The Enfluenee sf ahensbarbitone ow Maternal sad Perinatal Hepatic Drug-metabolizing Enzymes in the Rat1 Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/20/14 For personal use only.
J. U. BELL,^ kf.M. HANSELL, A N D D. J. ECOBICWON" Dcparbmenrs ofPharrnacology and Anuto~ny,Dalhousie University, Hallfi~x,Nova Scoria 838457 Received April 2, I975
BELI..9. U., HANSELL,M. M., and ECORHCHON, D. J. 1975. The influence 06 phenobarbitone on maternal and periwatal hepatic drug-metabolizi~~g enzymes in the rat. Can. J. PhysioE. Pharmarol. 53. 1147-1 157. Phenobarbitone (PB) (75 rng/kg) was administered orally Far three cc~nsecutivedays to pregnant or lactating rats at different pre- and postnatal stages in order that the perinatal animals would receive the agent either by transplacental passage or via the milk. Control animals received equivalent vsltnmes of saline. The darns. fetarses, and pups were killed 24 h after the last dose. Hepatic p-nitroanisole Odemethylase (OD), carboxylesterase (CE), and bromcssuifophthalein-glutathione (BSP-GSH) cc~njugatingenzyme activities in a 12 100 g - 20 min supernatant cpf a 2w w/v homogenate were measured. The morphc~logysf the developing rat liver in the absence and presence of PB was examined by electron microscopy. The results demonstrated that the transplacental passage of PB to rat fetuses at tern1 or 3 days preparturn had no effect den either the hepatic drug-metabolizing enzyme activities or on the ultrastsuctura! appearance sf the liver. Increased hepatic OD activity W B S observed in the pregnant animal brat no effect was observed in the lactating dam. Pkenubarbitc~nereceived by the sucklmg rat had two distinct effects. Compared to contrd~!activities. twofold increases in hepatic OD activity were observed in rat pups as early as 4 days after birth, associated with a masked proHiferaaIon in hepatic smooth endoplasmis reticralum. Hn contrast, BB-related significant increases in neonatal hepatic CE and BSP-GSH conjugating enzyme activities were not observed aantil2h days of age. Ten the 4-day-old treated pups, characteristic morphological changes included numerous smahl membrane whorls in addition to increased smooth endopjasmis reticulum and nn~icrobcpdiesin the liver.
Changes in kinetic parameters of hepatic drug-metabalizirtfg enzymes have been reported to occur in gerinatal laboratory animals both as a result sf tissue maturation and expssrrc to exogenous inducing chemicals QJsndorf et a!. 1959; Hart et wl. 1962; Dfgllner et a!. t 965; Pantuck et al. 1968; Gram et w&.1969; Mitoma and Lek'alEey 1970; Basu st ~ l 19'7% . ; Mrasner e l ak. 1974; BeIE and Ecobichsn 1375). Studies in mice (Peters et al. 1963; Krasner et al. 1974), rats (Feues and Liscio 1970; Henderson 1971; MacLeod et cab. 1972; Bell and Ecsbichonr 1375) , rabbits (Fouts and Adamson 1959; Hart et al. 19621, hamsters (Nebert and Gelbsin 1949), and swine (Short 2nd Davis 1970) have revealed that the perinates of these species are deficient in hepatic microsoma1 drug-metabolizing activity, appreciable 'This investigation was supported by Mediea1 Research Council sf Canada grants MA-368 1 , MA-4809. 'Present address: Division of Pbarrnacslsgy and Therapeutics, University of Calgary, Calgary, Alberta. "equest reprints from Dr. D. %.Ecobiehon, Bepartment of Pharmaccklogy, Dalhorasie University, Halifax, N.S. B3H 4H7,
Bevels sf activities beirng observed o t ~ % y2-3 weeks after birth. Many agents known to cause the induction of hepatic enzymes in nangregnant or male adult animals have &en administered to pregnant dams with increases in fetal hepatic enzymes being observed in some studies but not in others (Hart at a1. 1962; Fotats and Hart 1965 ; Dixon and Wilson 1968 ;Nebere and Gelboin 1969; Feuer 1973 ) . In several studies, agents were administered to pregnant dams but analysis of the perineatal hepatic enzyme activity was done 18-96 h after birth during which time the neonate had been nursed repeatedly, thereby receiving additional agent via the mil%=and reinforcing the agent received transplacentally (Hart eek a/, 196%;FOU~S and Hart 1865; Bantuck el al. 1968). The purpose of this study was to examine the sn togenesis sf three functionally diverse hepatic drug-metabolizing enzymes in untreated perinatal rats and in those acutely exposed to PB via either the transplacental route or via the milk and t s correlate the biochemical changes observed with m s ~ h s l o g i c a laherations in the liver.
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If48
CAN. J . PWYSHOL. PHARMACOL. VOE. 53. I945
Male and female Wistar strain rats (BioBreeding Laboratories, Ottawa, Canada) were housed on hardwood shavings in a room at 25 " C with a controlled Iight cycle consisting of 12 h darkness ( 7 pm - 7 am) and 12 h light (7 am - 7 pm). Mating was carried out by placing three female rats (150-200 g) with one male until such time as evidence of coitus was observed following daily sampling for sperm by vaginal lavage. Day 0 sf gestation was taken as that day when sperm were observed microscopica8ly. Zero and -3 day (18 days gestation) fetuses were removed by caesarean section from dams stunwed by a blow on the head and killed by cervical dislocation. For postnatal anlmals, the dams were allowed to give birth wormaily and the Bitter sizes were adjrasted to contain I0 pups to minimize nutritional variations between groups. The neonates and dams were killed at selected postparturition intervals by decapitation or cervical dislocation. Sodium phenobarbitone (Poulenc Etd., Montreal, Que. ] solution (25 mg/rnl) was administered orally to pregnant: or lactating rats by a stainless steel crarved feeding tube 416 gauge, 36 mm long with a 3 nlim ball tip). At selected times, a dose of 75 mg kg l day-' was administered for three consecutive days between 9 and 10 am and the dams and perinates were killed 24 h after the third dose. Control animals received comparable volumes of 0.9 % saline. A f e r cervical dislocation, the abdomen was quickly opened and samples of liver were removed for fixation and examination by electron microscopy. For electron microscopy, 1-mm cubes of liver were fixed in 1 % osmium tetroxide in 5-cslIidine buEer at 8 "C for 2 h, dehydrated in a series of graded alcohol solutions and prspylene oxide and embedded in Spurr low viscosity embedding medium. Ultrathin (silver) sections were cut on a Porter-Blum MT2B ultramicrotome, stained with karanyl acetate ;knd lead citrate and examined with a Philips 208 electron microscope. Follo~ringremoval of small samples for electron microscopy, the entire liver was weighed, and 5.0 g samples were minced with scissors and washed thoroughly to remove excess blood with 1.15 % KC1 buffered with 0.02 M Tris-HC1 at pH 7.4. With the younger animals, it was necessary to pool the livers s f a number of littermasates to provide suficient preparations for all of the assays. A11 procedures were carried out at 0-4 "C in an ice bath. The livers were homogenized with a Potter-Elvehjem glass hsmogenkzer and a motor-driven 'Teflon pestle, using suEcient cold 1.15% KC1 to produce a final homogenate concentration of 20% (wlv). The homogenates were centrifuged in a Somall RC2-B refrigerated centrifuge at 8 "C for 20 min a%1%100 g. The supernatant fraction was carefully removed via Pasteur pipette and was assayed for the enzymatic activities. The determinations of DNA md WNA were done by the colorimetric method of Schneider (1957) using aliquots of the whole homogenate. Standard curves were prepared with known concentrations of
calf thymus DNA (type I ) and yeast WNA (type XI) purchased from the Sigma Chemical Company, St. Louis, Mo. Protein concentrations Sw the 242 000 g/min supernatant were determined by the method s f Hartree (1972), using bovine serum albumin as the standard. ODa activity was measured at 37 " C using an incubation mixture containing glucose-6-phosphate ($0 ,umok), NABP (1 .O pmol), glucose-6-phosphate Behydrogenase (EG 1.I. 1-49) (0.5 U), nicotirsamide ( I W rrnol), 242 000 g/rnin supernatant ( I .O ml), variable concentrations of p-nitroanisale (1.25 X 1 0 - W 2.5 X IW3 M ) and suficient 0.05 Ad Tris-HC1 buffer9 pH 7.4, to yield a final volume s f 4.0 mI. Incubations were carried out for 5 min in a Dubnoff metabolic shaker (I35 cycles per minute) with air as the gas phase. The reaction was stopped after 5 min by adding 2.8 rnl sf 20% trichloroacetic acid and, after centrifuging to remove the denatured protein, the y-nitrophenol formed was measured by the method of Kato and Gillette (1865). The reaction rate was Hinear for the 5-min Incubatismn and product formation was proportional to the amount of protein added to the incubation mixture. Hepatic CE activity was determined spectrophotsmetrically, using a-waphthyl acetate as the substrate. The formation of free a-naphthol was measured at 322 nm, 37 "G,and pH 7.4 for 1.0 min, using substrate concentrations ranging from 1.0 X 10-a M to 2.0 X 10-:' ~%g(Ecobichsn 5970). Initial rates of hydrolysis were derived from the recorder tracings. BSP-GSH conjugating enzyme activity was determined spectrophotometrica1Iy as described by Goldstein and Consbes (1966). This assay was carried out at 37 "C and p H 8.2, conciitions under which the conjugated product had a greater absorbance at 330 wm than did equimolar concentrations s f free BSP. Enzymatic activity was determined on aiiquots sf the 24%000 g/min supernatant which had been dialyzed overnight at 4 " C against 0.1 M pyrophosphate BsuRer, p H 8.2. Initial rates of csnjergation were measured spectrophstometrica1Iy for 2.0 min with substrate concentrations ranging from 2.28 X lo-' M to 1.82 X IBE-5W. The conjugate formed was determined using a standard curve prepared with purified conjugate synthesized according to the method of \%elan ef al. (1970). For each enzyme, Lineweaver-Bork plots were drawn using initial reaction rates determined at each sarbstrate concentration and apparent K,, and V values were estimated from 'best fit' lines through the points. Since no attempt was made t o purify any of the enzymes, vrtlues stated are only "apparent' kinetic parameters. No significant statistical differences ( p > 0.05) in kinetic parameters determined by eye or by a computerized Fortran programme were observed (Bell and Ecobichsn 1975). Statistical analysis of significance was established 'Abbreviations: PB, phensbarbitone; OD, p-nitroanisole 6-demethylase; CE, carbsxylesterase (EC 3 . I .I. E ) ; BSP-GSM, bromssulfophthalein-glutathione; BSB, br~mssulfophthaHein~
1 149
BELL ET AE.: BNFEUENCE OF PHENOBAWBITONE ON ENZYMES IN RATS
TABLE 1. The effects of maternally-administered sodium phenobarbitone on perinatal body weights and hepatic WNA/BNA ratiosa WNAIBNA
Body weight (g)
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Perinatal age (days)
Control
Prenatal - 3
Treated
(3) 1.85 + (6) 5.76 2 (4) 8.39 2 (4) 12.00 5 44) 25.81 (5) 40.90 k
( 6 ) 2.42 f 0.09 (7) 6.22 0.22 47) 9 - 1 2 2 1.23 (7) 13.15 9 1.65 (7) 23.92 k 2.02 (6) 42.34 i-5.98
+
0
Postnatal 4 7 14 21
-
O.llh 0.20h 0.79 1.23 3.78 3.19
Control
Treated
46) 1 . 8 3 0.08 (7) 2.46 & 0 . 3 2
(3) 1.27 k 0.02" ( 6 ) 2.16 k 8.16 (4) 1.78 k 0.09 (4) 1.93 2 0.07 (4) 2.24 0 . 4 % ( 5 ) 3.13 8.25'
(7) 1.82 k 0.31 (7) 2.07 i-0-29 ( 4 ) 2.11 k 0.4% (6) 2.36 2 0.43
-
"The results are presented as mean values ? SD of the number of litters indicated in parentheses. "ndieates values which are significantly diKerent Prom control values, p < 0.05.
TABLE 2. The effects of pregnancy and sodium phensbarbitone administration during pregnancy on the apparent kinetic parameters sf OD in maternal rat liver" Vehicle-treated Group
PI
Nonpregnant Pregnant ( - 3 days) ( - 7' days) (- 14 days) (- 18 days) -
-
p p
-
v
K~
(7) 0.27 Ic_ 8.06 ( 5 ) 0 . 1 5 k 0.03' ( 5 ) 0.24 0.02 (4) 0.28 2 0.04 (4) 0.38 f 8.86 --
-
-
Pkenobarbitone-treated (X
IO-~M)
3.50 k 5 . 3 8 d6.10 k 3.52 4.00
1.30 I .29' 0.89 0.58 0.84
-
P%
v
(6) 0.59 -t (4) 0.36 & (4) 0.38 & (4) 0.56 (4) 0.55
iw)
K~ ( X 0. 8.0Sb
0.16
+ 0.1~5~ 0.17
6.14 2 0.94b 44.7 7 0 * 6 9 5.57 5 1.06 6.05 2 0.f4b 5.36 f 0.98 -
-
-
T h e vaIues presented are the mean r SD of the number of animals shown in parentheses. Tlne apparent 6' values are presented as nanomc~lesof p-nitrophenol formed per minute per milligram oP protein. b%'alues are significantly diFeererat from corresponding vehicle-treated animals, p < 0.05. CVafues are sign~ficantlydifferent from values for nonpregnant, vehicle-treated animals, p < 0 05.
at the 5 % Ievel of significance ( p < 0.05) using a Student's t test for the means of two independent samples.
Results As shown in Table I , in ufero exposure to maternally administered PB4 led to a significant ( p < 0.05) reduction in fetal body weight at both prenatal ages studied. Postnatal exposure to PB inn the milk caused no significant altcratiorns in body weights. Although not shown in Table I , a reduction in liver weight was sbserved at -3 days, the value (8.13 g 0.01) being significantly ( p < 0.05) lower than that observed foe control liver (0.26 g 0.02). The celHular content of RNA, as indicated by the RNA-DNA ratio was significantly higher than control values at 3 days before term and at 2 1 days sf postnatal age. Pregnancy has been shown to reduce hepatic enzyme levels (Feuer and LiscIIo 1970; Soyka and Long 1972). In the present study, only the hepatic microsomai OD was affected, no changes being observed in @E or BSP-GSH conjugating enzyme activities during pregnancy
*
tactation. As is shown in Table 2, pregnancy si~qificantlyreduced the apparent V and raised the apparent K , of OD at 3 days before parturition. At earlier stages of pregnancy and during lactation, thc levels of OD activity were not significantly different from those detected in nonpregnant females. Morphologically, the liver appeared little different from that of nsnpregnant female rats. Treatment with PB ( 7 5 mg/kg) for three consecutive days resulted in marked increases in hepatic 08 in nonpregnant and in pregnant rats at 3, 14, and 18 days before parturition. While PB increased OD activity at 7 days before delivery, the increase was not statistica%lysignificant due t s the variation observed. During the Zactational period, BB treatment increased maternal hepatic OD and @E only at 21 days after parturition. No significant changes were observed in hepatic BSP-GSH conjugating enzyme activity in pregnant and lactating females fsllowing treatment. As is shown in Fig. 1, none of the enzymes studied in the fetal liver responded to the effects of transpIacenatally received PB, the activities OP
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1150
CAW. J. PHYSlOL. PKIARMACOL. VOk. 53, 1975
respiratory depression, all of them dying within 20 rnin sf birth. Pw the newborn and older pups exposed to PB via the maternal milk, two distinct patterns of response were observed. On the one hand, the activity sf OD was markedly increased at each postnatal interval studied, the induced activities observed at days 4, '7, 14, and 21 being of comparable rnzgnitude. In contrast, no eRects of PI3 were observed on hepatic CE and BSP-GSH conjugating enzyme before 21 days sf age, a situation which clssely rcCIRBOXYLESTERASE fleeted the effect of PB on hepatie RNA (Table 1 ) . An examination sf the zpparent K , values for the three enzymes at the different stages sf development in the presence and absence of maternal PB treatment revealed no significant differences ( p > 0.05) for CE or BSP-GSH conjugating enzyme activities. For OD, the apparent K , values for PB-treated 14- and 21-day-old pups were 5.3 and 4.8 x My respectively, significantly (twofold ) higher than those of contrgsH pups csf the same age which 2 had values of 2-22 and 2.5 x respeew BSP COMJUGASE 6 tivcly. g u.e-, Figure 2 8 shows the ultrastructural appearance sf hepatscytes from fetal liver 3 days befsrc birth. The rough endoplasmic reticulum present was observed to have dilated cistemae and there was an abundance of free ribssomes. Smooth endoplasmic reticulum was sparse. Oecasional patches sf glycogen were seen as were occasional small rniersbodies and lipid droplets. 14 21 -3 0 4 7 CsrnslQerabEe hematspoietic tissue was visible ->rn ACE fdaysi at this stage of development csrnpaed 80 FIG. 1. The influence of phenobarbitaswe (PB) on parenchymal tissue. As is shown in Fig. %B,the the ontogenesis of rat hepatic OD, CE, and BSPGSH conjugating enzyme at various pre- and post- ~altrastructureof hepatscytes of -3 day fetuses natal stages sf developnment. The pregnant or lactat- receiving PB tramasplacentally was similar ts ing dams received PB orally (75 mg/kg) for t h e e that sf control tissue, At term, hepatocytes of consecrative days, the dams, fetuses, and pups being untreated perinates and fetuses receiving PB killed 24 h after the third dose. Apparent maxin~aam transplaeental%ywere essentially similar, though activities were determined on the 12 100 g - 20 rnin showing ultrastructural differences from -3 day supernatant fraction of a 20% w/v hsmsgenate s f liver. Values are expressed as the amount of product tissue. Large concentrations of glycogen were formed per minute per milligram protein and repreobserved and the rough endoplasmic reticulum sent the mean 9 SEM for six to seven individual present both in the form of tubules with samples. The asterisk ("1 signifies seatistica%lysignifiexpanded cisterlaae and in parallel arrays. Ssrne cant differences from control va%uesat p < 8.05. sn~sath enciopkasrnic reticulum was seen as being no different %ramthose defected in con- were free ribosomes, lipid droplets and microtrol fetal liver. As eonfirmatian that tbe fetal bodies. Hegatocytes from control, untreated 4-dayrats were indeed receiving PB from the dams via the placenta, a number sf delivered litters old pups demonstrated relatively mature structreated ior sampling at term exhibited severe ture (Fig. 3A). Abundant rough endoplasmic z
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BELL ET AL.: INFLUENCE OF PHENBBARBHTONE ON ENZYMES IN RATS
1154
PIG,2. Electron rnicrsgraphs of hepatic tissue from -3-day-old fetuses from either a vehicle-treated dam ( A ) s r one having received sodium phensbarbitone (75 mg/kg, po) for three consecutive days ( 2 ) . ( A ) The control animal shows hematspoietic eeIIs as well as a portion of a hepatwyte containing sparse rough endoplasmic reticulum with expanded cisterwae and abundant free ribosomes. (B) From a sodium phenobarbitcsne exposed animal, shows essentially the same ultrastrucerare ( A ) X 96 80 ( B ) X 10 250.
reticulum, generally in paraS%el arrays, was obserwcd and smooth enadoplasrnic reticulum was visible, accompanied by smaIl patches of glycogen, rnierobsdies, Iysssomes and Iipid droplets. Hepatocytes from 4-day-old pups receiwing PB via the milk showed marked altera-
tions in ~Itrastrueture (Fig, 3B). While the mitochondria and rough endoplasmie rctickalk~rn were normal the cells contained abundant smooth endsplasmic reticu%urn,p&icu%arly toward the periphery of the cell. Whorls o% smooth membranes were seen (Fig, 3@), sften
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CAN. J . PHYSIBL. PHARMACBL. V8L. 53, 1945
Fro, 3. Hepatic tissue from a control 4-day-old pup and Prom 4-day-old pups suckIed by dams receiving sodium phenobarbitone (75 rng/kg) orally for 3 days. ( A ) Shows a portion of a control hepatocyte containing normal ultrastructure X 9540. B and &: are portions of hepat~cytes from exposed 4-day-old pups showing accumulations of smooth endoplasrnic reticulum ( S ) microbodies and concentric membrane whorls (arrow). (B) x $250; ( C ) x 7050.
surrounding aggregations 0% smooth endoplasmic reticulum. The cells contained many microbodies and lipid droplets. Hepatocytes from control pups, 1- and 2weeks-old demonstrated normal mature sfrueture not unlikk that observed in the 4-day-old
control pups or in adults (Fig. 4A). The &erationns in hepatocyte ultrastructure from suckBing pups of csmgarable age receiving PB from thc dam via milk were similar to those observed at 4 days of zge in that there was an increase in smooth endoplasrnic reticulum often associated
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BELL ET AL.: INFLUENCE OF PHENOBAWBITONE ON ENZYMES IN RATS
Hepatic tissue from 14-day-old rat pups suckled by dams receiving either vehicle (75 mg/kg) orally for three consecutive days. ( A ) Shows a portion of a control hepratocyte with normal structure and patches of glycogen (G). (B) From sodium phenobarbitone-exposed 14-day-old pup shows accnma~lationsof smooth endoplasmie reticulum ( S ) and glycogen X 13 395. FIG.4.
01S Q ~ ~ phensbarbitone B P ~
with patches of glycogen, small membrane whorls, and many microbodies (Fig. 4B). A11 other organelles appeared nst-maio
Disenssisn The slow dcvelsprncnt of perinatd hepatic drug-metabolizing enzymes following birth has
been well dscumented (see Introduction). The marked influence of certain classes of foreign chemicds (xenobiotics) on hepatic enzyme induction in young and adult animals is well. known. The inability of the prepant dam to biotransform xenobiotics as efficiently as the nonpregnant animal has been recognized (King
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1 1 54
CAN. J. PHYSIOL. PHARMACOL. VOL. 53, I945
and Beckes 1963; Creavea and Parke 1965; Guarins el a&. 1969; Feuer and Liscis 1978; Soyka and Long 1972), Relatively little has been published about the influence which xenobiotics administered to the pregnant or lactating darn might have s n the kinetic parametcrs sf developing enzymatic patterns in the fetus and neonate. It can be concluded from the observations made in Figs. 9 2nd 2 that the transplacental passage of PBQbefare birth bad no effect. sn the nnorphology or enzymatic activities in the fetal rat kepatocyte either at term or at 3 days before birth. With the exception sf BSP-GSH conjugating enzyme, kow levels of activity were observed, The absence sf any influence of PB on the enzymatic activities was similar to the observations of Fonts and A d m s o n (%959), Hart ef al, ( 19621, and Dixon and Willson ( 1965) for rabbits. The appearance 0% the fetal hepaeocytes was similar to that reported by Ballner et a&.(1965 9, the cells containing rough endspkasmic reticulum, free ribosomes, snitschondria, patches of glycogen, but very little smooth endop%aismicreticulum. The msrphoEogy of the feral hepatocyte was not altered appreciably by PB treatment 0%the pregnant dm. Other studies, examining the microssma1 oxidation reactions of newborn liver between 18-86 h after birth, have noted sharp increases in activities over control vducs If the dams were treated during the latter pard of pregnancy with a xensbistic inducing agent (Fouts and Hart 1965; Pantuck el a&.1968). Such experjlmen& suffer from the disadvantage that, during the interval, the neonate would have been nursed several times, and would have received additional agent via this route. The present study OW 4-day-old neonates revealed two quite distinct effects of PB, depending upon the enzymes examined. Far the enzyme closely associated with the endoplasmic reticnHum, OD, there was a marked twsfsld increase at day 4 (Fig. I concomitant with a marked increase in the quantity of smooth endoplasmic reticuIram observed in the hepatocyte (Fig. 4). Comparable twofold increases in OD activity 2nd similar changes in hepatocyte morpksHogy were observed at all other postnatal ages studied. Unlike OD, both hepatic CE and BSP-GSH
>
conjugating enzyme showed n s response to BB received via the milk until 21 days sf age. Though marked increases in smooth endsplasmic reticulum were observed before then, significant ( p < 0.05) increases in 2ctivity were not. Factors other than proliferation of smooth endoplasmie reticulum may influewee changes in these enzymes since CE is not entirely localized En the endoplasmic reticulum (Schwark and Ecobichon 1968) and the BSP-GSM cowjugating enzyme is a soluble cytoplasmic prstein (Combes and Stakelum 1961) , Concentric membrane arrays or whorls within the cytoplasm of hepatscytes have been noted in several studies, generally following the chronic administration of high doses of exogenous enzyme inducing chemieaBs (Burger and Herdson $966; Nishizumi 1978; Nsrbaek and Allen 1872). In previous studies, whorls were rarely observed in 28-day-old pups acutely exposed to chksrobiphenyls or DDT (Hansetl and Eeobichsn 19494). In this study, whorls were rarely observed in older pups but were quite commonly found in hepatocytes of 4-dayold animals (Fig. 3 ) . This observation might be related to higher tissue coracewtratisns of BB as a result of the inability sf the younger pups to eEcicntly bistransfsrm and eliminate the agent. Chiesera et a&. Q 1967) suggested that the growing liver in the neonate or after partial Isepateebomy was partic~slarlypronc to devcloping these arrays. Norback and Alkeas (1972) noted that such membrane arrays often surrounded lipid droplets, suggesting that these chsnfiguraeisns provided sites for the storage and possible detoxification of lipophikic exsgenous, toxic chemicals. Jezequel ( 1974) suggested that whorls more likely represented a degradation of altered membranes. Several reports have dernsnskrated that hepatic levels of drug-metabolizing enzymes in female animals are significantly seduced during pregnancy (King and Becker 1963 ; Fehaer 1970; Soyka and Long 1972). During gestation, elevated levels of progesterone and reduced metabolites interfered campctitive%yor nosacompetitively with oxidative drug reactions, thereby reducing the activity and changing apparcnt K , values (Kardlsh and Feuer 1972; Soyka and Long 1972). In the present study (Table 2 ) , pregnancy significantly ( p < 0.85 )
BELL ET AE.: INFLUENCE OH: PHENOBAWBITBNE ON ENZYMES IN RATS
reduced OD activity and increased the apparent
fold increase in the mixed Eunctlsn oxidase group of enzymes which was associated marobserved for the other enzymes as might be phslogically with a visible increase in smooth expected since progesterone is intimately asso- endoplasmic reticulum. The other hepatic enciated with the mixed function oxidases local- zymes studied developed more slowly after ized in membranes sf the smooth endsplasmic birth and PB elicited no effect until 21 days reticulum (Kardish and Feuer H 972). Sodium after birth. The morphologiczl and enzymatic developphenobarbitone significantly ( p < 0.05) increased OD activity throughout pregnancy, con- mcnt sf rat liver has been extensively studied firming the results of Fahim et al. (1970). No by Greengard C 1974). At birth, the parenstatistically significant changes in OD activity chymal cells which are considered t~ contain were observed during lactation until day 21. the drug-detoxifying enzymes occupy 85 % of Changes in the kinetic parameters of the BSPthe Iiver mass. They tandergcs a twofold increase GSH conjugating enzyme were not stzttistica~~y in volume at birth and a further twofold insignificant. While iascreased @E activity was crease between the 12th and 28th days of age measured early in pregnancy, the significance (Greengard et a%.1972). Werzfcld et at. (1973) was difficult to assess due to the variations found that the synthesis sf smooth endspIasrnic obsemed. From the present study, it would reticulum occrarrcd between the 2nd and 12th appear that exposure to enzyme-inducing days sf age. Henderson ( 197 % ) showed a good agents during pregnancy will only affect the correlation between the lack of mixed function mixed function sxidase class of enzymes closely oxidases during the first 30 days after birth associated with thc smooth endoplasmic re- and rapid liver growth and suggested that the level of hepatic drug oxidation in thc develepticulum. It has been suggested that the transplacental ing rat was related to the rate of liver growth. and milk transfer of progesterone metabolites This concept was supported by liver regcnerato the perinate contributes to the delayed post- tion studies where decreased levels of drug natal development of hepatic rnlcrosornal drug- oxidation were associated with rapid cellular metabolizing enzymes (Mardish and Feuer proliiferationn (Henderson and Kersten E 978). 1972; Soyka and Long 1972; Feuer 1973). The present study supported this hypothesis at The administration of S~-pregnane-3a920a- Iease far mixed function esxidases though it dioB and 5p-pregnane-3p-ol-20-one to 18-day- would appear that the mechanisms for their old female rats markedly reduced anisrosomal synthesis can be activated earlier in the neocournarin 3-hydroxylase activity while pre- natal period by inducing chemicals. For drugmature weaning of litkcmates at 2 weeks sf metabolizing enzymes sf certain other classes, age caused a significant increase in drug- it would appear that more complete maturation metabolizing activity (Feuer H 973 ) , From the of the liver must occur before their synthesis present study it is obvious that the PB received can be induced. by the suckling pup in addition to progesterone J. W. T., and P..~RKE. D. V. W. and metabolites was suacieat to offset the in- BASU,T. K., BICKERSON, 1971. Effect of development on the activity of mihibiting influence sf the steroids, twofold inducca-ssssmal drug-metabolizing-enzymes in rat liver. tion of neonatal hepatic OD sccurriatg as early Biochem. J . 124, 19-24. as 4 days after birth. Similar effects have been BELL,9. U., and E c o ~ ~ c ~ D oo9.1975. ~ i , The development of kinetic garametrrs of hepatic drug-metabolizing enreported on hepatic microsomal cournarin zymes in perinatal rats. Can. 9.Bioshem. 53.433-437. 3-kydroxylase levels following the treatment 0% BURGER,P. C., and HERDSOM,P. B. E966. Phenobarbital newborn rat pazps intrapesitoneally with low induced fine structurat changes in rat liver. Am. J . doses of phenobarbitone (Feuer 19'909, In the PathoI. 48,793-802. drug-exposed neonate, two distinct effects can CHIESERA, E.. CLEMENTI, F., CONTI,F., and MELDOLESI, J . 1967. The induction of drug-metabolizing enzymes be observed. While increased OD activity was in the Iiver during growth and regeneration. A bioobserved in control rats between term m d 4 chernisaE and ultrastructural s%udy..&ab.Invest. 16, days of age, the 4-day-old livcr responded to 24-267. G . S. 1961. A liver enzyme the influence sf the drug by producing a two- COMBES,B., and STAKEHUM,
M,, close to term. Similar effects were not
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CAN. 5. PHYSLOE. PMAWMACOC. VOL. 53, 1975
that conjugates sulfobromophthlein sodium with glutathione. I. Clin. Invest. 44,981-988. CREAVEW, P. J., and PARKE.D. V. 1965. The effect of pregnancy on the miceosornal metabolism of foreign compounds. Proceeding of the Second Meeting of the Federation of European Biochemists, pp. 88-83. DALLNER, G., SEEKEVITZ, P., and PALADE,6 . E. 1965. Synthesis s f microssmal membranes and their enzymic constituents in developing rat liver. Biochem. Biophys. Res. Commun. % 135-141. I, D r x c t ~ ,R . L o ,and WILI.SON,%I. J. 1968. Metabolism of hexobarbital and zoxazolarnine by placentae and fetal liver saapernatant fraction and response to phensbarbital and chlordane treatment. Arch. Int. Pharmacodyn. 142,453466. ECQBHCHON, D. 9. 1970. Characterization of the esterases d canine serum. Can. 9. Biochem. 48,1359-1367. FAHIM. M. S.. MAIL, D. G., JONES,T.M . , FAHIM, Z.. and WHITT,F. D. 1970. Briag-steroid interaction in the pregnant rat, fetus and neonate. Am. 9. Obstet. G ~ n e c o l 107, . 1250-1258. FEUER, G. 1970. 3-Hydroxylation of soumarin or 4-methylsoumarin by rat-liver microssrnes and its induction by 4-methylcoumarin given orally. @hem. Biol. Interact. 2,283-216. 1973. The link between the mother and fetus in drug metabolism. Rev. Can. BioT. Suppl. 32, 113-121. FEUER,6..and L r s c ~ oA. , 1970. Effect of drugs on hepatic drug metaboIism in the fetus and newborn. Int. J. Clin. Pharmacsl. Ther. Toxisol. 3, 30-33. F o u ~ sI., W., and A e a ~ s sW. ~ H. , 1959. Drug metabolism in the newborn rabbit. Science, 129, $97-898. Fouas, 9. W., and HART, E. G. 1965. Hepatic drug metabolism during the perinatal period. Ann. N.Y. Acad. Sci. 123,245-20. G o r s s r ~ sJ., ~ ,and CBMBES,B . i966. Spectrophotometris assay s f the iiver enzyme that cagalyzes sulfobromophthalein-glutathione conjugation. J. Lab. CIin. Med. 67,863-872. GRAM,Tk. E., GUAWHNCZ, A. M s , SCPIROEDER, B. H.,and GHLE ETTE,J. W. 1969. Changes in certain kinetic properties of hepatic microsomal aniline hydrsxylase and ethyImfsapkine demethylase associated with postnatal development and maturation in male rats. Blochem. I. 113, $81-685. GREENGARD, 0 . 1974. Enzymic and movhcslogicaI elifferentiation of rat liver. Brz PeEinataE pharmacoiogy, problems and priorities. Edited by Jy.Dancis and S . C. Hwang. Raven Press, New York, N.Y. pp. 14-26. GREENGARD. O., ~ E D E R M A N Me, , and KNOX,B%I. E. 197%. Cytomorphometry of developing rat liver and its zipplication to enzymic differentiation. J. Cell Biol. 52. 269-272. Gviaan~s,A . M . , GRAM,T.E., SCHWOEDER, D. H., CALL, 5. B., and GILL.ETTE.J. W. 1969. Alterations in kinetic constants for hepatic rnicrosomal aniline hydroxylase and ethylmorphine Ndemethylase associated with pregnancy in rats. J . Pharmacasl. Exp. Ther. 168, 224-228. HANSELL, M .M., and ECOBPCKON, D. J. 1974. Effects of chemically pure cklorobiphenyls on the morphology of rat liver. T ~ x i c o l ,8hppI. . Pharmac01~28,418427, HART,L. G., ADAMSON, R. H., DIXON, R. L., and FBUTS,
9. R. 196%.Stimulation of hepatic micrssomal drug metabolism in the nevl~bornand fetal rabbit. J. Pharmacol. Exp. Thee. 137,103-106. MARTWEE,E. F. 1972. Determination d protein: A msdificatissn of the Lowry method that gives a linear photometric response. Anal. Biochem. 43,422427. H E ~ F E L DA, . , FEDERMAN, M., and GREENGARD, 0. 1973. Subcellular morphometric and biochemical analyses of developing rat kepatocytes. J. Cell Biol. 57.475483. HENDERSON, P. Tk. 1971. Metabolism of drugs in ra%liver during the perinatal period. Bischem. ~ h a r m a c s l 2@, . 1225-1232. HENDERSON, P. T., and KERSTEN, K. J. 1970. Metabolism of drugs during rat liver regeneration. Biochern. Pharmacol. 19,2343-2351. SEZEQUEL,A . M . 1974. UBtrastructural basis of drug metabolism. Hsr. 9. Med. Sci. 10, 380-3235. JONBORF,I. W. R., M a a c ~ ~8. e , P., and BRODIE,B. B. 1959. Inability of n e ~ ~ b omice m and guinea pigs to metabolize drugs. Biochem. Pharmacol. 1,352-354. KARDESH, R . , and FEUER,G. 1972. Relationship between maternal progesterones and the delayed drug metabolism in the neonate. Biol. Weonat. 2@,58-67. &TO, R., and GILLETTE, S o R. 1965. Effect of starvation on NADPH-dependent enzymes in liver microssmes s f male and female rats. J. Pharmacol. Exp. %herc158, 279-284. KING,J . E., and BECKEW, R. F. 1963. Sexdifferences in the response of rats to pentobarbital sodium 1. Males, nonpregnant females and pregnant females. Am. J . Ohstet. Gynecsl. %. 856-864. KMSNER,I., ERIKSSON, M., and YWFFE,S. J. 1974. Developmental changes in mouse liver alcohol dehydrsgenase. Biochem. PharmacoI. 23,5 19-522. MACLEOD,S. M., RENTON,K. W . , and EADE,N. I&. 197%. Development of hepatic microssrnal drug-oxidizing enzymes in immature male and femake rats. J. Pharrnacol. Exp. Ther. 183,489498. Mr-nohf.~,C., and LEVALLEY, S. E. 1970. Effect on newborn rats of perinatal exposure to phenobarbital. Arch. Bnt. Pharmacodyn. 887, 155-162. NEBERT,D. W, ,and G E L B ~ I N H ., V . 1969. Tkke trz viva and in s~itroinduction of aryl hydrocarbon hydrasxylabe in mammalian cells of different species, tissues. strains, and developmental and hormonal states. Arch. Biochem. Blophys. B34,76-89. Wns~naa~~r, M. 1970. Light and eiectrfsn rnicrosc~pestudy d chlorobiphenyl poisoning. Arch. Ewviron. Health, 21,620-632. N o a a ~ c D. ~ , H . , and ALLEN,J. R. 194%.Chlsriwated aromatic hydrocarbon induced modifications s f the hepatic endoplasmic reticulum: comncentraic membrane arrays. Environ. Health Perspectives, I, 137-143. PANTUCK, E., CONNEY, A . H . , and K U N T Z R ~ AW. N ,1968. Effect of phenobarbital on the metabolism of pentobarbital and meperidine in fetal rabbits and rats. Biochem. Pharmacol. 17. 1441-1447. PETERS, V . B., KELLY.G.W . , and D E M ~ ~ T Z E W R. M. , 1963- Cytoplasmic changes in fetal and neonatal hepatic cells of the mouse. Ann. N.Y. Acad. Sci. 811, 87-103.
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BELL ET AE.: EPJFLlJENCE OF PHENOBARBITONE ON ENZYMES IN RATS
1157
SCWNEEDER, W . C cfE19eDetermination of nascleic acids in ment of drug-metabolizing enzyme activity in swine. tissues by pentose analysis. In Methods in enzyrnul9. Pharmacol. Exp. Ther. 174,185-296. ogy. VoI. 3. E d i f ~ by d S. P. Cc~lowickand H. 0. KapSBYKA, L. F., and LONG,W. J . 197%.In iyifro inhibition of Ian. Academic Press Inc., New York, N.Y. pp. drug metabolism by metabolites of progesterone. J . 680-684. Pharmacol. Exp. Ther. 182,320-3219. SCHWARK, W. S., and Ecosac~oiv,D. J . 1968. SubceHular WHELAN.G., MBCH, J., and COMES, B. 1948. ,4 direct assessment s f the importance of conjugation for localization and drug-induced changes s f rat liver and kidney esterases. Can. J . Physiol. Pharrnacol. 4-6, kiliary transport of sulfobrcrmophthaIeiw sodium. S. 205-2 12, Lab. Clin. Med. 75,542-457. SHORT,C. R . , and Dnvns, L. E. 1970. Perinatal develop-
J. U. BELL,^ kf.M. HANSELL, A N D D. J. ECOBICWON" Dcparbmenrs ofPharrnacology and Anuto~ny,Dalhousie University, Hallfi~x,Nova Scoria 838457 Received April 2, I975
BELI..9. U., HANSELL,M. M., and ECORHCHON, D. J. 1975. The influence 06 phenobarbitone on maternal and periwatal hepatic drug-metabolizi~~g enzymes in the rat. Can. J. PhysioE. Pharmarol. 53. 1147-1 157. Phenobarbitone (PB) (75 rng/kg) was administered orally Far three cc~nsecutivedays to pregnant or lactating rats at different pre- and postnatal stages in order that the perinatal animals would receive the agent either by transplacental passage or via the milk. Control animals received equivalent vsltnmes of saline. The darns. fetarses, and pups were killed 24 h after the last dose. Hepatic p-nitroanisole Odemethylase (OD), carboxylesterase (CE), and bromcssuifophthalein-glutathione (BSP-GSH) cc~njugatingenzyme activities in a 12 100 g - 20 min supernatant cpf a 2w w/v homogenate were measured. The morphc~logysf the developing rat liver in the absence and presence of PB was examined by electron microscopy. The results demonstrated that the transplacental passage of PB to rat fetuses at tern1 or 3 days preparturn had no effect den either the hepatic drug-metabolizing enzyme activities or on the ultrastsuctura! appearance sf the liver. Increased hepatic OD activity W B S observed in the pregnant animal brat no effect was observed in the lactating dam. Pkenubarbitc~nereceived by the sucklmg rat had two distinct effects. Compared to contrd~!activities. twofold increases in hepatic OD activity were observed in rat pups as early as 4 days after birth, associated with a masked proHiferaaIon in hepatic smooth endoplasmis reticralum. Hn contrast, BB-related significant increases in neonatal hepatic CE and BSP-GSH conjugating enzyme activities were not observed aantil2h days of age. Ten the 4-day-old treated pups, characteristic morphological changes included numerous smahl membrane whorls in addition to increased smooth endopjasmis reticulum and nn~icrobcpdiesin the liver.
Changes in kinetic parameters of hepatic drug-metabalizirtfg enzymes have been reported to occur in gerinatal laboratory animals both as a result sf tissue maturation and expssrrc to exogenous inducing chemicals QJsndorf et a!. 1959; Hart et wl. 1962; Dfgllner et a!. t 965; Pantuck et al. 1968; Gram et w&.1969; Mitoma and Lek'alEey 1970; Basu st ~ l 19'7% . ; Mrasner e l ak. 1974; BeIE and Ecobichsn 1375). Studies in mice (Peters et al. 1963; Krasner et al. 1974), rats (Feues and Liscio 1970; Henderson 1971; MacLeod et cab. 1972; Bell and Ecsbichonr 1375) , rabbits (Fouts and Adamson 1959; Hart et al. 19621, hamsters (Nebert and Gelbsin 1949), and swine (Short 2nd Davis 1970) have revealed that the perinates of these species are deficient in hepatic microsoma1 drug-metabolizing activity, appreciable 'This investigation was supported by Mediea1 Research Council sf Canada grants MA-368 1 , MA-4809. 'Present address: Division of Pbarrnacslsgy and Therapeutics, University of Calgary, Calgary, Alberta. "equest reprints from Dr. D. %.Ecobiehon, Bepartment of Pharmaccklogy, Dalhorasie University, Halifax, N.S. B3H 4H7,
Bevels sf activities beirng observed o t ~ % y2-3 weeks after birth. Many agents known to cause the induction of hepatic enzymes in nangregnant or male adult animals have &en administered to pregnant dams with increases in fetal hepatic enzymes being observed in some studies but not in others (Hart at a1. 1962; Fotats and Hart 1965 ; Dixon and Wilson 1968 ;Nebere and Gelboin 1969; Feuer 1973 ) . In several studies, agents were administered to pregnant dams but analysis of the perineatal hepatic enzyme activity was done 18-96 h after birth during which time the neonate had been nursed repeatedly, thereby receiving additional agent via the mil%=and reinforcing the agent received transplacentally (Hart eek a/, 196%;FOU~S and Hart 1865; Bantuck el al. 1968). The purpose of this study was to examine the sn togenesis sf three functionally diverse hepatic drug-metabolizing enzymes in untreated perinatal rats and in those acutely exposed to PB via either the transplacental route or via the milk and t s correlate the biochemical changes observed with m s ~ h s l o g i c a laherations in the liver.
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If48
CAN. J . PWYSHOL. PHARMACOL. VOE. 53. I945
Male and female Wistar strain rats (BioBreeding Laboratories, Ottawa, Canada) were housed on hardwood shavings in a room at 25 " C with a controlled Iight cycle consisting of 12 h darkness ( 7 pm - 7 am) and 12 h light (7 am - 7 pm). Mating was carried out by placing three female rats (150-200 g) with one male until such time as evidence of coitus was observed following daily sampling for sperm by vaginal lavage. Day 0 sf gestation was taken as that day when sperm were observed microscopica8ly. Zero and -3 day (18 days gestation) fetuses were removed by caesarean section from dams stunwed by a blow on the head and killed by cervical dislocation. For postnatal anlmals, the dams were allowed to give birth wormaily and the Bitter sizes were adjrasted to contain I0 pups to minimize nutritional variations between groups. The neonates and dams were killed at selected postparturition intervals by decapitation or cervical dislocation. Sodium phenobarbitone (Poulenc Etd., Montreal, Que. ] solution (25 mg/rnl) was administered orally to pregnant: or lactating rats by a stainless steel crarved feeding tube 416 gauge, 36 mm long with a 3 nlim ball tip). At selected times, a dose of 75 mg kg l day-' was administered for three consecutive days between 9 and 10 am and the dams and perinates were killed 24 h after the third dose. Control animals received comparable volumes of 0.9 % saline. A f e r cervical dislocation, the abdomen was quickly opened and samples of liver were removed for fixation and examination by electron microscopy. For electron microscopy, 1-mm cubes of liver were fixed in 1 % osmium tetroxide in 5-cslIidine buEer at 8 "C for 2 h, dehydrated in a series of graded alcohol solutions and prspylene oxide and embedded in Spurr low viscosity embedding medium. Ultrathin (silver) sections were cut on a Porter-Blum MT2B ultramicrotome, stained with karanyl acetate ;knd lead citrate and examined with a Philips 208 electron microscope. Follo~ringremoval of small samples for electron microscopy, the entire liver was weighed, and 5.0 g samples were minced with scissors and washed thoroughly to remove excess blood with 1.15 % KC1 buffered with 0.02 M Tris-HC1 at pH 7.4. With the younger animals, it was necessary to pool the livers s f a number of littermasates to provide suficient preparations for all of the assays. A11 procedures were carried out at 0-4 "C in an ice bath. The livers were homogenized with a Potter-Elvehjem glass hsmogenkzer and a motor-driven 'Teflon pestle, using suEcient cold 1.15% KC1 to produce a final homogenate concentration of 20% (wlv). The homogenates were centrifuged in a Somall RC2-B refrigerated centrifuge at 8 "C for 20 min a%1%100 g. The supernatant fraction was carefully removed via Pasteur pipette and was assayed for the enzymatic activities. The determinations of DNA md WNA were done by the colorimetric method of Schneider (1957) using aliquots of the whole homogenate. Standard curves were prepared with known concentrations of
calf thymus DNA (type I ) and yeast WNA (type XI) purchased from the Sigma Chemical Company, St. Louis, Mo. Protein concentrations Sw the 242 000 g/min supernatant were determined by the method s f Hartree (1972), using bovine serum albumin as the standard. ODa activity was measured at 37 " C using an incubation mixture containing glucose-6-phosphate ($0 ,umok), NABP (1 .O pmol), glucose-6-phosphate Behydrogenase (EG 1.I. 1-49) (0.5 U), nicotirsamide ( I W rrnol), 242 000 g/rnin supernatant ( I .O ml), variable concentrations of p-nitroanisale (1.25 X 1 0 - W 2.5 X IW3 M ) and suficient 0.05 Ad Tris-HC1 buffer9 pH 7.4, to yield a final volume s f 4.0 mI. Incubations were carried out for 5 min in a Dubnoff metabolic shaker (I35 cycles per minute) with air as the gas phase. The reaction was stopped after 5 min by adding 2.8 rnl sf 20% trichloroacetic acid and, after centrifuging to remove the denatured protein, the y-nitrophenol formed was measured by the method of Kato and Gillette (1865). The reaction rate was Hinear for the 5-min Incubatismn and product formation was proportional to the amount of protein added to the incubation mixture. Hepatic CE activity was determined spectrophotsmetrically, using a-waphthyl acetate as the substrate. The formation of free a-naphthol was measured at 322 nm, 37 "G,and pH 7.4 for 1.0 min, using substrate concentrations ranging from 1.0 X 10-a M to 2.0 X 10-:' ~%g(Ecobichsn 5970). Initial rates of hydrolysis were derived from the recorder tracings. BSP-GSH conjugating enzyme activity was determined spectrophotometrica1Iy as described by Goldstein and Consbes (1966). This assay was carried out at 37 "C and p H 8.2, conciitions under which the conjugated product had a greater absorbance at 330 wm than did equimolar concentrations s f free BSP. Enzymatic activity was determined on aiiquots sf the 24%000 g/min supernatant which had been dialyzed overnight at 4 " C against 0.1 M pyrophosphate BsuRer, p H 8.2. Initial rates of csnjergation were measured spectrophstometrica1Iy for 2.0 min with substrate concentrations ranging from 2.28 X lo-' M to 1.82 X IBE-5W. The conjugate formed was determined using a standard curve prepared with purified conjugate synthesized according to the method of \%elan ef al. (1970). For each enzyme, Lineweaver-Bork plots were drawn using initial reaction rates determined at each sarbstrate concentration and apparent K,, and V values were estimated from 'best fit' lines through the points. Since no attempt was made t o purify any of the enzymes, vrtlues stated are only "apparent' kinetic parameters. No significant statistical differences ( p > 0.05) in kinetic parameters determined by eye or by a computerized Fortran programme were observed (Bell and Ecobichsn 1975). Statistical analysis of significance was established 'Abbreviations: PB, phensbarbitone; OD, p-nitroanisole 6-demethylase; CE, carbsxylesterase (EC 3 . I .I. E ) ; BSP-GSM, bromssulfophthalein-glutathione; BSB, br~mssulfophthaHein~
1 149
BELL ET AE.: BNFEUENCE OF PHENOBAWBITONE ON ENZYMES IN RATS
TABLE 1. The effects of maternally-administered sodium phenobarbitone on perinatal body weights and hepatic WNA/BNA ratiosa WNAIBNA
Body weight (g)
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Perinatal age (days)
Control
Prenatal - 3
Treated
(3) 1.85 + (6) 5.76 2 (4) 8.39 2 (4) 12.00 5 44) 25.81 (5) 40.90 k
( 6 ) 2.42 f 0.09 (7) 6.22 0.22 47) 9 - 1 2 2 1.23 (7) 13.15 9 1.65 (7) 23.92 k 2.02 (6) 42.34 i-5.98
+
0
Postnatal 4 7 14 21
-
O.llh 0.20h 0.79 1.23 3.78 3.19
Control
Treated
46) 1 . 8 3 0.08 (7) 2.46 & 0 . 3 2
(3) 1.27 k 0.02" ( 6 ) 2.16 k 8.16 (4) 1.78 k 0.09 (4) 1.93 2 0.07 (4) 2.24 0 . 4 % ( 5 ) 3.13 8.25'
(7) 1.82 k 0.31 (7) 2.07 i-0-29 ( 4 ) 2.11 k 0.4% (6) 2.36 2 0.43
-
"The results are presented as mean values ? SD of the number of litters indicated in parentheses. "ndieates values which are significantly diKerent Prom control values, p < 0.05.
TABLE 2. The effects of pregnancy and sodium phensbarbitone administration during pregnancy on the apparent kinetic parameters sf OD in maternal rat liver" Vehicle-treated Group
PI
Nonpregnant Pregnant ( - 3 days) ( - 7' days) (- 14 days) (- 18 days) -
-
p p
-
v
K~
(7) 0.27 Ic_ 8.06 ( 5 ) 0 . 1 5 k 0.03' ( 5 ) 0.24 0.02 (4) 0.28 2 0.04 (4) 0.38 f 8.86 --
-
-
Pkenobarbitone-treated (X
IO-~M)
3.50 k 5 . 3 8 d6.10 k 3.52 4.00
1.30 I .29' 0.89 0.58 0.84
-
P%
v
(6) 0.59 -t (4) 0.36 & (4) 0.38 & (4) 0.56 (4) 0.55
iw)
K~ ( X 0. 8.0Sb
0.16
+ 0.1~5~ 0.17
6.14 2 0.94b 44.7 7 0 * 6 9 5.57 5 1.06 6.05 2 0.f4b 5.36 f 0.98 -
-
-
T h e vaIues presented are the mean r SD of the number of animals shown in parentheses. Tlne apparent 6' values are presented as nanomc~lesof p-nitrophenol formed per minute per milligram oP protein. b%'alues are significantly diFeererat from corresponding vehicle-treated animals, p < 0.05. CVafues are sign~ficantlydifferent from values for nonpregnant, vehicle-treated animals, p < 0 05.
at the 5 % Ievel of significance ( p < 0.05) using a Student's t test for the means of two independent samples.
Results As shown in Table I , in ufero exposure to maternally administered PB4 led to a significant ( p < 0.05) reduction in fetal body weight at both prenatal ages studied. Postnatal exposure to PB inn the milk caused no significant altcratiorns in body weights. Although not shown in Table I , a reduction in liver weight was sbserved at -3 days, the value (8.13 g 0.01) being significantly ( p < 0.05) lower than that observed foe control liver (0.26 g 0.02). The celHular content of RNA, as indicated by the RNA-DNA ratio was significantly higher than control values at 3 days before term and at 2 1 days sf postnatal age. Pregnancy has been shown to reduce hepatic enzyme levels (Feuer and LiscIIo 1970; Soyka and Long 1972). In the present study, only the hepatic microsomai OD was affected, no changes being observed in @E or BSP-GSH conjugating enzyme activities during pregnancy
*
tactation. As is shown in Table 2, pregnancy si~qificantlyreduced the apparent V and raised the apparent K , of OD at 3 days before parturition. At earlier stages of pregnancy and during lactation, thc levels of OD activity were not significantly different from those detected in nonpregnant females. Morphologically, the liver appeared little different from that of nsnpregnant female rats. Treatment with PB ( 7 5 mg/kg) for three consecutive days resulted in marked increases in hepatic 08 in nonpregnant and in pregnant rats at 3, 14, and 18 days before parturition. While PB increased OD activity at 7 days before delivery, the increase was not statistica%lysignificant due t s the variation observed. During the Zactational period, BB treatment increased maternal hepatic OD and @E only at 21 days after parturition. No significant changes were observed in hepatic BSP-GSH conjugating enzyme activity in pregnant and lactating females fsllowing treatment. As is shown in Fig. 1, none of the enzymes studied in the fetal liver responded to the effects of transpIacenatally received PB, the activities OP
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CAW. J. PHYSlOL. PKIARMACOL. VOk. 53, 1975
respiratory depression, all of them dying within 20 rnin sf birth. Pw the newborn and older pups exposed to PB via the maternal milk, two distinct patterns of response were observed. On the one hand, the activity sf OD was markedly increased at each postnatal interval studied, the induced activities observed at days 4, '7, 14, and 21 being of comparable rnzgnitude. In contrast, no eRects of PI3 were observed on hepatic CE and BSP-GSH conjugating enzyme before 21 days sf age, a situation which clssely rcCIRBOXYLESTERASE fleeted the effect of PB on hepatie RNA (Table 1 ) . An examination sf the zpparent K , values for the three enzymes at the different stages sf development in the presence and absence of maternal PB treatment revealed no significant differences ( p > 0.05) for CE or BSP-GSH conjugating enzyme activities. For OD, the apparent K , values for PB-treated 14- and 21-day-old pups were 5.3 and 4.8 x My respectively, significantly (twofold ) higher than those of contrgsH pups csf the same age which 2 had values of 2-22 and 2.5 x respeew BSP COMJUGASE 6 tivcly. g u.e-, Figure 2 8 shows the ultrastructural appearance sf hepatscytes from fetal liver 3 days befsrc birth. The rough endoplasmic reticulum present was observed to have dilated cistemae and there was an abundance of free ribssomes. Smooth endoplasmic reticulum was sparse. Oecasional patches sf glycogen were seen as were occasional small rniersbodies and lipid droplets. 14 21 -3 0 4 7 CsrnslQerabEe hematspoietic tissue was visible ->rn ACE fdaysi at this stage of development csrnpaed 80 FIG. 1. The influence of phenobarbitaswe (PB) on parenchymal tissue. As is shown in Fig. %B,the the ontogenesis of rat hepatic OD, CE, and BSPGSH conjugating enzyme at various pre- and post- ~altrastructureof hepatscytes of -3 day fetuses natal stages sf developnment. The pregnant or lactat- receiving PB tramasplacentally was similar ts ing dams received PB orally (75 mg/kg) for t h e e that sf control tissue, At term, hepatocytes of consecrative days, the dams, fetuses, and pups being untreated perinates and fetuses receiving PB killed 24 h after the third dose. Apparent maxin~aam transplaeental%ywere essentially similar, though activities were determined on the 12 100 g - 20 rnin showing ultrastructural differences from -3 day supernatant fraction of a 20% w/v hsmsgenate s f liver. Values are expressed as the amount of product tissue. Large concentrations of glycogen were formed per minute per milligram protein and repreobserved and the rough endoplasmic reticulum sent the mean 9 SEM for six to seven individual present both in the form of tubules with samples. The asterisk ("1 signifies seatistica%lysignifiexpanded cisterlaae and in parallel arrays. Ssrne cant differences from control va%uesat p < 8.05. sn~sath enciopkasrnic reticulum was seen as being no different %ramthose defected in con- were free ribosomes, lipid droplets and microtrol fetal liver. As eonfirmatian that tbe fetal bodies. Hegatocytes from control, untreated 4-dayrats were indeed receiving PB from the dams via the placenta, a number sf delivered litters old pups demonstrated relatively mature structreated ior sampling at term exhibited severe ture (Fig. 3A). Abundant rough endoplasmic z
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BELL ET AL.: INFLUENCE OF PHENBBARBHTONE ON ENZYMES IN RATS
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PIG,2. Electron rnicrsgraphs of hepatic tissue from -3-day-old fetuses from either a vehicle-treated dam ( A ) s r one having received sodium phensbarbitone (75 mg/kg, po) for three consecutive days ( 2 ) . ( A ) The control animal shows hematspoietic eeIIs as well as a portion of a hepatwyte containing sparse rough endoplasmic reticulum with expanded cisterwae and abundant free ribosomes. (B) From a sodium phenobarbitcsne exposed animal, shows essentially the same ultrastrucerare ( A ) X 96 80 ( B ) X 10 250.
reticulum, generally in paraS%el arrays, was obserwcd and smooth enadoplasrnic reticulum was visible, accompanied by smaIl patches of glycogen, rnierobsdies, Iysssomes and Iipid droplets. Hepatocytes from 4-day-old pups receiwing PB via the milk showed marked altera-
tions in ~Itrastrueture (Fig, 3B). While the mitochondria and rough endoplasmie rctickalk~rn were normal the cells contained abundant smooth endsplasmic reticu%urn,p&icu%arly toward the periphery of the cell. Whorls o% smooth membranes were seen (Fig, 3@), sften
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CAN. J . PHYSIBL. PHARMACBL. V8L. 53, 1945
Fro, 3. Hepatic tissue from a control 4-day-old pup and Prom 4-day-old pups suckIed by dams receiving sodium phenobarbitone (75 rng/kg) orally for 3 days. ( A ) Shows a portion of a control hepatocyte containing normal ultrastructure X 9540. B and &: are portions of hepat~cytes from exposed 4-day-old pups showing accumulations of smooth endoplasrnic reticulum ( S ) microbodies and concentric membrane whorls (arrow). (B) x $250; ( C ) x 7050.
surrounding aggregations 0% smooth endoplasmic reticulum. The cells contained many microbodies and lipid droplets. Hepatocytes from control pups, 1- and 2weeks-old demonstrated normal mature sfrueture not unlikk that observed in the 4-day-old
control pups or in adults (Fig. 4A). The &erationns in hepatocyte ultrastructure from suckBing pups of csmgarable age receiving PB from thc dam via milk were similar to those observed at 4 days of zge in that there was an increase in smooth endoplasrnic reticulum often associated
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BELL ET AL.: INFLUENCE OF PHENOBAWBITONE ON ENZYMES IN RATS
Hepatic tissue from 14-day-old rat pups suckled by dams receiving either vehicle (75 mg/kg) orally for three consecutive days. ( A ) Shows a portion of a control hepratocyte with normal structure and patches of glycogen (G). (B) From sodium phenobarbitone-exposed 14-day-old pup shows accnma~lationsof smooth endoplasmie reticulum ( S ) and glycogen X 13 395. FIG.4.
01S Q ~ ~ phensbarbitone B P ~
with patches of glycogen, small membrane whorls, and many microbodies (Fig. 4B). A11 other organelles appeared nst-maio
Disenssisn The slow dcvelsprncnt of perinatd hepatic drug-metabolizing enzymes following birth has
been well dscumented (see Introduction). The marked influence of certain classes of foreign chemicds (xenobiotics) on hepatic enzyme induction in young and adult animals is well. known. The inability of the prepant dam to biotransform xenobiotics as efficiently as the nonpregnant animal has been recognized (King
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CAN. J. PHYSIOL. PHARMACOL. VOL. 53, I945
and Beckes 1963; Creavea and Parke 1965; Guarins el a&. 1969; Feuer and Liscis 1978; Soyka and Long 1972), Relatively little has been published about the influence which xenobiotics administered to the pregnant or lactating darn might have s n the kinetic parametcrs sf developing enzymatic patterns in the fetus and neonate. It can be concluded from the observations made in Figs. 9 2nd 2 that the transplacental passage of PBQbefare birth bad no effect. sn the nnorphology or enzymatic activities in the fetal rat kepatocyte either at term or at 3 days before birth. With the exception sf BSP-GSH conjugating enzyme, kow levels of activity were observed, The absence sf any influence of PB on the enzymatic activities was similar to the observations of Fonts and A d m s o n (%959), Hart ef al, ( 19621, and Dixon and Willson ( 1965) for rabbits. The appearance 0% the fetal hepaeocytes was similar to that reported by Ballner et a&.(1965 9, the cells containing rough endspkasmic reticulum, free ribosomes, snitschondria, patches of glycogen, but very little smooth endop%aismicreticulum. The msrphoEogy of the feral hepatocyte was not altered appreciably by PB treatment 0%the pregnant dm. Other studies, examining the microssma1 oxidation reactions of newborn liver between 18-86 h after birth, have noted sharp increases in activities over control vducs If the dams were treated during the latter pard of pregnancy with a xensbistic inducing agent (Fouts and Hart 1965; Pantuck el a&.1968). Such experjlmen& suffer from the disadvantage that, during the interval, the neonate would have been nursed several times, and would have received additional agent via this route. The present study OW 4-day-old neonates revealed two quite distinct effects of PB, depending upon the enzymes examined. Far the enzyme closely associated with the endoplasmic reticnHum, OD, there was a marked twsfsld increase at day 4 (Fig. I concomitant with a marked increase in the quantity of smooth endoplasmic reticuIram observed in the hepatocyte (Fig. 4). Comparable twofold increases in OD activity 2nd similar changes in hepatocyte morpksHogy were observed at all other postnatal ages studied. Unlike OD, both hepatic CE and BSP-GSH
>
conjugating enzyme showed n s response to BB received via the milk until 21 days sf age. Though marked increases in smooth endsplasmic reticulum were observed before then, significant ( p < 0.05) increases in 2ctivity were not. Factors other than proliferation of smooth endoplasmie reticulum may influewee changes in these enzymes since CE is not entirely localized En the endoplasmic reticulum (Schwark and Ecobichon 1968) and the BSP-GSM cowjugating enzyme is a soluble cytoplasmic prstein (Combes and Stakelum 1961) , Concentric membrane arrays or whorls within the cytoplasm of hepatscytes have been noted in several studies, generally following the chronic administration of high doses of exogenous enzyme inducing chemieaBs (Burger and Herdson $966; Nishizumi 1978; Nsrbaek and Allen 1872). In previous studies, whorls were rarely observed in 28-day-old pups acutely exposed to chksrobiphenyls or DDT (Hansetl and Eeobichsn 19494). In this study, whorls were rarely observed in older pups but were quite commonly found in hepatocytes of 4-dayold animals (Fig. 3 ) . This observation might be related to higher tissue coracewtratisns of BB as a result of the inability sf the younger pups to eEcicntly bistransfsrm and eliminate the agent. Chiesera et a&. Q 1967) suggested that the growing liver in the neonate or after partial Isepateebomy was partic~slarlypronc to devcloping these arrays. Norback and Alkeas (1972) noted that such membrane arrays often surrounded lipid droplets, suggesting that these chsnfiguraeisns provided sites for the storage and possible detoxification of lipophikic exsgenous, toxic chemicals. Jezequel ( 1974) suggested that whorls more likely represented a degradation of altered membranes. Several reports have dernsnskrated that hepatic levels of drug-metabolizing enzymes in female animals are significantly seduced during pregnancy (King and Becker 1963 ; Fehaer 1970; Soyka and Long 1972). During gestation, elevated levels of progesterone and reduced metabolites interfered campctitive%yor nosacompetitively with oxidative drug reactions, thereby reducing the activity and changing apparcnt K , values (Kardlsh and Feuer 1972; Soyka and Long 1972). In the present study (Table 2 ) , pregnancy significantly ( p < 0.85 )
BELL ET AE.: INFLUENCE OH: PHENOBAWBITBNE ON ENZYMES IN RATS
reduced OD activity and increased the apparent
fold increase in the mixed Eunctlsn oxidase group of enzymes which was associated marobserved for the other enzymes as might be phslogically with a visible increase in smooth expected since progesterone is intimately asso- endoplasmic reticulum. The other hepatic enciated with the mixed function oxidases local- zymes studied developed more slowly after ized in membranes sf the smooth endsplasmic birth and PB elicited no effect until 21 days reticulum (Kardish and Feuer H 972). Sodium after birth. The morphologiczl and enzymatic developphenobarbitone significantly ( p < 0.05) increased OD activity throughout pregnancy, con- mcnt sf rat liver has been extensively studied firming the results of Fahim et al. (1970). No by Greengard C 1974). At birth, the parenstatistically significant changes in OD activity chymal cells which are considered t~ contain were observed during lactation until day 21. the drug-detoxifying enzymes occupy 85 % of Changes in the kinetic parameters of the BSPthe Iiver mass. They tandergcs a twofold increase GSH conjugating enzyme were not stzttistica~~y in volume at birth and a further twofold insignificant. While iascreased @E activity was crease between the 12th and 28th days of age measured early in pregnancy, the significance (Greengard et a%.1972). Werzfcld et at. (1973) was difficult to assess due to the variations found that the synthesis sf smooth endspIasrnic obsemed. From the present study, it would reticulum occrarrcd between the 2nd and 12th appear that exposure to enzyme-inducing days sf age. Henderson ( 197 % ) showed a good agents during pregnancy will only affect the correlation between the lack of mixed function mixed function sxidase class of enzymes closely oxidases during the first 30 days after birth associated with thc smooth endoplasmic re- and rapid liver growth and suggested that the level of hepatic drug oxidation in thc develepticulum. It has been suggested that the transplacental ing rat was related to the rate of liver growth. and milk transfer of progesterone metabolites This concept was supported by liver regcnerato the perinate contributes to the delayed post- tion studies where decreased levels of drug natal development of hepatic rnlcrosornal drug- oxidation were associated with rapid cellular metabolizing enzymes (Mardish and Feuer proliiferationn (Henderson and Kersten E 978). 1972; Soyka and Long 1972; Feuer 1973). The present study supported this hypothesis at The administration of S~-pregnane-3a920a- Iease far mixed function esxidases though it dioB and 5p-pregnane-3p-ol-20-one to 18-day- would appear that the mechanisms for their old female rats markedly reduced anisrosomal synthesis can be activated earlier in the neocournarin 3-hydroxylase activity while pre- natal period by inducing chemicals. For drugmature weaning of litkcmates at 2 weeks sf metabolizing enzymes sf certain other classes, age caused a significant increase in drug- it would appear that more complete maturation metabolizing activity (Feuer H 973 ) , From the of the liver must occur before their synthesis present study it is obvious that the PB received can be induced. by the suckling pup in addition to progesterone J. W. T., and P..~RKE. D. V. W. and metabolites was suacieat to offset the in- BASU,T. K., BICKERSON, 1971. Effect of development on the activity of mihibiting influence sf the steroids, twofold inducca-ssssmal drug-metabolizing-enzymes in rat liver. tion of neonatal hepatic OD sccurriatg as early Biochem. J . 124, 19-24. as 4 days after birth. Similar effects have been BELL,9. U., and E c o ~ ~ c ~ D oo9.1975. ~ i , The development of kinetic garametrrs of hepatic drug-metabolizing enreported on hepatic microsomal cournarin zymes in perinatal rats. Can. 9.Bioshem. 53.433-437. 3-kydroxylase levels following the treatment 0% BURGER,P. C., and HERDSOM,P. B. E966. Phenobarbital newborn rat pazps intrapesitoneally with low induced fine structurat changes in rat liver. Am. J . doses of phenobarbitone (Feuer 19'909, In the PathoI. 48,793-802. drug-exposed neonate, two distinct effects can CHIESERA, E.. CLEMENTI, F., CONTI,F., and MELDOLESI, J . 1967. The induction of drug-metabolizing enzymes be observed. While increased OD activity was in the Iiver during growth and regeneration. A bioobserved in control rats between term m d 4 chernisaE and ultrastructural s%udy..&ab.Invest. 16, days of age, the 4-day-old livcr responded to 24-267. G . S. 1961. A liver enzyme the influence sf the drug by producing a two- COMBES,B., and STAKEHUM,
M,, close to term. Similar effects were not
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CAN. 5. PHYSLOE. PMAWMACOC. VOL. 53, 1975
that conjugates sulfobromophthlein sodium with glutathione. I. Clin. Invest. 44,981-988. CREAVEW, P. J., and PARKE.D. V. 1965. The effect of pregnancy on the miceosornal metabolism of foreign compounds. Proceeding of the Second Meeting of the Federation of European Biochemists, pp. 88-83. DALLNER, G., SEEKEVITZ, P., and PALADE,6 . E. 1965. Synthesis s f microssmal membranes and their enzymic constituents in developing rat liver. Biochem. Biophys. Res. Commun. % 135-141. I, D r x c t ~ ,R . L o ,and WILI.SON,%I. J. 1968. Metabolism of hexobarbital and zoxazolarnine by placentae and fetal liver saapernatant fraction and response to phensbarbital and chlordane treatment. Arch. Int. Pharmacodyn. 142,453466. ECQBHCHON, D. 9. 1970. Characterization of the esterases d canine serum. Can. 9. Biochem. 48,1359-1367. FAHIM. M. S.. MAIL, D. G., JONES,T.M . , FAHIM, Z.. and WHITT,F. D. 1970. Briag-steroid interaction in the pregnant rat, fetus and neonate. Am. 9. Obstet. G ~ n e c o l 107, . 1250-1258. FEUER, G. 1970. 3-Hydroxylation of soumarin or 4-methylsoumarin by rat-liver microssrnes and its induction by 4-methylcoumarin given orally. @hem. Biol. Interact. 2,283-216. 1973. The link between the mother and fetus in drug metabolism. Rev. Can. BioT. Suppl. 32, 113-121. FEUER,6..and L r s c ~ oA. , 1970. Effect of drugs on hepatic drug metaboIism in the fetus and newborn. Int. J. Clin. Pharmacsl. Ther. Toxisol. 3, 30-33. F o u ~ sI., W., and A e a ~ s sW. ~ H. , 1959. Drug metabolism in the newborn rabbit. Science, 129, $97-898. Fouas, 9. W., and HART, E. G. 1965. Hepatic drug metabolism during the perinatal period. Ann. N.Y. Acad. Sci. 123,245-20. G o r s s r ~ sJ., ~ ,and CBMBES,B . i966. Spectrophotometris assay s f the iiver enzyme that cagalyzes sulfobromophthalein-glutathione conjugation. J. Lab. CIin. Med. 67,863-872. GRAM,Tk. E., GUAWHNCZ, A. M s , SCPIROEDER, B. H.,and GHLE ETTE,J. W. 1969. Changes in certain kinetic properties of hepatic microsomal aniline hydrsxylase and ethyImfsapkine demethylase associated with postnatal development and maturation in male rats. Blochem. I. 113, $81-685. GREENGARD, 0 . 1974. Enzymic and movhcslogicaI elifferentiation of rat liver. Brz PeEinataE pharmacoiogy, problems and priorities. Edited by Jy.Dancis and S . C. Hwang. Raven Press, New York, N.Y. pp. 14-26. GREENGARD. O., ~ E D E R M A N Me, , and KNOX,B%I. E. 197%. Cytomorphometry of developing rat liver and its zipplication to enzymic differentiation. J. Cell Biol. 52. 269-272. Gviaan~s,A . M . , GRAM,T.E., SCHWOEDER, D. H., CALL, 5. B., and GILL.ETTE.J. W. 1969. Alterations in kinetic constants for hepatic rnicrosomal aniline hydroxylase and ethylmorphine Ndemethylase associated with pregnancy in rats. J . Pharmacasl. Exp. Ther. 168, 224-228. HANSELL, M .M., and ECOBPCKON, D. J. 1974. Effects of chemically pure cklorobiphenyls on the morphology of rat liver. T ~ x i c o l ,8hppI. . Pharmac01~28,418427, HART,L. G., ADAMSON, R. H., DIXON, R. L., and FBUTS,
9. R. 196%.Stimulation of hepatic micrssomal drug metabolism in the nevl~bornand fetal rabbit. J. Pharmacol. Exp. Thee. 137,103-106. MARTWEE,E. F. 1972. Determination d protein: A msdificatissn of the Lowry method that gives a linear photometric response. Anal. Biochem. 43,422427. H E ~ F E L DA, . , FEDERMAN, M., and GREENGARD, 0. 1973. Subcellular morphometric and biochemical analyses of developing rat kepatocytes. J. Cell Biol. 57.475483. HENDERSON, P. Tk. 1971. Metabolism of drugs in ra%liver during the perinatal period. Bischem. ~ h a r m a c s l 2@, . 1225-1232. HENDERSON, P. T., and KERSTEN, K. J. 1970. Metabolism of drugs during rat liver regeneration. Biochern. Pharmacol. 19,2343-2351. SEZEQUEL,A . M . 1974. UBtrastructural basis of drug metabolism. Hsr. 9. Med. Sci. 10, 380-3235. JONBORF,I. W. R., M a a c ~ ~8. e , P., and BRODIE,B. B. 1959. Inability of n e ~ ~ b omice m and guinea pigs to metabolize drugs. Biochem. Pharmacol. 1,352-354. KARDESH, R . , and FEUER,G. 1972. Relationship between maternal progesterones and the delayed drug metabolism in the neonate. Biol. Weonat. 2@,58-67. &TO, R., and GILLETTE, S o R. 1965. Effect of starvation on NADPH-dependent enzymes in liver microssmes s f male and female rats. J. Pharmacol. Exp. %herc158, 279-284. KING,J . E., and BECKEW, R. F. 1963. Sexdifferences in the response of rats to pentobarbital sodium 1. Males, nonpregnant females and pregnant females. Am. J . Ohstet. Gynecsl. %. 856-864. KMSNER,I., ERIKSSON, M., and YWFFE,S. J. 1974. Developmental changes in mouse liver alcohol dehydrsgenase. Biochem. PharmacoI. 23,5 19-522. MACLEOD,S. M., RENTON,K. W . , and EADE,N. I&. 197%. Development of hepatic microssrnal drug-oxidizing enzymes in immature male and femake rats. J. Pharrnacol. Exp. Ther. 183,489498. Mr-nohf.~,C., and LEVALLEY, S. E. 1970. Effect on newborn rats of perinatal exposure to phenobarbital. Arch. Bnt. Pharmacodyn. 887, 155-162. NEBERT,D. W, ,and G E L B ~ I N H ., V . 1969. Tkke trz viva and in s~itroinduction of aryl hydrocarbon hydrasxylabe in mammalian cells of different species, tissues. strains, and developmental and hormonal states. Arch. Biochem. Blophys. B34,76-89. Wns~naa~~r, M. 1970. Light and eiectrfsn rnicrosc~pestudy d chlorobiphenyl poisoning. Arch. Ewviron. Health, 21,620-632. N o a a ~ c D. ~ , H . , and ALLEN,J. R. 194%.Chlsriwated aromatic hydrocarbon induced modifications s f the hepatic endoplasmic reticulum: comncentraic membrane arrays. Environ. Health Perspectives, I, 137-143. PANTUCK, E., CONNEY, A . H . , and K U N T Z R ~ AW. N ,1968. Effect of phenobarbital on the metabolism of pentobarbital and meperidine in fetal rabbits and rats. Biochem. Pharmacol. 17. 1441-1447. PETERS, V . B., KELLY.G.W . , and D E M ~ ~ T Z E W R. M. , 1963- Cytoplasmic changes in fetal and neonatal hepatic cells of the mouse. Ann. N.Y. Acad. Sci. 811, 87-103.
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SCWNEEDER, W . C cfE19eDetermination of nascleic acids in ment of drug-metabolizing enzyme activity in swine. tissues by pentose analysis. In Methods in enzyrnul9. Pharmacol. Exp. Ther. 174,185-296. ogy. VoI. 3. E d i f ~ by d S. P. Cc~lowickand H. 0. KapSBYKA, L. F., and LONG,W. J . 197%.In iyifro inhibition of Ian. Academic Press Inc., New York, N.Y. pp. drug metabolism by metabolites of progesterone. J . 680-684. Pharmacol. Exp. Ther. 182,320-3219. SCHWARK, W. S., and Ecosac~oiv,D. J . 1968. SubceHular WHELAN.G., MBCH, J., and COMES, B. 1948. ,4 direct assessment s f the importance of conjugation for localization and drug-induced changes s f rat liver and kidney esterases. Can. J . Physiol. Pharrnacol. 4-6, kiliary transport of sulfobrcrmophthaIeiw sodium. S. 205-2 12, Lab. Clin. Med. 75,542-457. SHORT,C. R . , and Dnvns, L. E. 1970. Perinatal develop-
