Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern with consecutive differences in P450-mediated 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 beta-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 9000 g 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 2alpha-, 2beta-, 6beta-, 14alpha-, 15alpha-, 15beta-, 16alpha-, 16beta- 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 6alpha- and 7alpha-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.

Species differences in the interactions of cyclosporine A (CSA) and tacrolimus (TAC) with the cytochrome P450 (CYP) system in male rat and human liver were investigated in vitro by assessing effects on a series of model reactions for different CYP isoforms. CSA and TAC concentration dependently inhibited ethoxyresorufin O-deethylation, ethoxycoumarin O-deethylation and pentoxyresorufin O-depentylation and 7alpha- and 17-testosterone hydroxylation (TH) activities in both species. In rat liver no effect of CSA was seen on ethylmorphine N-demethylation and 2alpha- and 6beta-TH activities, but an inhibition due to TAC. Both CSA and TAC, however, distinctly decreased ethylmorphine N-demethylation and 2beta- and 6beta-TH activities in human liver. The same results were seen with 14alpha- and 15beta-TH activities. 2alpha-, 16alpha- and 16beta-TH activities were only inhibited in human liver with TAC, whereas only in this case 6alpha-TH activity was left unaffected. p-Nitrophenol hydroxylase activity was not influenced by either substance in both species. Thus, CSA mainly interacts in rat with the CYP isoforms 1A, 2A and 2B and in man with the CYP subtypes 1A, 2A, 2B, 2C and 3A. TAC seems to interfere predominantly in rat with the CYP isoforms 2A, 2B, 2C and 3A and in man with the CYP subtypes 1A, 2B, 2C and 3A. In summary, our results point to distinct species differences in the interactions with the CYP system with both substances, and although from literature CSA and TAC are known to be metabolized mainly by CYP 3A, according to our findings in rat liver CSA seems not to interact with this CYP subtype.

The present work was done to clarify the relevance of altered serum bile acid (BA) profile in healthy women after the administration of the depot oestrogen ethinyloestradiol propanolsulphonate (EES). In the serum of 20 healthy women before and two times after oral EES application, 11 free and 14 taurine- and glycine-conjugated BA were analysed by HPLC with postcolumn derivatisation and fluorescence detection. EES significantly enhanced the total serum BA concentration and that of taurine-conjugated BAs, more pronounced the secondary BAs taurodeoxycholic, tauroursodeoxycholic and taurolithocholic acid. These secondary BAs are produced in the intestine by bacteria due to 7alpha-dehydroxylation of the primary BAs cholic and chenodeoxycholic acid. Because of unchanged free BAs, also produced by intestinal bacteria due to deconjugation, the results were interpreted as a sign of disturbed transport of BAs into the liver. Inhibition of the liver Na(+)-bile salt co-transporter (Ntcp) in the sinusoidal membrane by ethinyloestradiol, formed from the prodrug EES, may be responsible for the altered BA profile in serum.

Para-nitrophenol (PNP) is a well-known substrate for both phase I (hydroxylation at cytochrome P450) and phase II reactions (glucuronidation and sulfation). HPLC separation of PNP conjugates has already been described, but not for respective studies with liver slices, which nowadays have proven to be a suitable model for metabolic studies. Therefore we adapted an HPLC method for the simultaneous measurement of PNP glucuronidation (PNP-G) and sulfation (PNP-S) in this in vitro system. Both activities are substantially maintained over an incubation period of 24 h. PNP-G activity, however, seems to be better preserved, as indicated by stable values for PNP-G but reduced PNP-S values after 48 h liver slice preincubation. 24 h exposure of the slices to beta-naphthoflavone or phenobarbital does not change PNP-G or PNP-S activities.

For the extensive use of precision-cut liver slices (particularly of human origin) for toxicological investigations successful cryopreservation is necessary. But so far, survival of thawed slices was limited to few hours. This was now overcome by modification of previous procedures. The concentration of DMSO as a cryoprotectant was enhanced to 30%, and washing steps after rapid thawing were omitted. The slices were frozen in liquid nitrogen, thawed at 38 degrees C and incubated immediately in Williams medium E. Protein and potassium contents were stable until 24 h. Glutathione content, amounting to nearly 50% of fresh slices, increased during incubation. High initial lactate dehydrogenase leakage dropped after medium change to less than half during 2-24 h. Testosterone hydroxylation and 7-ethoxycoumarin O-deethylation rates were similar to fresh slices, the latter reaction was inducible by beta-naphthoflavone within 24 h. Methylumbelliferone glucuronidation and p-nitrophenol glucuronidation and sulfation were well measurable and either maintained or decreased by about 50% until 24 h.Altogether, the results are encouraging for further experiments to standardise cryopreservation conditions and to investigate the suitability of this cryopreservation protocol with human liver slices.

7-Ethoxycoumarin (EC) is widely used as a model substrate for monooxygenase function, its O-deethylation representing cytochrome P450 (P450) activity mainly of 1A but also of 2B isoforms. Reports on investigations of its own capacity to induce or suppress P450 activities, however, have not been found in biomedical literature. To avoid the influence of in vivo pharmacokinetics, studies can well be undertaken with liver slice incubation. Therefore in the present investigation precision-cut rat liver slices from male 43-63-day-old male HAN:Wistar outbred rats were incubated at 30 degrees C in carbogen saturated William's Medium E for 24 h. EC was added previously to final concentrations of 10, 25, 50, 75 or 100 microM. After incubation, homogenate was prepared from slices and used for model reactions (7-ethoxyresorufin O-deethylation [EROD] and 7-pentoxyresorufin O-depentylation [PROD]). EROD, indicating activities of 1A isoforms, was enhanced by incubation with EC at 25 and 50 microM to about doublefold but showed control or lower values at 75 and 100 microM. Incubation with beta-naphthoflavone in comparison led to variable increases (3-5-fold of controls). For PROD as an indicator of the phenobarbital inducible P450 isoforms 2B1 and 2B2 no enhancement was found, but a decrease by incubation with 75 and 100 microM EC. To further investigate the correlation between enzyme activity and gene expression after slice incubation, P450 1A1 mRNA content was measured by RT-PCR. Induced gene expression for 1A1 was seen with different EC concentrations to a variable extent, though not as strong as with BNF. Similar incubation with 4-methyl-7-ethoxycoumarin revealed an even stronger induction of EROD activity with maxima at about 10-32 microM, reaching BNF values. In contrast incubation with 7-benzyloxycoumarin had no evident inducing or suppressing effect, neither on EROD nor on PROD activity.

Precision-cut rat liver slices (KRUMDIECK slicer, slice thickness 200-250 microm) were incubated in rollers containing modified William's medium E at 37 degrees C for 2, 24 and 48 hrs. Protein, DNA, potassium and glutathione concentrations did not decrease during 48 hrs. Lactate dehydrogenase (LDH) leakage into the medium was relatively marked during the first 2 hrs of incubation, from the 2nd to the 48th hr LDH leakage was very low. The same is true of the release of thiobarbituric acid-reactive substances. Albumin synthesis and transport into the medium decreased to about 70% after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation rate was relatively stable up to 48 hrs, whereas testosterone hydroxylation decreased significantly without alterations of the proportions of the 7 quantified hydroxylated metabolites. After exposure of the slices to beta-naphthoflavone for 6 hrs CYP1A1-mRNA expression, measured by competitive RT-PCR, was increased by a factor of at least 1000. Precision-cut liver slices are a useful tool for the study of various hepatic functions, drug metabolism and its induction in vitro.

The chemiluminescence (CL) amplifiers luminol (LM) and lucigenin (LC) react with different reactive oxygen species (ROS) in dependence on the ROS generating system used. With liver microsomes LMCL indicates predominantly superoxide anion radicals, whereas LCCL is mainly a measure for hydroxyl radical formation or of reactive organic radicals.With brain microsomes only LCCL, but not LMCL could be measured. For both brain microsomes from newborn (both sexes) and 60 day-old (male) rats LCCL is dependent on protein and NADPH concentration, activity in newborns being only 15 % compared with young adult rats. As compared with liver microsomes 10fold higher protein concentrations are needed to obtain comparable LCCL, whereas the NADPH demand is the same as with liver. A distinct ontogenetic development was demonstrated: low activities in the fetus, in newborn and 10-day-old rat are followed by higher activities with increasing age, after a maximum at an age of 60 days a decline was observed. Microsomal lipidperoxidation (LPO) was measured as formation of thiobarbituric acid reactive substances (TBARS) and was also dependent on protein and NADPH concentration. Unexpectedly, LPO with brain microsomes from newborn rats did not show any developmental variation.

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.