13. pp. 299-303. ~pPergamon Vol. Press Ltd., 1978. Printed in Great Britain.

0531-5565/78/1001-.0299502.00/0

. Geront.

INDUCTION OF HEPATIC MIXED FUNCTION OXIDASES IN SENESCENT RODENTS LINDA S. BIRNBAUM and MALCOLM B. BAIRD Masonic Medical Research Laboratory, Utica, NY 13503, U.S.A. (Received 27 January 1978)

INTRODUCTION ThE HEPATIC metabolism of lipophilic xenobiotics and endogenous substrates such as steroids and fatty acids is sensitive to a wide variety of effectors, including chemical treatment, environmental manipulation, sex, species and even strain of animal (Conney, 1967; Gillette et al., 1972; Gunsalus et al., 1975). The mixed function oxidase activities which are induced depend upon the class of compound used, barbiturate, steroid, or p o l y c y c l i c hydrocarbon. The specificity seems to reside in the multiple terminal oxidases of the pathway, the cytochrome P450 molecules (Levin et al., 1974). However, the other components, N A D P H cytochrome P450reductase and phospholipid, may also respond to the inducers (Gillette et al., 1972). An age-associated decrease in the ability of senescent rodents to metabolize drugs has been reported by this laboratory (Baird et al., 1971, 1975) and others (Kato and Takanaka, 1968a). The in vivo decline is paralleled by a drop in arylhydrocarbon and zoxazolamine hydroxylase activities in whole cell homogenates (Baird et al., 1975, 1976; Kato and Takanaka, 1968b). The basis for this decrement is unclear, since age-related loss of cytochrome P450 and/or N A D P H cytochrome c reductase is not always present (Baird et al., 1975; Kato and Takanaka, 1968a,b; Grinna and Barber, 1972; K a t o et al., 1970; Adelman, 1972). In this report, we demonstrate that the ability of senescent rodents to respond to both pregnenolone-16~-carbonitrile (PCN) and 3-methylcholanthrene (3MC), as well as phenobarbital (PB), is not impaired. We have examined this in terms of coordinated monoxygenase activities as well as microsomal protein components.

MATERIALS AND METHODS Male CFN rats were maintained in our aging colony under constant environmental conditions as previously described (Baird et al., 1975). Young rats were 3 months old; old rats were 28 - 30 months. Control rats were untreated (Baird et al., 1976a). Animals were induced with PB, PCN or 3MC as previously described (Birnbaum et al., 1976). At least 5 animals were used per age and treatment group. Rats were sacrificed and hepatic microsomes prepared by differential centfifugation (Bimbaum et al., 1976). Cytochromes P450 and bs were assayed spectrally according to the procedure of Omura and Sate (1964), assuming an extinction coefficient of 91mM-lcm-a for cytochrome P4s0and 185mM-lcm-I for cytochrome bs, and NADPH-and NADH-cytochrome c reductases were measured by the methods of Williams and Kamin (1962), using an extinction coefficient of 27.7mM-Icm-: for reduced cytochrome c. The demethylations of ethylmorphine and benzphetamine were assayed by measuring the formaldehyde produced with the Nash reagent (Cochin and Axelrod, 1959) according to the procedures of Lu et al. (1972). Benzo(a)pyrene hydroxylase activity was measured fluorometricaUy(Hamen and Fouts, 1972), and protein determined by the method of Lowry et al. (1951) using crystalline egg albumin as a standard. Pyrophosphate-washed microsomes (Welton and Aust, 1972) were analyzed by polya~vlamide gel electrophoresis in the presence of sodium dodecyl sulfate (Birnbaum et al., 1976). Cytochrome P450peptides were defined by their peroxidase activity (Welton and Aust, 1974). 299

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LINDA S. BIRNBAUM AND MALCOLM B, BAIRD

RESULTS A N D DISCUSSION The age-associated decline in hepatic microsomal enzyme activities in uninduced rats is shown in Table 1. (Although these values are expressed in terms of microsomal protein, the TABLEI. MICROSOMALENZYMEACTIVITIESIN CONTROLRATS Cytochrome Cytochrome NADPH NADH EthylB e n z p h e Benzo(a)Age Pa.,0* b~* reductase'~ reductaset morphine tamine pyrene N-deN-dehydrofnethylase~ methylase.~ xylase§ Young 1"26±0"0211 0.75±0.02 58.6~2.3 622-k30 3.90~0.45 5.64±0-31 138.5±10'5 Old 1 " 0 1 ± 0 . 0 8 0.85±0.03 58.4±1.8 601 !21 1.29±0.11 2-94-k0'49 91.84-23.4 Old/young 0.80 1'13 1.00 0.97 0.33 0.52 0'67 *nmol hemoprotein per mg microsomal protein. tnmol cytochrome c reduced per rain per mg microsomal protein. :~nmol forlmaldehyde produced per min per mg microsomal protein. §Fluorescent units per 15 min per mg microsomal protein. [[Mean ±SEM. results are the same if expressed as total activity per g wet weight liver or per 100 g body weight). There is a small, but signficant, decrease (Student's t test, p

Induction of hepatic mixed function oxidases in senescent rodents.

13. pp. 299-303. ~pPergamon Vol. Press Ltd., 1978. Printed in Great Britain. 0531-5565/78/1001-.0299502.00/0 . Geront. INDUCTION OF HEPATIC MIXED F...
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