Aryl hydrocarbon hydroxylase induction in mouse liver cells--relationship to position in the cell cycle

The inducibility of aryl hydrocarbon hydroxylase (AHH) by benzo[a]-pyrene (BaP) has been studied in synchronously grown cultures of mouse liver cells. These cells (NMuLi cl 8) have low basal levels of AHH which can be induced greater than 100-fold by BaP. Cells were synchronized in G1(G0) by serum starvation and in S by release from serum starvation in combination with excess thymidine. When released from G1(G0) by replating at lower cell density in fresh medium with 20% serum, cells began entering S with a lag of 12 h. Addition of BaP (1 microgram/ml) 8 h before serum stimulation, at the time of stimulation or 7.5 h after stimulation all gave similar induction kinetics: the AHH activity peaked as the cells began entering S regardless of when the BaP was added. Cells blocked in various parts of S by excess thymidine were inducible for AHH activity as efficiently as cells moving through S and into G2. These results indicate that the inducibility of AHH is greater when cells are actively proliferating and may be a contributing factor to why growing cells are more sensitive to mutagenesis and transformation than quiescent cells.     

Aryl hydrocarbon hydroxylase activity assayed in whole cell lysates using synchronous fluorescence spectroscopy.

Synchronous fluorescence spectrophotometry has been used to measure induced and constitutive levels of aryl hydrocarbon hydroxylase activity in lysates of C3H 10T1/2 mouse embryo fibroblasts. Without compromising sensitivity, the method was reproducible, eliminated the need to extract metabolites, and made the procedure simpler and less time consuming than other methods. Moreover, since the assay was tailored to directly measure 3-hydroxybenzo(a)pyrene, a metabolite produced by several cytochrome P-450s, it may be more generally applicable than dealkylation assays, which apparently detect only P-450-IA1.     

Aryl hydrocarbon hydroxylase of rat brain mitochondria: Properties of,and effect of inhibitors and inducers on,enzyme activity

Aryl hydrocarbon hydroxylase (AHH), a typical example of mixed-function oxidase system, was studied in rat brain mitochondria. The enzyme was found to require oxygen and NADH for optimal expression of the activity. Coaddition of NADPH in the incubation system containing NADH resulted in an additive effect on the enzyme activity. NADH- and NADPH-dependent mitochondrial AHH activity was linear with respect to protein concentration and incubation time. The enzyme exhibited a sharp optima at pH 7.6. Specific activity of NADH-dependent mitochondrial AHH in rat brain was 3–4 and 8–11 times higher than that of NADPH-dependent mitochondrial and microsomal enzyme activity, respectively. Of the species investigated, NADH-dependent mitochondrial AHH followed the order: mice ? guinea pig > rat, while NADPH-supported mitochondrial AHH was in the order: rat > guinea pig ? mice. Specific activity of NADH-dependent mitochondrial AHH in various rat brain regions was similar with the exception of olfactory lobes which exhibited 60% higher activity than other region. When total region activities were added approximately whole brain activity was recovered. The apparent K_m value of NADH-dependent mitochondrial AHH was 1.18 μm with benzo(a)pyrene as a substrate. This K_m value was five to six times lower than that of NADPH-dependent microsomal AHH in rat brain (6.66 μm). NADH-dependent mitochondrial AHH was inhibited by KCN in a concentration-dependent manner while NADPH-supported mitochondrial AHH did not reveal any sensitivity to cyanide. Brain microsomal NADH as well as NADPH-supported AHH was also inhibited by KCN in a concentration-dependent manner. Carbon monoxide inhibited NADH-dependent mitochondrial AHH activity (48%) and had no effect on NADPH-dependent mitochondrial enzyme. Mitochondrial NADH and NADPH-dependent AHH activities were induced by 3-methylcholanthrene (64–73%) and benzo(a)pyrene (91–92%) pretreatments while no induction occurred with phenobarbital administration. 1-Benzylimidazole, SKF 525 A, metyrapone, and α-naphthoflavone inhibited both basal and 3-methylcholanthreneinduced NADH-dependent mitochondrial AHH activity. α-Naphthoflavone was more effective in inhibiting 3-methylcholanthrene-stimulated rat brain NADH-dependent mitochondrial AHH. Mitochondrial NADH-dependent AHH activity increased gradually with the onset of development and attained a steady state after 49–56 days of age. An increase of eight- to ninefold in the specific enzyme activity was observed between 7- and 56-day-old rats. No significant increase in brain mitochondrial AHH activity was observed between 56- and 91-day-old rats.     

Comparison of aryl hydrocarbon hydroxylase and epoxide hydratase. Induction in primary fetal rat liver cell culture

The activity of aryl hydrocarbon hydroxylase (AHH) and/or epoxide hydratase (EH) is induced in primary fetal rat liver cell culture by benz-[alpha]anthracene (BA), phenobarbital (PB), cigarette smoke condensate (CSC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and trans-stilbene oxide (TSO). The response of the two enzymes to the different chemicals varies as follows: (a) AHH is induced by lower concentrations of BA, PB and CSC than those required to significantly induce EH; (b) AHH is selectively induced by TCDD and by low BA concentrations; (c) the kinetics of AHH induction by BA, PB and CSC is faster than that of EH; (d) TSO is a selective inducer of EH. As described earlier for AHH, RNA and protein synthesis and the continuous presence of the inducer are required in the early phases of EH induction. Later when the EH activity has reached a plateau, intact RNA and protein synthesis is not necessary to maintain the enzyme at its optimal value. The removal of the inducer determines a decay of the EH activity, allowing the estimation of a biological tau 1/2 of about 72 h. TSO prevents the AHH induction by PB, but not that mediated by BA and CSC. Added together with PB, BA, CSC or PB plus BA, TSO induces the EH activity in a more than additive manner. This effect is only seen after 6 days of continuous treatment. These results indicate that in this tissue culture model, the mechanism of AHH and EH induction can clearly be dissociated.     

Comparison of aryl hydrocarbon hydroxylase and epoxide hydratase. Induction in primary fetal rat liver cell culture

The activity of aryl hydrocarbon hydroxylase (AHH) and/or epoxide hydratase (EH) is induced in primary fetal rat liver cell culture by benz[a]anthracene (BA), phenobarbital (PB), cigarette smoke condensate (CSC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and trans-stilbene oxide (TSO). The response of the two enzymes to the different chemicals varies as follows: (a) AHH is induced by lower concentrations of BA, PB and CSC than those required to significantly induce EH; (b) AHH is selectively induced by TCDD and by low BA concentrations; (c) the kinetics of AHH induction by BA, PB and CSC are faster than that of EH; (d) TSO is a selective inducer of EH. As described earlier for AHH, RNA and protein synthesis and the continuous presence of the inducer are required in the early phases of EH induction. Later, when the EH activity has reached a plateau, intact RNA and protein synthesis is not necessary to maintain the enzyme at its optimal value. The removal of the inducer determines a decay of the EH activity, allowing the estimation of a biological $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 72 h. TSO prevents the AHH induction by PB, but not that mediated by BA and CSC. Added together with PB, BA, CSC or PB plus BA, TSO induces the EH activity in a more than additive manner. This effect is only seen after 6 days of continuous treatment. These results indicate that in this tissue culture model, the mechanism of AHH and EH induction can clearly be dissociated.     

Aryl hydrocarbon hydroxylase activity in the fungus Cunninghamella bainieri: evidence for the presence of cytochrome P-450.

The aryl hydrocarbon hydroxylase (AHH) enzyme from the fungus Cunninghamella bainieri has been characterized. It is NADPH dependent and exhibits a pH optimum near 7.8. It is inhibited by CO, SKF 525-A, and metyrapone, but cyanide shows no inhibitory effect. These data, together with the pattern of inhibition and stimulation shown by metal ions, suggest that the fungal AHH activity is due to a cytochrome P-450. About 25% of the hydroxylase activity remains in the supernatant while the remainder precipitates after centrifugation at 100,00g for 2.5 h. The 100,000g supernatant was further fractionated by (NH₄)₂SO₄ precipitation. A NADPH-dependent cytochrome c reductase is concentrated mainly in the 100,000g supernatant, and a cytochrome c oxidase is present mainly in the 100,000g pellet. The cytochrome c reductase is essential for AHH activity as shown by the inhibition of AHH activity with cytochrome c and dichloroindophenol. Solubilization of a portion of the 100,000g pellet in aqueous digitonin followed by dithionite reduction and addition of CO resulted in the observation of a maximum absorbance at 450 nm characteristic of cytochrome P-450.     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Effects of biogenic amines on the formation of adenosine 3', 5'-monophosphate in human thyroid slices.

The effects of various concentrations of biogenic amines on the formation of adenosine-3', 5'-monophosphate (cyclic AMP) and their interactions with other thyroid stimulators were investigated in human thyroid slices from normal and Graves' disease. Most of biogenic amines were found to have the stimulatory effects to some extent. Among the biogenic amines tested, histamine was the most potent thyroid stimulator, norepinephrine and serotonin, the intermediate in terms of cyclic AMP formation. The effect of histamine was almost as potent as TSH in thyroid slices from Graves' disease. This stimulatory effect of histamine was blocked by metiamide, a histamine H2-receptor antagonist, but not by chlorpheniramine, a histamine H1-receptor antagonist. The effect of norepinephrine was completely inhibitied by propranolol, but not by phentolamine. Polyphloretin phosphate did not inhibit norepinephrine- or histamine-induced cyclic AMP formation, while it significantly depressed cyclic AMP formation induced by prostaglandin E2. The maximal effect of histamine was additive to that of TSH. It is suggested that biogenic amines, histamine and norepinephrine, in particular, have the thyroid receptors different from that of TSH or prostaglandin E2 and could play an important role in thyroid physiology.     

Effects of biogenic amines on the formation of adenosine 3',5'-monophosphate in human thyroid slices.

The effects of various concentrations of biogenic amines on the formation of adenosine-3', 5'-monophosphate (cyclic AMP) and their interactions with other thyroid stimulators were investigated in human thyroid slices from normal and Graves' disease. Most of biogenic amines were found to have the stimulatory effects to some extent. Among the biogenic amines tested, histamine was the most potent thyroid stimulator, norepinephrine and serotonin, the intermediate in terms of cyclic AMP formation. The effect of histamine was almost as potent as TSH in thyroid slices from Graves' disease. This stimulatory effect of histamine was blocked by metiamide, a histamine H2-receptor antagonist, but not by chlorpheniramine, a histamine H1-receptor antagonist. The effect of norepinephrine was completely inhibited by propranolol, but not by phentolamine. Polyphloretin phosphate did not inhibit norepinephrine- or histamine-induced cyclic AMP formation, while it significantly depressed cyclic AMP formation induced by prostaglandin E2. The maximal effect of histamine was additive to that of TSH. It is suggested that biogenic amines, histamine and norepinephrine, in particular, have the thyroid receptors different from that of TSH or prostaglandin E2 and could play an important role in thyroid physiology.     

Variation in DNA swivel enzyme activity during the mammalian cell cycle.

Synchronized cells of a normal human lymphocytic cell line contain little swiven enzyme activity in G0 and G1 and high activity in Sphase. The level of activity in different growth phase appears to be related to the fraction of the population engaged in DNA replication. No endogenous inhibitor or activator of swiven activity could be demonstrated. The evidence implies that the enzyme may be present only during S phase; it is therefore a possible control factor for replication.