A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions

ANALYTICAL

107,

BIOCHEMISTRY

A Rapid Method

150- 155 (1980)

for Assaying Microsomal

the Metabolism of 7-Ethoxyresorufin Subcellular Fractions

ROBERTAJ.POHL

Received

The

formation

oxidase methanol Conditions inhibition tion

versus

tetraacetate

may and

of

resorutin

be assayed measuring

are described by the product. activity

are

in the

incubation

January

Il.

from

7-ethoxyresorufin

rapidly by the fluorescence

precipitating of resorufin

for minimizing Higher activity obtained

by

by

microsomal

including

bovine

mixed-function

protein in incubation in the supernatant after

of the instability plots of microsomal serum

albumin

mixtures centrifugation.

of resort&r and protein concentra-

with of

or ethylenediamine-

mixture.

’ Abbreviations used: ERF. 7-ethoxyresorufin: mixed-function oxidase: hplc. high-pressure chromatography; BSA. bovine serum albumin; /3-naphthoflavone: Hepes. J-(2.hydroxyethylj-l-piperazine-,l”-2-ethanesulfonic acid: DMSO. dimethyl oxide: AHH. aryl hydrocarbon hydroxylation.

MFO. liquid /3-NF. sulf-

vantageous than the observation of the course of a single reaction, as described by Burke and Mayer ( 1). Therefore, we developed the following procedure for measuring the amount of resorufin formed in a given period of time. MATERIALS

AND METHODS

7-Ethoxyresorufin and resorufin were obtained from Pierce Chemical Company (Rockford, Ill.). Bovine serum albumin (BSA). Fraction V, was obtained from Armour Pharmaceutical Company (Chicago. Ill.). Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, rhodamine B. N-2hydroxyethylpiperazine - N’ - 2 - ethanesulfonic acid (Hepes), tetrdsodium ethylenediaminetetraacetic acid (EDTA) and NADPH were obtained from Sigma Chemical Company (St. Louis, MO.). /3-Naphthoflavone (/3-NF) was obtained from Aldrich Chemical Company (Milwaukee, Wise.). Male Sprague-Dawley CD rats, 175210 g (bred at the National Institute of Environmental Health Sciences’ animal facility). were given 10 mg /3-NF in 0.4 ml dimethyl sulfoxide (DMSO) by esophageal intubation intermittently for 1.5 days (50

150 I30 I TO-06$02,00/O

FOUTS

1980

the

the effects and linear

The fluorometric measurement of the appearance of resorufin during metabolic oxidation of 7-ethoxyresorufin (ERF)’ by microsomal preparations was developed by Burke and Mayer (1) as a sensitive method for determining initial rates of mixed-function oxidase (MFO) activity. Metabolism of ERF proved to be mediated specifically by the cytochrome P-448 form of cytochrome P-450. This feature makes measurement of ERF activity valuable for qualitatively describing modifications of MFO activity induced by exposure of animals to certain environmental agents (2-4). Benzo[a]pyrene, commonly used to measure the activity of cytochrome P-448, is a less desirable substrate because it produces a complicated mixture of metabolites that is best studied by high-pressure liquid chromatography (hplc). In many cases the rapid assay of large numbers of different samples is more ad-

0003-26971801

R.

ANDJAMES

by

RAPID

RESORUFIN

mgikg body wt), on Days I. 2, 3, 7, 8, 9, 14, and 1.5. Controls received DMSO by esophageal intubation at the same times. Animals were killed 24 h after the last dose and their livers were removed and placed in ice-cold I. 15% KC1 with 0. I4 mM Hepes. pH 7.5 (KCL-Hepes). Microsomes were isolated from homogenized liver (5) and were suspended in KCL-Hepes. Aliquots were frozen and stored in liquid nitrogen. Protein concentration was assayed by the method of Lowry (I/ trl. (6) using BSA standards. Incubation mixtures contained Hepes, pH 7.8 (0. I M), glucose 6-phosphate (5 mM). glucose-6-phosphate dehydrogenase ( I-2 units), MgSO, (5 mM), BSA (1.6 mgiml). ERF (1.5 PM), and varying concentrations of microsomal protein. The reaction was initiated by addition of NADPH (0.6 nmol). Total volume was 1.25 ml in 16 x IOO-mm culture tubes for timed incubations in a Dubnoff metabolic incubator at 37°C. Microsomal protein concentration and incubation time were adjusted so that 0.02-0.25 nmol of resorufin was formed. Fluorescence readings twice as high as background are obtained when 0.02 nmol resorufin is produced. and this represents the minimum detectable quantity in our experience. The reaction was stopped by addition of 3.5 ml methanol. Precipitated protein was centrifuged down and the fluorescence of the supernatant was measured at 585 nm in a Turner 430 spectrofluorometer using an excitation wavelength of 550 nm. Blanks contained no NADPH. When the reaction was monitored fluorometrically (dynamic assay), a 2.5-m] incubation mixture was used and resorufin was added as an internal standard after the desired elapsed time. The fluorometer cell compartment was kept at 37°C. ERF and the microsomes were mixed together at least 5 min before initiating the reaction by the addition of NADPH (7). RESULTS Stuhility of ERF trrtti t~so~.~cfitr. Burke and Mayer ( 1) reported originally that both

ASSAY

151

ERF and resorufin were decomposed in aqueous solution by exposure to room lighting. This group later improved the procedure for synthesis of ERF and obtained a product not subject to photolysis (8). Therefore, we tested ERF and resorufin for sensitivity to fluorescent room lighting under our assay conditions as follows: First, solutions of ERF (1.5. 5.0. and IO FF;~) were made in methanol or in 0.1 ILI Hepes, pH 7.6, and solutions of resorufin (1.25 PM) were made in methanol or in a mixture of Hepes buffer, pH 7.6. in 60% methanol. Absorbance and fluorescence of the solutions did not change from initial readings when measurements were made at one-half hour intervals for 2 h during which the solutions stood on the laboratory bench in borosilicate glass tubes. Second. identical assays of ERF deethylation, incubated in amber or clear glass centrifuge tubes, gave identical results. These observations indicate that it is unnecessary to protect ERF or resorufin from light during assay by this procedure. Absorbance wavelength maxima and molar absorbancy index for ERF and resorufin were determined for use in establishing the exact concentration of each solution prepared and for monitoring the stability of solutions during any storage. The absorbancy index for ERF in both solvents (wavelengths: 470 nm in methanol, 480 nm in 0. I M Hepes, pH 7.6) was 16 mpvr-’ cm-‘. Resorufin. as supplied by the manufacturer. was not sufficiently stable for measurement of a reproducible absorbancy index. However. an absorbancy index (at 575 nm) of 40 rnh4-l cm--’ was calculated for resoruhn formed in incubation mixtures (methanolic supernatant). assuming complete conversion of ERF to resorufin. ERF fluorescence in both solvents was excited maximally by 470 nm light and fluorescence maximum was 560 nm. The resorufin excitation wavelength chosen was 550 nm (I-nm entrance slit in Turner 430 spectrofluorometer). fluorescence maximum was 585 nm.

152

POHL

AND

ERF reacts with NADPH in the presence of acid with the formation of a product which fluoresces at the same wavelength as resorufin (unpublished observation). Methods which use acid to precipitate protein or to facilitate extraction of resorufin into organic solvents give, therefore, artificially elevated results. A standard solution of resorufin in ethanol may not remain stable during storage in the refrigerator. A solution of rhodamine B should be used to standardize the assay of ERF metabolism as was suggested by Burke t>r al. (9). The fluorescent intensity (at excitation 550 nm, fluorescence 585 nm) of rhodamine B in the methanolic supernatant of incubation mixtures is three times that of resorufin on a molar basis (data not shown). Eflkct of the addition of BSA or EDTA to the incubation mixtures. Incubation mixtures containing buffer, MgSO.,, NADPH. NADPH-regenerating system, substrate, and microsomes were used in our original experiments. We noted, however, that graphs of ERF metabolized vs concentration of microsomes gave linear plots which did not extrapolate to the concentration axis at the origin (Fig. I). When BSA, which may provide a milieu for more homogeneous solution or dispersion of the hydrophobic substrate (IO), was added to the mixture, specific activity was increased and activity vs microsomal protein concentration plots extrapolated to the origin. The optimum concentration of BSA was 1.6 mgiml (BSA concentrations from 0.8 to 3.2 mgiml were tested). Similar effects were observed in the dynamic assay following addition of BSA to the incubation mixture. although the fluorescence of the metabolite was quenched by 65%. It was also observed that the addition of EDTA to the incubation mixtures (optimum concentration of 60 PM) corrected protein concentration vs activity plots to the same extent as did the addition of BSA (Fig. 1). Addition of EDTA to incubation mixtures containing the optimum concen-

FOUTS

FIG. 1. Effect of BSA and/or EDTA on ERF deethylase activity by rat hepatic microsomes. Control.- - -; /3-naphthoflavone treated. -. (A) Incubation mixtures were precipitated with methanol after 2 min (/5NF treated) or 4 min (control). (B) Metabolism in a Ruorometer cell was monitored by recording the appearance of resorufin. (0) No BSA or EDTA, (0) I.6 mg BSA/ml. (0) 60 PM EDTA, (B) BSA + EDTA.

tration of BSA did not further change the activity. Sen.siti\lity rind precision oj‘ the ttieosuretnrnt oj’ resoru$tz c~onc’etltrrltion by this procedure. An abbreviated standard curve for this assay is plotted in Fig. 2. The fluorescence of 0.1 nmol resorufin was four times higher than that of the blank. The variability of the data obtained from day to day, as seen in the standard curve, is greater than that seen within a single experiment (Table 1). The coefficients of variability of the data in Fig. 2 are 31. 26, and 25% for 0.1 0.25. and 0.5 nmol resorufin. respectively. Other j‘trctors c[j&ting ERF drethylusc uctit’ity Omission of MgSO, from the incubation mixtures decreased ERF deethylase activity 20-30% (data not shown). Resorufin is an inhibitor of ERF deethylase activity (Table 2). Plots of substrate concentration vs ERF deethylation activity in the presence or absence of added resorufin are given in Fig. 3. The shape of the curves obtained when microsomes from fiNF-treated rats were used is variable with peak activity shifting between substrate

RAPID

RESORUFIN

13

ASSAY TABLE INHIBITION

OF ERF

2

METABOLISAI

my RESORUFIN

nmoliminimg Microsomal protein’, Resort&V’ Control

(nmol)

FIG.

2. Standard

mean t SE concomitantly

curve.

Data

points

( ,I = 11) of standard with assays of ERF

represent

the

curves determined metabolism.

concentrations of 1.0 and I .5 PM. In contrast, the curves obtained when microsomes from control rats were used were typical of saturation plots. Addition of resorufin to mixtures before incubation decreased the apparent rate of the reaction at all substrate concentrations by either control or @-NF-treated rat microsomes. LineweaverBurk plots of the data from Fig. 3 can be drawn (not shown) but, even when estimating initial rates from curves obtained by dynamic assay, it is difficult to measure activity using substrate concentrations in the apparent K,,, range without depleting 20-30% of the substrate in the first few seconds. Therefore, variation in the shape of the curves obtained occurs and these plots cannot be analyzed. They do suggest that the inhibition by resorufin is not exclusively of the competitive type. TABLE PRECISION

OI-‘ ERF

D~~THYI

Microsomes”

ASE Ass.4~

Fluorescenceh

the

sample ” Coefficient

0.45

i- 0.03

7c;

0.63

2 0.03

5/;

NADPH read: were subtracted

fluorescence. of variability.

0.036 cro.020

1.20 0.94

ethanol

solvent

tubes containing N, before the were

wab resorufin remaining

added.

evaporated

from

solution under incubation

Microsomes

from

(0.25 mg protein) were incubated for 4 min: fi-NF-treated rats (25 pg protein) were ? min. ti A representative

from fi-NF

pooled treated.

livers

experiment of

four

rats

incubaa stream mixture

control

rat\

those from incubated

using

microsome\

each,

control

and

DISCUSSION The rapid method we have described for measuring the metabolism of ERF is useful for assessing induction of aryl hydrocarbon hydroxylation (AHH) activity in microsomes from animals exposed to xenobiotics. Its use for measuring discrete changes in kinetic parameters. responses to

CV’

I

of and

0.15 0.5

PROCFIIL’RF

7

absence f 0.02

4.67 3.5Y

I

” Hepatic microsomes from 15 &incubation mixture, incubated h Mean i- SD, ,I = 6 (duplicate in the (7) 0.07

0.132 0.058

components

for

treated

0 0. I

‘I The tion of

fl-NF

/3-NF-treated 3 min. assays). Blanks

rata. I-un

(I) 0.05 -t 0.01. before tabulating

FIG. 3. Plots of substrate (ERF) concentration VII ERF deethylation activity without (0.m) and with the addition of 0.2 nmol (a.A) or 0.5 nmol 1~i.0) of resorufin. Ethanol was evaporated from resorufin before adding the remainder of the incubation mixture for dynamic assay. The data plotted with open symbol\ were obtained using microsomes from control rat\ (I.0 mg symbols.

protein2.5 ml): from b-NF-treated

those plotted using closed rat\ (50 @:2.5 ml).

154

POHL

AND

thermal or other physical perturbations, etc., is compromised by the complexity of the interactions between the substrate and product with the enzymes in the incubation mixture. For example, the mechanism by which BSA increases activity of ERF deethylation (Fig. 1) is not clear. The addition of BSA to incubation mixtures has been used to “solubilize” substrates for AHH (10,ll) and its use to overcome the difficulty inherent in working with hydrophobic substrates has been discussed by Hansen and Fouts (10). They found that added BSA corrected the linearity of plots of microsomal protein concentration vs benzo[ (I]pyrene metabolized by increasing activity observed in incubation mixtures containing small amounts of microsomal protein. Our observation that the addition of EDTA to ERF deethylation mixtures increases activity as much as, but not more than, the addition of BSA, indicates that the sequestering of a polyvalent cationic inhibitor may be the mechanism involved. The operation of such a mechanism in the experiments of Burke c)t 01. (9) via precipitation of polyvalent cations by the phosphate buffer (12) in which their microsomes were suspended may account for their linear plots of activity vs microsomal protein concentration. Inhibition of ERF deethylation activity by product (Table 2, Fig. 3) is a further factor complicating interpretation of measured rates of activity. Inhibition by 0.1 nmol of resorufin is greater than 20%. Concentrations of product near the limit of detection by this method are, therefore, inhibitory. Also, optimum substrate concentration is so low that substrate depletion during incubation cannot be ignored. Ten percent of the substrate has been depleted by formation of less than 2 nmol of product. The effects of these two factors may be minimized by adjusting the microsomal concentration in the incubation mixture and shortening the incubation time so that approximately 0.1 nmol of resorufin is formed during the incubation. Assay of different concentrations

FOUTS

of each microsomal preparation gives a basis for evaluating optimization of incubation conditions for that preparation. At best, however, observed rates are not expressions of substrate-saturated. uninhibited reaction rates. The unusual shape of the curves obtained when substrate saturation of microsomal enzyme from /3-NF-treated rats was investigated (Fig. 3) may be explained by substrate inhibition of one form of cytochrome P-450 induced by treatment of rats with P-NF. Evidence for this possibility was recently reported by Warner and Neims (14) using cytochromes partially purified from hepatic microsomes of control, phenobarbital-treated and p-NF-treated rats. The enhanced microsomal ERF activity observed in the presence of magnesium is similar to that seen in many in \litr.o assays of MFO activity in microsomes (13). Burke and Mayer reported that no addition of magnesium to incubation mixtures was required ( 1). The reason for this difference from our results may be related to their use of phosphate buffer which can tie up magnesium whereas Hepes buffer does not ( 12). Although study of the kinetics of ERF deethylation is compromised by complications, assay of ERF deethylation is useful in studies of the substrate specificity of MFO and/or cytochrome P-450 isolated from different sources (2.14,15). Also, the induction of microsomal MFO activity by exposure of animals to polycyclic hydrocarbon-type inducers may be quickly and sensitively detected using the procedure described in this paper. REFERENCES I.

Burke. M. .Mrtnhd.

D., and Mayer. Di.S/‘“\. 2, 583-m.

R.

7‘.

t 1974)

l)rrc,g

2.

Burke, M. Ue/trhrd

D.. and Mayer. I)i\[lC!\. 3. 745-73.

R.

I’.

(1975)

fIrr/c

3. Bend, J. Dostal.

R.. L..

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G. L.. Ben-Zvi, I.. and Fout\. J. R. (1978) Bi,,/. G. .\fcj/.

I.lf/J. 18. ho-h?. E.. and Muller/‘/~~~~ur~~(~o/. 15,

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RESORUFIN

6. Lowry, 0. H., Rosebrough. N. J.. Farr. A. L.. and Randall. R. J. (1951) .I. Biol. Chc/,l. 193. ‘65-275. 7. Fouts, J. R. (1980) it! Fine-Needle Biopsy of the Rat Liver (Zbinden. G.. ed.). pp. 33-37. Pergamon. Oxford. 8. Mayer. R. T., Jermyn, J. W.. Burke, M. D.. and Prough. R. A. ( 1977) Pc\tic,. R;&u~~~I. /%~\i~~/. 7. 349-354. 9. Burke, M. D.. Prough. R. A., and Mayer, R. T. (1977) Drrrg ,Mrt~rho/. Disp”.s. 5, 1-x. 0. Hansen. A. R.. and Fouts. J. R. (1971) CIIV,U~ Ricd. Irltcr.rrc.f. 5. 167-1X’.

ASSAY

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I I. Alvares. A. P.. &hilling. G.. Kuntzman. R. ( 1970) Bir~lrr/,l. l449- 1455.

Garbut. A.. and Ph~~rr,rcic,r>/. 19,

12. Good. N. E., Winget. G. D. Winter. W.. Connolly. T. N.. Izawa. S.. and Singh. R. M. M. (1966) Bio(~/r(~t~ti.rtrv

5, 467-477.

13. Peter\. M. A.. and Pouts. Plr~rrf?frrc~d 19. 533&54-l. 14. Warner.

M..

and

.21(~tlrhr~/. Di\po\, 15. Johnson. 309.

J. R. (1970)

Neims. A. H. 7. 18% 193.

E. F. (1980)

J. Biol.

Ric~c~/rc,vi.

(19791

C/~c,rr.

255.

L)rrrc 304-