Pharmacokinetics/genotype associations for major cytochrome P450 enzymes in native and first- and third-generation Japanese populations: comparison with Korean, Chinese, and Caucasian populations.

Application of foreign clinical data across geographic regions can accelerate drug development. Drug disposition can be variable, and identification of factors influencing responsible pharmacokinetic/pharmacogenomic approaches could facilitate the universal application of foreign data and reduce the total amount of phase III clinical trials evaluating risks in different populations. Our objective was to establish and compare genotype (major cytochrome P450 (CYP) enzymes)/phenotype associations for Japanese (native and first- and third-generation Japanese living abroad), Caucasian, Chinese, and Korean populations using a standard drug panel. The mean metabolic ratios (MRs) for the four ethnic groups were similar except for a lower activity of CYP2D6 in Caucasians and CYP2C19 in Asians. Genotype, not ethnicity, impacted the MR for CYP2C9, CYP2C19, and CYP2D6; neither affected CYP1A2, CYP2E1, and CYP3A4/5 activities. We conclude that equivalent plasma drug concentrations and metabolic profiles can be expected for native Japanese, first- and third-generation Japanese, Koreans, and Chinese for compounds handled through these six CYP enzymes.

Validation of a flow cytometric acridine orange micronuclei methodology in rats.

Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to evaluation of much larger numbers of cells clearly demonstrate the usefulness of flow cytometry method in the routine micronucleus evaluation.

RNA expression in the early characterization of hepatotoxicants in Wistar rats by high-density DNA microarrays.

High-density microarrays are useful tools to study gene expression for the purpose of characterizing functional tissue changes in response to the action of drugs and chemicals. To test whether high-density expression data can identify mechanisms of toxicity and to identify an unknown sample through its RNA expression pattern, groups of male Wistar rats were administered 6 hepatotoxicants. The compounds chosen for this study were microcystin-LR (MLR), phenobarbital (PB), lipopolysaccharide (LPS), carbon tetrachloride (CT), thioacetamide (THA), and cyproterone acetate (CPA). These hepatotoxicants are known to induce adverse liver effects through different mechanisms. Liver mRNA was isolated and used to generate biotinylated cRNA for hybridization to a custom 1,600-rat gene DNA microarray. Treatment correlation matrices analyzed hybridization data from a hepatotoxicant-blinded sample, with gene expression coefficients (GEC) evaluated by means of hierarchical cluster analysis and visual representation as dendrograms. The experimental liver toxicity from the different treatments was confirmed by means of concurrent histopathology, liver enzymes, and bilirubin assays. This toxico genomic analysis identified multiple genes and groups of genes that were affected by the hepatotoxicants on study, indicating that high-density microarray expression data are useful to identify groups of genes involved in toxicity. In addition, the mRNA expression profile of an unidentified sample can be accurately identified when compared with the expression profiles resident in the data set. This study supports the use of gene expression-profiling technology to determine or to predict toxic liver effects.

EDTA-dependent platelet phagocytosis. A cytochemical, ultrastructural, and functional characterization.

Platelet satellitosis of polymorphonuclear cells is a phenomenon induced or enhanced by the anticoagulant EDTA. In contrast with previously reported studies, the subject in the present case did not demonstrate platelet satellitism but was profoundly pseudothrombocytopenic owing to platelet phagocytosis. Virtually all polymorphonuclear leukocytes and monocytes contained numerous ingested platelets in contrast with previous cases in which phagocytosis was observed only rarely and involved ingestion of single cells. The phenomenon was documented by immunocytochemical staining and transmission electron microscopy. Autoantibodies were detected in EDTA-anticoagulated blood. However, neither platelet antibody nor phagocytosis was present when heparin, acid-citrate dextrose, or citrate was used as an alternative anticoagulant. The antibody was not temperature dependent. Mixing studies showed the transfer of the phagocytosis phenomenon to healthy donors. Although platelet function assays are typically normal in EDTA-dependent platelet satellitism, this subject showed no secondary aggregation wave in response to adenosine diphosphate and depressed adenosine triphosphate release with collagen, adenosine diphosphate, and arachidonic acid.

Pharmacogenetic application in drug development and clinical trials.

Pharmacogenetics examines the genetic characteristics of individuals to understand variations in response to therapeutics. This approach has the potential to significantly affect the development of new medicines. The application of pharmacogenetic principles could yield significant time and resource savings within the drug development process. In preclinical drug development, pharmacogenetics could be applied to compound screening and identifying potential side effects before entering full clinical testing. Subpopulations of patients with different drug responses and underlying genetic markers could be stratified in clinical trials by analyzing their genotype. These data can improve clinical trial design and offer the possibility of optimized drug prescription based on patient genotype. Pharmacogenetics can guide the development of therapeutic interventions by identifying nonresponder patient groups. Advances in high-throughput genotyping technologies have added potential by facilitating the technical hurdles and improving drug development strategies, clinical trial design, and postmarket pharmaco-vigilance. Pharmacogenetics, thus, impacts all phases of drug development and will fundamentally change the practice of medicine in the near future.

Genotyping for Functionally Important Human CYP2D6*4 (B) Mutation Using TaqMan Probes.

The microsomal enzyme cytochrome P450 2D6 (CYP2D6), also known as debrisoquine 4-hydroxylase, is involved in the oxidative metabolism of many widely used drugs, including neuroleptics, tricyclic antidepressants, antiarrhythmics, and ß-adrenergic blocking agents (1). Polymorphisms of CYP2D6 are the best characterized examples of genetically mediated effects on a drug-metabolizing enzyme of clinical importance (2). When a drug that is a CYP2D6 substrate is taken by different individuals, it is not uncommon to observe large differences in plasma concentrations at steady state. This is explained, in part, by the three clinically distinct phenotypes associated with the CYP2D6 gene, normal metabolizers, poor metabolizers, and rapid metabolizers. In normal metabolizers, steady-state plasma drug concentrations fall within the desired therapeutic range and toxic effects are nonexistent or minimal. In fast-metabolizer individuals, desired concentrations are below therapeutic, and these patients generally do not respond at the recommended dosing regimen. In poor-metabolizer individuals, drug concentrations are above therapeutic level and undesired toxicity can be evoked.

Effects of phenylhydrazine or phlebotomy on peripheral blood, bone marrow and erythropoietin in Wistar rats.

This study was conducted to characterize better the response of rats to blood loss and hemolysis and to incorporate automated methods into the routine evaluations of those responses. Serial phlebotomies of 1.5-2.0 ml of blood per day for 5 days, or intraperitoneal injection of 50 mg kg(-1) phenylhydrazine (PHZ) for 3 days, were used to cause anemia associated with blood loss or hemolysis, respectively. Maximum decreases in red blood counts were observed on Day 3 in PHZ-treated animals (68%) and Day 4 in blood-loss animals (35%). In the routine complete blood count (CBC), hemoglobin, hematocrit/hemoglobin ratio and erythrocyte indices could be used to discriminate between the two treatments. Free plasma hemoglobin in PHZ-treated animals resulted in marked elevations of mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) with a 2:1 hematocrit/hemoglobin ratio rather than the anticipated 3:1 ratio. Although both groups of animals had elevated white blood cell counts, PHZ-treated animals also had monocytosis and basophilia. Reticulocyte counts were more sensitive than erythropoietin (EPO) concentrations in predicting erythroid changes. Maximum mean reticulocyte values were ca. 24% in serially phlebotomized animals and >99% in PHZ-treated rats. Plasma EPO levels were 4-10-fold higher than EPO levels in urine, kidney or liver. Flow cytometric differentials of rat bone marrow using 2, 7-dichlorofluorescin successfully predicted erythroid hyperplasia in both experimental groups. Erythrocyte indices returned to normal within 14 days and the remaining CBC parameters were normal within 28 days for both treatment groups. Reticulocyte counts remained slightly elevated on Day 28, but were normal when assessed at Day 56 in blood-loss and PHZ-treated animals.

Spontaneous and thiazolidinedione-induced B6C3F1 mouse hemangiosarcomas exhibit low ras oncogene mutation frequencies.

Hemangiosarcomasare uncommon malignant endothelial cell tumors in humans and experimental animal species. The mechanisms giving rise to these tumors are poorly understood even though the histotypes are comparable between humans and rodents. Activating mutations in cellular ras protooncogenes have been detected in sporadic and chemically induced human and rodent hemangiosarcomas. Ras activation significantly modulates tumor angiogenesis, suggesting that mutations in ras genes might be causally related to vascular tumorigenesis. To more clearly define the role of ras in experimental vascular tumorigenesis, mutations in the Ki- and Ha-ras genes were characterized in 63 hemangiosarcomas that arose unexpectedly in control and treated B6C3F1 mice during a two-year carcinogenicity study of the thiazolidinedione troglitazone. DNA was extracted from paraffin sections of mouse hemangiosarcomas, control liver, or positive control hepatocellular carcinomas with defined mutations in the Ki- or Ha-ras genes. Exons 1 and 2 of the Ki- and Ha-ras genes were independently amplified using primer extension preamplification/locus-specific heminested PCR, and PCR amplicons were directly sequenced to identify mutations in codons 12, 13, or 61. Activating mutations were detected in 3 of 63 hemangiosarcomas: a single G-->A transition in the second position of Ki-ras codon 13 in a tumor from a treated animal and two G-->T transversions in the second position of Ha-ras codon 13, one in a single tumor from a control animal and one in a tumor from a treated animal. These mutations are consistent with endogenous mutagenesis arising from oxidative DNA damage. The low frequency of mutation (<5%) indicates that ras mutations did not contribute significantly to hemangiosarcoma development and suggests that mutational ras activation may not be a necessary step in vascular tumorigenesis in mice.

High-throughput genotyping method for glutathione S-transferase T1 and M1 gene deletions using TaqMan probes.

A high-throughput genotyping method has been developed to detect gene deletion polymorphisms of glutathione-S-transferase theta and mu (GSTT1 and GSTM1). This method utilizes the 5'-nuclease activity of Taq polymerase in conjunction with fluorogenic TaqMan probes. In contrast to traditional allelic discrimination genotyping to detect single nucleotide polymorphisms, the current assay has been designed to detect gene deletion by utilizing custom-designed TaqMan probes in conjunction with an exogenous internal positive control probe. The TaqMan genotyping results were validated by a commonly used multiplex PCR technique. Screening of 71 unrelated individuals revealed gene deletion (null) genotype of 15.5% and 40.8% for GSTT1 and GSTM1, respectively. This TaqMan genotyping method is rapid, reproducible, and highly sensitive and could be applied toward fully automated large-scale genotyping.

p53 is not inactivated in B6C3F1 mouse vascular tumors arising spontaneously or associated with long-term administration of the thiazolidinedione troglitazone.

Hemangiomas and hemangiosarcomas are uncommon in rodents and humans and, as such, the mechanisms giving rise to these tumors are poorly understood. Inactivating mutations in the p53 gene have been detected in sporadic and chemically induced human and rodent hemangiosarcomas. Additionally, experimental ablation of p53 function in mice by targeted gene disruption increases the incidence of both spontaneous and carcinogen-induced vascular tumors. These findings implicate p53 disruption in vascular tumor development. In this study, we characterized p53 inactivation immunocytochemically and by gene sequencing in a large number of vascular tumors that developed in B6C3F1 mice during a long-term (2-year) study of the thiazolidinedione troglitazone. For comparative purposes, a murine hemangiosarcoma induced by polyoma middle-T antigen, which transforms endothelial cells via a p53-independent mechanism, five spontaneous human hemangiosarcoma specimens, and species-specific positive control tissues were also evaluated by immunocytochemistry for p53 inactivation. While 20% of the human hemangiosarcomas and all positive control tissues expressed significant levels of nuclear p53, indicating functional inactivation of the protein, none of the 161 mouse vascular tumors studied expressed detectable p53 protein. The absence of inactivating mutations was confirmed in eight of the histologically most malignant mouse hemangiosarcomas by sequencing exons 5 to 8 of the p53 gene. These results demonstrate that p53 inactivation did not play a role in development of the vascular tumors seen in the long-term study of troglitazone, and they indicate that loss of p53 function is not essential for vascular tumor development in mice.

Regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury.

Inflammatory lung injury is probably regulated by the balance between proteases and protease inhibitors together with oxidants and antioxidants, and proinflammatory and anti-inflammatory cytokines. Rat tissue inhibitor of metalloprotease-2 (TIMP-2) and secreted leukoprotease inhibitor (SLPI) were cloned, expressed, and shown to be up-regulated at the levels of mRNA and protein during lung inflammation in rats induced by deposition of IgG immune complexes. Using immunoaffinity techniques, endogenous TIMP-2 in the inflamed lung was shown to exist as a complex with 72- and 92-kDa metalloproteinases (MMP-2 and MMP-9). In inflamed lung both TIMP-2 and SLPI appeared to exist as enzyme inhibitor complexes. Lung expression of both TIMP-2 and SLPI appeared to involve endothelial and epithelial cells as well as macrophages. To assess how these endogenous inhibitors might affect the lung inflammatory response, animals were treated with polyclonal rabbit Abs to rat TIMP-2 or SLPI. This intervention resulted in significant intensification of lung injury (as revealed by extravascular leak of albumin) and substantially increased neutrophil accumulation, as determined by cell content in bronchoalveolar lavage (BAL) fluids. These events were correlated with increased levels of C5a-related chemotactic activity in BAL fluids, while BAL levels of TNF-alpha and chemokines were not affected by treatment with anti-TIMP-2 or anti-SLPI. The data suggest that endogenous TIMP-2 and SLPI dynamically regulate the intensity of lung inflammatory injury, doing so at least in part by affecting the generation of the inflammatory mediator, C5a.

High throughput genotyping for the detection of a single nucleotide polymorphism in NAD(P)H quinone oxidoreductase (DT diaphorase) using TaqMan probes.

AIMS: The two electron reduction of quinones to hydroquinones by NAD(P)H quinone oxidoreductase (NQO1) plays an important role in both activation and detoxification of quinone and similarly reactive compounds. A single nucleotide polymorphism at exon 6 leads to an amino acid change at codon 187 from proline to serine. The variant allele has been associated with decreased NQO1 enzyme activity and increased cancer risks. The aim of this study was to develop a rapid genotyping procedure for epidemiological and clinical research into the potential biological and toxicological implications associated with this genetic polymorphism. METHODS: A high throughput genotyping method using fluorogenic probes has been developed to screen this single nucleotide polymorphism. This assay utilises the 5' nuclease activity of Taq polymerase in conjunction with fluorogenic TaqMan probes. The TaqMan genotyping procedure was validated by a restriction fragment length polymorphism method and direct sequencing. RESULTS: This method can be used for the rapid screening of known polymorphisms in large populations. In a population of 143 unrelated individuals, Pro/Pro (wildtype), Pro/Ser (heterozygous), and Ser/Ser (mutant) genotypes were 69.2%, 26.6%, and 4.2%, respectively. CONCLUSIONS: This genotyping method is highly accurate and could be applied to automated large scale genotyping studies.

Technologies for detecting genetic polymorphisms in pharmacogenomics.

BACKGROUND: Pharmacogenomics is an emerging scientific discipline examining the genetic basis for individual variations in response to therapeutics. METHODS AND RESULTS: Genetic polymorphisms are a major cause of individual differences in drug response. Metabolic phenotyping can be accomplished by administering a probe drug or substrate and measuring the metabolites and clinical outcomes. However, this approach tends to be labor intensive and requires repeated sample collection from the individual being tested. Alternatively, genotyping allows determination of individual DNA sequence differences for a particular trait. Commonly used genotyping methods include gel electrophoresis-based techniques, such as polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism analysis, multiplex PCR, and allele-specific amplification. Fluorescent dye-based high-throughput genotyping procedures are increasing in popularity, including oligonucleotide ligation assay, direct heterozygote sequencing, and TaqMan (Perkin Elmer, Foster City, CA) allelic discrimination. High-density chip array and mass spectrometry technologies are the newest advances in the genotyping field, but their wide application is yet to be developed. Novel mutations/polymorphisms also can be identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. CONCLUSIONS: Rapid and accurate detection of genetic polymorphisms has great potential for application to drug development, animal toxicity studies, improvement of human clinical trials, and postmarket monitoring surveillance for drug efficacy and toxicity.

Use of acridine orange in: flow cytometric assessment of micronuclei induction.

The micronucleus assay is a widely accepted method for evaluation of clastogens and aneugens. In the current study, acridine orange (AO) supravital staining was adapted for flow cytometric usage to assess micronucleated cells in rat bone marrow and spleen. Cyclophosphamide was used as a positive control test compound and results were compared to manual scoring in Wright-stained slides. In bone marrow, both manual and flow cytometric methods demonstrated positive dose response-trends for micronucleated polychromatic erythrocytes (MNPCE). Significant elevations in MNPCE were observed at all doses of cyclophosphamide, and comparisons between methods in bone marrow were not statistically different. The flow cytometric method was more sensitive in spleen samples, showing dose- and time-related increases in micronuclei compared with manual scoring. AO proved to be a sensitive discriminator of RNA and DNA, allowing distinct separation of polychromatic erythrocytes (PCE), normochromic erythrocytes (NCE), total nucleated cells (TNC), and micronucleated populations within both PCE and NCE regions. These results support the use of AO-based flow cytometry to provide a rapid and sensitive indicator of micronuclei inducers.

Use of acridine orange in: flow cytometric evaluation of erythropoietic cytotoxicity.

Cytotoxic insult to bone marrow frequently impairs the proliferating and maturational abilities of erythroid cells. Typically, a ratio of enucleated, immature polychromatic erythrocytes (PCE) to mature normochromatic erythrocytes (NCE) is used to assess cytotoxicity in the micronucleus (MN) assay. The effects of cyclophosphamide (CP) on PCE/NCE ratio in rat bone marrow and spleen were assessed by a newly developed flow cytometric procedure using glutaraldehyde-fixed, acridine orange (AO)-stained cells, and compared to manual scoring of PCE/NCE in Wright stained slides. Comparison of methods showed that manual and flow cytometric determination of PCE were not statistically different. Several other parameters of cytotoxicity could be simultaneously assessed because the method allowed use of unfractionated whole bone marrow/spleen cell samples. Absolute numbers of total nucleated cells (TNC), a ratio of TNC to total erythrocytes (TE), and determination of RNA content within the PCE population demonstrated dose- and time-dependent effects with CP treatment. Shifts in RNA content were particularly sensitive, correctly identifying all CP-treated from control specimens, even in those samples where PCE/NCE ratio was similar. The AO methodology provided a more rapid, statistically-superior, and thorough approach in the assessment of bone marrow and spleen cytotoxicity than the conventional manual method of scoring PCE/NCE ratio alone.

Comparison of flow cytometric and manual bone marrow differentials in Wistar rats.

Preclinical drug trials frequently require the evaluation of animal bone marrow, a time-consuming process requiring the skills of a highly trained hematologist. In the present study, a flow cytometric technique was developed that could effectively replace the need for manual bone marrow differentials in rats. Peroxidase activity, measured indirectly with 2'7'-dichlorofluorescein, was coupled with the use of species-specific T- and B-lymphocyte antibodies and cell size to produce a flow cytometric analysis of rat bone marrow. Accurate identification of lymphocyte, proliferating and maturing erythroid and myeloid, and megakaryocyte populations was confirmed by cell sorting. Flow cytometry yielded differentials that were indistinguishable from manual differentials and published reference ranges. Enumeration of lymphocyte numbers with monoclonal markers is a key advantage of flow cytometric differentials because misidentification of lymphocytes in poorly prepared or stained bone marrow smears is a common problem. The most apparent advantage is increased throughput and reproducibility. Operator training for analysis using flow cytometry can be readily accomplished within a few days as opposed to the extensive training required for individuals performing manual bone marrow differentials. This methodology provides a high-volume, rapid, and relatively low-cost tool for the reliable evaluation of rat bone marrow differentials that has been heretofore unavailable.

Flow cytometric evaluation of bone marrow differentials in rats with pharmacologically induced hematologic abnormalities.

Previously, flow cytometric determination of peroxidase activity, cell size, and reactivity to lymphocyte antibodies were used to produce bone marrow differentials in untreated rats. In the present study, abnormal hematologic profiles were induced with erythropoietin (EPO), recombinant murine stem cell factor (rm-SCF), granulocyte-macrophage stimulating factor (GM-CSF), and cyclophosphamide (CP). Manual and flow cytometric data showed comparable levels of erythroid and myeloid hyperplasia in EPO- and rm-SCF/GM-CSF-treated animals, respectively. In CP-treated animals, flow cytometric data revealed significant decreases in cellularity at concentrations of CP > or = 5 mg/kg. In contrast, 20 mg/kg CP were necessary to induce microscopically apparent hypoplasia in histologic bone sections, showing that the automated methodology was a more sensitive indicator of bone marrow hypocellularity than was the more conventional manual method. Megakaryocyte counts were consistently higher by flow cytometer than by manual counts performed on cytocentrifuge preparations made from the same cell suspensions but were similar to megakaryocyte counts performed on histologic sections of femur, indicating that the automated methodology produced a more accurate reflection of true megakaryocyte numbers. Induction of hematologic abnormalities in the present study showed that manual bone marrow differentials can be replaced with the more efficient and reliable flow cytometric method in most preclinical toxicology studies.

Genetic analysis of multiple loci in microsamples of fixed paraffin-embedded tissue.

Molecular analysis of alterations in genomic DNA is essential for understanding mechanisms by which chemical agents induce or modify tumor development. The assessment of microsatellite polymorphisms, loss of heterozygosity, mutations, and gene rearrangement allows specific comparisons of tumors to premalignant lesions or normal tissue or between similar tumors seen in laboratory species and humans. Utilization of these techniques is frequently limited by minute quantities of available tissue, often restricted to small formalin-fixed tumors or biopsies in paraffin blocks. To address these limitations, we have combined recently developed methodologies for selective recovery, amplification, and analysis of DNA. These techniques provide sufficient materials of high quality for analysis of DNA alterations in microscale amounts of starting material. By combining whole genome amplification through primer extension preamplification with locus-specific heminested PCR, we are able to analyze multiple genetic loci from as little as 1 mm2 of a 3-micron-thick formalin-fixed paraffin section. From 10 to greater than 100 loci can be analyzed per tissue section, and locus-specific PCR products may be further evaluated by a variety of techniques (e.g., SSCP, sequencing). Integrating these methodologies into situations where evaluation of very small tissue samples is necessary provides a powerful approach for elucidating molecular events that may be causally related to chemically induced cellular transformation and tumorigenesis.

All-trans retinoic acid reduces membrane fluidity of human dermal fibroblasts. Assessment by fluorescence redistribution after photobleaching.

All-trans retinoic acid (RA) preserves human dermal fibroblast viability and stimulates proliferation in vitro. These effects are mediated, at least in part, by reducing the extracellular Ca2+ requirement. The same concentrations of RA that reduce the extracellular Ca2+ requirement also interrupt movement of Ca 2+ across the fibroblast plasma membrane. Based on these observations, we have examined the effects of RA on membrane properties that could influence Ca2+ movement. Fibroblasts were labeled with 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3 diazole)-amino-caproyl phosphatidyl-choline (a fluorescent phospholipid analogue) and examined for fluorescence redistribution after photobleaching (FRAP) with a pulse of intense light as a measure of membrane fluidity. Using this approach, we observed that membrane fluidity was higher when the cells were incubated in medium containing a low (sub-optimal) level of extracellular Ca2+ (0.15 mmol/L) than in a medium containing an optimal concentration (1.4 mmol/L). Treatment of the cells with 3 micromol/L RA reduced membrane fluidity of the cells under both high- and low-Ca2+ conditions. These findings demonstrate that RA has a direct effect on the plasma membrane of human dermal fibroblasts. This provides a possible mechanism for the previously identified inhibition of Ca2+ movement across the membrane of the same cells and for the previously identified protective effects against lysis under low-Ca2+ conditions.

Cellular hyperplasia in rats following continuous intravenous infusion of recombinant human epidermal growth factor.

In this study, we determined in vivo morphologic effects of continuous intravenous infusion of recombinant human epidermal growth factor (EGF) in adult Wistar rats. The EGF used consisted of the amino acid residues 1-48 of the human 53-amino-acid EGF molecule, purified from transfected Escherichia coli. Doses of 25, 100, or 250 micrograms/kg body weight were administered using Harvard digital syringe infusion pumps for 4 weeks. At necropsy, the submandibular salivary glands, Harderian glands, liver, kidneys (females only), and ovaries were enlarged and urinary bladders were thickened in 100- and 250-micrograms/kg rats. Numerous tissues of the 100- and 250-micrograms/kg rats contained hyperplastic epithelial cells, and selected organs also had mesenchymal cell proliferation. Epithelial proliferation was most pronounced in the trachea, nasal cavity, nasolacrimal duct, tongue, stomach, small intestine, large intestine, urinary tract, salivary gland ducts, and Harderian gland. Periportal hepatocytes were hypertrophic, correlating with increased liver weight. In addition, mesenchymal cell proliferation was evident in the gastric mucosa lamina propria and in heart valves in 100- and 250-micrograms/kg rats. Increased ovarian weight correlated with increased number and size of corpora lutea and an increased incidence of luteal cysts. Continuous systemic exposure of adult Wistar rats to high doses of EGF resulted in generalized epithelial hyperplasia and tissue-selective mesenchymal proliferation.