Michael Rost

Monooxygenation, cytochrome P450-mRNA expression and other functions in precision-cut rat liver slices

Precision-cut rat liver slices (KRUMDIECK slicer, slice thickness 200-250 microm) were incubated in rollers containing modified Williams 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.

Monooxygenation, conjugation and other functions in cryopreserved rat liver slices until 24 h after thawing

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.

Monooxygenation, cytochrome P4501A1 and P4501A1-mRNA in rat liver slices exposed to beta-naphthoflavone and dexamethasone in vitro

Precision-cut liver slices (0.5 mm) were incubated at 30 degrees C in a modified Williams Medium E for up to 48 hrs. During the incubation, K+ and GSH/GSSG concentrations did not decrease. Cytochrome P450-dependent dealkylation rates of 7-ethoxycoumarin (ECOD), 7-allyloxycoumarin (ACOD) and 7-ethoxyresorufin (EROD) decreased to 1/3, 1/2 or did not change at all, respectively, after a 48 hrs incubation period. Exposure of the slices to 25 microM beta-naphthoflavone (beta NF) resulted in about 3 times higher monooxygenation rates. An exposure to a combination beta NF and dexamethasone (10(-6)M) caused a marked induction (6 times higher rates) after 48 hrs. Simultaneously an increase in P4501A1 content was observed. P4501A1-mRNA expression (measured by RT-PCR) was distinctly increased following beta NF exposure for 6 or 24 hrs. DMSO (0.2%) and dexamethasone alone modified monooxygenation rates, but did not have significant effects on P4501A1 content or, in the case of DMSO, P4501A1 gene expression (for dexamethasone not determined). Liver slices are a useful and simple tool for the detection of a beta NF-like induction within a few hours after preparation of the slices.

Luminol-and lucigenin-amplified chemiluminescence with rat liver microsomes. Kinetics and influence of ascorbic acid, glutathione, dimethylsulfoxide, N-t-butyl-a-phenyl-nitrone, copper-ions and a copper complex, catalase, superoxide dismutase, hexobarbital and aniline.

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.

What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence? 1. Investigations with hydrogen peroxide and sodium hypochlorite.

Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H2O2 is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe2+ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co2+ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co2+. Cu2+ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration-reaction curve is not as steep. NaOCl enhances H2O2/Fe(2+)-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 10(4) (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity.

Para-nitrophenol glucuronidation and sulfation in rat and human liver slices

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.

Induction of cytochrome P450 1A1 in rat liver slices by 7-ethoxycoumarin and 4-methyl-7-ethoxycoumarin

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 Williams 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.

The ambiguous effect of ascorbic acid on chromate induced proteinuria in rats

The influence of ascorbic acid (AA, 5 g/kg body weight) on chromate (Cr, 10 mg/kg) induced proteinuria, which is a sensitive parameter of its nephrotoxicity, was investigated in adult female Wistar rats. The concentrations of Cr and ascorbic acid (AA) were determined in renal tissue. Cr nephrotoxicity is related to its intracellular reduction from Cr(VI) to Cr(III). Proteinuria was completely prevented by enhancement of extracellular reduction of Cr(VI) to Cr(III) followed by rapid renal excretion when Cr and AA were given concomitantly. With an interval up to 1 h between Cr and AA, proteinuria was decreased probably by the radical scavenging function of AA. At an interval of 3 h AA enhanced Cr toxicity by increased intracellular Cr reduction. If the interval was increased to 5 h or if Cr was given 24 h after AA, no influence of AA could be detected. Our results confirm that AA is a very effective reductant of Cr which can influence Cr nephrotoxicity in very high concentrations. It depends on the interval between Cr and AA administration whether or not there is a beneficial effect of AA in Cr nephrotoxicity.

Influence of ethinyloestradiol propanolsulphonate on serum bile acids in healthy volunteers

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.

Cyclosporine A and Tacrolimus: In vitro investigations on the differential interactions with the cytochrome P450 system in rat and human liver

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.