Metabolic activation of aromatic hydrocarbons in purified rat liver nuclei

'Proc. Nat. Acad. sci. USA Vol. 73, No. 2, pp. 457-461, February 1976

Biochemistry

Metabolic activation of aromatic hydrocarbons in purified rat liver nuclei: Induction of enzyme activities and binding to DNA with and without monooxygenase-catalyzed formation of active oxygen (nuclear and microsomal aryl hydrocarbon hydroxylase/nuclear and microsomal cytochrome P-450) ELEANOR C. ROGAN, PAULA MAILANDER, AND ERCOLE CAVALIERI Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Neb. 68105 Communicated by Melvin Calvin, December 4,1975 ABSTRACT Purified rat liver nuclei covalently bound low levels of seven aromatic ['4C~hydrocarbons to nuclear DNA. Induction with 3-methylcholanthrene increased the binding of six carcinogenic hydrocarbons, but did not raise the level of binding of noncarcinogenic anthracene. Removal of the nuclear envelope by Triton N-101 eliminated binding and aryl hydrocarbon hydroxylase activities and cytochrome P-450 from the nuclei. Binding of two stogcarcinogens, benzo[ajpyrene and to nuclear DNA was com7,12-dimethylbenz~alathacene, pared to the levels of aryl hydrocarbon hydroxylase and c.ytochrome P-450 in nuclei frm uninduced and benz~ajanthracene-, 3-methylcholanthrene-, and phenobarbital-induced rats. Microsomal hydroxylase and cytochrome P.450 were also assayed. Induction with 3-methylcholanthrene gave the largest increases in nuclear activities: 11 times as much hysdroxylase, 6 times as much cytochrome P45O, and 4 times as muhbinding of bohhdrocarbons. Benz[alanthracene and phenobarbital induedtese nuclear activities 0- to 4-fold. In the presence of added NADPH, binding of benzofajpyrene to DNA by nuclei increased rapidly for at least 20 min. When NADPH was not added, the reaction stopped at a low level in 5 min. When CO was bubbled through the reaction mixture with or without added NADPH, binding of benzo[aprne and 7,12-dimethylezaatrcene was partially inhiie,indicating that cohrm P450 plays a role in this activation. Since no nuclear hydroxylase activity was seen without added NADPH or in the presence of CO, activation and subsequent binding of hydrocarbons to nuclear DNA, at least in part, does not require the activated oxygen used in monooxygenase reactions.

thracene (BA), a weak carcinogen; 3-methylcholanthrene (MC), a strong carcinogen; and phenobarbital, a general monooxygenase inducer. In addition to the binding of hydrocarbons to DNA in purified nuclei which appears similar to that catalyzed by AHH, we have observed binding of benzo[a]pyrene (BP) and 7,12-dimethylbenz[alanthracene (DMBA) under conditions which would not favor monooxygenase activities (1)namely, without addition of NADPH to supply electrons for oxygen activation and recycling of cytochrome P-450 to the usual ferric form (12). We have extended our study of this binding with both BP and DMBA, demonstrating here that this binding is not due to endogenous NADPH. Similarly, a low level of binding is also seen in the presence of carbon monoxide, which interacts with cytochrome P-450, thereby inhibiting monooxygenase hydroxylations; this implies that cytochrome P-450 is essentially involved in binding activation. The absence of nuclear AHH activity without added NADPH or in the presence of CO indicates that formation of activated oxygen by monooxygenase (12) does not occur. The implications of these findings in relation to the mechanism of binding activation will be discussed. MATERIALS AND METHODS

Weanling (3-week-old) male Sprague-Dawley rats (the Eppley Colony) were treated one of four ways: no induction, one intraperitoneal injection of 25 mg of MC per kg body weight or 21 mg/kg of BA 24 hr before killing, or twicedaily injections of 36 mg/kg of phenobarbital for 3 days before killing. Nuclei were purified from the livers by the procedure of Berezney et al. (13). Microsomes were prepared by precipitation with calcium,, the "rapid" method of Schenkman and Cinti (14), starting with the first superna-

Metabolism of polycyclic aromatic hydrocarbons by microsomal monooxygenases is the cellular method for detoxification and disposal of these compounds. Monooxygenases also appear to play a role in the activation of hydrocarbons to carcinogenic forms. A common assumption, and one we share, is that the carcinogenic process is initiated by the chemical interaction of a carcinogen with DNA. We have previously reported the presence in rat liver nuclei of enzymes which activate hydrocarbons to forms which covalently bind to DNA (1). Such activation has been ascribed to the monooxygenase aryl hydrocarbon hydroxylase (AHH)

tant from the preparation of nuclei as follows: to 25 ml of supernatant was added 33 ml of sucrose-TKM (0.25 M sucrose, 0.025 M KCI, 0.005 M Mg9l2, 0.05 M Tris-HCI, pH 7.5; total volume, 7 ml/g wet weight liver) and the suspension was centrifuged for 10 min at 9750 X g. The resulting supernatant was mixed with 5 volumes of 0.0125 M sucrose, 8.8 mM CaC12, 5.5 mM Mg9l2. This suspension was centrifuged for 10 min at 1900 X g. The precipitated microsomes were finally suspended in 1 or 2 ml of 0.23 M TrissHCI-50% (vol/ vo1) glycerol, pH 7.5. Cytochrome P-450 was measured by its carbon monoxide binding difference spectrum after reduction with sodium dithionite on a Cary model 14 spectrophotometer according to Estabrook et al. (15), except that the buffer used was 0.23 M Tris-50% glycerol, pH 7.5, and the nuclei were sonicated. (1 min in a Ladd fixed intensity ultrasonic unit at 82 kHz) be-

(2). Monooxygenases are generally inducible in nuclei and microsomes by phenobarbital and with. specificity by some hydrocarbons (3-11). Carcinogenic hydrocarbons' may enhance

their own metabolism by inducing enzymes which show specificity for the hydrocarbon. We report here preliminary studies which compare the inductive effects of benz[a ]anAbbreviations: AHH, aryl hydrocarbon hydroxylase; A, anthracene; BA, benz[alanthracene; BP, benzo[alpyrene; DBA, dibenz[a,hlanthracene; DMBA, 7,12-dimethylbenz~ajanthracene; MBA, 7-methylbenz[alanthracene; MC, 3-methylcholanthrene; TKM, Tris.HCI-

KCI-M90l2.

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Proc. Nat. Acad. Sci. USA 73 (1976)

FIG. 1. Electron micrograph of Triton-treated MC-induced nuclei. Triton-treated nuclei were fixed in glutaraldehyde, mium tetroxide, embedded, and viewed in a Siemens 1A electron microscope.

fore the determination was made. Concentration was calculated from the difference between 480 and 450 nm using an extinction coefficient of 91 mM'1 cm-1 (16). AHH was assayed as described by Nebert and Gelboin (8). One unit is defined as 1 nmol of 3-hydroxybenzo[a]pyrene produced in 20 min of incubation at 37'. Measurements were made on an Aminco-Bowman spectrophotofluorometer or a Turner model 430 spectrofluorometer, exciting at about 394 nm and recording emission at about 520 nm using 3-hydroxybenzo[alpyrene (supplied by the National Cancer Institute) as the standard. The materials and procedures for assay of the binding of [14C]hydrocarbons to DNA in purified nuclei have already been published (1). Samples were counted by liquid scintillation to 1% standard error. Triton-treated nuclei were prepared from MC-induced nuclei suspended in 1 ml of sucrose-TKM. Half of the nuclei were removed, centrifuged, and resuspended in 1 ml of sucrose-TKM, 0.3% Triton N-101 (Sigma Chemical Co.). Both aliquots of nuclei were treated as follows (17): 10 strokes in a Dounce homogenizer, centrifugation at 1900 X g, resuspension in sucrose-TKM with or without 0.3% Triton N-101, 10 more strokes in a Dounce homogenizer followed by three washings in sucroseTKM (to remove the Triton). Both aliquots were finally suspended in 0.5 ml of sucrose-TKM and binding of [14C]BP was measured. Comparisons of binding and AHH in the presence of carbon monoxide were made on equal aliquots of the same preparation of nuclei. Incubations were carried out for 20 min at 370 in 100 X 14 mm glass tubes sealed with a rubber septum. CO was slowly bubbled through the mixture for 1

postflixed in os-

min before nuclei were added and the bubbling was continued for the first 5 min of incubation. Reaction conditions in these measurements of binding and AHH activity differed only in the concentration of substrate (0.2 mM [14C]BP or [14C]DMBA and 0.1 mM BP, respectively) and the presence of 3 mM MgCl2 in AHH assays.

RESULTS The presence of enzymes in the nuclear envelope capable of activating polycyclic hydrocarbons to species which reacted with DNA was demonstrated with Triton-treated MC-induced nuclei. Treatment with Triton removed the nuclear envelope and associated ribosomes, leaving the nuclear contents morphologically intact (Fig. 1). These nuclei did not bind [14C]BP to DNA or contain detectable AHH or cytochrome P-450, while control nuclei had normal levels of these enzymes. Uninduced nuclei activated and bound low levels of seven hydrocarbons to DNA. MC induction increased the amount of hydrocarbon bound to nuclear DNA for the six carcinogens of various potencies but did not increase binding of noncarcinogenic anthracene (A) (Table 1). Specificity of induction was studied by comparing the levels of binding of two strong carcinogensj BP and DMBA, with AHH and cytochrome P-450 in nuclei and microsomes after induction with BA, MC, and phenobarbital (Table 2). MC was the strongest inducer of nuclear activities: 11 times as much AHH, 6 times as much cytochrome P-450, and 4 times as much binding of BP and DMBA. BA induction tripled the level of nuclear AHH, increased cytochrome P-450 only

Biochemistry: Rogan et al.

Proc. Nat. Acad. Sci. USA 73 (1976)

459

Table 1. Binding of hydrocarbons to nuclear DNA pmol of hydrocarbon/pmol of DNA*

['4C ] Hydrocarbon

Uninduced

MC-induced (1)

DMBA MC BP MBA DBA BA A

3.8 ± 3.6 (1) 3.1 ± 0.2 2.2 ± 0.9 (1) 2.9 ± 0.8 0.5 ±0.4 1.8±1.1 3.3 ± 1.3

14.5 ± 5.5 7.6 ± 3.0 8.4 ± 5.2 5.4 ± 2.3 4.9 ±0.5 5.0±3.2 2.5 ± 1.2

UNINDUCED MICROSOMES

BA- MICROSOMES MC-MICROSOMES

PB-MICROSOMES

MBA, 7-methylbenz[a]anthracene; DBA, dibenz[a,h]anthracene. * Binding was measured in 10 min incubations as previously described (1). Uncertainty here and in Tables 2 and 3 is expressed as standard deviation.

50%, and doubled the level of binding of BP, while no significant increase was seen in the binding of DMBA. The effect of BA induction of microsomal enzymes was similar to that of MC; both hydrocarbons induced cytochrome P-448 in microsomes, rather than cytochrome P-450 (Fig. 2). This was in contrast to the cytochrome P-450 induced in microsomes by phenobarbital. Quantitatively the effect of phenobarbital induction was greatest on the level of microsomal and nuclear cytochrome P-450, 2.5- and 4-fold increases, respectively; but a negligible effect on AHH was found in both nuclei and microsomes, and binding of BP and DMBA was 1.5- to 2-times higher than in uninduced nuclei. The level of cytochrome P-450 in both uninduced and induced nuclei was too low and the spectrum too broad (Fig. 2) to determine whether maximum absorbance occurred at 448 or 450 nm. For both AHH and cytochrome P-450 the ratio of enzyme concentration in nuclei compared to that in microsomes rose significantly in MC-induced animals (Table 2). In BA- and phenobarbital-induced animals this ratio remained similar to the ratio found in uninduced animals. The kinetics of BP binding are shown in Fig. 3. Without added NADPH the binding activity was finished in about 5 min, while with added NADPH the reaction continued strongly for at least 20 min. The ability of nuclei to bind BP and DMBA to DNA without added NADPH was further explored (Table 3). When NADPH was not added to the incubation mixture, no AHH

410

450

500

WAVELENGTH, nm

FIG. 2. Cytochrome P-450 difference spectra. Microsomal protein: uninduced, 1.6 mg/ml; BA, 2.2 mg/ml; MC, 2.2 mg/ml; phenobarbital, 1.7 mg/ml. Nuclear protein: 3.9 mg/ml.

activity was detectable. Furthermore, when the binding assay was carried, out in the presence of CO, the amount of BP or DMBA bound was similar to that bound in the absence of added NADPH. In the presence of CO, AHH activity was reduced at least 100-fold and was variably detectable. Addition of NADPH did not overcome either the CO inhibition of AHH or the partial inhibition of binding. These experiments demonstrate that the binding of BP and DMBA seen without added NADPH is not attributable to endogenous NADPH and that some binding of BP and DMBA to nuclear DNA can occur without the activated oxygen used by AHH and other monooxygenases.

DISCUSSION The presence of monooxygenase and electron transport enzymes in cell nuclei has been demonstrated in recent years

Table 2. Monooxygenase activities in nuclei and microsomes Induced rats

Enzymic activity*

BA

Uninduced rats

MC

Binding of hydrocarbon to nuclear DNA, pmol/Mmol of DNA BP DMBA AHH, units/mg of protein Nuclei

Phenobarbital

2.2 ± 0.9 (1) 3.8 ± 3.6 (1)

5.2 ± 1.2 4.6 ± 1.4

8.4 ± 5.2(1) 14.5 ± 5.5 (1)

3.5 ± 1.4 7.9 ± 2.9

0.11 ± 0.07

0.36 ± 0.16 4.6 ± 0.8 0.078

1.2 ± 0.1 5.4 ± 1.1 0.22

0.08 ± 0.03 1.6 ±0.3 0.050

1.2 ± 0.1 Microsomes 0.092 Nuclei/microsomes Cytochrome P4 5.0, nmol/mg of protein 0.02 ± 0.02 Nuclei 1.00 ± 0.08 (450 nm) Microsomes 0.02 Nuclei/microsomes

0.08 ± 0 01 0.13 ± 0.05 0.03 ± 0.02 1.17 ± 0.09 (448 nm') 1.3 ± 0.2 (448 nm) 2.4 ± 0.6 (450 nm) 0.033 0.10 0.02.6

* Rats were induced and enzymes were assayed as described in Materials and Methods; incubations for binding and AHH measurements were carried out for 20 min. Wavelengths in parentheses indicate the absorption maximum of the microsomal cytochrome.

460

Proc. Nat. Acad. Sci. USA 73 (1976)

Biochemistry: Rogan et al. 12

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10 Time, Min

15

20

FIG. 3. Kinetics of BP binding in MC-induced nuclei. Binding of [14C]BP to nuclear DNA was measured at the indicated times as described in Materials and Methods. Each point is the average of two to seven determinations. Solid line, with added NADPH; broken line, without added NADPH.

(18). Washing of nuclei with the detergent Triton N-101 has been shown to remove the nuclear envelope (17). The simultaneous removal of binding and AHH activities and cytochrome P-450 clearly shows that the binding activity as well as the other enzymes are associated with the nuclear envelope. The amount of hydrocarbon bound to DNA in uninduced nuclei (Table 1) was generally low; however MC induction increased the level of binding for all hydrocarbons studied with the exception of anthracene, which is noncarcinogenic. The effect of MC induction on binding of BP and DMBA could be distinguished from that with induction by BA or phenobarbital (Table 2). Induction with BA or phenobarbital at best only gave about half the increase in binding observed with MC induction. The levels and induction of nuclear AHH activity we observed were similar to those reported by others (5, 6, 18) for normal nuclei and those induced with MC or phenobarbital. Again MC had a much greater inductive effect than BA, and phenobarbital did not increase the level of nuclear AHH. The basal level of nucleTable 3. Binding of BP and DMBA to.DNA without activated oxygen

Binding of [1'C ] hydrocarbon*, pmol bound/ ,umol of DNA AHH*, pmol - -o product/mg BP DMBAt of protein Complete -NADPH +CO +CO-NADPH Background:

11.0 ±0.6 2.6 ± 1.1 1.8 ± 0.4 2.1 ± 0.8 0.6 ±0.2

10.2 ± 2.3 3.4 ±1.2 1.8 ± 0.2

400-665 0 (