Biomedical subjects

L Aicher

Publications and source records attributed to L Aicher.

10 recordsLinked to original sources

PubMed1998

Cyclosporine A-induced decrease in calbindin-D 28 kDa in rat kidney but not in cerebral cortex and cerebellum.

Recently, we reported that in rat, cyclosporine A (CsA) markedly decreases the levels of calbindin-D (CABP-D) 28 kDa in kidney. CABP-D 28 kDa is a calcium-binding protein which is highly expressed in calcium-transporting tissues such as kidney or brain. In this study, we investigated whether, in addition to the kidney, CsA also has an effect on CABP-D 28 kDa in rat brain. Three groups of male Wistar rats received 15 mg/kg/day or 50 mg/kg/day of CsA orally for 12 days, whereas controls received vehicle solution for the same period. CABP-D 28-kDa protein and CsA were quantified in homogenates of kidney, cerebral cortex and cerebellum, and the localization of CABP-D 28 kDa was assessed in the different tissue sections by immunohistochemistry. In kidney, CABP-D 28 kDa was strongly and dose dependently decreased, and was located in tubular epithelial cells. In brain, CABP-D 28 kDa was not changed and was mainly located in pyramidal cells of the cortex and in cerebellum exclusively in Purkinje cells. High CsA concentrations were measured in kidney, more than 17-fold greater than those found in cortex. In cerebellum, CsA was below the limit of detection. These data suggest that at clinically relevant doses, CsA may not affect CABP-D 28-kDa levels in brain.

Animals↗

PubMed1998

New insights into cyclosporine A nephrotoxicity by proteome analysis.

Using two-dimensional gel electrophoresis (2-DE), we recently discovered an association between decreased calcium-binding protein, calbindin-D 28 kDa, urinary calcium wasting and intratubular corticomedullary calcifications in rat kidney. This observation prompted us to investigate kidney tissues of other species, including man. In this paper we show that in dogs and monkeys, which are generally devoid of cyclosporine A (CsA)-mediated nephrotoxicity, renal calbindin levels were not affected by the CsA treatment whereas in CsA-treated human kidney-transplant recipients with renal vascular or tubular toxicity, a marked decrease in renal calbindin-D 28 kDa protein level was found in most of the kidney biopsy sections. The present results strongly suggest that calbindin is a marker for CsA-nephrotoxicity. The discovery of calbindin-D 28 kDa being involved in CsA toxicity has evolved from the application of 2-DE and has not been reported previously, proving that proteomics can provide essential information in mechanistic toxicology. Considering the current improvements in proteome methods it is expected that high throughput proteomics will become an indispensable tool in preclinical safety testing.

Animals↗

PubMed1998

Changes in the liver protein pattern of female Wistar rats treated with the hypoglycemic agent SDZ PGU 693.

SDZ PGU 693 acts as a hypoglycemic agent by stimulating glucose utilisation in insulin-sensitive peripheral tissues, such as skeletal muscle and fat. In a 28 day toxicity study the compound was found to induce hepatocellular hypertrophy in Wistar rats treated with 300 mg/kg/day. To gain insights into the pathomechanism of these alterations, aliquots of liver samples from control and treated female Wistar rats were separated by two-dimensional protein gel electrophoresis and the digitized images of the protein patterns were searched for protein abundance changes. Significant treatment-related quantitative changes (P < 0.001) were found in 29 liver proteins. Major increases were observed in several microsomal proteins, including NADPH cytochrome P-450 reductase, cytochrome b5 and serine protease inhibitor. The changes in the cytochrome related enzymes, both known co-factors of the P-450 enzyme system, strongly suggest that SDZ PGU 693 induces microsomal proliferation and induction of the P-450 enzyme system. Decreases were observed in a series of mitochondrial proteins, such as F1ATPase-delta subunit and ornithine aminotransferase precursor as well as in several cytosolic proteins such as the liver fatty acid binding protein, arylsulfotransferase and the senescence marker protein-30. The changes in F1ATPase-delta subunit and liver fatty acid binding protein together suggest a down-regulation of the mitochondrial liver fatty acid metabolism, likely reflecting the pharmacological action of the compound. These results show that SDZ PGU 693 produces a complex pattern of gene expression changes which give insights into the molecular mechanisms of both its pharmacological action and a toxic response.

Animals↗

PubMed1998

Hepatocellular effects of cyclosporine A and its derivative SDZ IMM 125 in vitro.

The novel immunosuppressive drug O-hydroxyethyl-D(Ser)8-cyclosporine (SDZ IMM 125) and cyclosporine A (CyA) were compared in different in vitro models with respect to hepatocellular side effects. SDZ IMM 125 was less lipophilic than CyA and also decreased liposomal membrane anisotropy less. Furthermore, SDZ IMM 125 increased Na+ and Ca++ permeability across the liposomal membranes significantly more than CyA. The uptake of CyA and SDZ IMM 125 into freshly isolated rat hepatocytes was neither saturable, Na+ dependent or temperature sensitive, nor could it be inhibited vice versa, indicating passive diffusion. The diffusion coefficient of CyA was about two times higher than that of SDZ IMM 125, reflecting its higher lipophilicity. In primary hepatocyte monolayers the cellular concentrations of CyA were about two times higher than that of SDZ IMM 125. As an indicator of cholestasis the saturable uptake of cholyltaurine into isolated cells was found to be apparently competitively inhibited to the same extent by both compounds. In isolated perfused rat livers SDZ IMM 125 caused a significantly greater decrease in bile flow than did CyA. Release of lactate dehydrogenase from hepatocyte primary cultures and from isolated perfused livers were determined as parameter of cell damage. In both systems the cytotoxicity of SDZ IMM 125 was significantly higher than that of CyA. The data suggest that SDZ IMM 125 causes greater cholestatic and cytotoxic effects than CyA at equimolar cellular exposure.

Animals↗

PubMed1997

Decrease in kidney calbindin-D 28kDa as a possible mechanism mediating cyclosporine A- and FK-506-induced calciuria and tubular mineralization.

The use of the immunosuppressant cyclosporine A (CsA) is limited by its adverse renal effects. Most recently, we reported that the drug markedly decreases the levels of the calcium-binding protein calbindin-D 28kDa in kidneys of male Wistar rats. In the present study, the potential relationship between drug-induced nephrotoxicity and the decrease in kidney calbindin-D 28kDa was investigated. Four groups of male Wistar rats were treated for 10 or 31 days with either the immunosuppressant CsA (50 mg/kg/day), FK-506 (5 mg/kg/day), rapamycin (5 mg/kg/day) or with the nonimmunosuppressive cyclosporine derivative 3'keto-[Bmt1]-[Val2]-CsA (SDZ PSC-833) (50 mg/kg/day), and the effects on calcium homeostasis, kidney histology and renal calbindin-D 28kDa were examined. Similar effects were found with CsA and FK-506; both drugs strongly reduced kidney calbindin-D 28kDa protein levels, increased urine calcium excretion, caused intratubular calcification, and induced basophilic tubules. In contrast, rapamycin and SDZ PSC-833 caused no decrease in renal calbindin-D 28kDa levels, no noticeable alterations in calcium metabolism, and no renal calcification. The results provide evidence for a link between decreased renal calbindin, increased calcium urine excretion, and intratubular kidney calcification. The present data show no correlation between the decrease in renal calbindin and the induction of basophilic tubules; however, it needs to be investigated if these apparently independent kidney effects may have a common origin upstream of calbindin expression.

Animals↗

PubMed1996

Cyclosporine A decreases the protein level of the calcium-binding protein calbindin-D 28kDa in rat kidney.

Despite the widespread use of cyclosporine A (CsA), its mechanism of action and side effects are not yet completely understood. There exists a large body of evidence suggesting that disturbance of calcium homeostasis is a critical step in the cascade of cellular and molecular events induced by the drug. As recently shown in our laboratory by two-dimensional protein gel electrophoresis (2-DE) analysis of kidney homogenates, CsA induced numerous changes in several kidney proteins. One kidney protein in particular was shown to be strongly down-regulated by the drug. In this work we report the identification of the strongly decreased kidney protein as calbindin-D 28kDa, a vitamin D-dependent calcium-binding protein associated with calcium handling by cells. The assignment of the down-regulated protein spot is based on its internal amino acid sequence analysis and its specific reaction with a monoclonal antibody raised against calbindin-D 28kDa. In kidney homogenates of male Wistar rats treated with 50 mg/kg/d CsA for up to 28 days, calbindin levels were measured by ELISA and were shown to be continuously decreased with prolonged CsA treatment. To our knowledge, this is the first report describing the effect of CsA on kidney calbindin-D 28kDa protein levels. Further studies are needed to elucidate whether the CsA-mediated down-regulation of the calcium-binding protein calbindin-D 28kDa may be a critical factor for the renal adverse effects induced by this drug.

Amino Acid Sequence↗

PubMed1995

Protein variability in male and female Wistar rat liver proteins.

In the emerging field of mechanistic toxicology, growing attention is being paid to the interpretation of sex-related toxic responses. The Wistar rat outbred stock is a frequently used rodent for toxicity testing. Outbred strains, which display a relatively high degree of genetic variability between the individual animals are often used in risk assessment, as they are considered to best approximate the variability present in a human population. In this study two-dimensional (2-D) protein gel electrophoresis was applied to investigate the liver protein patterns of male and female Wistar rats and to search for (i) sex-related and (ii) interindividual qualitative and quantitative differences in protein expression. Among the sexes, six proteins were detected that were shown to be exclusively present in male rats and one that was present only in females. A male-specific protein was tentatively assigned to alpha 2u globulin. Seven protein spots showed statistically significant abundance changes (p < 0.001) between males and females, one was tentatively assigned to heme oxygenase 1 and another to the 23 kDa morphin-binding protein. Four sets of protein spots were detected that showed positional shifts in the individual patterns and are likely to represent polymorphic proteins inherent in the Wistar rat. These results form a valuable basis for future investigations of drug-induced changes in the male and female Wistar rat liver pattern.

Alpha-Globulins↗

PubMed1994

Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants.

Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy-Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability.

Amino Acid Sequence↗

PubMed1994

Developmental expression of human microsomal epoxide hydrolase.

Microsomal epoxide hydrolase (mEH) is a critical biotransformation enzyme that catalyzes the hydrolysis of a large number of epoxide intermediates, which arise frequently from the oxidation of pharmaceutical and environmental compounds by the cytochrome P450 mixed function oxygenase system. The enzyme mEH has been implicated directly as a key determinant of certain chemically initiated cancers and developmental toxicities. To evaluate mEH expression in human tissues and provide a framework for assessing the relative risk in the fetus to potential developmental toxins, in the current study, the authors characterized mEH in human tissues as a function of gestational age. Analyses included enzymatic activity determinations, immunochemical quantitation of protein levels and RNA hybridization assays. With respect to activity, hepatic mEH enzymatic levels were strongly correlated with increasing gestational age (r = .82, P < .001). Similarly, mEH activity levels in the liver were highly correlated with protein contents (r = .93, P < .001). However, mEH enzymatic activity in the fetal lung did not exhibit similar concordance nor did measured RNA levels appear to correlate with enzymatic activity levels in fetal or adult tissue samples. Of the fetal tissues surveyed, the liver and adrenal glands exhibited the highest levels of detectable mEH RNA, followed by the lung and kidney. These data suggest that post-transcriptional regulatory pathways may be important in determining constitutive levels of mEH functional activity and that fetuses during early gestation may be uniquely sensitive to the presentation of epoxide-containing xenobiotics compared with fetuses at later stages of development.

Aging↗

PubMed1993

Human peripheral lymphocytes as indicators of microsomal epoxide hydrolase activity in liver and lung.

In this study, we have applied an improved assay for the determination of microsomal epoxide hydrolase activity to assess enzymatic levels in human lung, liver, and blood lymphocytes. The assay is fluorescence-based and monitors the epoxide hydrolase-mediated conversion of (+/-)-benzo[a]pyrene-4,5-epoxide to (+/-)-trans-benzo[a]pyrene-4,5-dihydrodiol, using a high pressure liquid chromatography separation system. Approximately a 40-fold range in microsomal epoxide hydrolase activities was detected in blood lymphocytes collected from 70 individual donors. In 38 individuals who were sampled twice after a 3-month interval, the repeatability of an individual's lymphocyte epoxide hydrolase activity was highly correlated (r = 0.80, p < 0.02). In addition, within the same individual there appeared to be a strong correlation between lymphocyte and liver epoxide hydrolase activity (r = 0.92, p = 0.02), and some correlation between liver and lung activity (r = 0.58, p = 0.05). Activities were assessed in lymphocytes from a styrene-exposed worker population but no significant associations between blood concentrations of styrene and epoxide hydrolase activity levels were observed. Neither were any correlations detected in these workers between epoxide hydrolase activities and age, years on the job, alcohol consumption, sex, or smoking status. The results of our study suggest that blood lymphocytes are a useful sentinel cell for epoxide hydrolase activity determinations in individuals, as these measures are relatively stable over time and appear to reflect activity levels in other target organs.

Adult↗