Determination of CYP activity in precision-cut liver slices: whether to use intact slices or slice homogenate.

Despite CYP induction in vitro in precision-cut liver slices (LS) is well documented, there are no standardised assays for determining CYP activity as a major end-point. In this paper, short-term assays with intact and homogenised LS from male and female rats were directly compared. We obtained similar results for 7-ethoxycoumarine O-deethylation (ECOD) with LS from both sexes: higher basal activities were measured in LS homogenate, whereas slightly stronger induction by BNF was found with intact LS. CYP3A-dependent basal and dexamethasone (Dex)-induced 2beta-, 15beta- and 6beta-testosterone hydroxylation (TH) rates were higher in both intact and homogenised LS from male compared to female rats. CYP3A induction in vitro could likewise be detected in intact and homogenised LS preferentially by determining 2beta- and 15beta-TH, with higher induction factors observed in LS from females. 6beta-TH seems to be less inducible in intact LS of males. In vivo pretreatment of liver donors with BNF and Dex did not substantially disturb the subsequent in vitro induction of ECOD and TH, respectively.

Cryopreserved precision-cut rat liver slices: morphology and cytochrome P450 isoforms expression after prolonged incubation.

Precision-cut liver slices are an accepted in vitro system for toxicological investigations. However, cryopreservation of slices would make a more efficient utilisation, particularly of human liver tissue possible. In the present study sections of cryopreserved male rat liver slices were examined immunohistochemically for cytochrome P450 (CYP) isoforms expression after prolonged incubation and after exposure to typical inducers. Morphologically, with just thawed slices no major alterations were seen, but remarkable cell damage was observed even after 2 h of incubation mainly in the middle of the slices and in the periportal and intermediate regions of the lobules. After 24 h of incubation, viable cells were only observed at the edges of the slices or around bigger vessels. In the viable cells of the cryopreserved liver slices after 2 h of incubation CYP expression pattern was similar to that in normal liver specimens: a low CYP1A1, but a strong CYP2B1 and 3A2 expression predominantly in the central and intermediate lobular zones. After 24 h, the immunostaining for CYP2B1 and 3A2 in the viable cells was reduced, but that for CYP1A1 was increased. Incubation with beta-naphthoflavone further elevated CYP1A1 and 2B1 expression. Phenobarbital caused an enhanced CYP2B1 and 3A2 and dexamethasone and pregnenolone 16 alpha-carbonitrile an increased CYP3A2 immunostaining. These results show that also in cryopreserved liver slices and after a prolonged incubation, a distinct expression pattern and an in vitro induction of phase I enzymes can be demonstrated immunohistochemically.

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices.

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 microM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 microM pregnenolone 16 alpha-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.

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.

Integrated optical pickup system for axial dual focus.

We propose an optical pickup that acquires data from both layers of a dual-layer digital versatile disk simultaneously. An adaptive optical element that uses liquid crystals creates two axial foci separated by a spacing of 55 mum, which is the distance between the two layers. The spacing between the foci can be varied by the adaptive element. The separation of the reflected light into TE and TM polarized light, corresponding to each of the layers, is made by dielectric gratings that are characterized by high aspect ratios. Electron-beam lithography and reactive ion etching techniques were used to produce the submicrometer structures. All fabricated elements were assembled in a pickup system, whose properties were measured.

Optimal oxygen tension conditions for viability and functioning of precision-cut liver slices.

Optimal oxygenation of culture media is important for the successful use of liver slices as an in vitro tool for studying liver function. For this reason the influence of 20, 40, 70 and 95% O2 concentration on the viability and metabolism of liver slices was investigated. The slices were incubated in the roller system at 37 degrees C under continuous gassing for 2, 24 and 48 hrs. Protein, DNA and potassium contents were maintained or even increased over time without influence by O2 concentrations. The albumin secretion of slices incubated at 40-95% O2 did not differ, but was much lower at 20% O2. A slight non-significant decrease in albumin secretion after 24 hrs of cultivation could be observed, whereas a much steeper decline was found in all groups after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation (ECOD) did not differ between the various O2 concentrations, but declined from 2 to 48 hrs of incubation. It can be concluded that O2 concentration of 20% is not sufficient to maintain all cell functions of incubated rat liver slices, wheras 40, 70 and 95% are useful O2 concentrations to retain all parameters investigated.

Induction of cytochrome P450 2B1-mRNA and pentoxyresorufin O-depentylation after exposure of precision-cut rat liver slices to phenobarbital.

Precision-cut rat liver slices were prepared from male Wistar rats with a Krumdieck slicer and cultured in William's medium E for up to 24 h. In untreated control slices, CYP2B1-mRNA concentration, which was quantified by competitive RT-PCR, did not decrease during this time. After exposure of the slices to 100 microM phenobarbital, CYP2B1-mRNA increased by about 10- or 60-fold after 6 or 24 h, respectively. The extent of this in vitro induction was similar to that after in vivo administration of 60 mg/kg phenobarbital. Pentoxyresorufin O-depentylation (PROD) was also inducible in vitro after 24 h, but to a lesser extent than the corresponding CYP-mRNA. Precision-cut liver slices proved to be a simple and reliable in vitro system for the sensitive detection of an induction by phenobarbital.

Use of Fresh and Cryopreserved Human Liver Slices in Toxicology with Special Reference to In vitro Induction of Cytochrome P450.

Human liver slices were prepared with a Krumdieck slicer from macroscopically healthy surgical waste after partial hepatectomy. They were incubated without or with the addition of the inducer beta-naphthoflavone (BNF) (25 mum) either immediately after preparation (fresh slices) for up to 24 hours or after cryopreservation in liquid nitrogen (thawed slices) for up to 6 hours. Potassium concentration was well maintained in fresh and thawed slices over 24 and 6 hours, respectively, but at lower levels than in rat liver slices. Albumin secretion showed relatively large interindividual differences. Both parameters were lower in thawed slices than in fresh ones, but indicated a certain number of viable cells. In untreated fresh slices CYP1A1-mRNA was not detectable; however, it increased distinctly within 6 hours of exposure to BNF. The amounts of induced CYP1A1-mRNA differed by a factor of more than 100 among six human livers and were lower than in fresh rat liver slices. Even in thawed human slices, CYP1A1-mRNA expression could be induced in vitro by BNF, although at a very low level and preferentially in those specimens with comparably high inducibility already before freezing.

Application of cryopreserved precision-cut liver slices in pharmacotoxicology--principles, literature data and own investigations with special reference to CYP1A1-mRNA induction.

Principle steps necessary for cryopreservation of precision-cut liver slices as currently applied by different groups are summarized including own results concerning mode of freezing. Now we use rapid freezing by immersion in liquid nitrogen after exposure to 10% DMSO as the cryoprotectant for rat liver slices. The results indicate well-maintained cytochrome P450 (CYP)-dependent deethylation rates in slice homogenate after short-term incubation. ECOD rate in intact thawed slices was even higher than in fresh ones after 2 h incubation. In contrast to fresh slices all parameters except protein content decreased to marginal levels during long-term incubation of thawed slices for 24 h. The first preliminary experiments on albumin secretion by thawed rat liver slices, measured between the 2nd and the 4th hour of incubation, showed partial maintenance of this liver specific differentiated function. Trials to induce CYP1A1 in thawed rat liver slices in vitro by beta-naphthoflavone (BNF) resulted in increased expression of CYP1A1-mRNA within 6 h as shown by RT-PCR and quantified by competitive RT-PCR. The decline of deethylation rates, determined in slice homogenates, and of viability within 24 h incubation was not prevented by exposure to BNF or DMSO. The results derived from one sample of cryopreserved human liver slices indicate a quite acceptable maintenance of function up to 6 h, if the same protocol as developed for rat liver slices was used.

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 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.

Immunohistochemical localization of cytochrome P450 1A1 in precision-cut rat liver slices after in vitro exposure to beta-naphthoflavone.

Mono- and polyclonal antibodies have been used to study the localization and distribution of cytochrome P450 1A1 (CYP1A1) in cultured precision-cut liver slices with various immunohistochemical methods. Neither in non-incubated slices nor in slices incubated in the absence of beta-naphthoflavone (BNF) for 24 hrs was CYP1A1 immunohistochemically detectable. After incubation in the presence of BNF (25 microM), however, CYP1A1 was well visible in parenchymal and biliary epithelial cells. CYP1A1 was not evenly distributed, but was localized predominantly in hepatocyte layers near the surfaces of the slices. This distribution could be due to the preferential uptake of BNF by outer cell layers or due to functional changes of inner cells. Together with results obtained with other methods (e.g. RT-PCR) this investigation also demonstrates that precision-cut liver slices are a useful tool for the detection of in vitro induction of CYP1A1.

Serum concentrations of iodine, thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH) and insulin-like growth factor 1 (IGF-1) during the last trimester of pregnancy, during labour, and in early puerperium of women with normal pregnancy or with intrauterine growth retardation (IUGR).

In women with intrauterine growth retardation (IUGR), insulin-like growth factor 1 (IGF-1) concentrations tended to reduce during the last trimester of pregnancy. Parameters of thyroid function in maternal serum were not distinctly influenced by IUGR, except for high concentrations of iodine. Triiodothyronine (T3) concentrations in cord blood of normal pregnancies was significantly lower than maternal concentrations, but was relatively high when the fetus was growth retarded. The results are discussed in connection with changes of thyroid function and changes of IGF-1 during pregnancy.

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 William's 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.

Ethoxycoumarin O-deethylation (ECOD) activity in rat liver slices exposed to beta-naphthoflavone (BNF) in vitro.

Precision-cut rat liver slices (0.5 mm) were incubated at 30 degrees C in William's Medium E up to 24 hrs. Our incubation conditions seem to be suitable for maintaining slice function, indicated by constant contents of tissue protein, potassium and glutathione. Thiobarbituric acid reagible substances (TBARS) released into the incubation medium did not significantly increase. Addition of DMSO (0.2 % v/v) or BNF (50 microM) to the incubation medium had no influence on most parameters described above except for increased TBARS release. If ECOD activity was determined in intact liver slices without addition of any cofactor, but substrate only, the main amount of metabolite was found in the medium, and the amount of metabolite retained within the tissue could be neglected. In slices incubated for 24 hrs, no significant changes of ECOD activity occurred for control and DMSO groups, compared with slices incubated for 2 hrs, but in the BNF group activities were more than 3.5 times as high. If ECOD activity was determined in slice homogenate, i.e. with addition of cofactors, decreased activities were measured in all groups after 24 hrs of incubation. This decrease was highest in the control group, lowest in the BNF group. We conclude that intact liver slices can be used as a simple tool to investigate in vitro enzyme induction of BNF type.

Postnatal development of body mass and of hepatic xenobiotics biotransformation of male rats with low body mass at birth.

Body mass gain of male rats with low body mass at birth was lower from birth up to the 5th postnatal day, but higher from the days 5 to 30 in comparison with male litter mates with normal body mass at birth. Absolute body and liver masses were significantly lower up to the 30th postnatal day, but fully improved at adult age. No differences of ethoxycoumarin O-deethylation activities existed between both groups at all ages. Ethylmorphine N-demethylation activity was significantly lower in newborn males with low body mass, but had been fully developed as early as the 5th postnatal day. We conclude that postnatal adaptation of male rats with spontaneous growth retardation at birth is slightly delayed only during the first days of life.

Lipid peroxidation in brain tissue of newborn rats derived from two models of disturbed pregnancy.

Two models of disturbed pregnancy, both causing decreased fetal body mass, were used: the endotoxin-model (treatment of pregnant rats with heat-denaturated bacterial material) and the stress-model (chronic restraint of prenatally lithium-treated pregnant dams). Fetuses were delivered by Caesarean section in the morning of the 21st gestational day. After disturbed pregnancy lipid peroxidation (iron-stimulated formation of thiobarbituric acid reagible substances) was not changed in 9,000 g supernatant of total brain. The values of all groups were higher after determination in frozen/thawed tissue in comparison to freshly prepared material. The results do not indicate an enhanced vulnerability of brain tissue after both models of disturbed pregnancy.

Enhanced effect of chronic stress on pregnancy outcome in Uje:WIST rats by prenatal treatment with lithium.

Female Uje:WIST rats were exposed to lithium (20 mmol LiCl/l drinking water, lithium levels about 1 mmol/l serum) for at least three weeks before mating and continuously up to the end of gestation. Female F1-offspring were raised without further treatment and mated at an age of 90-120 days with untreated males. The effect of chronic restraint stress during gestation on pregnancy outcome was estimated. Moderate changes (higher frequency of prolonged gestation, decreased body mass of the newborn pups) resembled those which were induced by considerably longer stress exposure (extended to the premating period) of prenatally untreated female Uje:WIST rats. An enhanced stress sensitivity following prenatal lithium treatment is discussed.

Delayed fetal development in two models of disturbed pregnancy of rats. Effect on mortality, body mass development, blood urea nitrogen (BUN) and urinary ammonium concentrations during the first week of extrauterine life.

To induce pregnancy disturbance, two models were used ("endotoxin-model" and "stress-model"), both causing decreased fetal body mass. Fetuses were delivered by Caesarean section in the morning of the 21st gestational day. Postnatal mortality rate during rearing for one week amounted to 12% in controls and was enhanced in the endotoxin- and stress-models (to approximately 25% and approximately 30%, respectively). During this period body mass gain of surviving pups was significantly delayed in the stress-model (approximately 140% of birth mass), compared with controls or pups of the endotoxin-model (160% and 155%, respectively). Additionally, decreased BUN concentrations were registered in newborns as well as 7-day-old pups of the stress-model. Urinary ammonium concentrations were not changed significantly. Possible alterations of metabolic processes in pups of the stress-model are discussed.

Influence of low birth weight on subsequent reproductive performance of female rats.

Female rats which were born underweight had lower absolute body mass throughout the whole experimental period than female litter mates with normal birth weight (controls). Their reproductive performance observed during three consecutive pregnancies was not different from that of controls except for lower litter size in the 1st pregnancy. Thus at least for rats, low spontaneous birth weight of females does not indicate subsequent poor pregnancy outcome.

Placental morphology and concentrations of glutathione (GSH and GSSG) and lipid peroxides (LPO) in two models of disturbed pregnancy of Uje:WIST rats.

Treatment of pregnant rats with heat-denaturated bacterial material (endotoxin-model) or exposure to chronic restraint of prenatally lithium-treated pregnant dams (stress-model) were used as two models of disturbed pregnancy, both causing decreased fetal body mass. The quotient of fetal/placental mass was lowered in the endotoxin-group only. Placental mass and protein content were not changed significantly in both experimental groups, although a tendency to smaller placentae was noticed in the stress-group. Placental histology of the stress-group did not differ from untreated controls. In the endotoxin-group an altered structure of the placental barrier was observed. Small decreases of GSH and GSSG in the endotoxin-group and of GSSG in the stress-group without significant changes of the GSH/GSSG relationship were measured in homogenates of the placental labyrinth. Moderate enhancement of LPO concentration occurred in the endotoxin-group and more distinctly in two litters of the stress-group, the latter being connected with high GSSG concentrations and low fetal/placental mass-quotients.