Age Related Changes in the Induction of Hepatic Mixed-Function Monooxygenase System in Miniature Pigs: Effects of Pentachloroanisole, Pentachlorophenol and Phenobarbital

Agins, Alan P., Ph.D., University of Rhode Island, 1982. Age Related Changes in the Induction of the Hepatic Mixed-Function Monooxygenase Sys tern in Miniature Pigs: Effects of Pentachloroanisole, Pentachlorophenol and Phenobarbital. Major Professor: Dr. George C. Fuller. Pentachloroanisole (PCA), an environment a 1 degradation product of the biocide pentachlorophenol (PCP), has been detected in the food chain. The metabolic fate of PCA was examined in miniature pig hepatic microsomes, in vitro. The compound was shown to be a substrate for a cytochrome P450-dependent demethylation reaction, which results in the regeneration of the parent compound, PCP. A disproportionately large increase in PCA demethylase activity (PCADM) following pretreatment with phenobarbital suggests that the compound is preferentially metabolized by specific-inducible form(s) of cytochrome P450. A comparison of the effects of PCA and purified PCP on the hepatic MFO system of miniature pigs was conducted at various stages of postnatal development. Phenobarbital was utilized as a positive control for induction. PCA, PCP and phenobarbital (10 mg/kg/day X 4 days, P.O.) were administered to piglets at 1, 4, and 8 weeks of age and the levels of cytochromes P450 and b 5 , and the activities of NADPH-Cytochrome c reductase, aniline hydroxylase (ANOH), p-nitroanisole demethylase (NADM), and PCA demethylase were determined. In one week old piglets, PCA produced significant increases in all parameters measured, with the greatest effect (300% of control) on its own in vitro metabolism. The pleiotropic response evoked by PCA was similar to that of phenobarbital, but of lesser magnitude . PCP produced small increases in only P450 and nitroanisole demethylase . The qualitative differences in the induction patterns produced by PCA and PCP suggests that the two compounds exert different effect on MFO . By eight weeks of age, the magnitude of induction by PCA was diminished . Furthermore, although specific activities for ANOH, NADM, and PCADM in phenobarbital treated pigs were similar at 1 and 8 weeks of age, examination of catalytic activity profiles suggested an age dependent decrease in the induction of specific forms of cytochrome P450 . On further investigation, Eadie-Hofstee plots from kinetic experiments with ANOH and PCADM exhibited biphasic patterns suggestive of multiple forms of P450 catalyzing the same reaction . By integrating the effects of age and treatment on the various kinetic species for each substrate, a minimum of four forms of cytochrome P450 are suggested to exist in miniature pig hepatic microsomes . Of the four forms, two are inducible by phenobarbital and one of these forms appears to display age-dependency in the magnitude of induction . These data indicate that MFO induction by exogenous chemicals varies qualitatively as well as quantitatively with age .

interest in developmental toxicity studies has grown from both theoretical considerations and practical experience. In pharmacology, it has long been recognized that an effect in the infant cannot always be predicted by extrapolation of adult response. Yet, with the exception of teratology studies, the vast majority of toxicological research is conducted in relatively mature animals. Since the human infant is often unavoidably exposed to the same chemical milieu as the adult population, responses of the neonate need to be examined and defined.
It appears that within this framework, perinatal enzymology may play a central role. Chemically induced perturbations of enzyme ontogenetic profiles, alterations in isoenzyme patterns, or other modifications to key metabolic pathways may result in subtle biochemical lesions in the absence of apparent morphological or functional abnormalities. Furthermore, such changes may have profound effects on maturational processes or future health.
One enzyme system, the microsomal mixed-function monooxygenase (MFO) system, plays a critical role in the metabolism of foreign compounds such as drugs, pesticides and carcinogens. The enzyme system also has important homeostatic functions through its metabolism of steroids, heme, fatty acids and a number of other endogenous substrates.
2 Additionally, the multicomponent enzyme system, in many cases, is extremely sensitive to induction by exogenous chemicals. This adaptive mechanism, once thought to be strictly beneficial for the detoxification of xenobiotics, has received much attention over the last decade with the knowledge that many chemicals, particularly carcinogens, require metabolic activation prior to exhibiting their detrimental effects.
This study attempts to examine temporal changes in the response The metabolism of foreign compounds by hepatic microsomes was first described by Mueller and Miller (1949) . These investigators showed that both the oxidative N-demethylation and the reduction of the azo linkage of aminoazo dyes were catalyzed in vitro by microsomes derived from rat liver homogenates . In 1955, Brodie and coworkers extended these initial studies by utilizing various drugs as substrates, and after compiling the research efforts from numerous laboratories (Brodie et ~· , 1958) proposed that the microsomal fraction of cells was responsible for a vast number of reactions involved in drug metabolism . The reactions had a strict requirement for both molecular oxygen and pyridine nucleotides as reducing agents , which lead to the classification of this enzyme system as a mixed-function oxidase (Mason,195 7) or monooxygenase (Hayaishi, 1962) . Further direct support for this terminology was provided by Posner et al. (1961) who showed that the oxygen molecule inserted into a hydroxylated product was derived from 18 0 2 and not water .
Although investigators in the early 1950s had successfully isolated and characterized various individual components of microsomes , a 4 direct connection to drug metabolizing capacity remained obscure. In the latter part of the decade, Klingenberg (1958) and Garfinkel (1958) reported that an additional component, a cytochrome not y et accounted for, existed in microsomes. The ability of this cytochrome to undergo unique spectral changes in the presence of carbon monoxide led to the early name of "CO Binding Pigment". Omura and Sato ( 1964) further characterized this new cytochrome and labeled it Cytocrhome P450 due to the location of the Sor et peak of the reduced, carbon monoxide complexed material. The reactivity of Cytochrome P450 with carbon monoxide provided a powerful tool for experimentation linking microsomal electron transport to drug metabolism. Estabrook et ~·, (1963) provided firm evidence that Cytochrome P450 was a crucial component for hydroxylation reactions in adrenal cortex particles and subsequently extablished its role as the "terminal oxidase" of the microsomal drug-metabolizing enzyme system (Cooper~~., 1965).
In 1968, Lu and Coon successfully solubilized and resolved the enzyme system into fractions containing Cytochrome P450, NADPH-Cytochrome P450-Reductase, and a heat stable factor, subsequently shown to be phosphatidylcholine. The ability to reconstitute catalytic activity toward a variety of drug, steroid and fatty acid substrates was a major milestone and opened the way for more refined approaches to mechanistic and functional studies.
In the two decades following the initial studies of microsomal mediated metabolism an extremely large body of literature has been generated from research in the areas of pharmacology, toxicology, biochemistry, endocrinology, molecular biology and genetics.

Components of the Microsomal MFO System
The microsomal fraction of liver contains at least three flavoproteins; NADPH-Cytochrome P450-Reductase, NADH-Cytochrome b 5 -Reductase and Amine Oxidase, two heme proteins; Cytochrome P450 and Cytochrome b 5 , and a non-heme iron protein; Stearyl-CoA Desaturase. In the presence of the microenvironment of the smooth endoplasmic reticulum these components work either independently or in concert in an electron transfer capacity. The following discussion will be limited to those components investigated in the present study.
NADPH-Cytochrome P450-Reductase NADPH-Cytochrome P450-Reductase (NADPH Dehydrogenase. EC 1. 6 . 2 . 4) was first observed in whole liver extracts by Horecker (1950) . Phillips and Langdon (1962) and Williams and Kamin (1962) identifi ed the microsomal fraction as the origin of this enzyme and upon purification revealed that the protein is capable of reducing a wide variety of both one and two electron acceptors . The nomenclature for this enzyme varies between laboratories and is dependent on both the acceptor used and the condition for the assay. Artificial acceptors such as cytochrome c and ferricyanide are routinely utilized due to the ease of measurement, the extended linearity and the higher turnover in these assays . Although Cytochrome P450 is known to be the native acceptor, the term NADPH-Cytochrome c Reductase is often used interchangeab ly.
One major difference is that phosphatidylcholine is required for 6 reduction of Cytochrome P450 in reconstituted systems but not for electron transfer to artificial acceptors (Strobel and Digman, 1978) .
The mechanism by which the enzyme transfers electrons to Cytochrome P450 has been extensively investigated . It has been demonstrated that the protein contains equimolar amounts of FAD and FMN (Yasukochi and Masters, 1976). Vermillion and Coon (1978), using FMN-depleted reductase demonstrated that the enzyme containing only FAD remains capable of being readily reduced by NADPH and suggested that the FMN moiety probably interacts directly with Cytochrome P450 .
Although conclusive evidence for the exact mechanism is still lacking, the overall sequence of electron transfer from NADPH to FAD to FMN to P450 is consistent with the observed biphasic reduction kinetics of the enzyme. Furthermore, this scheme appears to be thermodynamically favorable as a function of reduction potential differences of the two flavin moieties (Oprian ~al . , 1979) .
In addition to its essential role in transferring reducing equivalents to Cytochrome P450, the reductase has also been reported to have P450-independent catalytic activity. Hernandez et ~· , (1967) showed that Cytochrome c Reductase catalyzed the reduction of azo dyes without benefit of P450 . Furthermore, the participation of this flavoprotein in the initiation of microsomal NADPH-dependent lipid peroxidation has been reported (Pederson et~. , 1973 (Strittmatter and Velick, 1956a) . Although the physiological function of this enzyme system was not apparent at that time, approximately a decade later Holloway and Wakil (1970) implicated Cytochrome b 5 in microsomal fatty acid desaturation reactions . It is now well established that this microsomal electron transport system is composed of NADH-Cytochrome b 5 -Reductase, Cytochrome b 5 and a cyanide-sensitive terminal desaturase which functions to convert Steryl-CoA to Oleyl-CoA (Prasad and Joshi, 1979) .
Less well established has been the role of these components with respect to the "drug-metabolizing" enzyme system. The synergistic effect of NADH on NADPH-dependent reactions led early investigators to postulate a permissive role for Cytochrome b 5 . With the knowledge that Cytochrome P450 mediated oxidations required two separate electron transfers, it was suggested that the second electron may be denoted via Cytochrome b 5 (Estabrook and Cohen, 1969) . Lu and coworkers (1974) and Imai and Sato (1977) demonstrated that in reconstituted metabolic systems, Cytochrome b 5 was not an obligatory component for activity, although in both studies synergistic effects were seen. It was also suggested by Lu et al. (1974) that the role of Cytochrome b 5 may be dependent on such factors as tissue, sex, age and the particular substrate utilized . More recently, Imai (1979), using various purified forms of Cytochrome P450, showed that Cytochrome b 5 was required for maximal activity with some forms of P450, but had little or no effect 8 on other forms in reconstituted systems . Sugiyama (1979) reported the purification of a unique form of Cytochrome P450 from rabbit liver .
This form of P450 had a high affinity for Cytochrome b 5 and required its presence for reconstitution of catalytic activity with nitroanisole as substrate . Further support for the role of Cytochrome b 5 in P450-dependent reactions has come from the finding that in addition to NADH-Cytochrome b 5 -Reductase, NADPH Cytochrome P450 Reductase can efficiently reduce Cytochrome b 5 (Enoch and Strittmatter, 1979) . Thus it appears that Cytochrome b 5 serves a central role in microsomal electron transport by interacting with two separate reductases, a desaturase and some forms of Cytochrome P450 .

Cytochrome P450
Cytochrome P450 is the dominant heme protein in microsomes . The mammalian liver is the richest source of the hemoprotein, however, Cytochrome P450 is also found in kidney, lung, skin, intestinal tract, adrenal gland, placenta, ovary and blood platelets (Hodgson and Dauterman, 1980) . cytochrome P450 as the terminal oxidase of the MFO system displays functional heterogeneity in the various tissues, yet although many of the extrahepatic tissues are capable of supporting low levels of xenobiotic metabolism, empirically, the liver is the major site for such metabolism . The Cytochrome P450 in kidney, for example, appears to be quite active in the omega oxidation of fatty acids (Masters et ~· , 1980), while the P450 in adrenal gland mitochondria is mainly responsible for the metabolism of steriod hormones (Sih, 1969 390 -410 nm . A third type of difference spectrum was also described as a reverse Type I . This spectrum is characterized by a peak at 420 nm and a trough between 385 -390 nm and thus appears to be a mirror image of a Type I spectrum . Whereas Type I producing compounds represent a large, structurally diverse group including drugs, pesticides and steriods, Type II compounds tend to be primary amines, pyridines and imidazole compounds (Mailman et ~., 1974) . Gigon and coworkers ( 1968)  In conjunction with optical difference spectra studies, electron paramagnetic resonance (EPR) spectroscopy had shown that Cytochrome P450 can exist in a high-spin (Fe 3 + heme: S = 5/2), a low-spin (Fe 3 + heme: S 1/2) or most commonly in a mixed spin state (Jefcoate, 1978). A Type I change correlates with an increase in high-spin character upon binding of the substrate, while a Type II change reflects a conversion of native high-spin P450 to a low-spin complex of the heme and the ligand . In this respect, two modes of compound interaction with P450 are now recognized as "substrate" binding and "ligand" binding (Testa and Jenner, 1981) .
From the integration of diverse physical and chemical studies, a structural model of the active site of Cytochrome P450 has been proposed (Lipscomb and Gunsalus, 1973;Rein et al . , 1976) .
In these models, the active site contains an iron protoporphyrin IX moiety in a large, relatively accessible hydrophobic pocket in the apoprotein . The heme is loosely anchored to the site by a combination of hydrophobic forces and covalent bonds to the central iron ion . The fifth iron ligand has been reported to be a thiolate anion contributed by a cycteine residue of the protein (Dolphin et al., 1979) . This anionic mercaptide linkage has been implicated for both the unique specral properties of this cytochrome and in having important electronic effects on oxygen activation in the normal cycle of catalysis (Collman and Sorrell, 1975) . The sixth axial ligand ll has been reported to be either oxygen, in the form of water or a hydroxyl group from a proximal amino acid (Griffin and Peterson, 1975) or nitrogen in the form of histidine. Although conclusive evidence is lacking, it is generally accepted that the sixth linkage is relatively weak, but functions to hold the heme iron in a square octahedral, hexacoordinated configuration. Displacement of the weak bond by stronger ligands such as amines and pyridines results in a heme-ligand complex typified by a Type II spectrum and low-spin cytochrome. The "locking" of Cytochrome P450 into a low-spin state appears to be responsible for some of the inhibitory actions exerted by such compounds (Testa and Jenner, 1981).
In contrast to ligand binding, substrate binding involves mainly hydrophobic interactions between non-polar regions of the protein and the substrate. This binding results in a dissociation of the native sixth ligand linkage and a change in the configuration of the heme molecule from octahedral, hexacoordinated to square pyramidal, pentacoordinated (White and Coon, 1980). This change also results in a conversion to a high-spin state, which increases the redox potential of the system and creates a more favorable electron flow sequence to Cytochrome P450 (Slingar ~ ~., 1979).
The mechanism of binding and subsequent activation of molecular oxygen by Cytochrome P450 is quite complex and has recently been reviewed (White and Coon, 1980). Essentially, following the binding of oxygen to the reduced P450-substrate complex, a second electron transfer results in the creation of peroxide anion. This reactive protein-complexed intermediate then reacts with substrate to form a hydroxylated compound and water. In addition to this mechanism, evidence has accumulated indicating that in some cases, Cytochrome p450 acts in an oxidase or peroxygenase capacity resulting in the production of hydrogen peroxide or other peroxy compounds. The hemolytic cleavage of the oxygen-oxygen bond may lead directly to insertion of a hydroxyl group into a substrate independent of the NADPH/0 2 pathway (Coon, 1981).
Induction of Monooxygenase Activity Conney and Burns (1959) first demonstrated the phenomenon of drug induced synthesis of liver microsomal enzymes. This finding served to substantiate a number of earlier assumptions concerning the MFO system and became a powerful tool for the study of induced enzyme synthesis in general. Extensive reviews on MFO induction have been published (Conney, 1967;Mannering, 1968).
Historically, inducers of MFO have fallen into one of two categories. One group, containing numerous drugs and xenobiotics of diverse chemical structure, is best typified by phenobarbital. The other class consists primarily of polycyclic aromatic hydrocarbons of which 3-methylcholanthrene is generally recognized as the prototype (Conney, 1967). The effects of these two types of inducers are similar in some respects in that they lead to increased protein synthesis, as determined by amino acid incorporation both in vivo and 13 in vitro (Kato ~ ~·, 1965;Gelboin, 1964) . The increase in protein synthesis can be blocked by the metabolic inhibitor, ethionine (Conney et al . , 1956), and at the level of translation by puromycin, however, -the inhibition by Actinomycin D indicates that induction by both types of inducers involves DNA-dependent RNA synthesis (Nebert and Gelboin, 1969). There are however, major differences between the two classes of inducers.
Phenobarbital is a much more comprehensive inducer of MFO components than 3-MC. Among the effects produced by phenobarbital are an increase in both the total liver protein and the specific protein content in microsomes (Conney et~. , 1960), increased levels of Cytochrome P450, Cytochrome b 5 , NADPH-Cytochrome P450 Reductase, and numerous Cytochrome P450-dependent reactions (Kuriyama et ~., 1969;Conney, 1967). In addition, a marked proliferation of the smooth endoplasmic reticulum can be observed by electron microscopy (Fouts and Rogers, 1965) which is consistent with the observed increase in microsomal protein and phospholipid content following induction (Orrenius and Ericsson, 1966) .
In contrast to phenobarbital, 3-MC and cogeners induce fewer components of microsomes. There are, however, two major qualitative changes observed in microsomes fol lowing 3-MC treatment . A shift in the wavelength of the reduced CO peak to 448 nm and a substantial increase in the activity of Aryl Hydrocarbon Hydroxylase, have been associated with the de ~ synthesis of a new hemoprotein, Cytochrome P448 (Alvares et~., 1967;Kuntzman et~. , 1969) .
A third class of inducers, the polychlorinated biphenyls, came to interest during the 1970s as a result of their increasing contamination of the environment (Alvares et ~., 1973). These compounds, typified by Aroclor 1254, exhibited potent inductive effects which were consistent with both phenobarbital and 3-MC effects. On further examination, it was concluded that this combined effect was probably due to the complex mixture of isomers in various preparations (Stonard and Greig, 1976).
Although most inducers of MFO usually require multiple administrations with fairly high doses to produce their maximal effect, the extremely toxic compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was found to be an inducer of the 3-MC type and was more potent than any other inducer on a dose-per-unit body weight basis (Poland and Glover, 1974). Lucier et al. (1975) demonstrated that a single oral dose of TCDD at 3 µg/kg to pregnant rats caused marked elevations in both maternal and progeny hepatic MFO parameters. These effects were still present months later.
Although the number of xenobiotics capable of induction is quite large they have in the past been conveniently placed into one of the two classes. It is, however, becoming evident as assay capabilities and separation techniques expand, that many compounds exhibit unique induction properties. The effects of pregnenolone-16-a-carbonitrile (Elshourbagy and Guzelian, 1980) and isosafrole  cannot be definitively categorized as either 3-MC or phenobarbital type of induction.

15
The mechanism(s) by which inducers lead to increased synthesis of MFO components remains somewhat obscure. The documented increase in DNA-dependent RNA synthesis during the induction process has led investigators to propose that inducers play some role as derepressors of regulatory or other genes (Nebert et ~·, 1981). One early theory to account for the ability of such a diverse structural group of compounds to invoke similar effects was proposed by Marshall and McLean ( 1971). These authors suggested that induction was mediated through an "endogenous factor", which was normally inactivated by Cytochrome P450. Compounds capable of binding to P450 would block inactivation, resulting in increased cellular levels of the factor and subsequent induction. This theory is supported by the empirically derived evidence that a very large proportion of inducers are either substrates or inhibitors of P450 capable of producing Type I binding spectra . Identification of a specific "endogenous factor" however, has yet to be accomplished.
The discovery of a hepatic cytosolic "species" in mice, which stereospecifically and reversibly bound TCDD, led to the postulation of a specific receptor for the induction of Aryl Hydrocarbon Hydroxylase (Poland et ~·, 1976). This receptor was shown to bind a variety of halogenated dibenzo-dioxins and furans with different affinity which closely correlated with their potencies as inducers. Furthermore, although the inductive polycyclic aromatic hydrocarbons competed with TCDD for binding, phenobarbital, pregnenolone-16-a-carbonitrile and steroid hormones displayed no specific binding. This cytosolic species, now termed the Ah receptor, is recognized as the major product of regulatory gene ( s) in the murine Ah locus (Nebert et al., 1981). The receptor has also been detected in rats and rabbits (Kahl et al., 1980). Excellent reviews of the research on the Ah locus and -genetic control of induction have recently been published (Nebert et al., 1981(Nebert et al., , 1982. Of great interest has been Nebert's comparison of the Cytochrome P450 and immune systems and his hypothesis that mammalian tissues have the genetic capacity to produce hundreds or thousands of inducible forms of P450 in response to different chemical stimuli.

Developmental Aspects of Monooxygenase Activity
Since the first reports by Jondorf et ~· (1959) and Fouts and Adamson (1959), investigators of the perinatal development of MFO activity have flourished in the literature. Extensive reviews by Short et al. (1976), Neims et al. (1976 and most recently Klinger et al. (1981) have indicated that although variations exist between laboratories with respect to species, sex and substrates utilized, the general patterns of development are similar in most mammalian species. Essentially the monooxygenases are apparently absent or barely detectable in fetal organs, especially in the fetal liver, until just before birth. After birth, in most species, development of specific microsomal components and metabolic pathways generally follow one of three profiles: (1) activity rises rapidly from birth and plateaus at maturity, (2) activities peak shortly after birth, often exceeding adult levels, and subsequently decrease to adult levels, or (3) activity remains low during early postnatal development and rises rapidly coincident with the onset of sexual maturity.
A number of factors have been postulated to explain the deficiency of MFO activity during late fetal and early postnatal life . Wilson (1969) suggested that high levels of somatotropin during development inhibited MFO . Soyka and Long (1972) found that progesterone inhibited MFO in vitro and suggested that this maternally derived inhibitor was responsible for decreased activity in fetal and neonatal animals . Short et al . (1976) and Kuenzig et al. (1975) (Klinger et ~. , 1981) . Furthermore, these authors suggested that qualitative differences in Cytochrome P450, or in the ratio of different P450 subpopulation may be responsible for the many variations observed in developmental profiles .
Induction of monooxygenase activity during perinatal development appears to be highly dependent on both the inducer and species utilized. In laboratory animals with long gestation periods, such as the guinea pig, phenobarbital type inducers are effective in increasing MFO activity in the late gestational fetus (Kuenzig et ~·, 1975).
Species less developed at birth, such as the rat, are generally refractory to phenobarbital induction in utero (Guenther and Mannering, 1977;Cresteil et~., 1979), but rapidly become responsive after birth. Shubert and Netter (1981) have further demonstrated that the onset of inducibility is independent of parturition, but is largely determined by the time passed since conception. In contrast, inducers of Cytochrome P448, such as TCDD and 3-MC, are quite effective transplacental inducers (Guenther and Mannering, 1977;Lucier et~., 1975).
Early studies on the postnatal inducibility of MFO activity indicated that younger animals were more responsive to phenobarbital induction when measured as percent increase of specific activity over controls (Basu et ~·, 1971). Although such studies did not receive a great deal of attention at that point in time, more recently interest has been rekindled with the knowledge of the heterogeneity of Cytochrome P450. Atlas et al. (1977) showed that various P450 (P448) subspecies follow different developmental patterns in rabbit, as well as altered sensitivity to induction. Cresteil and coworkers (1979) reported age dependent changes in the catalytic activity of aniline hydroxylation and nitroanisole demethylation as a function of induction by either phenobarbital or 3-MC. Klinger et al. (1981)  Heterogeniety of Cytochrome P450 Species differences in the rates of metabolism of several narcotics (Axelrod, 1956) and the induction of specific metabolic pathways at the expense of others in a single species (Conney !::!_ al., 1959) led to the early proposal of more than one liver microsomal drug metabolizing system. With the discovery that Cytochrome P450 was the terminal oxidase of the enzyme system (Cooper et al., 1965)  induced microsomes and that in 3-MC treated animals (Sladek and Mannering, 1969). It was generally accepted during this period that phenobarbital caused only a quantitative increase in the form of Cytochrome P450 normally present in uninduced microsomes, however, Grasdalen et ~· (1975) demonstrated that phenobarbital induced microsomes displayed different characteristics from controls and indicated that metyrapone was more selective for the former while SKF 525A was more selective for uninduced form(s). Jonen et al. (1974) also indicated that metyrapone had a greater affinity for phenobarbital induced P450. Thus the early evidence indicated that phenobarbital induction led to qualitative as well as quantitative changes in microsomes. Napthoflavone (7,8-Benzoflavone) strongly inhibits many reactions induced by 3-MC (Burke ~ ~·, 1977), yet stimulates the activity of native and phenobarbital induced Cytochrome P450 (Cinti, 1978). Tetrahydrofuran has recently been shown to be a potent inhibitor of a form of P450 found in uninduced microsomes, but has little or no effect in phenobarbital induced forms of P450 (Hultmark et ~., 1979). An extremely thorough review of the types of mechanisms of P450 inhibitors was recently published (Testa and Jenner, 1981).
Further evidence for the presence of multiple forms of Cytochrome P450 in liver microsomes has been deduced from kinetic studies with various substrates. Although the metabolism of many compounds appears to follow normal Michalis-Menten kinetics, with resulting linear double reciprocal plots, a number of substrates such as aniline (McCoy, 1980), aminopyrine (Kotake, 1981) 7-ethoxycoumarin (Greenlee 21 and Poland, 1978;Boobis et ~·, 1981) and phenacetin (Boobis et al., 1981) have been shown to exhibit biphasic kinetic profiles indicative of multiple enzymes acting on the same substrate (Segel, 1975). Multiple forms have also been implicated (Shiverick and Neims, 1979) for the developmental changes observed in the hydroxylation of testosterone at the 6S, 7a and 16a positions in rats .
The ability to solubilize and resolve microsomal enzyme components (Lu and Coon, 1968), has enabled a finer approach to studying the multiplicity of Cytochrome P450. A number of criteria have been utilized and include determination of molecular weights by SDS-PAGE, differences in spectral characteristics, differences in catalytic activities of purified forms, immunological properties, peptide mapping, and amino acid sequencing (Lu and West, 1980). By assimilating the research from numerous laboratories, the authors have indicated that thus far, depending on treatment, there are five to seven forms of P450 that have been isolated from rabbit, five to six forms from rat, four to six forms from mice, and at least two forms from pigs.
An undetermined number may be present in human liver microsomes.

Pentachlorophenol and Pentachloroanisol
Pentachlorophenol (PCP) and the lower chlorinated phenols, tetraand tri-chlorophenol have been used as fungicides, herbicides, insecticides, and precursors in the synthesis of other pesticides since the early 1930s. The literature on PCP is abundant. A review of the toxicology and occurrence of PCP in the environment up to 1967 was 22 published by Bevenue and Beckman (1967) . More recently, Ahlborg and Thunberg (1980) compiled an extensive review of the literature from 1967 on, including some aspects not covered by the previous authors .
The acute toxicity of PCP has generally been attributed to the uncoupling effect of the compound on oxidative phosphorylation (Weinbach, 1954) . The clinical symptoms associated with acute poisoning, including increased respiratory rate and volume, progressive neuromuscular weakness and increased body temperature are consistent with such a mechanism of action . Of greater concern, however, are the potential chronic effects of chlorinated phenols . The occurrence of PCP in water and the food chain (Ahlborg and Thunberg, 1980) coupled with the detection of this compound in the urine of a diverse population of non-occupationally exposed persons, suggests that PCP is quite ubiquitous in the environment .
Subacute and chronic toxicity studies (Knudsen et ~· ;Schwetz et ~· , 1977) have led to variable and inconsistent results, which have now been attributed to differences in the purity of the PCP preparations utilized (Ahlborg and Thunberg, 1980) . Specifically, the presence of chlorinated dibenzodioxins and dibenzofurans in tecnical grades of PCP, and the documented wide spectrum of toxic manifestations of these agents (Kimbrough, 1972) precludes analysis of specific cause and effect relationships . Goldstein and coworkers (1977) demonstrated that technical grade PCP produced hepatic porphyria and dramatic increases in microsomal drug metabolizing activity in an eight month feeding study in female rats . The qualitative and quantitative 23 nature of these effects were consistent with the effects of a number of chlorinated dioxins. In the same study, purified PCP was shown to be devoid of any hepatic effects with the exception of a moderate increase in glucuronyl transferase activity at the highest dosage level. This study implied that many of the hepatic and extrahepatic effects reported in earlier studies were probably due to contaminants.
Kimbrough and Linder (197 S) reported that purified PCP, in addition to increasing liver size, produced an enlargement of hepatocytes, a slight increase in smooth endoplasmic reticulum and lipid vacuoles .
The cause of these effects, however, is not known .
In contrast to studies on enzyme induction, a number of investigators have examined the potential for PCP and other chlorinated phenols to inhibit MFO activity . Arrhenius et ~·, (1977) showed that in vitro, PCP selectively inhibited the C-oxygenation of dimethylaniline (P450-dependent) thus favoring the N-oxygenation (P450independent). The author concluded that these results were due to either a specific attack on the P450 enzyme or a disturbance in the transfer of electrons to P450 . Carlson (1978) reported inhibition of EPN detoxification and nitroanisole demethylation in vitro by various trichlorophenol isomers . The observed inhibition appeared to be noncompetitive and was not demonstrated in microsomes obtained from treated animals . It thus appears that PCP and cogeners may be similar to various other phenols and alcohols in their interaction with Cytochrome P450 (cf Testa and Jenner, 1981) . Furthermore, a competitive aspect for inhibition may be consistent with the finding that PCP is a substrate for Cytochrome P450 (P448) mediated dechlorination (Ahlborg, 1978) • Pentachloroanisol (PCA), the methyl ether of PCP, has been found in lake sediments and fish tissues (Kuehl et ~· , 1978), in shellfish (Miyazaki, 1981) and in the blood and milk of cows exposed to commercial grades of PCP (Firestone et ~. , 1979) . The occurrence of PCA in the environment has been attributed solely to the degradation of PCP by microorganisms in soil and wood (Kaufman, 1978). Cserjesi and Johnson (1972) reported the capacity of three species of Trichoderma to methylate PCP in liquid cultures . Curtis et ~· (1972) reported elimination of PCA was similar to that for PCP in rodents. They concluded that PCA must be demethylated prior to excretion and that this step was probably rate limiting for clearance .

Miniature Pigs
The development of a strain of genetically small pigs was initiated at the Hormel Institute of the University of Minnisota in 1949 (England, 1954). Since then, other breeds of miniature pigs have been established in the United States and abroad.
The use of the pig in biomedical research received much attention in the 1960s by virtue of its similarities to the human in renal, cardiovascular, and digestive tract anatomy and physiology, dental characteristics, eye structure, and skin morphology . In addition to these traits, the pig is capable of developing many human pathological conditions including atherosclerosis, gastric ulcer and obesity (Pond and Houpt, 1978). The young pig has found greatest utility in studies of nutrition. Since the digestive physiology and nutrient requirements of newborn pigs is remarkably similar to human infants, baby pigs have been used to develop and evaluate some human infant formulas (Book and Bustard, 1974) . Although the pig has received increasing popularity as a laboratory model, housing and handling constraints continue to restrict its use to larger, well equipped facilities .
The use of the swine in drug toxicity studies was advocated by Earl et al . (1964). Investigators of drug metabolism pathways in miniature pigs, however, have been extremely limited. In an attempt to 26 find a suitable "metabolic" replacement for the dwindling supply of rhesus monkeys, Litterst et al . (1976) conducted a comparative study using the miniature pig as one of five species . Based on an arbitrary scale, the authors concluded that the miniature pig was the most comparable species . Freudenthal et al. (1976) further characterized some parameters of the MFO system in miniature pigs ranging in age from two to eight months . They concluded that the two month old pig demonstrated adult levels of activity . Early postnatal development of the MFO system has been reported for the domestic, Duroc pig (Short and David, 1970;Short and Stith, 1973)  Flasks containing cofactors and substrate (or blank) were preincubated for 10 minutes at 37°C to allow temperature equilibration.
The reaction was initiated by the addition of microsomes (approximately 2-3 mg protein) and the mixtures (in a total volume of 2.5 ml) were incubated aerobically, with vigorous shaking in a Dubenof f Metabolic shaking incubator for 10 minutes. The reaction was terminated with 0.5 mls of 50/. TCA and the protein precipitated by centrifugation. A 1.0 ml aliquot of the clear supernatant was transferred to a clean test tube and 0.5 mls of Nash reagent (3.9 M Ammonium acetate; 0.039 M acetylacetone) was added. The resulting colored product was measured in an Abbott Bichromatic Analyzer (ABA-100) using a peak wavelength of 415 nm and sideband wavelength of 450 nm.
An internal calibration factor, previously determined from formaldehyde standards, was utilized and results were obtained directly as nMoles HCHO/ml. Activity was calculated by subtracting substrate blank values from their corresponding sample value, which was then converted to nMoles HCHO/min/mg protein.

Determination of PCP Formation
Proof of the formation of pentachlorophenol (PCP) as a result of the demethylation of PCA was established qualitatively as follows: The PCA demethylation assay (above) was modified slightly such that the final volume was 1.0 ml. Assays were conducted in screw cap culture tubes and contained 0.2 mM PCA and microsomes from phenobarbital treated piglets. The reaction was terminated after 20 minutes by the addition of 1.0 ml 6 N HCl, followed by 5.0 mls hexane. Tubes were vortexed for 1 minute and then centrifuged to separate the aqueous and organic layers. The hexane phase was removed to a clean glass Vial and the extraction was repeated. The combined hexane extracts were evaporated to dryness under a stream of nitrogen at room temperature . Analysis for PCP (and PCA) was performed using a Waters Associates Liquid Chromatograph . The HPLC parameters were as fol lows:

Aniline Hydroxylation
The para-hydroxylation of aniline was determined by measuring the formation of the product, p-aminophenol, according to the method of Imai et al . ( 1965) . Incubation conditions were identical to those for PCA demethylation except semicarbazide was omitted . Aniline-HCl, di s solved in 0 . 1 M Tris-HCl, was added to give a final concentration of 5.0 mM . Blanks received buffer alone . Reactions were terminated after 10 minutes by the addition of 1 . 5 mls 50/o TCA and the protein precipitated by centrifugation . To a 1 . 0 ml aliquot of the clear supernatant, 0 . 5 mls of 10% Na 2 co 3 was added to neutralize the acid . One ml of 2% phenol in 0 . 2 N NaOH was then added to each tube and the color allowed to develop at 37°c for 40 minutes. The resulting colored product was read in an Abbott Bichromatic Analyzer using a 650 nm primary and 550 nm sideband wavelength filter . An internal calibration factor, previously determined from p-aminophenol standards was utilized and 35 results were obtained directly as nMoles pAP/ml . Activity was converted to nMoles pAP/min/mg protein.

p-Nitroanisole Demethylation
The activity of p-Nitroanisole demethylase was determined by a modification of the procedure described by Netter and Seidel ( 1964) in which the production of p-nitrophenol is followed directly in the incubation mixture at 420 nm at pH 7 . 8 . Each individual sample was run in the presence and absence of NADP and the absorbance differences recorded at 5 minute intervals at 36 415 nm and 450 nm. Activity was determined by subtracting the NADP blank value from its corresponding sample value and comparing that value to a p-nitrophenol standard curve generated along with the assay. Activity was converted to nMoles pNP/min/mg protein.
Cytochromes P450 and B 5 The content of Cytochrome P450 in microsomes was determined according to the method of Omura and Sato ( 1964).

Statistical Methods
The statistical analyses for this study were performed using the

In Vitro Metabolism of Pentachloroanisole
Since pentachlorophenol has been detected in biological fluids of fish (Glickman et ~· , 1977) and of mice (Vodicnik et ~·, 1980) treated with pentachloroanisole, PCA was investigated as a substrate for the microsomal mixed-function monooxygenase system . The demethylation reaction, one of the many diverse pathways involved in xenobiotic metabolism, is easily measured since the by-product, formaldehyde, can be conveniently quantitated colorimetrically. In the case of PCA, the quantity of formaldehyde formed should represent the stoichiometric conversion of the substrate to PCP.
In order to establish monooxygenase involvement, cofactor requirements were determined as well as the effects of various MFO inhibitors . The results of these experiments are shown in Table 1  Activity was measured as the rate of formaldehyde production in microsomes from phenobarbital induced minipigs as detailed in Materials and Methods. Activity in the complete system was 5.45 nmoles/min/mg protein .
cThe final concentration of all inhibitors was 5 x l0-5 M.
In order to verify that the formaldehyde produced in the assay was a result of the oxidative demethylation of PCA to PCP, some incubation mixtures were subjected to organic extraction and the residues were analyzed by HPLC . Using the HPLC parameters described in the In conjunction with catalysis of PCA, the interaction of the substrate with cytochrome P450 was investigated using difference spectroscopy. Pentachloroanisole when added to microsomal suspensions produced a typical Type I binding spectrum with a peak absorbance at 385 nm and a trough at 419 nm (Figure 3). Of particular interest, however, is the inability of the compound to produce discernable spectral changes when added to uninduced microsomal preparations.  In all cases, optimal assay conditions were previously determined and the reactions we re within linear ranges with respect to time and protein concentrations used.
The specific activities as a function of age are shown in Table 2. In all cases maximal levels are attained by four weeks of age . Furthermore, the activities in one week old piglets ranged from 65 to 80% of the eight week old activity.  Since a vast number of compounds metabolized by the hepatic mixedfunction monooxygenase system are also capable of inducing the enzyme system, pentachloroanisole was investigated for its induction potential. Furthermore, since pentachlorophenol is the product of microsomal demethylation, this compound was also investigated in order to determine if the parent compound (PCA) or the metabolite (PCP) was responsible for induction. Phenobarbital was utilized in this study as a positive control.
At the dose of 10 mg/kg/ day for four days, PCA and PCP produced no overt signs of toxicity in any of the age groups. Similarly, phenobarbital treated piglets remained alert and active . All piglets, during treatment situations, consumed their normal ration of milk diet and either maintained or gained weight. At the time of the sacrifice, livers from treated pigs were inspected grossly and appeared normal with respect to color and texture. ANOH and NADM activities were increased 5.5-and 9-fold, respectively, over controls. A dramatic 14-fold increase in PCADM was observed.
The observed increases in the different enzyme activities are paralleled by an increase in the concentration of cytochrome P450 in microsomes from treated pigs. Phenobarbital produced a three-fold increase in P450 (Table 5), while PCA and PCP produced much smaller, but significant increases in the concentration of the hemoprotein.
The wavelength of maximum absorbance in all microsomal preparations was 450 nm. The microsomal concentration of cytochrome b 5 and the activity of NADPH-cytochrome-c Reductase are also shown in Table 5.  Phenobarbital produced similar increases in b 5 and the reductase (37% and 32/ 0 , respectively) over controls. It is interesting to note that PCA produced almost identical increases in both parameters and these correlated with the increase in P450 concentration in that treatment group .

Treatment Effects on Hepatocellular Morphology
Since induction of cytochrome P450 and related metabolic activities by phenobarbital has been associated with hepatocellular changes in smooth endoplasmic reticulum content, electron microscopy was used in an attempt to correlate biochemical and morphological effects.
Furthermore, qualitative similarities in the induction profiles of phenobarbital and PCA suggested that the latter compound may be a "phenobarbital type" of inducer. is apparent, with PCA producing small increases in ANOH and NADM activity and its major effect on its own metabolism (Table 6). PCP, as in one week old piglets, appears to increase only NADM activity .
Phenobarbital treatment in four week old piglets results in significant increases in all six parameters measured . ANON, NADM, and PCADM were 4 . 5, 5.4 and 15-fold, respectively, greater than control activities ( Table 6).
The levels of cytochrome P450, b 5 and cytochrome c Reductase were not significantly changed by PCA or PCP, however, phenobarbital again produced a 3-fold increase in P450 and significant, albeit smaller increases in the other two parameters (Table 7).

Biochemical Effects in Eight Week Old Piglets
The results in eight week old piglets were for the most part similar to those in younger animals (Tables 8 & 9), however, the extent of induction by PCA appears to be less than that in one week old piglets. Although PCA produces a significant 35% increase in NADM activity, this is approximately half the increase (67%) observed in the younger animals . Similarly, PCA treatment produced only a 75% increase in its own metabolism (PCADM) as compared to a 3-fold increase in the one week old group . ANOH activity is no longer significantly different from controls . PCP treatment, as with the earlier groups, had no significant effect on ANOH, however, unlike the younger animals the compound did produce a small increase in PCADM .
The effects of phenobarbital compared relatively well with those seen in the two previous age groups . PCADM activity was approximately 13 -fold greater and ANOH 4 . 6 -fold greater than controls (Table 8) .
NADM activity, however, was increased 4 . 7-fold by phenobarbital which is approximately half the 9-fold increase observed in one week old piglets .
As with the four week old piglets, the only significant increases in cytochrome P450, b 5 and Reductase in eight week old piglets were produced by phenobarbital (Table 9) .

Catalytic Activities
The relative ease by which cytochrome P450 can be quantitated in microsomal suspensions imparts the ability to analyze data from  (6) PCA (6) PCP (6) Phenobarbital (  Similar, yet less pronounced alterations in catalytic activity were observed for ANOH (Table 11) and NADM (Table 12) . In both cases, however, only phenobarbital produced any significant effects.

Kinetic Analysis
Changes in various catalytic activity profiles following chemical induction of    analyses were conducted . Based on the assumption that there are multiple forms of cytochrome P450, which may differ in their affinities toward a particular substrate, it should be possible to detect the activities of different forms by kinetic anaylsis providing the affinity differences are large enough.
When aniline concentrations were varied from 5 uM to 5 mM, a biphasic pattern was observed when plotted by the Eadie-Hofstee method ( Figure 10) . Kinetic constants for the two phases were deter- Similar analyses were conducted with microsomes from 1, 4, and 8 week old uninduced piglets . A summary of the kinetic constants are listed in Table 13 . In uninduced microsomes, the high affinity component, designated Form I, remains relatively constant with respect to affinity and maximum velocity over the four ages . In contrast, the low affinity component, Form II, appears to undergo age dependent changes in both parameters.
The developmental profiles of the two forms of ANOH are depicted in Figure   12 .     to 65/o of the total activity . There appears to be a leveling off in both components to eight weeks followed by a further 2.5-fold increase in the activity of Form II by sixteen weeks of age .
The effects of phenobarbital on the two forms of aniline hydroxylase are shown in Table 14. At all ages, phenobarbital has little or no effect on the activity of the high-affinity component, Form I . The low-affinity component, Form II, is increased at all ages . Of particular interest is the inverse relationship between age and the total increase in activity. In one week old piglets, phenobarbital produced a 12.8-fold increase in the activity of the low-affinity component .
By eight weeks of age, the increase is approximately half that value (6 . 4-fold) and by sixteen weeks phenobarbital produces only a threefold increase in this component .
In addition to phenobarbital, the effects of PCA on the two forms of aniline hydroxylase were investigated in four week old piglets . A comparison of the kinetic parameters are shown in Table   15 . As with phenobarbita 1, the high-affinity component was unchanged while the low-affinity component was slightly increased in PCA treated piglets . Furthermore, while phenobarbital does not appear to alter the apparent K m of the low-affinity component, PCA treatment reduced the apparent K by approximately 50/o • m Similar kinetic analyses were conducted using PCA demethylase .
As with aniline hydroxylase, Eadie-Hofstee plots appeared biphasic aKinetic parameters were determined as detailed in Materials and Methods. Experimental data was obtained from duplicate determinations on pooled microsomes from three pigs (1, 4, and 8 weeks) or two pigs (16 weeks).
bPhenobarbital was administered at 10 mg/kg P.O. for four consecutive days.
cV b/V is the ratio of the V for phenobarbital induced pigs to the V for uninduced pigs (Table 13) When the data are arranged to illustrate age related increases in the two components as a result of phenobarbital induction (Table   16), it is interesting to note that while the low-affinity form is increased to a greater extent over bas a 1 levels, the magnitude of this increase remains relatively constant with age . In contrast, the magnitude of the increases in the high-affinity component produced by phenobarbital appears to decrease with age . In this respect, an interesting correlation exists between these data and that seen in Table 10 for catalytic activity of PCA demethylase . In Table 16, there appears to be a greater than SOI. decrease in the magnitude of phenobarbitals effect between one and eight weeks of age (5 . 4-fold vs . 2 . 5-fold) . Similarly, the catalytic activity of PCADM (Table 10) decreases from 2 . 15 to O. 91 between one and eight weeks of age in phenobarbital treated pigs .

DISCUSSION
In support of those studies reporting the detection of pentachlorophenol in biological fluids following treatment with pentachloroanisol e (Glickman et ~· , 1977;Vodicnik et ~·, 1980), the present investigation has shown that PCA is indeed a substrate for microsomal mixed-function oxidation. As evidenced by the requirement for NADPH and the inhibition by carbon monoxide and other documented inhibitors of MFO (Table 1), PCA is metabolized via a cytochrome P450-dependent demethylation reaction resulting in the formation of PCP (Figur~ 1) .
Since PCP has been shown to be metabolized to lower chlorinated compounds such as tetrachlorohydroquinone and trichlorophenol (Ahlborg and Thunberg, 1978), the peak at 2 . 3 minutes in Figure l Whereas it is often assumed that biodegradation of pesticides and other environmental contaminants renders them inactive, PCA represents one biodegraded product that can be reactivated to its parent compound, PCP, in species possessing the proper metabolic capacity .
Furthermore, as indicated by Glickman et al. (1977), PCP exhibits a higher degree of bioaccumulation and is more persistent than PCP in fish. Thus, the compound may present a more difficult assessment with respect to contamination of the food chain .
Initial kinetic analysis of PCA demethylation provided some interesting results . The activity in uninduced microsomes (< 0 . 5 nmoles/min/mg protein) is much lower than that reported in the literature for other 0-demethylated substrates such as nitroanisole in various laboratory species. The possibility of species differences, however, has been discounted since a similarly low rate of PCA metabolism has been observed in uninduced rat liver (Agins et ~· , 1982) . An unusually large increase in activity following phenobarbital induction, in this case greater than ten-fold, suggested the existence of a specific-inducible form of cytochrome P450 that preferentially metabolized PCA. In support of this assumption are the sensitivity of PCA demethylat ion in phenobarbita 1 induced microsomes to metyrapone inhibition (Table 1) and the results from substrate binding spectra ( Figure 3). The inability to detect discernable spectral changes in uninduced microsomes is most likely a function of the low concentration or absence of the PCA specific form of cytochrome P450 . Following phenobarbital induction, a binding spectra becomes readily apparent, which correlates with the increase in demethylase activity . Furthermore, the spectral dissociation constant (K ) of approximately 15 uM s indicates that PCA has a relatively high affinity for the inducible form of cytochrome P450 (Figure 4).
In conjunction with these findings, it is interesting to note that Hultmark and coworkers (1979)  subpopulations.
The finding that PCA was a substrate for cytochrome P450dependent metabolism prompted an investigation of the compound as a potential inducer of the MFO system . Furthermo"re, since it has been reported that younger animals, in many cases, respond to a greater extent than their adult counterparts (Basu ~ ~· , 1971;Klinger et ~. , 1981) the present investigation attempted to focus on age related differen ces in induction.
The choice of the miniature pig for this study was based on a number of factors . The minipig is the largest, non-altricious animal giving multiple births . As such it is well suited for perinatal studies, because unlike the rat and most laboratory species, the neonate is mobile minutes after birth . This imparts the ability to wean and raise piglets with minimal effort and handling . Furthermore, the size of the neonate permits easy, direct oral administration of test compounds, thereby avoiding indirect maternal influences .
Although a large number of studies have been conducted on the postnatal development of the hepatic MFO system in various laboratory 86 species, similar studies in the miniature pig have not appeared in the literature. Short and Davis (1970) reported that in the domestic Duroc pig, MFO activity rises rapidly and in a linear fashion from birth to approximately four weeks of age . From this poiTLt, activity either plateaus or continues to increase at a much reduced rate.
Similar patterns have been reported for rabbits, rats and mice (Gram and Fouts, 1966) . As seen in Tables 3 and 4, · the developmental pattern of MFO in miniature pigs is consistent with those in other laboratory animals . Oxidative metabolism appears to reach maximum at four weeks of age. Furthermore, activities in the one week old piglet range from 65% to 80% of activity in eight week old pigs . Although the present investigation did not include newborns, values of 0.14 nmoles/mg protein for cytochrome P450 and 0 . 22 nmoles/min/mg protein for aniline hydroxylase were obtained from piglets less than 24 hours old in a previous study (Agins, unpublished results Although differing in magnitude, the qua lit at ive nature of PCA' s induction is quite similar to that of phenobarbital. In both cases, the greatest effect was seen in PCADM followed by NADM and then ANOH (Table 4) . Similarly, PCA increased the microsomal concentration of cytochromes P450 and b 5 and the activity of cytochrome c reductase (Table 5) . Interestingly, the increases in the latter two parameters were identical in both phenobarbital and PCA treated pigs. The increase in cytochrome P450 concentration in phenobarbital treated pigs, however, was much greater than in PCA treated animals . In explaining this apparent disparity (i.e . similar effects of the two compounds on some microsomal components and differences in others) an important issue requires clarification.
The increased concentration of microsomal proteins following treatment may be a result of an increased rate of synthesis, a decreased rate of degradation or a combination of both . In this respect, Kuriyama et al. (1969) showed that the net increase in cytochrome c reductase following phenobarbital treatment was a 88 function of both increased synthesis and decreased degradation . In contrast, the increased concentration of cytochrome b 5 seen after treatment was only due to a decrease in the rate of decay of this protein .
The authors additionally showed that after a short lag, cytochrome P450 synthesis followed that of reductase .
Thus, it is 1 ikely that phenobarbita 1 and PCA produce a true induction of reductase and P450 . That differences exist in the magnitude of P450 induction would suggest that phenobarbital is a more efficient or stronger inducer . In view of the proposed heterogeneity of cytochrome P450 populations (Lu and West, 1980), however, the greater effect of phenobarbital may be a result of more than one form of cytochrome P450 induced .
In favor of this assumption, at least two forms of cytochrome P450 have been shown to be induced by phenobarbital in mice (Huang et ~·, 1976), rats (Bornheim andFranklin, 1982) and more closely related to the present study, pigs (Tsuji et ~·, 1980). In the latter study, the authors reported that at least two and possibly three forms of P450 were induced by phenobarbital in adult male pigs .

89
In addition to the biochemical effects produced by PCA and phenobarbital, morphological changes in hepatic ultrastructure were also comparable. While in the one week old control piglet, the major portion of endoplasmic reticulum is associated with ribosomes ( Figures   5 and 6), both PCA and phenobarbital produced a substantial proliferation of smooth endoplasmic reticulum (Figures 7 and 8), consistent with the documented effect of phenobarbital on this organelle (Fouts and Rogers, 1965). On a morphological basis, PCP did produce an increase in the cellular content of smooth ER (Figure 9) . This finding while consistent with a study by Kimbrough and Linder (1975), would not appear to be consistent in view of the correlation between the biochemical and morphological data for PCA and phenobarbital . Cresteil et al. (1980), however, proposed three effects of exogenous chemicals on endoplasmic reticulum: (1) induction of metabolic activities without proliferation of membranes as produced by polycyclic hydrocarbons,  This may be the result of either a decrease in the synthesis of a particular form or a diluting effect of increased synthesis of non-specific forms .
In support of this proposal, Thomas and coworkers (1980) reported that the major phenobarbita 1 inducible form of P450 (P450-b) in rats accounted for 70°/o of the total P450 in immature animals, while only 3Slo in adult microsomes. Furthermore, a minor form of P450 (P450-a) present in uninduced microsomes, was induced by both phenobarbital and 3-MC in immature, but not adult rats.

Since a number of investigations have suggested that biphasic
Michaelis-Menten kinetics are indicative of more than one species of P450 catalyzing the same reaction (Greenlee and Poland, 1978;Boobis ~ ~. , 1981;McCoy, 1980), attempts were made to further investigate the possibility of age dependent changes in the induction of P450 subpopulations utilizing kinetic experiments . Based on the assumption that sufficiently large differences in affinities toward a particular substrate may enable detection of the activities of different P450 forms, aniline concentrations were varied over three orders of magnitude . When plotted by the Eadie-Hofstee method, aniline hydroxylase activity could, in fact, be resolved into two kinetic components ( Figure 10) . While the affinities of the two forms differed by greater than 100-fold, the differences in maximum velocities varied by only three-fold . Furthermore, although the absolute values for the kinetic constants differ slightly, these results are in agreement with those reported by McCoy (1980) for aniline hydroxylase activity in hamster liver microsomes.
By conducting similar kinetic analyses at various ages, a developmental profile was obtained for the two forms of aniline hydroxylase activity ( Figure 12) . While the high-affinity component At this point, it is important to mention that while two aniline hydroxylase activities could be resolved in control and i n duced animals in this study, the possibility exists that the detection of additional kinetic species may be obscured by the predominance of a low-affinity/high-capacity enzyme or similarities in enzyme affinity towards a particular substrate . Thus, the observed activity of the low-affinity component in phenobarbital induced pigs may be the cumulative activities of more than one form of cytochrome P450. This possibility may help to explain the changes in the magnitude of phenobarbital ' s effect as a function of age (Table 14) . As previously discussed, Thomas and coworkers (1980) described a minor form of cytochrome P450 (P450a) which was induced by phenobarbital in immature, but not adult rats . If the minor inducible form in pig liver (P450 A) (Tsuji et~. , 1980), which was more active towards aniline, is under similar temporal control, it could explain the greater effect of phenobarbital on one week versus older animals . Furthermore, the age-dependent induction of a more highly active form of aniline hydroxylase would explain the significant increase in catalytic activity in one week old piglets (Table 11) and the absence of a significant effect in older animals.
In view of the qualitative similarities between PCA and phenobarbital induction, an attempt was made to further characterize the PCA inducible form using similar kinetic analysis. As with phenobarbital, PCA had no effect on the high-affinity component of aniline hydroxylase (Table 15). PCA did however appear to increase the activity of the low-affinity component slightly as well as decrease the apparent K • Based on the earlier suggestion that the low-affinity component m in phenobarbital induced microsomes probably represents more than one form of P450, it is tempting to speculate that the difference in apparent K 's reflects the induction of only one of these forms by PCA. Tween 80 alone, as substrate carrier followed shortly thereafter, it was later learned that acetone produced inhibitory effects similar to ethanol in uninduced microsomes (Hultmark ~ ~·, 1979). By widening the substrate concentration range and avoiding organic solvent vehicles, PCA demethylase activity did in fact display a biphasic nature ( Figure 13). The summary of the kinetic data for the two forms of PCA demethylase as a function of age and treatment (Table 16) provided some interesting correlations with a number of results throughout the entire study.
The close similarity in the apparent K values

98
Whether this form is similar to the "ethanol sensitive" constitutive form in rats (Hultmark ~ ~·, 1979) remains to be determined . It may be worth noting, however, that the kinetic parameters in uninduced adult pigs from earlier experiments (Figure 2) are remarkably similar to those for uninduced adult pigs ' high-affinity component (Table   16) . Thus, the presence of acetone in the early PCA demethylase assay may have inhibited the expression of the low-affinity constitutive form .
In agreement with the age-dependent changes in the degree of phenobarbital's effect on aniline hydroxylase activity, a similar phenomenon was observed for PCA demethylase . The magnitude of phenobarbital ' s effect on the low affinity component of PCADM remains relatively constant with age . However, since the constitutive and phenobarbital ind u cible forms of P450 responsible for the low-affinity activity are probably different, these results may be misleading.
Although uninduced microsomes may contain a small amount of the phenobarbital inducible form of P450 Tsuji et ~. , 1980), kinetic analysis was not able to separate out the contribution of the component in uninduced pigs . Therefore, the magnitude of phenobarbital's effect is most likely underestimated.
For the same reasons above, age-dependent changes in the inducibility of this particular form would not be detectable.
In contrast, the high-affinity component does undergo age related changes in the degree of induction . The increase in activity over basal level in eight week old pigs is approximately 50% of the effect observed in one week old animals . This effect is consistent with that 99 seen in aniline hydroxylase activity where a similar 50' 7o decrease in the inducibility of the low-affinity component was observed between one and eight weeks of age .
In summary, based on the biphasic kinetic characteristics of two substrates, as a function of age and treatment, it would appear that at least four forms of cytochrome P450 exist in hepatic microsomes of miniature pig. They are: (1) the high-affinity component of aniline hydroxylase, present in uninduced and induced microsomes, but not altered by phenobarbital or PCA induction, The mechanism(s) by which temporal alterations are manifested in the induction of specific cytochrome P450 subpopulations are presently not known. However, in view of the intimate relationship between endogenous substances, particularly steroid hormones, and the mixedfunction monooxygenase system, it is quite possible that these agents may play a role in cellular regulation of P450 subtypes .

CONCLUSIONS
(1) Pentachloroanisole is metabolized in vitro to pentachlorophenol by hepatic microsomes from miniature pigs . The requirement for NADPH, inhibition by carbon monoxide, and production of a substrate binding spectrum indicate that the compound is catalyzed via a cytochrome P450-dependent demethylation reaction.
(2) The relatively large increase in the rate of PCA deme thylation following phenobarbital induction strongly suggests that PCA is preferentially catalyzed by a phenobarbital inducible form(s) of cytochrome P450 . The very low levels of activity, co u pled with the inability to detect a substrate binding spectrum in uninduced microsomes suggests that the substrate-specific form of P450 is absent or present in very low concentration in uninduced microsomes . (3) The postnatal developmental pattern of hepatic mixedfunction monooxygenase activity in miniature pigs is similar to that in other laboratory species . In most parameters measured, an increase between one and four weeks of age is followed by a plateau to eight weeks of age . The additional rise in activity for aniline hydroxylase and PCA demethylase between eight and sixteen weeks of age, observed in kinetic experiments, may be associated with the attainment of sexual maturity in miniature pigs .
(4) Pentachloroanisole treatment produced increases in all MFO parameters measured in one week old minipigs . The qua 1 itative nature of PCA ' s induction is similar to that of phenobarbital . The greatest effect, a three-fold increase in its own metabolism, suggests that the compound induces a specific form of cytochrome P450, similar to that induced by phenobarbital .
(5) Pentachlorophenol treatme n t produced increases in some, but not all MFO parameters in one week old piglets. The overall qualitative differences produced by PCA and PCP suggests that the two compounds induce different species of cytochrome P450 which exhibit overlapping substrate specificity in some cases . Kimbrough, R.D. and Lindner, R.E.: The effect of technical and 99/. pure pentachlorophenol on the rat liver; Light microscopy and ultrastructure. Toxicol. Appl. Pharmacol. 33: 131-132, 1975.