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.

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 [CCl4], 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 9,000 g 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 administration caused a decrease in EROD and EMND activities, ECOD activity remained unaffected, and PROD activity was even increased. CCl4 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. CCl4 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 CCl4 treatment. An elevation in LPO products was also seen in the spleens of the control rats due to CCl4 administration. Tissue GSH and GSSG content in both livers and transplant containing spleens were strongly reduced after BBZ treatment. After CCl4 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, CCl4 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.

Transplantation of fetal liver tissue suspension 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.

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 various mitogens (fluorene [FEN], fluorenone [FON] and 2-acetylaminofluorene [AAF]) or cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ] and carbon tetrachloride [CCl4]) or the respective solvents 24 or 48 hours before sacrifice. The expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers was assessed by immunohistochemistry and P450 mediated monooxygenase functions in spleen and liver 9000 g supernatants by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (EMND). The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a moderate P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. Correspondingly, regular EROD, ECOD and EMND activities were observed. Each of the three mitogens increased the P450 1A1 expression in the hepatocytes of the perivenous zones of the liver lobules. FON administration caused an additional P450 1A1 immunostaining in the periportal areas, and AAF treatment a P450 1A1 expression in bile duct epithelia. Also the staining for P450 2B1 and 3A2 in the hepatocytes of the perivenous and intermediate zones of the liver lobules was intensified after treatment with any of the mitogens. The three model reactions were significantly increased within the livers after FEN and FON administration, whereas after AAF treatment only ECOD was enhanced, EROD remained unaffected and EMND was decreased. The cytotoxin AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ only produced a perivenous necrosis of single cells, whereas CCl4 caused complete necrosis of the centrilobular parenchyma. Immunohistochemically, AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 was even decreased in the periportal areas. Due to AAL treatment EROD and EMND activities were not affected and ECOD activity was increased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. After BBZ treatment, EROD and EMND activities were decreased, ECOD activity was not affected. CCl4 administration caused a strong reduction in the expression of all three P450 isoforms and in the activity of all three model reactions. Spleens of control rats displayed almost no P450 isoforms expression and P450 mediated monooxygenase functions, without as well as after treatment with the mitogens or cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants.