity is mediated through non-specific partitioning into lipid bilayers. As part of a systematic analysis of mechanisms of PAH developmental toxicity in zebrafish, we show here that three tetracyclic PAHs (pyrene, chrysene, and benz[a]anthracene) activate the AHR pathway tissue-specifically to induce distinct patterns of CYP1A expression. Using morpholino knockdown of ahr1a, ahr2, and cyp1a, we show that distinct embryolarval syndromes induced by exposure to two of these compounds are differentially dependent on tissue-specific activation of AHR isoforms or metabolism by CYP1A. Exposure of embryos with and without circulation (silent heart morphants) resulted in dramatically different patterns of CYP1A induction, with circulation required to deliver some compounds to internal tissues. Therefore, biological effects of PAHs cannot be predicted simply by quantitative measures of AHR activity or a compound's hydrophobicity. These results indicate that current models of PAH toxicity in fish are greatly oversimplified and that individual PAHs are pharmacologically active compounds with distinct and specific cellular targets.     

Blockade of the aryl hydrocarbon receptor pathway triggered by dioxin, polycyclic aromatic hydrocarbons and cigarette smoke by Phellinus linteus

Environmental pollutants including halogenated and polycyclic aromatic hydrocarbons activate the aryl hydrocarbon receptor (AhR) and thereby cause a wide range of pathological changes. Development of AhR antagonists will be useful for prevention and treatment of diseases related to AhR activation. Towards this end, we aimed in the present study at seeking for potential inhibitors of the AhR pathway in mycelial extracts using the dioxin responsive element-based sensing via secreted alkaline phosphatase (DRESSA). Through the screening of 13 mycelia, extracts prepared from Phellinus linteus, Cordyceps militaris and Hericium erinaceum inhibited activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene or 3-methylcholanthrene. Subsequent studies revealed that only Phellinus linteus suppressed activation of AhR and AhR-dependent gene expression triggered by all of these agonists. Cigarette smoke is known to contain a number of halogenated and polycyclic aromatic hydrocarbons. We found that Phellinus linteus has the potential to block activation of AhR and AhR-dependent gene expression triggered by cigarette smoke. Furthermore, the inhibitory effect of Phellinus linteus on the AhR pathway was independent of; 1) depression of AhR or AhR nuclear translocator, and 2) induction of AhR repressor. We conclude that Phellinus linteus contains potent inhibitor(s) of AhR activation and may be useful for prevention of pathologies associated with aberrant activation of AhR.     

Tumor necrosis factor-alpha modulates effects of aryl hydrocarbon receptor ligands on cell proliferation and expression of cytochrome P450 enzymes in rat liver "stem-like" cells.

Various liver diseases lead to an extensive inflammatory response and release of a number of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). This cytokine is known to play a major role in liver regeneration as well as in carcinogenesis. We investigated possible interactions of TNF-alpha with ligands of the aryl hydrocarbon receptor (AhR) and known liver carcinogens, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and coplanar 3,3',4,4',5-pentachlorobiphenyl (PCB 126). These compounds have been previously found to disrupt cell cycle control in contact-inhibited rat liver WB-F344 cells, an in vitro model of adult liver progenitor cells. TNF-alpha itself had no significant effect on the proliferation/apoptosis ratio in the WB-F344 cell line. However, it significantly potentiated proliferative effects of low picomolar range doses of both TCDD and PCB 126, leading to an increase in cell numbers, as well as an increased percentage of cells entering the S-phase of the cell cycle. The combination of TNF-alpha with low concentrations of AhR ligands increased both messenger RNA (mRNA) and protein levels of cyclin A, a principle cyclin involved in disruption of contact inhibition. TNF-alpha temporarily inhibited AhR-dependent induction of cytochrome P450 1A1 (CYP1A1). In contrast, TNF-alpha significantly enhanced induction of CYP1B1 at both mRNA and protein levels, by a mechanism, which was independent of nuclear factor-kappaB activation. These results suggest that TNF-alpha can significantly amplify effects of AhR ligands on deregulation of cell proliferation control, as well as on expression of CYP1B1, which is involved in metabolic activation of a number of mutagenic compounds.     

Modulation of the in vivo covalent binding of the carcinogen benzo(a)pyrene to rat liver DNA by selective induction of microsomal and nuclear aryl hydrocarbon hydroxylase activity.

The influence of microsomal (mAHH) and nuclear (nAHH) aryl hydrocarbon hydroxylase activity on the covalent binding of tritiated benzo(a)pyrene to rat liver DNA was evaluated in vivo. Induction of mAHH was obtained after phenobarbitone treatment (180% of control), which increased DNA binding to 210%, but left the nAHH unchanged. mAHH and nAHH were slightly induced with dieldrin (130% and 120%), but the binding remained unchanged. The increasing effect of mAHH as well as the possibly decreasing effect of nAHH induction on the binding became obvious when the data of 11 individual rats were used to solve the equation Binding = aX(mAHH) + bX(nAHH) + c. Multiple linear regression analysis resulted in positive values for a and c, a negative value for b, and a multiple correlation coefficient R = 0.82. An influence of other enzymes involved in the metabolism of benzo(a)pyrene cannot be excluded. The study shows clearly that the binding of a foreign compound to DNA in vivo is not only dependent on microsomal enzyme activities but also on nuclear activities even if the latter are considerably lower than those of microsomes.     

Glucocorticoid regulation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferases, and NAD(P)H:quinone oxidoreductase in cultured fetal rat hepatocytes

The regulation of polycyclic aromatic hydrocarbon-inducible enzymes, cytochrome P450IA1, NAD(P)H:quinone oxidoreductase, and glutathione S-transferases, by glucocorticoids was investigated using primary fetal rat hepatocyte culture. Treatment of cells in culture with 1,2-benzanthracene (100 microM, 72 hr) resulted in 60-, 2-, and 6-fold increases in cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone reductase activities, respectively. The inductive effect of 1,2-benzanthracene on cytochrome P450IA1 and glutathione S-transferase (1-chloro-2,4-dinitrobenzene conjugation) activities was potentiated approximately 3- and 2- to 3-fold, respectively, when dexamethasone (0.01-1 microM) was included in the culture medium. In contrast, 1 microM dexamethasone was found not to potentiate the induction of NAD(P)H:quinone oxidoreductase activity by 1,2-benzanthracene. Treatment of cultured hepatocytes with dexamethasone alone, at concentrations of up to 100 microM, resulted in a 2- to 4-fold increase in glutathione S-transferase and NAD(P)H:quinone oxidoreductase activity. Both the induction of glutathione S-transferase activity by high concentrations of dexamethasone alone and the potentiation of 1,2-benzanthracene induction by lower concentrations of dexamethasone were observed for other steroids of the glucocorticoid class in conjunction with a variety of polycyclic aromatic hydrocarbons. Western immunoblot analyses indicated that low concentrations of dexamethasone (0.1-1 microM) potentiated 1,2-benzanthracene-dependent induction of cytochrome P450IA1, glutathione S-transferase Ya/Yc subunit and NAD(P)H:quinone oxidoreductase content. Additionally, increased glutathione S-transferase activity in response to concentrations of dexamethasone exceeding 1 microM was associated with concomitant increases in Ya/Yc and Yb subunit content. Potentiation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone oxidoreductase protein content by low concentrations of glucocorticoids and induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase by high concentrations of glucocorticoids alone indicates the importance of these endogenous compounds in the regulation of some hepatic enzymes involved in xenobiotic metabolism.     

Effect of aryl hydrocarbon hydroxylase induction on the in vivo covalent binding of 1-nitropyrene, benzo[a]pyrene, 2-aminoanthracene, and phenanthridone to mouse lung deoxyribonucleic acid

The effect of aryl hydrocarbon hydroxylase induction on the covalent binding of 1-nitropyrene (1-NP), benzo[a]pyrene (BaP), 2-aminoanthracene (2-AA), and phenanthridone (PNDO) to mouse lung DNA was investigated. Cytochrome P-450-dependent monooxygenases were induced in mouse lung by intratracheal instillation of BaP, Aroclor-1254, or coal gas condensate (CGC) 24 hr before instillation of [3H]BaP, [3H]-2-AA, [14C]-1-NP, or [14C]PNDO. All inducing agents increased the amount of radioactivity of [3H]BaP, [3H]-2-AA, and [14C]-1-NP or metabolites bound to DNA. However, pretreatment with BaP resulted in the highest amounts of radiolabels covalently bound to DNA. At 4 hr after instillation of radiolabels in BaP-induced mice, the amounts of [3H]BaP, [3H]-2-AA, and [14C]-1-NP bound to DNA were increased 5.4-, 5.2-, and 160-fold above that of control levels; the amount of 1-NP bound to DNA was fifty times higher than the amount bound by BaP. Labeled compounds were still bound to DNA 1 week after administration. [14C]PNDO was not bound to DNA in uninduced or induced mice. Based on the amount of labeled compounds bound to DNA, pretreatment of mice with BaP and CGC induced enzymes with similar specificities; however, enzymes induced by Aroclor were less effective in the metabolism of labeled compounds to DNA-bound products. These data show that specific cytochrome P-450-dependent monooxygenases are inducible in mouse lung and suggest that pre-exposure to inducing agents may be important in the potential toxicity to proximal tissues in direct contact with inhaled xenobiotics.     

Correlation between gene expression of aryl hydrocarbon receptor (AhR), hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1) and 1B1 (CYP1B1), and inducibility of CYP1A1 and CYP1B1 in human lymphocytes.

The relationships between gene expression of aryl hydrocarbon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1), 1B1 (CYP1B1), CYP1A1, and the inducibility of CYP1A1 and CYP1B1 were determined in 32 cultivated human lymphocytes. Cytochrome P450 induction was performed by incubating lymphocytes with benzanthracene. The relative gene expression levels were determined by quantitative real-time RT-PCR assay. We found that gender is an important confounding factor for gene expression in cultivated lymphocytes. AhR, CYP1A1 and CYP1B1 levels in noninduced lymphocytes were significantly higher in female nonsmokers than in male nonsmokers (p < 0.05). Nevertheless, CYP1A1 and CYP1B1 inducibility was lower in female nonsmokers. CYP1A1 inducibility was higher in male smokers than in male nonsmokers (p < 0.05). After controlling for gender and cigarette smoking, AhR levels positively correlated with CYP1B1 levels and CYP1A1 inducibility (p < 0.01 and p = 0.03, respectively). Arnt levels also correlated with CYP1B1 levels in induced lymphocytes (p < 0.01). However, AhR levels were negatively correlated with CYP1B1 inducibility. These data indicate that AhR expression associates with individual variation of CYP1A1 inducibility and CYP1B1 expression in cultivated lymphocytes. Furthermore, gender and cigarette smoking are important confounding factors for gene expression levels in cultivated lymphocytes.