Effects of nerve growth factor on cholinergic brain neurons

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on choline Cheryl F. Dreyfus hTerve growth ~a&f~rfNGFj is a dully churacfe~zed molecule, well known for ifs actions in the dj~ferent~at~on and mainfenunce of perjpherul ner~rons. However, recent studies suggest fhaf ifs actions are not ii~~~~~to fke peri~ke~, but may extend to tke CMS. in part~curur, this fropkic agent appears to affect de~e~opmenf and swvioal of a variety of bruiti cell populations. Noteworthy are ifs actions on cholinergic neurons fkal degenerate in Alzheimer’s disease and Nuntington’s chorea. However, studies of NGF receptor sites suggest that effects of NGF may also extend to non-ckolinergic cell groups. Cheryl Dreyfus summarizes these data and points to future work necessary to define further the underl@g mechanisms of action and to examine the ~ln~f~on of NGF on diverse brain popuiaf~onsNerve growth factor (NGF), a fully defined polypeptide, is well known for its action in the differentiation, maintenance and function of sympathetic and sen~~~,~~y~~~f(~S~~~~~~~~! over, recent studies sugg’est that the action of this molecule is not limited to the PNS, but extends to the CNS, where it may play a critical role in the development and survival of neuronal population&*. In particular, effects of NGF on cholinergic neurons of both the basal forebrain (a population that degenerates in Alzheimer’s dementia71 and the striaturn (a population affected in Huntington’s choreas] are being defined. New work has suggested that these actions of NGF may extend to other brain systems as well. The purpose of this review is to summarize these data and indicate the action of this well characterized molecule in the CNS. More comprehensive reviews have appeared elsewhereP6.

The chemical nahwe of NGF

The structure Of bidogically active NGF has been well characterized. The mole&e has been sequenced and consists of two identical chains of 118 amino acids each1c9. This NGF dimer is part of a multisubunit complex termed 7s NGF. 7s NGF contains, in addition to the NGF dimer (called the @subunit), two molecules of acidic protein {the cy-

subunit), and two molecuies Of arginine esteropeptidase (the ysubunit). The m~tisubu~it complex therefore has the composi~on ff~y&t which is stabilized by zinc ions. Studies that have defined the molecular structure and sequence of NGF have resulted in the development of sensitive radioimmunoassays the for protein, and northern blot and nuclease protection assays to determine levels of its mRNA in structures throughout the PNS and CNS. In the brain such studies have linked NGF to the development and mai~ten~ce ~?f populations of cholinergic neurons.

Action of NGF on cholinergic populations of basal forebrain NGF affects a variety of chol-

inergic populations of the forebrain, including those of the nucleus basalis, substantia innominata and striatum@. However, the most compelling evidence that NGF functions in the CNS comes fmm studies suggesting that it may be essential for the development and function of neurons of the basal forebrain. In particular, basal forebrain neurons of the medial septum and the diagonal band of Broca that project to the hippocampus have been examined in detail. These studies have begun to define a physiological role for the trophic factor in viva, and have delineated the actions of exogenous NGF on projecting basal forebrain cholinergic ~pulations. Evidence that NGF may play a physiotogical role in the basal forebrain system comes from a

number of observations. Firstly, both NGF and specific receptors for the factor are present in the mature and developing basal forebrain-hippO~~pa1 system. Moreover, NGF and its receptors develOp in concert with the choiinergic innervation of the hippocampus, suggesting that the trophic agent may foster the establishment of this brain complex. NGFa~di~r~ep~r~b~ forebrain-hippocampus Initial evidence for the presence of NGF in the basal forebrainhippocampal system was noted over ten years ago: lesions of basal forebrain afferents to the hippocampus resulted in sprouting of adrenergic sympathetic neurites into the denervated hippocampus’“-‘2. It ivas postulated that NGF (or an NGF-like substance) was released from the hippocampus, since the adrenergic neurites were known to be responsive to NGF, and since further examination revealed that the sprouting response was blocked by anti-NGF antiseruma. Subsequent studies have supported this view. NGF protein and mRNA have now been detected in the hippocampot~,s““‘4. Mureover, insitu hyb~dizatiOn techniques suggest that granule cells and pyramidal cells may be the source of the trophic factor”. How does NGF, synthesized in the hippocampus, interact with projecting basal forebrain neurons? Present data indicate that hippocampal NGF may bind to specific NGF receptors on basal forebrain terminals in the hippocampus. Ligand binding studieP g and studies Of NGF receptors using a monoclonal antibody to the receptor4d6*19 and NGF receptor mRNA6~0 have suggested that NGF receptors exist and are synthesized in the basal forebrain system. Moreover, these receptors resemble those of the periphery es of low and exist as two sub and high affinityb,‘6*’ P. While the function of low affinity binding is unclear, biological actions of NGF are associated almost exclusively with high affinity binding”s. Thus, it is of great interest that autoradiographic simultaneous analysis of NGF binding and identifiimrnuno~t~herni~~ cation of cholinergic neurons have determined that high affinity NGF

TiPS - April 1989 [Vol. 101 and its receptor coincides with the innervation of the hippocampus by the basal forebrain cholinergic axons20,2s25, suggesting that NGF produced by the target hippocampus may interact with NGF receptors on basal forebrain axons to influence the ingrowth and establishment of basal forebrainhippocampal connections. In summary, a number of observations now suggest that NGF, produced in the hippocampus, binds to specific receptors on basai forebrain cells, is retrogradely transported to basal forebrain cell bodies and may regulate the function of projecting basal forebrain cholinergic neurons. What might these functions be? To address this question a variety of studies have begun to define effects of exogenous NGF on the basal forebrain.

FQ. 1. Sfmuftaneousmdioautographic vfsua/izationofkighafMyNGFbindingsites and mullochemica tocakation of choline acetyltransferase in cho!!nergic neurons of %sociated cultures of embryonic day 17 rat basal forebrain. A subpopulation of cimlinergic neumns exhibits high affinity NGF binding sites (arrows). Cholinergic neurons were idenMed using a monoctonat antibody directed against choline acetyltmnsfarase (provided by Sakatierra and Crawfordj. To identify high affinity NGFbindiirg sites, cuttun?swere incubated with ‘-?-labelred NGF(0.2nM) for 1 h at37”C, then washed in non-radioactive NGF (0.2~. 4”c, 3Om:o) to dissociatelow arXnity binding sites.

binding sites are associated with the choline@ population in the basal forebrain (Fig. 1)21. In this study cholinergic neurons were identified using a monoclonal antibody generated against choline acetyltransferase (CAT), the acetyl-holine-synthesizing enzyme. High affinity sites were distinguished using techniques exploiting the dissimilar dissociation constants for high and low affinity sites. It is likely, therefore, that NGF exerts its effects (see below) on cholinergic populations directly, by binding to high affinity receptors. In the PNS, NGF, produced in target areas, binds to specific receptors and undergoes selective retrograde transport from terminals to cell bodies’*2. In the basal forebrain system NGF appears to act similarly. Thus, 12Ylabeled NGF injected into the hippocampus is transported from basal forebrain terminals to cell bodi#. Moreover, while relatively high levels of NGF and NGF mRNA are detected in the hippo-

campus, relatively high levels of the protein, but little of the mRNA, are found in the basal forebraini3,‘*. NGF synthesis in the hippocampal region is apparently responsible for the protein found in the basal forebrain cell bodies. In aggregate, these data suggest that NGF specifically binds to high affinity receptors and is retrogradely transported to cell bodies of projecting basal forebrain neurons, the presumed site of action. NGF and the developing system Related studies of the developing basal forebrain-hippocampal complex further support the concept that NGF may play a physiological role in this system. The postnatal development period is distinguished by dramatic increases in hippocampal-associated NGF protein and NGF mRNA. In addition, basal forebrain-associated NGF receptor and NGF receptor mRNA levels are increased. Interestingly, this maturation of NGF

Actions of exogenous NGF Both culture studies26-2s and experiments in vivo29*30 indicate that exogenous NGF elicits increases in a host of cholinergic traits in fetaPG2*, neonata129”0 and adulta9s30 tissues. Most notably, administration of NGF results in an elevation in activity of choline acetyltransferase (CAT), the acetylcholine synthesizing enzyme4-6*2G30. Moreover, in explant culture, this increase in CAT activity is associated with a marked enhancement of CAT-staining intensity2’. Consequently, increases in CAT activity may be associated with an increase in enzyme protein. Further, exposure to the NGF results in an elevation of another cholinergic trait, the catabolic enzyme acetylcholinesterase2’, co-localized with CAT in basal forebrain cells. Multiple traits associated with the same neuron appear to be equally affected by NGF. Related work suggests that the action of NGF may extend beyond effects on the cholinergic phenotype to other neuronal traits. Its application to dissociated basal forebrain cultures results in increased fiber outgrowth from acetylcholinesterase-positive cells2’. These actions on neuronal morphology, as well as on cholinergic function, suggest that the trophic agent may provide general stimulatory signals influencing the cholinergic cell as a whole. It should be emphasized that these actions of NGF also occur in the mature

TiPS - April 2989 CVol. 201 brain. NGF, therefore, may act to affect basal forebrain function throughout life. Additional studies are defining the effect of NGF on cell survival in the brain. In the peripheral system NGF has dramatic effects on the survival of developing sympathetic and sensory neurons’“: in the absence of the growth factor neurons die. In the brain, as noted above, NGF appears to act preferentially on two cholinergic populations which degenerate in Alzheimer’s or Huntington’s disease. Therefore, a number of studies have explored the possibility that lack of this factor may result in cell death and that the presence of the factor may enhance cell survival. Thus far, however, information indicating that NGF may play a critical role in cell survival, although promising, is not complete. In studies parallel to those performed on peripheral neurons, developing basal forebrain cells have been exposed to anti-NGF antibodies in aivo and in culture, to deplete these neurons of the growth factor, and the effects on the numbers of cholinergic neurons have been examined. Injection of the antibody into the developing brain, however, has not produced consistent resultsz9. It is possible that the injected antibodies cannot penetrate to the most critically effective sites of action. Alternatively, the brain system may depend on a number of growth factors for survival. Effects may be evident only in a sparse system, depleted of additional environmental signals. This.hypothesis is supported by the recent report that ’ NGF enhances survival of cholinergic cells in a sparse culture environment in which many of the supportive factors produced by glial cells or other neuronal populations are deficient”. In the adult, effects of NGF on cholinergic cell survival are more dramatic: in the rat brain subjected to the ~ansection of the ascending cholinergic projection to the hippocampus, NGF prevents a loss of acetylcholinesteraseand CATpositive neurons in the medial septum and vertical limb of tit,: diagonal band of Broca31,32.These lesions are associated with learning deficits33. However, chronic infusion of NGF reduces both the cholinergrc cell loss and the learn-

147 ing deficits. The data indicate that NGF may enhance adult cell survival, as well as affect brain function, in the critical basal forebrain cell group. Emerging view of NGF actions on projectingneurons A growing body of literature now suggests that NGF may influence the development and mature function of projecting cholinergic neurons of the basal forebrain. The emerging view of how this may be effected is illustrated in Fig. 2. NGF, synthesized in the target hippocampus, binds to and is incorporated into cholinergic terminals of the basal forebrain. It appears to interact with high affinity receptors on these terminals and is retrogradely transported to cell bodies. NGF interacts with basal forebrain cholinergic neurons to regulate function, development and survival. This model should, however, be viewed with caution. The story of the action of NGF on the basal forebrain system is by no means complete. For example, signals regulating the synthesis and release of NGF in the hippocampus are unknown. Similarly, cellular mechanisms underlying the action of NGF on basal forebrain cholinergic function are undefined. Furthermore, although existing data most strongly support the concept that target regions may provide trophic factors essential for the normal function of projecting cells, it is also possible that the local environment may produce critical signals as well. Current study is directed to further definition of these issues.

tnfiu-tce of NGF outside basal forebrain Recent work indicates that the action of NGF may not be limited to basal forebrain cholinergic neurons that project to the hippocampus; it may, in fact, extend to diverse populations of both cholinergic and non-cholinergic neurons. Furthermore, actions of NGF may vary at critical developmental periods. The subsequent sections summarize these observations and suggest that the trophic influences of NGF may be more extensive than previously believed. Sfrii&??l The most convincing evidence that effects of NGF extend not only to projecting, but also to other neuronal populations comes from studies of its action on striatal cholinergic neurons. These cells are intemeurons that lie entirely within the striatum, and differ from basal forebrain neurons that project to distant target sites. Nevertheless, administration of NGF to striatal cells in uivcr30or in culture34 results in a dramatic increase in CAT activity. A variety of data suggest that the NGF effect may be a physiological one. NGF and its mRNA4e5,’ ,‘a have been localized to the striatum, suggesting that it is synthesized there. In addition, NGF binding sites have been localized to the striatal region in the adult anima116*17.At least a portion of these receptors are of thIz high affinity binding subFurthermore, morpho:::a; studies indicate that NGF binding is associated with acetylcholinesterase-positivecek~~‘, suggesting that, as in the basal fore-

Basal Forebrain

Fig, 2. The proposed interaction neurons of the basal forebrain.

behveen

PIGF, produced in the hippocampus.

and

TiPS - April 1989 [Vol. 201

1-H brain, the high affinity NGF receptors may be associated with cholinergic neurons. Thus, both non-projecting and projecting cholinergic populations appear to rqond to NGF via high affinity binding sites; NGF and high affinity NGF receptors exist in both systems; both systems respond to application of exogenous trophic factor. Although the basal forebrain and striatum respond to NGF, marked differences in their responsiveness have been noted with maturity. NGF appears to affect cholinergic function in the basal forebrain throughout life. By contrast, studies on cholinergic cells of the striatum indicate that these neurons lose responsiveness to NGF with increased development. fnjections of NGF that increase CAT activity in the striatum of rats of postnatal day 1 are ineffective in regulating CAT levels in postnatal day 22 --:m*@). These differences are PII.II,UI” unrelated to neurotransmitter phenotype, and may be associated with distinctive characteristics of the physiology and environment of the two brain populations. Therefore, the actions of NGF described for projecting cholinergic neurons appear not to apply to all NGF-~s~nsive cells in the CNS, and further study is necessary to define the actions of NGF on diverse cell groups. NGF receptors on zzon-cholinergic populafiozzs The suggestions that NGF may act on non-cholinergic cell populations are based on binding and immunocytochemical studies which have detected the receptor on widespread, non-cholinergic populations of the adult and developing brain. ~thou~ the presence of receptors does not necessarily imply function3s, and further study is necessary to determine whether NGF has an actiorl on these cell groups, it should be noted that nerve growth factor binding sites are evident on the cochlear nuclei, the prepositus hypoglossal nucleus, the suprageniculate nuclei and the dorsal part of the lateral lemniscus in the adult4*4,*6J7_Moreover. in developing animals high levels of NGF receptors have been described in the olfactory bulbt5 and the cerebellum25~36. Interestingly, the

high levels of NGF receptors in the cerebellum are observed only transiently, indicating that the role of NGF in this region may be short lived and directed towards a specific function during a narrowly defined developmental period. It should be noted, also, that NGF receptors are not limited to neuronal populations. Glial cells36 and meningeal cells= also appear to have receptor sites. This diversity in cell populations displaying NGF binding sites suggests that the actions of NGF in the mature and developing brain are more extensive than previously believed. However, as mentioned above, further studies are essential to determine whether these binding sites are actually functional, as well as to identify the responsive populations, and to begin to evaluate the importance of NGF to the physiology of the diverse cell groups. !I!

cl

q

The action of NGF is best defined in the basal forebrain cholinergic system, where it may regulate function and cell survival. This brain region degenerates in Alzheimer’s dementia and effects of NGF on this system have raised the possibility that NGF may be a useful therapeutic agent in approaching this degenerative disease3’. However, caution is advised. Thus far, no evidence exists that NGF-related deficiencies are a cause of this disease3s. Moreover, recent binding studies raise the possibility that the actions of NGF are not limited to the basal forebrain system, but may also extend to other cholinergic and noncholinergic cell groups. Since both neurons and non-neuronal popuations exhibit binding, the function of NGF on diverse ceil groups may vary. Furthermore, receptor studies indicate that NGF may selectively affect distinct brain populations at critical stages during development and maturity. Because application of exogenous NGF may affect brain populations other than those of the basal forebrain, future experiments are necessary to pursue this work and further identify the mechanisms by which NGF may regulate brain function. Only then will we fully

appreciate its role during fetal development, neonatal life, adulthood and senescence.

Acknowledgements The work described was supported in part by NIH grants NS 20788, NS 10259, HD 23315, DA 05132, and a grant from the Alzheimer’s Disease and Related Disorders Association, Inc. I thank MS Mary F. Ginsburg, Dr Paulette Bemd, MS Midori Yokoyama, Mr Edward K. O’Malley, Dr Stella Elkabes, Dr Joshua Adler, Dr Emanuel DiCicco-Bloom and Dr Ira B. Black for valuable discussion and critical review of this paper.

References 1 Greene. L. A. andShooter. E. M. (1980) Annu. I&u. Nercmsci. 3, 355-402 . .

2 Thoenen, H. and Barde, Y-A. (1980) PhysioJ. Rev. 60, 1284-1335 3 Levi-Montalcini, R. (1987) EMBOI. 6, 1145-1157

4 Whittemore, S. R. and Se&r, A. (1987) Brain Res. Rev. 12,439-464 5 Thoenen. H.. Bandtlow. C. and Heumann, R. (1987) Rev. h~siol. Biothem. PhannaceJ. lB9,146-178 6 Springer, J. E. (1988) Exp. Neural. 102, 354-365 7 Rossor, M. N., Iversen, L. L., Reynolds, G. I’., Mountjoy, C. Q. and Roth, M. (1984) Br. Med. j. 288,961~964 8 ConnealIy, P. M. (1984) Am. j. Hum. &net. 36,506-526 9 Hague-AngeIetti, R. and Bradshaw, R. A. (1971) Pror. Nafi Acad. Sci. USA 68, 2417-2420 10 Loy, R. and Moore, R. Y. (1977) Exp. Neural. 57,645-650 11 Crutcher, K. A., Brothers, I.. and Davis, J. N. (1979) Exp. Nertrof. 66, 778-783 12 Stenevi, U. and Bjorkhmd, A. (1978) Neurosci. Lett. 7, 219-224 13 Korsching, S., Auberger, G., Heumann, R., Scott, J. and Thoenen, H. (1985) EMBO 1.4,1389-1393 14 Shelton, D. L. and Reichardt, L. F. (1986) Proc. Nat1 Acud. Sci. USA 83.27%27l8 15 Ayer-LeLievre, C., Olson, L., Ebendal, T., Seiger, A. and Persson, H. (1988) Scienre‘i40,1339-1341 16 Richardson. I’. M.. Veree fssa. V. M. K. and RioReIIe, R. J: (198%)f. N&us& 6, 2312-2321 17 Raivich, G. and Kreutzberg, G. W. (1987) Nerrruscieuce20. 23-36 18 Ben-&P., Martinez, I-I. J., Dreyfus, C. F. and Black, I. B. (1988) Neuroscience 26, 121-129 19 Hefti. F., Hartikka, J., Salvatierra, A., Weiner, W. J. and Mash, D. C. (1986) Netcrosci. Left. 69. 37-41 20 Buck, C. R., Martinez, H. J., Black, I. B. and Chao, M. V. (1987) Proc. Nati Acnd. Scz’.USA 84.3060-3063 21 Dreyfus, C.-F., Bemd, I’., Martinez, H., Rubin, S. J. and Btack, 1. B. Exp. Neural. (in press) 22 Schwab, M. E., Otten, U., Agid, Y. and

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149

Thoenen, H. (1979) Brain RP~. 473-483 23 Large, T. H. et al. (1986) Science

168, 234,

352-355

24 Auberger, G., Heumann, R., Hellwig, R., Korsching, S. and Thoenen, F. (1987) Dev. Biol. 120, 322-328 25 Yan, Q. and Johnson, E. M., Jr (1988) J. Neurosci. 8, 3481-3498 26 Honegger, P. and Lenoir, D. (1982) Dev. Brain Res. 3, 229-238 27 Martinez, H. J., Dreyfus, C. F., Jonakait, G. M. and Black, 1. 8. (1987) Brain Res. 412,295-301

28 Hartikka,

J. and Hefti, F. (1988) J. Neuro-

sci. 8,2967-2985 29 Gnahn, H., Hefti, F., Heumann, R., Schwab, M. E. and Thoenen, H. (1983) Dev. Brain Res. 9, 45-52 30 Johnston, M. V., Rutkowski, J. L.,

31

32 33 34

Wainer, 6. H., Long, J. 8. and Mobley, W. C. (1987) Neurochem. Res. 12,985-994 Kromer, L. F. (1987) Science 235,214-216 Williams, L. R. et al. (1986) Proc. Nat! Acad. Sci. USA 83,9231-9235 Stein, D. G. and Will, B. E. (1983) Brain Res. 261,127-131 Martinez, H. J., Dreyfus, C. F., Jonakait,

Regulation of sexual differentiation in drug and steroid metabolism Peter G. Zaphiropc@x, Agneta Mode, Gunnar Norstedt and Jan-Ake Gustafsson Certain members of the cytochrome P-450 family are expressed at different levels in the livers of male and female rats. Although little is known of the functional significance of these sex differences, progress has been made towards the understanding of the endqcrine control of hepatic sex differences in cytochrome P-450 levels. Jan-Ake Gustafsson and colleagues describe a subpopulation of hepatic sexually differentiated P-450.9 that is regulated by sex differences.in growth hormone (GH) secretory pattern. This secretory pattern is in turn regulated by gonadal steroids. These studies demonstrate a novel action of GH and suggest that the hormonal secretory rhythm is pivotal in determination of biological effects. During the last few decades it has become evident that the liver is a sexually differentiated tissue. This which has been phenomenon, studied most extensively in the rat, is evident in several liver functions and can be measured as sex differences in the level of a variety of hepatic proteins ranging from hormone receptors and secretory proteins to enzymes (Table I; for reviews see Refs 1 and 2). Among the enzymes exhibiting sexually dimorphic levels are members of the cytochrome P-450 family which catalyse the oxidation of a vast array of drugs, xenobiotics and steroids3. The sex-specific expression of individual forms of P-450 cytochromes results in sex differences in the metabolism of

P. G. Zaphiropoulos Mode

is Lecturer,

is Assistant Professor, A. G. Norstedt

is Associate

Professor, and \-A. Gustafsson is Professor nnd Chairman of the Department of Medical Nutrition, Karolinska Institufe. Huddinge University Hospital F69, S-141 86 Huddinge, Sweden.

many compounds. However, the unambiguous assignment of specific metabolic reactions to individual P-450s on the basis of catalytic activity alone is difficult. The recent development of specific antibodies to individual forms of P-450 has led to a clearer picture of their sexually differentiated forms. Most P-450-catalysed reactions are more efficient in male than in female rat liver. For example, P-450 isozymes predominantly present in maie livers are P-450 pcn2, P-450 g (br1.h enzymes ;~~:s?s.sin~ steroid bfi-hydrn~~:~,lase ;nc61=ity) anr- P-430 16~~(active in 16c+hydroxyla!ion :>f. for example, testosterone). A r.otable exception is P-450 15fi (active on steroid sulfates) which is not present in the adult male but accounts for up to 40% of the P-450 in the female. The developmentally regulated cytochrome P-450 f is also present at higher levels in females than in males but the difference is not more than twofold4.

G. M. and Black, I. B. (1985) Proc. Nafl Acnd. Sci. USA 82,7777-7781

35 Yan, Q.. Snider, W. D., Pinzone, J. J. and Johnson, E. M., Jr (1988) Neuron !, 335-343

36 Schatteman, C. C., Gibbs, L., Lanahan, A. A., Claude, I’. and Bothwell, M. (1988) /. Neurosci. 8.860-873 37 Au! hC Working Group on Nerve Growth Factor and Alzheimer’s Disease (1989) Science 243,ll 38 Coedert. M., Fine, A., Hunt, S. P. and Ullrich, A. (1986) Mol. Bruiz Res. 1, 85-92

The physiological significance of sex differences in the liver is largely unknown but one may speculate that these differences are important in maintaining appropriate endocrine balance and perhaps in handling metabolic demands which may be different during certain conditions (e.g. during growth, pregnancy and lactation). The pharmacological and toxicological consequences of sex differences in the liver are not clear but it is known that several diseases occur in a sex-dependent manner and that the metabolism of many carcinogens and mutagens is sexually differentiated. A significant role for a sex-dependent liver metabolism in the development of diseases, particularly those related to dietary and environmental toxins, is therefore highly likely. Though the functional significance of hepatic sexual dimorphism is an open question, knowledge is accumulating regarding the endocrine factors responsible for sex differences in the liver. The pronounced (more than tenfold) sex differences of the male-specific P-450 1611and the female-specific P-450 158 have turned these isozymes into valuable markers in regulatory studies of hepatic sexual differentiation5T6. This line of research has revealed a novel endocrine system where gonadal steroids indirectly regulate sexspecific functions of the liver via the pituitary gland. Influence of gonadal steroids Sex differences in hepatic steroid metabolism are developmentally regulated and become manifest in adult animals7. This sexual differentiation is, however, predetermined during the neonatal period when testicular androgens imprint the ability to express a male pattern of metab-

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