Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 mediated monooxygenase functions and on oxidative state

Exp Toxic Pathol 200 I; 52: 529-538 URBAN & FISCHER http://www.urbanfischer.de/journals/exptoxpath

Institute of Pharmacology and Toxicology, Friedrich Schiller University lena, Germany

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 mediated monooxygenase functions and on oxidative state* A. Lupp, M. TRALLS, U. FUCHS, and W. KLINGER With 5 figures Received: November 20, 1999; Accepted: December 2, 1999 Address for correspondence: Dr. med. A. Lupp, Institute of Pharmacology and Toxicology, Friedrich Schiller University lena, Nonnenplan 4, D - 07743 lena, Germany; Fax: +49-3641-938702, e-mail: [email protected] Key words: Fetal hepatocytes; Spleen; Transplantation; Cytochrome P450; Monooxygenase functions; Oxidative state; Allyl alcohol; Bromobenzene; Carbon tetrachloride; Thioacetamide. Abbreviations: AAL: allyl alcohol; BBZ: bromobenzene; b.wt.: body weight; CCI 4 : carbon tetrachloride; DMSO: dimethylsulfoxide; ECOD: ethoxycoumarin O-deethylation; EMND: ethylmorphine N-demethylation; EROD: ethoxyresorufin O-deethylation; GSH: reduced glutathione; GSSG: oxidized glutathione; LPO: lipid peroxidation; P450: cytochrome P450; PROD: pentoxyresorufin O-depentylation; TAA: thioacetamide.

Summary Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCI 4 ], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on P450 mediated monooxygenase functions in liver and spleen 9,000g supernatants were assessed by measuring the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD), and ethylmorphine N-demethylation (EMND). Additionally, the influence on the oxidative state was investigated by assessing the liver and spleen tissue content of lipid peroxidation (LPO) products and of reduced and oxidized glutathione (GSH; GSSG). The livers of both solvent treated transplant recipients and control rats displayed regular EROD, ECOD, PROD and EMND activities. After AAL treatment EROD and EMND activities within the livers were not affected, but ECOD and PROD activities were increased. BBZ adminis-

* Part of the results was presented as a poster at the 16th European Workshop on Drug Metabolism 1998 in Kopenhagen, Denmark (proceedings published in Exp Toxic Pathol 1999; 51: 375-388).

tration caused a decrease in EROD and EMND activities, ECOD activity remained unaffected, and PROD activity was even increased. CCI 4 and TAA administration caused a strong reduction in the activity of all four model reactions. Spleens of control rats displayed almost no P450 mediated monooxygenase functions, independent whether the rats had been treated with the cytotoxins or not. In the transplant containing spleens, however, significant EROD and ECOD, but hardly any PROD or EMND activities were seen. After AAL administration EROD activity was not affected in the transplant containing spleens, but ECOD activity was increased. BBZ treatment led to a decrease in EROD and an elevation in ECOD activity. CCI 4 and TAA strongly reduced the activity of both of these model reactions. The tissue content of LPO products within livers and transplant containing spleens was significantly increased after BBZ and CCI 4 treatment. An elevation in LPO products was also seen in the spleens of the control rats due to CCI 4 administration. Tissue GSH and GSSG content in both livers and transplant containing spleens were strongly reduced after BBZ treatment. After CCl 4 administration only a significant decrease in liver GSSG contents was seen. TAA treatment caused a reduction in the GSH and GSSG content in the spleens of both transplant recipients and control rats, but not in the livers. From these results it can be concluded, that the effects of cytotoxins like AAL, BBZ, CCI 4 or TAA on P450 dependent monooxygenase functions and on oxidative state are exerted in the ectopic intrasplenic liver cell transplants in a similar way as in normal orthotopic liver. 0940-2993/01/52/06-529 $ 15.0010

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Introduction Previously we had been able to demonstrate that transplanted syngenic fetal rat liver cells are able to survive in spleens of adult transplant recipients for at least I year (Lupp et al. 1998a, b, c; 1999a). Similar to normal orthotopic liver, the transplanted ectopic hepatocytes had displayed P450 isoforms expression, P450 dependent monooxygenase functions and glycogen storage. With respect to these parameters developmental changes had been observed in the transplants. P450 isoforms expression and P450 mediated monooxygenase functions had been inducible, as in normal orthotopic liver, by pretreatment of the rats with ~-naphthoflavone, phenobarbital or dexamethasone. Recently we reported about the effects of different cytotoxins on the intrasplenic transplants in comparison to normal liver (Lupp et al. 2000). Four compounds with characteristic zonal toxic effects within normal orthotopic liver had been examined: allyl alcohol (AAL), exerting toxic effects within the livers primarily on the periportal hepatocytes (BELINSKY et al. 1984; KLINGER et al. 1986), and bromobenzene (BBZ), carbon tetrachloride (CCI 4 ) and thioacetamide (TAA), all three leading to cell damage mainly in the perivenous zones of the liver lobules (see e.g. ASHWORTH et al. 1965; BRODIE et al. 1971; GROOTHUIS et al. 1983; KLINGER et al. 1986; JAHN et al. 1993). AAL is metabolized, mainly by alcohol dehydrogenase, to the toxin acrolein (OHNO et al. 1985; ATZORI et al. 1989), but also the involvement of certain P450 isozymes in its metabolization is discussed (KLINGER et al. 1986). BBZ and CCl 4 are activated to reactive species by the P450 system, BBZ mainly by phenobarbital sensitive isoforms and CCI 4 by 2B 1/2 and 2E I subtypes (see e.g. ANDERS and POHL 1985; LAU and MONKS 1988; ALLIS et al. 1996; GRUEBELE et al. 1996; KIM et al. 1996; WANG et al. 1998). TAA is predominantly activated by microsomal flavin-containing monooxygenases (DE FERREYRA et al. 1983; CHIELI and MALVALDI 1984; SANZ et al. 1998). Both TAA and its metabolite thioacetamide-S-oxide are known to inhibit heme biosynthesis thereby affecting P450 concentration (MATSUURA et al. 1983; SATYABHAMA and PADMANABAN 1984; SATYABHAMA et al. 1986; BHAT and PADMANABAN, 1988; SALONPAA et al. 1995). Besides TAA also the other three cytotoxins are known to lead to a decline in liver P450 content and/or monooxygenase functions (see e.g. GUMBRECHT and FRANKLIN 1983; KLINGER et al. 1986; BEYHL and MAYER 1987; SESARDIC et al. 1989; JAHN et al. 1993; TANAKA et al. 1994). Thus, besides effects on morphology and glycogen content, in our previous studies also the influence of the four cytotoxins on the expression of three P450 isoforms (I A I, 2B I and 3A2) had been examined in the transplants in comparison to normal liver. In parallel to these previous investigations, the aim of the present study was to further examine the influence of the four cytotoxins on P450 mediated monooxygenase 530

Exp Toxic Pathol 52 (2001) 6

functions within intrasplenic liver cell transplants in comparison to normal liver. Effects on P450 mediated monooxgenase functions were measured by four model reactions for different P450 subtypes (CORREIA 1995; LEWIS 1996): ethoxyresorufin O-deethylation (EROD; mainly IA); ethoxycoumarin O-deethylation (ECOD; predominantly I A, 2A, 2B, 2C); pentoxyresorufin O-depentylation (PROD; primarily 2B); ethylmorphine Ndemethylation (EMND; mainly 3A). Additionally, effects of the four cytotoxins on the oxidative state within livers and spleens were assessed by measuring the tissue content of lipid peroxidation (LPO) products and of reduced and oxidized glutathione (GSH; GSSG).

Material and methods Animals: Fisher 344 inbred rats from our own institute's breed were used. The animals were housed in plastic cages under standardized conditions (light-dark cycle 12/12 h, temperature 22 ± 2°C, humidity 50 ± 10%, pellet diet Altromin 1316, water ad libitum). Donor-fetuses were taken from pregnant Fisher inbred rats at the 21 st day of gestation. The fetal livers were immediately removed, pooled and minced by razor blades in 4 °C cold Hank's balanced salt solution (l:2 w/v) until a homogeneous suspension was obtained. Recipients were syngenic 60-90 days old male rats. After laparotomy, spleens of the recipients were injected 0.2 ml of the fetal liver tissue suspension in longitudinal direction. Subsequently the abdomen was closed again with two sutures on top of each other. Age matched control animals received no surgery. No immunosuppressant was given to the transplant recipients. Pretreatment of the rats with the cytotoxins: Four months after transplantation the recipients and age matched control rats were divided into four groups as follows (n =6-10 per group and treatment): (I) transplant recipients treated with the cytotoxins; (2) transplant recipients treated with the respective solvents; (3) control rats treated with the cytotoxins; (4) control rats treated with the respective solvents. • AAL: administered once orally as a 2% solution in DMSO at a volume of 3 mllkg b.wt. Solvent treated rats received once orally 3 mllkg b.wt. DMSO. Animals were sacrificed 24 hours after the treatment. • BBZ: given once as a 1:4 (v/v) dilution in com oil at a volume of 1.5 mllkg b. wt. intraperitoneally. Solvent treated rats received once 1.5 mllkg b.wt. com oil intraperitoneally. Animals were sacrificed 24 hours after the treatment. • CCI 4 : given once as a I: 10 (v/v) dilution in com oil at a volume of 0.5 mllkg b.wt. intraperitoneally. Solvent treated rats received once 0.5 mllkg b. wt. com oil intraperitoneally. Animals were sacrificed 24 hours after the treatment. • TAA: given once intraperitoneally at a dosage of 50 mglkg b.wt., dissolved in 5 mllkg b.wt. 0.9% NaCl. Solvent treated rats received once intraperitoneally 5 mllkg b.wt. 0.9% NaCI. Animals were sacrificed in ether anesthesia 48 hours after the treatment.

Preparation of 9,000 g supernatants: The livers and spleens were homogenized in 0.1 M sodium phosphate buffer pH 7.4 (1:3 w/v). Subsequently, the homogenate was centrifuged at 9,000 x g for 20 min at 4°C. The protein content of the 9,000 g supernatants was determined with the Biuret method as modified according to KLINGER and MOLLER (1974). Monooxygenase functions: EROD was measured according to POHL and FOUTS (1980) with modifications, and PROD as described by LUBET et al. (1985), fluorimetrically determining the metabolite resorufin. ECOD was performed by assessing the main metabolite 7-hydroxycoumarin fluorimetrically (AITIo 1978), and EMND by determining the reaction product formaldehyde photometrically (KLINGER and MOLLER 1977). The activity of all three model reactions was referred to the protein content of the 9,000 g supernatants. Monooxygenase functions within the transplant containing spleens were measured in the 9,000 g supernatants of the entire organs. Oxidative state: For the determination of the tissue content of lipid peroxidation (LPO) products as thiobarbituric acid reactive substances (TBARS) liver and spleen samples were homogenized with 19 volumes of ice cold saline and the analysis was carried out fluorimetrically as described by YAGI (1987). Tissue content of glutathione in its reduced (GSH) and oxidized (GSSG) state was determined according to ELLMAN (1959) and HISSIN and HILF (1976), respectively. Liver and spleen samples were homogenized with 11 volumes of 0.2 M sodium phosphate buffer (5 mM EDTA; pH 8.0) and 4 volumes of 25% metaphosphoric acid. After centrifugation (12,000 g, 4°C; 30 min) GSH was determined in the supernatants photometrically and GSSG was measured fluorimetrically. Statistics: The number of animals investigated per group comprised n = 6-10. All results are expressed as arithmetic means + S.E.M. For statistical analysis the Mann-Whitney test (p ~ 0.05) was applied.

Results Protein content After AAL and BBZ treatment no influence on the protein content of the livers and spleens of transplant recipients and of control rats was observed (not shown). CCI 4 and TAA administration, however, led to a decrease in the protein content of the livers of both groups of rats by about 17%. No effect was seen on transplant containing and control spleens (not shown).

Monooxygenase functions Monooxygenase activities in the livers were in no case influenced by the intrasplenic transplants. In all experiments the livers of solvent treated animals displayed regular EROD, ECOD, PROD and EMND activities (see figs. 1-3).

Almost no monooxygenase functions were detectable in the spleens of the control rats, independent of a treatment of the animals with the solvents or with the cytotoxins (see figs. 1-3). In contrast, and as already demonstrated previously (Lupp et al. 1998c; 1999a), in all experiments significant EROD and ECOD activities were measured in the transplant containing spleens after treatment of the rats with the solvents only. These activities in the 9,000 g supernatants of the entire organs amounted to about 1% or 5%, respectively, of the values observed in the livers of the solvent treated rats (see figs. 1-3). However, in the transplant containing spleens almost no PROD and EMND activities were seen. AAL treatment: EROD activity within the livers and transplant containing spleens was not affected due to AAL administration (fig. lA). In contrast, after AAL treatment ECOD activity was significantly enhanced in the livers by about 55% and in transplant containing spleens even by 195% (fig. 2A). Similarly to ECOD activity in the livers also PROD activity was increased due to AAL treatment by about 40%. In the transplant containing spleens, however, the (very low) PROD activity was only slightly, but not significantly, elevated (not shown). In both livers and transplant containing spleens EMND activity was not influenced (fig. 3A). BBZ treatment: Within the livers and transplant containing spleens EROD activity was significantly decreased due to BBZ administration by about 55% or 40%, respectively (fig. 1B). ECOD activity in the livers was not affected. In transplant containing spleens, however, the ECOD activity was increased by 65% (figure 2B). Liver PROD activity was significantly enhanced due to BBZ treatment by about 100%. The (very weak) PROD activity seen in the transplant containing spleens was not affected (not shown). Similar to EROD activity, also EMND activity was reduced in the livers by about 50% after BBZ administration. In contrast, a significant increase in the activity by about 75% was observed in the spleens of both transplant recipients and control rats (fig. 3B). CCI 4 treatment: In the CCl 4 treated rats the previously described severe tissue damage (Lupp et al. 2000) is apparently mirrored by the changes in the enzyme activities, even as referred to the (lower) protein content of the livers. EROD activity within the livers was strongly decreased by about 80% and also in the intrasplenic transplants a significant reduction of the activity by 65% was observed (fig. 1C). Quantitatively the same effects were seen with the ECOD activity: in the livers CCl 4 caused a reduction of the activity by about 80% and in the transplant containing spleens by 75% (fig. 2C). Exp Toxic Pathol52 (2001) 6

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Fig. 1. Ethoxyresorufin O-deethylation activity in 9,000 g supernatants of livers and spleens of control rats and of transplant recipients after administration of the solvents or of allyl alcohol (A), bromobenzene (B), carbon tetrachloride (C), or thioacetamide (D) 4 months after surgery. Ordinate scale: amount of the reaction product resorufin. Data are given as arithmetic means + S.E.M. Asterisks mark statistically significant differences from the values of the solvent treated rats (*p ~ 0.05; Mann-Whitney test; n = 6-8 for each group). Fig. 2. Ethoxycoumarin O-deethylation activity in 9,000 g supernatants of livers and spleens of control rats and of transplant recipients after administration of the solvents or of allyl alcohol (A), bromobenzene (B), carbon tetrachloride (C), or thioacetamide (D) 4 months after surgery. Ordinate scale: amount of the reaction product 7-hydroxycoumarin. Data are given as arithmetic means + S.E.M. Asterisks mark statistically significant differences from the values of the solvent treated rats (*p ~ 0.05; Mann-Whitney test; n = 6-8 for each group). Exp Toxic Pathol 52 (200 I) 6

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almost no PROD activity was visible, independent whether the rats had been treated with TAA or not (not shown). Similarly, EMND activity was only decreased in the livers by about 65%. The (already very low) EMND activity within the transplant containing spleens was not affected due to TAA treatment (fig. 3D).

Oxidative state Tissue content of LPO products: The transplants within the spleens did not affect the concentration of LPO products within the livers independent whether the rats had been treated with the solvents or with the cytotoxins. In general the tissue content of LPO products in spleens was about two times as high as in livers with slightly, but not significantly higher values in transplant containing organs. No effect on tissue LPO products content within livers and spleens was seen after AAL and TAA treatment (fig. 4A, 40). BBZ administration led to a significant increase in the concentration of LPO products within the livers by 534

Exp Toxic Pathol 52 (200 1) 6

about 30% and in the transplant containing spleens by about 60%. No effect was seen on the spleens of the control rats (fig. 4B). After CCl4 administration a strong augmentation in the content of LPO products was seen in the livers by about 230% and in the spleens of both transplant recipients and control animals by about 100% (fig. 4C). Tissue GSH and GSSG content: Between transplant recipients and control rats no significant differences in liver GSH and GSSG values were observed, neither after treatment with the solvents nor after administration of the cytotoxins. Spleen GSH and GSSG concentrations in general did not significantly differ from the values of the respective livers. There were no significant differences between the GSH and GSSG contents of transplant containing spleens and control organs. Liver and spleen GSH or GSSG content was not affected due to AAL administration (fig. 5A). After BBZ treatment the GSH and GSSG concentrations within the livers were strongly reduced by about 70% or 50%, respectively. Also within the transplant

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containing spleens aSH and GSSa contents were diminished by about 50% or 15%, respectively. No effect was seen on the spleens ofthe control rats (fig. 5B). CCl4 administration caused no effect on liver GSH and a slight reduction in liver GSSa values by about 10%. GSH and GSSG contents of the spleens of both transplant recipients and control rats were not affected due to CC1 4 treatment (fig. 5C). In the livers GSH and GSSG content were not affected due to TAA treatment. In contrast, a significant decrease in the values of the two parameters by about 20% or 10%, respectively, was observed similarly in the spleens of both transplant recipients and control rats (fig. 50).

Discussion The present study was conducted in parallel to previous investigations on the effects of different cytotoxins with characteristic zonal toxic effects in normal orthotopic liver on morphology, P450 isoforms expression

and glycogen content in intrasplenic liver cell explants in comparison to normal liver (Lupp et al. 2000). For that purpose AAL, exerting necroses in the periportal zones of the liver lobules, and BBZ, CC1 4 and TAA, leading to perivenous cell damages in the liver lobules, had been examined in these previous studies. The aim of the present investigation was to further investigate the effects of these cytotoxins in the ectopic intrasplenic liver cell explants in comparison to normal orthotopic liver with respect to P450 mediated monooxygenase functions (in parallel to the already studied effects on P450 isoforms expression). Additionally, possible effects of the cytotoxins on the oxidative state in both transplant containing spleens and livers were investigated by assessing the tissue content of LPO products and ofGSH and GSSG. As shown previously (Lupp et al. 1998c; 1999a), also in the present investigation the normal orthotopic livers displayed regular monooxygenase functions as assessed by EROO, ECOO, PROD and EMNO activities. Also in the present study the transplanted liver cells within the spleens seemed to exert no (regulative) influence on the Exp Toxic Pathol 52 (200 I) 6

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respective livers, since in no case differences in monooxygenase activities and oxidative state between the livers of transplant recipients and control rats were observed. As in our previous findings (Lvpp et al. 1998c; 1999a), the transplant containing spleens displayed significant EROD and ECOD, but no EMND activity. This lack of EMND activity, however, might be explained by a possible minor sensitivity of this model reaction for low P450 contents, since a noticeable testosterone 6Phydroxylase activity can be observed in the transplant containing spleens, which additionally is inducible by dexamethasone, indicating the presence of 3A isoforms (own preliminary results). Also the only minor PROD activity found in the transplant containing spleens may be due to a low sensitivity of this model reaction. AAL is known to produce damages in periportal hepatocytes (BELINSKY et al. 1984; KLINGER et al. 1986). In our previous study only minor such effects had been seen (Lvpp et al. 2000). As had been expected from literature (ASHWORTH et al. 1965; BRODIE et al. 1971; GROOTHVIS et al. 1983; KLINGER et al. 1986; JAHN et al. 1993) in our previous investigations BBZ, CCI 4 and TAA had exerted their toxic effects mainly in the perivenous zones of the liver lobules (Lvpp et al. 2000). Only weak tissue damaging effects had been seen after BBZ, whereas TAA, and especially CCI4 , had caused strong necrogenic effects. Similar to liver also in the intrasplenic transplants fatty degeneration and necrosis of hepatocytes had been seen after treatment with any of the four cytotoxins. Also in the transplant containing spleens the toxic effects of the AAL and BBZ treatment had been not very prominent, whereas the effects seen after TAA and CCI4 administration had been much more noticeable. All four cytotoxins are known to lead to a decline in liver P450 content and/or monooxygenase functions (GVMBRECHT and FRANKLIN 1983; SATYABHAMA and PADMANABAN 1984; KLINGER et al. 1986; BEYHL and MAYER 1987; SESARDIC et al. 1989; JAHN et al. 1993; TANAKA et al. 1994). In the present study this expected reducing effect on monooxygenase functions was strongest after administration of CCI4 and TAA and was seen in the livers in the activities of all four model reactions. These results correspond well with our previous findings (Lvpp et al. 2000), where in the livers CCI 4 and TAA had led to a remarkable reduction in the expression of all three P450 isoforms tested. One of the reasons for these effects on both P450 subtypes expression and monooxygenase functions due to CCI 4 or TAA treatment is probably the remarkable cell damage described in the previous investigations (Lvpp et al. 2000) with secondary loss of P450 isozymes by leakage. On the other hand CCI 4 may also cause a direct (suicidal) impairment of the P450 enzymes, especially (but probably not solely) of the P450 2B and 2E subtype which are responsible for its activation (see e.g. RECKNAGEL and GLENDE 1973; SESARDIC et al. 1989; MANNO et al. 1992; MOODY 1992; ALLIS et al. 536

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1996; GRVEBELE et al. 1996; KIM et al. 1996). The same seems also to apply for the intrasplenic transplants. In comparison to CCI 4 and TAA treatment, after BBZ administration only EROD and EMND activities were significantly lower in the livers, ECOD was not affected, and PROD activity was even increased. Also these results fit well to our previous investigations (Lvpp et al. 2000), where only the perivenous P450 IAI expression had been diminished, and the P450 2B I and 3A2 isoforms expression had been mainly decreased by a numerical reduction of the immunostained cells due to fatty degeneration and necrosis, but not due to a change in the intensity of staining. Similar to liver, in the intrasplenic transplants only EROD was decreased and ECOD was increased. This, again, corresponds well with the previously noticed decrease in P450 IA I expression and the only less frequent but not less intense staining for P450 2B I and 3A2 due to hepatocyte fatty degeneration and necrosis (LvPP et al. 2000). After AAL treatment in the livers EROD and EMND activities remained unaffected and ECOD and PROD activities were enhanced, fitting well to the data obtained from our previous investigations. Only the number of P450 3A2 expressing hepatocytes had been reduced within the livers but not the intensity of staining. No influence had been seen on the P450 IAI expression, and the staining for P450 2B I was even more pronounced (Lvpp et al. 2000). Similar to liver, in the transplant containing spleens EROD activity remained unaffected, but ECOD activity was enhanced, again corresponding to the previous findings, where the P450 IAI expression had not been affected due to the treatment, and the staining for P450 2B I and 3A2 had been only less frequent due to the cell damage but not less in intensity. Thus, the present results support the previously made assumption (Lvpp et al. 2000), that BBZ and AAL at subnecrogenic dosages display an induction at least of the P450 2BI (and probably also IAI and 3A2) isoforms expression. The decrease in monooxygenase activities due to AAL and BBZ observed in literature (especially with model reactions for P450 2B and 3A subtypes) must be attributed rather to a marked cell damage with consecutive leakage of hepatocellular enzymes into the bloodstream (possibly because of a even higher dosing of the poisons in those studies) than to direct (inhibitory) effects on P450 isozymes. After treatment with BBZ a strong depletion of tissue GSH and GSSG content was seen in both livers and transplant containing spleens. This was most probably due to massive conjugation reactions of reactive BBZ intermediates (especially the 3,4-epoxide) with GSH because of the rather high dosage of BBZ (see e.g. ZANNONI et al. 1982; LAv and MONKS, 1988; FORT et al. 1996; WANG et al. 1998). Additionally, a significant increase in tissue LPO content was seen in livers and transplant containing spleens after BBZ administration. Probably due to the extensive conjugation reactions already the GSH threshold concentration was reached, the

remaining reactive BBZ metabolites thus causing tissue damage. Slight necrogenic effects in livers and transplant containing spleens due to BBZ treatment had been also observed in our previous investigations (Lupp et al. 2000). CCl 4 administration caused a very strong increase in the concentration of LPO products in the livers and spleens of both transplant recipients and control rats, reflecting the severe tissue damage previously seen in livers and transplant containing spleens (Lupp et al. 2000). In contrast, GSSG content was only slightly reduced and GSH concentration even unaffected in both livers and spleens. Thus, the toxic effects of CCl 4 may be mainly exerted and started at the (activating) P450 system without giving GSH a chance to "scavenge" the resulting trichloromethyl radical metabolites. From the results of the present investigation it can be concluded, that the effects of cytotoxins like AAL, BBZ, CCl 4 or TAA on P450 dependent monooxygenase functions and on oxidative state are exerted in the ectopic intrasplenic liver cell transplants in a similar way as in normal orthotopic liver.

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