aldrin epoxidation catalyzed by purified rat-liver cytochromes p-450 and p-448 high selectivity for...

Eur. J. Biochem. I l l , 545-551 (1980) 8 by FEBS 1980

Aldrin Epoxidation Catalyzed by Purified Rat-Liver Cytochromes P-450 and P-448 High Selectivity for Cytochrome P-450

Thomas WOLFF, Helmut GREIM, Mou-Tuan HUANG, Gerald T. MIWA, and Anthony Y . H. LU

Abteilung fur Toxikologie, Gesellschaft fur Strahlen- und Umweltforschung, Neuherberg, Department of Biochemistry and Drug Metabolism, Hoffmann-LaRoche Inc., Nutley, and Merck Sharp and Dohme Research Laboratories, Rahway

(Received August 1, 1980)

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent K , for aldrin in the complete system was 7 f 2 pM. SKF 525-A, at a concentration of 250 pM, inhibited aldrin epoxidation by 65 %, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 pM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5 % ethanol.

When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3 % of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent K, for aldrin was 27 k 7 yM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The Is0 was 0.05, 0.2 and 800 mM, respectively.

These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent K , and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.

A variety of compounds of widely different chem- ical structure are oxidized by the monooxygenase system in the endoplasmic reticulum which contains cytochrome P-450 as the terminal oxidase. The broad substrate specificity of this enzyme system has been partially attributed to the existence of multiple cytochrome P-450 species of different but overlapping substrate specificities [l - 51. This concept of the multiplicity of cytochrome P-450 was first supported by findings suggesting the occurrence of two major groups of microsomal hemoproteins typified by their response to inducing agents like phenobarbital and 3-methylcholanthrene, by spectral properties and by differences in substrate specificity [6 - 81. Compelling

Ahhrevintions. Lau2PtdCho, dilauroylphosphatidylcholine;

Enzymes. NADPH-cytochrome c reductase (EC 1.6.2.4); glu- SKF 525-A, diethylaminoethyl 2,2-diphenyl-valerate.

cose-6-phosphate dehydrogenase (EC 1.1.1.49).

evidence was provided by reconstitution of active monooxygenase complexes with several different cytochrome species isolated and purified from microsomal preparations; for review see [9].

A number of compounds forming metabolites susceptible to simple and sensitive estimation tech- niques have gained wide use as substrates for studies of the properties of monooxygenases in various mammalian tissues. Aldrin epoxidation, which is known to be mediated by monooxygenase [lo- 121, as yet has not been utilized as routine assay although this reaction promises to have some remarkable advantages. Dieldrin, the principle metabolite, undergoes further metabolism very slowly [13]. It can be quantified with high sensitivity by electron-capture gas chromatography using single hexane extracts of the incubation mixture [13]. Recently, it was demonstrated that the assay requires minimal amounts of liver microsomes or liver tissue [13,14].

546 Aldrin Epoxidation Catalyzed by Cytochromes P-450 and P-448

According to previous investigations [13], micro- soma1 aldrin epoxidation is inducible by treatment with phenobarbital in vivo and is reduced after 3-methylcholanthrene treatment of animals. The apparent K, was not altered after 3-methylcholanthrene treatment which resulted in the induction of cytochrome P-448. Development of enzyme activity during matura- tion was similar to that of ethylmorphine N-de- methylation, a reaction primarily mediated by cytochrome P-450 [l 51. These results provided indirect evidence that aldrin is specifically metabolized by cytochrome P-450 species mainly present in the microsomes of phenobarbital-treated animals and by the constitutive enzymes of untreated animals.

To elucidate which forms of microsomal cytochrome P-450 are involved, we decided to study the activity of purified cytochrome fractions on aldrin epoxidation in reconstituted monooxygenase systems. The enzyme systems used were reconstituted from cytochrome P-450 purified from rat liver microsomes after phenobarbital treatment or cytochrome P-448 purified from hepatic inicrosomes of 3-methylcholanthrene-treated rats, and purified NADPH -cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Enzyme activity, requirements for the components, substrate affinity and sensitivity towards inhibitors, such as SKF 525-A, 7,8-benzoflavone and ethanol, were analyzed in the two reconstituted monooxygenase systems.

MATERIALS AND METHODS

Ckemicais

Enzyme and co-enzymes were purchased from Boehringer (Mannheim, FRG); aldrin (1,2,3,4,10,10- hexachloro-l,4,4a,5,8,8a-hexahydro-l,4-endo-5,8-exodimethanonaphthalene) and dieldrin (1,2,3,4,10,10- hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-1,4- endo-5,8exodimethanonaphthalene), purity 99 %, from Riedel-de Haen (Seelze, FRG); n-hexane ( zur Riick- stantlsunal-yse) from Merck (Darmstadt, FRG) ; 7,8- benzoflavone (2-naphthoflavone) from Ega-Chemie (Steinheim, FRG); and sodium cholate, purity 98 %, froin Sigma Chemie (Miinchen, FRG). SKF 525-A was a gift of Dr H. Sies (Institut fur Biochemie, Uni- versitat Munchen). Dilauroylphosphatidylcholine was obtained from Serdary Research Laboratories (On- tario, Canada). All other chemicals were obtained from Merck (Darmstadt) and were of analytical grade.

Purification of Monooxygenase Components

Cytochrome P-450 from phenobarbital-treated rats and cytochrome P-448 from 3-methylcholanthrene- treated rats were prepared as described by West et al. [16]. These preparations generally contained 12 - 16 nmol cytochrome P-450/mg protein. For the ex-

periments described in this paper, cytochrome P-450 preparations containing 12 and 16 nmol/mg protein and cytochrome P-448 preparations containing 6.3 and 15.1 nmol/mg protein were used. Samples of NADPH-cytochrome c reductase with a specific activity of 31000 and 26000 units/mg protein were prepared as described by Yasukochi and Masters [17]. One unit of NADPH-cytochrome c reductase activity is defined as the amount of enzyme catalyzing the reduction of cytochrome c at an initial rate of 1 nmol/ min at 22°C under the assay conditions of Phillips and Langdon [18]. All the proteins and the lipid factor, Lau2PtdCho, dissolved in chloroform, were stored

For assay of enzyme activity, an aliquot of the Lau2PtdCho stock solution was dried under nitrogen. The residue was sonicated after addition of 0.05 M Tris buffer, pH 8.0, containing 1 mM EDTA, by the microtip of a Branson sonifier until no change in the opalescence of the suspension occurred. Final Laul- PtdCho concentration was 1 mg/ml. When this suspension became turbid after storing at room temperature, sonication was repeated before use.

at - 80 "C.

Incubation Procedure

The standard incubation contained the enzymes and lipid components in amounts given in the legends of the figures and tables. 0.1 M potassium phosphate buffer, pH 7.4, was added to a final volume of 1 ml. The NADPH-regenerating system consisted of 8 pmol glucose 6-phosphate, 1 .5 units glucose-6-phosphate dehydrogenase and 0.5 ymol NADP'. Assay volumes of 0.2 ml or 0.1 ml containing aliquots of the enzymes and cofactors were also used. After equilibration to 37°C the reaction was started by addition of 0.2 or 0.05 ymol aldrin in 20 ~l or 5 p1 methanol and ter- minated after 5 min by vigorous shaking with 5 ml n-hexane. Following a short centrifugation to facilitate phase separation, the supernatant was diluted 10- %-fold to obtain an appropriate dieldrin concentration for gas chromatography analysis. When low dieldrin concentrations were assayed, the reaction mixture was extracted with 2 ml hexane and used without further dilution.

Analytical Procedures

Dieldrin was separated and quantified by electron- capture gas chromatography on packed glass columns using aliquots of the hexane extracts without further purification. The recovery of dieldrin, when added to the assay sytem, was 90 & 5% after a single hexane extraction. Details of the procedure are given in [I 31. The liquid phase was 3 % XE-60 on chromosorb WAW DMCS (80- 100 mesh) which gives an optimal separation of aldrin and dieldrin. Column temperature was generally 200 "C. For determination of low dieldrin

T. Wolff, H. Greim, M.-T. Huang, G. T. Miwa, and A. Y . H. Lu 541

amounts the oven temperature was lowered to 170 "C. Activity of benzo[a]pyrene hydroxylation was assayed according to Wiebel et al. [19].

Activity of Epoxide Hydrolase towards Dieldrin

Although the dieldrin molecule possesses the epoxide ring, it is a poor substrate for epoxide hydrolase as revealed by its metabolic stability during incubation with liver microsomes [13]. Thus, in reconstituted cytochrome P-450 or P-448 systems, which apparently do not contain expoxide hydrolase, essen- tially no hydrolysis of the dieldrin formed will occur.

Effect of Solvent

The effect of methanol, the solvent of aldrin, on epoxidase activity has been analyzed using microsomes from control [26], phenobarbital-treated or 3-methylcholanthrene-treated rats (unpublished results). While the enzyme activity of controls was not affected at concentrations of 1 - 10 (v/v) methanol in the incubation medium, the phenobarbital-induced activity was linearily enhanced by 80% under the same conditions. By extrapolation it can be calcu- lated that the induced epoxidase activity in the absence of methanol will be only slightly decreased to 85% of the activity in the presence of 2 % (v/v) methanol, the highest solvent concentration used in our assays. Epoxidase activity in microsomes from rats pretreated with the inducer of cytochrome P-448, 3-methylcholanthrene, showed a reponse to methanol similar to controls.

Evuluution of Data

Substrate affinity for epoxidation dependent on cytochrome P-450 and P-448 was determined using monooxygenase systems as given in the legend of Fig.1 and 2. The apparent K , values were obtained by extrapolation of Lineweaver-Burk plots. Aldrin concentration varied over 2 -200 pM.

RESULTS

Cytochromr-P-450-Dependent Aldrin Epoxidution by the Reconstituted Rat Liver Monooxygenase System

Dieldrin formation in the reconstituted system was dependent on the presence of an NADPH-regenerating system (Table 1). Cytochrome P-450 and NADPH- cytochrome C reductase were essential components in this system. The sequence of addition of the protein and lipid components had no influence on epoxidation activity. When both the lipid factor Lau2PtdCho and sodium cholate were omitted, the epoxidation rate decreased to about 12 % of the activity in the complete system. Sodium cholate, which has been shown to activate cytochrome P-450 reactions [9], stimulated the epoxidation reaction more than two-fold. When

Table 1 , Requirements of cytoLhrome-P-450-dependent uldrin epori- dution The complete system was the same as given in the legend of Fig. 1. Enzyme activity in this system, as expressed by the turnover number, was 2.2 mol dieldrin min-' (mol cytochrome P-450)-'. For enzyme assay see Materials and Methods. Glc6P = glucose 6-phosphate

Assay conditions Enzyme activity

Complete system (control)

dehydrogenase)

dehydrogenase) + NADH

minus (NADPH + Glc6P + Glc6P-

minus (NADPH + Glc6P + GlcP-

minus cytochrome P-450 minus reductase minus LauzPtdCho minus sodium cholate minus (LauzPtdCho + sodium cholate) under C0/02 (1 : 1) plus MgCl2 (5 mM)

control

100

0

14 0 0

22 40 12 6

107

the incubations were carried out under a 1 : 1 mixture of oxygen and carbon monoxide, enzyme activity was markedly reduced by more than 90 %.

Metabolic rate linearly increased with cytochrome P-450 content (Fig. 1 A) and reached maximum at a concentration of 0.5 pM cytochrome P-450. Similarly, increasing amounts of NADPH-cytochrome c reductase considerably stimulated enzyme activity until the system was saturated in the presence of approximately 1000 units reductase (Fig. 1 B). The effect of various lipid concentrations is shown in Fig.1C with a maximum of enzyme activity at 5 pg Lau~PtdCho/ml. The optimal amount of lipid varied with substrate concentration: at a concentration of 0.05 mM aldrin, a threefold higher amount of LauzPtdCho was required for maximal activity than at 0.2 mM aldrin (data not shown). Enzyme activity also depended on the concentration of sodium cholate (Fig. 1 D). In the presence of Lau2PtdCho, epoxidation rate was stimulated twofold when the sodium cholate concentration was increased up to 500 pg/ml.

The turnover number of the system containing 0.34 nmol hemoprotein, 920 units cytochrome c reductase, 5 pg LauzPtdCho and 200 pg sodium cholate in 1 ml was 1.8 k 0.3 mol dieldrin min-' (mol P-450)-'. The apparent K , for aldrin was 7 f 2 pM. The cytochrome component in this system was used after storage for 3 months. Another sample of cytochrome P-450 stored for several weeks exhibited higher but inconsistent turnover numbers between 3 and 10.

Aldrin Epoxidution by u Cytoclzrame-P-448- Containing Monooxygerzuse System

Epoxidase activity was low in a reconstituted monooxygenase system containing cytochrome P-448 isolated from liver microsomes of 3-methylcholan-

548

.- - m P

% 200 - 0

Aldrin Epoxidation Catalyzed by Cytochromes P-450 and P-448

-Lau2 F'td Cho 0

- 800 L

-!

E

8 200

600 E -

400 0

m U x

.- L

._

c U .- L

0 4

o 0.1 a2 0.3 0.4 0.5 0.6 0.7 Cytochrome P-450 JwM)

0 10 20 30 40 50 LaupPtdCho (Lgirnl)

I B I

0 500 1000 1500 2000 Cytochrorne c reductase (unitsirnl)

I

0 100 200 300 400 500 Sodium cholate (pg/rnl)

Fig. 1. Dependence of aldrin epoxidution rute on the concentration of cytochrome P-450 ( A ) , NADPH-cytochrome c reductase ( B ) , LauzPtdCko ( C ) and sodium ckolate (0). The 1-ml incubation mixtures contained, unless otherwise indicated, 0.67 nmol cytochrome P-450, 920 units NADPH-cytochrome c reductase (1 unit = 1 nmol cytochrome c reduced/min, assayed at 22 "C), 5 pg LauzPtdCho, 200 pg sodium cholate and 0.2 pmol aldrin

threne-treated rats. The rate of dieldrin formation under optimal conditions in a system consisting of 0.21 nmol cytochrome P-448, 920 units cytochrome c reductase, 10 pg Lau2PtdCho and 200 pg sodium cholate in 1 ml was 3.5 pmol/min at a substrate concentration of 0.1 mM. This is equivalent to a turnover number of 18 pmol dieldrin min-' (nmol P-448)-' and corresponds to 1 % of the activity of the cytochrome P-450 system.

A cytochrome P-448 concentration of 0.25 pM and a little over 1000 units of NADPH-cytochrome c reductase were necessary for maximal epoxidase activity (Fig.2A, B). Addition of LauzPtdCho did not stimulate the activity as observed for the cytochrome P-450 system (Fig. 2C). In the absence of LauzPtdCho, the turnover number was somewhat increased to 25 pmol dieldrin min-' (nmol cytochrome P-448)-'. With another sample of the hemoprotein a turnover of 60 pmol dieldrin min-' (nmol P-448)-' was determined under these conditions. No enhancement of dieldrin formation by addition of sodium cholate was found in either the presence or absence of Lau2- PtdCho. In the complete system, the apparent K , for aldrin was 27 f 7 pM i.e. threefold more than that in the P-450 system. Enzyme activity under a 1 : 1 atmosphere of CO:O2 was lowered to 33% of the activity under normal atmosphere.

As a positive control, the cytochrome P-448 system was active in hydroxylation of benzo [ulpyrene as shown in Table 2. Hydroxylation rate in this system was higher than in the cytochrome P-450 system.

Effect of Inhibitors

The two inhibitors of monooxygenase-dependent reactions, SKF 525-A and 7,8-benzoflavone, affected the rate of aldrin epoxidation differently in the two monooxygenase systems (Fig. 3). 7,8-Benzoflavone had no effect in the cytochrome P-450 system at concentrations up to 250 pM (Fig. 3A). In the cytochrome P-448 system, 100 pM of the flavone inhibited epoxidation by 50 %. Further addition of 7,8-benzoflavone did not increase the extent of inhibition.

SKF 525-A, on the other hand, had a similar effect in both systems (Fig.3B). Approximately 200 pM SKF were necessary to inhibit the reaction by 50%. At low concentrations the inhibitor was slightly more inhibitory in the cytochrome P-448 system.

Aldrin epoxidase in the two monooxygenase systems markedly differed in its sensitivity towards ethanol (Fig. 3 C). While the cytochrome-P-448-dependent enzyme was half-inhibited by 5 % ethanol, which is equivalent to 800 mM, epoxidase in the P-450 system was enhanced more than threefold by this

T. Wolff, H. Greim, M.-T. Huang, G. T. Miwa, and A. Y. H. Lu

:c 5 -

E 4 - - E '

549

1" A

0 0.1 0.2 0.3 0.4 0.5 0.6 Cytochrome P-448 (KM) Cytochrome c reductase (unitslml)

4 $'1 0 0 10 20 30 40 50

Lau7PtdCho (Fg/ml)

Fig. 2. Dependence of aldrin epoxidcition activity on the concentration of' cytoclirome P-448 ( A ) , NADPH-cytochrome c recluctase ( B ) and Luu2PtdClzo ( C ) . The assay system, unless otherwise indicated, consisted of 0.21 nmol cytochrome P-448, 920 units NADPH-cytochrome c reductase, 200 pg sodium cholate and 50 nmol aldrin in a l-ml assay volume. The values were corrected for the blank of 2.5 pmol dieldrin produced in the absence of enzyme

Table 2. Metabolism ofhenzo[ a]p.yrene and aldrin in the cytochrome P-448 and P-450 reconstituted systems Incubation mixtures contained 0.34 nmol P-450 or 0.14 nmol P-448, 920 units cytochrome c reductase, 5 pg LauzPtdCho and 200 pg sodium cholate in a final volume of 1 ml. Concentration of benzo[a]- pyrene was 0.1 mM, of aldrin 0.05 mM. Numbers represent the means of two separate experiments. Enzyme activities (turnover numbers) are expressed as mol 3-hydroxy-benzo[a]pyrene or dieldrin formed during incubation (mol cytochrome)-' min-'. For enzyme assay see Materials and Methods

Assay Turnover number of ~~

cytochrome cytochrome P-448 P-450 system system

Benzo[a]pyrene hydroxylation 3.9 0.36 Aldrin epoxidation 0.026 1.75

ethanol concentration. At 10 ethanol the enhanced activity was slightly decreased but was still more than twofold the control activity.

DISC U SSION

The aim of this study was to evaluate the role of cytochromes P-450 and P-448 in the enzymatic for-

mation of dieldrin from aldrin. Earlier investigations using rat liver microsomes had suggested that this reaction is predominantly mediated by cytochrome P-450 [13]. The present results clearly indicate that aldrin epoxidation is primarily a cytochrome-P-450- dependent reaction. This is demonstrated by the high rate of dieldrin formation in the monooxygenase system reconstituted from cytochrome P-450 and by the low rate of epoxidation mediated by cytochrome P-448. The latter was less than 3 of the cytochrome- P-450-dependent activity when the highest value determined for cytochrome P-448 was compared to the lowest value obtained in the cytochrome P-450 system. The low activity observed in the cytochrome P-448 monooxygenase system was not due to insufficient enzymatic function of cytochrome P-448 since this system supported the hydroxylation of benzo [alpyrene, a reaction known to be mediated preferentially by cytochrome P-448 [20,21].

The remarkably low capability of cytochrome P-448 to metabolize aldrin may be a consequence of structural features of the substrate. Metabolic activity of cytochrome P-448 seems to be restricted to substrates of planar structure like benzo[a]pyrene [20,21], cthoxyresorufin [22], 2-acetaminofluorene [23] and 7-ethoxycoumarin [24]. Possibly, it is the nonplanar

550

150 - - - ?

s +- c

."

.- c

E a

c 5 0 .-

9 4

0

Aldrin Epoxidation Catalyzed by Cytochromes P-450 and P-448

B

lGoy\ ,;I , I I I 0 10 5 0 100 2 5 0

7,8- Benzoflavone (1M)

Ethanol (M) 0 0.10 0.25 0.50 1.0 2.0 3.0

0 0.5 I 2 3 4 5 10 Ethanol (yo, V / V )

Fig. 3. Eflects of 7,8-henzoflavone ( A ) and SKF 525-A ( B ) and etkunol ( C ) on the cytochrome-P-450-dependent and cytochrome-P-448- dependent aldrin epoxidation. The composition o f the cytochrome P-450 and cytochrome P-448 systems used werc the same as described under Fig. 1 and 2, respectively. Aldrin concentrations was 50 pM. 7,8-Benzoflavone was added in 20 1111 ethanol. Control activities of the P-450-dependent and P-448-dependent reactions were 1.20 and 0.015 mol dieldrin min-' (mol cytochrome)-', respectively. In the presencc of 20 p1 ethanol the corresponding numbers were 2.2 and 0.012 mol mm-' mol-'. (0-0) Cytochrome P-450; (A-A) cytochrome P-448

and bulky structure of aldrin which limits its metab- obilism by cytochrome P-448 to such a low extent.

The two monooxygenase systems, markedly differ- ing in their metabolic activity towards aldrin, also differed in the conditions necessary to obtain optimal enzyme activity. Epoxidation by the cytochrome P-450 system required a concentration of the henioprotein higher than in the cytochrome P-448 system. The lipid factor LauzPtdCho showed a differential effect on the two enzyme systems. This lipid, which is known to activate a variety of hydroxylation reactions in reconstituted microsomal monooxygenases [9], markedly increased the epoxidation rate when added to a mixture of cytochrome P-450 and cytochrome c reductase but did not alter the reaction rate in the cytochrome P-448 system. Further addition of sodium cholate which has been shown to stimulate demethyla- tion of benzphetamine in a cytochrome P-450 system [9] also enhanced the epoxidation rate in this system.

The cytochrome-P-448-dependent epoxidation, on the other hand, was not affected by sodium cholate or, as observed in the absence of LauzPtdCho, was slightly depressed.

It appears questionable whether the lack of a stimulatory effect by lipid is typical for a reaction mediated by cytochrome P-448 in reconstituted systems. The rate of the 2-hydroxylation of biphenyl, another cytochrome P-448 reaction, was reported to be decreased by addition of a lipid [22] whereas benzo[n]- pyrene hydroxylation in this system required the presence of LauzPtdCho for maximal activity [9]. In the case of aldrin epoxidation, the presence of small amounts of microsomal lipids or some kind of aggre- gation of the highly lipophilic substrate which might behave like lipid vesicles could also be responsible for the ineffectiveness of lipid addition on aldrin epoxidation in the monooxygenase system containing cytochrome P-448.

T, Wolff, H. Greim, M.-T. Huang, G . T. Miwa, and A. Y. H. Lu 551

Qualitative differences between the two aldrin- epoxidizing enzymes are further demonstrated by the variation of the values for the apparent K , and by the differential sensitivity towards ethanol and 7,8- benzoflavone, an inhibitor of cytochrome-P-448-dependent reactions [25]. It is interesting to note that ethanol enhances the aldrin epoxidation in our monooxygenase system, which was reconstituted from the microsomal cytochrome of phenobarbital-treated rats, but inhibits this reaction when the constitutive enzyme was assayed in microsomes of untreated rats [26].

In conclusion, aldrin apparently does not belong to the group of substrates that are metabolized by all forms of microsomal cytochrome P-450. It is highly selective for the phenobarbital-inducible cytochrome P-450 species and a poor substrate for the cytochrome P-448 species induced by 3-methylcholanthrene. Determination of the turnover number, the apparent K,,, and of the sensitivity towards 7,8-benzoflavone and ethanol of aldrin epoxidase offers a new possibility to monitor the activity of phenobarbital-induced cytochrome P-450 and 3-methylcholanthrene-induced cytochrome P-448 in mammalian tissues. Additionally, the differential effect of ethanol probably provides for a discrimination between constitutive cytochrome P-450 forms present in untreated animals and those form(s) induced by pretreatment with phenobarbital.

The authors thank Ms Heide Wanders for expert technical help and Dr F. Wiebel for performing the benzo[u]pyrene hydroxylation assays and for discussion of the manuscript. The secretarial help of Ms Judy Byers is gratefully acknowledged.

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T. Wolff and H. Greim, Abteilung fur Toxikologie, Gesellschaft fur Strahlen- und Umweltforschung (Munchen) mbH, lngolstadter LandstraBe 1, D-8042 Neuherberg, Federal Republic of Germany

M. T. Huang, Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche, Inc., Nutley, New Jersey, USA 07110

G . T. Miwa and A. Y. H. Lu, Merck, Sharp and Dohme Research Laboratories, New Jersey, USA 07065

aldrin epoxidation catalyzed by purified rat-liver cytochromes p-450 and p-448 high selectivity for...

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