Induction of aryl hydrocarbon hydroxylase in rodent lung by cigarette smoke: a potential short-term bioassay.

Aryl hydrocarbon hydroxylase (AHH) activity was increased several fold in the lungs of hamsters, mice and rats after inhalation of cigarette smoke. Hamsters had low basal activity of lung AHH but showed the greatest inducibility (the ratio of induced to noninduced enzyme). Inducibility was lower but the actual levels of enzyme activity, both basal and induced, were higher in mice and highest in rats. Several criteria were met which could qualify the lung AHH assay as a short-term bioassay to aid in the measurement of cigarette toxicity. (1) Within a relatively narrow range, AHH activity increased with the number of cigarettes smoked. (2) In tests with standard reference cigarettes and three commercial brands, cigarettes which delivered high levels of total particulate matter (TPM) in the smoke induced AHH in lung to the highest degree; measures which reduced the TPM, primarily filtering methods, reduced the enzyme-inducing effect. (3) Also, with regard to filtering methods, the type of cigarettes which have been reported to cause the most extensive damage to the respiratory tract of animals in chronic inhalation studies caused the greatest enzyme induction in acute experiments. (4) Assay results can be obtained rapidly; maximum induction occurs within 24 hr after smoke exposure.     

The induction of rat lung aryl hydrocarbon hydroxylase by diluted smoke from commercial cigarettes

Induction of pulmonary aryl hydrocarbon hydroxylase (AHH) by cigarette smoke in small rodents is well established. However, studies on dose-response relationships particularly at the lower ends of such relationships are few indeed. Availability of the British-American Tobacco Co. (B.-A.T.)-Mason inhalation system, in which precisely-calibrated dilutions of cigarette smoke can be released into an inhalation chamber, enables excellent dose-response relationships to be established. Using this inhalation system and cigarettes delivering tar (total particulate matter, water and nicotine free) equal to 1, 4 and 20 mg/cigarette, it has been shown that induction of AHH in male Sprague-Dawley rat lung is an extremely sensitive system to inhaled cigarette smoke. AHH induction was observed at a 200-s exposure to 40 puffs of a 1 : 5 dilution of smoke from an ultra mild cigarette delivering 1 mg tar. At this low concentration, it is not even possible to accurately weigh the TPM from diluted smoke in the exposure chamber. This extremely sensitive rat pulmonary enzyme system may prove valuable in the biological testing of modern low tar cigarettes.     

Interaction of certain metal ions with aryl hydrocarbon hydroxylase of rat lung microsomes

The effect of varying concentrations of cadmium, copper, zinc, and selenite on the activity of aryl hydrocarbon hydroxylase (AHH) of rat lungs was studied in vitro. All the metals resulted in a strong inhibition of enzyme activity. Copper and zinc were more inhibitory to rat lung AHH than cadmium and selenite. There was an additive inhibition of AHH activity when copper or zinc was added in the presence of cadmium. EDTA or glutathione exerted a protective effect on cadmium-induced inhibition of AHH activity. Prior incubation of the microsomes with N-ethylmaleimide, a thiol-blocking agent, had no effect on the inhibition of AHH activity caused by cadmium. Addition of cadmium along with zinc-thionein resulted in increased inhibition of rat lung AHH.     

Flavone modulators of rat hepatic aryl hydrocarbon hydroxylase

The cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) metabolizes a wide variety of endogenous and exogenous compounds to nontoxic metabolites and/or toxic products. We have utilized a series of 18 flavone modulators of AHH to distinguish and probe for different cytochrome P-450 isozymes in liver microsomes from control and 3-methylcholanthrene (MC)-injected rats. some flavones (maackiain acetate, flavanone, mollisacacidin, embinin, sciadopitysin) activated, while most of the tested compounds inhibited the MC-induced type of AHH. Although all flavones either inhibited or had little effect on the constitutive AHH in microsomes from control rats, the degree of inhibition varied greatly: some flavones (chrysin, chrysoeriol, baicalein, maackiain acetate, isoliquiritigenin, sciadopitysin) inhibited over 75% of the AHH. The various flavones we screened may prove useful in defining the cytochrome P-450 content of tissues and for probing the active sites of individual isozymes. The modulatory effects of the naturally occurring flavones assume additional importance in that they may be factors in animal and human responsiveness to cytochrome P-450 substrates.     

Effects of cigarette smoke on rat lung and liver ornithine decarboxylase and aryl hydrocarbon hydroxylase activities and lung benzo(a)pyrene metabolism

The response of rat lung and liver ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) activities and lung benzo(a)pyrene (BP) metabolism was studied after exposing the rats to cigarette smoke. A close analysis of the time curves for ODC and AHH activities in rat lung and liver after a single exposure to cigarette smoke resulted in no clear correlation between the two parameters. Prolonged treatment (10 days) produced an increase in pulmonary ODC activity; hepatic ODC activity was unaffected. 10-day treatment was ineffective in raising AHH activity above values observed after a single treatment. BP metabolism, as determined in isolated perfused lungs by the appearance of organic- and water-soluble metabolites in the perfusion medium, the amount of covalently bound metabolites in lung tissue and the disappearance of unchanged 3-H BP from the perfusate, was markedly increased in response to cigarette smoke treatment. The data presented indicate that induction of AHH activity and increased metabolism of BP do not necessarily require a pre-existing increase in ODC activity.     

Induction of aryl hydrocarbon hydroxylase by 3-methylcholanthrene in liver,lung and kidney of gonadectomized and sham-operated wistar rats

Detailed dose-response curves have been obtained for the induction of aryl hydrocarbon hydroxylase (AHH) m liver, lung and kidney by intraperitoneally administered 3-methylcholanthrene (3-MC) in Wistar rats. The effects of gonadectomy also were studied. Lung was the tissue most sensitive to induction, followed by liver, and then kidney in all cases. Although liver exhibited the greatest overall activity, the per cent increase over control AHH levels was much higher in the two extrahepatic tissues. Gonadectomy did not affect either control or induced AHH activity in any of the three organs in the female, or in the male lung. However, castration of the males decreased control liver enzyme levels and increased these levels in kidney. The AHH levels reached after maximal 3-MC induction were the same in castrated and sham-operated male rat livers. A different pattern was seen in male kidney enzyme levels where significantly increased maximal induction was observed in castrated animals.     

Blood monocyte aryl hydrocarbon hydroxylase activity in psoriasis.

The activity of aryl hydrocarbon hydroxylase (AHH) in monocytes from 13 healthy control subjects and in 16 patients with discoid psoriasis was measured. The mean basal monocyte AHH activities of the control and psoriatic groups were 3.4 +/- 0.47 and 4.46 +/- 0.55 respectively (pmol 3-OHBP h-1 10(-6) cells). This difference was not significant. The mean induced monocyte AHH activities in the control and psoriatic groups were 56.4 +/- 10.8 and 77.8 +/- 16.0 pmol 3-OHBP h-1 10(-6) cells respectively but this difference was not significant. The mean induction ratios in the control and psoriatic groups were 16.5 +/- 1.8 and 20.4 +/- 3.6 respectively. Again, this difference was not significant. We conclude therefore, that monocyte aryl hydrocarbon hydroxylase activity is not reduced in psoriasis.     

Induction of rat-hepatic microsomal cytochrome P-450 and aryl hydrocarbon hydroxylase by 1,3-benzodioxole derivatives

Several 1,3-benzodioxoles (BD) and related compounds were studied in relation to their ability to generate metabolite complexes with hepatic cytochrome P-450 following administration in vivo to rats. BD derivatives that formed stable metabolite complexes with cytochrome P-450 were considerably more effective inducers of cytochrome P-450 and aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (AHH) activity than derivatives that did not form stable complexes. Linear regression analysis showed that AHH activity was well correlated (r = 0.980) with total (i.e. complexed plus uncomplexed) cytochrome P-450 content and was not correlated with levels of uncomplexed cytochrome P-450. Aminopyrine N-demethylase (APDM) activity in hepatic microsomes from rats treated with 1,3-benzodioxoles was moderately correlated in a linear relationship with uncomplexed levels of cytochrome P-450 and not with total cytochrome P-450.     

Induction of rat-hepatic microsomal cytochrome P-450 and aryl hydrocarbon hydroxylase by 1,3-benzodioxole derivatives

1. Several 1,3-benzodioxoles (BD) and related compounds were studied in relation to their ability to generate metabolite complexes with hepatic cytochrome P-450 following administration in vivo to rats.

2. BD derivatives that formed stable metabolite complexes with cytochrome P-450 were considerably more effective inducers of cytochrome P-450 and aryl hydrocarbon (benzo[α]pyrene) hydroxylase (AHH) activity than derivatives that did not form stable complexes.

3. Linear regression analysis showed that AHH activity was well correlated (r = 0.980) with total (i.e. complexed plus uncomplexed) cytochrome P-450 content and was not correlated with levels of uncomplexed cytochrome P-450.

4. Aminopyrine N-demethylase (APDM) activity in hepatic microsomes from rats treated with 1,3-benzodioxoles was moderately correlated in a linear relationship with uncomplexed levels of cytochrome P-450 and not with total cytochrome P-450.     

Modulation of rat hepatic aryl hydrocarbon hydroxylase by various flavones and polycyclic aromatic hydrocarbons

The microsomal cytochrome P-450-dependent aryl hydrocarbon hydroxylase is important in the detoxification of polycyclic hydrocarbons as well as their activation to cytotoxic or carcinogenic derivatives. We have studied compounds that can modify the activity of this enzyme system. Three types of flavones are distinguished on the basis of their effect on the constitutive and polycyclic hydrocarbon-induced rat hepatic enzyme activity: (a) the 5,6- and 7,8-benzoflavones and their more hydrophobic derivatives inhibit the induced enzyme and increase or do not affect the constitutive enzyme activity; (b) derivatives typified by the 4'-hydroxylated benzoflavones similarly decrease both induced and constitutive activities; (c) polyhydroxyflavones inhibit the constitutive enzyme more than the induced enzyme. Two polycyclic hydrocarbons, 9-chloro-7H-dibenzo(a,g)carbazole and 6-aminochrysene, both potent inhibitors of the enzyme system, affect the constitutive and induced enzyme similar to compounds in groups a and b, respectively. The various activity-modulating compounds are useful reagents for distinguishing closely related enzymes present in a variety of different tissues and species under different conditions.     

Placental aryl hydrocarbon hydroxylase activity and placental calcifications.

Induction of aryl hydrocarbon hydroxylase (AHH) activity in the placenta as a result of maternal exposure to polycyclic aromatic hydrocarbons contained in cigarette smoke has been well documented. Furthermore, calcifications are more prevalent in the placentas of pregnant smokers than in those of non-smokers. The present study examines whether this latter relationship could be explained by the induction of AHH activity in the placenta. AHH levels were determined at birth in 141 unselected pregnant women admitted for delivery. Macroscopic placental examination was performed for vascular lesions, abnormalities of placental shape, of the cord and parameters of placental maturity such as basal and parenchymatous calcifications. Significant increases in the prevalence of calcifications of the placental basal plates and parenchyma with the induction of placental AHH were found. A similar significant association between smoking and AHH activation was also observed. These findings remained unchanged when controlling for smoking status assessed both by questionnaire and presence of cotinine in mother's urine. Moreover, the apparent association between smoking 'factor' and calcifications disappeared when controlling for AHH induction. Therefore, the association between smoking and placental calcifications previously related could be mediated by the AHH induction.     

Analysis of the aryl hydrocarbon hydroxylase assay

The assay method and the properties of aryl hydrocarbon hydroxylase were studied with rat liver microsomes. The assay could be carried out by two methods: (1) the microsomes (initially at 15–20°) were pre-incubated at 37° and then benzo[a]pyrene and NADPH were added to initiate the assay; and (2) benzo[a]pyrene was added to microsomes at 15–20°, pre-incubated at 37°, and then NADPH was added to initiate the reaction. The aryl hydrocarbon hydroxylase activity obtained by method 1 was usually 60–120 per cent higher than that obtained by method 2. Several lines of study suggested that the microsomes can exist in two states. Binding of benzo[a]pyrene at temperatures lower than 20° would limit the microsomes to a low activity state whereas binding at temperatures above 25° would enable the microsomes to exist in a high activity state. The molecular basis of these activity states remains to be investigated. The effects of acetone and methanol, both having been widely used as the solvents for benzo[a]pyrene, on aryl hydrocarbon hydroxylase have also been studied. The aryl hydrocarbon hydroxylase activities of control and phenobarbital-induced microsomes were higher with acetone as the solvent for benzo[a]pyrene as compared with methanol. The hydroxylase activity of 3-methylcholanthrene-induced microsomes, however, was slightly higher with methanol.