Laboratory study of PCB transport from primary sources to settled dust.

Dust is an important sink for indoor air pollutants, such as polychlorinated biphenyls (PCBs) that were used in building materials and products. In this study, two types of dust, house dust and Arizona Test Dust, were tested in a 30-m(3) stainless steel chamber with two types of panels. The PCB-containing panels were aluminum sheets coated with a PCB-spiked primer or caulk. The PCB-free panels were coated with the same materials but without PCBs. The dust evenly spread on each panel was collected at different times to determine its PCB content. The data from the PCB panels were used to evaluate the PCB migration from the source to the dust through direct contact, and the data from the PCB-free panels were used to evaluate the sorption of PCBs through the dust/air partition. Settled dust can adsorb PCBs from air. The sorption concentration was dependent on the congener concentration in the air and favored less volatile congeners. When the house dust was in direct contact with the PCB-containing panel, PCBs migrated into the dust at a much faster rate than the PCB transfer rate due to the dust/air partition. The dust/source partition was not significantly affected by the congener's volatility. For a given congener, the ratio between its concentration in the dust and in the source was used to estimate the dust/source partition coefficient. The estimated values ranged from 0.04 to 0.16. These values are indicative of the sink strength of the tested house dust being in the middle or lower-middle range.

Chamber study of PCB emissions from caulking materials and light ballasts.

The emissions of polychlorinated biphenyl (PCB) congeners from thirteen caulk samples were tested in a micro-chamber system. Twelve samples were from PCB-contaminated buildings and one was prepared in the laboratory. Nineteen light ballasts collected from buildings that represent 13 different models from five manufacturers were tested in 53-L environmental chambers. The rates of PCB congener emissions from caulking materials and light ballasts were determined. Several factors that may have affected the emission rates were evaluated. The experimentally determined emission factors showed that, for a given PCB congener, there is a linear correlation between the emission factor and the concentration of the PCB congener in the source. Furthermore, the test results showed that an excellent log-linear correlation exists between the normalized emission factor and the vapor pressure (coefficient of determination, r(2)⩾0.8846). The PCB congener emissions from ballasts at or near room temperature were relatively low with or without electrical load. However, the PCB congener emission rates increased significantly as the temperature increased. The results of this research provide new data and models for ranking the primary sources of PCBs and supports the development and refinement of exposure assessment models for PCBs.

Effect of cisplatin and BCNU on MMP-2 levels in human glioblastoma cell lines in vitro.

Matrix metalloproteinases (MMPs) play an important role in various physiological and pathological conditions such as tissue remodeling, and cancer cell invasion and metastasis. The aim of this study was to determine the effect of the antitumor compounds cis-dichlorodiammine platinum (ii) (cisplatin) and 1, 3 bis (2-chloroethyl)-1-nitrosourea (BCNU) on 72-kDa type IV collagenase activity (MMP-2) in human gliomas. Human glioblastoma cell lines were treated with cisplatin (25 microM), and BCNU (50 microM), and the levels of MMP-2 were estimated in serum-free conditioned medium and in cell extracts at different time intervals. Gelatin zymography revealed increased levels of MMP-2 in serum-free conditioned medium and in cell extracts of untreated glioblastoma cell cultures during a 72-h period. In contrast, MMP-2 levels were significantly decreased in cisplatin-treated cells both in conditioned medium and cell extracts. However, no significant changes of MMP-2 levels were noted in BCNU-treated cells. Quantitative analysis of MMP-2 enzyme activity by densitometry and amount of MMP-2 protein by ELISA showed significantly decreased levels of MMP-2 in cisplatin-treated cells compared to BCNU and untreated glioblastoma cells. The results indicate that decreased levels of MMP-2 might represent an additional mechanism by which cisplatin provides its antineoplastic effects.

Cisplatin but not BCNU inhibits urokinase-type plasminogen activator levels in human glioblastoma cell lines in vitro.

Glioblastomas extensively invade the surrounding normal brain tissue, with a concomitant expression of various proteolytic enzymes, in particular urokinase-type plasminogen activator (uPA). In this study we used cis-diamminedichloroplatinum (cisplatin) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), commonly used anti-cancer drugs for the treatment of glioblastomas, to study the expression of uPA in three human glioblastoma cell lines in vitro. Cells were treated with 25 microM cisplatin and 50 microM BCNU, and uPA levels were estimated by fibrin zymography during a 72-h time course. Treatment of glioblastoma cells with cisplatin resulted in significantly decreased levels of uPA in serum-free conditioned medium and cell extracts, compared to BCNU-treated and untreated cell lines. Quantitative levels of uPA enzyme activity assessed by scanning laser densitometry and uPA protein by ELISA using antibody against uPA showed decreased levels of uPA in cisplatin-treated glioma cell lines relative to BCNU and untreated cell lines. Our results suggest that anti-tumor compound, cisplatin, may exert its anti-neoplastic effects by inhibiting uPA in malignant glioblastomas.

Plasminogen activator inhibitor-1 in brain tumors: relation to malignancy and necrosis.

Tumor necrosis is a common feature of malignant neoplasms. The pathogenesis of tumor necrosis remains poorly documented. Recent evidence has shown a correlation between the presence of tumor necrosis and low content of tissue plasminogen activator in brain tumors and significantly higher levels of plasminogen activator inhibitor-1 (PAI-1) in human glioblastomas. We subjected fresh brain tumor tissue samples (n = 197) to an enzyme-linked immunosorbent assay to determine PAI-1 content. The results were correlated with the presence of edma and necrosis on imaging studies. The samples studied were from normal brain (n = 10), low-grade gliomas (n = 26), meningiomas (n = 47), acoustic neuromas (n = 18), glioblastomas (n = 45), metastases (n = 45), and areas of tumor necrosis (n = 6). The benign tumor samples (n = 96) had 3.5 times less PAI-1 than did the malignant tumors (n = 101). Tumor necrosis samples contained 3.8 times more PAI-1 than did the nonnecrotic malignant tumor samples (P < 0.000001). The benign meningioma samples showed a similar ratio compared with their malignant counterparts (0.35 versus 1.59 ng/mg, respectively, P = 0.0004). Regression analysis results showed a strong correlation between PAI-1 and necrosis (r = 0.47, P < 0.0000028) and, to a lesser extent, brain edema (r = 0.26, P = 0.001). A negative correlation between PAI-1 and tissue plasminogen activator levels almost reached statistical significance (P = 0.07). There was no correlation between PAI-1 content and the tumor size, duration of symptoms, or the sex or age of the patients. The results of this study indicate that malignant transformation is associated with a significant increase in PAlI1 and that PAI-1 may play an integral role in the pathogenesis of tissue necrosis, perhaps via the inhibition of tissue plasminogen activator and the promotion of microthrombosis.

Overexpression and localization of cathepsin B during the progression of human gliomas.

Degradation of the extracellular matrix is a prerequisite for acquisition of the invasive phenotype. Several proteinases released by invading tumor cells appear to participate in the focal degradation of extracellular matrix proteins. Using an enzyme-linked immunosorbent assay, enzymatic assays, Western and Northern blotting techniques, we determined whether increased levels of the cysteine protease cathepsin B correlated with the progression and invasion of human gliomas. The amount of cathepsin B activity and protein content were highest in glioblastomas, lower in anaplastic astrocytomas and lowest in normal brain tissue and low-grade gliomas. There were significantly higher amounts of M(r) 25,000 and 26,000 bands in glioblastoma and anaplastic astrocytoma than in normal brain and low-grade glioma tissue extracts as determined by Western blotting with anti-cathepsin antibodies. In addition, cathepsin B transcripts were overexpressed in anaplastic astrocytoma (about two- to three-fold), in glioblastoma (about eight- to 10-fold), compared with normal brain tissue and low-grade glioma. Immunohistochemical staining for cathepsin B showed intense immunoreactivity in tumor and endothelial cells of glioblastomas and anaplastic astrocytomas but only weak immunoreactivity in low-grade glioma and normal brain tissues. Therefore, we conclude that cathepsin B expression is greatest in highly malignant astrocytomas, especially in glioblastomas, and is correlated with the malignant progression of astrocytomas.

Expression of tissue inhibitors of metalloproteinases: negative regulators of human glioblastoma invasion in vivo.

Tissue inhibitors of metalloproteinases (TIMPs) are negative regulators of matrix metalloproteinases (MMPs) which degrade major components of the extracellular matrix. The aberrant expression of TIMPs is believed to represent an important modulating factor in the invasive capacity of human tumors. In the present study we analyzed the expression of TIMPs in human brain tumor tissue samples by an enzyme-linked immunosorbent assay (ELISA) and by Northern blotting analysis. Quantitation of TIMP-1 and TIMP-2 by ELISA demonstrated low levels of TIMP-1 and TIMP-2 proteins in glioblastomas, and moderate levels in anaplastic astrocytomas compared with normal brain tissues low-grade gliomas and metastatic tumors (renal and breast carcinomas and melanomas). Northern blot analysis of TIMP-1 transcripts demonstrated higher expression in meningioma, normal brain tissues and other metastatic tumors than in anaplastic astrocytoma and glioblastoma. Two distinct transcripts of 1.0 and 3.5 kb were observed for TIMP-2 mRNA in normal brain tissue and in tumor extracts. In addition, TIMP-2 mRNA expression was lower in glioblastoma and anaplastic astrocytoma than in meningioma, normal brain tissues and metastatic tumors. These findings suggest that down-regulation of both TIMP-1 and TIMP-2 contributes significantly to the invasive potential of human glioblastoma multiforme and anaplastic astrocytomas.

Plasminogen activator inhibitor-1 in the pathogenesis of delayed radiation damage in rat spinal cord in vivo.

The pathophysiology of radiation-induced damage to the central nervous system (CNS) is poorly understood. Preliminary data suggest that fibrinolytic inhibitors are involved in the development of necrosis. In this study, cervical spinal cord irradiation was studied in 90 rats by measuring plasminogen activator inhibitor (PAI)-1 on Days 2, 7, 30, 60, 90, 120, 130, or 145 after irradiation. Paralysis due to radiation necrosis developed in all animals kept alive for 140 to 150 days. Assay of PAI-1 was by Western blot, enzyme-linked immunosorbent assay (ELISA), and complex formation with 125I-labeled urokinase. No PAI-1 was detected in normal spinal cord tissue or in irradiated spinal cord up to Day 90. However, PAI-1 was detected at Day 120 and was marked by elevated ELISA levels at the time of paralysis. Western blot showed detectable PAI-1 (51 kD) at Day 120 and very significant levels at the time of paralysis. Complex formation with 125I-labeled urokinase was also detected at Day 120 with similar results. Immunohistochemical studies showed that PAI-1 was highly concentrated within and immediately adjacent to zones of necrosis at 145 days and was absent in normal tissue. This study adds considerable weight to the proposal that PAI-1 is closely associated with the pathogenesis of CNS radiation necrosis.

Elevated levels of plasminogen activators in the pathogenesis of delayed radiation damage in rat cervical spinal cord in vivo.

The pathophysiology of the cellular basis of radiation-induced demyelination and white-matter necrosis of the central nervous system (CNS) is poorly understood. Preliminary data suggest that tissue damage is partly mediated through changes in the proteolytic enzymes. In this study, we irradiated rat cervical spinal cords with single doses of 24 Gy of 18 MV photons or 20 MeV electrons and measured the levels of plasminogen activators at days 2, 7, 30, 60, 90, 120, 130 and 145 after irradiation, using appropriate controls at each time. Fibrin zymography revealed fibrinolytic bands representing molecular weights of 68,000 and 48,000 in controls and irradiated samples; these bands increased significantly at days 120, 130 and 145 after irradiation. Inhibition of these enzymatic bands with specific antibodies against tissue-type plasminogen activator (tPA) and amiloride, an inhibitor for urokinase plasminogen activator (uPA), confirmed that these bands were tPA and uPA. Enzymatic levels quantified by densitometry showed a twofold elevation in the levels of tPA and more than a tenfold increase in uPA after 120 days' irradiation. Activity of uPA was increased threefold by day 2 and increased steadily with time compared to nonirradiated control samples. Enzyme-linked immunosorbent assay (ELISA) also showed a threefold increase in the tPA content in the extracts of irradiated rat cervical spinal cords at days 120, 130 and 145. This study adds additional information to the proposed role of plasminogen activators in the pathogenic pathways of radiation damage in the CNS.

Modulation of fibrinolysis by ionizing radiation.

Radiation-induced damage to the central nervous system is believed to be targeted to glial or endothelial cells or both, although the pathophysiology of this process is still poorly understood. A series of experiments were, therefore, conducted, including irradiation to primary rat astrocytes (in vitro) and rat spinal cords (in vivo). The levels of plasminogen activators (uPA and tPA) and their inhibitors (PNI and PAI-1) were determined by fibrin zymography, ELISA, amidolytic activity assay, complex formation, and Western blot analysis. Fibrin zymography revealed the presence of M(r) 48,000 (uPA) and M(r) 68,000 (tPA) lytic bands that were increased in irradiated samples. Three- to four-fold higher levels of tPA and 8- to 10-fold higher levels of uPA were detected in irradiated samples. Western blot analysis confirmed the presence of a 51-kDa band (PAI-1) in irradiated samples. PAI-1 is undetectable in nonirradiated spinal cord. Serum-free medium and cell and spinal cord extracts of nonirradiated samples showed a 43-kDa band (PNI), the intensity of which is decreased in irradiated samples. Four- to five-fold decreased levels of PNI were detected in irradiated serum-free media and cell extracts, but no levels of PNI were detected in irradiated spinal cord extracts. This study provides additional information regarding the proposed roles of plasminogen activators and their inhibitors in the development of CNS damage after irradiation.

Decreased levels of glia-derived nexin/protease nexin I in irradiated rat spinal cord in vivo.

The pathophysiology of the cellular basis of radiation induced demyelination and white matter necrosis of the Central Nervous System (CNS) is poorly understood. There have been no previous studies that have shown the effect of irradiation on glia-derived nexin. In this study, rats were given cervical spinal cord irradiation, and glial derived nexin or protease nexin I (GDN/PNI) was measured on days 2, 7, 30, 60, 120, 130, and 145 after irradiation. The level of GDN/PNI significantly decreased after irradiation compared to levels in control spinal cord, and there was no detectable levels of GDN/PNI by the time paralysis developed. This study adds considerable weight to the proposal that GDN/PNI has an important role in the pathogenesis of CNS radiation damage.

Increased levels of plasminogen activator inhibitor-1 (PAI-1) in human brain tumors.

Considerable interest in the roles of serine proteases and serine protease inhibitors (serpins) in regulating physiologic and pathologic tissue remodeling has led to studies that indicate their critical participation in development and diseases of the brain. Plasminogen activator inhibitor-1 (PAI-1) is the most significant regulator of fibrinolysis in plasma, but little is known of the levels or activities of this important serpin in normal brain and brain tumors. For this reason, we estimated qualitative and quantitative levels of PAI-1 in normal human brain and various brain tumors. Western-blot results indicated that a 51 kDa band recognized with polyclonal anti-PAI-1 was more prominently in metastatic and glioblastoma than in meningiomas and low-grade gliomas; normal human brain lacked any detectable band. Reverse zymography also showed high levels of PAI-1 in malignant brain tumors. The complex formation with 125I-urokinase demonstrated that PAI-1 complex levels were increased in metastatic and glioblastoma when compared with low-grade gliomas and meningiomas. Since PAI-1 acts as a modulator of fibrinolysis, a better understanding of the balance between serine proteases and PAI-1 is likely to enhance our knowledge of brain tumor biology.

Characterization of the serpin, alpha 1-antichymotrypsin, in normal human cerebrospinal fluid.

Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 +/- 2.8 SEM) was analyzed for the presence of the serpin alpha 1-antichymotrypsin (alpha 1-ACT). A chymotrypsin-specific chromogenic substrate (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68-kDa protein migrating identical to authentic human plasma alpha 1-ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma alpha 1-ACT. These studies showed the formation of complexes at 37 degrees C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma alpha 1-ACT, were formed at the lower temperature whereas a single higher Mr band was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human alpha 1-ACT under these same conditions. alpha 1-ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpin: protease complex receptors increase during in vitro myogenesis.

In the course of studies on the regulation of plasminogen activator-mediated extracellular matrix degradation in muscle we found the presence of a factor, a cellular inhibitor of serine proteases having features similar to the serpin protease nexin I (PNI). This factor was present in the medium and at maximum concentration following fusion of skeletal muscle cells in culture. The ability of the PNI homologue in mouse muscle to inhibit ECM degradation by urokinase in myoblast medium was compared to that of human PNI purified from human fibroblasts. Stable (to SDS) 1:1 molar ratio complex formation between PNI and proteases, the proposed means by which these enzymes are regulated and removed, was also detected. Cell surface receptors for protease:PNI complexes, the specific binding sites for inactive complex internalization, were found on multinucleated myotubes, while little or no receptor activity was detected on myoblasts. These data suggest that developmental regulation of a) increased PNI proteolytic inhibitory activity expression and b) the appearance of protease:inhibitor complex receptors on muscle cell surfaces during myogenesis may constitute important regulatory features of muscle surface proteolytic activity. They complement previous studies of proteoglycan metabolism in muscle, which itself contains molecules capable of regulating the activity of myotube surface proteases.

LCAT inhibitors interfere with the enzymatic determination of cholesterol and triglycerides.

5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB) and p-chloro-mercuriphenylsulfonic acid (PCMPS) are well-known sulfhydryl inhibitors that are used to inhibit lecithin: cholesterol acyltransferase (LCAT). They were each found to interfere with the enzymatic assays of cholesterol and triglycerides. DTNB falsely reduced the measured plasma cholesterol content, and falsely increased triglyceride readings. The interference with the triglyceride assay could be largely prevented by blanking for glycerol. PCMPS had only a slight effect on the cholesterol assay, but falsely lowered the triglyceride readings to a great extent, even with glycerol-blanking. Thus, these inhibitors should be avoided when plasma samples are to be enzymatically analyzed for cholesterol or triglycerides.