Single pulmonary nanopolystyrene exposure in late-stage pregnancy dysregulates maternal and fetal cardiovascular function.

Large-scale production and waste of plastic materials have resulted in widespread environmental contamination by the breakdown product of bulk plastic materials to micro- and nanoplastics (MNPs). The small size of these particles enables their suspension in the air, making pulmonary exposure inevitable. Previous work has demonstrated that xenobiotic pulmonary exposure to nanoparticles during gestation leads to maternal vascular impairments, as well as cardiovascular dysfunction within the fetus. Few studies have assessed the toxicological consequences of maternal nanoplastic (NP) exposure; therefore, the objective of this study was to assess maternal and fetal health after a single maternal pulmonary exposure to polystyrene NP in late gestation. We hypothesized that this acute exposure would impair maternal and fetal cardiovascular function. Pregnant rats were exposed to nanopolystyrene on gestational day 19 via intratracheal instillation. 24 h later, maternal and fetal health outcomes were evaluated. Cardiovascular function was assessed in dams using vascular myography ex vivo and in fetuses in vivo function was measured via ultrasound. Both fetal and placental weight were reduced after maternal exposure to nanopolystyrene. Increased heart weight and vascular dysfunction in the aorta were evident in exposed dams. Maternal exposure led to vascular dysfunction in the radial artery of the uterus, a resistance vessel that controls blood flow to the fetoplacental compartment. Function of the fetal heart, fetal aorta, and umbilical artery after gestational exposure was dysregulated. Taken together, these data suggest that exposure to NPs negatively impacts maternal and fetal health, highlighting the concern of MNPs exposure on pregnancy and fetal development.

Chromium induces a persistent activation of mitogen-activated protein kinases by a redox-sensitive mechanism in H4 rat hepatoma cells.

Chromium is an important industrial metal, an environmental pollutant, and a human carcinogen. To investigate the mechanisms of chromium-induced carcinogenesis, activation of mitogen-activated protein (MAP) kinases ERK1 and ERK2 was examined in rat hepatoma cells following exposure to hexavalent chromium (Cr(VI)). Cr(VI) was found to activate both forms of MAP kinase in a dose- and time-dependent manner. In contrast to the protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate, which induced a transient activation of MAP kinases, Cr(VI) caused persistent activation of these enzymes. Furthermore, unlike phorbol 12-myristate 13-acetate, the ability of chromium to activate MAP kinases was found to be independent of PKC since chromium-induced MAP kinase activation occurred in PKC-depleted cells. Stimulation of ERK1 and ERK2 was associated with the ability of Cr(VI) to increase cellular peroxide levels as determined using the H2O2-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate and flow cytometry. Furthermore, the activation of these kinases by chromium was enhanced in cells treated with the glutathione-depleting agent, L-buthionine-[S,R]-sulfoximine, and attenuated in cells pretreated with an agent that elevates cellular levels of glutathione (i.e., N-acetyl-L-cysteine). The ability of chromium to modulate MAP kinase activity in this manner suggests a mechanism of chromium-induced carcinogenesis that involves the persistent stimulation of cellular regulatory pathways.

Recombinant human interleukin-3 (rhIL-3) enhances the mobilization of peripheral blood progenitor cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF) in normal volunteers.

To identify a precisely timed and safe protocol for progenitor cell mobilization, we studied the effects of rhIL-3 and rhG-CSF administration to normal volunteers. rhG-CSF 5 micrograms/kg/d was administered subcutaneously (s.c.) for 7 consecutive days either alone or preceded by rhIL-3 5 micrograms/kg/d s.c. for 4 consecutive days in sequential or partially overlapping schedules. The combined cytokines were well-tolerated--adverse effects were similar to those of the individual agents. Total white blood cell (WBC) and neutrophil counts rose briskly in response to rhG-CSF, and peak mean values were similar between treatment cohorts. Mean platelet counts were modestly elevated during rhG-CSF treatment only in the cohorts receiving rhIL-3 and rhG-CSF. Mean circulating CD34+ cells peaked on day 5 in the rhG-CSF group (38.9+/-14.3/microliter), day 6 in the sequential rhIL-3/rhG-CSF group (56.4+/-12.4/microliter), and day 6 in the partial overlap group (46.1+/-10.9/microliter). On day 3, mean CD34+ cell counts of the subjects who received sequential treatment were markedly higher than observed in the other groups (p<0.05) and were estimated to have been sufficient for collection of adequate grafts by single 10-L leukapheresis procedures in 60% of subjects. Circulating clonogenic cells (CFU-GM and/or BFU-E) were substantially higher in the sequential group than the rhG-CSF group on days 3-6 but were only minimally elevated above baseline in the partial overlap group. The numbers of circulating CD34+/Lin-/Thy-1+ cells (putative stem cells) were increased substantially, especially in the sequential group. On the basis of this pilot trial, we conclude that priming with rhIL-3 is a safe and well-tolerated method for enhancing the mobilization of human blood progenitors and stem cells by rhG-CSF.

Pharmacodynamics of daily subcutaneous recombinant human interleukin-3 in normal volunteers.

Normal volunteers received subcutaneous injections of recombinant human interleukin-3 (rhIL-3) on 4 consecutive days to characterize toxicity, pharmacokinetics, and hematopoietic effects. Dosages were 2.5, 5.0, and 7.5 micrograms/kg/day (n = 6 subjects per group). Adverse effects consisted predominantly of flu-like symptoms such as fever and headache. Mean area under the serum concentration-time curve and maximum serum concentration were linearly related to dose. Serum clearance was not apparently related to dose. Clearance increased slightly but significantly between days 1 and 4. Rapid but modest elevations in neutrophil and eosinophil counts were observed during treatment. Mean platelet counts rose modestly, peaking on day 10. Increases of CD34+ cell counts were correlated with increases of colony-forming unit-granulocyte macrophage (peak, day 7).

In vivo effects of chromium.

The production of reactive oxygen species on addition of hexavalent chromium (potassium dichromate, K2Cr2O7) to lung cells in culture was studied using flow cytometer analysis. A Coulter Epics Profile II flow cytometer was used to detect the formation of reactive oxygen species after K2Cr2O7 was added to A549 cells grown to confluence. The cells were loaded with the dye, 2',7'-dichlorofluorescein diacetate, after which cellular esterases removed the acetate groups and the dye was trapped intracellularly. Reactive oxygen species oxidized the dye, with resultant fluorescence. Increased doses of Cr(VI) caused increasing fluorescence (10-fold higher than background at 200 microM). Addition of Cr(III) compounds, as the picolinate or chloride, caused no increased fluorescence. Electron paramagnetic resonance (EPR) spectroscopic studies indicated that three (as yet unidentified) spectral "signals" of the free radical type were formed on addition of 20, 50, 100, and 200 microM Cr(VI) to the A549 cells in suspension. Two other EPR "signals" with the characteristics of Cr(V) entities were seen at field values lower than the standard free radical value. Liver microsomes from male Sprague-Dawley rats treated intraperitoneally with K2Cr2O7 (130 mumole/kg every 48 hr for six treatments) had decreased activity of cytochromes P4503A1 and/or 3A2, and 2C11. Hepatic microsomes from treated female Sprague-Dawley rats, in contrast, had increased activities of these isozymes. Lung microsomes from male Sprague-Dawley rats had increased activity of P4502C11.

Mechanism of action of psoralens: isobologram analysis reveals that ultraviolet light potentiation of psoralen action is not additive but synergistic.

The combination of psoralens and ultraviolet light (UVA, 320-400 nm), referred to as PUVA, inhibits proliferation of a variety of cell types. In the present studies, we used S-180 cells to investigate the mechanism underlying the antiproliferative actions of PUVA. We found that inhibition of growth of S-180 cells by PUVA was dependent on the concentration of psoralen as well as the dose of UVA light. Neither the psoralens nor UVA light by themselves inhibited cell growth. Several clinically important psoralen analogs inhibited cell growth. The potent phototoxin 4,5',8-trimethylpsoralen was the most active psoralen analog tested, followed by 5-methoxypsoralen and 8-methoxypsoralen. The angular furocoumarin, 5-methylangelicin, was the least active inhibitor of growth. Multivariate (isobologram) analysis of the growth-inhibition curves revealed that combinations of psoralens and UVA light were not simply additive but synergistic. Similar results were observed when inhibition of DNA synthesis was used as an endpoint for the biological effects of PUVA. These studies are the first to demonstrate that psoralens and UVA light act synergistically. Our results suggest that the synergism between the psoralens and UVA light may be an important property of PUVA that contributes to its therapeutic efficacy in proliferative diseases.

Effects of genistein and structurally related phytoestrogens on cell cycle kinetics and apoptosis in MDA-MB-468 human breast cancer cells.

We have studied the effects of phytoestrogens (genistein, quercetin, daidzein, biochanin A and kaempferol) on proliferation, cell cycle kinetics, and apoptosis of MDA-MB-468 breast cancer cells. Genistein and quercetin inhibited cell growth with IC50 values of 8.8 and 18.1 muM, respectively, while the other phytoestrogens were less effective. Flow cytometric analysis showed G2/M cell cycle arrest with 25 muM and higher concentrations of genistein. At 100 muM, genistein, quercetin and kaempferol caused accumulation of 70, 60 and 35% of cells, respectively, in G2/M phase by 24 h. In contrast, biochanin A and daidzein were ineffective. APO-BRDU analysis revealed apoptosis with 10 muM genistein (19.5%), reaching 86% at 100 muM. Apoptosis by genistein was confirmed by Hoechst 33342 staining and fluorescence microscopy. With 100 muM quercetin, 47% of the cells were apoptotic, while the other bioflavonoids had little effect. Genistein treatment resulted in a biphasic response on cyclin B1: 70% increase in cyclin B1 level at 25 muM, and 50 and 70% decrease at 50 and 100 muM, respectively. In contrast, the action of quercetin involved an increase in cyclin B1 level. Genistein had no effect on cdc2 level up to 50 muM concentration; however, there was a decrease in the phosphorylated form of the protein at 100 muM. Quercetin had no effect on cdc2 levels. Our results suggest that the action of genistein and quercetin involves G2/M arrest and apoptosis in MDA-MB-468 cells. Biochanin A and daidzein, although structurally related to genistein, did not share this mechanism. Thus, structurally related phytoestrogens have discrete target sites and mechanisms in their growth inhibitory action on breast cancer cells.

Effects of a bis(benzyl)spermine analog on MCF-7 breast cancer cells in culture and nude mice xenografts.

We studied the effects of a polyamine analog, N,N'-bis{3-[(phenylmethyl)amino]propyl}-1,7-diaminoheptane (MDL 27695) on MCF-7 cells, as part of an attempt to develop new drugs for breast cancer treatment. Using [H-3]-thymidine incorporation assay and long-term growth curves, we found that MDL 27695 inhibited the growth of MCF-7 cells in a dose-dependent manner in the low mu M range. G1 synchronized cells progressing in cell cycle showed delayed and inefficient entry into S phase in the presence of 4 mu M MDL 27695. Consistent with a G1 arrest, MDL 27695 significantly reduced estradiol-mediated increase in the expression of cyclin D1. HPLC analysis showed that treatment of MCF-7 cells with MDL 27695 reduced the accumulation of natural polyamines, putrescine, spermidine, and spermine, by 43, 38, and 45%, respectively, at 8 h after the initiation of cell cycle. This decrease in polyamine levels was not associated with a decrease in the activity of polyamine biosynthetic (ornithine decarboxylase, ODC; s-adenosylmethionine decarboxylase, SAMDC) or catabolizing (spermidine/spermine acetyltransferase, SSAT) enzymes. However, there was a 40% decrease in the uptake of putrescine and spermidine, in cells treated with MDL 27695. Our studies also showed that MDL 27695, at a dose of 20 mg/kg, caused a significant inhibition of tumor growth in nude mice harboring MCF-7 cell derived tumors, without overt symptoms of toxicity. These data indicate that the polyamine analog MDL 27695 is an efficient inhibitor of MCF-7 breast cancer cell growth in vitro and in vivo. Our results suggest that polyamines are critical factors in cell cycle regulation of breast cancer cells and potential targets for therapy.

Differential effects of estradiol and its analogs on cyclin D1 and CDK4 expression in estrogen receptor positive MCF-7 and estrogen receptor-transfected MCF-10AEwt5 cells.

Estradiol stimulates the growth of a majority of human breast tumors containing the estrogen receptors (ERs), proteins that mediates estrogen function. Opposing effects of estradiol have been found in cells expressing endogenous ER and those containing a transfected ER. To understand the role of estradiol structure in diverse estrogenic responses related to cell cycle regulation, we evaluated the effects of estradiol and its analogs on cell cycle progression, and the expression of cyclin D1 and the cyclin dependent kinase 4 (CDK4) in ER-positive MCF-7 and ER-transfected MCF-10AEwt5 cells. Four analogs of estradiol, with a re-positioned or a deleted hydroxyl group, were used. Our results show that estradiol and all of the analogs facilitated cell cycle progression of MCF-7 cells. In contrast, only estradiol inhibited the cell cycle progression of MCF-10AEwt5 cells significantly. Western blot analysis revealed that cyclin D1 protein increased to the maximal level by 6 h after the initiation of cell cycle from G1 phase of MCF-7 cells. The least effective analog in inducing cyclin D1 was 3-hydroxyestratriene. However, this analog was most effective at inducing CDK4, contributing to its efficacy in facilitating MCF-7 cell cycle. In contrast to MCF-7 cells, the level of cyclin D1 protein was not influenced significantly by estradiol or its analogs in MCF-10AEwt5 cells. Sucrose gradient sedimentation analysis of ER from MCF-7 cells showed that the major peak of [3H]-estradiol bound to ER could be displaced by a 10-fold excess of unlabelled estradiol or any of the analogs. In contrast, several analogs were less effective than unlabelled estradiol in competitive displacement of [3H]-estradiol bound to ER from MCF-10AEwt5 cells. These data indicate that the induction of cyclin D1 is an important part of the growth stimulatory effects of estrogens in MCF-7 cells, but it may not be involved in growth inhibition of MCF-10AEwt5 cells. Our results also show that estrogenic compounds interact with ER from MCF-10AEwt5 cells with altered ligand binding affinity, possibly due to the absence or dysfunction of certain transcription factors or ER-associated proteins that co-regulate ER function.

Flow cytometric determination of metallothionein levels in human peripheral blood lymphocytes: utility in environmental exposure assessment.

Metallothioneins (MT) are ubiquitous, low-molecular-weight proteins that exhibit high binding affinities for heavy metal ions. The expression of these cysteine-rich proteins is induced in response to various types of chemical and physical stresses and therefore can be used to assess human exposure to cytotoxic environmental agents. In the current study, MT levels of human peripheral blood lymphocytes were determined using an MT-specific antibody and flow cytometry. Treatment of human whole blood ex vivo with CdCl2 was found to induce a concentration- and time-dependent increase in lymphocyte MT levels at concentrations as low as 0.3 microM and within a 12-h period. Interestingly, differences were observed in the magnitude of cadmium-induced MT levels in the lymphocytes of six human test subjects. Two members of the study population exhibited CdCl2-induced cellular MT levels that were up to twofold greater than the lymphocytes of other human subjects. While the lymphocytes of most test subjects exhibited a symmetric (unimodal) distribution of cadmium-induced MT-specific fluorescence, the cells of two individuals displayed a heterogeneous (nonuniform) distribution of MT levels. Dual-parameter flow cytometric analysis using phenotype-specific antibodies indicated that variations in the responsiveness of subpopulations of lymphocytes to CdCl2 were responsible for the heterogeneous distribution of MT-specific cellular fluorescence. T-helper (CD4-positive) and T-suppressor/cytotoxic (CD8-positive) lymphocytes expressed higher cellular levels of MT than other lymphocyte subpopulations (i.e., B lymphocytes, natural killer cells). Our results suggest that MT protein levels of peripheral blood lymphocytes, as determined by this flow cytometric method, may be used to assess human exposure to toxic metals and to characterize various quantitative/qualitative aspects of the response of individuals to cadmium and possibly to other types of environmental stresses.

Effects of cadmium on metallothionein levels in human peripheral blood leukocytes: a comparison with zinc.

Metallothioneins (MT) are low-molecular-weight, cysteine-rich proteins that are induced in response to a variety of chemical stresses and therefore can be used to assess human exposure to environmental agents. In the current study, flow cytometry was used to characterize the basal and cadmium-induced expression of MT in the three major leukocyte populations of human peripheral blood. In the analysis, monocytes were the most sensitive leukocytes to this toxic metal, with significant increases in cellular MT levels being detected at concentrations of cadmium as low as 0.1 microM (24 h). The lymphocyte population also exhibited pronounced treatment-associated elevations in cellular MT, while the granulocyte population was found to be nonresponsive. Although both CdCl2 (3 microM) and ZnCl2 (50 microM) induced MT expression in monocytes to a similar degree and did not affect the expression of this protein in granulocytes, cadmium but not zinc treatment induced dramatic increases in MT levels of lymphocytes. Our results indicate that cellular MT protein levels, as determined by this flow cytometric method, may be used to characterize the differential responsiveness of the major human leukocyte subpopulations to transitional metals. It is evident from the current work that the responsiveness of all peripheral blood leukocyte populations should be analyzed in exposure assessment studies.

Metals and metallothionein in the liver of raccoons: utility for environmental assessment and monitoring.

The relationship between metallothionein levels and concentrations of several metals and radionuclides was examined in liver tissues of raccoons (Procyon lotor, n = 47) from the Department of Energy's Savannah River Site in South Carolina to determine the applicability of metallothioneins as an initial screening device for exposure assessment in free-living mammals and environmental monitoring. Using a fluorescent marker and a cell sorter to measure metallothionein, a significant positive correlation was found across animals between levels of metallothioneins and concentrations of selenium (Pearson's r = .30), mercury (Pearson's r = .3 1), and copper (Pearson's r = .30) in liver tissue. Arsenic, cobalt, silver, thallium, and tin were below detection limits in most or all liver samples. Other metals, including cadmium, chromium, radiocesium (137-Cs), copper, lead, manganese, strontium, and vanadium, showed only weak and nonsignificant correlations with metallothionein. Concentrations of mercury were correlated with concentrations of selenium (Pearson's r = .73), manganese (Pearson's r = .56), and strontium (Pearson's r = .57). In an a posteriori test, there was a still unexplained positive correlation between mercury (Pearson r = .56), selenium (Pearson r = .54), and radiocesium (Pearson's r = .38) concentrations and background cellular autofluorescence, and a negative correlation of strontium with the latter (Kendall tau = -.38). Background cellular autofluorescence may represent a generalized cellular stress response, or a yet unidentified biomarker. To better understand which metals contribute to the induction of metallothionein, principle component analysis (PCA) was performed. The first three principle components explained 78% of the variance, with highest loadings being from mercury and radiocesium. Metallothionein levels did not correlate well with the principal components from the metals and radiocesium, while autofluorescent background levels tended to correlate better.

Seasonal deposition of housedusts onto household surfaces.

Seasonal differences in the particle size fractions and mass loadings of household dust deposited on indoor surfaces were examined in four New Jersey homes. Housedust was collected during a 30-day period on non-electrostatic polyethylene sample plates on which a glass slide had been placed. In each home two samples were collected at a height of 1.5 m and two were collected at a height of 0.3 m above the floor. Dust samples were obtained from each home during a summer and winter collection period. Particle size measurement was completed using an adaptation of a Meridian ACAS 570 Interactive Laser Cytometer. Results indicated that the dust mass deposited on household surfaces during the summer was greater than during the winter. The arithmetic mean mass deposition rate for all houses was 0.37 +/- 0.13 microgram/cm2/day during the summer and 0.22 +/- 0.13 microgram/cm2/day during the winter. The total number of particles deposited, however, was greater during the winter than during the summer. The increase in winter time particle number was caused by greater numbers of particles with an equivalent spherical diameter < 2.5 microns. The most probable source of these particles was winter time combustion emissions within the residences and the subsequent particle deposition on household surfaces. The greater mass loadings measured on the low sampling plates during the summer were associated with a greater number of particles with an equivalent spherical diameter > 5 microns. In the winter, however, the particle mass and number loadings were similar at both heights. These results suggested that ventilation of the house during the summer allowed resuspended particles to enter which led to the higher levels of settled dust. Measurement of contaminant levels in housedust for exposure estimation therefore, should account for the seasonal and height differences in dust mass, and collect representative fractions of housedust that are available for human contact. Furthermore, since over 99% of the particles on indoor surfaces were < 50 microns any indirect sampling technique for dermal exposure estimation should have collection efficiencies similar to the hand of particles < 50 microns.

Characterization of hypoxia-dependent peroxide production in cultures of Saccharomyces cerevisiae using flow cytometry: a model for ischemic tissue destruction.

Peroxide production in cultures of Saccharomyces cerevisiae was measured using the H2O2-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) and flow cytometry. Aeration of cultures of S. cerevisiae exposed to a period of hypoxia was found to induce elevated levels of peroxide that were 100-fold higher than the levels observed in cultures maintained under exclusively aerated or hypoxic conditions. Simultaneous viability analysis, using the fluorescent DNA-intercalating dye propidium iodide, indicated that the increase in peroxide generation preceded cell damage and death. Various agents were found to influence the effect of peroxides on cell viability. The addition of ethanol to hypoxic stationary cultures dramatically increased the rate of cell death without further increasing the amount of peroxide produced, while glucose inhibited peroxide production and decreased the rate of cell death. Surprisingly, elevated peroxide levels of hypoxic/reaerated cultures were maintained upon addition of KH2PO4, although the cells remained viable for extended periods of time when compared to control and other test cultures. Similarities between our observations and those of other investigators using anoxic/reperfused organs suggest that hypoxic/reaerated yeast cultures may be a useful model system to study ischemia-dependent tissue destruction of mammals.

Purification of tyrosinase to homogeneity based on its resistance to sodium dodecyl sulfate-proteinase K digestion.

A two-step method was used to purify enzymatically active tyrosinase to apparent homogeneity from B16/C3 mouse melanoma cells. The purification was based on our finding that tyrosinase was highly resistant to sodium dodecyl sulfate-proteinase K digestion. Following treatment with proteinase K, tyrosinase was separated from all detectable contaminants using DEAE-52 ion-exchange chromatography. Using this procedure, tyrosinase was purified to high specific activity and in yields greater than 50%. The purified enzyme was then used to generate high titers of specific polyclonal antibodies in rabbits. These antibodies were used to immunoprecipitate all tyrosinase isozymes from murine melanoma tumor extracts and cultured B16/C3 cells metabolically labeled with [35S]methionine.

Effects of chromium on basal and insulin-induced tyrosine phosphorylation in H4 hepatoma cells: comparison with phorbol-12-myristate-13-acetate and sodium orthovanadate.

Chromium, in its various forms, is recognized both as a human carcinogen and as a nutrient essential in glucose homeostasis. Although the genotoxicity of this element is associated with its carcinogenic properties, the manner in which chromium mediates its epigenetic effects on cells, including its ability to potentiate insulin action, is not known. In the current studies, Western blotting with antiphosphotyrosine antibodies was used to study the effects of chromium on protein tyrosine phosphorylation in intact H4 rat hepatoma cells. Treatment of cells with hexavalent chromium [Cr(VI)] was found to induce the tyrosine phosphorylation of three prominent sets of proteins, having median molecular masses of 210, 125, and 87 kDa. Cr(VI) pretreatment also inhibited the insulin-induced tyrosine phosphorylation of the major substrate of the insulin receptor kinase, insulin receptor substrate-1, and its subsequent association with the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3'-kinase. Furthermore, Cr(VI) was found to alter the pattern of other p85-binding (insulin-induced) phosphoproteins that were distributed throughout the soluble and particulate fractions of cells. Virtually all of the alterations in basal and insulin-induced phosphorylations associated with Cr(VI) treatment were also observed in cells treated with the protein kinase C (PKC) agonist phorbol-12-myristate-13-acetate. However, the effects of Cr(VI) were determined to be independent of PKC activity, because they were sustained in PKC-depleted cells. The pattern of phosphoproteins induced by Cr(VI) also had similarities to the pattern generated in response to the phosphatase inhibitor sodium orthovanadate. However, several specific differences, including the ability of vanadate to increase insulin receptor beta subunit autophosphorylation [i.e., an effect not observed with Cr(VI)], indicated that these agents modulate phosphorylation by distinct mechanisms. The ability of Cr(VI) to alter the phosphorylation state of key regulatory proteins in a manner similar to that of other biologically active agents suggests a mechanism by which this element can modulate the growth and metabolism of cells.

Characterization of a photoalkylated psoralen receptor in HeLa cells.

Psoralens in combination with ultraviolet light are potent modulators of epidermal cell growth and differentiation. Responsive cell types contain specific, saturable, high-affinity binding sites for the psoralens. These binding sites become covalently modified by the psoralen molecule following ultraviolet light exposure. In the present studies the psoralen receptor, labeled with [3H]8-methoxypsoralen, was visualized in the cytoplasmic and plasma membrane fractions of HeLa cells following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor had an apparent molecular mass of approximately 22,000 daltons and was shown to be sensitive to protease, but not nuclease treatment. The radiolabeled receptor could not be visualized in nuclear extracts of cells. Covalent binding of the radioligand to the receptor protein was inhibited by excess unlabeled 8-methoxypsoralen, indicating that covalent psoralen-receptor binding was saturable. In addition, the covalently modified receptor was found to persist in cells for over 5 h. The presence of a cellular protein that exhibits specific affinity for the psoralens and becomes photoalkylated by these compounds, together with previous data showing that the psoralens have direct effects on the cell surface membranes, supports our model that some of the biological effects of photoactivated psoralens are receptor-mediated.

Inhibition of calcium signalling in murine splenocytes by polyamines: differential effects on CD4 and CD8 T-cells.

Transmembrane Ca2+ influx is recognized as a universal second messenger that transduces T-cell activation signals to cytoplasm and nucleus, thereby stimulating transcription and cell division. To examine the role of endogenous factors that regulate mitogenic Ca2+ signalling of T-cells, we measured the concanavalin (Con) A-induced increase in cytoplasmic free calcium ([Ca2+]i) in spleen cells of BALB/c mice, using flow cytometry with an indicator dye, Indo-1 acetoxymethyl ester (Indo-1/AM). Con A is a polyclonal activator of T-cells. Unstimulated splenocytes had a [Ca2+]i of 100 nM. [Ca2+]i increased with Con A in a dose-dependent manner up to a concentration of 50 micrograms/ml. In the presence of 50 micrograms/ml Con A, [Ca2+]i was 350 nM. Natural polyamines (putrescine, spermidine and spermine) inhibited Con-A-induced Ca2+ influx in a dose-dependent manner. Putrescine was the most effective polyamine in desensitizing the Ca2+ signal, and decreased [Ca2+]i from 350 nM in the absence of putrescine to 250 nM in the presence of 100 microM putrescine. This effect was not mimicked by structurally related homologues or inorganic cations, suggesting a specific structural effect of the polyamine. H.p.l.c. analysis showed that polyamines were internalized during incubation of cells in vitro. In experiments using monoclonal anti-CD4 and anti-CD8 antibodies, we found a differential effect of putrescine on Ca2+ influx in CD4 and CD8 subpopulations of T cells. For CD4+ cells, [Ca2+]i decreased from 625 nM to 420 nM in the presence of 500 microM putrescine, whereas [Ca2+]i was not affected by putrescine in CD8+ cells. These data suggest that natural polyamines have cell-specific effects on mitogen-stimulated Ca(2+)-influx in T-cell subsets.

Characterization of the inflammatory response to biomaterials using a rodent air pouch model.

Using a rodent air pouch, the inflammatory responses to biomaterials with distinct physical properties and chemical compositions were compared. The polymers examined were expanded poly(tetrafluoroethylene) (ePTFE), silicone, low-density polyethylene (LDPE), poly(L-lactic acid) (PLLA), poly(desaminotyrosyl-tyrosine ethyl carbonate) [poly(DTE carbonate)], and poly(desaminotyrosyl-tyrosine benzyl carbonate) [poly(DTBzl carbonate)]. We found that implantation of disks (4.5-4.8 mm) of these materials into rodent air pouches for 2 days had no effect on the number or type of cells recovered relative to sham controls. With each of the materials, macrophages were the predominant cell type identified (60-75%), followed by granulocytes (20-25%) and lymphocytes (10%). Implantation of poly(DTE carbonate), ePTFE, LDPE, or poly(DTBzl carbonate) into the pouches for 2 days caused an increase in release of superoxide anion by the pouch cells. Cells from pouches containing poly(DTE carbonate) also released more hydrogen peroxide and were more phagocytic. In contrast, PLLA and silicone had no effect on the functional activity of cells recovered from the pouches. Prolonging the implantation time of poly(DTE carbonate) or PLLA to 7 days did not alter the number or type of cells isolated from the pouches. However, cells from pouches containing poly(DTE carbonate) for 7 days continued to produce increased quantities of superoxide anion relative to sham control pouch cells. These results suggest that the air pouch model is a highly sensitive method and therefore useful for evaluating the functional responses of inflammatory cells to biomaterials.