Regulation of aryl hydrocarbon (benzo-(A)-pyrene) hydroxylase activity in mammalian cells. Induction of hydroxylase activity by N6,O2'-dibutyryl8 adenosine 3':5'-monophosphate and aminophylline.

Treatment of hamster BHK cells with N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), aminophylline, theophylline, or papaverine increased the level of aryl hydrocarbon (benzo(a)pyrene) hydrolxylase activity. The highese increase, 100-fold, was obtained with Bt2cAMP plus aminophylline or theophylline. N2,O2-Dibutyryl guanosine 3':5'-monophosphate gave a lower induction than Bt2cAMP. The level of hydroxylase activity started to decrease 6 hours after treatment with the inducer and was reduced to almost the uninduced level after 24 hours. Repeated addition of Bt2cAMP and aminophylline did not prevent this decrease. The hydroxylase can also be induced by treating cells with benz(a)anthracene, and the level of this induced activity was maintained for 24 hours. Aminophylline gave a 2- to 8-fold stimulation of the induction by benz(a)anthracene. The enzyme activity induced by Bt2cAMP, aminophylline, and benz(a)anthracene converted benzo(a)pyrene to similar alkali-extractable metabolities with a fluorescence spectra similar to that of 3-hydroxybenzo(a)pyrene. These induced enzyme activities also showed a similar heat stability. Induction by Bt2cAMP and aminophylline, like induction by benz(a)anthracene, required continued protein synthesis and only an initial period of RNA synthesis. Compared to the benz(a)anthracene-induced hydroxylase with a Km of 4.3 muM, the hydroxylase induced by Bt2cAMP and aminophylline showed a Km of 0.14 muM, and was 100-fold more sensitive to inhibition by 7,8-benzoflavone. Increasing the serum concentration in the culture medium stimulated the induction by aminophylline but did not stimulate induction by benz(a)anthracene. The results indicate that aryl hydrocaarbon (benzo(a)pyrene) hydroxylase can be induced by compounds that increase the level of adenosine 3':5'-monophosphate and that this induction and induced enzyme activity differs from that caused by benz(a)anthracene.

N*,O'-Dibutyryl guanosine 3'S'-monophosphate gave a lower induction than Bt,cAMP. The level of hydroxylase activity started to decrease 6 hours after treatment with the inducer and was reduced to almost the uninduced level after 24 hours. Repeated addition of Bt,cAMP and aminophylline did not prevent this decrease. The hydroxylase can also be induced by treating cells with benz(a)anthracene, and the level of this induced activity was maintained for 24 hours. Aminophylline gave a 2-to &fold stimulation of the induction by benz(a)anthracene. The enzyme activity induced by Bt,cAMP, aminophylline, and benz(a)anthracene converted benzo(a)pyrene to similar alkali-extractable metabolites with a fluorescence spectra similar to that of 3hydroxybenzo(a)pyrene.
These induced enzyme activities also showed a similar heat stability. Induction by Bt,cAMP and aminophylline, like induction by benz(u)anthracene, required continued protein synthesis and only an initial period of RNA synthesis. Compared to the benz(a)anthracene-induced hydroxylase with a K, of 4.3 pM, the hydroxylase induced by Bt,cAMP and aminophylline showed a K, of 0.14 FM, and was loo-fold more sensitive to inhibition by 7&benzoflavone. Increasing the serum concentration in the culture medium stimulated the induction by aminophylline but did not stimulate induction by benz(u)anthracene.
The results indicate that aryl hydrocarbon (benzo(a)pyrene) hydroxylase can be induced by compounds that increase the level of adenosine 3'S'-monophosphate and that this induction and induced enzyme activity differs from that caused by benz(a)anthracene.
Treatment with different concentrations of Bt,cAMP and Bt,cAMP plus 0.7 mM aminophylline showed that there was an increase in induction up to 2 mM Bt,cAMP ( Fig. la). Treatment with aminophylline or papaverine only, showed an optimal induction at 0.7 to 1.5 mM and 17 pM, respectively ( Fig. 1, b and c). Theophylline showed an optimal induction at 0.1 to 0.3 mM. There was a decreased induction at higher doses presumably due to cytotoxicity (23).
Kinetics of Inrluctiorz-Treatment of the BHK cells with Bt,cAMP, aminophylline, or Bt,cAMP plus aminophylline showed that after a 2-hour lag period, aryl hydrocarbon hydroxylase activity increased for 4 hours followed by a decline to almost basal activity after an additional 18 hours. The results with Bt,cAMP plus aminophylline are shown in Fig. 2~2. This decrease was not prevented by repeated treatment of the cell cultures every 3 hours with the inducing agents. Aryl hydrocarbon hydroxylase activity can also be induced by treating the BHK cells with benz(a)anthracene.
In this case there was a lag period of less than 1 hour, the level of the hydroxylase activity increased for about 6 hours, and this increased level was maintained for an additional 18 hours (Fig.  26). Optimal induction was obtained with 1.3 pM or higher concentrations of benz(a)anthracene. The addition of aminophylline gave a further 2-to 8-fold increase in enzyme induction (Fig. 3) and 50%, respectively, whereas addition of actinomycin D at 3 hours showed no inhibition of enzyme induction (Fig.  4).

Cycloheximide
(1.5 pg/ml) blocked enzyme induction when added together with or up to 3 hours after treatment with the inducer.
These results indicate that, as with induction by benz(a)anthracene (24), induction by Bt,cAMP and aminophylline requires continued protein synthesis and only an initial period of RNA synthesis.
Properties of Induced Enzyme-The fluorescence spectrum of the alkali-extractable metabolites produced by the hydroxylase activity was determined for the uninduced and induced enzyme activities (Fig. 5). The results indicate that the hydroxylase in control cells and in cells induced by Bt,cAMP plus aminophylline or by benz(a)anthracene, metabolize benzo(a)pyrene to alkali-extractable metabolites with a fluorescence spectrum similar to that of 3-hydroxybenzo(a)pyrene.
The ase activity (25, 26), was tested for its effect on the enzyme activity induced by Bt,cAMP plus aminophylline and by benz(a)anthracene.
Although both were inhibited, the Bt,cAMP plus aminophylline-induced enzyme was about 100 times more sensitive than the benz(a)anthracene-induced enzyme (Fig. 6) reciprocal plots of benzo(a)pyrene concentration and initial reaction velocity has indeed shown that the Bt,cAMP plus aminophylline-induced enzyme had a 30-fold higher affinity to benzo(a)pyrene (K, = 0.14 FM) than the benz(a)anthraceneinduced enzyme (K, = 4.2 fiM) (Fig. 7). The addition of Bt,cAMP plus aminophylline to homogenates of the benz-(a)anthracene-induced enzyme did not alter either the K, of the hydroxylase for the substrate or the sensitivity of the hydroxylase to 7&benzoflavone. The K, of the uninduced hydroxylase activity could not be determined because of its low activity in untreated BHK cells.
Induction of Enzyme Activity by Fetal Calf Serum-Induction by Bt,cAMP and aminophylline was tested in the presence of various concentrations of fetal calf serum (0 to 50%) in the culture medium. The addition of serum, without these compounds, induced aryl hydrocarbon hydroxylase activity, and there was a 30-fold increase with 50% serum (Fig. 8). Induction by Bt,cAMP and aminophylline can occur in the absence of serum. However, the degree of induction was increased in the presence of serum, and the highest induction was also with 50% serum (Fig. 8). The kinetics of enzyme induction with 10% serum showed that the increased enzyme activity declined to the basal level within 12 hours after addition of fresh serum. Three hourly treatments with fresh 10% serum prevented this decline in contrast to the effect of 3 hourly treatments with aminophylline.
Although Bt,cAMP plus aminophylline-inducible and basal enzyme levels were, therefore, dependent on concentration of serum, there was no effect of serum concentration on enzyme inducibility with benz(a)anthracene. DISCUSSION We ha+e previously shown that the treatment of a variety of mammalian cultured cells with Bt,cAMP and aminophylline increased the metabolism of benzo(a)pyrene to water-soluble products (19). The present results showed that this increase in benzo(a)pyrene metabolism is due to the induction by these compounds of aryl hydrocarbon hydroxylase activity. These results suggest that CAMP is a regulator of aryl hydrocarbon hydroxylase in mammalian cells. The kinetics of the hydroxylase induction has shown a maximal enzyme activity at 6 hours, followed by a decline to the basal enzyme level 24 hours after the addition of Bt,cAMP and aminophylline. This can explain the lack of detectable induction in measurements made 24 hours after addition of Bt,cAMP (17,18 7 (left). Double reciprocal plots of benzo!a)pyrene concentration and initial reaction velocity of the induced enzyme. Cell homogenates from BHK cells treated with 1 mM Bt*cAMP plus 0.7 mM aminophylline (a) or 4.2 PM benz(a)anthracene (b) for 6 hours were incubated with the indicated amount of benzo(a)pyrene, and the hydroxylase activity was assayed as described under "Methods" (V), enzyme activity expressed by the arbitrary intensity of fluorescence. (S), concentration of benzo(a)pyrene.
FIG. 8 (right). Effect of various concentrations of fetal calf serum on basal and Bt,cAMP plus aminophylline-inducible aryl hydrocarbon hydroxylase activity. BHK cells were washed twice with medium (without serum), fresh culture medium containing the indicated concentration of fetal calf serum was added with (0) or without (0) 1 mM Bt,cAMP plus 0.7 mM aminophylline, and the cells were incubated for 6 hours before the enzyme assay. enzyme but not of the benz(a)anthracene-inducible enzyme. This suggests a different mechanism for induction of the hydroxylase by these different compounds.
The differences in the K, values have shown that the CAMP-induced aryl hydrocarbon hydroxylase is more efficient than benz(a)anthracene-induced enzyme in metabolizing carcinogenic polycyclic hydrocarbons at a low substrate concentration. This CAMP-induced hydroxylase may, therefore, be of importance in chemical carcinogenesis, since our environment contains small amounts of chemical carcinogens.
Since Bt,cGMP also induces aryl hydrocarbon hydroxylase activity in BHK cells, and aminophylline and papaverine are known to inhibit cyclic phosphodiesterase which decompose all 3'-5' cyclic nucleotides (30), other cyclic nucleotides such as cGMP may also be regulators of this hydroxylase activity.