Microsomal rat liver UDP glucuronyltransferase: Effects of piperonyl butoxide and other factors on enzyme activity

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(IF

B[OCHEMiSTICY AND BIOPHYSICS

145, 520-530 (1971)

Microsomai Rat Liver UDP Glucuronyltransferase: Effects of Piperonyl Butoxide and Other Factors on Enzyme Activity G. W. L U C I E R , O. S . . ~ [ c l ) A N I E L ,

AND H. B . . ~ [ A T T H E W S

National Ltstitute of Et~vironmental Health Sciences, Departme~d of Health, Education, and Welfare, P. O. Box 12233, Research Triangle Park, North Carolina 27709

Received April 1, 1971; accepted May 5, 1971 Activation of rat liver microsomal U1)P glucuronyltransferase by detergent or ageing was not accompanied by a change in K,, for 1-naphthol or UDPGA. Divalent cations (Mg2+, Mn ~§ and Cos+) stimulated glucuronic acid conjugation of 1-naphthol and p-nitrophenol in both unactivated and activated microsomes, however, Zn2+ strongly inhibited UDP glucuronyltransferase. A total activation factor of 30-fold resulted when both Triton X-100 and MgC12 were added to the incubation medium. Triton X-100 and MgCI~ also stimulated UDP glucuronyltransferase from rabbit liver microsomes with a total activation factor of 20-fold. The UI)P glucuronyltransferases conjugating 1-naphthol, testosterone, and a dieldrin metabolite were inhibited itt vitro by insecticide synergist chemicals. Piperonyl butoxide had I~0values of 7.3 X 10-6 Mand 9.0 >( 10-~ Mfor testosterone and 1-naphthol glucuronyltransferase, respectively. Inhibition of enzyme from detergent-activated microsomes was considerably less than that of enzyme from unactivated or aged microsomes. Pretreatment of male or female rats with piperonyl butoxide for 3 days resulted in a two-fold induction of the UDP glucuronyltransferase conjugating 1-naphthol arid p-nitrophenol. 1-Naphthol and p-nitrophenol were shvwB to be glucuronidated by the same enzyme by analysis of the mixed substrate method, divalent cation requirements, activation by detergent or ageing, submicrosomal distributions, levels of induction by piperonyl butoxide, and gravitational studies. This work was greatly facilitated by the use of a new, rapid, and sensitive radioassay for UDP glucuronyltransferase which is described here. U D P glucuronyltransferase plays an imp o r t a n t role in the metabolism and excretion of numerous xenobiotics and endogenous compounds, but studies of these enzymes in rat liver have been complicated b y the low activity and i m p u r i t y of the enzyme preparations (1). Leuders and Kuff (2) used Triton X-100 to obtain a 10-fold activation of p-nitrophenyl glucuroxfide formation in rat liver microsomes. However, the K ~ for U D P G A could not be determined in unactivated microsomes due to a lack of sensitivity in the p-nitrophenol assay (2). Therefore, it was not resolved whether T r i t o n X-100 activation involved an increase in the n u m b e r of active sites or an increase in affinity of enzyme for U D P G A .

Insecticide synergists are believed to exert their p r i m a r y action b y serving as alternative substrates for the mixed function oxidase enzymes (3-5). Over a longer period of time, m a n y of these compounds have been shown to induce mixed function oxidase activity (6, 7). However, there is evidence t h a t the action of insecticide synergists m a y involve other enzymes which function in the metabolism of xenobiotics (5, 8), including the glucuronyltransferases (9). I n this paper we report K,~ values for both activated and u n a c t i v a t e d enzyme preparations obtained by the use of a sensitive assay technique. The effect of divalent cations and other factors on U D P glucuronyltransferase was studied in order to 520

RAT LIVER UDP GLUCURONYLTRANSFERASE establish o p t i m a l r e a c t i o n conditions a n d to gain i n s i g h t i n t o possible e n z y m e m u l t i pIicity. W e also e v a l u a t e d i n vitro i n h i b i t i o n a nd i n vivo i n d u c t i o n of h e p a t i c U D P g l u c u r o n y l t r a n s f e r a s e in rats b y s e v e r a l insecticide synergists. MATERIALS AND METHODS

Chemicals. The following biochemicals were obtained from Sigma Chemical Company, St. Louis, Mo., and used without further purification; UDPGA (~mmonium salt, 98-100%), UDPG (sodium salt, 98-100%), UDPG dehydrogenase (bovine liver, 200 units/rag), 1-naphthol (recrystallized, 99 + %), testosterone, p-nitrophenol (spectrophotometer grade), and phenolphthalein. Insecticide synergists used in this study were analytical standards and they are illustrated in Fig. 1. In addition piperonyl butoxide (80%) was further purified to obtain a 99 -{-% pure compound (10). All the synergists used are methylenedioxyphenyl compounds, except for MGK-264, which is an N-alkyl compound. Testosterone-4-*4C (58.2 mCi/mmole) and 1-naphthol-l-14C (19.6 mCi/mmole) were purified by thin layer chromatography prior to use. Dieldrin-F-l-~*C, a hydroxylated dieldrin metabolite, was extracted from rat feces and purified following oral administration of dieldrin-~4C (72 mCi/mmole) to male and female rats (11). The structure of this compound is uncertain, although the basic dieldrin molecule is intact. Animals and preparation of microsomes. Male and female Charles River rats (CD strain, 125150 g) and male New Zealand rabbits were used for preparation of microsomes and for all other studies. In enzyme induction studies, either male or female rats were treated orally with 150 mg/kg/ day for 3 days with piperonyl butoxide in 0.2 ml DMSO. Control rats were administered 0.2 ml

0

MGK-264

Sulfoxide

Piperonyl 8uloxicfe

0,""~

OJ'~'l ,,~

o...9 o..j

Tropitol

Safmlr

Piperonaf

Methylenedioxybenzene

FIG, 1. Structttral formulas synergists used in this study.

of

insecticide

521

DMSO on the same schedule. Rats were treated daily at 5 PM and were sacrificed 16 hr after the last treatment. Rabbit or rat liver mierosomes were prepared according to the procedure of Matthews et al. (11) and were resuspended in 50 mM Tris-HC1 buffer (pH 7.4 at 37 ~ at a concentration of approximately 8rag protein/ml of microsomal suspension. Microsomal subfractions were obtained by the method of Mulder (12) and the fractions were resuspended in Tris-HC] buffer. To activate microsomes, a portion of the microsomal suspension was rehomogenized with Triton X-100 to make a 0.20% mixture (12). Enzyme assays. A new and rapid method was used to assay O-glucuronide formation of relatively apolar radioactive substrates containing a free hydroxyl group. One of the labeled substrates (testosterone4-14C, 0.0025 t~Ci; 1-naphthol-l-14C, 0.0050 t~Ci; or dieldrin F-1-14C, 0.0020 /,Ci) was added to a liquid scintillation vial in a benzene or acetone solution. In the 1-naphthol assay various substrate concentrations were obtained by addition of unlabeled 1-naphthol (amounts of unlabeled substrate added to the incubation medium are given with the results). When measuring testosterone or dieldrin F-1 glucuronidation no unlabeled substrate was added to the incubation medium due to substrate solubility difficulties. Hence testosterone or dieldrin was approximately 0.4/tM in the incubation medium. Following evaporation of solvents under nitrogen, 1.0 ml of 50 mM Tris-HC1 buffer was added and the incubation medium made 0.6 mM with respect to UDPGA (the K,~ for UDPGA using 0.5 mM 1-naphthol in the incubation mixture was 0.34 raM). The incubation medium was brought to 37 ~ and activated microsomes equivalent to 0.4 mg protein or unactivated microsomes equivalent to 2.0 mg protein were added. In some experiments involving 1-naphthol conjugation the incubation medium was made 10 mM with respect to MgCl=. In the in vitro inhibition studies, the insecticide synergists were added to the incubation medium in 20sl DMSO before addition of microsomes. DMSO at this concentration had no measurable effect on UDP glucuronyltransferase activity. All incubation mixtures were incubated under nitrogen for 6 rain at 37 ~ in a metabolic shaker (Precision Scientific Company). The reaction was stopped by the addition of 10 ml of nonaqueous scintillation fluid (13) followed by vigorous shaking of the scintillation vials. Unmetabolized substrate partitioned into the scintillation fluid, whereas all glucuronide formed remained in the aqueous phase. Nonreacted substrate was measured by liquid scintillation counting and glucuronide formation was determined by difference based on blanks without UDPGA in the reaction medium.

5'22

LUCIER, McDANIEL, AND MATTHEWS

p-NitropheFiyl glucuronide formation was measured by a modification of the procedure of HollmanandTouster (14). Theineubationmixture consisted of 0.9 m~i p-nitrophenol, 1.0 mM UDPGA, and 2.0 mg of microsomal protein in 1.0 ml of 50 mM Tris-HC1 buffer (pH 7.4 at 37~ In some cases the incubation mixture contained 10 FIlMMgC12. The contents were incubated under nitrogen for 6 rain and the reaction stopped by the addition of 1.5 ml 3.5% trichloroacetic acid. Microsomal protein was then sedimented by centrifugation, and the supernatant made basic with 0.5 ml 1 N KOH. Disappearance of substrate was measured spectrophotometrically at M05 against blanks incubated without UDPGA. UDPG dehydrogenase activity was determined by the method of Zeidenberg et al. (15) and microsomal protein assayed according to Lowrey (16). Phenolphthalein glucuronide formation was measured by the procedure of Talalay et al. (17) as modified by Gram et al. (18). Two milligrams microsomal protein was used and the incubation time was 6 min. RESULTS

Radioassay for UDP glucuronyltransferase. The

increase i n the

rate

of

1-naphthyl

glueuronide formation, using 0.5 m.~i 1-naphthol, was linearly proportional to the a m o u n t of a c t i v a t e d or u n a e t i v a t e d mierosomes added up to 2.0 mg protein. For all substrates tested reaction rates were linear with respect to time for at least the first 10 rain. I n parallel experiments, results o b t a i n e d using the rapid m e t h o d were similar to those o b t a i n e d from the more laborious c o n v e n tional extraction procedures (Table I). I n c u b a t i o n mixtures held u n d e r carbon monoxide or oxygen gave the same results as those held u n d e r nitrogen. T h u s , oxidative reactions were n o t a factor i n the assay procedure. Hydrolysis of 1 - n a p h t h y l glueuronide b y 5-glueuronidase yielded 1 - n a p h l h o l as det e r m i n e d b y t h i n layer r a d i o e h r o m a t o g r a m s (4:1 ether: hexane as the mobile phase).

Effects of Trilon X-IO0 on UDP glucurongltransferase. A d d i t i o n of T r i t o n X-100 (0.2 %) to hepatic microsomal suspensions increased the c o n j u g a t i o n of p - n i t r o p h e n o l b y app r o x i m a t e l y 10-fold, thus confirming the results of other workers (2, 12). W e found a similar effect b y detergent o n glucoronide

TABLE I DETERMINATION OF ~'[ICROSOMAL GLUCURONID.kTION OF 1 - N A P H T H O L , D I E L D R I N - F - I , AND TESTOSTERONE BY THE RAPID RADIOASSAY AND BY EXTRACTION PROCEDURES a % Administered radioactivity SubstrateC

Rapid assay ~glanka

1-Naphthol CO N2 02 Dieldrin F-1 CO N2 02 Testosterone CO N~ 02

Remaining in sample

Extraction methodb Conjugated

Blankd

Ether extract

Conjugated

100.7 4- 1.4 99.2 • 0.9 99.3 4- 0.9

72.64-2.9 73.74-3.4 73.44-1.3

28.1 25.5 25.9

102.4• 99.84-3.3 100.74-3.1

74.2• 70.74-5.4 73.94-3.0

28.2 ~.1 26.8

98.7 + 4.4 99.6 4- 3.7 98.4 4- 5.3

68.44-5.3 66.34-1.9 66.24-2.5

30.3 33.3 32.2

97.04-3.3 96.84-4.9 100.54-4.0

66.04-6.3 64.54-5.1 67.74-5.0

31.0 32.3 32.8

101.4 • 2.O 100.3 4- 1.9 100.t 4- 1.2

70.4• 66.34-2.3 65.84-4.2

31.0 34.0 34.3

Each value expressed as the mean -4- SD, derived from at least 12 determinations using four male rats. Substrate concentrations were; 1-naphthol 0.5 mM, dieldrin F-1 0.4 tiM, and testosterone 0.4 gM. Divalent cations were omitted from the incubation medium and other incubation conditions are described in the Materials and Methods. b The incubation mixture was extracted 5 times with 3 ml of anhydrous ether and radioactivity measured by scintillation counting of the combined ether extracts. c Substrates were incubated under carbon monoxide, oxygen, or nitrogen. Blanks were incubated without UDPGA in the reaction medium.

RAT LIVER UDP GLUCURONYLTRANSFERASE

523

TABLE II TRITON X-100 ACTIVATION OF RAT AND RABBIT LIVER MICROSOMALUDP GLUCURONYLTRANSFERASEa R a t liver 1-Naphthol

Males UM DAM AM AMT Females UM D.4,M

5.3 53.4 64.8 55.6

:h • • •

0.6 2.0 5.3 3.2

2.4 4- 0.6 50.5 • 2.1

p-Nitrophenol

2.0 23.7 32.1 24.7

4. 444.

Rabbit liver Phenolphthalein

0.9 3.0 3.1 2.3

1.9 4. 1.2 26.9 :t: 1.5 ---

1.3 4- 0.8 22.4 4- 2.9

1.4 4- 1.0 24.6 4. 2.7

1-Naphthol

7.4~0.8 59.74.1.7

p-Nitrophenol

4.5• 30.1•

a Activity of each substrate expressed in nmoles conjugated (means 4- SD)/min/mg microsomal protein. Each value for rat liver microsomes obtained from eight determinations using four animals and each value for rabbit liver microsomes obtained from eight determinations using two animals. Substrate concentrations were 0.5 mM 1-naphthol, 0.9 mM p-nitrophenol, or 0.5 mM phenolphthalein. Divalent cations were omitted from the incubation mixtures. Abbreviations of microsomal fractions are: UM, unactivated microsomes; DAM, detergent-activated microsomes; AM, microsomes at 4 ~ for 6 days; AMT, aged microsomes treated with Triton X-100 (0.2% v/v). f o r m a t i o n of 1 - n a p h t h o I a n d p h e n o l p h t h a l e i n ( T a b l e I I ) . A c t i v i t y of r a b b i t liver microsomes was also e n h a n c e d n e a r l y 10-fold b y T r i t o n X - 1 0 0 t r e a t m e n t ( T a b l e I I ) . T h e K,, for U D P G A using 1 - n a p h t h o l (0.5 m~1) as t h e s u b s t r a t e was 0.34 mM in b o t h a c t i v a t e d a n d u n a c t i v a t e d m i c r o s o m e s a n d t h e Km for 1 - n a p h t h o l a t s a t u r a t i n g c o n c e n t r a t i o n s of U D P G A was 0.41 m ~ in b o t h p r e p a r a t i o n s (Fig. 2). V . . . . for 1 - n a p h t h o l c o n j u g a t i o n in a c t i v a t e d m i c r o s o m e s was 12 t i m e s t h a t in u n a c t i v a t e d p r e p a r a t i o n s on a p e r m i l l i g r a m p r o t e i n basis. T e s t o s t e r o n e g l u c u r o n i d a t i o n was a p parently inhibited by detergent treatment, b u t results were h i g h l y v a r i a b l e a n d m i g h t h a v e b e e n affected b y t h e e x t r e m e l y low w a t e r s o l u b i l i t y of s u b s t r a t e . F o l l o w i n g c e n t r i f u g a t i o n of d e t e r g e n t - a c t i v a t e d m i c r o s o m e s a t 105,000g for 60 min, 54 % of t h e U D P g l u c u r o n y l t r a n s f e r a s e conj u g a t i n g 1 - n a p h t h o l a n d p - n i t r o p h e n o l was r e c o v e r e d in t h e s u p e r n a t a n t fraction, w i t h t h e r e m a i n d e r of t h e a c t i v i t y r e s e d i m e n t e d i n t o t h e m i e r o s o m a l pellet. In contrast approximately 16 % of t e s t o s t e r o n e U D P glue u r o n y l t r a n s f e r a s e a c t i v i t y was in t h e supern a t a n t fraction. T h u s i t a p p e a r s t h a t a c t i v a tion with Triton X-100 involved partial solubilization. P r i o r to in vitro a c t i v a t i o n , U D P glucuronyltransferase activity from male rat

-5

0

I

5

I

I0

I

15

~lrnM UOPSA)

I

20 5

5

[~j(rn

/ nap

~0hM} 15

Fin. 2. Kinetic determinations of 1 naphthol glucuronyltransferase from unactivated (K]), detergent-activated (A), and 6-day aged microsomes ( 9 When determining Km for UDPGA, the incubation mixture contained 0.5 mM 1-naphthol, 10 mM MgCI~, varying concentrations of UDPGA, 0.4 mg protein from aged or detergent-activated microsomes or 2.0 mg protein from freshly prepared unactivated microsomes in a total volume of 1.0 ml Tris-ItC1 buffer (pH 7.4). When determining K~, for 1-naphthol, the incubation mixture contained varying concentrations of 1-naphthol, 0.6mM UDPGA, and other incubation contents as described above. The data represent the average from at least four experiments. In all cases, enzyme was obtained from male rat livers. livers was twice t h a t f r o m female liver p r e p a rations. A c t i v a t i o n of female r a t liver microsomes was a p p r o x i m a t e l y 20-fold for b o t h p - n i t r o p h e n y l a n d 1 - n a p h t h y l glucurotfide

524

LUCIER, McDANIEL, AND MATTHEWS

formation compared 1o a 10-fold increase for male rat liver microsomes (T'~ble II). Therefore, after detergent activation, the relative enzyme activities from liver mierosomes of either sex were nearly identical,

l~ffects of storage on UDP glucuronyltransferase. The effects of storage at 4 ~ on microsomal UDP glucuronyltransferase activity with 1-naphthol or p-~itrophenol as substrates are illustrated in Fig. 3. Of particular interest is the spontaneous enzyme activation, which was previously reported by Leuders and Kuff (2). In the present study, a gradual decrease in activity was observed up through 30 hr after preparation followed by a strong activation of the enzyme through the sixth day. By the fifth day after preparation, the microsomes were more active than freshly prepared microsomes. Activation by ageing did not involve a change in affinity of enzyme for U D P G A or 1naphthol (Fig. 2). Unlike detergent-acti-

vated prepara.tions, spontaneous activation was not accompanied by partial solubilization of UDP glueuronyltr'msferase. Triton X-100 was slightly inhibitory towards aged mierosomes (Table II).

Effects of diralent cations on e~lzgme aclivity. Div'dent cations (Mg 2+, Mn 2+, and Co ~+) at 10 m~ stinmlated p-nitrophenyl and 1-naphthyl glueuronide formation in rabbit and rat liver microsomes (Table I I I ) . On the other hand Zn ~+ was inhibitory. Manganese ions were most effective of the three, resulting in more than a threefold enhancement of rat liver U D P glueuronyL transferase. Stimulation of activity was optimal at 10 m~i while all of the cations were inhibitory at 60 mM and above. U D P glueuronyltransferase from both the supernatant fraction and resedimented fraction of detergent-activated rat liver mierosomes was enhanced by divalent cations (Table III), and stimulation of activity was similar when using either activated or unactivated prepa7O rations. In vitro inhibition of UDP glucuronyl~o lransferase by insecticide synergist chemicals. / F Inhibition values for sulfoxide, piperonyl "~ 5o butoxide (80 and 99%), tropital, safrole, piperonal, methylenedioxybenzene, and } .~. MGK-264 are listed in Tables IV and V. / ..... With testosterone as the substrate, MGK264 was the most potent inhibitor of U D P glucuronyltransferase, followed by sulfoxide, E 10 piperonyl butoxide, and tropital. Activity was inhibited 81% at 10-SM MGK-264 / I 2 5 4 5 6 7 8 9 10 while inhibition levels for piperonal, safrole, Doys (flier Preperotion or methylene dioxybenzene were much lower. FIG. 3. Effect of storage at 4~ on the hepatic Values for each inhibitor were not signifimicrosomal UDP glueuronyltransferase con- cantly different for microsomes from either jugating 1-naphthol [(9169 Triton X-100- male or female rats. Inhibition values of treated microsomes, (Q-Q) untreated microtestosterone glucuronyltransferase from desomes] and p-nitrophenol [(A-A) Triton X-100treated mierosomes, (A-A) untreated micro- tergent-activated microsomes were not obsomes]. Incubation mixtures, when measuring 1- tained, since activity was highly variable naphthyl glucuronide formation, contained 0.5 mM following addition of detergent. Dieldrin-F1-naphthol, 0.6 mM UDPGA, and 0.4 mg micro- 1-~4C glucuronidation was inhibited at apsomal protein in 1.0 ml 50 mM Tris-HCl buffer proximately the same level as testosterone (pH 7.4). Incubation mixtures, when measuring glucuronide formation with all the inhibitors p-nitrophenyl glucuronide formation, contained tested. 0.9 mMp-nitrophenol, 1.0 mM UDPGA, and 2.0 mg Results were somewhat different when microsomal protein in 1.0 ml Tris-HC1 bttffer. The measuring glucuronide formation of 1-naphdata represent an average from at least four experiments. In all cases, enzyme was obtained thol in the presence of insecticide synergists (Table V). Sulfoxide was the most potent from male rat livers. .

.

.

.

.

.

.

RAT L I V E R U D P G L U C U R O N Y L T R A N S F E R A S E

525

TABLE III E F F E C T OF D I V A L E N T CATIONS ON MICROSOMAL U D P Control

p-Nitrophenol DAM SDAM PDAM SER RER RLM 1-Naphthol DAM SDAM PDAM SER REI% UM RLM

MgCh

22.4 23.5 19.7 18.9 30.6 27.8

44444. •

1.9 2.4 2.3 3.1 1.8 2.2

57.0 48.9 56.3 39.5 60.3 55.9

54.4 53.0 57.7 40.8 68.4 5.2 70.1

444. 44• 4-

2.3 2.1 2.0 1.8 2.5 0.7 3,3

143.8 144.1 149.2 122.0 171.5 16.6 150.1

GLUCURONYLTRANSFERASE a

MgS04

MnCh

CoCh

4.8 5.3 3.7 4.6 4.5 3.6

55.1 4- 5.4 49.0 4. 3.3 53.6 4. 5.0

67.2 4- 4.0 65.1 4. 4.7 64.9 • 5.1

35.7 4- 4.0 36.7 4- 3.1 38.3 4- 3.3

4.04-0.9 3.34-0.6 2.94-0.8

4- 4.5 4- 5.3 -4- 5.1 4- 6.9 4- 5.8 4- 1.0 4- 4.5

142.8 4. 5.1 147.0 4. 4.2 144.3 4- 4.4

159.2 4. 5.5 158.3 4. 4.9 166.4 4. 5.3

80.8 4. 3.1 89.4 4- 3.0 85.1 4- 2.5

6.1 4. 0.8 5.5 4. 0.4 5.6 4. 0.5

17.5 4- O.7

23.8 4- 2.7

11.0 4. 0.4

<1

4. 4. 4444-

ZnCh

" Activities expressed in nmoles conjugated (means 4. S D ) / m i n / m g protein. The incubation mixture contained 0.9 mM p-nitrophenol or 0.5 mM 1-naphthol. D i v a l e n t cations, when used, were at 10 mM and incubation time was for 6 min or less. Male rat or rabbit livers were used as the enzyme source. The abbreviations of microsomal fractions are: DAM, d e t e r g e n t - a c t i v a t e d rat liver microsomes; SDAM, s u p e r n a t a n t fraction of d e t e r g e n t - a c t i v a t e d rat liver microsomes; P D A M , resedimented pellet f r a c t i o n of d e t e r g e n t - a c t i v a t e d rat liver microsomes; SER, smooth endoplasmic reticulum fraction of r a t liver microsomes; R E R , rough endoplasmic reticulum fraction of rat liver microsomes; UM, u n a c t i v a t e d rat iver microsomes at 0 day; RLM, d e t e r g e n t - a c t i v a t e d r a b b i t liver mi crosomes. T A B L E IV PERCENTAGE INHIBITION OF GLUCURONIDATION a OF TESTOSTERONE-I4C AND D I E L D R I N - F - I - l t C BY INSECTICIDE SYNERGIST CHEMICALS (10-4 M) Enzyme source Inhibitor

Testosterone UM b

Piperonyl butoxide (80%) Piperonyl butoxide (99%) Sulfoxide Tropital Safrole Piperonal Methylenedioxybenzene MGK-264

84 =t= 2.0 88 4- 1.3 89 4- 1.4 43 4- 1.2 25 -r 0.9 9 4- 0.8 7 4- 2.1 98 4. 2.0

Dieldrin-F-1 AM c

-87 4- 2.8 -----95 4. 4.1

UM b

71 71 74 56 30 7 5 89

4. 4444. 4. 4. 4.

3.0 1.4 1.5 1.3 1.6 0.9 2.4 3.7

AM c

-70 4- 2.2 -----92 -4- 1.4

I n c u b a t i o n mixtures as described in the Materials and Methods section. I n c u b a t i o n t i m e was 10 min. Values (means + SD) are calculated as percentage inhibition of control a c t i v i t y and are an average of at least 12 determinations. Male rat liver microsomes were used as the enzyme source. b U n a c t i v a t e d microsomes at 0 day. Microsomes aged for 6 days at 0% i n h i b i t o r o f 1 - n a p h t h o l g l u c u r o n i d a t i o n , followed by piperonyl butoxide, tropital, and M G K - 2 6 4 . T h e 150 o f p i p e r o n y l b u t o x i d e i n h i b i t i o n of t e s t o s t e r o n e g l u c u r o n y l t r a n s -

f e r a s e (7.3 X 10 -6 ~.i) w a s a l m o s t 10 t i m e s that for 1-naphthol glucuronyltransferase i n h i b i t i o n (9.0 X 10 -5 M). R e s u l t s w i t h 1naphthol resembled those with testosterone

526

LUCIEll, McDANIEL, ANI)-'qMATTHt.]WS TABLE V PERCENTAGE

~ N l t I B I T I O N OF 1 - N A P H T H O L

G L U C U R O N Y L T R A N S F E R A S E '~ BY I N S E C T I C I I ) E

SYNERGIST CHEMICALS Inhibitor

Piperonyl butoxide (80%) Piperonyl butoxide (99%) Sulfoxide Tropital Safrole Piperonal Methylenedioxybenzene MGK-264

(10 4 M ) Enzyme source

-

UM b

58.1 63.4 74.0 39.3 16.3 5.7 5.1 58.5

444+ 4444-

4.2 2.4 1.7 1.7 0.8 2.4 3.4 2.3

DA~-24.3 27.5 31.4 13.0 5.8

44444<5 <5 11.3 +

2.1 3.3 4.0 0.6 2.3

1.9

-

sr)A:,i d -

-!).9 4- 1.3 -. . . . -. . 6.7 4. 2.2

-

~DA.~;

-

-33.2 + 1.2 -. . . . . -. . 14,8 4- 1.7

"A M ; : - - - -

-t~2.5 4- 1.9 --

-55.9 4- 2.0

= Incubation mixture contained 0.2 mM 1-naphthol. Other incubation conditions are described in the Materials and Methods section. Male rat livers were used as the enzyme source. Calculation of inhibition values as described in Table IV using 12 determinations. b Unactivated microsomes at 0 day. c Detergent-activated inicrosomes. d Supernatant fraction of detergent-activated microsomes. e Resedimented pellet fraction of detergent-activated microsomes. / Micosomes aged for 6 days at 4~

as the s u b s t r a t e i n t h a t safrole, piperonal, a n d m e t h y l e n e d i o x y b e n z e n e were n o t effective inhibitors. E n z y m e from detergenta c t i v a t e d microsomes was not strongly inhibited, although sulfoxide a n d p i p e r o n y l butoxide did exhibit a low level of i n h i b i t i o n (Table V). P a r t i a l l y solubilized e n z y m e (105,000g s u p e r n a t a n t from detergent-act i v a t e d microsomes) was n o t as strongly i n h i b i t e d b y p i p e r o n y l butoxide as e n z y m e from the r e s e d i m e n t e d pellet. I n h i b i t i o n values o b t a i n e d with aged microsomes as the e n z y m e source a n d 1-naphthol a n d testosterone as s u b s t r a t e s were similar to the values for u n a c t i v a t e d microsomes (Tables I V a n d V). P i p e r o n y l butoxide appeared to cause a mixed t y p e of i n h i b i t i o n of u n a c t i v a t e d 1n a p h t h o l glucuronyltransferase as shown b y analysis of L i n e w e a v e r - B u r k e plots (Fig. 4). D e t e r m i n a t i o n of Ki for p i p e r o n y l b u t o x i d e (1.2 X 10-~ .~i) also i n d i c a t e d a mixed t y p e of inhibition. N e i t h e r piperonyl butoxide nor M G K 264 interfered with in vitro f o r m a t i o n of U D P G A b y e n z y m a t i c oxidation of U D P G . Mixed substrate study. T h e mixed substrate m e t h o d is one of the criteria used to j u d g e w h e t h e r one e n z y m e catalyzes two different enzymic reactions or if two differ-

6O

I

50 40

o

-~> 3o

10 -5

5 rO [~1 (mM I-nclphthol)

15

FIG. 4. Lineweaver-Burke double reciprocal plot of inhibition of 1-naphthol glucuronyltransferase by 10-4M piperonyl butoxide. Piperonyl butoxide-treated incubations ( O - O ) ; control ( 0 - 0 ) . The data are an average of four experiments. ent enzymes are responsible (19). I n this m e t h o d each s u b s t r a t e x~ill act as a comp e t i t i v e i n h i b i t o r towards the other if one e n z y m e is catalyzing two reactions s i m u l t a n e o u s l y . T h u s , a t s a t u r a t i n g substrate concentrations, the t o t a l rate of reaction will be less t h a n t h e s u m of each when m e a s u r e d separately if only one en-

RAT LIVER UDP GLUCURONYLTRANSFERASE zyme is operative. I n this experiment the sum of p-nitrophenol (0.5 mM) and 1-naphthol (0.5 mM) glucuronidation was 185.7 n m o l e s / m i n / m g protein (139.6 for 1 naphthol and 46.1 for p-nitrophenol) when measured separately. However, when measured together p-nitrophenol glucuronidation was reduced to 22.8 nmoles and 1-naphthol to 103.5 nmoles giving a total of 126.3 n m o l e s / m i n / m g protein. These results indicate t h a t 1-naphthol and p-nitrophenol were glucuronidated b y the same enzyme.

Induction of UDP glucuronyltransferase. Piperonyl butoxide p r e t r e a t m e n t of male or female rats resulted in an induction of the U D P glucuronyltransferase conjugating p-nitrophenol and 1-naphthol (Table VI). Induction was approximately twofold and is slightly higher in females t h a n in males. A twofold induction was also obtained in aged and unactivated microsomes from piperonyl butoxide-treated rats, and the inductive effect was not altered when livers were homogenized in Tris-HC1 buffer containing 5 mM MgC12. Induction was not accompanied by a change in K~ for 1-naphthol or U D P G A . DISCUSSION There are several advantages of the radioactive assay method described here for U D P glucuronyltransferase activity: (1) T h e method is extremely sensitive,

527

with lower detection limits of less t h a n 10 ng for glucuronidation of 1-naphthol-~4C or testosterone-14C. Sensitivity of the radioactive assay is approximately 500 times greater t h a n the standard spectrophotometric assays (1, 14, 17). ( 2 ) T h e Km for U D P G A using 1-naphthol as the substrate was 0.34 mM, while the Km for U D P G A when measuring p-nitrophenyl glucuronide formation was 1.4 mM (12) making it possible to use considerably lower concentrations of U D P G A in the 1-naphthol assay. (3) The radioactive assay involves limited transfer of liquids and is therefore more rapid and less variable t h a n spectrophotometric assays or radioactive assays which require separate extraction procedures. ( 4 ) D u e to the sensitivity of the assay, shorter incubation times are required to measure glucuronide formation, which is a distinct advantage in kinetic studies. Leuders and Kuff (2) were unable to determine if activation of microsomal preparations by detergent or by ageing resulted in an alteration of enzyme affinity for U D P G A . In this study, using the 1-naphthol-14C assay, no difference in the K~ of U D P G A was observed between unactivated, detergent-activated, or spontaneously activated preparations. Thus, activation of U D P glucuronyltransferase apparently involves an increase in the n u m b e r of avail-

TABLE VI IND'(TCTION OF UDP GLUCURONYLTRANSFERASE IN MALE AND FEMALE RATS a FOLLOWING ORAL TREATMENTS WITH PIPERONYL BUTOXIDE (150 MG PER KG PER DAY FOR 3 DAYS) Males Control

Body weight Liver weight Microsomal protein b p-Nitrophenol c 1-Naphthol ~

188 9.1 15.2 24.1 54.5

44444-

Females Induced

6.3 0.9 1.1 2.2 2.7

197 9.9 18.7 44.3 88.6

44444-

Control

7.5 0.6 1.0 2.6 2.0

173 7.6 13.8 21.3 50.9

4- 6.9 4- 0.4 -4- 1.1 4- 2.0 4- 1.7

Induced

178 9.0 17.6 44.0 89.4

44444-

8.4 0.7 0.6 3.4

2.2

a Values (means 4- SD) obtained using nine rats of each sex. b Milligrams microsomal protein per gram liver. c Glucuronidation measured in nmoles (means 4- SD)/min/mg microsomal protein. Incubation medium contained 0.9 mM p-nitrophenol, 1.0 mM UDPGA, 2.0 mg microsomal protein (detergentactivated microsomes) in a final volume of 1.0 ml. d Glucuronidation measured in nmoles (means 4- SD)/min/mg microsomal protein. Incubation medium contained 0.5 mM 1-naphthol, 0.6 mM UDPGA, 0.4 mg microsomal protein (detergent-activated microsomes) in a final volume of 1.0 ml.

52S

LUCIER, McDANIEL, AND MATTHEWS

able active sites for glucuronidation rather than an increase in affinity of enzyme for substrate or UDPGA, An alternative explanation for activation is that an enzymesubstrate-activator complex dissociates faster than the enzyme-substrate complex. However, it is doubtful if this is the case for detergent activation. Contrary to the reported results of Leuders and Kuff (2), our results indicate that over one-half of the enzyme conjugating 1-naphthol and p-nitrophenol was apparently solubilized by detergent. This difference might have resulted from differences in rehomogenization techniques following Triton X-100 addition. In our studies, if microsomes were not rehomogenized following Triton X-100 addition, only slight solubilization was detected. The degree of "solubilization" was directly proportional to the amount of detergent added to the microsomal preparations. However, it should be noted that gravitational criteria alone do not constitute proof of true solubilization (enzyme completely surrounded by water molecules). The enzyme may still be membrane bound after detergent treatment, but the normal membrane environment of UDP glucuronyltransferase is obviously changed by Triton X-100. Several agents have previously been shown to stimulate in vitro UDP glucuronyltransferase activity. Stevenson et al. (20) reported that diethylnitrosamine enhanced the rate of o-aminophenyl glucuronide formation in rat liver homogenates ~4thout changing the affinity of enzyme for substrate. Activation by ATP and UDP-N-acetylglucosamine (21) was related to reduced UDPGA degradation. Stimulation of UDP glucuronyltransferase by Triton X-100 is apparently more similar to that of diethylnitrosamine, since detergents had no effect on the rate of UDPGA degradation (2). Divalent cations have been usually omitted from the incubation medium when measuring p-nitrophenyl glucuronide formation (2, 12, 18). Storey (22), using mouse liver homogenates, and Tomlinson and Yaffe (23), using rabbit liver microsomes, reported that MgC12 inhibited p-nitrophenyl glucuronide formation. In

contrast, we found that MgC12 (10 raM) resulted in almost a threefold stimulation of unactivated or activated rat liver UDP glucuronyltransferase measured by either 1-naphthol or p-nitrophenol giucuronidation. We also report a twofold stimulation of 1-naphthol and p-nitrophenol glucuronidation by MgCl~ in detergent-activated rabbit liver microsomes. The contrasting reports might be related to differences in microsomal preparation procedures. The function of divalent cations in glucuronide formation in rat liver is not clear. Peters and Fours (24) conducted an extensive study on the stimulation of hepatic mixed function oxidase enzymes by divalent cations but reached no definite conclusion as to a mechanism of action. The increase in overall ionic strength of the incubation medium resulting from addition of MgCI~ is not related to UDP glucuronyltransferase stimulation, since increases in the molar concentration by addition of KCI to the reaction mixture slightly decreased enzyme activity. Divalent cations exert a stimulatory effect on spontaneously activated, detergent-activated (both supernatant fraction and resedimented fraction), unactivated microsomes, and enzyme from smooth or rough endoplasmic reticulum, which suggests that the cation effect is a general phenomenon. Since MgSO4 enhances enzyme activity to the same level as MgC12, activation does not appear to be caused by an associated anion. Induction of UDP glucuronyltransferase by piperonyl butoxide might have been related to mobilization of endogenous Mg 2+. However, the effects of the various cations studied were qualitatively similar in both piperonyl butoxideinduced and control rats. There has been much discussion as to the possible multiplicity of hepatic UDP glucuronyltransferase (18, 21, 25, 26). Results from the present study indicate 1-naphthol and p-nitrophenol are giucuronidated by the same enzyme. This view is supported by the following evidence: (1) competitive inhibition between 1-naphthol and p-nitrophenol as determined by the mixed substrate method; (2)similar effects on glucuronidation of p-nitrophenol and 1-naph-

RAT LIVER UDP GLUCURONYLTRANSFERASE

thol by ageing or Triton X-100 treatment of microsomes; (3)similar effects on glucuronidation of either substrate by addition of divalent cations to the incubation medium; (4) similar submicrosomal distribution of enzyme activity towards both substrates; (5) similar levels of enzyme induction by piperonyl butoxide; (6) similar gravitational distribution of UDP glucuronyltransferase towards either 1-naphthol or p-nitrophenol in detergent-activated microsomes. The inhibition of UDP glucuronyltransferase by insecticide synergists using detergent-activated microsomes is too low to indicate biological significance. However, levels of inhibition are much higher for unactivated enzyme. At this point, it is not known which preparation more resembles the in vivo condition. The inability of insecticide synergists to inhibit detergent-activated enzyme might, be related to disruption of membrane integrity of UDP glucuronyltransferase since the enzyme in the supernatant fraction is not as strongly inhibited by piperonyl butoxide as it is in the resedimented fraction. In spontaneously activated microsomes, where no alteration in gravitational properties is evident, glucuronide formation of 1-naphthol and testosterone is inhibited at the same level observed for unactivated preparations. Thus, it appears that a particular membrane environment is required for effective inhibition of enzyme activity. The inhibitory action of piperonyl butoxide on UDP glucuronyltransferase is not related to inhibitor impurities, since 99% piperonyl butoxide is a better inhibitor than 80 % piperonyl butoxide. Methylenedioxybenzene and piperonal are not effective inhibitors of enzyme activity suggesting that the methylenedioxyphenyl group is not the sole inhibitory moiety. The structural requirements necessary for enzyme inhibition remain unclear, but both side chain and methylenedioxybenzene moieties appear to be important. MGK264, not being a methylenedioxyphenyl compound, may act on a different site of UDP glucuronyltransferase. Synergism of carbamate and organophosphate insecticides may be related to

529

inhibition of both conjugative and mixed function oxidase enzymes. Of particular interest are the carbamate insecticides which are rapidly hydroxylated, conjugated, and eliminated in the in. vivo detoxication process (27-29). Some of the hydroxylated metabolites are cholinesterase inhibitors and require conjugation to be detoxified and eliminated. If these hydroxylated metabolites were protected by synergist administration, the toxicity of the administered compound should be increased. Previous studies (12, 15) reported phenobarbital pretreatment of rats effected a twofold induction of UDP glucuronyltransferase. The increase in activity occurred primarily in the smooth endoplasmic reticulum. In contrast, Gram el al. (18) using three different substrates found no inductive effect of phenobarbital on the submierosomal fractions of rabbit liver UDP glueuronyltransferase. The difference in findings might be related to variation of species response to inducing agents. The studies are difficult to compare since Mulder calculated enzyme activity on a per gram liver basis while Gram measured specific activity related to microsomal protein. The latter method is generally considered more desirable in induction studies (30). In the present study, piperonyl butoxide induction of glucuronidation was approximately twofold when calculating enzyme activity in relation to microsomal protein. ACKNOWLEDGMENTS The advice and encouragementof Dr. Robert G. Owens throughout the course of this study are greatly appreciated. Thanks are extended to Mrs. Merritt Long and Mr. Clyde Williams for their skilled technical assistance. REFERENCES

1. DUTTON,G. J., AND STOREY, [. D. E., Methods Enzymol. 5,159 (1962). 2. LEUDERS, K. K., AND KUFF, E. L., Arch. Biochem. Biophys. 120,198 (1967). 3. ANDERS, M. W., AND MANNERING, G- J., Mol. Pharmacol. 2,319 (1966). 4. WILKINSON, C. F., .a*NDHICKS, L. J., J. Agr. Food Chem. 17, 829 (1969).

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LUCIEI~, Mcl)ANIEL, AND MATTHEWS

5. CASIDA, J. E., J. Agr. Food Chem. 18, 753 (1970). 6. MATTHEWS,H. B., SKRINJARIC-~POLJAI~.,~'I. L., AND CASI~)A,J. E., Life Sci. 9, 1039 (1970). 7. MATTHEWS, H. B., SKRINJ ARIC-SPOL.1Ale, M. L., AND C XSlDA,J. E., Biochem.Pharmacol., in press. 8. LVClFm, G. W., AND MENZ~:R, ]{. E., J. Agr. Food Chem. 18, 698 (1970). 9. MEnrZNDAL~:, H., :kiD ])OROUGH, W., Entomological Society of America Convention, Miami Beach, Florida, Dec., 1970. 10. FISHnEL~, L., Unpublished data (1970). 11. MATTHEWS, I-I. B., McKINNV;Y, J. D., AND LUCIER, G. W., d. Agr. Food Chem., in press. 12. MULDER, G. J., Biochem. J. 117,319 (1970). 13. LUClER, G. W., AND MENZER, l{. E., Or, Agr. Food Chem. 16,936 (1968). 14. HO~LmAN, S., AND TOUSTER, O., Biochim. Biophys. Acta 26, 338 (1962). 15. ZIEDENBERG,P., ORRENIUS, S., AND ERNSTER, L., J. Cell Biol. 32,528 (1967). 16. ]LowREY, O. I{., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, S. J., J. Biol. Chem. 193,265 (1951). 17. TALALAY,P., FISHMAN, W. H., :~.ND~{UGGINS, C., J. Biol. Chem. 166,757 (1946).

18. (ht:xx~, T. E., HANsI.:X, A. R., AND FOUTS, J. R., Biochem. J. 106,587 0968). 19. DIXON, M., AND Wgnn, E. C., in "Enzymes," 2nd ed., p. 86. Academic Press, New York (1964). 20. STEV~.ZNSON, I., (h~,.:I':N~VOOD, 1)., ANn McEw,,:N, J., Biochim. Bioph!ls. Res. Commttn. 32,866 (1968). 21. POG~'~LL,B. M., AND LELOIa, L. F., J. Biol. Chem. 236,293 (1961). 22. S'rom.zu J. D. E., Biochem. J. 95,209 (1965). 23. TOMLINSON,G. A., ANt)YAFF~:,S. J., Biochem. J. 99,507 (1966). 24. PETERS, M. A., AriD FOt'TS, J. R., Biochem. Pharmacol. 19,533 (1970). 25. DUTTON, G. J., A~;D L~WES, J., Biochem. J. 98, 30P (1966). 26. MOWAT, A. P., AND ARRAS, I. M., Biochim. Biophys. Acta 9.12, 65 (1970). 27. DOROUGH,H. W., AND CASIDA, J. E., J. Agr. Food Chem. 12,294 (1964). 28. KNAAK,J. B., TALLANT,M. J., BARTLEY,W. J., AND SULLIVAN,L. J., J. Agr. Food Chem. 13, 537 (1965). 29. LEELING, iN]'. C., AND CASII)A, J. E., J. Agr. Food Chem. 14,281, (1966). 30. FOISTS,J. R., Personal communication (1970).