-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Analytical and Bioanalytical Chemistry 08/2008; 392(6):1167-72. · 3.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Toxicology in Vitro 01/2003; 16(6):749-58. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Toxicology 08/2002; 176(3):187-93. · 3.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Toxicology 04/2001; 161(1-2):103-9. · 3.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 09/2000; 52(4):335-8. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Toxicology 05/2000; 144(1-3):93-7. · 3.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 10/1998; 50(4-6):440-9. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 10/1998; 50(4-6):507-13. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 10/1998; 50(4-6):514-8. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Obstetrics and Gynaecology 06/1997; 17(4):340-3. · 0.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 08/1996; 48(5):433-8. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 06/1993; 45(2-3):145-8. · 2.78 Impact Factor
-
R Glöckner
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 06/1993; 45(2-3):99-100. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 03/1993; 45(1):35-7. · 2.78 Impact Factor
-
R Glöckner
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 01/1993; 44(7):421-3. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 01/1993; 44(7):371-3. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Experimental and Toxicologic Pathology 10/1992; 44(5):287-93. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In undisturbed pregnancies of the rat skeletal development coincides with gestational age, and in that case a prolonged gestation is connected with an intrauterine progress of skeletal ossification. In disturbed pregnancy associated with fetal retardation also perinatal ossification is delayed. In the rat this delay can obviously be compensated by a prolonged pregnancy. For estimation of skeletal ossification a simple score has been developed. A total score of all selected perinatal ossification centres (sternebrae, bones of the fore- and hindpaw, caudal vertebrae) was ascertained and compared with a reduced score obtained by scoring the lower part of the body only (caudal vertebrae, hindpaw). With this reduced scoring system the same qualitative relations between control and retarded fetuses and newborns of different gestational age could be established as with the total score. The estimation of the appearance of only one representative ossification centre of the forepaw may give a rapid survey of skeletal development, but seems to be uncertain for detailed characterization.
Anatomischer Anzeiger 02/1991; 173(4):209-14.
-
[show abstract]
[hide abstract]
ABSTRACT: On Day 21 of gestation ( = expected day of delivery), GGT activity of the mammary gland is low 4-6 h before beginning of delivery. It is significantly higher immediately (up to 3 h) before delivery or after labor. The extent of prepartal increase is individually different. The activity measured early in the morning cannot be used to predict the remaining time to beginning of spontaneous parturition.
Journal of Experimental Animal Science 02/1991; 34(2):77-80.
-
[show abstract]
[hide abstract]
ABSTRACT: In the rat, hepatic glutathione (GSH) levels increase prenatally and reach maximum concentrations at term. They are independent of the kind of delivery (cesarean section, spontaneous labor). Brain levels are distinctly lower and do not show a perinatal peak. In both organs a slight increase of GSH concentrations can be observed in the first 3 h after birth. Different litter sizes representing different intrauterine development conditions do not influence GSH levels, which is in contrast to the recently reported effect of litter size on prenatal development of hepatic gamma-glutamyltranspeptidase (GGT) activity. We suggest that perinatal GGT activity and GSH levels are regulated by different mechanisms.
Biology of the Neonate 02/1991; 59(5):287-93. · 1.90 Impact Factor
