Influence of recipient gender on intrasplenic fetal liver tissue transplants in rats: cytochrome P450-mediated monooxygenase functions

Toxicology 197 (2004) 199–212

Influence of recipient gender on intrasplenic fetal liver tissue transplants in rats: cytochrome P450-mediated monooxygenase functions Amelie Lupp∗ , Sabine Hugenschmidt, Michael Rost, Dieter Müller Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Nonnenplan 4, D-07743 Jena, Germany Received in revised form 20 October 2003; accepted 7 January 2004

Abstract Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern with consecutive differences in P450mediated monooxygenase activities, which have been shown to be due to a differential profile of growth hormone (GH) secretion. Parallel to previous investigations on P450 isoforms expression, the aim of the present study was to elucidate the influence of recipient gender on P450-mediated monooxygenase activities in intrasplenic liver tissue transplants in comparison to orthotopic liver. Fetal liver tissue suspensions of mixed gender were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting transplant-recipients and age-matched controls were treated with ␤-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the vehicles and sacrificed 24 or 48 h thereafter. P450-dependent monooxygenase activities were assessed by a series of model reactions for different P450 subtypes in liver and spleen 9000g supernatants. In spleens of male and female control rats only very low monooxygenase activities were detectable, whereas with most model reactions distinct activities were observed in transplant-containing organs. Livers and transplant-containing spleens from male rats displayed higher basal ethoxycoumarin O-deethylase and testosterone 2␣-, 2␤-, 6␤-, 14␣-, 15␣-, 15␤-, 16␣-, 16␤- and 17-hydroxylase activities than those from females. On the other hand, like the respective livers, spleens from female transplant-recipients demonstrated more pronounced p-nitrophenol- and testosterone 6␣- and 7␣-hydroxylase activities than those from male hosts. With nearly all model reactions gender-specific differences in inducibility by BNF, PB or DEX could be demonstrated in livers as well as in transplant-containing spleens. These results further confirm that the P450 system of intrasplenic liver tissue transplants and the respective orthotopic livers is similarly influenced by recipient gender. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hepatocytes; Spleen; Transplantation; Cytochrome P450; Gender

Abbreviations: BNF, ␤-naphthoflavone; b.wt., body weight; DEX, dexamethasone; DMSO, dimethylsulfoxide; ECOD, ethoxycoumarin O-deethylation; EROD, ethoxyresorufin O-deethylation; P450, cytochrome P450; PB, phenobarbital; PNPH, p-nitrophenol-hydroxylation; PROD, pentoxyresorufin O-depentylation; TH, testosterone hydroxylation ∗ Corresponding author. Tel.: +49-3641-938718; fax: +49-3641-938702. E-mail address: [email protected] (A. Lupp). 0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.01.003

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A. Lupp et al. / Toxicology 197 (2004) 199–212

1. Introduction Hepatocellular transplantations have emerged as an alternative treatment option to whole or partial liver transplantations for the treatment of acute or chronic liver failure, as a temporary relief after extensive liver resections, or for the treatment of an enzyme deficiency (Mito and Kusano, 1993; Habibullah et al., 1994; Strom et al., 1999; Bilir et al., 2000). The advantages are a minimal surgical intervention with a lesser morbidity, a major cost effectiveness and the preservation of the native liver. Furthermore, cells of one donor can be used for several recipients. Hepatocytes can be cryopreserved, which offers the possibility of cell banking and, finally, they can be modified genetically ex vivo before transplanting them (back) into the recipient. On the other hand, liver cell transplantations into an ectopic site like the spleen can provide an excellent tool to study humoral and nerval as well as other topic influences on liver cell differentiation, multiplication and function. Recently, we have demonstrated cytochrome P450 (P450) isoforms expression and respective P450mediated monooxygenase functions in intrasplenic fetal liver tissue transplants (Lupp et al., 1998a, c). We have been able to show developmental changes both in P450 isoforms expression and in P450-dependent monooxygenase functions with time after grafting pointing to a differentiation of the transplanted fetal cells into adult ones at least at 2 months after grafting. Comparable results were reported also by other authors (Maganto et al., 1990; Kato et al., 1994, 1995, 1996). In further studies we have additionally demonstrated that substances with characteristic effects on normal orthotopic liver, like inducers of the P450 system, mitogens or cytotoxins, exert a similar influence also on the intrasplenic liver tissue transplants (Lupp et al., 1998b, 1999a,b). We then have tried to elucidate the influence of systemically acting hormones on the intrasplenic transplants in comparison to orthotopic livers. Male and female rats have, for instance, a differential GH secretion profile leading to a sex-specific cytochrome P450 isoforms expression pattern in orthotopic livers (see e.g. Westin et al., 1990; Legraverend et al., 1992a,b; Waxman, 1992; Shapiro et al., 1995; Waxman et al., 1995; Shimada et al., 1997; Agrawal and Shapiro, 2000, 2001; Pampori

et al., 2001). Thus, in a further study (Lupp et al., 2003) we have investigated the influence of the recipient gender on the P450 isoforms expression pattern in intrasplenic liver tissue transplants in comparison to the orthotopic livers. For this, fetal liver tissue suspensions of mixed sex have been transplanted into the spleen of adult male or female syngenic recipients and 4 months after grafting evaluated for the expression of the P450 subtypes 1A1, 2B1, 2E1, 3A2 and 4A1 by means of immunohistochemistry. These investigations have revealed that the transplants seemed to be influenced in the same way by the sex-specific GH secretion profile as the livers, since, like in the respective livers, in the transplants which had been inoculated into male recipients the male predominant P450 isoforms 2B1, 3A2 and 4A1 (Lewis, 1996) were more strongly expressed than in grafts from female hosts, whereas the opposite was the case with the expression of P450 2E1, which is a female prevalent P450 subtype (Lewis, 1996). Also sex-specific differences in the inducibility of the P450 system by typical inducers, like ␤-naphthoflavone (BNF), phenobarbital (PB) or dexamethasone (DEX) (Correia, 1995; Lewis, 1996; Lake et al., 1998), occurred in the same way both in livers and intrasplenic transplants. In these previous investigations, however, the P450 isoforms expression was assessed only semi-quantitatively. Additionally, the presence of P450 enzymes must not necessarily also correlate with the function since other factors such as the NADPH–cytochrome P450 reductase and cofactors are also required. Thus, the aim of the present study was to further examine the influence of the recipient gender, with quantitative measures of P450-mediated monooxygenase functions, on intrasplenic liver tissue transplants at 4 months after transplantation in comparison to orthotopic liver. Effects on P450-mediated monooxygenase activities were measured by a series of model reactions for different P450 subtypes (Correia, 1995; Lewis, 1996): ethoxyresorufin O-deethylation (EROD; mainly P450 1A), ethoxycoumarin O-deethylation (ECOD; predominantly P450 1A, 2A, 2B, 2C), pentoxyresorufin O-depentylation (PROD; chiefly P450 2B), p-nitrophenol-hydroxylation (PNPH; predominantly P450 2E), and testosterone hydroxylation (TH) at different positions (2␣ [P450 2C11, but also P450 2C6], 2␤ [P450 3A2], 6␣ [P450 2A1], 6␤ [P450 3A1, but also P450 1A1, 1A2, 2A2, 2C11,

A. Lupp et al. / Toxicology 197 (2004) 199–212

2C13, 2D1, 3A2], 7␣ [P450 2A1, but also P450 2C13], 15␣ [P450 2A2, but also P450 2C12, 2C13], 15␤ [P450 2C12], 16␣ [P450 2A2, 2B1, 2B2, 2C6, 2C7, 2C11, 2C13], 16␤ [P450 2B1, 2B2], 17 [P450 2B1, 2C6, 2C11]). Of the P450 isoforms involved P450 1A1 and 2C6 exhibit no sex-specificity, P450 2A2, 2B1, 2B2, 2C11, 2C13, 2D1, 3A1 and 3A2 are male predominant/specific and P450 1A2, 2A1, 2C7, 2C12 and 2E1 are female prevalent/specific. Thus, higher ECOD, PROD, and 2␣-, 2␤-, 6␤-, 15␣-, 15␤-, 16␣-, 16␤- and 17-TH activities are to be expected with male, and more elevated EROD, PNPH and 6␣- and 7␣-TH activities with female rats (Lewis, 1996). Parallel to the previous investigations also sex-specific differences in the inducibility of the P450-dependent monooxygenase functions were evaluated by additionally pretreating the animals with BNF, PB or DEX, or the respective vehicles before sacrifice.

2. Material and methods 2.1. Animals The study was conducted under the license of the Thuringian Animal Protection Committee. All animals received care according to the criteria outlined in the “German Law on the Protection of Animals” and in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). Fischer-344 inbred rats from our own institute’s breed were used. The animals were housed in plastic cages under standardized conditions (12-h light:12-h dark cycle, temperature 22±2 ◦ C, humidity 50±10%, pellet diet Altromin 1316, water ad libitum). Donor-fetuses were taken from pregnant Fischer344 inbred rats at the 21st day of gestation. The fetal livers were immediately removed, pooled and minced by razor blades in 4 ◦ C cold Hank’s balanced salt solution (1 + 1; w + v) until a homogenous suspension was obtained. In the mean the litters consisted of 3.63 ± 1.93 (mean ± S.E.M.) female and 3.54 ± 1.94 male fetuses, with no statistically significant difference in the amount of either sex within the litters (Mann–Whitney test; P > 0.05; n = 356). Thus,

201

with high probability it can be assumed that fetal liver tissue suspensions were originating from fetuses of originally half of either sex. Cell viability of the fetal hepatocyte clusters was assessed by the trypan blue exclusion method and amounted to 99%. Recipients were syngenic 60–90-day-old male or female Fischer-344 inbred rats. After laparotomy, 0.2 ml of the fetal liver tissue suspension (approximately 8.6 × 106 viable cells, mainly in clusters) were injected through a 21-gauge needle into the spleens of the recipients in longitudinal direction. Subsequently, the abdomen was closed again with two sutures one on top of the other. Age-matched control animals received no surgery. No immunosuppressant was given to the grafted rats. 2.2. Treatment Four months after surgery, the male and female transplant-recipients as well as the respective age-matched control rats were treated either with BNF, PB or DEX, or the respective vehicles (dimethylsulfoxide (DMSO) or 0.9% NaCl). In total 128 rats (eight animals for each treatment and gender, both in transplant-recipients and controls) were used. The inducers were given orally: • BNF: once 50 mg/kg body weight (b.wt.), dissolved in 2.5 ml/kg b.wt. DMSO. • PB: once 50 mg/kg b.wt., dissolved in 2.5 ml/kg b.wt. 0.9% NaCl. • DEX: in the morning of three consecutive days 4 mg/kg b.wt., dissolved in 2.0 ml/kg b.wt. 0.9% NaCl. The animals were sacrificed in ether anesthesia 24 (BNF, DEX) or 48 h (PB) after the (last) treatment. 2.3. Preparation of 9000g supernatants After sacrifice in ether anesthesia, the livers and spleens were removed quickly and homogenized in 0.1 M sodium phosphate buffer pH 7.4 (1:3 w/v). For preparation of 9000g supernatant, the homogenate was centrifuged at 9000 × g for 20 min at 4 ◦ C. The protein content of the 9000g supernatants was determined with the Biuret method as modified according to Klinger and Müller (1974).

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2.4. Monooxygenase functions EROD was measured according to Pohl and Fouts (1980) with modifications, PROD as described by Lubet et al. (1985), fluorimetrically determining the metabolite resorufin. With ECOD the main metabolite 7-hydroxycoumarin was measured fluorimetrically (Aitio, 1978). PNPH was performed according to Chang et al. (1997), determining the p-nitrocatechol metabolite spectrophotometrically. Testosterone hydroxylase activities were assessed by an HPLC method. Reactions were carried out at 37 ◦ C for 10 min (liver 9000g samples) or 40 min (spleen 9000g samples) in 1.01-ml incubation mixtures containing 100 ␮l 9000g supernatant (dilution 1:5 with liver samples), 200 ␮l 0.1 M MgCl2 , 200 ␮l 0.025 M glucose-6-phosphate, 300 ␮l 0.1 M sodium-phosphate buffer (pH 7.4), 10 ␮l 0.05 M testosterone and 200 ␮l 2.5 mM NADPH, and were stopped with 1.5 ml 0.31 M trichloroacetic acid. The samples were then centrifuged at 5000 × g. Two milliliter of the clear supernatant to which 15 ␮l corticosterone (0.26 mM in ethanol) had been added as internal standard were given onto LC-18 columns (SupelcleanTM LC-18 SPE tubes, 1 ml; Supelco, Supelco Park; Bellefonte, PA, USA) for isolation of testosterone and its hydroxylated metabolites by solid phase extraction (conditioning of the columns: 2 ml water, 2 ml methanol, 2 ml water; loading of the columns: 2 ml supernatant (flow ≤0.5 ml/min), 2 ml water, drying of the columns; elution: 2 ml methanol). After the elution of the substances from the column with 2 ml methanol, the solvent was evaporated and the samples were dissolved in 250 ␮l of the mobile phase (65.7% water, 27.3% methanol, 7% tetrahydrofuran). HPLC method: equipment: JASCO Labor- und Datentechnik GmbH, Gro␤-Umstadt, Germany; injection volume: 20 ␮l; column: Kromasil 100, C18, 5 ␮m; 300 mm × 4.0 mm; ternary low pressure gradient; flow rate: 1 ml/min; gradient: 0–5 min: 65.7% water, 27.3% methanol, 7% tetrahydrofuran; 5–45 min: linear gradient to 32.8% water, 60.2% methanol, 7% tetrahydrofuran; 45–53 min: linear gradient to 29.2% water, 63.8% methanol, 7% tetrahydrofuran; 53–55 min: linear gradient to 65.7% water, 27.3% methanol, 7% tetrahydrofuran; 55–60 min: 65.7% water, 27.3% methanol, 7% tetrahydrofuran; retention times (min): 6␣-hydroxytestosterone (HT): 10.5;

15␤-HT: 11.4; 7␣-HT: 12.0; 15␣-HT: 12.3; 6␤-HT: 13.9; 14␣-HT: 14.8; 16␣-HT: 16.1; 16␤-HT: 20.0; 2␣-HT: 22.2; 2␤-HT: 23.6; 11␤-HT: 24.0; corticosterone: 25.6; androstenedione: 28.4; testosterone: 33.5; detection: UV/VIS-detector UV 975; wavelength 254 nm; evaluation software: BORWIN V4, JASCO Labor- und Datentechnik GmbH, Gro␤-Umstadt, Germany. The activities of the model reactions were referred to the protein content of the 9000g supernatants. Activities within the transplant-containing spleens were measured in the 9000g supernatants of the entire organs. 2.5. Statistics The animals investigated per treatment group comprised n = 8. The results are expressed as arithmetic mean ± S.E.M. For the statistical analyses the Mann–Whitney test (P ≤ 0.05) was applied.

3. Results All data of the model reactions for P450-mediated monooxygenase functions measured in the present study are presented in Tables 1–6. Additionally, ECOD, PNPH and testosterone 6␤and 7␣-hydroxylase activities in livers and spleens of male and female control rats and transplant-recipients both after treatment with the vehicles and after administration of the inducers are depicted in Figs. 1–4. 3.1. Basal monooxygenase activities 3.1.1. Livers In no case differences were noticed between basal liver activities (i.e. after treatment with the vehicles only) of control rats and transplant-recipients. Basal ECOD and 2␣-, 2␤-, 6␤-, 14␣-, 15␣-, 15␤-, 16␣- and 17-TH activities were distinctly higher in the livers of male than in those of female rats. 16␤-TH activity was more pronounced in males than in females only with transplant-recipients. In contrast, basal PNPH and 7␣-TH activities in the livers of female rats significantly exceeded those of male rats. 6␣-TH was significantly higher in female than in

Table 1 EROD, ECOD, PROD and PNPH activities in 9000g supernatants of livers from male (M) or female (F) Fischer-344 rats, controls and transplant-recipients, at 4 months after transplantation DMSO/0.9% NaCl

BNF

PB

Controls

Transplantrecipients

Controls

Controls

Transplantrecipients

Transplantrecipients

154.0 ± 23.4 224.8 ± 31.8

155.3 ± 30.0 200.3 ± 23.2

2347.6 ± 77.5∗ 2348.1 ± 99.1∗

1940.7 ± 68.6∗+# 2323.4 ± 82.4∗

1746.7 ± 122.3∗# 827.0 ± 51.5∗

1551.9 ± 57.9∗# 737.5 ± 46.2∗

440.4 ± 19.5 399.4 ± 24.4∗

440.2 ± 26.3# 340.0 ± 16.9∗

32.7 ± 1.9 37.1 ± 3.9

216.3 ± 43.0∗ 151.7 ± 14.8∗

110.9 ± 4.5∗+ 124.3 ± 5.6∗

765.5 ± 74.5∗# 476.5 ± 28.1∗

721.7 ± 66.3∗# 391.5 ± 34.6∗

172.5 ± 27.9∗# 74.4 ± 10.3∗

194.1 ± 35.3∗# 65.6 ± 10.3∗

88.0 ± 3.1# 101.5 ± 3.9

120.4 ± 4.2∗# 151.7 ± 9.0∗

121.5 ± 5.2∗# 141.6 ± 6.8∗

90.0 ± 1.7∗# 120.2 ± 5.0∗

110.4 ± 5.4∗ 121.6 ± 4.4∗

111.1 ± 7.0∗ 124.0 ± 6.2∗

119.1 ± 3.9∗ 127.1 ± 4.0∗

120.1 ± 17.8 194.9 ± 46.8

130.5 ± 17.4 167.7 ± 16.4

ECOD

M F

419.6 ± 11.9# 297.1 ± 10.6

408.9 ± 8.8# 280.0 ± 6.2

PROD

M F

29.6 ± 2.5 37.1 ± 5.4

PNPH

M F

87.9 ± 3.8 98.3 ± 3.3

Before sacrifice, the rats were pretreated with the vehicles DMSO or 0.9% NaCl or with BNF, PB or DEX. Activities are expressed as nmol/(g protein × min). Data are given as arithmetic mean ± S.E.M., n = 8; ∗ P ≤ 0.05 vs. DMSO/0.9% NaCl; + P ≤ 0.05 vs. controls; # P ≤ 0.05 vs. female rats (Mann–Whitney test).

Table 2 EROD, ECOD, PROD and PNPH activities in 9000g supernatants of control and transplant-containing spleens from male (M) or female (F) Fischer-344 rats at 4 months after transplantation DMSO/0.9% NaCl

BNF

Controls

Transplantrecipients

Controls

PB Transplantrecipients

DEX

Controls

Transplantrecipients

Controls

Transplantrecipients

EROD

M F

0.65 ± 0.06 0.66 ± 0.06

0.85 ± 0.06+ 0.91 ± 0.06+

0.79 ± 0.08 0.80 ± 0.05

1.26 ± 0.15∗+ 1.29 ± 0.14∗+

0.77 ± 0.06 0.77 ± 0.03

1.19 ± 0.10∗+ 1.02 ± 0.10+

1.00 ± 0.05∗ 0.86 ± 0.05

1.00 ± 0.06 1.43 ± 0.31

ECOD

M F

0.43 ± 0.17 0.69 ± 0.16

2.40 ± 0.44+# 1.11 ± 0.28

0.53 ± 0.15 0.71 ± 0.17

5.18 ± 1.00∗+# 1.56 ± 0.41

0.51 ± 0.14 0.70 ± 0.21

5.35 ± 0.71∗+# 2.10 ± 0.68

0.41 ± 0.11 0.41 ± 0.16

3.68 ± 0.88+ 3.46 ± 0.22∗+

PROD

M F

0.49 ± 0.05 0.26 ± 0.16

0.44 ± 0.06 0.45 ± 0.03

0.43 ± 0.06 0.47 ± 0.05

0.54 ± 0.09 0.56 ± 0.07

0.38 ± 0.08 0.36 ± 0.04

1.05 ± 0.19∗+ 0.77 ± 0.10∗+

0.61 ± 0.08 0.52 ± 0.06

0.63 ± 0.10 0.67 ± 0.04∗

PNPH

M F

1.58 ± 0.29 2.28 ± 0.25

3.81 ± 0.41+# 7.04 ± 0.39+

1.47 ± 0.31# 3.08 ± 0.39

1.19 ± 0.25# 2.50 ± 0.28

4.44 ± 0.24+# 6.96 ± 0.46+

1.72 ± 0.26 2.11 ± 0.52

4.91 ± 0.38+# 7.00 ± 0.41+

6.50 ± 0.27∗+# 10.55 ± 0.70∗+

203

Before sacrifice, the rats were pretreated with the vehicles DMSO or 0.9% NaCl or with BNF, PB or DEX. Activities are expressed as nmol/(g protein × min). Data are given as arithmetic mean ± S.E.M., n = 8; ∗ P ≤ 0.05 vs. DMSO/0.9% NaCl; + P ≤ 0.05 vs. controls; # P ≤ 0.05 vs. female rats (Mann–Whitney test).

A. Lupp et al. / Toxicology 197 (2004) 199–212

447.4 ± 106.3∗ 364.1 ± 94.9

M F

465.8 ± 112.8∗ 389.1 ± 95.9∗

Controls

736.7 ± 220.8∗ 911.7 ± 285.4∗

EROD

764.5 ± 227.4∗ 999.0 ± 359.4∗

Transplantrecipients

DEX

DMSO/0.9% NaCl

BNF

PB

DEX

Controls

Transplantrecipients

Controls

Transplantrecipients

Controls

Transplantrecipients

Controls

Transplantrecipients

M F

2214.8 ± 90.1# 84.99 ± 11.65

2160.5 ± 79.8# 86.52 ± 11.23

2031.3 ± 183.6# 65.12 ± 8.75

1977.0 ± 104.0# 52.42 ± 2.25∗

1798.8 ± 36.4∗# 58.18 ± 1.80∗

1687.3 ± 68.5∗# 54.04 ± 3.65∗

1472.1 ± 121.5∗# 93.10 ± 6.3

1383.1 ± 96.7∗# 109.86 ± 3.45+

2␤

M F

104.0 ± 5.0# 29.17 ± 14.09

124.3 ± 18.0# 23.17 ± 14.45

111.9 ± 9.4# 32.61 ± 8.15

119.9 ± 6.8# 25.28 ± 6.46

440.7 ± 46.7∗# 126.04 ± 18.24∗

484.3 ± 26.3∗# 139.78 ± 22.06∗

810.5 ±113.4∗ 1090.0 ± 55.83∗

826.6 ± 91.9∗# 1174.9 ± 97.52∗

6␣

M F

10.22 ± 2.51# 24.42 ± 4.83

16.08 ± 3.68 26.54 ± 2.96

18.33 ± 4.07# 28.92 ± 2.27

13.55 ± 3.68 22.21 ± 4.87

36.06 ± 1.85∗ 43.83 ± 3.88∗

45.41 ± 4.56∗ 37.99 ± 4.87

10.93 ± 2.06# 35.03 ± 8.72

6.76 ± 4.48# 29.13 ± 3.41

6␤

M F

1207.6 ± 56.5# 230.5 ± 54.6

1354.1 ± 50.7# 150.3 ± 25.4

1204.9 ± 68.3# 182.3 ± 16.5

1333.3 ± 69.8# 160.7 ± 13.4

3779.7 ± 300.5∗# 332.8 ± 56.8

4452.2 ± 215.1∗# 400.3 ± 41.5∗

4395.4 ± 541.7∗# 1865.3 ± 249.8∗

4666.2 ± 334.1∗# 2489.6 ± 240.2∗

7␣

M F

100.1 ± 7.9# 431.9 ± 69.5

116.2 ± 9.2# 505.6 ± 57.5

209.3 ± 14.8∗# 902.6 ± 38.6∗

210.6 ± 10.0∗# 837.4 ± 49.6∗

87.7 ± 23.8# 799.6 ± 40.6∗

54.4 ± 11.5∗# 746.2 ± 64.6∗

187.1 ± 15.7∗# 651.1 ± 29.3∗

Before sacrifice, the rats were pretreated with the vehicles DMSO or 0.9% NaCl or with BNF, PB or DEX. Activities are expressed as nmol/(g protein × min). Data are given as arithmetic mean ± S.E.M., n = 8; ∗ P ≤ 0.05 vs. DMSO/0.9% NaCl; + P ≤ 0.05 vs. controls; # P ≤ 0.05 vs. female rats (Mann–Whitney test).

Table 4 Testosterone 14␣-, 15␣-, 15␤-, 16␣-, 16␤- and 17-hydroxylase activities in 9000g supernatants of livers from male (M) or female (F) Fischer-344 rats, controls and transplant-recipients, at 4 months after transplantation DMSO/0.9% NaCl Controls

BNF Transplantrecipients

PB

DEX

Controls

Transplantrecipients

Controls

Transplantrecipients

Controls

Transplantrecipients

14␣

M F

160.0 ± 12.9# 0.857 ± 0.292

134.9 ± 9.9# 1.599 ± 1.220

116.5 ± 11.5∗# −0.364 ± 1.299

96.1 ± 8.0∗# −1.087 ± 1.472

385.0 ± 36.5∗# 17.795 ± 5.851∗

403.8 ± 32.6∗# 14.900 ± 3.956∗

134.2 ± 24.0# 1.739 ± 0.802

123.8 ± 16.7# 0.877 ± 2.949

15␣

M F

60.95 ± 4.69# 7.239 ± 3.211

63.86 ± 3.01# 5.806 ± 2.529

43.11 ± 6.22∗# 4.822 ± 2.690

56.04 ± 2.02# 6.745 ± 2.102

128.99 ± 4.94∗# 7.195 ± 1.596

147.21 ± 7.52∗# 7.635 ± 2.023

157.75 ± 18.36∗# 10.094 ± 1.291

159.45 ± 15.64∗# 9.904 ± 2.056

15␤

M F

58.08 ± 8.10# 28.33 ± 6.14

82.69 ± 9.23# 23.19 ± 3.64

61.45 ± 3.37# 20.52 ± 2.30

77.00 ± 3.44+# 20.93 ± 1.70

149.44 ± 14.33∗# 46.17 ± 4.83*

178.47 ± 15.29∗# 56.46 ± 6.08∗

376.85 ± 58.99∗ 273.59 ± 27.51∗

421.08 ± 52.91∗ 382.99 ± 41.68∗

16␣

M F

2098.3 ± 179.2# 100.77 ± 11.92

2190.9 ± 97.8# 87.67 ± 7.85

1984.4 ± 179.4# 13.84 ± 4.45∗

1936.2 ± 65.7# 9.21 ± 2.34∗

3428.4 ± 101.8∗# 442.37 ± 75.89∗

3599.4 ± 174.5∗# 497.57 ± 76.20∗

1978.9 ± 155.3# 182.66 ± 18.85∗

1932.1 ± 108.4# 186.08 ± 23.39∗

16␤

M F

102.5 ± 6.5 89.28 ± 5.08

111.4 ± 3.9# 86.03 ± 10.06

116.6 ± 15.0# 79.25 ± 1.54

115.5 ± 7.3# 78.44 ± 1.33

1477.9 ± 50.3∗# 501.83 ± 101.50∗

1680.6 ± 104.9∗# 588.94 ± 66.69∗

306.6 ± 41.6∗# 189.56 ± 15.90∗

320.0 ± 61.0∗ 219.96 ± 11.70∗

17

M F

789.6 ± 28.1# 206.5 ± 24.7

809.7 ± 60.8# 195.9 ± 16.6

760.5 ± 10.7# 192.2 ± 32.4

776.9 ± 22.8# 172.5 ± 17.4

1306.4 ± 114.7∗# 436.8 ± 45.6∗

1292.5 ± 85.1∗# 485.3 ± 62.2∗

849.6 ± 85.1# 262.0 ± 29.9

810.8 ± 104.5# 232.3 ± 21.2

Before sacrifice, the rats were pretreated with the vehicles DMSO or 0.9% NaCl or with BNF, PB or DEX. Activities are expressed as nmol/(g protein × min). Data are given as arithmetic mean ± S.E.M., n = 8; ∗ P ≤ 0.05 vs. DMSO/0.9% NaCl; + P ≤ 0.05 vs. controls; # P ≤ 0.05 vs. male rats (Mann–Whitney test).

A. Lupp et al. / Toxicology 197 (2004) 199–212

2␣

205.5 ± 21.5∗# 667.6 ± 49.4∗

204

Table 3 Testosterone 2␣-, 2␤-, 6␣-, 6␤- and 7␣-hydroxylase activities in 9000g supernatants of livers from male (M) or female (F) Fischer-344 rats, controls and transplant-recipients, at 4 months after transplantation

Table 5 Testosterone 2␣-, 2␤-, 6␣-, 6␤- and 7␣-hydroxylase activities in 9000g supernatants of spleens from male (M) or female (F) Fischer-344 rats at 4 months after transplantation DMSO/0.9% NaCl Controls 2␣

M F

2␤

M F

6␣

BNF Transplantrecipients

Controls

PB Transplantrecipients

Controls

152.0 ± 13.9 167.2 ± 10.5

304.1 ± 19.5+# 211.3 ± 16.9+

169.5 ± 7.5 173.1 ± 12.9

7.94 ± 1.81# 3.23 ± 1.01

35.36 ± 6.66+# 14.79 ± 2.16+

4.47 ± 1.99 5.11 ± 1.36

32.10 ± 7.63+ 18.53 ± 1.70+

5.29 ± 1.73 2.01 ± 1.56

M F

40.72 ± 7.92 40.17 ± 16.97

66.84 ± 7.46+# 104.28 ± 8.23+

56.03 ± 7.16 45.66 ± 12.48

79.18 ± 12.57 125.62 ± 27.18+

6␤

M F

44.96 ± 2.97 39.11 ± 5.23

245.45 ± 22.77+# 138.91 ± 12.70+

50.22 ± 7.65 39.64 ± 5.79

7␣

M F

32.46 ± 10.19+# 172.68 ± 11.83+

32.10 ± 7.91∗ 9.65 ± 8.98

0.20 ± 10.12 6.36 ± 5.97

Transplantrecipients 248.6 ± 20.6+ 207.6 ± 17.5

Controls

Transplantrecipients

158.3 ± 11.7 175.0 ± 13.7

251.5 ± 17.6+# 197.0 ± 9.8

54.47 ± 2.21∗+# 17.06 ± 2.46+

5.47 ± 2.72 4.82 ± 1.97

106.01 ± 14.44∗+ 123.68 ± 20.69∗+

57.93 ± 3.65 49.76 ± 18.00

122.49 ± 8.74∗+# 171.16 ± 15.93∗+

40.85 ± 4.06 56.08 ± 6.37

117.88 ± 33.83+ 168.63 ± 12.71∗+

274.78 ± 38.31+# 144.89 ± 15.56+

62.69 ± 11.08 41.17 ± 4.94

391.5 ± 35.19∗+# 233.69 ± 11.76∗+

65.94 ± 5.51∗ 46.69 ± 7.86

831.3 ± 92.61∗+ 792.65 ± 153.3∗+

29.37 ± 3.87# 329.09 ± 56.05∗+

45.82 ± 5.74∗# 21.33 ± 5.70

45.71 ± 6.15# 338.22 ± 18.82∗+

13.91 ± 6.72 18.87 ± 9.05

82.19 ± 18.86∗+# 521.17 ± 54.86∗+

Before sacrifice, the rats were pretreated with the vehicles DMSO or 0.9% NaCl or with BNF, PB or DEX. Activities are expressed as pmol/(g protein × min). Data are given as arithmetic mean ± S.E.M., n = 8; ∗ P ≤ 0.05 vs. DMSO/0.9% NaCl; + P ≤ 0.05 vs. controls; # P ≤ 0.05 vs. female rats (Mann–Whitney test).

Table 6 Testosterone 14␣-, 15␣-, 15␤-, 16␣-, 16␤- and 17-hydroxylase activities in 9000g supernatants of control and transplant-containing spleens from male (M) or female (F) Fischer-344 rats at 4 months after transplantation DMSO/0.9% NaCl Controls

BNF Transplantrecipients

Controls

PB Transplantrecipients

Controls

DEX Transplantrecipients

Controls

Transplantrecipients

14␣

M F

5.89 ± 2.71 0.981 ± 0.970

17.19 ± 1.70+# 2.963 ± 1.875

5.27 ± 1.68 3.348 ± 2.130

13.90 ± 6.48 2.882 ± 2.880

18.11 ± 4.80# 4.836 ± 1.583

43.11 ± 5.24∗+# 16.280 ± 5.338∗

11.39 ± 3.50 5.912 ± 2.946

13.82 ± 1.36# 5.054 ± 1.994

15␣

M F

33.27 ± 2.90 34.66 ± 2.09

49.90 ± 2.86+# 37.41 ± 3.25

36.28 ± 4.23 35.56 ± 5.20

47.62 ± 2.55+ 48.01 ± 11.26

30.23 ± 3.31 26.08 ± 3.24∗

74.95 ± 8.50∗+ 50.96 ± 11.80

31.88 ± 4.36 34.41 ± 2.82

79.94 ± 9.04∗+ 80.34 ± 18.31∗+

15␤

M F

3.681 ± 3.498 2.245 ± 1.081

7.673 ± 0.723 6.265 ± 1.415+

0.371 ± 0.371# 2.174 ± 0.462

6.894 ± 0.731+ 5.718 ± 1.360+

3.616 ± 1.542 3.615 ± 0.470

14.098 ± 1.656∗+ 12.534 ± 3.883+

4.222 ± 1.362 8.582 ± 4.202

80.82 ± 10.43∗+ 88.60 ± 11.585∗+

16␣

M F

17.88 ± 4.08 11.26 ± 3.96

39.69 ± 4.39+# 17.15 ± 1.89

14.22 ± 4.87 16.39 ± 8.36

38.53 ± 6.89+ 24.41 ± 7.05

27.77 ± 3.90 14.91 ± 5.19

195.34 ± 28.89∗+ 155.29 ± 49.40∗+

23.18 ± 1.01 11.14 ± 10.21

60.46 ± 7.74∗+# 206.3 ± 24.27∗+

16␤

M F

194.9 ± 6.7 184.9 ± 5.4

247.4 ± 17.3+# 193.4 ± 8.7

180.3 ± 9.5 177.7 ± 19.6

252.4 ± 28.5+ 228.8 ± 12.5∗

177.8 ± 11.0 191.3 ± 17.6

406.1 ± 59.2∗+ 298.9 ± 42.2∗+

170.5 ± 13.8 185.3 ± 9.8

302.5 ± 47.6+ 282.8 ± 16.9∗+

17

M F

45.20 ± 4.57 39.80 ± 4.50

99.40 ± 6.25+# 59.68 ± 6.20+

38.09 ± 4.04 47.03 ± 8.94

99.91 ± 9.57+# 70.12 ± 8.51

43.16 ± 3.96 33.78 ± 4.39

218.3 ± 10.83∗+# 157.33 ± 24.01∗+

38.51 ± 3.67 39.76 ± 4.11

168.3 ± 7.22∗+# 135.10 ± 9.20∗+

205

Before sacrifice, the rats were pretreated with the vehicles DMSO or 0.9% NaCl or with BNF, PB or DEX. Activities are expressed as pmol/(g protein × min). Data are given as arithmetic mean ± S.E.M., n = 8; ∗ P ≤ 0.05 vs. DMSO/0.9% NaCl; + P ≤ 0.05 vs. controls; # P ≤ 0.05 vs. female rats (Mann–Whitney test).

A. Lupp et al. / Toxicology 197 (2004) 199–212

318.7 ± 42.8+# 215.2 ± 8.3+

171.4 ± 11.6 159.2 ± 16.7

DEX

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Liver

Spleen

control rats transplant recipients

male rats

10

3000 #

* + *

2500 2000

# #

*

*

1500 1000 #

#

#

7-hydroxycoumarin [nmol / (g protein x min)]

7-hydroxycoumarin [nmol / (g protein x min)]

3500

500 0

#

8

+

*

6

*

+ 4

#

+

2

BNF

PB

DEX

DMSO/NaCl

treatment Liver

PB

Spleen

control rats transplant recipients

female rats

BNF

DEX

treatment control rats transplant recipients

female rats

3500

10

3000

#

* * *

2500 2000 1500

#

#

* *

1000 #

#

* *

#

0 DMSO/NaCl

BNF

PB

DEX

treatment

7-hydroxycoumarin [nmol / (g protein x min)]

7-hydroxycoumarin [nmol / (g protein x min)]

#

+

0 DMSO/NaCl

500

control rats transplant recipients

male rats

8 6

+ 4

#

*

# 2

#

0 DMSO/NaCl

BNF

PB

DEX

treatment

Fig. 1. ECOD activities in 9000g supernatants of livers or spleens of male or female control rats and transplant-recipients 4 months after surgery and after administration of the vehicles 0.9% NaCl or DMSO or after treatment with BNF, PB or DEX before sacrifice. Ordinate scale: amount of the reaction product 7-hydroxycoumarin. Data are given as arithmetic mean ±S.E.M. Asterisks mark statistically significant differences from the values of the vehicle-treated rats, crosses mark statistically significant differences between transplant-recipients and controls and rhombs between male and female animals (∗,+,# P ≤ 0.05; Mann–Whitney test; n = 8 for each group).

male livers with control rats only. No gender differences were seen in liver EROD and PROD activities (Tables 1, 3, 4; Figs. 1–4). 3.1.2. Spleens With male rats basal EROD, ECOD and PNPH as well as 2␣-, 2␤-, 6␣-, 6␤-, 7␣-, 14␣-, 15␣-, 16␣-, 16␤- and 17-TH activities were significantly higher in transplant-containing organs than in control spleens. No differences were seen in PROD and 15␤-TH activities between male control rats and male transplant-recipients. Spleens of female transplant-recipients displayed more pronounced ac-

tivities than the respective organs of female control rats only with EROD, PNPH and 2␣-, 2␤-, 6␣-, 6␤-, 7␣-, 15␤- and 17-TH, but not with ECOD, PROD and 14␣-, 15␣-, 16␣- and 16␤-TH (Tables 2, 5, 6; Figs. 1–4). In spleens of control rats no differences were seen in the activities between males and females, with the only exception of 2␤-TH activity, which was higher in male than in female animals. Similar to the livers, with transplant-recipients, however, basal values of male rats were significantly higher with ECOD and 2␣-, 2␤-, 6␤-, 14␣-, 15␣-, 16␣-, 16␤- and 17-TH activities and of female animals with PNPH and 6␣- and 7␣-TH

A. Lupp et al. / Toxicology 197 (2004) 199–212

Liver

16

180

14

#

#

150

* *

120

#

*

*

#

* *

90 60 30 0

12 #

10

+

8 6

BNF

PB

+ #

DMSO/NaCl

Spleen

control rats transplant recipients

female rats

*

150

#

#

*

*

*

#

14

#

* *

90 60 30

p-nitrobrenzcatechine [nmol / (g protein x min)]

p-nitrobrenzcatechine [nmol / (g protein x min)]

#

180

0

DEX

16

210

120

PB

treatment control rats transplant recipients

female rats

#

+

#

BNF

treatment Liver

#

+

4

0

DEX

*

#

2 DMSO/NaCl

control rats transplant recipients

male rats

210 p-nitrobrenzcatechine [nmol / (g protein x min)]

p-nitrobrenzcatechine [nmol / (g protein x min)]

Spleen

control rats transplant recipients

male rats

207

+

*

12 10

#

#

#

8

+

+

+

6 #

4

#

2 DMSO/NaCl

BNF

PB

DEX

treatment

0

DMSO/NaCl

BNF

PB

DEX

treatment

Fig. 2. p-Nitrophenol hydroxylase activities in 9000g supernatants of livers or spleens of male or female control rats and transplant-recipients 4 months after surgery and after administration of the vehicles 0.9% NaCl or DMSO or after treatment with BNF, PB or DEX before sacrifice. Ordinate scale: amount of the reaction product p-nitrobrenzcatechine. Data are given as arithmetic mean ± S.E.M. Asterisks mark statistically significant differences from the values of the vehicle-treated rats, crosses mark statistically significant differences between transplant-recipients and controls and rhombs between male and female animals (∗,+,# P ≤ 0.05; Mann–Whitney test; n = 8 for each group).

activities. No gender differences were seen in EROD, PROD and 15␤-TH activities of transplant-containing spleens (Tables 2, 5, 6; Figs. 1–4). 3.2. Induction experiments 3.2.1. Livers Only occasionally significant differences in the values between control rats and transplant-recipients were noticed, as with ECOD, PROD and 15␤-TH activities in male rats after BNF treatment. In this instance ECOD and PROD activities were lower in transplant-recipients than in control rats, whereas the

opposite was the case with 15␤-TH activity (Tables 1, 3, 4; Figs. 1–4). BNF treatment strongly increased EROD and ECOD and to a lesser extent also PROD, PNPH and 7␣-TH activities in the livers of both male and female animals. Inducibility of EROD and ECOD activities was higher in females than in males. As with basal values, liver PNPH and 7␣-TH activities of female animals still exceeded those of male rats also after BNF administration. With both male and female animals 2␤-, 6␣-, 6␤-, 15␤-, 16␤- and 17-TH activities were not affected by BNF treatment. Even a decrease was seen with 2␣-TH activity in female

208

A. Lupp et al. / Toxicology 197 (2004) 199–212

Liver

Spleen

control rats transplant recipients

male rats

male rats 1400

6000

#

5000

#

4000

*

*

#

#

* *

3000 #

#

#

#

1000 0

6β-hydroxytestosterone [pmol / (g protein x min)]

6β-hydroxytestosterone [nmol / (g protein x min)]

7000

2000

1200

+

*

1000 800 #

400

#

+

DMSO/NaCl

BNF

PB

0

DEX

* *

DMSO/NaCl

1200

5000 4000

#

3000

#

2000

* #

DMSO/NaCl

#

BNF

*

# PB

*

DEX

treatment

6β-hydroxytestosterone [pmol / (g protein x min)]

6000

#

DEX

control rats transplant recipients

female rats 1400

#

PB

Spleen

7000

#

BNF

treatment control rats transplant recipients

female rats

6β-hydroxytestosterone [nmol / (g protein x min)]

#

+

200

Liver

0

+

600

treatment

1000

control rats transplant recipients

+

*

1000 800 600 400 200 0

# #

#

+

+

DMSO/NaCl

BNF

+

* PB

DEX

treatment

Fig. 3. Testosterone 6␤-hydroxylase activities in 9000g supernatants of livers or spleens of male or female control rats and transplant-recipients 4 months after surgery and after administration of the vehicles 0.9% NaCl or DMSO or after treatment with BNF, PB or DEX before sacrifice. Ordinate scale: amount of the reaction product 6␤-hydroxytestosterone. Data are given as arithmetic mean ± S.E.M. Asterisks mark statistically significant differences from the values of the vehicle-treated rats, crosses mark statistically significant differences between transplant-recipients and controls and rhombs between male and female animals (∗,+,# P ≤ 0.05; Mann–Whitney test; n = 8 for each group).

transplant-recipients, with 14␣-TH activity in both male transplant-recipients and controls, with 15␣-TH activity in male controls, and with 16␣-TH activity in both female transplant-recipients and controls (Tables 1, 3, 4; Figs. 1–4). After PB administration a distinct elevation in ECOD, PROD and 2␤-, 6␣-, 6␤-, 14␣-, 15␤-, 16␣-, 16␤- and 17-TH activities was observed with female and especially with male rats, the values of the male animals strongly exceeding those of the female rats. To a lesser extent also EROD activity was increased in males, with females in transplant-recipients only. 15␣-TH activity was elevated in male rats only. No effect on 15␣-TH activity was seen in female animals.

A slight increase in activity was also observed with both males and females with PNPH and 7␣-TH, the values of female animals (as with basal values) still being higher than those of male rats. 2␣-TH activity, in contrast, was decreased in both males and females (Tables 1, 3, 4; Figs. 1–4). DEX treatment caused a strong increase in 2␤-TH activity in the livers of male and especially of female rats. A distinct elevation in 6␤- and 15␤-TH activities was noticed for both sexes. In these two cases, however, the values of males still exceeded those of the female animals. To a lesser extent also PROD activities were enhanced after DEX administration, again (in parallel to uninduced values), the activities of male rats

A. Lupp et al. / Toxicology 197 (2004) 199–212

Liver

Spleen

control rats transplant recipients

male rats

male rats

1000 800 600 #

400 #

#

#

#

#

* *

* *

BNF

PB

DMSO/NaCl

#

#

7α-hydroxytestosterone [pmol / (g protein x min)]

7α-hydroxytestosterone [nmol / (g protein x min)]

800

1200

0

*

700 600 500 400

+

200

#

100

+

0

DEX

#

300

DMSO/NaCl

treatment Liver

#

#

BNF

PB

Spleen

* DEX

control rats transplant recipients

female rats 900

1400

800

1200

#

1000

#

800 #

#

#

#

* *

* *

#

#

* *

400

7α-hydroxytestosterone [pmol / (g protein x min)]

7α-hydroxytestosterone [nmol / (g protein x min)]

#

treatment control rats transplant recipients

female rats

600

control rats transplant recipients

900

1400

200

209

600

#

500

+

400

*

300 200

200

100

0

0

DMSO/NaCl

BNF

PB

DEX

treatment

#

+

700 #

*

+

*

#

+ # DMSO/NaCl

BNF

PB

DEX

treatment

Fig. 4. Testosterone 7␣-hydroxylase activities in 9000g supernatants of livers or spleens of male or female control rats and transplant-recipients 4 months after surgery and after administration of the vehicles 0.9% NaCl or DMSO or after treatment with BNF, PB or DEX before sacrifice. Ordinate scale: amount of the reaction product 7␣-hydroxytestosterone. Data are given as arithmetic mean ± S.E.M. Asterisks mark statistically significant differences from the values of the vehicle-treated rats, crosses mark statistically significant differences between transplant-recipients and controls and rhombs between male and female animals (∗,+,# P ≤ 0.05; Mann–Whitney test; n = 8 for each group).

still exceeding those of the female animals. ECOD, 7␣- and 16␣-TH activities were increased only in female, but not in male rats, whereas 15␣-TH activity was enhanced in male rats only. PNPH and 16␤-TH activities were only slightly increased with both groups of animals. 2␣-TH activity was decreased in males but not in females. No effect was seen on EROD, 6␣-, 14␣- and 17-TH activities (Tables 1, 3, 4; Figs. 1–4). 3.2.2. Spleens No influence of any of the three inducers was observed on the activities of control spleens of both male and female rats, with the only exception of an increase

in EROD and 6␤-TH activity after DEX treatment in male rats (Tables 2, 5, 6; Figs. 1–4). In transplant-containing spleens BNF treatment caused an increase in EROD and PNPH activities with both sexes similar to that seen with respective livers. As it was the case for basal PNPH activities, also after induction with BNF values of females were higher than those of male rats. ECOD activity was significantly enhanced in male, but not in female animals, yielding even higher values in males, whereas the already higher basal 7␣-TH activity of females was elevated in female animals only. 16␤-TH activity was also only increased in female but not in male rats.

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No effect due to the BNF treatment was observed on PROD and on 2␣-, 2␤-, 6␣-, 6␤-, 14␣-, 15␣-, 15␤-, 16␣- and 17-TH activities with both sexes (Tables 2, 5, 6; Figs. 1–4). After PB administration PROD and 6␣-, 6␤-, 14␣-, 16␣-, 16␤- and 17-TH activities were increased in the transplant-containing spleens of both sexes, with higher PROD, 6␤-, 14␣-, 16␣-, 16␤- and 17-TH activities in males and more pronounced 6␣-TH activities in female rats (as it was the case also for the basal values). EROD, ECOD, 2␤-, 15␣- and 15␤-TH activities were enhanced in male animals and 7␣-TH activities in females only. With both sexes PNPH and 2␣-TH activities of transplant-containing spleens remained unaffected by PB treatment (Tables 2, 5, 6; Figs. 1–4). DEX administration led to an increase in 2␤-, 6␤-, 7␣-, 15␣-, 15␤-, 16␣- and 17-TH activities with both sexes, with still significantly higher 17-TH-activities in male and more elevated 2␤-, 7␣- and 16␣-TH values in female rats. A significant increase in ECOD, PROD and 6␣- and 16␤-TH activities occurred in female animals only. No effect due to DEX with both male and female rats was observed on EROD, PNPH and 2␣- and 14␣-TH activities (Tables 2, 5, 6; Figs. 1–4).

4. Discussion Parallel to previous investigations on P450 isoforms expression (Lupp et al., 2003), the aim of the present study was to elucidate the influence of the recipient gender also on P450-mediated monooxygenase activities in intrasplenic liver tissue transplants in comparison to orthotopic livers. For this purpose, fetal liver tissue suspensions of mixed sex were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting, basal and induced P450-mediated monooxygenase activities were measured in liver and spleen 9000g supernatants by a series of model reactions for different P450 subtypes. Regular basal EROD, ECOD and PROD activities as described in our previous investigations (Lupp et al., 1998c, 1999a) and (in the case of PNPH and TH) in the range of literature data (Fujita et al., 1995; Niwa et al., 1995; Shimada et al., 1997; Morel et al., 1999) were seen in the orthotopic livers of both transplant-recipients and control rats. As to be

expected from literature (Niwa et al., 1995; Lewis, 1996; Shimada et al., 1997) higher ECOD, PROD and 2␣-, 2␤-, 6␤-, 14␣-, 15␣-, 15␤-, 16␣-, 16␤- and 17-TH activities are measured in the livers of male than of female rats and more elevated EROD, PNPH and 6␣- and 7␣-TH activities in the livers of female than of male rats. In comparison to the livers only negligible monooxygenase activities were seen in the control spleens as shown already previously (Lupp et al., 1998c, 1999a). In transplant-containing organs, however, distinct activities were observed with most model reactions. As seen in the respective livers, spleens possessing transplants of either sex hosted by male rats displayed higher basal ECOD and 2␣-, 2␤-, 6␤-, 14␣-, 15␣-, 16␣-, 16␤- and 17-TH activities than those hosted by female animals. On the other hand, more distinct PNPH and 6␣- and 7␣-TH activities were seen with spleens of female than with those of male transplant-recipients. In the present study PNPH and TH activities in transplant-containing spleens have been demonstrated for the first time. Respective literature data are not available so far. No sex-specific differences could be demonstrated in the present study with EROD, PROD and 15␤-TH activities since only very low values were measured with these model reactions in the graft-containing organs of both male and female transplant-recipients. It can be concluded, however, that the sex-specific differences in basal P450-mediated monooxygenase activities as seen in the livers could clearly also be established in the intrasplenic transplants. Thus, with respect to constitutive P450-mediated monooxygenase functions the intrasplenic transplants seem to be influenced in the same way by the sex-specific factors such as the GH secretion profile as the livers. These results correspond well to our previously reported immunohistochemical data (Lupp et al., 2003). As to be expected from literature and from our previous results (Traber et al., 1989; Kato et al., 1996; Lupp et al., 1999a), the characteristic effects of the three inducers on the model reactions for P450-mediated monooxygenase activities are exerted in a similar way in livers and intrasplenic transplants. Moreover, as to be expected from our immunohistochemical data (Lupp et al., 2003), the gender-specific differences in inducibility of the different model reactions by BNF, PB or DEX as seen in livers could be demonstrated similarly also in the

A. Lupp et al. / Toxicology 197 (2004) 199–212

transplant-containing spleens. This, again, proves that P450-mediated monooxygenase functions in the intrasplenic transplants are controlled in the same way by sex-specific influences such as the GH secretion profile as those in the livers. Irrespective of a treatment with the inducers, with both sexes no differences were noticed in monooxygenase activities between the livers of transplantrecipients and control rats. Thus, the transplanted liver cells within the spleens do not seem to exert any regulative influence on the respective livers. In summary, the results of the present investigation further prove that the P450 system of intrasplenic liver tissue transplants is influenced by the recipient gender like that of the respective orthotopic livers. For a possible therapeutic use of ectopic transplants in patients a profound knowledge of the characteristics of these transplants in comparison to the orthotopic liver is of great importance. In line with previously reported results also in the present investigation a striking similarity between the ectopically transplanted and the respective cells of the orthotopic livers was observed with respect to different properties including response to the influence of xenobiotics or endogenous factors. Altogether the results support the clinical evidence that intrasplenic liver cell transplants in some case may be suitable as a therapeutic alternative for whole liver transplantation.

Acknowledgements We gratefully thank Mrs. H. Stadler for excellent technical assistance.

References Agrawal, A.K., Shapiro, B.H., 2000. Differential expression of gender-dependent hepatic isoforms of cytochrome P-450 by pulse signals in the circulating masculine episodic growth hormone profile on the rat. JPET 292, 228–237. Agrawal, A.K., Shapiro, B.H., 2001. Intrinsic signals in the sexually dimorphic circulating growth hormone profiles of the rat. Mol. Cell. Endocrinol. 173, 167–181. Aitio, A., 1978. A simple and sensitive assay of 7-ethoxycoumarin deethylation. Anal. Biochem. 85, 488–491. Bilir, B.M., Guinette, D., Karrer, F., Kumpe, D.A., Krysl, J., Stephens, J., McGavran, L., Ostrowska, A., Durham, J., 2000.

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Hepatocyte transplantation in acute liver failure. Liver Transpl. 6, 32–40. Chang, T.K.H., Crespi, C.L., Waxman, D.J., 1997. Spectrophotometric analysis of human CYP2E1-catalyzed p-nitrophenol hydroxylation. In: Phillips, I.R., Shephard, E.A. (Eds.), Methods in Molecular Biology: Cytochrome P450 Protocols, vol. 107. Humana Press, Totowa, pp. 147–152. Correia, M.A., 1995. Rat and human liver cytochrome P450: substrate and inhibitor specificities and functional markers. In: Ortiz de Montellano, P.R. (Ed.), Cytochrome P450: Structure, Mechanism and Biochemistry. Plenum Press, New York, London, pp. 607–630. Fujita, I., Sindhu, R.K., Kikkawa, Y., 1995. Hepatic cytochrome P450 enzyme imprinting in adult rat by neonatal benzo[a]pyrene administration. Pediatr. Res. 37, 646–651. Habibullah, C.M., Syed, I.C.H., Qamar, A., Taher-Zu, Z., 1994. Human fetal hepatocyte transplantation in patients with fulminant hepatic failure. Transplantation 58, 951–952. Kato, K., Hodgson, W.J.B., Lutton, J.D., Abraham, N.G., 1994. Developmental expression of cytochrome P450s within intrasplenically transplanted fetal hepatocytes from spontaneously hypertensive rats. Cell Transplant. 3, 15–21. Kato, K., Hodgson, W.J.B., Abraham, N.G., Onodera, K., Matsuda, M., Kasai, S., Mito, M., 1995. Effect of 70% partial hepatectomy on developmental expression of cytochrome P450 IVA1 within intrasplenically transplanted fetal hepatocytes from spontaneously hypertensive rats. Intern. Hepatol. Commun. 3, 254–259. Kato, K., Hodgson, W.J.B., Abraham, N.G., Onodera, K., Imai, M., Kasai, S., Mito, M., 1996. Expression and inducibility of cytochrome P450 IIIA family within intrasplenically transplanted fetal hepatocytes. Cell Transplant. 5, 117–122. Klinger, W., Müller, D., 1974. The influence of age on the protein concentration in serum, liver and kidney of rats determined by various methods. Z. Versuchstierk. 16, 146–153. Lake, B.G., Renwick, A.B., Cunninghame, M.E., Price, R.J., Surry, D., Evans, D.C., 1998. Comparison of the effects of some CYP3A and other enzyme inducers on replicative DNA synthesis and cytochrome P450 isoforms in rat liver. Toxicology 131, 9–20. Lewis, D.F.V., 1996. Cytochromes P450: Structure, Function and Mechanism. Taylor & Francis, London. Legraverend, C., Mode, A., Westin, S., Ström, A., Eguchi, H., Zaphiropoulos, P.G., Gustafsson, J.A., 1992a. Transcriptional regulation of P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion. Mol. Endocrinol. 6, 259–266. Legraverend, C., Mode, A., Wells, T., Robinson, I., Gustafsson, J.A., 1992b. Hepatic steroid hydroxylating enzymes are controlled by the sexually dimorphic pattern of growth hormone secretion in normal and dwarf rats. FASEB J. 6, 711–718. Lubet, R.A., Mayer, R.T., Cameron, J.W., Nims, R.W., Burke, M.D., Wolff, T., Guengerich, F.P., 1985. Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in rat. Arch. Biochem. Biophys. 238, 43–48.

212

A. Lupp et al. / Toxicology 197 (2004) 199–212

Lupp, A., Lucas, N., Lindström-Seppä, P., Koponen, K., Hänninen, O., Danz, M., Klinger, W., 1998a. Developmental expression of cytochrome P450 isoforms after transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats. Exp. Toxicol. Pathol. 50, 41–51. Lupp, A., Lucas, N., Lindström-Seppä, P., Koponen, K., Hänninen, O., Danz, M., Klinger, W., 1998b. Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of ␤-naphthoflavone, phenobarbital and dexamethasone on cytochrome P450 isoforms expression and on glycogen storage. Exp. Toxicol. Pathol. 50, 173– 183. Lupp, A., Trautmann, A.K., Krausse, T., Klinger, W., 1998c. Developmental changes of cytochrome P450 dependent monooxygenase functions after transplantation of fetal liver tissue suspension into spleens of adult rats. Exp. Toxicol. Pathol. 50, 239–244. Lupp, A., Lau, K., Trautmann, A.K., Krausse, T., Klinger, W., 1999a. Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: inducibility of cytochrome P450 dependent monooxygenase functions by ␤-naphthoflavone, phenobarbital and dexamethasone. Exp. Toxicol. Pathol. 51, 65–74. Lupp, A., Tralls, M., Fuchs, U., Lucas, N., Danz, M., Klinger, W., 1999b. Transplantation of fetal liver tissue suspensions into the spleens of adult syngenic rats: effects of various mitogens and cytotoxins on cytochrome P450 (P450) isoforms expression and on P450 mediated monooxygenase functions. Exp. Toxicol. Pathol. 51, 375–388. Lupp, A., Hugenschmidt, S., Danz, M., Müller, D., 2003. Influence of recipient gender on cytochrome P450 isoforms expression in intrasplenic fetal liver tissue transplants in rats. Toxicology 188, 171–186. Maganto, P., Traber, P.G., Rusnell, C., Dobbins, W.O., Keren, D., Gumucio, J.J., 1990. Long-term maintenance of the adult pattern of liver-specific expression for P-450b, P-450e, albumin and alpha-fetoprotein genes in intrasplenically transplanted hepatocytes. Hepatology 11, 585–592. Mito, M., Kusano, M., 1993. Hepatocyte transplantation in man. Cell Transplant. 2, 65–74. Morel, G., Cossec, B., Lambert, A.M., Binet, S., 1999. Evaluation of rat hepatic 2E1 activity in function of age, sex and inducers: choice of an experimental model capable of testing the hepatotoxicity of low molecular weight compounds. Toxicol. Lett. 106, 171–180.

Niwa, T., Kaneko, H., Naritomi, Y., Togawa, A., Shiraga, T., Iwasaki, K., Tozuka, Z., Hata, T., 1995. Species and sex differences of testosterone and nifedipine oxidation in liver microsomes of rat, dog and monkey. Xenobiotica 25, 1041– 1049. Pampori, N.A., Agrawal, A.K., Shapiro, B.H., 2001. Infusion of gender-dependent plasma growth hormone profiles into intact rats: effects of subcutaneous, intraperitoneal, and intravenous routes of rat and human growth hormone on endogenous circulating growth hormone profiles and expression of sexually dimorphic hepatic cyp isoforms. Drug Metab. Dispos. 29, 8–16. Pohl, R.J., Fouts, J.R., 1980. A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions. Anal. Biochem. 107, 150–155. Shapiro, B.H., Agrawal, A.K., Pampori, N.A., 1995. Gender differences in drug metabolism regulated by growth hormone. Int. J. Biochem. Cell Biol. 27, 9–20. Shimada, M., Murayama, N., Nagata, K., Hashimoto, H., Ishikawa, H., Yamazoe, Y., 1997. A specific loss of growth hormone abolished sex-dependent expression of hepatic cytochrome P450 in dwarf rats: reversal of the profiles by growth hormone treatment. Arch. Biochem. Biophys. 337, 34–42. Strom, S.C., Chowdhury, J.R., Fox, I.J., 1999. Hepatocyte transplantation for the treatment of human disease. Semin. Liver Dis. 19, 39–48. Traber, P.G., Maganto, P., Wojcik, E., Keren, D., Gumucio, J.J., 1989. Induction of P-450IIB genes within the rat liver acinus is not dependent on the chemical inducer or on the acinar organization. J. Biol. Chem. 264, 10292–10298. Waxman, D.J., 1992. Regulation of liver specific steroid metabolizing cytochromes P-450: cholesterol 7␣-hydroxylase, bile acid 6␤-hydroxylase, and growth-hormone-responsive steroid hormone hydroxylases. J. Steroid Biochem. Mol. Biol. 43, 1055–1072. Waxman, D.J., Ram, P.A., Pampori, N.A., Shapiro, B.H., 1995. Growth hormone regulation of male-specific rat liver P-450s 2A2 and 3A2: induction by intermittent growth hormone pulses in male but not female rats rendered growth hormone deficient by neonatal monosodium glutamate. Mol. Pharmacol. 48, 790– 797. Westin, S., Ström, A., Gustafsson, J.A., Zaphiropoulos, P.G., 1990. Growth hormone regulation of the cytochrome P-450 IIC subfamily in the rat: inductive, repressive and transcriptional effects on P-450f (IIC7) and P-450PB1 (IIC6) gene expression. Mol. Pharmacol. 38, 192–197.