ontogenic development of steroid 16α-hydroxylase as a tool for the study of the multiplicity of...

Eur. J. Biochem. 120, 213-220 (1981) Q FEBS 1981

Ontogenic Development of Steroid 16a-Hydroxylase as a Tool for the Study of the Multiplicity of Cytochrome P-450

Franqoise PASLEAU, Claudine KOLODZICI, Pierre KREMERS, and Jacques E. GIELEN

Laboratoire de Chimie Medicale, Institut de Pathologie, Universiti. de Liege

(Received June 23/August 10, 1981)

1. Activities of progesterone, testosterone, pregnenolone and dehydroepiandrosterone 16a-hydroxylase are un- detectable in the fetal rat liver. During the neonatal period, the four enzymic activities increase in parallel to the concentration of cytochrome P-450. Until puberty, they develop similarly in male and female rat livers. From the 40th to the 55th day, the four steroid 16a-hydroxylase activities increase rapidly i n the male rat liver. The sexual differentiation of the steroid I6a-hydroxylation observed in adult male and female rats takes place around the 55th day.

2. In the adult rat liver, steroid 16a-hydroxylase is supported by two forms of cytochrome P-450 (form I and form II), which differ in their relative affinities for the various steroid substrates, and by their relative proportions in male and female rat livers. These two forms of cytochrome P-450 are also present in the young male and female rat livers, but are roughly equal in proportion. The transition from the immature to the adult repartition of the two forms occurs during puberty and is correlated with the sexual differentiation of the steroid lba-hydroxylase activities.

3. During the critical phases of the rat ontogenic development, the in v i m interactions between benzo[u]- pyrene and steroids were compared at the level of two rat liver monooxygenases: steroid 16a-hydroxylase and aryl hydrocarbon hydroxylase. (a) In the immature male and female rat livers, progesterone 16a-hydroxylase, and to a lesser extent, pregnenolone 16a-hydroxylase are inhibited by benzo[a]pyrene. Progesterone 16a- hydroxylase is also inhibited by metyrapone. (b) In the young rat, aryl hydrocarbon hydroxylase cannot be inhibited by steroids and appears to be supported by a single form of cytochrome P-450. The transition from the immature to the adult situation occurs around the 40th day.

Sexual differences in the metabolism of exogenous compounds (ethylmorphine [I], aminopyrine [2], diazepam [3]) and endogenous (glutathione [4], histidine [5], steroid hormones [6 - 81) have been demonstrated in different species [I, 2,7] for quite some time. In general, sexual differences developed during a post-pubertal period and cannot be observed in young animals. The low capacity of newborn animals to metabolize most lipophilic compounds is well known [9, 101. However, Gustafsson has demonstrated the physiological importance of testosterone during the early post-natal period in determining the evolution of steroid metabolism in the adult rat liver [6,11,12]. Similar ‘imprinting by androgens’ has also been described for drug-metabolizing enzymes [13,14].

In the particular case of the rat steroid I6a-hydroxylases [I 51, we previously suggested that sexual differentiation could _ _ _ ~

Enzymes. Dehydroepiandrosterone I6a-hydroxylase, pregnenolone 16r-hydroxylase, progesterone lha-hydroxylase, testosterone Iha-hydroxylase, aryl hydrocarbon hydroxylase, ethylmorphine N-demethylase, benzphetamine hydroxylase, hexobarbital hydroxylase: flavoprotein- linked rnonooxygenase (EC 1.14.34.1); NaDPH-cytochrome reductase (EC 1.6.2.4).

Triviai Names. Dchydrocpiaudrosterone, 38-hydroxy-5-androsten-I 7- one; hexobarbital, 5-(1 -cyclohexen-I -yl)-I ,5 dimethyl-2,4,6-( 1 H, 3H, 5H)- pyrimidinetrione; methylcholanthrene, 1,2-dihydro-3-methyl-benz[il- aceanthrylene; metyrapone, 2-methyl-1,2-di-3-pyridyl-l-propanone; a-naphthoflavone, 7,8-benzoflavone; pregnenolone, 38-hydroxy-5-pregnen-20-one; progesterone, 4-pregnen-3,20-dione; testosterone, 178-hydroxy-4-androsten-3-one ; tetrahydrofuran, diethylene oxide.

be correlated with the existence of two different forms of cytochrome P-450 (form I and form 11) in the adult liver. These forms present different stereospecificities towards different steroid substrates [lb], and appear to be spccifically involved in endogenous hormone metabolism ; indeed, the steroid 16a-hydroxylase activities are not inhibited by xenobiotics such as metyrapone, x-naphthoflavone, benzo[a]- pyrene, hcxobarbital or 7-ethoxycoumarin [I 5,171.

It was thus quite interesting to investigate the quantitative and qualitative properties of the steroid-hormone-metabolizing system during the period of ontogenic development of the animal, and to compare them to those of the adult monooxygenase system. In the present publication, we demonstrate a 10-fold physiological induction of the male rat liver steroid 16a-hydroxylase activities during puberty. Steroid 16~-hydroxylase heterogeneity (form I and form 11) is already present in the immature male and female rats. Sexual differentiation appears to be associated with a modification of both the qualitative properties of the cytochrome P-450 forms responsible for the 16~-hydroxylase activity, and the quantitative distribution of form I and form I1 in the adult male and female rat liver.

MATERIALS AND METHODS

[ 1 6w3H] Progesterone (26 Ci/mrnol), [ 1 6ce3 HI pregnenolone (26 Ci/mmol) and [G-3H]benzo[a]pyrene (0.5- 1 Ci/ mmol) were provided by the Institut National des Radio-

elements (1.R.E). [I 6a-3H]Testosterone and [IG~-~H]dehydroepiandrosterone were synthesized as previously described [I 81.

200

Anrmalr

Sprague-Dawley (IFA-CREDO, Centre des Oncins, Lyon, France) rats werc used. Two females were caged with one male for three days. Pregnant aniinals were then maintained in individual cages placcd in an air-conditioned room at 21 i 1 <C, under controlled illumination (the light period extended from 6 h to 18 h), and were fed rat food pellets ad lihizum (UAR-A03 Villemoisson, France).

'r 400 200

Sanzp le Prepara I ions

Rats were killed by decapitation. Liver homogenisation and microsome isolation were performed as described in an earlier publication [18]. The samples were stored at -20 'C. Protein concentrations were determined according to the method of Lowry et al. [19], using bovine serum albumin as the standard. The concentration of microsomal cytochrome P-450 was measured according to Omura and Sato [20].

Enzynzr Assay

The measurement of 16a-hydroxylase activity was performed by incubating specifically 16-tritiated steroids and by counting the tritium released in the medium. These methods have been described in detail elsewhere [I 8,211. The unlabelled compounds used as potential inhibitors were dissolved in acetone and added to the tritiated steroid substrate acetone solution containing Tween 80 (0.5 mg). The solvent was then evaporated under a nitrogen stream. Thc substrate, Tween 80

and inhibitor residue was then dissolved in a I-ml incubation mixture as described earlier [I 8,211.

The aryl hydrocarbon (benzo[a]pyrene) liydroxylase activity was measured by incubating [G-3H]benzo[a]pyrene as described by Van Cantfort et al. [22]. The steroids tested as aryl hydrocarbon hydroxylase inhibitors were introduced with Tween 80 into the incubation medium as already de-' scribed [IS]. All assays were run in triplicate on at least three series of three to fifteen rats. The results are expressed as the mean values and the standard deviation (0) from the mean values.

RESULTS Ont ogenesis of Sir void I6a- Hydrosyluse

Fig. 1 represents the ontogenesis of progesterone, pregnenolone, testosterone and dehydroepiandrosterone 16a-hydroxylase activities measured in v i m in male and female rat liver microsomes. During the last days of fetal life, the cytochrome P-450 content is very low (about 0.13 nmol/mg protein). Steroid 16a-hydroxylase activity could not be detected in male and female rat livers before birth. During the neonatal period (first to third day), the four enzymic activities (Fig. 1) increased rapidly in parallel to the cytochrome P-450 concentration (Table 1 ) . They then stabilized at a level which corresponded approximately to a mean enzymic activity of 200, 40, 75 and 400 pmol x min-' x mg protein-' for progesterone, pregnenolone, testosterone and dehydroepiandrosterone 16a- hydroxylases, respectively. From birth until weaning (21 st day), sexual differences could not be detected. Between the 21 st and 32nd days, hepatic steroid 161-hydroxylase activities decreased progressively in both sexes, and increased rapidly thereafter in male rat liver microsomes to reach the

6oo t A 400 d

t i4 200

Age (days)

Fig. 1. Ontogenic development of progesrrrone ( A ) , pregnenolone ( B ) , tesroslcrone ( C ) and del7~(lr-oc.piai i ( l~(~,~~e~on~~ (0) 16cc-hydroxylases meusured in vitro in male (.--- 0 ) and femalc I'A -A) ral liver micro.wrr7e.y. The enzymic activitics represent the mean values (i S.D.) of the results obtained with series of 3 - 15 rats

T'tble 1 Oniogeilic c,ro/urron of c J lochrome P-450 torltrtil In nzuh aizd flw7~2k~ tar h e r mrc to\omes The cytociiloine P-450 concentintioris 'ire expressed in ninol Y mg protein

Sex Cytochrome P-450 present .it

'

~ ~ -~ -

2 ddys I days 10 days 29 ddyi 40 days 60 ddys

ninoi Y nig protein ~ .. ~

i 0 23 0 015 048 k 0015 071 ri: 0046 0 75 & 0 040 085 f 0025 0 85 f 0 112 0 18 & 0 010 0 33 & 0 030 0 61 0 011 0 6 5 k 0 052 0 56 i- 0 055 0 7 5 * 0 099

adult level around the 55th day. We observed parallel induction kinetics for the four steroid 16a-hydroxylases in the pubescent male rat liver, with ratios of 10:6:4:4 between the adult and immature progesterone, testosterone, pregnenolone and dehydroepiandrosterone 16a-hydroxylase activities respectively. In adult females, the four 16%-hydroxylase activities were similar or slightly lower than those in immature animals. Similarly to steroid 16~-hydroxylase, cytochrome P-450 concentrations increased rapidly aftcr birth, but already attained the adult level in 10-day-old male and female rat livers (Table 1). No further modifications were observed during the subsequent development of the animal, even during critical periods of life such as weaning or puberty.

Multiplicity of' Steroid Ihcc-Hj&o.xylusc in thc Imrnartire Rut

In the second phase, we investigated whether the cytochrome P-450 multiplicity, observed in the adult rats in the case of steroid 16a-hydroxylase [I 5,161, was already present in the young rats, or if it appeared at puberty and was thus correlated to sexual differentiation. In the adult rat liver, two cytochrome P-450 forms (form I and forin 11) were responsible for the steroid hydroxylations at the 1 6 ~ position [16]. Adult 16%-hydroxylase inhibition by unlabelled pregnenolone led us to identify the two cytochrome P-450 forms [16]. Form I was unable to hydroxylate pregnenolone, for which form 11 had the highest affinity. Form I1 was quantitatively dominant in the males; form I was dominant in the females and had the highest affinity for testosterone.

If only one cytochrome P-450 form was responsible for testosterone, progesterone, pregnenolone and dehydroepiandrosterone 1 ba-hydroxylases in the immature rat liver, then each of the four steroid substrates had to inhibit the three other substrate hydroxylation, and the inhibitory power had to be proportional to their relative affinity for the enzyme. To test this hypothesis, each 16-tritiated substrate was incu- bated in the presence of increasing amounts of the other unlabelled potential substrates (Fig. 2 and 3) . We obtained identical results for male and female 10-day-old rats (Fig. 2). The comparison of the inhibition curves presented in Fig.2 seemed complex and suggested the existence of more than one cytochrome P-450 form for the steroid 16~-hydroxylase activity. The hypothesis of a unique cytochrome P-450 that should have the highest affinity for testosterone (such as the adult form I) or for pregnenolone (such as adult form 11) could not be supported in the case of the immature rat liver. Indeed, pregnenolone was a weak inhibitor of the activity of testosterone (Fig. 2A), progesterone (Fig. 2B), and dehydroepiandrosterone (Fig. 2C) Iba-hydroxylases for an inhibitor/ substrate concentration ratio of 5 ([I]/[S] = 5). Testosterone could not inhibit more than 507; of the activity of pro-

I O' i 5 10 1 5 10

[Il/lSl

Fig. 2. Inhihitioti of iestosrerone ( A ) , progi~stewnc~ ( B ) , dchjdrocpion- drosterorie (C) und pregnenolone ( D ) 16cc-lijdroxjlases by ri~.sto.sic~rone (A----A), progesterone iO-~- - -o ) , d~h~di.oc,piandro.stri.onr. i.-- --.) and pregnetiolot7e [@ --a), measured in vitro in 10-day-old mule rui liver microsornes. The ordinate expresses the percentage of the non- inhibited control enzymic activity. The abscissa cxprcsscs the ratio between inhibitor and substrate concentrations ([I]/[S])

gesterone (Fig. 2B) and pregnenolone (Fig. 2 D) 16%-hydroxylases for an inhibitor/substrate concentration ratio of 5 ([I]/[S] = 5); however, progesterone inhibited 70';; of the testosterone 16a-hydroxylase activity (Fig. 2A) with the same value of the [I]/[S] ratio. These conflicting values and the biphasic shape of the inhibition curves led us to surmise that at least two cytochrome P-450 forms were also responsible for the steroid I6a-hydroxylation in the immature rat liver.

We also compared the inhibition of testosterone, progesterone and dehydroepiandrosterone 16a-hydroxylases in v i m by pregnenolone in young and adult rats of both sexes (Fig. 3). In 10-day-old male and female rats, pregnenolone inhibited each of the three 16%-hydroxylase activities with an intensity falling half-way between adult male and female levels; inhibition curves for testosterone and progesterone I6a-hydroxylases stabilized on a plateau for an inhibitor/substrate concentration ratio higher than 1. Form I could be re- presented by the enzymic activity which could not be inhibited by pregnenolone. It thus appeared that, similar to the adult situation, two forms ofcytochrome P-450 (form I and form 11) were also present in the young male and female rat livers. They seemed to be roughly equal in proportion on the basis of the enzymic activities that they supported.

216

I

A

loo=

I c

Fig. 3. Inhibition of teslosrrrotie (A), progesrerone ( B ) and dehydroepiandrosterone (CJ I6m-hydroxylases by pregnenolone, measured in vitro on adult male (0---0), adult female (a ~- -a) and 10-day-old male and female (A-----A) rat liver microsomes. The ordinate expresses the percentage of activity compared to the non-inhibited control enzyme. The abscissa expresses the ratio between inhibitor and substrate concentrations ([I]/[S])

We then attempted to characterize the period of life during which the shift from the immature to the adult pattern of steroid 16a-hydroxylase distribution occurred. We measured the inhibition of testosterone and progesterone 16a- hydroxylases by pregnenolone on 10,21,32,40 and 60-day-old male and female rats (Fig. 4). Until the 32nd day, the inhibition pattern did not change; it was characteristic of young animals and similar in male and female rat livers. Sex differences were observed on the 40th day : testosterone and progesterone 16a-hydroxylases were largely inhibited by pregnenolone in the male rat liver, whereas they became insensitive to pregnenolone in the female rat liver, as was the case with adult animals (60th day) [16].

Inceracsions of Scerord and Exogenous Lipophilic Suhssrate during Ontogenic Developmenr

We investigated the possible competition between steroid and exogenous compounds, such as metyrapone, a-naphthoflavone and benzo[a]pyrene for the various forms of cytochrome P-450 present in young male and female rat livers. We had previously observed the high specificity of the steroid I6a-hydroxylase [I 71, so that endogenous metabolism could be preserved from competition due to various xenobiotics (metyrapone, a-naphthoflavone, benzo[a]pyrene, hexobarbital, 7-ethoxycoumarin) in adult rats. a-Naphthoflavone (Table 2) did not inhibit more than 40% of the activity of testosterone and progesterone 16a-hydroxylases, for an inhibitor/substrate concentration ratio of 2.5 ([I]/[S] = 2.5) in male and female rat livers. These results are similar to those obtained for adult male and female rat livers [15].

Conversely, metyrapone (Table 2) inhibited more than 60 of the progesterone 16a-hydroxylase activity in the young

I A

50

21 0

10

r c 60

Fig. 4. Inhibition of testosterone (A) andprogesrerone (B) 16cr-hydroxylases b.v pregnenolone, measured in vitro on 10, 21, 32, 40 and 60-day-old male (B) and female (0) rat liver micro.somes. The ordinate expresses the percentage of activity compared to the non-inhibited control enzyme

animal of both sexes. This was quite different to the adult situation, for which each of the four 16a-hydroxylases was insensitive to metyrapone, even for inhibitor/substrate concentration ratios higher than 1 [15].

We measured steroid 16~-hydroxylase inhibition by benzo- [alpyrene in vitro, in immature rat liver microsomes (Table 3). Testosterone and dehydroepiandrosterone 16a-hydroxylases were not inhibited by benzo[a]pyrene in immature or pubescent rat livers of both sexes. On the other hand, progesterone 16a-hydroxylase, and to a lesser extent, pregnenolone 16a- hydroxylase, were inhibited by benzo [alpyrene in male and female rat livers, until the 21st day of life with a maximal effect for an inhibitor/substrate concentration ratio of 1 ([I]/ [S] = 1). We then observed a transition period around the 32nd day which led to the adult situation characterized by the lack of inhibition of the steroid 16a-hydroxylase by the exogenous compounds [17]. Conversely, aryl hydrocarbon hydroxylase activity was not inhibited, or was poorly inhibited, by steroids in virro in 10-day-old male or female rat liver microsomes (Table 4). On the 21st and 32nd days, a slight inhibition of aryl hydrocarbon hydroxylase activity by unlabelled steroids was observed in male as well as in female rats. On the 40th day, the results were identical to those already described for adult rats [17], i.e. a significant inhibition of the ary hydrocarbon hydroxylase activity by steroids, more in the male than in the female rat.

DISCUSSION

We followed the evolution of steroid 16a-hydroxylase in the rat liver from birth to adulthood. We did not find significant qualitative differences in the evolution of the en-

21 7

Table 2. Inhihirion of steroid 16a-hydroxylase by a-naphthojlavone or meiyrapone, measured in vitro on 10-day-old male and,female rat livers The results are expressed as a percentage of the corresponding non-inhibited enzymic activity for inhibitor/substrate concentration ratios of 0.5 and 5

Inhibition of activity of

testosterone progesterone dehydro- pregnenolone I6a-hydroxylase 16a-hydroxylase epiandrosterone 1 ha-hydroxylaae

~~ ~ ~- ~ ~- _.~ Inhibitor [Il/[SI

16a-hydroxylase

7"

a-Naphthoflavone

Metyrapone

0.5 2.5

0.5 2.5

- --____ -

69 f 4.7 62 f 3.4

70 k 8.4 63 k 4.4

64 f 4.6 59 f 9.8

48 f 3.0 31 f 3.5

~ -

304 k 0.7 82 f 4.2

98 k 4.9 88 f 2.8

~

92 + 8.3 8 6 f 1 1

94 f 5 8 59 k 3.0

Table 3. Inhibition of testosierone, progesterone, dehydroepiandrosterone and pregnenolone 16a-hydroxylases by benzo[a]pyrene, measured in vitro on 10, 21, 32 and 40-day-old male and female rar livers The results are expressed as a percentage of the corresponding non-inhibited enzymic activity, for an inhibitor/substrate concentration ratio of 5

Sex Inhibition of activity of -

testosterone progesterone 16a-hydroxylase I6a-hydroxylase

dehydroepiandrosterone pregnenolone 16a-hydroxylase 16a-hydroxylase

10

21

32

5 P 5 0 s 3

104 & 13.8 99 f 18.0

37 f 6.0 38 & 7.3

88 _+ 9.8 89 f 10.8

65 _+ 3.1 64 f 5.0

94 f 14.0 29 k 8.8 89 f 5.3 59 k 6.2 92 k 10.8 30 f 6.6 93 f 10.5 61 k 10.5

81 f 6.2 50 f 17.0 86 f 7.5 84 k 7.4 75 k 6.5 37 k 3.2 91 k 13.0 85 f 11.3

40 s 86 f 8.3 92 f 7.8 94 f 14.0 78 k 3.8 ? 79 k 6.8 68 f 9.0 91 f 10.7 93 f 6.8

Table 4. hhibiiion of aryl hydrocarbon hydroxylase hy testosterone, progesrerone, dehydroepiandrosterone and pregnenolone measured in vitro on 10, 21, 32 and 40-day-old male and female rat livers The values express the percentage of activity compared to the non-inhibited control enzyme, for an inhibitor/substrate concentration ratio of 5

Age Sex Inhibition of activity by

testosterone progesterone dehydroepiandrosterone pregnenolone

days % ~ ~ ~ ~ _ _ _ -~ ~. -___ _______ ~

10 s 85 k 11 3 7 8 f 9 9 77 f 8.3 9 5 k 6 2 9 82 f 13.8 84 i 1 0 3 76 k 10.3 9 1 f 9 3

21

32

60 f 11.4 68 f 7.3 64 f 11.0 92 + 17.0 67 f 13.3 75 f 13.4 67 15.0 92 f 14.3

80 f 7.8 69 f 11.5 47 f 7.1 55 & 13.0 67 f 8.8 67 f 11.8 58 + 12.0 88 k 8.4

40 s 46 f 12.4 33 f 9.9 36 i 7.9 53 f 15.0 P 59 f 6.4 52 f 9.6 50 f 9.8 76 f 1.9

zymic activities when (16-3H)-labelled testosterone, progesterone, pregnenolone and dehydroepiandrosterone were used as substrates. By referring to the quantitative evolution of the rates of hydroxylation, we were able to focus on different critical periods of life: first, the enzymic activities develop rapidly at birth and during the early postnatal days; then they stabilize until the 21st day and no sexual difference can be

detected in the immature rats; from weaning onwards, the four 16a-hydroxylase activities decrease slightly but significantly for about one week. The 16a-hydroxylase differentiation finally takes place in the male rat liver from the 30th to the 55th day. However, this increase in enzymic activity could not be correlated to a physiological change in the microsomal cytochrome P-450content which stabilized during

218

10-Day-old d and $! rats.

Form I

0 c

I-"

Adult d

IFITRl P-450 &> Adult Q *

Form II

h' L I U

Steroids

I

Fig. 5 . Tentative expplunarory scheme show3ing the interucrion of /he wrr/ou\ \irh.\trtrre.s nrrh rhe monoo.\~~geizusc's. The following abbreviations are used : Testo, testosterone; Pregn, pregnenolone; BzP, benzo[a]pyrene: MTR. metyrapone; ANF, a-naphthoflavone. Form I, form 11, P-448 and P-450 represent the different cytochrome P-450 species. The large arrow (4) precedes the molecule that is considered as the substrate with the highest affinity for a given cytochrome P-450 form. The double arrows (++) indicate the molecule(s) that could compete with the substratc for the same cytochrome P-450 species (+)

the 2nd week of life (Table 1). This ontogenic evolution could not be compared to the developmental profile observed for different drug monooxygenases : elthylmorphine demethylase [23], aryl hydrocarbon hydroxylase [24], benzphetamine hydroxylase [24], or hexobarbital hydroxylase [25] present a nearly linear increase in activity from birth to the 40th day in parallel to the other constituents of the microsomal oxidative system (cytochrome P-450, cytochrome hS, cytochrome P-450 reductase).

The 16a-hydroxylase differentiation corresponded to the maturation of the reproduction tract and the hypothalamopituitary axis [26]. Nevertheless, it was not possible to demonstrate a direct correlation between the ontogenesis of different hormonal parameters (luteinizing hormone, follicle stiniu- lating hormone, prolactine, testosterone, estrogen) and the evolution of steroid 16%-hydroxylase.

We measured the concentration of these hormones in the plasma of male and female rats in parallel to the steroid 16x-hydroxylase activities (unpublished data) ; our results correlated with those previously published [27 - 301.

Therefore, we compiled a comprehensive review of the literature available on this particular subject. Different authors and particularly Gustafsson and co-workers have studied the sexual difference in hepatic steroid metabolism. They showed that the enzymic differentiation at puberty in the male rat liver is predetermined by a gonadal androgen exposure early in postnatal life (imprinting) [I 1,12,31,32]. In the absence of androgens, the imprinting does not occur and the female phenotype develops at adult age [32- 351. The post-pubertal castration of the male rat has relatively little effect on adult hepatic steroid metabolism.

Similar neonatal imprintings have also been described for the turnover of different cytochrome P-450 species in the rat liver I361 and for enzymes involved in the hepatic metabolism

of exogenous compounds [13,14,37]. The imprinting process does not result from a direct action of androgens at the hepatic level; it rather depends upon an androgen (or estrogen) sensitizing action on the hypothalamopituitary axis [6,13,14,

Kramer and co-workers suggested that growth hormone may be responsible for at least some of the actions of estradiol on hepatic metabolism in male rats [42,43]. On the other hand, they showed that follitropin and lutropin directly affect drug metabolism in gonadectomized rats [44]. These results were of particular interest, as the secretion of both lutropin and follitropin is acutely influenced by testosterone and estradiol [45 -471.

In all cases, the regulation of the hepatic steroid metabolism differentiation was not yet totally clear. It probably reflects a complex endocrine regulation involving the coopera- tion of hypophyseal, gonadal and other endocrine secretion products (excluding thyroid or adrenal glands [39]) which could modulate the phenotype expression of the liver by way of specific hormonal receptors [48].

We then determined that two forms of cytochrome P-450 were already active for steroid 16a-hydroxylase in the immature rat. However, contrary to the adult situation, the two forms seem to be present in similar concentrations in the young rat livers. The transition from the immature to the adult repartition of the two forms occurred during puberty (Fig. 4), just prior to the sexual differentiation of the steroid 1 6a-hydroxylase activities.

On the other hand, the low capacity of newborn animals to metabolize lipophilic compounds is well known [9,10]. The difference between adult and newborn mixed-function oxidase systems was not only quantitative but also qualitative: V, and K, modifications [l , 49 - 51 1, different cytochrome P-450 half- lives and stability [36], alterations in the type-I and type-I1

38-41].

spectral interactions [49,52], different sensitivities to inhibitors (a-naphthoflavone, metyrapone, carbon monooxide, SKF-525 A 125) [52]. In the case of the steroid 16~-hydroxylase, the 'young cytochrome P-450' presented different sensitivities to exogenous inhibitors (metyrapone, bcnzo[a]- pyrene), when compared to the adult forms (Tables 2 and 3). Such age-dependent sensitivity to inhibitors was also described for aryl hydrocarbon hydroxylase activity [53,54].

Fig. 5 represents an attempt to summarize all of the information accurnulated on the evolution of the cytochrome P-450 forms responsible for the steroid hormone metabolism and on the possible competition of endogenous and exogenous substrates for their respective enzymes. Firstly, no qualitativc of quantitative differences could be detected between male and female steroid I6a-hydroxylase activities in immature rats. Two forms of cytochrome P-450 were responsible for the steroid 16~-hydroxylations. Progesterone and pregnenolone 16a-hydroxylases were inhibited by benzo[a]pyrene, whereas steroid (testosterone, dehydroepiandrostcrone) were not. Contrarily to the adult situation, metyrapone significantly inhibited the progesterone 16a-hydroxylase in the young rat livers. Similar results were described for aryl hydrocarbon hydroxylase activity [53]. We also demonstrated that the aryl hydrocarbon hydroxylase activity was not inhibited by steroids, even for high inhibitor concentrations such as those for adult methylcholanthrene-induced rat liver [17]. However, the induced form was inhibited by a-naphthoflavone [I 51, but the young form was not 1531. Secondly, during the transition period covering the 32nd to the 40th day, form I became progressively dominant in the female and form I1 in the male rat livers (Fig. 4). The four 1Ga-hydroxylase activities also became insensitive to exogenous inhibitors (metyrapone, a-naphthoflavone, tetrahydrofuran, benzo[a]pyrene, hexobarbital, 7-ethoxycoumarin) [15,17]. This qualitative differentiation preceded the physiological increase in the steroid 16a-hydroxylase activities in the male rat livers, that developed between the 40th and the 55th day of life. This delayed differentiation of the enzymic activity could be correlated to a non-synchronized evolution of the various com- ponents of the monooxygenase system. The NADPH-cytochrome P-450 reductase activity could be rate-limiting for the oxidation of different drugs [55]. Its stimulation by the substrate was also age-dependent in the case of ethylmorphine N-demethylase [56]. In adult control rat livers, the aryl hydrocarbon hydroxylase activity was apparently supported by two cytochrome P-450 forms. One form is inhibited by steroids and appeared to be present in a higher concentration in the male rat liver [17]. The other form is not inhibited by steroids; it is dominant in the female rat liver. This observation could be correlated to the work of Wiebel et al. [54] who found a preferential inhibition of female rat liver aryl hydrocarbon hydroxylase activity by a-naphthoflavone.

The authors are very grateful to Ms Martine Verbys and to Ms Janice Lynn Delaval for their collaboration in the preparation of this manuscript. This work was financially supported by grant 1072 from the Council for Tobacco Rcsearch, USA, Inc. and by grants 39001.79, 34522.81, 34523.81 and 35424.81 from the Fonds de la Recherche Scieniifque MPdic.de.

REFERENCES I . Van den Berg. A. P., Noordhoek, J., Savenije-Chapel, E. M. &

Koopman-Kool. E. (1978) Biochem. Pharmacol. 27. 627-633. 2. Stegeman, J . J. & Chevion, M . (2980) Biochem. Plzarmacol. 20,

533- 558.

3. Nau, H. & Liddiard, C . (1979) Biochem. Pharmacol. 2Y, 447-449. 4. Chhabra, R. S. CQ Fouts, J . R . (1974) Drug. Mrtab. Di.rp0.c.. 2, 375-

5. 6.

7.

8.

9.

10.

11 .

12.

13. 14.

15.

16.

17.

18.

19.

21.

22.

23.

24.

25.

26. 27. 28. 29.

30. 31. 32. 33.

34. 35.

36.

37.

38.

39.

40.

41.

42. 43.

379. Fiegelson, M. (1973) Enzyme (Basel) 15, 169-197. Gustafsson, J . A,, Eneroth, P., Pousette, A,, Skett, P., Sonnenschein.

C., Stenberg, A. & Ahlen, A. (1977) J . Scc~oid Biochem. 8, 429- 443.

Conney, A. H., Levin, W., Jacobson, M. & Kuntzman, R. (1969) in Microsomrs and Drug Oxidation (Gillette, R., Conney, A. H.. Cosmides, J., Estabrook, W., Fouts, R. & Mannering, J . , eds) pp. 279 - 294, Academic Press, New York.

Lemm, U. & Wenzel, H. (1978) Hoppe Seyler's Z. Pliysiol. ('hem. 359. 81 - 88.

Hart, J. G. & Timbrell, J. A. (1979) Biochem. Pharmucol. 28, 3015- 3017.

Mancini, R. E., Sonawane, R. & Yaffe, S. J. (1980) Res. Commun. Clieni. Puthol. Pharmacol. 27, 603 - 606.

Einarsson, K., Gustafsson, J. A. & Stenberg, A. (1973) J . Bid. Chem. 248,4987-4997.

Gustafsson, J. A. & Ingelman-Sundberg, M. (1974) J . Bid. Cheni.

Chung, L. W. K. (1977) Biwhem. Pliurnzacol. 26, 1979- 1984. Lamartiniere, C. A,, Dieringer, C. S., Kita, E. & Lucier, G . W. (1979)

Kremers, P., Pasleau, Fr. & Gielen, J. E. (1978) Biocliem. Bioplzyx

Pasleau, Fr., Kremers, P. & Gielen, J. E. (1980) J . Steroid Biochem.

Pasleau, Fr., Kremers, P. & Gielen, J. E. (1981) C'hem. Bid . Interuci.

Kremers, P.. De Graeve, J., Azhir, A. & Gielen, J. E. (1978) Eur. J .

Lowry, O., Rosebrough, N., Farr, A. & Randall, R. (1951) J . Biol.

De Graeve, J., Kremers, P. & Gielen, J , E. (1977) Eur. J . Biochcm.

Van Cantfort, J., De Graeve, J. & Gieien, J. E. (1977) Biochem.

Short, C. R. & Stith, R . D. (1973) Bioclwm. P/iurmacol. 22, 1309-

Fouts, J. R. & Deveraux, T. R. (1972) J . Pharniacol. E . y . Tlzer.

Short, C. R. & Davies, L. E. (1970) J . Pharniucol. E ~ Y ~ . Tlier. 174,

Greenstein, B. D. (1978) J . Reprod. Fertil. 52, 419-426. Ojeda, S. R. & Jameson, M. E. (1977) Endocrinology, 100, 881 -889. Ojeda, S. R . & Ramirez, V. D. (1972) Endocrinology, YO, 466-472. Mcijs-Roelofs, H. M. A,, de Grcef, W. J. & Uilenbrock, J. T. J . (1975)

Hoger, W. H. (1977) Endocrinology, 100, 1027- 1032. KrduhS, I. & Clayton, R. B. (1968) J . Bid . Chrm. 243. 3546-3547. Tabei, T. c!L Leroy Heinrichs, W. (1973) Gynecol. Invest. 4, 229-236. Berg, G. A. & Gustafsson, J. A. (1973) J . Bid . Chem. 248, 6559-

Gustafsson, J. A. & Stenberg, A. ( 1 974) J . Biol. Chrm. 240,711 - 719. Lax, E. R., Hoff, H. G., Ghraf, R., Schroder, E. & Schriefers, H.

Levin, W., Ryan, D., Kuntzman, R. Rr Conney, A . H. (1975) Mol.

El Defrawy El Masry, S. & Mannering, G. J . (1974) Drug Meiah.

Gustafsson, J. A. & Stenberg, A. (1976) Proc. Nail Acad. &i. USA,

Kramer, R. E., Greiner, J. W., Rumbaugh, R . C., Sweeney, T. D.

Gustafsson, J . A. & Stenberg, A . (1975) Endocrinology, Yh, 501 -

Gustafsson, J. A. & Stenberg, A. (1975) A(.tu Endocrinol. 78, 545-

Kramer, R. E. & Colby, H. D. (1976) J. Endocrinol. 71, 449-450. Kramer, R. E., Greiner, J. W., Rumbaugh, R. C., Sweeney, T. D.

& Colby, H. D. (1978) J . Pharmacol. Exp. Ther. 204, 247-254.

24Y, 1940 - 1945.

Biochem. .I. I80, 313-318.

Res. Comniun. 84, 706-71 2.

13, 733 - 736.

34,279 - 286.

Biochem. 82, 529- 533.

Chem. 193, 265-275.

74, 561 - 566.

Bi0phj.s. Res. Commun. 79, 505 - 512.

1319.

123,458 - 468.

185-196.

J . Endocrinol. 64, 329- 336.

6567.

(1 974) Hoppe-Seyler's Z . Physiol. Cheni. 355, 1325 - 1331,

Pharmacoi. 11, 190-200.

Dispos. 2, 279 - 284.

73, 1462- 1465.

& Colby, H. D. (1979) J . Pharmacol. E x p . Thrr. 208, 19-23.

504.

553.

220

44. Krdmer, R. E., Greiner, J. W. & Colby, H. D. (1977) Biochern.

45. Kamel, E. & Frankel, A. I. (1979) Endocrinology, 104, 1461 - 1466. 46. Lieberman, M. E., Maurer, R. A. & Gorski, J. (1978) Proc. Nail

47. Miller, W. L. & Gorski, J. (1977) Endocrinology, 100, 1306-1316. 48. Lax, E. G., Ghraf, R., Schriefers, H., Herrmann, H. & Petutschnigk,

49. Iba, M. M., Soyka, L. F. & Schulman, M. P. (1977) Mol. Pharmacol.

50. Miiller, D. & Klinger, W. (1978) Pharmazie, 33, 397-400.

Pharmacol. 26,66 -69.

Acad. Sci, USA, 75, 5946 - 5949.

D. (1976) Acla Endocrinol. 82, 774-784.

13, 1092 - 1104.

51. El Defrawy El Masry, S., Cohen, G. M. & Mannering, G. J. (1974)

52. Tredger, J. M. & Chhabra, R. S. (1980) Drug. Metab. Dispos. 8,

53. Owens, I. S. & Nebert, D. W. (1975) Mol. Pharmacol. 11, 94- 104. 54. Wiebel, F. J. & Gelboin, H. V. (1975) Biochern. Pharmacol. 24,

55. Kuenzig, W., Kamm, J. J., Boublik, H., Jenkins, F. & Burns, J. J .

56. Cohen, G. M. & Mannering, G. J. (1974) Drug Melab. Dispos. 2,

Drug. Metab. Dispos. 2, 267-278.

16-21.

1511-1515.

(1974) J . Pharmacol. Exp. Ther. 191, 32-44.

285 - 292.

F. Pasleau, C. Kolodzici, P. Kremers, and J. E. Gielen, Laboratoire de Chimie Medicale, Institut de Pathologie, Universite de Libge au Sart-Tilman, B-4000 Liege, Belgium

ontogenic development of steroid 16α-hydroxylase as a tool for the study of the multiplicity of...

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