Aryl Hydrocarbon Hydroxylase Activity Research Articles

Failure of Ah Receptor to Mediate Induction of Cytochromes P450 in the CYP1 Family in the Human Hepatoma Line SK-Hep-1

The Ah receptor mediates the induction of cytochrome P450 1A1 (CYP1A1) and toxicities of 2,3,7,8-tetrachlorodibanzo-p-dioxin (TCDD). It has been detected in tissues of many species and in murine and human hepatoma lines. We show that the human hepatoma line SK-Hep-1 has cytosolic Ah receptor detectable by specific binding of [3H]TCDD. Concentrations of Ah receptor were low (mean = 43 ± 3 fmol/mg cytosol protein compared to 430 fmol/mg protein in Hepa-1); the estimated number of receptor sites per cell is ∼9000, compared to 35,000 in Hepa-1. Ah receptor in SK-Hep-1 cells was physicochemically similar to Ah receptor in C57BL/6 mouse liver and in other human hepatoma lines studied to date except that binding affinity for TCDD, the most avidly bound ligand, was lower (estimated Kd was 14 nM by Woolf plot analysis). Translocation of the Ah receptor–ligand complex to the nucleus was shown; binding of the activated Ah receptor–ligand complex to an XRE in the 5′-upstream region of the CYP1A1 gene was demonstrated by gel-shift analysis. However, after SK-Hep-1 cells were incubated with typical PAHs including 3-methylcholanthrene, benzanthracene, and dibenz(a,h)anthracene, each over a wide range of concentrations, no induction of aryl hydrocarbon hydroxylase activity was detectable. On Northern analysis, no message for human CYP1A1 was detected in mRNA prepared from noninduced SK-Hep-1 cells or from cells treated for 24 h with 13 μM dibenz(a,h)anthracene. Further analysis by RT-PCR did not detect the induction of CYP1A1, CYP1A2, or CYP1B1 message in response to 10−7 M TCDD, 10−5 M benzanthracene, or 10−5 M 3-methylcholanthrene. Transient transfection of reporter constructs containing either a minimal promoter or the CYP1A1 promoter fused to a reporter gene (luciferase) did not show any expression in response to increasing concentrations of TCDD up to 10−8 M. Estimation of the size of the transcripts for AhR and ARNT protein revealed normal sizes, 2.7 and 2.4 kb, respectively. Together, these data suggest that SK-Hep-1 cells express an Ah receptor defective at the level of trans-activation of gene expression. SK-Hep-1 is the first human hepatoma line described with a demonstrable defect in CYP1A1 or its regulation.

Activation of c-Ha-ras by benzo(a)pyrene in vascular smooth muscle cells involves redox stress and aryl hydrocarbon receptor.

Repeated cycles of vascular injury by benzo(a)pyrene (BaP) increase the onset and progression of atherosclerotic lesions in laboratory animals. This atherogenic response is partly mediated by activation of cis-acting antioxidant/electrophile response elements that enhance c-Ha-ras transcription in vascular smooth muscle cells (vSMCs). Activation of antioxidant/electrophile responsive cis-acting elements may depend on metabolism of BaP by cytochrome P450s to intermediates that induce oxidative stress and modulate gene expression. To test this hypothesis, we evaluated mitogen-activated c-Ha-ras expression in vSMCs treated with BaP or its metabolic intermediates alone, and in combination with agents that modulate cellular redox status. BaP (0.3 and 3 microM), BaP-3, 6-quinone (0.3 microM), or hydrogen peroxide (50 microM) enhanced serum-activated c-Ha-ras. Ellipticine (0.01 nM), a known inhibitor of cytochrome P450 metabolism and aryl hydrocarbon receptor (AhR) antagonist, inhibited c-Ha-ras induction by BaP (3 microM). Serum challenge of G(0) synchronized cultures of vSMCs with DL-buthionine-(S,R)-sulfoximine (0.1 mM), a depletor of cellular glutathione, increased c-Ha-ras mRNA levels during the early phase of the mitogenic response. Combined BaP/DL-buthionine-(S, R)-sulfoximine challenge was cytotoxic to the cells and inhibited c-Ha-ras expression, whereas up-regulation of antioxidant capacity by N-acetylcysteine (0.5 mM) precluded BaP-induced ras expression. BaP increased formation of reactive oxygen species and depleted cellular glutathione, but these changes did not correlate with the kinetics of c-Ha-ras induction. BaP did not enhance c-Ha-ras expression in vSMCs from AhR knockout mice, although aryl hydrocarbon hydroxylase activity was constitutively expressed in these cells. These results suggest that c-Ha-ras activation in vSMCs by BaP involves a redox-sensitive mechanism that is coupled to AhR receptor-dependent functions.

Bioavailability and biotransformation of 3,4,3′,4′-tetrachlorobiphenyl (TCB) in in situ intestinal preparations of uninduced and TCB induced channel catfish

Diet is a major route of exposure of polychlorinated biphenyls for aquatic animals living in contaminated environments. This study investigated systemic bioavailability and intestinal metabolism of [ 14C]- 3,4,3′,4′-tetrachlorobiphenyl (TCB) in channel catfish, relative to TCB micelle solubility and TCB preexposure, using in vitro methods and in situ isolated intestinal preparations. Results showed that micelle fatty acid composition affected TCB solubilization in vitro, as well as systemic bioavailability of [ 14C]-TCB delivered in situ. Systemic bioavailability of [ 14C]-TCB was reduced by TCB dietary preexposure at two doses. TCB was scantily metabolized in the catfish intestine to 2-OH-TCB in the in situ preparation. High-dose TCB preexposure compositely induced aryl hydrocarbon hydroxylase (AHH) activity compared to other treatments, but had no significant effect upon intestinal cytochrome P450 content and glutathione-S-transferase (GST) activity. These results suggest that TCB partitioning and bioavailability may be influenced by dietary fatty acid composition and TCB preexposure. Furthermore, these results indicate that the intestine can minimally influence the metabolite profile of absorbed TCB.

Effect of Micelle Fatty Acid Composition and 3,4,3',4'-Tetrachlorobiphenyl (TCB) Exposure on Intestinal [14C]-TCB Bioavailability and Biotransformation in Channel Catfish in Situ Preparations

Polychlorinated biphenyls are transferred in the diet along aquatic food chains. This study investigated the effect of dietary micelle composition and 3,4,3',4'-tetrachlorobiphenyl (TCB) exposure upon the subsequent systemic bioavailability and intestinal metabolism of [(14)C]-TCB in a catfish in situ intestinal preparation. Initial in vitro experiments examined the solubility of [(14)C]-TCB in micelles of varying fatty acid composition. Micelles composed of single fatty acids demonstrated greater [(14)C]-TCB solubility with those fatty acids of longer chain length. Similarly, micelles of the long-chain fatty acid, linoleic acid, solubilized more [(14)C]-TCB than mixed micelles formulated from equal amounts of myristic (14:0), palmitic (16:0), stearic (18:0), or linoleic (18:2) acids. Systemic bioavailability of [(14)C]-TCB (60 microM) from an in situ perfused intestinal preparation was 2.2-fold greater when delivered to the intestine in linoleic acid micelles as compared to the mixed micelle preparation. Catfish exposed in vivo to either 0.5 or 5.0 mg TCB/kg feed for 10 days resulted in a 45 to 47% decrease in the subsequent systemic bioavailability of [(14)C]-TCB in the in situ intestinal preparation. Total intestinal cytochrome P450 content was not significantly affected by TCB preexposure. Immunodetectable CYP1A was found only in the 5.0 mg TCB/kg diet treatment. Corresponding intestinal aryl hydrocarbon hydroxylase (AHH) activities were 2.46 +/- 1.16, 2.43 +/- 1.58, and 11.35 +/- 10.25 pmol/min/mg protein for the control, 0.5, and 5 mg TCB/kg diet groups, respectively. [(14)C]-TCB in the in situ preparation was metabolized to only a small degree upon a single pass through the intestinal mucosa of the catfish. High variability and low rates of metabolism precluded the association of the magnitude of metabolism with dietary TCB pretreatment. Analysis of tissue sample extracts demonstrated 4 minor peaks, 3 of which were tentatively identified by co-elution with standards as 2-OH-3,4,3',4'-TCB, 4-OH-3,5,3',4'-TCB, and 5-OH-3, 4,3',4'-TCB. A fourth remains unidentified. Histological changes in the intestine such as thinning of the submucosa and increased numbers of goblet cells were evident at the 5.0 mg TCB/kg diet dose. These results suggest that TCB intestinal bioavailability may be linked to micelle composition as well as TCB exposure history. Furthermore, single pass intestinal metabolism appears to be a minor contributor to the biotransformational modification of dietary TCB.

The relationship between aryl hydrocarbon hydroxylase activity and DNA adducts measured by 32P-postlabelling assay in lymphocytes of lung cancer patients

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the 32P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 108 nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min-1 10-6 cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n = 42, r = 0.25, p = 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n = 13, r = 0.70, p < 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n = 17, r = 0.44, p = 0.07) and GSTP1-AA genotype (n = 29, r = 0.37, p = 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes.

Aryl hydrocarbon hydroxylase activity in F-344 rats subchronically exposed to benzo(a)pyrene and fluoranthene through diet

In order to investigate the relationship between aryl hydrocarbon hydroxylase (AHH) activity and exposure to benzo[a]pyrene [B(a)p] and fluoranthene (FLA), AHH activities in liver tissues of male and female F-344 rats were determined. Based on a range-finding study, doses of 0, 5, 50, and 100 mg/kg B(a)p or 0, 150, 750, and 1500 mg/kg FLA were administered in the animal diet over a 90-day period. After dosing, animals were sacrificed, liver tissues were removed, and microsomes were isolated. AHH activities were determined by reverse-phase HPLC coupled with fluorescence detection using 3-hydroxy B(a)p, and trans-2,3-dihydroxy-1,10-epoxy-1,2,3,10b tetrahydrofluoranthene as the standards. A dose-dependent increase in enzyme activity was observed with increased B(a)p or FLA exposure in both males and females. Our results also demonstrate that B(a)p-exposed females possess a higher AHH activity than males, but there is no significant sex difference with regard to enzyme activity in the case of FLA at higher doses. Overall, our findings suggest that long-term exposure to the parent compound results in elevated levels of AHH activity, which may contribute to the formation of toxic reactive metabolites and subsequent symptoms in target organs.

Synthetic peptide mimics of a predicted topographical interaction surface: the cytochrome P450 2B1 recognition domain for NADPH-cytochrome P450 reductase.

In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B 1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116-134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.

Evidence for and against the presence of polynuclear aromatic hydrocarbon and 2,3,7,8-tetrachloro- p-dioxin binding proteins in the marine mussels, Bathymodiolus and Modiolus modiolus

Chemosynthetic mussels were collected in the vicinity of gas and petroleum seeps in the Gulf of Mexico. Aryl hydrocarbon hydroxylase (AHH) and glutathione S-transferase (GST) activities in the hepatopancreas and gill, respectively, were elevated in mussels collected at the site more highly contaminated with polynuclear aromatic hydrocarbons (PAHs). The aryl hydrocarbon receptor (AhR) and the 4S PAH binding protein (PBP) are ligand activated transcription factors which regulate expression of various genes including cytochrome P450 1A. The presence of these proteins was investigated in PAH-exposed mussels. RT-PCR analysis revealed only a 45.2% nucleotide similarity between the AhR2 from Fundulus heteroclitus and from mussel mRNA transcripts containing a putative member of the PAS gene family. Furthermore, in gel electrophoretic mobility shift and protein cross-linking assays utilizing mussel cytosol which was incubated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), no specifically-bound retarded band with a [ 32P]dioxin responsive element was clearly indicated. Likewise, sucrose density gradient analysis of cytosol incubated with [ 3H]TCDD did not give a specifically-bound 8-10S peak associated with the AhR complex. In contrast, incubation of mussel cytosol with [ 3H]benzo(a)pyrene (BaP) gave a 4S peak which was not displaced by TCDD but was nonspecifically decreased by excess BaP or benzo(ghi)perylene. The potential role of the 4S PAH binding protein in the induction of CYP1A-dependent activity in these species is currently unclear. Mussels collected from the North Sea were treated with BaP (5 mg/kg) or TCDD (20 μg/kg) for 48 h to investigate induction of enzyme activities in mussels from a pristine location. Western blot analysis indicated the presence of a 33 kDa protein when a PAH binding protein antibody was used, but no detectable induction of AHH or GST activity was observed in the treated mussels.

EFFECT OF LOW-LEVEL SHORT-TERM o-XYLENE INHALATION ON BENZO[a]PYRENE (BaP) METABOLISM AND BaP-DNA ADDUCT FORMATION IN RAT LIVER AND LUNG MICROSOMES

o -Xylene is a commonly used solvent that alters mixed-function oxidase (MFO) activity in an organ- and isozyme-specific pattern following intraperitoneal (ip) administration. Similar MFO alterations have been observed after ip or inhalation exposure to other methyl benzenes. These MFO alterations shifted the metabolism of the carcinogen benzo[a]pyrene (BaP) toward formation of toxication metabolites in lung. The purpose of this study was to determine whether o-xylene inhalation caused similar MFO changes and whether these alterations were reflected in altered BaP metabolism and BaP-DNA adduct formation. o-Xylene (300 ppm, 6h) decreased the activity of arylhydrocarbon hydroxylase (AHH) in lung. CYP2B1 activity (benzyloxyresorufin O -dealkylase; BROD), which is responsible for metabolism of BaP to relatively nontoxic metabolites, was decreased in lung, as was, to a lesser extent, CYP1A1 (ethoxyresorufin O-dealkylase; EROD), which is responsible for metabolism of BaP to reactive/toxic metabolites. The BROD/EROD ratio, an indirect indicator of the pattern of BaP toxication/detoxication, was decreased in lung, suggesting that BaP metabolism is shifted toward toxication. No MFO alterations were observed in liver. In lung microsomes, o-xylene increased formation of 7,8-BaP-diol, while 9,10-BaP-diol, 3-OH BaP, and 9-OH BaP were decreased. In liver, o-xylene increased 9-OH BaP formation, while 4,5- and 9,10-diols as well as total diols were decreased. The toxication/detoxication ratios for BaP individual and total metabolite groups were increased in lung microsomes and unaltered in liver. The major BaP-DNA adduct, BaP diol epoxide-N 2-deoxyguanosine, was increased in lung but decreased in liver microsomes from o-xylene-exposed rats. Four minor BaP-DNA adducts were formed in lung and three in liver, only one of which (liver adduct 3) was decreased. The o-xylene-induced increase in BaP adduct formation in lung and decrease in liver indicate that coexposure to organic solvents such as the methyl benzenes may alter the carcinogenesis of BaP, or other PAHs, in an organ-specific fashion.

Oil shale processing as a source of aquatic pollution: monitoring of the biologic effects in caged and feral freshwater fish.

The biologic effects of the oil shale industry on caged rainbow trout (Oncorhynchus mykiss) as well as on feral perch (Perca fluviatilis) and roach (Rutilus rutilus) were studied in the River Narva in northeast Estonia. The River Narva passes the oil shale mining and processing area and thus receives elevated amounts of polycyclic aromatic hydrocarbons (PAHs), heavy metals, and sulfates. The effects of the chemical load were monitored by measuring cytochrome P4501A (CYP1A)-dependent monooxygenase (MO) activities [7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase (AHH)] as well as conjugation enzyme activities [glutathione S-transferase (GST) and UDP-glucuronosyltransferase] in the liver of fish. CYP1A induction was further studied by detecting the amount and occurrence of the CYP1A protein. Histopathology of tissues (liver, kidney, spleen, and intestine) and the percentage of micronuclei in fish erythrocytes were also determined. Selected PAHs and heavy metals (Cd, Cu, Hg, and Pb) were measured from fish muscle and liver. In spite of the significant accumulation of PAHs, there was no induction of MO activities in any studied fish species. When compared to reference samples, AHH activities were even decreased in feral fish at some of the exposed sites. Detection of CYP1A protein content and the distribution of the CYP1A enzyme by immunohistochemistry also did not show extensive CYP1A induction. Instead, GST activities were significantly increased at exposed sites. Detection of histopathology did not reveal major changes in the morphology of tissues. The micronucleus test also did not show any evidence of genotoxicity. Thus, from the parameters studied, GST activity was most affected. The lack of catalytic CYP1A induction in spite of the heavy loading of PAHs was not studied but has been attributed to the elevated content of other compounds such as heavy metals, some of which can act as inhibitors for MOs. Another possible explanation of this lack of induction is that through adaptation processes the fish could have lost some of their sensitivity to PAHs. Either complex pollution caused by oil shale processing masked part of the harmful effects measured in this study, or oil shale industry did not have any severe effects on fish in the River Narva. Our study illustrates the difficulties in estimating risk in cases where there are numerous various contaminants affecting the biota.

Oil Shale Processing as a Source of Aquatic Pollution: Monitoring of the Biologic Effects in Caged and Feral Freshwater Fish

The biologic effects of the oil shale industry on caged rainbow trout (Oncorhynchus mykiss) as well as on feral perch (Perca fluviatilis) and roach (Rutilus rutilus) were studied in the River Narva in northeast Estonia. The River Narva passes the oil shale mining and processing area and thus receives elevated amounts of polycyclic aromatic hydrocarbons (PAHs), heavy metals, and sulfates. The effects of the chemical load were monitored by measuring cytochrome P4501A (CYP1A)-dependent monooxygenase (MO) activities [7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase (AHH)] as well as conjugation enzyme activities [glutathione S-transferase (GST) and UDP-glucuronosyltransferase] in the liver of fish. CYP1A induction was further studied by detecting the amount and occurrence of the CYP1A protein. Histopathology of tissues (liver, kidney, spleen, and intestine) and the percentage of micronuclei in fish erythrocytes were also determined. Selected PAHs and heavy metals (Cd, Cu, Hg, and Pb) were measured from fish muscle and liver. In spite of the significant accumulation of PAHs, there was no induction of MO activities in any studied fish species. When compared to reference samples, AHH activities were even decreased in feral fish at some of the exposed sites. Detection of CYP1A protein content and the distribution of the CYP1A enzyme by immunohistochemistry also did not show extensive CYP1A induction. Instead, GST activities were significantly increased at exposed sites. Detection of histopathology did not reveal major changes in the morphology of tissues. The micronucleus test also did not show any evidence of genotoxicity. Thus, from the parameters studied, GST activity was most affected. The lack of catalytic CYP1A induction in spite of the heavy loading of PAHs was not studied but has been attributed to the elevated content of other compounds such as heavy metals, some of which can act as inhibitors for MOs. Another possible explanation of this lack of induction is that through adaptation processes the fish could have lost some of their sensitivity to PAHs. Either complex pollution caused by oil shale processing masked part of the harmful effects measured in this study, or oil shale industry did not have any severe effects on fish in the River Narva. Our study illustrates the difficulties in estimating risk in cases where there are numerous various contaminants affecting the biota.

Formation and persistence of nucleotide alterations in rats exposed whole-body to environmental cigarette smoke.

The assessment of pathological effects produced by environmental tobacco smoke in humans is controversial in epidemiological studies. On the other hand, animal models are poorly sensitive to smoke carcinogenicity. We designed an experimental study assessing the tissueselective formation and persistence of DNA adducts in smoke-exposed rats. Sprague-Dawley rats were exposed for 6 h per day, 5 days per week, to environmental smoke resulting from a mixture of sidestream and mainstream smoke generated from Kentucky 2R1 reference cigarettes. The total particulate matter was in the range of 73-93 mg/m(3). DNA adducts were measured by (32)P-post-labelling in rat organs (lung, heart, liver, bladder and testis), tissues (dissected tracheal epithelium) and cells [isolated bronchoalveolar lavage (BAL) cells]. A time-related increase of (32)P-post-labelled DNA modifications was detectable by autoradiography, in the form of massive diagonal radioactive zones and individual spots. Top levels were reached after 4-5 weeks of exposure. The ratio of smoke-induced DNA adducts to the background levels detected in sham-exposed rats was 11.2 in the tracheal epithelium, 10.4 in BAL cells, 7.3 in the heart, 6.3 in the lung, 5.1 in the bladder, 1.9 in the testis and 1. 1 in the liver. Appearance of DNA adducts in the lung was also revealed by synchronous fluorescence spectrophotometry. Smoke-related oxidative damage was demonstrated by a significant enhancement of 8-hydroxy-2'-deoxyguanosine in lung DNA. In parallel, there was a time-related induction of lung microsomal arylhydrocarbon hydroxylase activity, an elevation in cytosolic glutathione S-transferase activity, and a moderate but progressive and significant depletion of reduced glutathione. After discontinuing exposure to environmental cigarette smoke for 1 week, DNA adduct levels significantly dropped in the lung, tracheal epithelium, heart and bladder. The decrease was evident but not statistically significant in BAL cells, and was negligible in the heart. The selective localization and the differential persistence of these promutagenic nucleotide modifications in rat organs, tissues and cells suggest that exposure to environmental cigarette smoke, at least under the high exposure regimens used in experimental studies, may pose a potential risk of developing mutation-related diseases.

Benzo(a)pyrene diol-epoxide-DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) in human white blood cells from smokers and non-smokers

Benzo(a)pyrene diol-epoxide (BPDE)-DNA adducts and aryl hydrocarbon hydroxylase (AHH) activity were determined in white blood cells (WBCs) obtained from non-smokers, ex-smokers and smokers. DNA adduct levels ranged from non-detectable (ND) to 8.8 per 10 8 nucleosides in all samples analysed. The mean level in WBC from smokers (1.93±0.55 mean±SE) was similar to that from non-smokers (1.18±0.40) and ex-smokers (1.83±0.50). The distribution frequency of AHH activity in non-smokers was different from that in smokers, while 50% of non-smokers had AHH activity ranging from ND to 20 fmol mg -1 min -1 , This low AHH activity was not found in smokers; no correlation was found between AHH activity and tobacco consumption (r=0.097; P = 0.76; n=12). On the contrary, AHH activity was correlated with BPDE-DNA adduct levels in WBCs from the same subjects (r=0.457; P=0.019; n=26). Our findings indicate that there are limitations to the use of WBCs as surrogate cells for determining internal PAH exposure in target organs.

The Existence of the 4S Polycyclic Aromatic Hydrocarbon-Protein Binding in 14-Day-Old Chick Embryo Liver

Cytochrome P-450IA1, the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH), is regulated by two high-affinity binding proteins, the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein which primarily binds PAHs and the 8S Ah (dioxin) receptor which binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to determine whether the 4S protein existed in 14-day-old chick embryo liver when AHH activity is maximal to determine if they are linked as is the 8S Ah receptor and to confirm the existence of the dioxin receptor by investigating their ligand binding characteristics in the presence and absence of sodium molybdate, an agent that stabilizes steroid hormone receptors and partially stabilizes the dioxin receptor. Competitive ligand binding studies were performed with liver cytosol from livers of male 14-day-old chick embryos using [3H]-benzo[a]pyrene (B[a]P) or [3H]-TCDD in the presence and absence of a 200-fold excess of B[a]P, benzo[e]pyrene (B[e]P), 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed using sucrose gradient analysis. In the absence of molybdate, the 4S PAH-binding protein had high affinity for B[a]P, B[e]P, 3-MC, but very low affinity for TCDBF; the Ah receptor exhibited high affinity for TCDBF. In the presence of sodium molybdate, the Ah receptor was stabilized while the 4S PAH-binding protein was relatively unaffected. These results affirm the existence of two distinct PAH-binding proteins in 14-day-old chick embryo liver cytosol and suggest a linkage of the 4S protein to AHH.

Correlations between cytochrome P450, haem synthesis enzymes, and aromatic compounds in flounder (Platichthys flesus) from the Baltic Sea

Concentrations of cytochrome P450 (CYP), activities of aryl hydrocarbon hydroxylase (AHH), haem synthetase (HEM-S), and 5-aminolevulinic acid synthetase (ALA-S) in liver as well as fluorescent aromatic compounds (FACs) in bile of flounder (Platichthys flesus) were studied in samples collected during a synoptic survey along a transect in the Baltic in autumn 1994. Cytochrome P450 concentrations and activities of the haem synthesis enzymes HEM-S and ALA-S were highest in the northwestern areas and among these parameters significant (p<0.01) positive correlations were found. Highest activities of microsomal AHH were measured in flounder from areas near major ports in the centre of the transect. Concentrations of FACs in bile correlated significantly with AHH activities. The results indicate that CYP concentrations and ALA-S and HEM-S activities, which are generally considered the principal biomarkers of carcinogenic pollution, may give a different perspective on carcinogenesis in fish.

INHIBITION OF RAT LUNG MIXED-FUNCTION OXIDASE ACTIVITY FOLLOWING REPEATED LOW-LEVEL TOLUENE INHALATION: POSSIBLE ROLE OF TOLUENE METABOLITES

Toluene is a commonly used solvent that has been shown to alter mixed-function oxidase (MFO) activity, in an organ- and isozyme-specific pattern, following intraperitoneal administration. The purpose of this study was to determine whether similar changes occurred following repeated, low-level inhalation exposure, and to investigate the role of toluene metabolites in these alterations. Exposure to 375 ppm toluene, 6 h/d for up to 5 d, resulted in significant inhibition of the activity of pulmonary arylhydrocarbon hydroxylase (AHH), cytochrome P-4502B1 (CYP2B1), and CYP4B1, but not CYP1A1. After exposure to lower toluene levels (125 ppm, 6 h/ d, 3 d), the activities of lung AHH, CYP2B1, and CYP4B1 were also significantly decreased, but in a dose-related manner. MFO activity was not consistently altered in liver. Control pulmonary or liver microsomes were incubated with various concentrations (0.01-10 m M) of toluene or its metabolites and CYP2B1, CYP1A1, and/or CYP4B1 activities were subsequently determined. Benzaldehyde produced a significant dose-related inhibition in the activity of all three lung P-450s examined (IC50 10-3 M). Toluene was found to be a more potent inhibitor of lung CYP2B1 and CYP1A1 (IC50, 10-4 M) than benzaldehyde, but neither toluene nor benzyl alcohol was an effective inhibitor of lung CYP4B1. Toluene and its metabolites were weaker inhibitors of CYP1A1 than of CYP2B1. For CYP2B1 and CYP1A1, the order of inhibitory potency was toluene > benzaldehyde > benzyl alcohol and suggests that both the parent molecule and its metabolites may act in concert to inhibit catalytic activity of these cytochromes. The MFO inhibition seen after repeated low-level toluene inhalation exposure could result in altered metabolic profiles of other xenobiotics in an organ-specific fashion.

The relationship between CYP1A1 aryl hydrocarbon hydroxylase activity and lung cancer in a Japanese population.

Because aryl hydrocarbon hydroxylase (AHH) is considered to be responsible for the activation of benzo(a)pyrene and other polyaromatic hydrocarbons in cigarette smoke to carcinogens, it is important to examine CYP1A1 (AHH) activity in the determination of susceptibility to lung cancer. We investigated AHH activity in peripheral mitogen-treated lymphocytes in 108 lung cancer patients and 95 healthy control individuals. Non-induced AHH activity was detectable in all the samples. AHH inducibility (3-methylcholanthrene-induced/non-induced AHH activity) showed a very wide interindividual variation as well as non-induced AHH activity. No significant associations were found between adjusted AHH activity and histologic type of tumor among lung cancer patients. Adjusted AHH inducibility of genotype C [geometric mean and 95% confidence interval (CI); 15.56 and 11.69-20.71] in MspI polymorphism was significantly higher than those of the other two genotypes (P = 0.0001), while no significant difference was observed between genotypes A (4.76 and 3.82-5.93) and B (5.60 and 4.57-6.86). On the other hand, non-induced AHH activity of genotype Val/Val (0.121 and 0.082-0.178 pmol/min/10(6) cells) in isoleucine-valine (Ile-Val) polymorphism was significantly higher than those of genotypes Ile/Ile (0.042 and 0.034-0.052 pmol/min/10(6) cells) and Ile/Val (0.040 and 0.030-0.053 pmol/min/10(6) cells) (P < 0.0001). Even after controlling for age, cigarettes smoked per day and season of the year, high AHH inducibility (7.0 < versus 0 < < or = 3.0: OR and 95 %CI, 12.4 and 2.88-53.4) was an independent risk factor for lung cancer. The data indicate that high AHH inducibility may strongly associate with the susceptibility to lung carcinogenesis.

Modifications in the carcinogen-metabolizing capacity of mouse liver treated with N-nitroso compounds.

One tenth of the LD50 as a single dose of various N-nitroso compounds (N-nitrosodimethylamine; NDMA, N-nitrosodiethylamine; NDEA, N-nitrosoethylpropylamine; NEPA, N-nitrosodipropylamine; NDPA, N-nitrosomethylethylamine; NMEA, N-nitroso-methylbutylamine; NMBA and N-nitrosoethylbutylamine; NEBA) was administrated into male mice. This dose markedly increased the hepatic contents of cytochrome P450 and cytochrome b5 and activities of NADPH-cytochrome c reductase and aryl hydrocarbon hydroxylase (AHH). The highest increase in the activity of cytochrome P450 (+142% relative to the control value) was shown in animals treated with either N-nitrosoethylpropylamine or N-nitrosodiethylamine. On the other hand, the lowest increase in the activity (+16%) was revealed in animals treated with N-nitrosodimethylamine (not significant compared to the control value). Cytochrome b5 content was increased by 190% of the control value in mice treated with N-nitrosomethylbutylamine, while N-nitrosodibutylamine induced the lowest increase (+20%). The maximum increase (+182%) in the activity of aryl hydrocarbon hydroxylase was shown in animals which received N-nitrosomethylbutylamine, while the lowest increase (+23%) in animals which received N-nitrosodiethylamine. The activity of hepatic AHH was also increased above the control value in animals treated with NDMA, NEBA NDPA, NMEA and NDBA by 138, 98, 90, 89 and 69%, respectively. Identically, NADPH-cytochrome c reductase activity was increased in animals which received NEPA, NMBA, NDMA, NMEA, NDPA, NEBA and NDEA by 202, 150, 110, 95, 94, 77 and 37%, respectively.

Quantitation of CYP1A expression in two flatfish species showing different prevalences of contaminant-induced hepatic disease

English sole ( Pleuronectes vetulus) and starry flounder ( Platichthys stellatus) are two sympatric flatfish species which show markedly different responses to chemical contaminant exposure. Whereas English sole develop hepatic neoplasms, the prevalences of which are strongly linked to exposure to polycyclic aromatic hydrocarbons (PAHs), evidence of neoplasia is virtually nonexistent in starry flounder, even those residing in areas with very high levels of PAH in the sediments. Because PAHs are activated to genotoxic forms by the action of cytochrome P450 1A (CYP1A) in teleosts, we are examining the hypothesis that variation in the hepatic expression of this major inducible cytochrome P450 isozyme may contribute to the differential contaminant response. These two species were captured in the Duwamish Waterway, a contaminated area of Puget Sound, Washington. Catalytic activity of CYP1A [determined as aryl hydrocarbon hydroxylase (AHH) activity] was measured in the liver to quantitatively assess the relative induction of CYP1A in these two species, and AHH activity was significantly higher in English sole than in starry flounder ( p = 0.015). Cellular expression of CYP1A was determined by immunohistochemical localization of tissues, using an avidin-biotin peroxidase complex method, with polyclonal rabbit anti-cod CYP1A. The results showed a markedly reduced expression of CYP1A in hepatocytes of starry flounder, which is consistent with the apparent resistance of this species to the development of hepatocellular neoplasia. This reduced expression of CYP1A in hepatocytes of contaminant-exposed fish was previously seen in oyster toadfish from the Elizabeth River, Virginia, and this species is also apparently resistant to hepatocellular neoplasia.

Cytochrome P450 system and heme synthesis enzymes activity in flounder liver as biomarkers of marine environments pollution

The total cytochrome P450 (CYP) levels, aryl hydrocarbon hydroxylase (AHH), aminopyrine N-demethylase (APND), 5-aminolevulinic acid synthase (ALA-S), and heme synthase (HEM-S) activities were analyzed in liver of flounder ( Platichthys flesus) collected from urban and nonurban embayments of the Baltic Sea off Tallinn area to show the response of biomarkers to anthropogenic contaminants, especially carcinogenic PAHs, in marine environments. CYP, APND, ALA-S, and HEM-S values were higher in flounder from the urban sampling site. This correlates well with higher level of PAHs in tissues of flounder from the same site. AHH activities were quite high but have not shown statistically significant site difference although a slight tendency to be higher in flounder liver from the urban embayment was noticed. This study has confirmed the capability of AHH assay to demonstrate toxic contamination of marine environments in relation to the Baltic flounder as well as in previously tested species from other areas. Analyses of APND, ALA-S, and HEM-S activities are recommended as useful biomarkers for evaluation of general toxic load of environmental contamination.