Aryl Hydrocarbon Hydroxylase Induction Research Articles

Effects of polchlorinated paraffins on hepatic, renal and intestinal biotransformation rates in comparison to the effects of polychlorinated biphenyls and naphthalenes

AbstractPolychlorinated paraffins (PCPs), biphenyls (PCBs) and naphthalenes (PCNs) were compared as inducers of drug‐metabolizing enzymes in different rat tissues. Two different chain lengths (C11 or C20) and chlorination degrees (40 or 70% chlorine) of PCPs, two preparations of PCNs (50–70% chlorine), and one PCB preparation (54% chlorine) were used. The compounds were administered intraperitoneally to rats, and the activities of various drug‐metabolizing enzymes were analysed in hepatic and renal microsomes and in the post‐mitochondrial supernatant of the small intestinal mucosa. PCB and PCNs were potent inducers of both hepatic and renal drug‐metabolizing enzyme activities, whereas PCPs produced only minor changes in these activities. When compared with each other, PCNs were somewhat stronger inducers than PCB. The enhancement of enzyme activities in the liver was different from that of the kidney. The maximal induction of aryl hydrocarbon hydroxylase was 4.8‐fold in the liver and 7.2‐fold in the kidney, that of ethoxy‐coumarin deethylase 12‐fold in the liver and 53‐fold in the kidney, while the activity of UDP glucuronosyl‐transferase was increased only in the liver (maximally 4.5‐fold). In small intestinal mucosa, there were only moderate changes in the activities of drug‐metabolizing enzymes.

Hydrocarbon-based oils as inducers of cutaneous aryl hydrocarbon hydroxylase

Petroleum-based oils applied to the skin of rats induced cutaneous aryl hydrocarbon hydroxylase activity. A heavy oil like Kuwait Crude induced benzo(a)pyrene (BaP) hydroxylase and diphenyloxazole (PPO) hydroxylase activities to levels obtained with Aroclor 1254, while a light oil like Fuel Oil No. 2 was less effective. Intraperitoneal injection of these oils to rats also induced hepatic aryl hydrocarbon hydroxylase activities. Inhibition of these activities by α-naphthoflavone, but not by metyrapone, suggests that the cytochrome induced is of the P448 type.

Dissociation between ornithine decarboxylase activity and aryl hydrocarbon hydroxylase induction in cell cultures treated with benz[ a]anthracene and inhibitors of ornithine decarboxylase

The induction of aryl hydrocarbon hydroxylase and ornithine decarboxylase by benz[ a]-anthracene in the presence or absence of ornithine decarboxylase inhibitors was studied in three different cell culture systems. An almost complete abolishment of ornithine decarboxylase activity by 1,3-diamino-2-propanol or α-difluoromethyl ornithine before the addition of the inducer did not affect appreciably the induction of aryl hydrocarbon hydroxylase by benz[ a]anthracene in human embryo, HeLa and Reuber H-II-4-E cells in culture. These results suggest that the induction of aryl hydrocarbon hydroxylase does not require ornithine decarboxylase activity per se and can be expressed in the absence of continuous polyamine synthesis.

Influence of Mycoplasma arginini infection on the induction of aryl hydrocarbon hydroxylase by TCDD in rat hepatoma cell cultures

Mycoplasma arginini was eliminated from a rat hepatoma cell line (H-4-II-E) by plating at low cell density and treatment with chlortetracycline (250 μg/ml), kanamycin (25 μg/ml), tylosin (100 μg/ml), 3% M. arginini antiserum and 5% fresh guinea-pig serum. The induction of AHH activity in the cell culture was measured in response to increasing concentrations of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The ED 50 values (estimated doses that produce 50% maximum enzyme induction) were calculated to be 0·256, 0·452 and 0·344 pmol TCDD/plate for original, mycoplasma-free and reinfected cells, respectively. Although the absence of M. arginini in the rat hepatoma cell line makes the cells slightly less responsive to AHH induction by TCDD, this decrease does not detract from the use of the method to screen food extracts and environmental samples for the presence of certain toxic planar organic compounds.

Aryl hydrocarbon hydroxylase activity in cultured lymphocytes of psoriatic patients.

To evaluate further the metabolic capacity of psoriatics, the activities and inducibility of aryl hydrocarbon hydroxylase (AHH) were measured in cultured mitogen-stimulated lymphocytes from the peripheral blood of 68 psoriatic and 39 control patients. There was no significant difference in the mean basal and induced AHH activities between the psoriatics and the controls, although the former showed a slight tendency towards a higher inducibility ratio. In both, the basal and induced AHH values decreased significantly with increasing age. The cigarette smokers in both groups had significantly higher AHH activities than the nonsmokers. Antipsoriatic treatment (Anthralin, PUVA) did not seem to influence the lymphocyte AHH activities. No association was demonstrable between the AHH activities and the duration of the disease.

The Ah Locus, A Multigene Family Necessary for Survival in A Chemically Adverse Environment: Comparison With the Immune System

Publisher Summary This chapter discusses the drug-metabolizing enzyme systems, presents the Ah system, and provides data for multiple Ah-structural gene products. A cytosolic receptor is regarded as the major product of the Ah-regulatory genes. Sucrose-density gradient analysis following dextran–charcoal treatment is among the most reliable methods for characterizing an Ah receptor. The aryl hydrocarbon hydroxylase (AHH) fluorescent assay is simple and extremely sensitive. This assay, using benzo[α]pyrene as the substrate in vitro, is most commonly used as the biochemical marker for the Ah locus in laboratory animal and human studies. When males and females of four inbred strains were housed together and allowed to breed randomly for 46 to 48 months, the AHH inducibility by 3-methylcholanthrene of weanlings between 18 and 22 generations approximated the distribution found normally for out-bred or random-bred mouse strains. These data indicate the involvement of multiple Ah regulatory genes and a “natural selection” tendency of the induction process to “drift” toward lower intensity in populations having heterogeneous genetic input.

Expression and subcellular distribution of mouse cytochrome P 1-450 mRNA as determined by molecular hybridization with cloned P 1-450 DNA

Cytochrome P 1-450 (P 1-450) is defined as that cytochrome most closely associated with 3-methylcholanthrene (MC)-induced aryl hydrocarbon hydroxylase (AHH) activity. Recently a cloned DNA sequence (clone 46) was shown to represent a portion of the P 1-450 structural gene [Negishi et al. , Proc. Nat. Acad. Sci. U.S.A. 78 : 800–804 (1981)]. Poly(A +)-enriched RNA was isolated from total liver homogenate, membrane-bound polysomes and from free polysomes at various times after MC treatment of Ah -responsive C57BL 6N (B6) and Ah -nonresponsive DBA 2N (D2) inbred mice. The poly(A +)-enriched RNA was separated by methylmercury-agarose gel electrophoresis and hybridized to nick-translated [ 32P]DNA from clone 46. By means of this RNA-DNA hybridization, only 6% of total polysomal P 1-450 mRNA exists in free polysomes after 24 h of MC treatment. The data indicate that the endoplasmic reticulum is the principal site of synthesis for this integral microsomal protein. Studies of induction kinetics following MC treatment provided the evidence of the rapid increase of total liver and membrane bound P 1-450 mRNA preceding the synthesis of apo-P 1-450 and the increase of AHH activity.

The Ah regulatory gene product. Survey of nineteen polycyclic aromatic compounds' and fifteen benzo[a]pyrene metabolites' capacity to bind to the cytosolic receptor

The capacity of 19 polycyclic aromatic compounds and 15 benzo[a]pyrene metabolites to displace [1,6- 3 H]2,3,7,8- tetrachlorodibenzo-p- dioxine ([ 3 H]Tcdd) from the mouse liver cytosolic Ah receptor was examined. We compared our data with various parameters taken from previously published results: the capacity of seven polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase (AHH) activity in human cell cultures, the capacity of 10 polycyclic hydrocarbons to induce azo dye N-demethylase activity in rat liver, the capacity of 6 polycyclic hydrocarbons to shorten zoxazolamine paralysis times in the intact rat, and the capacity of 15 benzo[a]pyrene metabolites to induce AHH activity in rat hepatoma H-4-II-E cultures. An excellent correlation is seen between the capacity to displace the radioligand from the Ah receptor and the capacity to induce these monooxygenase activities. Differences in the rate of cellular uptake and formation of alkali-extractable metabolites of dibenzo[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene in Hepa-1 mouse hepatoma cell cultures do not account for differences in the capacity of these three polycyclic hydrocarbons to displace [ 3H]TCDD from the Ah receptor.

Lymphocyte aromatic hydrocarbon responsiveness in acute leukemia of childhood

Aryl hydrocarbon hydroxylase (AHH) activity and inducibility were examined in mitogen-stimulated cultured lymphocytes from children with acute leukemia in remission, with nonleukemic malignancies, and with no family or personal history of malignant disease. Neither morphological differences nor differences in mitogen responsiveness were observed among the three sources of cells studied. Levels of constitutive and dibenzanthracene-induced AHH activity were found to be similar among the three groups by analysis of variance. However, when results were analyzed in terms of inducibility ratios, it was found that cells from leukemic children were significantly less inducible (p less than 0.005) than cells from unaffected children or children with nonleukemic malignancies. The reason for this difference became apparent when statistical criteria were employed for the pheontypic separation of individuals who were highly aromatic hydrocarbon responsive and minimally responsive. A significantly larger proportion (p less than 0.001) of leukemic children than unaffected children or children with nonleukemic malignancy were found to be minimally aromatic hydrocarbon responsive. Moreover, in patients with acute lymphoblastic leukemia relapsing while on therapy, longer durations of the first remission were correlated (r = 0.63, p less than 0.05) with the highly inducible AHH phenotype.

Lymphocyte Aromatic Hydrocarbon Responsiveness in Acute Leukemia of Childhood

Aryl hydrocarbon hydroxylase (AHH) activity and inducibility were examined in mitogen-stimulated cultured lymphocytes from children with acute leukemia in remission, with nonleukemic malignancies, and with no family or personal history of malignant disease. Neither morphological differences nor differences in mitogen responsiveness were observed among the three sources of cells studied. Levels of constitutive and dibenzanthracene-induced AHH activity were found to be similar among the three groups by analysis of variance. However, when results were analyzed in terms of inducibility ratios, it was found that cells from leukemic children were significantly less inducible (p less than 0.005) than cells from unaffected children or children with nonleukemic malignancies. The reason for this difference became apparent when statistical criteria were employed for the pheontypic separation of individuals who were highly aromatic hydrocarbon responsive and minimally responsive. A significantly larger proportion (p less than 0.001) of leukemic children than unaffected children or children with nonleukemic malignancy were found to be minimally aromatic hydrocarbon responsive. Moreover, in patients with acute lymphoblastic leukemia relapsing while on therapy, longer durations of the first remission were correlated (r = 0.63, p less than 0.05) with the highly inducible AHH phenotype.

Induction of arylhydrocarbon hydroxylase and blast transformation in human blood lymphocytes

Variation in arylhydrocarbon hydroxylase(AHH) inducibility in human peripheral blood lymphocytes was studied. AHH inducibility and the degree of blast transformation were determined simultaneously using [ 3H]benzo(a)pyrene and [ 3H]thymidine, respectively. AHH inducibility in terms of induction ratio(induced level to basal) or induction ratio per unit of blast transformation varied at different culture time and at different phytohemagglutinin concentrations within the same individuals. However, the ratio of absolute induced AHH activity and unit of blast transformation gave persistent value for the same individuals, indicating that AHH inducibility of human lymphocytes should be expressed in this manner in the study of cancer susceptibility.

Rapid vertical tube rotor gradient assay for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the Ah receptor.

The Ah receptor, which regulates induction of aryl hydrocarbon hydroxylase (cytochrome P1-450), can be assayed using [3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. Sucrose density gradient (SDG) centrifugation is the most reliable method for detecting and quantitating Ah receptor; however, previous gradient assays in swinging bucket rotors (SBR) have required overnight centrifugation to achieve adequate separation of specific receptor peaks from nonspecific binding to other cytosolic components. These lengthy runs limit the number of assays which can be performed and could permit excessive dissociation of radioligand from the receptor. Thus, we tested brief (2-h) SDG centrifugation assays in a vertical tube rotor (VTR) as a possible alternative to prolonged SBR runs. Two-hour VTR runs yield gradient profiles very similar to those obtained with 16-h runs in an SBR and the calculated sedimentation coefficient (in low ionic strength buffer) is about 9.6 S for the Ah receptor assayed in either rotor. The concentration of Ah receptor in cytosols from liver, lung, kidney, and testis of C57BL/6J mice is the same by VTR analysis as when measured by SBR analysis. Thus, extensive dissociation of [3H]TCDD from the Ah receptor does not appear to occur in cytosols from a variety of tissues even with run times up to 16 h. Assays performed by rapid VTR centrifugation appear equally valid to those obtained by overnight SBR centrifugation.

Genetic linkage between the AH locus and a major gene that inhibits susceptibility to audiogenic seizures in mice.

Mice of the DBA/2 (D2) strain are highly susceptible to sound-induced seizures at 21 days of age; whereas, mice of the C57BL/6 (B6) strain are resistant to these seizures. Although the difference in susceptibility to audiogenic seizures (ASs) between these two strains is inherited as a multiple-factor trait, an association was observed between susceptibility to ASs and the Ah locus. The Ah locus controls the inducibility of aryl hydrocarbon hydroxylase (AHH) activity by a number of aromatic hydrocarbons. B6 mice carry the Ahb allele and have inducible AHH activity; whereas, D2 mice carry the Ahd allele and have noninducible activity. Inducibility is inherited as a Mendelian dominant trait in crosses between these strains. Mice carrying the Ahb allele are generally less susceptible to ASs sat 21 days of age than are mice carrying the Ahd allele. The combined results from B6 X D2 recombinant inbred strains, congenic strains (where the Ahb allele was placed into the D2 genome and the Ahd allele placed into the B6 genome), the B6D2F1 X D2 backcross generation, and a random survey of various inbred strains, suggest that the association between these two traits is due to genetic linkage, rather than to pleiotrophy or to chance. A major gene that inhibits susceptibility to ASs appears to be closely linked to the Ah locus. This gene has been designated Ias, for inhibition of ASs. A large portion of the genetic variability of AS susceptibility may be due to the segregation of Ias.

Genetic regulation of bilirubin-UDP-glucuronosyltransferase induction by polycyclic aromatic compounds and phenobarbital in mice. Association with aryl hydrocarbon (benzo[a]pyrene) hydroxylase induction.

Genetic regulation of bilirubin-UDP-glucuronosyltransferase induction by polycyclic aromatic compounds and phenobarbital in mice. Association with aryl hydrocarbon (benzo[a]pyrene) hydroxylase induction.

A relationship between cord blood and maternal blood lymphocytes and term placenta in the induction of aryl hydrocarbon hydroxylase activity

Correlations between lymphocyte aryl hydrocarbon hydroxylase (AHH) inducibility in cord blood and maternal lymphocytes and placental AHH activity were studied in 15 smokers and 11 non-smokers. Placental AHH activity was extremely low in the non-smokers regardless of the lymphocyte AHH induction ratio, but was elevated to a variable extent in the smokers, in whom it showed a statistically significant correlation ( r = 0.75, P < 0.01) with cord blood lymphocyte AHH inducibility. The correlation between maternal lymphocyte AHH inducibility and placental AHH activity was poor ( r = 0.04, not significant). These findings suggest that AHH induction in man may be ‘systemically’ regulated and that the genetic background will determine the extent of induction at a given level of exposure to polycyclic aromatic hydrocarbons.

Sex dependent effect of chronic ethanol consumption in rats on hepatic microsome mediated mutagenicity of benzo[ a]pyrene

The effect of chronic ethanol consumption by rats on hepatic microsomal metabolism of the procarcinogen benzo[ a]pyrene (B[ a]P) was investigated both with respect to induction of microsomal arylhydrocarbon hydroxylase (AHH) activity and activation of B[ a]P to a mutagen. In female rats, chronic ethanol ingestion produced a 42% increase in AHH activity ( P < 0.01), as measured in isolated microsomes, and also resulted in a significantly enhanced capacity ( P < 0.01) of these microsomes to activate B[ a]P to a mutagen detectable in the Ames bacterial mutagenesis assay. Hepatic microsomes from male rats on the other hand did not exhibit any significant differences, either in AHH activity or in their capacity to activate B[ a]P to a mutagen after chronic ethanol feeding.

Physiological significance of benzo(alpha)pyrene adsorbed to carbon blacks: elution studies, AHH determinations.

Desorption of benzo(alpha)pyrene from commercial carbon blacks by tissue fluids in vitro is compared to arylhydrocarbon hydroxylase induction in mice at three levels of carbon black exposure. Less than 0.005% of the adsorbed benzo(alpha)pyrene content determined by soxhlet extraction in toluene is eluted by human plasma, swine serum, swine lung homogenate and swine lung washings. Statistically significant differences in elution efficiency are observed by the various tissue fluids and carbon blacks. There is no detectable increase in AHH level in mouse lung or liver tissues even at the highest carbon black exposure and consumption rate of 1g/g bdywt/year.

Aryl hydrocarbon hydroxylase induction and its relationship to the toxicity of halogenated aryl hydrocarbons

Aryl hydrocarbon hydroxylase induction and its relationship to the toxicity of halogenated aryl hydrocarbons

Genetic Differences in Susceptibility to Chemically Induced Myelotoxicity and Leukemia

The Ah locus represents a complex "cluster" of genese controlling the induction of numerous drug-metabolizing enzyme "activities" by polycyclic aromatic compounds. Allelic differences at the Ah locus are reflected in the large differences in inducibility of cytochrome P1-450 and benzo[a]pyrene metabolism in numerous tissues when the mice receive the chemical daily in their diet. This experimental model system offers to the hematologist and clinical pharmacologist a means to study genetic differences in toxic chemical depression of the bone marrow, as well as a potential model to study aplastic anemia and leukemia explainable on a single-gene basis. The genetically "responsive" individual who is at increased risk for cancer caused by subcutaneous or topical or intratracheal polycyclic hydrocarbons is at decreased risk for toxicity of the bone marrow and leukemia caused by oral benzo[a]pyrene (when compared with the genetically "nonresponsive" individual receiving the same dose of the same xenobiotic). In other words, tissue sites in direct contact with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dct with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dct with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dose but the route of administration and the tissue in which the malignancy or toxicity develops are therefore very important in the interpretation of data from tumorigenesis or toxicity experiments involving P1-450 inducers such as polycyclic hydrocarbons. There exists sufficient evidence that heritable variation of the Ah locus occurs in man. Growing evidence indicates that persons with higher aryl hydrocarbon hydroxylase inducibility in their cultured mitogen-activated lymphocytes may have a statistically significantly increased risk for certain types of cancer and drug toxicity. It remains to be determined at the present time, however, whether this genotype can be used as a biochemical marker in the individual patient for predicting increased susceptibility to certain types of environmentally caused cancers or toxicity in man.

Genetic differences in susceptibility to chemically induced myelotoxicity and leukemia.

The Ah locus represents a complex "cluster" of genese controlling the induction of numerous drug-metabolizing enzyme "activities" by polycyclic aromatic compounds. Allelic differences at the Ah locus are reflected in the large differences in inducibility of cytochrome P1-450 and benzo[a]pyrene metabolism in numerous tissues when the mice receive the chemical daily in their diet. This experimental model system offers to the hematologist and clinical pharmacologist a means to study genetic differences in toxic chemical depression of the bone marrow, as well as a potential model to study aplastic anemia and leukemia explainable on a single-gene basis. The genetically "responsive" individual who is at increased risk for cancer caused by subcutaneous or topical or intratracheal polycyclic hydrocarbons is at decreased risk for toxicity of the bone marrow and leukemia caused by oral benzo[a]pyrene (when compared with the genetically "nonresponsive" individual receiving the same dose of the same xenobiotic). In other words, tissue sites in direct contact with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dct with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dct with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dose but the route of administration and the tissue in which the malignancy or toxicity develops are therefore very important in the interpretation of data from tumorigenesis or toxicity experiments involving P1-450 inducers such as polycyclic hydrocarbons. There exists sufficient evidence that heritable variation of the Ah locus occurs in man. Growing evidence indicates that persons with higher aryl hydrocarbon hydroxylase inducibility in their cultured mitogen-activated lymphocytes may have a statistically significantly increased risk for certain types of cancer and drug toxicity. It remains to be determined at the present time, however, whether this genotype can be used as a biochemical marker in the individual patient for predicting increased susceptibility to certain types of environmentally caused cancers or toxicity in man.