Biomedical subjects

W Klinger

Publications and source records attributed to W Klinger.

At least 19 recordsLinked to original sources

PubMed2003

Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing?

Malignant hyperthermia (MH) is a condition that manifests in susceptible individuals only on exposure to certain anaesthetic agents. Although genetically heterogeneous, mutations in the RYR1 gene (19q13.1) are associated with the majority of reported MH cases. Guidelines for the genetic diagnosis for MH susceptibility have recently been introduced by the European MH Group (EMHG). These are designed to supplement the muscle biopsy testing procedure, the in vitro contracture test (IVCT), which has been the only means of patient screening for the last 30 years and which remains the method for definitive diagnosis in suspected probands. Discordance observed in some families between IVCT phenotype and susceptibility locus genotype could limit the confidence in genetic diagnosis. We have therefore assessed the prevalence of 15 RYR1 mutations currently used in the genetic diagnosis of MH in a sample of over 500 unrelated European MH susceptible individuals and have recorded the frequency of RYR1 genotype/IVCT phenotype discordance. RYR1 mutations were detected in up to approximately 30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive families. In five families a mutation-positive/IVCT-negative individual was observed, and in the other five families a mutation-negative/IVCT-positive individual was observed. These data represent the most comprehensive assessment of RYR1 mutation prevalence and genotype/phenotype correlation analysis and highlight the possible limitations of MH screening methods. The implications for genetic diagnosis are discussed.

Chromosomes, Human, Pair 19↗

PubMed2002

Estradiol, testosterone, dehydroepiandrosterone and androstenedione: novel derivatives and enantiomers. Interactions with rat liver microsomal cytochrome P450 and antioxidant/radical scavenger activities in vitro.

Interactions of 27 steroids, among them 17 derivatives such as ethers, sulfates and amidosulfonates derived from 17 beta- and 17 alpha-estradiol, from testosterone and alpha- and beta-dihydrotesosterone and from dehydroepiandrosterone with rat liver microsomal cytochromes P450 (P450) were investigated in vitro by assessing binding to P450 and effects on P450 mediated monooxygenase functions as measured by different model reactions: ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD) and ethylmorphine N-demethylation (EMND). With the exception of 17 alpha-estradiol-3-dimethylamidosulfonate, estrone, its -3-methylether and -3-amidosulfonate and testosterone, all other steroids displayed type I or reverse type I binding to P450. All steroids inhibited EROD activity in micromolar concentrations. An additional strong inhibition of ECOD and EMND activities was only demonstrated for the androgens and progestins. Estriol, estrone and mestranol displayed less inhibitory actions on the model reactions than estradiol. No major differences in comparison to the parent compounds were noted with the other derivatives. The only exceptions were 17 beta-(8,9-dehydro-14 alpha,15 alpha-methylene)estradiol, which displayed stronger effects than estradiol, and dehydroepiandrosterone-3-sulfate, which was less effective than dehydroepiandrosterone. Possible antioxidant properties of the steroids were examined by the stimulated lipid peroxidation (LPO), H2O2 production, and lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) using rat liver microsomes. Additionally, the influence on rat whole blood chemiluminescence (WB-CL) was assessed. All the estrogens, but not their methylethers and amidosulfonates inhibited LPO in micromolar concentrations. The effects on the other oxidase model reactions or on WB-CL were less distinct. Only ethinylestradiol and 17 beta-(8,9-dehydro-14 alpha,15 alpha-methylene)estradiol displayed a strong inhibitory action on all model reactions. With the exception of dehydroepiandrosterone-3-sulfate, which in general had only weak effects, the androgen and progestin derivatives, in contrast, strongly decreased H2O2 formation and LM- and LC-CL, but were mostly ineffective on LPO and WB-CL.

7-Alkoxycoumarin O-Dealkylase↗

PubMed2001

Cytochrome P450 (P450) isoforms expression, P450 concentration, monooxygenase activities, reactive oxygen species formation, lipid peroxidation, and glutathione content in wild catch carp and tench liver--influence of a two weeks exposure to phenobarbital.

Carps, both sexes, 3 years old, weighing about 1 kg, and tenches of both sexes, 6 years old, weight about 250 g, were caught from a Thuringian lake without industrial pollution in November 1995 (fish without food uptake, water temperature at about 10 degrees C) and kept for 2 weeks in basins with clean water and addition of 0, 0.1, 1.0 or 10.0 mg/l phenobarbital-Na (PB). The concentration of PB was controlled during and at the end of the exposure period. The animals were fed pellets, but no food uptake was observed. After 24-48 h in fresh water the fish were sacrificed and the following hepatic parameters were immediately determined biochemically: monooxygenase functions: cytochrome P450 (P450) content, ethylmorphine N-demethylation (EN), ethoxycoumarin O-deethylation (ECOD), ethoxyresorufin O-deethylation (EROD), 7-benzyloxy-4-methyl-coumarin O-debenzylation (BCDB); oxidase function indicators: microsomal Fe2+/NADPH dependent hydrogen peroxide formation (H2O2), microsomal Fe2+/NADPH dependent luminol and lucigenin amplified chemiluminescence (LMCL, LCCL), microsomal Fe2+/NADPH dependent lipid peroxide formation (LPO); oxidative state: lipid peroxidation products (TBARS) and GSH and GSSG. Additionally, the expression of three P450 isoforms, 1A1, 2B and 3A, was assessed immunohistochemically in tissue samples from brain, gill, heart, spleen, liver, gut and ovary of both fish species and in kidney of tenches. PB did not influence body or liver weights, but increased liver P450 concentration in both species by 50-100%, though not significantly. Carp: PB increased both EN and EROD significantly, but not ECOD and BCDB; H2O2 and TBARS were enhanced significantly. LPO, LMCL and LCCL were not significantly influenced. Tench: PB increased all monooxygenase reactions (EN, ECOD, BCDB and EROD), though only significantly ECOD; H2O2 was elevated only after treatment with 0.1 mg/l PB, whereas LPO was decreased (!) after treatment by all three concentrations, though significantly only after 1.0 mg/l PB. LMCL was depressed (not significantly), but LCCL increased 5fold. TBARS were significantly enhanced. P450 1A1 subtype expression was concentration dependently elevated by PB in gill and liver of both fish and in the heart and kidney of tenches, P450 2B and 3A isoforms expression was induced in brain, gill, heart, liver and gut of both fish and in the kidney of tenches. In summary, the increased activities of the monooxygenase reactions tested and the elevated expression of all three P450 isoforms investigated in certain tissues indicate an induction of the P450 families 1, 2 and 3 by PB in fish.

Animals↗

PubMed2001

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.

7-Alkoxycoumarin O-Dealkylase↗

PubMed2000

Influence of subchronic administration of catechol estrogens on the formation of reactive oxygen species in rat liver microsomes.

Metabolic pathways of estrogens are the formation of catechol estrogens (CE; 2- and 4-hydroxy-estrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. In previous investigations subchronic administration of estrogens showed prooxidative and antioxidative activities in rat liver microsomes (BARTH et al. 1999). To find out whether or not catechol metabolites are responsible for prooxidative activity, we checked 2- and 4-hydroxy-estradiol (2OH-E2 and 4OH-E2) and the non-catechol metabolite 6alpha-hydroxy-estradiol (6alpha-OH-E2) for formation of reactive oxygen species in liver microsomes of 30-day-old male Wistar rats after 5 days treatment (1, 10 mg/kg b. wt. orally, once a day). The results were compared with those after treatment of the rats with estradiol (E2), estradiol valerate (E2V) and ethinylestradiol (EE2). In liver homogenates glutathione and lipid peroxides were determined, in microsomes NADPH-Fe++-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) were investigated. In liver 9000 x g supernatants monooxygenase activities were measured. The two catechol estrogens did not show any antioxidative activity, whereas 6alpha-OH-E2 significantly diminished lipid peroxides in the liver as well as LPO and LUM-CL in liver microsomes. Among estrogens, only EE2 showed antioxidative activity. Both CE inhibited ethoxycoumarin O-deethylation. Peroxidative activity as enhanced LUC-CL was found after 2OH-E2 (1 mg/kg b.wt.) and E2, but 10 times higher doses of both CE did not change LUC-CL. Microsomal H2O2 generation was enhanced by E2, E2V and both CE, not by 6alpha-OH-E2. The lower level of H2O2 enhancement caused by CE in comparison to E2 and E2V together with unchanged LUC-CL after high CE doses did not unequivocally prove the CE to be mainly responsible for the prooxidative activities of E2 and E2V in liver microsomes, at least in 30-day-old male rats. Unchanged GSH in the liver after CE administration supports this hypothesis.

Administration, Oral↗

PubMed2000

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 isoforms expression and on glycogen content.

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 the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers were assessed by immunohistochemistry. Additionally, effects on glycogen content within the hepatocytes were examined. In the livers AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ led to a perivenous necrosis of single cells only, whereas CCl4 and TAA caused complete necrosis of the centrilobular parenchyma. Treatment with each of the four cytotoxins led to necrosis and fatty degeneration of single or groups of hepatocytes within the intrasplenic transplants. This effect was most pronounced with CCl4 and TAA. 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 strong P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. 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 in the periportal areas was even decreased. 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. TAA and, more pronounced, CCl4 administration caused a strong reduction in the expression of all three P450 isoforms. Spleens of control rats displayed almost no P450 isoforms expression, independent of the treatment with the 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. AAL administration slightly reduced the P450 2B1 and 3A2 expression in the transplants. BBZ and, much more pronounced, CCl4 and TAA treatment diminished the staining for all three P450 isoforms. AAL administration led to a marked decrease in the glycogen content of the hepatocytes of the periportal zones of the liver lobules, whereas after BBZ, CCl4 and TAA treatment a strong perivenous reduction in the glycogen content was seen. Similarly, within the intrasplenic transplants a remarkable decline in the glycogen content of the hepatocytes was caused by the treatment with each of the four cytotoxins. Especially after AAL and BBZ treatment the glycogen depletion within both livers and transplants was much more pronounced than the effects on morphology or P450 isoforms expression. It can be concluded that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA seen in normal orthotopic liver are exerted in a similar way also in intrasplenic liver cell transplants.

Animals↗

PubMed1999

A reduced K+ current due to a novel mutation in KCNQ2 causes neonatal convulsions.

Benign familial neonatal convulsions (BFNC) is a rare dominantly inherited epileptic syndrome characterized by frequent brief seizures within the first days of life. The disease is caused by mutations in one of two recently identified voltage-gated potassium channel genes, KCNQ2 or KCNQ3. Here, we describe a four-generation BFNC family carrying a novel mutation within the distal, unconserved C-terminal domain of KCNQ2, a 1-bp deletion, 2513delG, in codon 838 predicting substitution of the last seven and extension by another 56 amino acids. Three family members suffering from febrile but not from neonatal convulsions do not carry the mutation, confirming that febrile convulsions and BFNC are of different pathogenesis. Functional expression of the mutant channel in Xenopus oocytes revealed a reduction of the potassium current to 5% of the wild-type current, but the voltage sensitivity and kinetics were not significantly changed. To find out whether the loss of the last seven amino acids or the C-terminal extension because of 2513delG causes the phenotype, a second, artificial mutation was constructed yielding a stop codon at position 838. This truncation increased the potassium current by twofold compared with the wild type, indicating that the pathological extension produces the phenotype, and suggesting an important role of the distal, unconserved C-terminal domain of this channel. Our results indicate that BFNC is caused by a decreased potassium current impairing repolarization of the neuronal cell membrane, which results in hyperexcitability of the central nervous system.

Amino Acid Sequence↗

PubMed1999

Influence of oestrogens on formation of reactive oxygen species in liver microsomes of differently aged male Wistar rats.

Metabolic pathways of oestrogens are the formation of catechol oestrogens (CE; 2- and 4-hydroxy-oestrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. We checked the oestrogens oestradiol (E2), oestradiol valerate (E2V) and ethinyloestradiol (EE2) for formation of reactive oxygen species in vitro and ex vivo in male Wistar rats in dependence on age. In liver microsomes of 10-, 30-, 60- and 270-day-old rats the influence of E2, E2V and EE2 (10(-7)-10(-3) M) on NADPH-Fe(++)-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) was investigated. The same parameters, additionally P450 content and monooxygenase activities were measured in liver 9000 x g supernatants after subchronic administration of the oestrogens (1, 10 mg/kg b. wt. orally). The most important results are the strong inhibitory capacities of the oestrogens in vitro on LPO in the order of E2V < E2 < EE2, most pronounced in 10-, 60- and 270-day-old animals. In microsomes of 30-day-old rats with the highest control LPO the antioxidative effect of the oestrogens was lower. Whereas the H2O2 generation was not changed by E2, enhanced by E2V, but diminished by EE2 in all age groups, CL(LUC) and CL(LUM) were inhibited in the order of E2 < E2V < EE2. Also after subchronical treatment of the rats the antioxidative action of the oestrogens was evident, microsomal LPO was inhibited in the order of E2 < E2V < EE2. All oestrogens inhibited ethylmorphine N-demethylation. But enhanced H2O2 generation and increased CL(LUC) also indicate a formation of reactive oxygen species by these oestrogens. Obviously in vitro the antioxidative phenolic structure of the oestrogens dominates, whereas after in vivo administration the dose- and age-dependent biotransformation produces prooxidative in addition to antioxidative structures.

Aging↗

PubMed1999

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.

2-Acetylaminofluorene↗

PubMed1999

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: inducibility of cytochrome P450 dependent monooxygenase functions by beta-naphthoflavone, phenobarbital and dexamethasone.

In the present study the effects of beta-naphthoflavone (BNF), phenobarbital (PB) and dexamethasone (DEX) on cytochrome P450 (P450) dependent monooxygenase functions were investigated in intrasplenic liver cell explants in comparison to adult liver. Fetal liver tissue suspensions were transplanted into the spleens of 60-90 days old adult male syngenic Fisher 344 inbred rats. 2, 4 or 6 months after surgery, transplant recipients and age matched controls were orally treated with BNF (1x50 mg/kg body weight (b.wt.)), PB (1x50 mg/kg b.wt.), DEX (for 3 days 4 mg/kg b.wt. per day), or the respective solvents (dimethylsulfoxide or 0.9% NaCl). The animals were sacrificed 24 (BNF, DEX) or 48 (PB) hours after the last treatment. P450 mediated monooxygenase functions were measured in spleen and liver 9000 g supernatants by three model reactions for different P450 subtypes: ethoxyresorufin O-deethylation (EROD; 1A), ethoxycoumarin O-deethylation (ECOD; 1A, 2A, 2B), and ethylmorphine N-demethylation (END; 3A). Spleen weights were significantly higher in transplanted rats, compared to controls, at all three time points after surgery. Induction with PB or DEX, and in some cases also with BNF, lead to a significant increase in liver weights of transplant recipients and control rats independent of the time after transplantation. In contrast, there was no influence on spleen weights due to BNF or PB. At all time points after surgery, with DEX a marked decrease in body weights, weights of adrenal glands and of lymphatic organs like thymus glands and spleens was observed, with the weights of the transplant containing spleens being still higher in comparison to control organs. Spleens of control animals displayed nearly no P450 mediated monooxygenase functions neither without nor with induction. After transplantation, however, significant EROD and ECOD, but hardly any END activities were seen in the host organs at all three time points after surgery. In transplant containing spleens EROD and ECOD were significantly increased after BNF or PB treatment at all three time points after surgery, and ECOD after DEX administration, but at 4 and 6 months after transplantation only. END was only induced after DEX treatment at 6 months after transplantation. With the livers of both transplant recipients and control rats EROD and ECOD were increased after BNF induction and EROD, ECOD, and END after PB treatment at all three time points after transplantation. After DEX administration END was significantly enhanced only at 2 and 4 months after transplantation, ECOD was decreased at 2 and 4 months, and EROD was diminished at all three time points after surgery. Transplantation of fetal liver tissue suspensions into the spleens did not influence monooxygenase functions and their inducibility within the respective livers of the animals. These results demonstrate that transplanted liver cells originating from syngenic fetal liver tissue suspensions display P450 dependent monooxygenase functions which are, simi lar to normal adult liver, inducible by BNF, PB and DEX. Both monooxygenase functions and their inducibility within the transplant containing spleens display quantitative and qualitative developmental changes.

Animals↗

PubMed1998

Ciprofibrate--racemate and enantiomers: effects of a four-week treatment on male inbred Fischer rats. A biochemical and morphological study.

Ciprofibrates (racemate and both enantiomers, Raccip, R- and Scip) were administered orally in doses of 1 and 10 mg/kg once daily over 28 days to male inbred Fischer 344 rats, age 90-110 days at the beginning of the experiment. Body mass gain was observed in all groups. The 1 mg groups showed almost no difference to the control group. The 10 mg groups exhibited less body mass gain, most pronounced in the Scip group. Liver masses were increased in a dose dependent manner up to more than 200%, only the 10 mg Scip group was not significantly different from the 1 mg group which exhibited an increase in liver weight to about 175%. Also the kidney weights increased to 130%, whereas thymus and spleen weights were decreased in the high dose groups. Liver microsomal cytochromes P450 (P450) concentrations were not altered in the 1 mg groups and distinctly lowered in the 10 mg groups. Ethoxyresorufin and ethoxycoumarin O-deethylations were lowered in all experimental groups in a dose dependent manner, after administration of the high doses down to 30% of the control levels or less. Pentoxyresorufin O-depentylation, however, was increased in all 1 mg groups. In the high dose groups it was not altered. Ethylmorphine N-demethylation was decreased after administration of the high doses by about 50%, but only Scip decreased this reaction also after administration of the low dose. NADPH/Fe2+-stimulated microsomal luminol and lucigenin amplified chemiluminescence was increased, whereas hydrogen peroxide formation was depressed even by the low doses to 50% of the normal values, to about 25% by the high doses. Microsomal lipid peroxidation, however, was only slightly or not influenced. Glutathion concentrations (in the reduced and the oxidized form) were increased in a dose dependent manner by about 20 to 30%, the concentration of lipid peroxides was not significantly influenced. Thus, the effects of the enantiomers were not different and were similar to those of the racemate. In serum, cholesterol and triglycerides were only moderately lowered. Albumin concentrations were significantly enhanced in all groups, total proteins after 1 mg/kg Raccip only. Serum bilirubins were not altered, and among the indicator enzymes for liver damage only ALAT, alkaline phosphatase and the dehydrogenases were increased, in no case higher than twofold. Histologically distinct effects were seen after administration of both doses, more pronounced after 10 mg/kg, but with no differences between the enantiomers and Raccip: marked hypertrophy of the hepatocytes, reduced staining of the nuclei, strongly acidophilic granulated cytoplama, no basophilia of the cell bodies, loss of glycogen. These changes were most pronounced around the central veins. Hepatocyte apoptoses also were observed. By immunohistochemistry an increased staining was seen for all P450 isoforms tested (1A1, 2B1, 2E1, 3A2 and 4A1), predominantly perivenously and most pronounced after administration of the high doses without differences between Rcip, Scip or Raccip (preliminary results). By electron microscopy a moderate proliferation of peroxisomes after treatment with 1 mg/kg Cips with a ratio between mitochondria and peroxisomes of about 1:1 (controls: 10:1) was observed, and the peroxisomes were a more heterogeneous population. The relative portions of glycogen and both forms of the ER decreased. Treatment with 10 mg/kg Rcip, Scip or Raccip led to a strong increase in the number of peroxisomes, in some hepatocytes the ratio between mitochondria and peroxisomes was 1:3 with an increased heterogeneity among the peroxisomes evidenced by a broad range of electron densities. Most peroxisomes lacked a nucleoid. Thus, the biochemical effects differed only slightly and the morphological effects of the enantiomers were not different and were similar to those of the racemate.

Administration, Oral↗

PubMed1998

Chromatographic resolution of ciprofibrate and interaction of the racemate and both enantiomers with rat liver microsomes in vitro.

The enantiomers of ciprofibrate may be achieved by enantioselective HPLC separation of its methylesters using a OD - Daicel column. Ciprofibrates (racemate and both enantiomers) bind to oxidized cytochrome P-450 in rat liver microsomes according type II like aniline or most probably as inversed type I, but less pronounced and with a general shift to the left. Ethylmorphine N-demethylation, ethoxycoumarin and ethoxyresorufin O-deethylation are all inhibited by the ciprofibrates, most effectively ethoxyresorufin O-deethylation by S(-)-ciprofibrate even in microM concentrations. Microsomal luminol and lucigenin amplified chemiluminescence indicating the formation of reactive oxygen species, microsomal hydrogen peroxide formation and NADPH/Fe stimulated lipid peroxidation were inhibited in a concentration dependent manner in concentration ranges between mM and microM. This might be due to distinct scavenger activities of all 3 compounds: the zymosan stimulated chemiluminescence of whole blood was completely inhibited in mM concentrations and influenced significantly down to concentrations of 10 microM, whereas burst and phagocytosis tests with human polynuclear leucocytes were not influenced.

Aniline Compounds↗

PubMed1998

Influence of hypoxia and hypoxia/hypercapnia upon brain and blood peroxidative and glutathione status in normal weight and growth-restricted newborn piglets.

Glutathione (reduced (GSH) and oxidized (GSSG)), lipid peroxidation products (TBAR) and in vitro production of reactive oxygen species (ROS, by means of stimulated lipid peroxidation, H2O2 formation and amplified chemiluminescence (CL) in 9000 xg brain supernatants) were studied in the cerebellum (C) and temporoparietal area (TP) of the brain of normal weight (NW) and spontaneously intra-uterine growth-restricted newborn piglets (IUGR) after 1 hour hypoxia (fractional inspired oxygen concentration (FiO2) 8%), and in combination with 10% CO2, followed by 3 hours recovery (FiO2 30%). The strong GSH depletion accompanied by an increased concentration of GSSG and TBAR, more distinct in IUGR, is the most important result in the brain after hypoxia and reoxygenation. Hypercapnia-related acidosis seems to protect the brain of IUGR from hypoxia/reoxygenation induced injury by reducing GSH depletion as well as GSSG and TBAR increases. But stimulated lipid peroxidation and H2O2 formation in 9000 xg supernatants of C and TP were found to be higher in acidosis and hypercapnia. Decreased or unchanged amplified CL, demonstrating lower in vitro production of ROS, cannot explain the GSH depletion after hypoxia and reoxygenation. The scarce changes in erythrocyte GSH and GSSG as well as plasma TBAR concentrations did not reflect the findings in the brain. Nevertheless, the changes in the brain support the hypothesis that oxidative stress plays a role in neuronal damage after hypoxic stress, but the brain of IUGR did not reveal a special response to moderate hypoxia.

Acid-Base Equilibrium↗

PubMed1998

Investigation on possible antioxidative properties of the NMDA-receptor antagonists ketamine, memantine, and amantadine in comparison to nicanartine in vitro.

Possible antioxidative properties of three N-methyl-D-aspartate (NMDA)-receptor antagonists, the anesthetic ketamine and the antiparkinson drugs memantine and amantadine were investigated in vitro on the microsomal cytochrome P450 (P450) system of rat livers and on rat whole blood chemiluminescence in comparison to nicanartine, a substance with known antiatherosclerotic, hypolipemic and antioxidative capacity. For this purpose, the effects on NADPH- and iron-stimulated lipid peroxidation (LPO), hydrogen peroxide (H2O2) production, and NADPH- and iron-stimulated lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) were examined using rat liver microsomes. Additionally, the influence on LM amplified whole blood chemiluminescence after zymosan activation of polymorphonuclear leukocytes (WB-CL) was investigated. Furthermore, binding to P450 and effects on P450 mediated monooxygenase function, as measured by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (END), were assessed. Nicanartine concentration dependently reduced LPO and H2O2 production already at a concentration of 1 microM, whereas LC and LM amplified CL and WB-CL were not affected. EROD and END were concentration dependently diminished starting at 1 microM, and ECOD already at 0.1 microM. Ketamine decreased LPO, H2O2 production and LM and LC amplified CL, starting at 100 microM. WB-CL was significantly diminished already at 10 microM. EROD and ECOD were inhibited at 10 and 100 microM and END at 100 microM. With memantine a concentration dependent inhibition of LPO and WB-CL was seen at 100 and 1000 microM and a reduction of LC and LM amplified CL only at 1000 microM. H2O2 production was not affected. EROD and ECOD were significantly diminished by a concentration of 100 microM. No effect was observed on END. Amantadine significantly reduced LPO and WB-CL, but only at 1000 microM. H2O2 production and LC and LM amplified CL were not affected. EROD was significantly diminished at 100 microM, whereas no influence was seen on ECOD and END. Nicanartine displayed type II or reverse type I, ketamine, memantine and amantadine type I substrate binding to P450. The highest binding affinity to P450 was seen with nicanartine, followed by ketamine, memantine and then amantadine. These results demonstrate, that all four substances seem to act as radical scavengers and/or as inhibitors of the oxidative function of P450. All four substances seem to interfere with the monooxygenase function of P450. This may result in a possible influence on the biotransformation of endogenous as well as of foreign compounds. The effects of nicanartine were much more pronounced than those of ketamine, memantine, and amantadine.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1998

Influence of different transplantation methods on liver cell survival in spleens of rats.

Isolated hepatocytes, liver tissue suspensions, or liver tissue cylinders from biopsies were transplanted into the spleens of adult male rats. Donors were syngenic fetuses, syngenic or allogenic adult rats, or autologous material was obtained from the rat's own liver. The outcome of the different transplantation procedures was evaluated at 1 and 6 months after surgery. Additionally the influence of a 30% hepatectomy (HX) on the result of the transplantation was investigated. When fetal material was transplanted, the best results in sequence with respect to the number of viable hepatocytes within the spleens were obtained (1) after transplantation of syngenic fetal liver tissue suspensions, (2) syngenic fetal liver tissue cylinders and (3) syngenic fetal isolated hepatocytes. HX only improved the results with transplantation of syngenic fetal isolated hepatocytes. After transplantation of syngenic fetal liver tissue suspensions and isolated hepatocytes, but not after transplantation of syngenic fetal liver tissue cylinders, the number of hepatocytes was higher at 6 months than at 1 month after surgery. Concerning syngenic adult liver material, only transplantation of isolated hepatocytes lead to a remarkable and increasing number of surviving hepatocytes at both 1 and 6 months after surgery. These results were further improved by HX. With syngenic adult liver, the other transplantation methods yielded no or nearly no viable hepatocytes in the spleens. In comparison to the results after transplantation of syngenic fetal liver tissue suspensions, transplantation of syngenic adult isolated hepatocytes was less efficient, but still yielded more viable hepatocytes than the transplantation of syngenic fetal isolated hepatocytes. After transplantation of autologous liver tissue suspensions, autologous liver tissue cylinders or allogenic adult liver material only few surviving hepatocytes were observed. At 1 month after transplantation of syngenic fetal liver material, syngenic adult isolated hepatocytes or autologous liver tissue cylinders into the spleens 40-80% of the explants consisted of bile ducts independent from the transplantation method. At 6 months after surgery the bile ducts were much less and in some cases no longer visible. After transplantation of autologous liver tissue suspensions or allogenic adult liver material only very few bile ducts were seen, but anyhow in those cases only poor results were obtained. Thus, with respect to transplantation outcome and long-term liver cell survival, intrasplenic transplantation of both syngenic fetal liver tissue suspensions and syngenic adult isolated hepatocytes seem to be the most suitable methods and should be chosen for further investigations on explant morphology and function.

Animals↗

PubMed1998

Influence of hypoxia and hyperthermia upon peroxidative and glutathione status in growth-restricted newborn piglets.

Normal weight (NW) and spontaneously intra-uterine growth-restricted newborn piglets (IUGR) were submitted to 1 hour hypoxia and hyperthermia followed by 3 hours reoxygenation. Glutathione (GSH, GSSG), lipid peroxidation products (TBAR) and cytochrome P450 dependent production of reactive oxygen species were studied by determination of H2O2 production and amplified chemiluminescence (CL) in brain and peripheral organs of NW and IUGR. A severe decrease of GSH was accompanied by enhanced H2O2 formation and lipid peroxidation in the brain of both NW and IUGR after hypoxia/hyperthermia and reoxygenation. In the other organs studied, only muscles showed enhanced lucigenine amplified CL and TBAR production. Nearly the same results in NW and IUGR reveal no higher risk of IUGR.

Animals↗

PubMed1998

Developmental expression of cytochrome P450 isoforms after transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats.

In the present study, the developmental expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, and the ability to store glycogen was investigated in intrasplenic liver cell explants in comparison to adult and fetal liver. Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher inbred rats. Animals were sacrificed at 3 days, 1, 2, 4 weeks, 2, 4, 6 months and 1 year after transplantation. Spleens and livers of transplant recipients were compared to those of sham operated and control rats. Three days after transplantation little bulks of hepatocytes and only few bile ducts were seen in the red pulp of the transplant containing spleens. A massive hypertrophy and proliferation of bile ducts and also an augmentation in the number of hepatocytes were observed 4 weeks after transplantation. One month later, however, the bile ducts had become more and more atrophic, while instead the number of hepatocytes continuously increased. One year after surgery large masses of hepatocytes with apparent cord structure and only few but well preserved bile ducts were seen. Within the livers of adult rats, P450 1A1 was only slightly expressed by some hepatocytes around the central veins. P450 2B1 and 3A2 isoforms expression was much stronger, but also predominantly located in the hepatocytes of the central zone of the liver lobule. Hepatocytes of fetal livers displayed a moderate P450 1A1 expression. In some cells also a very mild staining for P450 2B1 and 3A2 was observed. Within the hepatocytes of the intrasplenic liver cell explants P450 1A1 was still expressed 3 days after transplantation, disappeared at 1 week after surgery, but reappeared at 4 weeks after transplantation. After 2, 4 and 6 months no staining for P450 1A1 was detectable any more. One year after transplantation again a slight P450 1A1 expression appeared. With P450 2B1 and 3A2 a mild to moderate expression was seen already at 3 days after transplantation. Four weeks after surgery nearly all of the hepatocytes were stained for P450 2B1 and 3A2, but there were marked differences between the individual cells in the extent of the expression of these two P450 subtypes, like it was also the case with normal adult liver. Within hepatocytes of the fetal livers strongly stained glycogen granules were seen, which, in comparison to adult livers, were rather coarse-grained. Three days after transplantation the glycogen granules in the transplanted hepatocytes were still coarse-grained, but from 1 week after transplantation on, they became more and more fine-grained. As it was also the case with normal adult liver cells, there were marked differences between the individual transplanted hepatocytes in their glycogen content. These results demonstrate that transplanted liver cells originating from syngenic fetal liver tissue suspensions can survive in host organs like the spleen for at least 1 year. They proliferate, differentiate, are able to store glycogen, and express different P450 isoforms, like normal adult liver cells.

Animals↗

PubMed1998

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of beta-naphthoflavone, phenobarbital and dexamethasone on cytochrome P450 isoforms expression and on glycogen storage.

In the present study, the effect of beta-naphthoflavone (BNF), phenobarbital (PB) and dexamethasone (DEX) on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, and on glycogen storage was investigated in intrasplenic liver cell explants in comparison to adult liver. Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher inbred rats. Four months after surgery, transplant recipients and age matched controls were orally treated with BNF (1 x 50 mg/kg body weight (b.wt.)), PB (1 x 50 mg/kg b.wt.), DEX (for 3 days 4 mg/kg b.wt. per day), or the respective solvents (dimethylsulfoxide or 0.9% NaCl). The animals were sacrificed 24 (BNF, DEX) or 48 (PB) hours after the last treatment. The livers of both solvent treated transplant recipients and control rats displayed only in few liver lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, which was all mainly located in the hepatocytes around the central veins (zone III, according to Rappaport). After BNF administration a P450 1A1 expression was induced in the hepatocytes of the peripheral regions of the liver lobules (zone I, according to Rappaport), whereas the staining of the hepatocytes around the central veins disappeared. Also the staining for P450 2B1 in the hepatocytes of zone III became slightly more pronounced. Following PB treatment the P450 1A1 expression in the hepatocytes of the central regions (zone III), as seen in few lobules after solvent treatment only, was reduced, whereas the staining for P450 2B1 and 3A2 was more pronounced in the hepatocytes of the intermedial and central regions of the liver lobules (zone II and III). DEX treatment diminished P450 1A1 and 2B1 expression within the livers of both transplant recipients and control rats. In contrast, the staining for P450 3A2 was enhanced in all regions of the liver lobules. Transplantation of fetal liver tissue suspensions into the spleens did not influence the inducibility of P450 isoforms expression within the respective livers of the animals. Spleens of control rats displayed no P450 isoforms expression without as well as with induction. In the explant containing spleens, however, similar to normal liver, the transplanted hepatocytes displayed nearly no P450 1A1, but a strong P450 2B1 and 3A2 expression. After BNF treatment a staining for P450 1A1 was induced and also the P450 2B1 expression was slightly more pronounced. PB treatment caused an increase in the staining for P450 2B1 and 3A2 and DEX administration for P450 3A2 within the transplanted hepatocytes. Additionally, after DEX treatment some bile ducts of the explants displayed a slight staining for P450 1A1, 2B1 and 3A2. All hepatocytes within the livers of both solvent treated transplant recipients and control rats displayed a slightly PAS-positive cytoplasma and, in most cases, homogeneously distributed, fine-grained, strongly PAS-stained granules indicating glycogen storage. No regional variance in the glycogen content of the hepatocytes was seen within the liver lobules, but there was a marked difference between the individual hepatocytes of the same lobular region in the extent of glycogen accumulation. The hepatocytes within the explants displayed the same type of glycogen storage as did the adult liver cells. BNF treatment did not display any effect on the glycogen accumulation in livers and intrasplenic liver cell explants. After PB administration, only in livers, but not in the transplants, the glycogen content in the hepatocytes around the central veins was slightly reduced. DEX treatment lead to an excessive storage of fat within the hepatocytes of both livers and spleens. Thus, the glycogen was displaced, leading to a "spoke-wheel" like pattern of glycogen storage. Additionally, within the hepatocytes of both livers and liver cell explants a higher amount of glycogen seemed to be stored and the granules appeared to be more coarse-grained. (ABSTRACT

Animals↗