Aryl Hydrocarbon Hydroxylase Induction Research Articles

The Toxicological Effects of Halogenated Naphthalenes: A Review of Aryl Hydrocarbon Receptor-Mediated (Dioxin-like) Relative Potency Factors

There is no doubt that chloronaphthalenes (PCNs) and their brominated counterparts (PBNs) are dioxin-like compounds, but there is less evidence for mixed bromo/chloronaphthalenes (PXNs). In this article we review information relating to the dioxin-like potency of PCNs and PBNs obtained in vivo, in vitro, and in silico. The aim was to help and improve the quality of data when assessing the contribution of these compounds in the risk analysis of dioxin-like contaminants in foods and other sample types. In vivo and in vitro studies have demonstrated that PCN/PBN congeners are inducers of aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase, and luciferase enzymes that are features specifically indicative of planar diaromatic halogenated hydrocarbons such as dioxin and dioxin-like compounds. PCNs in the environment are of multisource origin. The limited data on PBNs in the environment suggest that these also appear to originate from different sources. The toxicological data on these compounds is even scarcer, most of it directed toward explaining the exposure risk from accidental contamination of feed with the commercial PBN containing product, Firemaster BP-6. The occurrence of PBNs and PXNs is possible as ultra-trace environmental and food-chain contaminants produced at least from combustion processes at unknown concentrations. Available toxicological and environmental data enable a focus on PCNs as dioxin analogues to an extent that specific local or regional environmental influences could result in a risk to human health. There is the possibility that they may act synergistically with the better-known classic dioxin and other dioxin-like compounds. PBNs and PXNs are much less studied than the dioxins, but are known to be products of anthropogenic processes that contaminate the environment. A continuously increasing use of bromine for manufacture of brominated flame retardants over the past three decades is anticipated as a stream of “brominated” wastes, that when degraded (combusted), will release PBNs and PXNs. This calls for advanced analytical methods and greater interest toxicologically to understand and control pollution and exposure by PBNs and PXNs. Particular congeners of bromonaphthalene in single studies were found to be much more toxic than their chlorinated counterparts. In addition, brominated/chlorinated naphthalenes also seem to be more potent toxicants than PCNs.About 20% of PCN congeners exhibit a dioxin-like toxicity with relative potencies varying between around 0.003 and 0.000001, but additional and more rigorous data are needed to confirm these figures. Recent food surveys have estimated a small but relevant human exposure to these compounds in foods, giving an additional source of dioxin-like toxicity to those compounds already covered by the World Health Organization–Toxic Equivalency Factors (TEFs) scheme. Given the additivity of response postulated for other dioxin-like compounds, it would seem unwise to ignore this additional contribution. Few data available showed that PBN congeners also exhibit a dioxin-like toxicity and are even more potent than PCN congeners, but the relative potency values were not derived for them until now. There are no toxicological data available for PXNs.

Commentary: From phenotype, to genotype, to gene-environment interaction and risk for complex diseases

sulfotransferase: Its probable role in carcinogenesis and in protein(methion-5-yl) binding in rat liver. Cancer Res 1970;30:577. 48 Irving CC, Russell LT. Synthesis of the o-glucuronide of N-2-fluorenylhydroxylamine. Reaction with nucleic acids and with guanosine 5-monophosphate. Biochemistry 1970;9:2471. 49 Cardona RA, King CM. Activation of the o-glucuronide of the carcinogen N-hydroxy-FAA by enzymatic deacetylation in vitro: Formation of FA-tRNA adducts. Biochem Pharmacol 1976;25:1051. 50 Kadlubar FF, Miller JA, Miller EC. Hepatic microsomal N-glucuronidation and nucleic acid binding of N-hydroxy arylamines in relation to urinary bladder carcinogenesis. Cancer Res 1977;37:805. 51 Kellermann G, Shaw CR, Kellermann ML. Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma. New Engl J Med 1973;289:934. 52 Kellermann G, Kellermann ML, Jett JR, Moses HL, Fontana RS. Aryl hydrocarbon hydroxylase in man and lung cancer. Human Genetics 1978;(Suppl. 1):161. 53 Higginson J. The role of the pathologist in environmental medicine and public health. Am J Pathol 1967;86:460. 54 Brody H. The systems view of man: implications for medicine, science and ethics. Perspect Biol Med 1973;17:71.

Assessment of carcinogenic potential of repeated fish fried oil in mice

Our prior studies have shown that single topical treatment of repeated fish fried oil extract (RFFE), containing various polycyclic aromatic hydrocarbons (PAHs), to the dorsal epidermis of mice caused enhancement of DNA damage along with higher expression of p53 and p21WAF1 proteins and cell-cycle arrest. In the present study carcinogenic potential of repeated fish fried oil (RFFO) and RFFE was assessed. Single topical application of RFFO (100 microL/animal) and RFFE (100-500 microg/animal) to Swiss albino female mice resulted in significant induction (1.8- to 7.4-fold) of ornithine decarboxylase activity. Twice weekly topical application of methylcholanthrene (MCA) for 24 wk or single topical application of 7,12-dimethylbenzanthracene (DMBA) or RFFO or RFFE, as initiator followed by twice weekly application of 12-O-tetradecanoyl phorbol myristate acetate (TPA) as promoter for 24 wk, resulted in development of skin papillomas after 6, 7, 18, and 9 wk, respectively. The cumulative number of tumors in MCA, DMBA/TPA, RFFE (200 microg)/TPA, and RFFE (500 microg)/TPA groups were 276, 168, 34, and 58 after 24 wk while negligible or minimal initiating activity was noticed in RFFO/TPA group. No tumors were found in animals either given twice weekly topical application of RFFO or a single initiating dose of DMBA followed by twice weekly application of RFFO. Histopathology of skin of animals treated with RFFE/TPA showed marked proliferation of epidermal layers along with abnormal mitosis and multinucleated tumor appearance. Skin of animals in groups RFFO/TPA and DMBA/RFFO showed sloughing and regeneration of epidermal layers, oedema along with proliferation of fibroblasts. Histochemical localization of gamma-glutamyl transpeptidase was found to be substantially higher in skin of mice treated with RFFO/TPA and RFFE/TPA. Animals treated with RFFO/TPA, DMBA/RFFO, and RFFE/TPA resulted in significant induction of cutaneous aryl hydrocarbon hydroxylase (AHH) (421-432%), ethoxyresorufin-O-deethylase (252-316%), and glutathione S-transferase (133-245%) activities. Animals treated with RFFO/TPA, DMBA/RFFO, and RFFE/TPA led to significant reduction in glutathione content (39-44%) with a concomitant increase in lipid peroxidation (254-492%). Animals treated with RFFO/TPA and RFFE/TPA led a significant decrease in catalase (43-69%) and superoxide dismutase (20-31%) activities while glutathione reductase activity was found to be diminished (23-51%) in RFFO, RFFO/TPA, DMBA/RFFO, and RFFE/TPA treated groups. These results suggest that RFFE possess skin tumor initiating activity and that it may have weak promoting activity as well, which may involve free radicals.

Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on “Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes”

to study the effects of drugs and other environmental toxicants on the liver, many believe that the use of hepatocyte-derived cultures offers advantages over studies in the liver of intact animals. Cell cultures can be manipulated more easily, and they are far less expensive to maintain than a

Intestinal bioavailability and biotransformation of 3,3′,4,4′-tetrachlorobiphenyl (CB 77) in in situ preparations of channel catfish following dietary induction of CYP1A

Previous studies with the catfish in situ perfused intestinal preparation have demonstrated a significant decline in the intestinal bioavailability of a coplanar polychlorinated biphenyl (PCB), 3,3′,4,4′-tetrachlorobiphenyl (CB 77)( 14C-TCB) dose in animals pre-exposed in vivo to TCB. This response was accompanied by CYP1A induction in the intestine, but little effect upon the oxidative metabolism of the subsequent in situ dose of [ 14C]-TCB. To ascertain the basis of these responses and the intestine specific contributions, the intestinal bioavailability and metabolism of [ 14C]-TCB were examined in the in situ intestinal preparation following in vivo exposure to β-naphthoflavone (BNF; 0, 10 or 50 mg BNF/kg diet for 10 days), BNF was selected as a known inducer of CYP1A and as a compound with a structure unlikely to influence or directly partake in diffusion based TCB concentration gradients. Appreciable amounts of [ 14C]-TCB molar equivalents (Meq) reached the perfused circulation of the intestinal preparation for all treatments. While BNF pre-exposure elicited induction of CYP1A activities aryl hydrocarbon hydroxylase (AHH) (9.2–12.5-fold) and elicited modest morphological changes (muciparous) in the intestine these changes were not associated with alterations in [ 14C]-TCB Meq bioavailability. [ 14C]-TCB metabolism in the intestinal mucosa ranged between 0.54 and 1.27%, for all treatments. As with bioavailability, intestinal metabolism of [ 14C]-TCB was not significantly influenced in either extent or profile by induction of CYP1A activity as associated with BNF treatment. Four metabolites were found in mucosal sample extracts of which three were tentatively identified as 2-OH-TCB, 4-OH-3,3′,4′,5-TCB, and 4,4′-diOH-3,3′,5,5′ tetrachlorobiphenyl. A fourth unknown metabolite presented chromatographic characteristics suggestive of another dihydroxylated metabolite. These data when examined alone and compared to the literature suggest that the intestine may metabolize [ 14C]-TCB slowly and independent of CYP1A, resulting in somewhat different profiles than published for other organs. In addition, it is likely that previous [ 14C]-TCB bioavailability findings in the perfused intestine may be based on TCB concentration gradients rather than biotransformation.

Disruption of glomerular cell-cell and cell-matrix interactions in hydrocarbon nephropathy

Environmental chemicals play an etiological role in greater than 50% of idiopathic glomerular diseases. The present studies were conducted to define mechanisms of renal cell-specific hydrocarbon injury. Female rats were given 10 mg/kg benzo(a)pyrene (BaP) once a week for 16 wk. Progressive elevations in total urinary protein, protein/creatinine ratios, and microalbuminuria were observed in rats treated with BaP for up to 16 wk. The nephropathic response involved early reductions in mesangial cell numbers and fibronectin levels by 8 wk, coupled to transient increases in podocyte cellularity. Changes in podocyte numbers subsided by 16 wk and correlated with rebound increases in mesangial cell numbers and fibronectin levels, along with increased alpha-smooth muscle actin and Cu/Zn superoxide dismutase and fusion of podocyte foot processes. In culture, mesangial cells were more sensitive than podocytes to hydrocarbon injury and expressed higher levels of inducible aryl hydrocarbon hydroxylase activity. Naïve mesangial cells exerted a strong inhibitory influence on podocyte proliferation under both direct and indirect coculture conditions, and this response involved a mesangial cell-derived matrix that selectively inhibited podocyte proliferation. These findings indicate that hydrocarbon nephropathy in rats involves disruption of glomerular cell-cell and cell-matrix interactions mediated by deposition of a mesangial cell-derived growth-inhibitory matrix that regulates podocyte proliferation.

Role of cytochrome P4501B1 in benzo[a]pyrene bioactivation to DNA-binding metabolites in mouse vascular smooth muscle cells: evidence from 32P-postlabeling for formation of 3-hydroxybenzo[a]pyrene and benzo[a]pyrene-3,6-quinone as major proximate genotoxic intermediates.

Benzo[a]pyrene (BP), a polycylic aromatic hydrocarbon (PAH), is a potent atherogen and carcinogen in laboratory animals. Since genotoxic mechanisms may contribute to the development of atherosclerosis by PAHs, we have tested the hypotheses that: 1) BP induces DNA adducts in mouse aortic smooth muscle cells (SMCs); 2) 3-hydroxybenzo[a]pyrene (3-OH-BP) and benzo[a]pyrene-3,6-quinone (BPQ) are proximate genotoxic metabolites; and 3) cytochrome P4501B1 (CYP1B1) mediates the activation of BP and its metabolites to ultimate genotoxic intermediates. Cultured mouse aortic SMCs were treated with BP, 3-OH-BP, or BPQ for 24 h, and DNA adduct formation was analyzed by (32)P-postlabeling. In some experiments, cells were pretreated with the CYP1B1 inhibitor 1-ethynylpyrene (EP) prior to exposure to BP or its metabolites. BP, 3-OH-BP, and BPQ induced formation of several DNA adducts that were not observed in dimethylsulfoxide-treated cells. Re- and cochromatography experiments indicated that 3-OH-BP and BPQ were proximate genotoxic metabolites of BP. DNA adduct formation was strongly inhibited by EP, a specific inhibitor of CYP1B1. BP treatment of SMCs resulted in induction of aryl hydrocarbon hydroxylase (AHH) activity and CYP1B1, but not CYP1A1, apoprotein. EP also blocked AHH induction by BP. In conclusion, the results of this study support the hypothesis that in SMCs, which are target sites for the development of atherosclerosis, the major bioactivation pathway of BP entails CYP1B1-mediated formation of the 3-OH-BP and BPQ, which are proximate genotoxic metabolites that may in turn get transformed to ultimate DNA-binding metabolites, which may contribute to atherogenesis by PAHs.

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.

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.

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.

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.

Functional characterization of the human CYP1A1 negative regulatory element: modulation of Ah receptor mediated transcriptional activity.

The mechanisms that underly the regulation of human CYP1A1 have merited considerable attention because of their association both with toxic outcomes and the etiology of several cancers. Previous work conducted in this laboratory has identified a negative regulatory element (NRE) in the 5' region of this gene that appeared to modulate CYP1A1 transcriptional activity. This NRE is present in two functional copies, a high affinity 21-bp palindrome centered at position -784, and an additional element found within a GC-rich region between position -728 and -558. In this report, the regulatory function of the NREs in the context of the CYP1A1 promoter was evaluated. This was accomplished by substituting mutated elements for the corresponding wild-type element in a vector that contained human CYP1A1 sequences positions -1140 to +59 directing the transcription of the chloramphenicol acetyltransferase reporter gene. Expression vectors containing specific mutations in each or both NREs were characterized. We show that eliminating the binding of the CYP1A1 repressor protein to one or both repressor motifs results in a significant 2- to 3-fold increase in the inducibility of CYP1A1 promoter activity. Although mutation of both sites appeared to result in an increase in inducibility over that observed with only one site mutated, the effect was not additive. Such aberrant transcriptional activity correlates with the highly inducible aryl hydrocarbon hydroxylase phenotype that is a reported marker for individuals predisposed to lung cancer. Mutation of the NRE, or more likely, the cognate repressor protein(s), may provide a genetic basis for this phenotype.

Biomonitoring exposure to environmental tobacco smoke (ETS): a critical reappraisal.

1 The most frequently used biomarkers for exposure to environmental tobacco smoke (ETS) are cotinine and thiocyanate in body fluids, carboxyhaemoglobin in red blood cells (COHb) and carbon monoxide in the expired air. Although not ideal, cotinine in blood, saliva or urine is an established biomarker for ETS exposure within the past 1-3 days. Comparison with cotinine concentrations in cigarette smokers reveals that passive smokers take up less than 1/100 of the nicotine dose of smokers. 2 Biomonitoring data available for the ETS-related exposure to genotoxic substances comprise uptake of benzene, polycyclic aromatic hydrocarbons (PAH), aromatic amines, tobacco-specific nifrosamines (TSNA), electrophilic compounds giving rise to urinary thioethers, mutagens causing urinary mutagenic activity and the formation of various DNA adducts. With the exception of TSNA, these biomarkers are related to chemicals occurring ubiquitously in the environment and in the food. As a consequence, the background levels in unexposed nonsmokers are high compared to the observed increases (if any) associated with ETS exposure. 3 Some markers of biological effects, which, by definition, are non-specific with regard to the underlying exposure, have also been investigated in relation to ETS exposure. These markers comprise cytogenetic effects, aryl hydrocarbon hydroxylase (AHH) induction, urinary hydroxyproline excretion and various factors indicative of cardiovascular risks. The available data suggest that passive smoking is associated with a small induction of placental AHH and also with effects on cardiovascular risk markers. The latter findings in particular may be confounded by other risk factors, which have been observed to be more frequent in passive smokers than in unexposed nonsmokers.

Interpatient variability: genetic predisposition and other genetic factors.

Polymorphisms and other genetic factors related to enzymes metabolizing drugs and xenobiotic chemicals are well known. This article focuses on selected molecular mechanisms and introduces some of the clinical implications arising from genetically determined interpatient variability or expression in some of these enzymes. Selected are the polymorphic enzymes of cytochromes P-450 (CYP) as examples of phase I enzymes and methyl transferases, n-acetyl transferases, and glutathione-s-transferases as examples of phase II enzymes. The polymorphism surrounding arylhydrocarbon hydroxylase induction is briefly described. Phase I enzymatic reactions are predominantly oxidative, whereas phase II reactions often couple with the byproducts of phase I. Overall, in poor metabolizers, whether phase I or phase II, there is limited metabolism in most patients unless another major metabolic pathway involving other enzymes exists. Drug metabolism also depends on whether the parent compound is a prodrug that forms an active metabolite, and poor metabolizers under this condition will form only trace amounts of an active compound. Therefore, the clinical significance of genetic polymorphisms and other genetic factors may be related to substrate, metabolite, or the major elimination pathway.

Environmental Factors and Aryl Hydrocarbon Hydroxylase Activity (CYP1A1 Phenotype) in Human Lymphocytes

Since aryl hydrocarbon hydroxylase (AHH) is considered to be responsible for the activation of benzo(a)pyrene (BP) and other polyaromatic hydrocarbons in cigarette smoke to carcinogens, it is important to examine AHH activity in the determination of susceptibility to lung cancer. Lymphocytes from healthy male adults (239) of non-smokers and smokers were cultured in vitro and assayed for non-induced and 3-methylcholanthrene (MC)-induced AHH activity and AHH inducibility (MC-induced AHH activity/non-induced AHH activity). A day-to-day variation in AHH activity was not observed while a seasonal variation was apparent. Very wide differences in non-induced AHH and MC-induced AHH activities were observed. The association of some selected environmental factors and AHH activity was studied. Age was related to non-induced AHH activity (Spearman's rank correlation coefficients (r), r = 0.185, p < 0.005) and AHH inducibility (r = -0.329, p < 0.001). Coffee consumption was associated with non-induced (age-adjusted r = 0.138, p < 0.05) and MC-induced AHH activity (age-adjusted r = 0.173, p < 0.01). Cigarette smoking was correlated with non-induced AHH activity (age-adjusted r = 0.191, p < 0.005) and AHH inducibility (age-adjusted r = -0.191, p < 0.005). No significant association was observed for any other selected factors, including alcohol intake or broiled food consumption. In conclusion, AHH activity might be affected by cigarette smoking and coffee consumption, and was dependent on the age of the donor. Day-to-day and seasonal variation analyses showed that this assay method was reproducible and reliable and AHH inducibility might be a useful biomarker in cancer epidemiology. As those factors may affect the AHH activity, a careful control of those factors to AHH activity is necessary in epidemiological studies on the association between AHH inducibility in human lymphocytes and lung cancer.

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities, Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b 5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23–2.1 nmol/min/mg and 0.5–11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major ( Abudefduf saxatilis) and Bermuda chub ( Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6 β- and 16 β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.

Expression of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt) in adult rabbits known to be non-responsive to cytochrome P-450 1A1 (CYP1A1) inducers.

Induction of aryl hydrocarbon hydroxylase by aryl hydrocarbons occurs only in neonatal rabbits and not in adult rabbits [Kahl, G. F., Friederich, D. E., Bigelow, S. W., Okey, A. B. & Nebert, D. W. (1980) Dev. Pharmacol. Ther. 1,137-162]. In the present study, we isolated cDNA clones encoding aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt) from adult rabbits. The deduced amino acid sequences of rabbit AhR and Arnt showed 80% and 94% identities with those of human AhR and Arnt, respectively. Rabbit AhR mRNA was predominantly expressed in the lung and liver. In contrast, rabbit Arnt mRNA was expressed at almost the same level in all tissues except for the heart, liver, and small intestine. Gel shift analysis showed that the AhR. Arnt complex could bind to the consensus xenobiotic-responsive element, which indicates that AhR expressed in adult rabbit liyers possessed binding activity to the consensus xenobiotic-responsive element in vitro, although aryl hydrocarbons did not induce the activity of AHH in adult rabbits. We propose that the incapability of adult rabbits to induce cytochrome P-450 1A1 (CYP1A1) is caused by factors other than AhR and Arnt.

Bioassay for determining 2,3,7,8-tetrachlorodibenzo- p-dioxin equivalents (TEs) in human hepatoma HepG2 cells

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benz[ a]anthracene (BA) highly induce cytochrome P4501A1, determined by aryl hydrocarbon hydroxylase (AHH) activity, in human hepatoma HepG2 cells within 24 h. AHH activity induced by TCDD and TCDF persists for at least 48 h. In contrast, AHH activity induced by BA rapidly declines, although the amounts applied are 4–5 orders of magnitude higher than those of TCDD or TCDF. AHH induction in HepG2 cells differs from that in rat hepatoma cells H4IIEC3/T in two aspects: (1) HepG2 cells are 20 times less sensitive to the test compounds than H4IIEC3/T cells. (2) TCDF-induced AHH activity does not persist in the rat cells. The results suggest that human HepG2 cells, because of their low sensitivity, are inferior to rat H4IIEC3/T cells for determining TCDD equivalents in environmental samples. They may be useful for investigating species dependent differences in the toxicokinetics of individual polyhalogenated aromatic hydrocarbon congeners.

Atypical cytochrome P450 induction profiles in glomerular mesangial cells at the mRNA and enzyme level: Evidence for CYP1A1 and CYP1B1 expression and their involvement in benzo[A]pyrene metabolism

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) modulates growth factor-related gene expression and proliferation of renal glomerular mesangial cells (GMCs) in vitro. Because many of the toxic and biochemical effects of this polycyclic aromatic hydrocarbon are mediated through oxidative metabolism, the present studies were conducted to examine the patterns of cytochrome P450IA1 (CYP1A1) and P450IB1 (CYP1B1) inducibility in mesangial cells and the molecular consequences of this response. Exposure of cultured GMCs to BaP (30 μM) or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, 10 nM) for 24 hr induced CYP1 A1 mRNA levels, a response abolished by cotreatment with 10 μM cycloheximide. The pattern of hydrocarbon inducibility was atypical in that BaP was a more effective inducer of CYP1A1 gene expression than TCDD, and both hydrocarbons induced aryl hydrocarbon hydroxylase (AHH) activity, but not ethoxyresorufin-O-deethylase activity. Cotreatment with α-naphthoflavone (αNF, 1 μM) or ellipticine (ELLIP, 0.1 nM) only partially inhibited the induction of AHH activity by BaP (30 μM). BaP and TCDD also induced expression of the CYP1B1 protein and the pattern of induction was comparable to that observed for CYP1A1. Treatment of GMCs with 30 μM BaP was associated with the formation of eight DNA adducts, and their occurrence could be inhibited by pretreatment with αNF (1 μM), but not ELLIP (0.1 nM). These results demonstrate that CYP1A1 and CYP1B1-related activities are induced in GMCs by BaP and TCDD and this induction is associated with metabolism of BaP to reactive intermediates that bind covalently to DNA.

The relationship between aryl hydrocarbon hydroxylase and polymorphisms of the CYP1A1 gene.

We examined the relationship between aryl hydrocarbon hydroxylase (AHH) and the frequency of a Msp I mutation in the 3′‐flanking region of cytochrome P450 (CYP) 1A1 (Mspl polymorphism) and another mutation in exon 7 (Ile‐Val polymorphism) in 84 healthy male subjects in Fukuoka, Japan. AHH inducibility (3‐methylcholanthrene (MC)‐induced AHH activity/non‐induced AHH activity) was correlated with the MspI polymorphism (P<0.0001) and age class (P=0.015), whereas no correlation was found for the Ile‐Val polymorphism (P=0.509). Age‐adjusted AHH inducibility (mean±SE) of the predominant homozygote (genotype A), the heterozygote (genotype B) and a homozygote rare allele (genotype C) genotypes was 4.89±0.36, 4.82±0.29 and 13.61±1.44, respectively. The genotype C showed much higher AHH inducibility than genotypes A and B (P<0.001), while no significant difference was observed between genotypes A and B. Non‐induced AHH activity was also correlated with these polymorphisms. The AHH activity of a homozygous mutant Val/Val genotype (0.076±0.010 pmol/min/106 cells) was significantly higher (P<0.05) than that of the wild‐type homozygous Ile/Ile (0.044±0.004 pmol/min/106 cells) and heterozygous Ile/Val (0.047±0.007 pmol/min/106 cells) genotypes. Our study suggests that the genotypes C and Val/Val, which are more frequent in smoking‐related lung cancer, are closely related with high AHH inducibility and high non‐induced AHH activity, respectively. Thus, the positive relationship between AHH inducibility and lung cancer is supported by our study. If our results are confirmed and the assessment of genotype becomes feasible on a population basis, identification of smokers who have genetically high susceptibility to lung cancer (genotype C or Val/Val) may become important for the prevention of lung cancer.