Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. I: The cytochrome P-450 dependent monooxygenase system

145

Mechanirms of Ageing and Development, 48 (1989) 145-155 Elsevier Scientific Publishers Ireland Ltd.

EFFECTS OF AGING AND CALORIC RESTRICTION ON HEPATIC DRUG METABOLIZING ENZYMES IN THE FISCHER 344 RAT. I: THE CYTOCHROME P-450 DEPENDENT MONOOXYGENASE SYSTEM

JULIAN E.A. LEAKEY”*dv*,HELEN C. CUNNYasd, JOHNNY BAZARE Jr.a, PEGGY J. WEBB”, RICHIE J. FEUERSb, PETER H. DUFFY’ and RONALD W. HARTCvd “Division of Reproductive and Developmental Toxicology, bDivision of Genetic Toxicology and cOffice of Director, National Center for Toxicological Research, Jefferson, AR 72079, and bDepartment of Pharmacology and Interdisciplinary Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (U.S.A.) (Received September 23rd, 1988) SUMMARY

The effects of long-term caloric restriction on the hepatic cytochrome P-450 dependent monooxygenase system were investigated in the 22-month-old Fischer 344 rat. Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver. Caloric restriction also increased hepatic microsomal testosterone 6/3_hydroxylase, lauric acid 12-hydroxylase and 4-nitrophenol hydroxylase activities over corresponding values in both ad fibitum fed 22-month and 60-day-old control male rats. This suggests that cytochrome P-450 isozymes, P-450 pcn,Br2, P-452 and P45Oj may be induced by caloric restriction. Such changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat. Key words: Cytochrome P-450; Monooxygenases; tion; Aging; Nutrition

Drug metabolism; Caloric restric-

INTRODUCTION

The efficiency of drug metabolism and elimination is well known to decrease as a function of age [1,2] and as a consequence many drugs are administered to elderly patients at reduced doses [3,4]. In the rat, several hepatic drug metabolizing enzyme activities have been shown to decrease with senescence [4-71. Since caloric restriction in rodents has been shown to increase life-span and to decrease the onset and *To whom all correspondence and reprint requests should be addressed. Printed and Published in Ireland

10], the present s t u d y was designe n in the m a l e F i s c h e r 344 rat c o u l d e t a b o l i z i n g e n z y m e activities. In 1 i o n on the h e p a t i c c y t o c h r o m e P bed. T h e h e p a t i c c y t o c h r o m e P-, he p r i m a r y r o u t e o f o x i d a t i v e m e dietary x e n o b i o t i c s a n d e n d o g e n o t lbolites e x h i b i t i n g either increaser m p o u n d a n d m a y also result in th~ ally toxic " a c t i v e o x y g e n " species • such as h y d r o x y l or supe~

ine gethe ent ent ra ds. 5ed of als

te rat, the h e p a t i c c y t o c h r o m e P-450s exist as a f a m i l y o f stru~ ks, at least seventeen o f which have been s h o w n to differ dit in pri )]. These i s o z y m e s exhibit o v e r l a p p i n g s u b s t r a t e specificiti~ s] ~y o f m o n o o x y g e n a s e substrates, b u t several monooxygenasc mono( ntly specific for i n d i v i d u a l i s o z y m e s t h a t t h e y m a y be utiliz, ',e o f those i s o z y m e s ( T a b l e I).

ted ure the are the

ISOZYMES OF RAT LIVER CYTOCHROME P-450 lsozyme

Gene designation

Specific monooxygenase activity

P-450a P-450b P-450c P-450d P-450e

IIA1 IIBI IA 1 IA2 lIB2

Testosterone 7a-Hydroxylase 7-Pentoxyresorufin-O-dealkylase 7-Ethoxyresoru fin-O-deer hylase 2-Naphthylamine-N-hydr oxylase 7-Pentoxyresoru fin-O-dealkylase

P-450f P-450g P-450h P-450i P-450j

IIC7 IICI 1

P-450k P-450pcn, P-450pcn 2 P-450db~ P-450db:

IIC6 IliA 1 IIIA2 IID1 lID2

P-452 P-450cr~7*

IVAI

IIEI

16a-Progesterone-6-oxidation Testosterone-2a-hydroxylation 5a-Androstane-3a, 17/3-disulfate-15a-hydroxylase 4-Nitrophenol hydroxylase Testosterone-6/3-hydroxylase, Testosterone-6/3-hydroxylase,

Lauric Acid-12-hydroxylase Cholesterol-Ta-hydroxylase

8,19,24,42 and 431. Table composed from references [15,18,

sozymes exhibit differential regul factors. Several isozymes which ltive forms are readily inducible b and d, which are inducible by poly, P-450b and e, which are inducibl hich is inducible by ethanol, ketoJ me P-452, which is inducible by

th fly .~S.

~.ic Ires

ad

ve tive concentrations of the variou es hange with age. For instance, in immature rats the isozymes es t, pcn~ and pcn 2 are the major forms present. DurinlLg pubert) ~cames P-450h and P-450i begin to develop in male and fema vhereas the relative concentrations of cytochromes P-450a, :n 2 he [25]. Thus, the predominant forms of hepatic cyl'tochrome ~re male rat appear to be cytochromes P-450h, k and pcn pc 2 and th he :ats appear to be cytochromes P-450i, k and a [25]. In males, Tie g old age ar ration o f cytochrome P-450h decreases again during c 'ile ,tochrome develops [5]. Thus the senescent male rat liver cyt, to be demasculinized. ~ment and reg he al hormonal factors are involved in the developmen rowth horm is ent cytochrome P-450 isozymes. For example gro~ 26--28], directs the expression by the pituitary in sexually dimorphic pulses [26--281 suppresses the expression of both cytochromes P-450h and P-450i [29--31] and also su Glucocorticoids, androgens and thyof cytochromes hromes P-450pcn~ and P-450pcn 2 [32]. Glucocorticoi 'tochrome ,°-450 isozymes rmones also play a role in the regulation of cytoc] roid hormones [33,34]. the expression of infl Whilee it is well established that nutritional factors also influence effec of caloric restriction hepatic drug metabolizing enzyme activities [35,36], the effects n on the hepatic cytochrome P-450 isozyme profiles have not been systematically ,I-I l--/pr~ ~0vP c i ~ r r l h ~ t h ~ P f f a t ' t ~ o f l n n ~ _ t a r m ~ u l n r i t ~ r~ examined. Here we describe the effects of long-term caloric restriction on monooxyic for cytochrome P-450 isozymes. genase activities, that are specific

MATERIALS AND METHODS

Reagents Ethoxy- and pentoxyresofin were synthesized by the method of Prough et al. [37] from resorufin (Matherson, Coleman lemann and a Bell, Norwood, OH.) and the appropriate iodoalkanes (Aldrich Chemical Co., Milwaukee, WS.). Ethylmorphine was synthesized from morphine (obtained from "rom NIDA, Bethesda, MD) by the method o f Baizer and Ellner [38]. [4-~4C]Testosterone .~rone and [1J4C]lauric acid were obtained from the Amersham Corp., Arlington Hei~ eights, IL. Glucose-6-phosphate dehydrogenase was obtained from Calbiochem, La ~a Jolla, CA. Organic solvents (hplc grade) were

1 Co., Phillipsburg, NJ. All othet f r o m sigma Chemical Co., St Lou

Lnd

sed at the N C T R in a specific previously [39]. They were maintai f r o m 0600 to 1800 h. All rats recei were divided into a control group t ch received 60% of the amount co group, starting at 14 weekss ooft age and continuing continuin for the d aent. Thirty days before sacrifice the rats were phase ~hase shifted I e of feeding of the restricted animals was synchronize~d to the t rose when the control rats were most active in feeding [39].

ree

ht~rd ad the the ~at Lhe

9reparation ge. were sacrificed by decapitation either at 60 days or 22 rr Is of liver (1.5--2 g) were removed, rinsed in ice cold saline, bl led mogenized in a buffer (3.5 vols.) designed to stabilize stabili micro ins ~lOortions of livers from two animals were combined for f, each s rewere rapidly frozen and stored at - 70 °C until assa)y. Micros, from the quick-thawed homogenates by differential cen as ed by Leakey et al. [40] using a Beckman L5-75 ultra ultracentrifuge. The washed buffer with 3mal pellets were suspended in 2.0 ml of 50 m M tris--acetate tr microsomal 20% glycerol ycerol (pH 7.4). Microsomal protein concentrations were determined by the tl standard. methodI of Lowry et al. [41] using bovine serum albumin as the E n z y m e assays Totaltl cytochrome P-450 and cytochrome B-5 were assayyed by an adaptation of spectrophotometer. D the metthod of O m u r a and Sato [42] using a Beckman DU7 rome P-420 concentraUc concentrations 1¢ were less !than 0.02 nnmol m o l / m g protein in all samCytochrome 50 reductase pies assayed. Cytochrome P-450 redu .~ductase activity was assayed using cytochrome c Langdon [43]. by the method of Phillips and Lan 7-Ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-dealkylase activities on of the direct fluorometric methods of Lubet et al. were measured by an adaptation :ture contained an N A D P H regenerating system con[44]. The 1-ml incubation mixture id, 0.5 mM N A D P and 0.3 I U / m l isocitrate dehydrosisting of 1.25 m M isocitric acid. as) and substrate concentrations of 1 and 4/aM for genase (all final concentrations ethoxy- and pentoxy-resorufint, respectively. Resorufin (0.25 nmol) was used as the milar direct fluorometric system was used to measure standard for both assays. A similar se activity. The ethoxycoumarin concentration was 7-ethoxycoumarin-O-deethylase ibrated with 0.25 nmol umbelliferone. This procedure 0.6 mM and the assay was calibrated lod of Ullrich and Weber [45]. was a modification of the method

m)mycin, aminopyrine, benzphetarr ylation of phenacetin were assaye ca10, et al. [46] using substrate concenm and an N A D P H regenerating syst ing 6-phosphate and 2 I U / m l gluco, ate ons). 4f Reinke and Moyer [47] was used kn incubation volume of 125/al w ha 1M and the same glucose 6-phq ~ed was s H regenerating system described above. The reaction reactfi the n of 125/al o f 1 M trichloroacetic acid to each tube. After ceJ to precipitated protien, 200/al aliquots of each superm ,ernatant wer ~00 roM sodium hydroxide and the absorbance at 520 nm was me~ letrically. Activities were calculated by the use of ast assay stand )n10 nmol o f 4-nitrocatechol. •osomal metabolism o f testosterone was assayed bymme t h o d s de )ur ory (Cunny H . C . et aL, unpublished data) from modificatio~ mod th~hiverick and Neims [48] and W o o d et aL [49]. Microsomal Micro hy of tcid was assayed using 200/aM [l-t4C]lauric acid as substrate su a cathe hplc method o f Bains et aL [24] to separate the 111- and E ted dites. All of the monooxygenase assays described above uti 1M um phosphate buffer (pH 7.4) for the stock assay buffer and incubation times were rere adjusted to give linear reaction rates when using 40--400/ag of microsomal protein.

Statistical analysis istical evaluation of differences between groups was achieved by Duncan's Statistical Multiple le range test using a SAS General Linear Models p~ program on an IBM A T personal computer. RESULTS

As expected, long-term caloric oric restriction greatly reduced both liver and body weight in the Fischer 344 rat by 22 months of age when compared with the 22month-old pair-fed controls (Table "able II). II) However, the liver weight as a percentage of body weight was not significantly lntly changed by caloric restriction, although it did decrease with age. Microsomal lal recovery (as protein wt per liver wet wt) also decreased slightly with age in the control rats and caloric restriction reduced this effect. The microsomal content o f total cytochrome P-450 was significantly reduced in the 22-month-old rats as compared ared to the 60-day-old animals, but there were no significant changes in either cytochro tochrome B-5 concentrations or cytochrome P-450

STRICTION ON BODY WEIGHT AND TOCHROME/>-450 COMPLEX ontrol ) Days ¢ = 3)

~g) '010) very (rag/g) {)(nmol/mg) nmol/mg) {)-reductase(nmol/min/mg)

3.7 8.83 4.12 17.57 0.68 0.49 177.3

±. 15.1A _-. 1.00A _+ 0.29A +_. 1.35A --- 0.06~' ± 0.07 A ± 20.8A

22 M o n t h s (N = 5)

459.2 12.48 2.72 13.44 0.52 0.48 176.6

± 7.8" __. 0.15~ ± 0.04" _ 1.01a ± 0.01" ± 0.02A ± 15.9A

~HT

R~ 22 (N

25 1 25

{pressed as mean ± S.E.M. Microsomal (MS) recovery is express >ressed as mg n er wet weight. Different superscript letters denote significantly different v antly different from 22-month controls.

tein 05t.

.se activities. Activities t o w a r d s 7 - e t h o x y r e s o r u f i n , 7-pentox,. md In I I tI .h I I --Uo I Uh ct , .o, UnI I It.rI UoI l t O . L 3 L U J-(-.~ 33 .)J reid were s i g n i f i c a n t l y d e c r e a s e d in the 2 2 - m oruth-old r/0 o f the values m e a s u r e d in 6 0 - d a y - o l d a n i m a l s , res pectively (Table IIl). 4did not significantly N i t r o p haenol e n o l a n d t e s t o s t e r o n e 7a- a n d 6/3-hydroxylase activities activit c h a n g e with age. L a r g e a g e - r e l a t e d c h a n g e s were o b s e r v e d in ir the m a j o r p a t h w a y s o f 6 a - h y d r o x y l a t i o n o f testosh e p a t i c t e s t o s t e r o n e m e t a b o l i s m . M i c r o s o m a l 2a- a n d 116(~-1 terone, m a r k e r s o f m a l e - s p e c i f i c c y t o c h r o m e P-450h were si g n i f i c a n t l y d e c r e a s e d in Testosterone-5ov the 2 2 -m o n t h - o l d c o n t r o l rats to < 15°/0 o f their 60-day values. i c r o s o m a l r e d u c t a s e [29], on, which is c a t a l y z e d b y a f e m a l e - p r e d o m i n a n t m mic reduction, was increased reased 30-fold in the 2 2 - m o n t h - o l d rats c o m p a r e d to 60-day-old rats. m on~r o he..'a ~ ng,e ~ -_ rv e o lloat ,t,ea d rl o h,~nn aaany changes in c y t o c h r o m e P-450 C a l o ,ri,,'. r i c rao'l".',io~"L,',,'.",, r e s t r i c t i o n raA,~o,~tt reduced m a n , ~ ef tthe ies. T o t a l c y t o c h r o m e P - 4 5 0 content did n o t decrease related m o n o o x y g e n a s e activities. with age in the restricted a n i mlals a l s ( T a b l e II), t e s t o s t e r o n e 2a- and 1 6 a - h y d r o x y l a s e 33--50°70 o f their 60-day values, whereas t e s t o s t e r o n e activities were o n l y r e d u c e d to 5 a - r e d u c t a s e activity i n c r e a s e d o n l y 15-fold (Table liD. In a d d i t i o n caloric r e s t r i c t i o n a p p e a r e d to induce several m o n o o x y g e n a s e activities. H e p a t i c 4 - n i t r o p h e naol, o l , t e s t o s t e r o n e 6~- a n d lauric acid 12-hydroxylase activities were all higher in thee restricted rats t h a n in either the 60-day or the 22m o n t h - o l d c o n t r o l rats ( T a b l e III). ic r e s t r i c t i o n on several m o n o o x y g e n a s e activities t h a t T h e effects o f age a n d caloric re also i n v e s t i g a t e d (Table IV). T h e rates o f N - d e m e were n o t isozyme-specific, were b e n z p h e t a m i n e a n d d o x y l a m i n e , a n d the t h y l a t i o n o f a m i n o p y r i n e , e t h y¢lmorphine, lmor[ dl d e c r e a s e d significantly with age, but d i d n o t change O - d e e t h y l a t i o n o f p h e n a c e t i n all

TRICTION

ON ISOZYME-SPECIFIC

?ontrol 0 Days ~/ = 3)

22 Months (N = 5)

.10

0.23

± 0.05 ^

N-

I~

R~ 2~ (1~

± 0 . 0 9 AB

(

'resorufin-Olase

[b & e]

0.015 ± 0.001 ^

0 . 0 0 5 4- 0.001 s

(

esorufin-Olase

[c & d]

0.071 ± 0 . 0 0 4 A

0.037 ± 0.003 B

q

m e 16a-

[h & b]

2.87

± 0.19 ~

0.07

_ 0.05 B

I

[h]

2.49

± 0.12 ^

0.30

-

~enol hydroxylase

[J]

0.56

± 0.07 s

0.43

± 0.07 A

(

~ne 6/3xylase

[pcn~J

0.37

± 0.09 A

0.21

± 0.12 A

0.75

± 0.09 a•

± 1.64 c*

~ylase me 2a-

0.13 B

~ylase

Lauric acid 12-hydroxylase

[P-452]

7.17

± 0.84 ^

3.45

± 0.58 a

12.21

Testosterone 5a-reductase

--

0.7

± 0.2 ^

19.9

± 2.2 a

10.4

± 1.0 c*

Activities ( n m o l ' m i n -j per mg o f m i c r o s o m a l protein), are expressed as m e a n ± S.E.M. Different supermea script letters denote significantly different values ( P < 0 . 0 5 ) . *Significantly different from 22-month controls.

riction. Testosterone 17-oxidation and erythromycinsignificantly due to caloric restriction. N-demethylation also decreased:1 with age and this decrease was reduced in the caloriithoxycoumarin-O-deethylase and lauric acid l lcally restricted animals. 7-Ethox, hange with age but were significantly increased in the hydroxylase activities did not chan red to both control groups. restricted animals when compared DISCUSSION

Although caloric restriction has previously been shown to alter hepatic ae rat [50], no systematic study has previously been monooxygenase activities in the :h cytochrome P-450 isozymes are affected by such undertaken to determine which restriction. The present study was a preliminary, two time-point study designed to

STRICTION

ON

NON-SPECIFIC

)l

~mine-N-demethylase ae-N-demethylase ne- 17-oxidase

SE

Restric~ 22 M o n :N = 5

s"

22 M o n t h s

~)

(iv = 5)

_+ 0 . 4 0 " __ 0 , 3 0 A

3 , 5 4 ___ 0 . 4 4 B 2 . 1 4 __. 0 , 3 7 B

3 , 2 4 -~ 3 . 0 4 -~

8 . 9 5 + 0.71A 5.11 --. 0.71A 2 . 4 0 ___ 0 , 1 3 A

2.60 _ 0.37 B

2 . 9 9 _~

1.07 + 0 . 1 2 ~ 0 . 8 7 + 0.198

1.21 "~ 1.79 -¢

a-O-deethylase

0 . 4 6 _+ 0 . 5 5 A

5.71 -

oumarin-O-deethylase

0 . 3 3 +_ 0 . 0 7 A 0 . 6 4 -4- 0 , 1 5 A

0.43 + 0.05 ~

7.9

5.0

ycin-N-demethylase ,4 1 1 - h y d r o x y l a s e

MON

+--- 0 . 4 At~

e x p r e s s e d ( n m o l ' m i n -t p e r m g o f m i c r o s o m a l

0.30 ~

0.28 + 0.05 a ----- 0 . 7 A

7 . 2 2 "~ 0.68 0 . 4 4 "~ I1.0

-~

p r o t e i n ) , a r e e x p~rreessssee d a s m e a n

*Significantly d c r i p t l e t t e r s d e n o t e s i g n i f i c a n t l y d i f f e r e n t v a l u e s ( P < 0 . 0 5 ) . *Sig~

:

er~

.~2-

ltrols,

ae what effects long-term caloric restriction has on o~ U ll aOt s p~lJl~,,k, e c t t l t r i l l LI l k. ff LOu -P-450 isozyme specific and non-specific monooxy~ ygenase activities in the aging male rat. The observed 3bserved changes in isozyme-specific monooxygen~ enase activities described above sulggest that the age-related decrease in hepatic cytochr, 'tochrome P-450h concentrations hass been delayed by caloric restriction and that hepatic ttc concentrations of isozymes c3~ochromes P-450pcn, P-450j and P-452 have been iinduced. The effects of both aging and caloric restriction are less dramatic for the non-specific monoox,ygenase activities than for the isozyme specific activit activities such as testosterone 2a-hydroxylase. This is probablyy due to the non-specific activities being catalyzed by different isozymes that exhibit reci ecipocally changing concentrations. Caloric restriction could influence luence the expression of hepatic cytochrome P-450 isozymes through a number of mechanisms. For instance alterations could be produced by a general slowing down :n of the aging process, an alteration of plasma cones of the hormones regulating the expression of these centrations and circadian profiles :cts of the reduced intake of the different classes of isozymes [51], or by direct effects nutrient (e.g., fatty acids) whichh are known to influence hepatic cytochrome P-450 concentrations [35,36]. Caloric restriction has been shown to alter plasma concentrations and circadian rhythms of corticosterone, th yroid hormones and growth hormone [51]. These ~y a role in the regulation of cytochrome P-450 isohormones are all known to play ;nt delay in the age-related demasculinization of the zymes [25,30--34]. The apparent

ific

elated to prolonged maintenance c one 6/3-hydroxylase activities ma, tions since glucocorticoids can in liver [52] and plasma corticoster( creased in calorically restricted also not surprising since this isozy: 23] and these calorically restricte prising, however that induction of mcentrations should be associated with increased life-s pan. Cyt~ es such as cytochrome P-450j have been implicate )licated in the ride and hydroxyl radicals [11] and these radicals are ar~ though! n cellular aging [10]. inclusion, these preliminary studies have demonstrate demonstrated that loJ ion reduces the age-related demasculinization of the hepatic tem in the 22-month-old male Fisher 344 rat. In ad addition, c hig osa), P-450j and P-452 are possibly maintained at levels i 22-month-old rats allowed free access to food. The Tl maint on of these isozymes, which are important in the metabolism me~ as exogenous compounds suggests that the ageing, caloricall . l l g ; l l l l k , a l 9 thal tlldll better adapted to metabolize potentially toxic kchemicals chem nterpart.

to 10-

raFhe ble be p$50 of jor

)ric ppose /or 3us

rat IL;~ ~ U

um

lltllll4gll

REFERENCES 1

2 3 4

5 6

7 8 9 l0 11 12

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s of

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