Evaluation of the dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to TiO2, chrysotile, crocidolite or amosite asbestos in a 90-day quantitative inhalation toxicology study - Interim results Part 2: Histopathological examination, Confocal microscopy and collagen quantification of the lung and pleural cavity.

The interim results from this 90-day multi-dose, inhalation toxicology study with life-time post-exposure observation has shown an important fundamental difference in persistence and pathological response in the lung between brake dust derived from brake-pads manufactured with chrysotile, TiO2 or chrysotile alone in comparison to the amphiboles, crocidolite and amosite asbestos. In the brake dust exposure groups no significant pathological response was observed at any time. Slight macrophage accumulation of particles was noted. Wagner-scores, were from 1 to 2 (1 = air-control group) and were similar to the TiO2 group. Chrysotile being biodegradable, shows a weakening of its matrix and breaking into short fibers & particles that can be cleared by alveolar macrophages and continued dissolution. In the chrysotile exposure groups, particle laden macrophage accumulation was noted leading to a slight interstitial inflammatory response (Wagner-score 1-3). There was no peribronchiolar inflammation and occasional very slight interstitial fibrosis. The histopathology and the confocal analyses clearly differentiate the pathological response from amphibole asbestos, crocidolite and amosite, compared to that from the brake dust and chrysotile. Both crocidolite and amosite induced persistent inflammation, microgranulomas, and fibrosis (Wagner-scores 4), which persisted through the post exposure period. The confocal microscopy of the lung and snap-frozen chestwalls quantified the extensive inflammatory response and collagen development in the lung and on the visceral and parietal surfaces. The interim results reported here, provide a clear basis for differentiating the effects from brake dust exposure from those following amphibole asbestos exposure. The subsequent results through life-time post-exposure will follow.

Evaluation of the exposure, dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to TiO2, chrysotile, crocidolite or amosite asbestos in a 90-day quantitative inhalation toxicology study - Interim results Part 1: Experimental design, aerosol exposure, lung burdens and BAL.

This 90-day repeated-dose inhalation toxicology study of brake-dust (BD) (brakes manufactured with chrysotile) in rats provides a comprehensive understanding of the biokinetics and potential toxicology in the lung and pleura. Exposure was 6 h/d, 5d/wk., 13wks followed by lifetime observation (~20 % survival). Control groups included a particle control (TiO2), chrysotile, commercial crocidolite and amosite asbestos. Aerosol fiber distributions of the chrysotile, crocidolite and amosite were similar (fibers L > 20 µm/cm3: chrysotile-Low/High 29/72; crocidolite 24; amosite 47 fibers/cm3; WHO-fibers/cm3: chrysotile-Low/High 119/233; crocidolite 181; amosite 281 fibers/cm3). The number of particles/cm3 in the BD was similar to that in the chrysotile, crocidolite & amosite exposures (BD 470-715; chrysotile 495-614; crocidolite 415; amosite 417 particles/cm3). In the BD groups, few fibers L > 20 µm were observed in the lungs at the end of exposure and no fibers L > 20 µm at 90d post exposure. In the chrysotile groups, means of 204,000 and 290,000 fibers(L > 20 µm)/lung were measured at 89d. By 180d, means of 1 and 3.9 fibers were counted on the filter corresponding to 14,000 and 55,000 fibers(L > 20 µm)/lung. In the crocidolite and amosite groups mean lung concentrations were 9,055,000 and 11,645,000 fibers(L > 20 µm)/lung at 89d. At 180d the means remained similar with 8,026,000 and 11,591,000 fibers(L > 20 µm)/lung representing 10-13% of the total lung fibers. BAL determined the total number of macrophages, lymphocytes, neutrophils, eosinophils, epithelial-cells and IL-1 beta, TNF-alpha and TGF-beta. At the moderate aerosol concentrations used in this study, neutrophil counts increased ~5 fold in the amphibole asbestos exposure groups. All other groups and parameters showed no important differences at these exposure concentrations. The exposure and lung burden results provide a sound basis for assessing the potential toxicity of the brake dust in comparison to the TiO2 particle control and the chrysotile, crocidolite and amosite asbestos control groups. The BAL results provide an initial indication of the differential response. Part 2 presents the presentation and discussion of the histopathological and confocal microscopy findings in this study through 90 days post exposure.

Nitrofen induces a redox-dependent apoptosis associated with increased p38 activity in P19 teratocarcinoma cells.

Nitrofen is a diphenyl ether herbicide that produces a spectrum of fetal abnormalities in rodents. To characterize the molecular mechanisms of nitrofen-mediated birth defects at the cellular level, we explored its effects on undifferentiated P19 teratocarcinoma cells. Nitrofen induces a time-dependent cell death of P19 cells that is associated with increases in TUNEL-positivity and caspase-3 cleavage suggesting that nitrofen induces P19 cell apoptosis. In addition, the increase in TUNEL-positive cells was inhibited with zVAD-fmk, suggesting that nitrofen induces a caspase-dependent apoptosis. Nitrofen treatment was associated with increased p38 MAP kinase activity, though pretreatment of cells with multiple p38 inhibitors did not affect nitrofen-mediated caspase-3 cleavage, suggesting caspase-3 cleavage is p38-independent. Nitrofen induced a dose-dependent increase in reactive oxygen species (ROS), which was accompanied by a decrease in the ratio of reduced/oxidized glutathione, indicating that nitrofen alters the cellular redox state of these cells. Furthermore, pretreatment of cells with N-acetyl cysteine gave a dose- and time-dependent reduction of caspase-3 cleavage, supporting the observations that caspase-3 cleavage is cell-redox-dependent. Therefore, nitrofen induces P19 cell apoptosis that is cell-redox-dependent and is associated with increases in p38 activity and ROS and may play a role in nitrofen-mediated birth defects.

Nondestructive indices of trace element exposure in squamate reptiles.

Compared with birds, mammals, fish, and even amphibians, very little is known about the effects of contaminants on reptiles. Recent evidence that many reptile populations may be declining has stimulated demand for toxicological studies of reptiles as well as development of nondestructive sampling techniques useful for assessing and monitoring contaminant exposure. The current study experimentally evaluated the utility of shed skins, tail clips, and blood samples as nondestructive indices of trace element exposure in banded water snakes, Nerodia fasciata. For 13.5 months, snakes were either fed fish from a coal ash-contaminated site or uncontaminated food from a reference site. Snakes fed contaminated prey accumulated As, Cd, Se, Sr, and V in various organs (i.e. liver, kidney, and/or gonads). Moreover, non-parametric discriminant function analysis revealed that snakes could be placed in two groups that reliably reflected their experimental diet based upon Se, Sr, and As concentrations in tail clips, blood, and/or shed skins. We suggest that nondestructive sampling techniques, particularly analyses of blood and tail clips, may be easily applied in evaluations of contaminant exposure in the field and laboratory and may prevent excessive destructive sampling of potentially threatened reptile species.

Decreased mitogen activated protein kinase activities in congenital diaphragmatic hernia-associated pulmonary hypoplasia.

BACKGROUND/PURPOSE: The mechanisms that cause pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) currently are unknown. The authors proposed that the reduced size and immaturity of these lungs may be associated with differences in the levels of mitogen activated protein (MAP) kinase phosphorylation (extracellular signal regulated protein kinases, ERK-1 and -2). METHODS: ERK-1 activities were measured using immune-complex kinase assays on fetal whole-lung lysates obtained from both nitrofen and olive oil-treated (control) pregnant rats. In addition, ERK-1 and ERK-2 functional activities were estimated by semiquantitative Western blot analysis, using an antibody specific for the diphosphorylated (dp-ERK, activated) forms of the enzymes. RESULTS: ERK-1 activities, measured using immune-complex kinase assays, were reduced in CDH lungs compared with olive oil-treated controls (P <.02). In addition, dp-ERK-1 and dp-ERK-2 levels were found to be reduced in CDH lungs compared with controls (dp-ERK-1, P =.003; dp-ERK-2, P =.04), whereas ERK-1 and ERK-2 protein levels were unchanged. CONCLUSIONS: The lower values of ERK-1 activity and reduced amounts of dp-ERK-1 and dp-ERK-2 in lung tissue from CDH animals, suggests that ERK-1 and ERK-2 activities are reduced in pulmonary hypoplasia associated with CDH. The observed reduction in ERK-1 and ERK-2 activities implicates attenuated cell signaling upstream of the ERK-1 and -2 enzymes.

Immunogenicity and protective efficacy of the alpha C protein of group B streptococci are inversely related to the number of repeats.

Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates. Alpha C protein is a protective cell surface-associated protein of GBS. This protein contains a repeat region flanked by N and C termini. Variable expression of tandem repeating units of alpha C proteins had been found among clinical isolates of GBS. We examined the effect of the number of repeats on the immunogenicity of the alpha C protein and its ability to elicit protection from GBS infection in a neonatal mouse model. Mice were immunized with purified alpha C proteins of constructs containing various numbers of repeats (n = 1, 2, 9, and 16) and the N- and C-terminal regions. Both the N-terminal and the repeat regions contain protective and opsonic epitopes. Antibody responses to the alpha C protein constructs with various numbers of repeats were tested with enzyme-linked immunosorbent assay plates coated with either native, nine-repeat alpha C protein or "repeatless" N-terminal antigen. An inverse relationship was found between the number of repeats and the immunogenicity of the alpha C protein; this effect was most pronounced on titers of antibody to the N-terminal region. An inverse relationship was also observed between the number of repeats and protective efficacy, i.e., mouse dams immunized with 5 microg of one- or nine-repeat alpha C protein transferred protective immunity to 65 or 11% of their pups, respectively (P < 0.0001). Thus, the presence of multiple repeats appears to lessen the antibody response to the complete alpha C protein, and especially the antibody response to its N-terminal region, and suggests a mechanism whereby repeat elements contribute to the evasion of host immunity.

Characterization of two distinct opsonic and protective epitopes within the alpha C protein of the group B Streptococcus.

Group B Streptococcus (GBS) is a major cause of neonatal sepsis, meningitis in early infancy, postpartum endometritis, and serious invasive infections in adults in the United States. We previously cloned, sequenced, and characterized the alpha antigen gene, bca, and showed that the alpha C protein of GBS is a trypsin-resistant, surface-associated polypeptide that contains a signal sequence, a unique N terminus, nine identical tandem repeats, and a C-terminal membrane anchor structure. Polyclonal antiserum raised to the recombinant alpha C protein and an opsonic monoclonal antibody, 4G8, raised to the native protein from GBS have been shown to be protective in a mouse model. The binding site of 4G8 has now been localized to the tandem repeat region of the alpha C protein. To determine whether the N terminus of the alpha C protein contains additional opsonic and/or protective epitopes, the sequence corresponding to the alpha C protein N terminus was subcloned into a pET vector, the expressed peptide from Escherichia coli was purified by Ni2+ affinity chromatography, and rabbit polyclonal antibodies were raised to the purified recombinant peptide. Antibodies to the alpha C protein N terminus were shown to be opsonic by an in vitro opsonophagocytosis assay. In addition, 69% of newborn mouse pups from mothers passively immunized with the antiserum to the recombinant N-terminal polypeptide of the alpha C protein were protected against lethal challenge with GBS A909. These data indicate that at least two distinct regions of the alpha C protein, the N terminus and the tandem repeat region, contain opsonic and protective epitopes.

Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein.

Group B streptococci (GBS) are the most common cause of neonatal sepsis, pneumonia, and meningitis. The alpha C protein is a surface-associated antigen; the gene (bca) for this protein contains a series of tandem repeats (each encoding 82 aa) that are identical at the nucleotide level and express a protective epitope. We previously reported that GBS isolates from two of 14 human maternal and neonatal pairs differed in the number of repeats contained in their alpha C protein; in both pairs, the alpha C protein of the neonatal isolate was smaller in molecular size. We now demonstrate by PCR that the neonatal isolates contain fewer tandem repeats. Maternal isolates were susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum, whereas the discrepant neonatal isolates proliferated. An animal model was developed to further study this phenomenon. Adult mice passively immunized with antiserum to the alpha C protein were challenged with an alpha C protein-expressing strain of GBS. Splenic isolates of GBS from these mice showed a high frequency of mutation in bca--most commonly a decrease in repeat number. Isolates from non-immune mice were not altered. Spontaneous deletions in the repeat region were observed at a much lower frequency (6 x 10(-4)); thus, deletions in that region are selected for under specific antibody pressure and appear to lower the organism's susceptibility to killing by antibody specific to the alpha C protein. This mechanism of antigenic variation may provide a means whereby GBS evade host immunity.

Large, identical, tandem repeating units in the C protein alpha antigen gene, bca, of group B streptococci.

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C protein alpha antigen of GBS is thought to have a role in both virulence and immunity. We previously cloned the C protein alpha antigen structural gene (named bca for group B, C protein, alpha) into Escherichia coli. Western blots of both the native alpha antigen and the cloned gene product demonstrate a regularly laddered pattern of heterogeneous polypeptides. The nucleotide sequence of the bca locus reveals an open reading frame of 3060 nucleotides encoding a precursor protein of 108,705 Da. Cleavage of a putative signal sequence of 41 amino acids yields a mature protein of 104,106 Da. The 20,417-Da N-terminal region of the alpha antigen shows no homology to previously described protein sequences and is followed by a series of nine tandem repeating units that make up 74% of the mature protein. Each repeating unit is identical and consists of 82 amino acids with a molecular mass of 8665 Da, which is encoded by 246 nucleotides. The size of the repeating units corresponds to the observed size differences in the heterogeneous ladder of alpha C proteins expressed by GBS. The C-terminal region of the alpha antigen contains a membrane anchor domain motif that is shared by a number of Gram-positive surface proteins. The large region of identical repeating units in bca defines protective epitopes and may play a role in generating phenotypic and genotypic diversity of the alpha antigen.

Cloned alpha and beta C-protein antigens of group B streptococci elicit protective immunity.

Streptococcus agalactiae (group B streptococci [GBS]) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C proteins of GBS play a role in immunity, but their number, size, structure, function, and virulence properties have not been well characterized. A recombinant library of DNA fragments from GBS strain A909 (type Ia/C) was prepared in the plasmid pUX12, a specially constructed Escherichia coli expression vector. The library was screened with a rabbit antiserum shown to be protective for passive immunity to GBS infection in a mouse lethality model. Clones were divided into two distinct groups on the basis of DNA-DNA cross-hybridization, restriction enzyme analysis, and the expression of antigenic proteins in E. coli. A characteristic clone from each group was chosen for further study. Clone pJMS23 expresses gene products that biochemically and immunologically correspond to the trypsin-resistant, C-protein alpha antigen. Clone pJMS1 expresses a gene product that binds to immunoglobulin A and is similar to the trypsin-sensitive, C-protein beta antigen. Antisera raised in rabbits against E. coli containing each of the plasmid clones were able to elicit protective immunity in mice challenged by GBS strains carrying the C proteins but not by non-C-protein-bearing strains. Southern blot analysis shows no DNA homology between the clones, and there is no immunological cross-reactivity between the antigens they express. Therefore, pJMS23 and pJMS1 encode two different C proteins that define unique protective epitopes.