Bridging inhaled aerosol dosimetry to physiologically based pharmacokinetic modeling for toxicological assessment: nicotine delivery systems and beyond.

One of the challenges for toxicological assessment of inhaled aerosols is to accurately predict their deposited and absorbed dose. Transport, evolution, and deposition of liquid aerosols are driven by complex processes dominated by convection-diffusion that depend on various factors related to physics and chemistry. These factors include the physicochemical properties of the pure substance of interest and associated mixtures, the physical and chemical properties of the aerosols generated, the interplay between different factors during transportation and deposition, and the subject-specific inhalation topography. Several inhalation-based physiologically based pharmacokinetic (PBPK) models have been developed, but the applicability of these models for aerosols has yet to be verified. Nicotine is among several substances that are often delivered via the pulmonary route, with varied kinetics depending upon the route of exposure. This was used as an opportunity to review and discuss the current knowledge and state-of-the-art tools combining aerosol dosimetry predictions with PBPK modeling efforts. A validated tool could then be used to perform for toxicological assessment of other inhaled therapeutic substances. The Science Panel from the Alliance of Risk Assessment have convened at the "Beyond Science and Decisions: From Problem Formulation to Dose-Response Assessment" workshop to evaluate modeling approaches and address derivation of exposure-internal dose estimations for inhaled aerosols containing nicotine or other substances. The discussion involved PBPK model evaluation criteria, challenges, and choices that arise in such a model design, development, and application as a computational tool for use in human toxicological assessments.

The impact of genome evolution on the allotetraploid Nicotiana rustica - an intriguing story of enhanced alkaloid production.

BACKGROUND: Nicotiana rustica (Aztec tobacco), like common tobacco (Nicotiana tabacum), is an allotetraploid formed through a recent hybridization event; however, it originated from completely different progenitor species. Here, we report the comparative genome analysis of wild type N. rustica (5 Gb; 2n = 4x = 48) with its three putative diploid progenitors (2.3-3 Gb; 2n = 2x =24), Nicotiana undulata, Nicotiana paniculata and Nicotiana knightiana. RESULTS: In total, 41% of N. rustica genome originated from the paternal donor (N. undulata), while 59% originated from the maternal donor (N. paniculata/N. knightiana). Chloroplast genome and gene analyses indicated that N. knightiana is more closely related to N. rustica than N. paniculata. Gene clustering revealed 14,623 ortholog groups common to other Nicotiana species and 207 unique to N. rustica. Genome sequence analysis indicated that N. knightiana is more closely related to N. rustica than N. paniculata, and that the higher nicotine content of N. rustica leaves is the result of the progenitor genomes combination and of a more active transport of nicotine to the shoot. CONCLUSIONS: The availability of four new Nicotiana genome sequences provide insights into how speciation impacts plant metabolism, and in particular alkaloid transport and accumulation, and will contribute to better understanding the evolution of Nicotiana species.

Genomic impact of cigarette smoke, with application to three smoking-related diseases.

There is considerable evidence that inhaled toxicants such as cigarette smoke can cause both irreversible changes to the genetic material (DNA mutations) and putatively reversible changes to the epigenetic landscape (changes in the DNA methylation and chromatin modification state). The diseases that are believed to involve genetic and epigenetic perturbations include lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular disease (CVD), all of which are strongly linked epidemiologically to cigarette smoking. In this review, we highlight the significance of genomics and epigenomics in these major smoking-related diseases. We also summarize the in vitro and in vivo findings on the specific perturbations that smoke and its constituent compounds can inflict upon the genome, particularly on the pulmonary system. Finally, we review state-of-the-art genomics and new techniques such as high-throughput sequencing and genome-wide chromatin assays, rapidly evolving techniques which have allowed epigenetic changes to be characterized at the genome level. These techniques have the potential to significantly improve our understanding of the specific mechanisms by which exposure to environmental chemicals causes disease. Such mechanistic knowledge provides a variety of opportunities for enhanced product safety assessment and the discovery of novel therapeutic interventions.

Assembly of macromolecular pores by immune defense systems.

Immune defence systems (complement, cytolytic lymphocytes) make use of transmembrane pores assembled from up to 20 soluble monomers in a highly regulated process to induce cell death. Inhibitors of pore formation have been found which protect blood, endothelial and epithelial cells from the destructive effect of complement lesions. Recently, a pore-forming protein showing immunological crossreactivity to complement C9 has been found in the protozoan parasite Trypanosoma cruzi, thereby extending this protein family and generalizing its means of generating non-selective membrane permeability.

The apoptosis endonucleases: cleaning up after cell death?

The term apoptosis describes the predictable structural changes associated with many forms of programmed cell death. One of the first visible events of apoptosis is the collapse of the nucleus. Nuclear degradation is manifested by digestion of chromatin into nucleosome-sized fragments or multiples of these. This digestion of DNA is enzymatic, and several attempts have been made to characterize apoptosis-specific endodeoxyribonucleases. Although there are strong candidates for such enzymes, direct evidence for their role in apoptosis is yet to be provided.

Tandem mass spectrometry protein identification on a PC grid.

We present a method to grid-enable tandem mass spectrometry protein identification. The implemented parallelization strategy embeds the open-source x!tandem tool in a grid-enabled workflow. This allows rapid analysis of large-scale mass spectrometry experiments on existing heterogeneous hardware. We have explored different data-splitting schemes, considering both splitting spectra datasets and protein databases, and examine the impact of the different schemes on scoring and computation time. While resulting peptide e-values exhibit fluctuation, we show that these variations are small, caused by statistical rather than numerical instability, and are not specific to the grid environment. The correlation coefficient of results obtained on a standalone machine versus the grid environment is found to be better than 0.933 for spectra and 0.984 for protein identification, demonstrating the validity of our approach. Finally, we examine the effect of different splitting schemes of spectra and protein data on CPU time and overall wall clock time, revealing that judicious splitting of both data sets yields best overall performance.

Systems toxicology meta-analysis of in vitro assessment studies: biological impact of a candidate modified-risk tobacco product aerosol compared with cigarette smoke on human organotypic cultures of the aerodigestive tract.

Systems biology combines comprehensive molecular analyses with quantitative modeling to understand the characteristics of a biological system as a whole. Leveraging a similar approach, systems toxicology aims to decipher complex biological responses following exposures. This work reports a systems toxicology meta-analysis in the context of in vitro assessment of a candidate modified-risk tobacco product (MRTP) using three human organotypic cultures of the aerodigestive tract (buccal, bronchial, and nasal epithelia). Complementing a series of functional measures, a causal network enrichment analysis of transcriptomic data was used to compare quantitatively the biological impact of aerosol from the Tobacco Heating System (THS) 2.2, a candidate MRTP, with 3R4F cigarette smoke (CS) at similar nicotine concentrations. Lower toxicity was observed in all cultures following exposure to THS2.2 aerosol compared with 3R4F CS. Because of their morphological differences, a smaller exposure impact was observed in the buccal (stratified epithelium) compared with the bronchial and nasal (pseudostratified epithelium). However, the causal network enrichment approach supported a similar mechanistic impact of CS across the three cultures, including the impact on xenobiotic, oxidative stress, and inflammatory responses. At comparable nicotine concentrations, THS2.2 aerosol elicited reduced and more transient effects on these processes. To demonstrate the benefits of additional data modalities, we employed a newly established targeted mass-spectrometry marker panel to further confirm the reduced cellular stress responses elicited by THS2.2 aerosol compared with 3R4F CS in the nasal culture. Overall, this work demonstrates the applicability and robustness of the systems toxicology approach for in vitro inhalation toxicity assessment.

About the involvement of deoxyribonuclease I in apoptosis.

Cell death by apoptosis is involved in a large variety of developmental events and physiological processes requiring a reduction in cell count. Nuclear collapse, one of the first visible changes denoting irreversible commitment to cell death by apoptosis, is frequently accompanied by chromatin degradation into nucleosome-sized fragments of multiples thereof. The identity of the endonuclease responsible for this DNA digestion has attracted some interest in recent years and several candidate endonucleases have been proposed. The scope of this article is to summarise the present knowledge about deoxyribonuclease I, one of the candidate enzymes.

The chemokine information source: identification and characterization of novel chemokines using the WorldWideWeb and expressed sequence tag databases.

The chemokine superfamily is a large group of more than 30 small proteins. Many of these were originally identified because of their role in the selective recruitment and activation of leukocytes during inflammation. More recently, some of the chemokine receptors and ligands have been implicated in the mechanism of viral infection for primate lentiviruses such as HIV-1. From the original identification of interleukin-8 (IL-8; the most studied member of the superfamily), the number of new family members has mushroomed over the last few years. Two events have dramatically altered the speed at which sequence information concerning novel chemokines has become available to the scientific community. First, many groups have been obtaining large amounts of sequence information from cDNA libraries by sequencing the clones at random, generating expressed sequence tags (ESTs). Although these ESTs are relatively short, typically less than 500 bases, this amount of sequence is usually sufficient to obtain the entire open reading frame for chemokines. Second, there has been a rapid growth in the use of the WorldWideWeb by bioinformatics groups. The Web was originally set up by the European Centre for Particle Physics (CERN) in Geneva as a method of transferring data between collaborating groups throughout the world. It has enabled biologists throughout the world to have almost instantaneous access both to the databases containing the EST sequences and to the automated tools that are required for searching such databases. With such methods we have been able to rapidly identify more than 10 new human chemokines from public domain databases. In addition to the known categories of chemokines, which are named C, CC, and CXC based on the spacings of N-terminal cysteine residues, we have been able to identify the first member of a novel chemokine subfamily, with a novel CXXXC cysteine spacing. Furthermore, we can subdivide the CC chemokines into monocyte chemotactic protein and macrophage inflammatory protein families based on their sequence identity levels, but also their clustering on the human genome, as identified on other Web sites. The rapid availability of all this data has reduced the amount of time spent on conventional gene identification, enabling us to move quickly on to trying to understand the biology and physiological relevance of these molecules. The novel chemokine sequences obtained and alignments with existing members of the superfamily are now contained within a Chemokine Information Source on an open access server, allowing further searching of chemokine sequences and increasing the availability of such data to the scientific community.

Selectivity and antagonism of chemokine receptors.

The chemokine superfamily can be subdivided into two groups based on their amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation and, so far, two human receptors have been cloned. The CC chemokines tend to be involved in chronic inflammation, and recently we have cloned a fourth leukocyte receptor for this group of ligands. Understanding what makes one receptor bind its range of agonists is important if we are to develop potent selective antagonist. We have started to investigate the molecular basis of this receptor selectivity by looking at why CC chemokines do not bind to the CXC receptors in several ways. First, we looked at the role of the three-dimensional structure of the ligand, and have solved the three dimensional structure of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined for the CC chemokine macrophage inflammatory protein-1 beta, and it has a completely different dimer interface to that of the CXC chemokine interleukin-8 (IL-8). However, the monomer structures of all the chemokines are very similar, and at physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Mutations of one of the residues in this region, Leu-25 in IL-8, to tyrosine (which is conserved at this position in CC chemokines) enables the mutant IL-8 to bind CC chemokine receptor-1 (CC-CKR-1) and introduces monocyte chemoattractant activity. Using other mutations in this region, we can show a direct interaction with the N-terminus of CC-CKR-1. Third, we have found that modification of the amino terminus of RANTES by addition of one amino acid makes it into an antagonist with nanomolar potency. Taken together, this data suggests a two-site model for receptor activation and for selectivity between CC and CXC chemokines, with an initial receptor contact provided by the main body of the chemokine, and activation provided by the amino terminal region.

Comparative molecular modelling of the Fas-ligand and other members of the TNF family.

A number of proteins with significant similarity to the tumour necrosis factor (TNF) have been identified over the last years. Upon interaction with their cognate receptor (members of the TNF-receptor family), all members of this protein family induce either cell death or proliferation/differentiation of the receptor-bearing cells. One of the last identified members of the TNF family is the apoptosis-inducing ligand of the Fas-receptor, termed Fas-ligand (FasL). Here we report the cloning and sequencing of the mouse cDNA for the FasL. Using knowledge-based protein modelling, we demonstrate that all members of the TNF family form trimeric complexes, and define the residues located at the subunit interfaces. The resulting structurally corrected multiple sequence alignment allows the identification of residues potentially involved in receptor recognition, and should help design mutagenesis experiments for structure-function relationship studies.

Structural and functional characterization of complement C8 gamma, a member of the lipocalin protein family.

Human complement component C8 exhibits an unusual structure in that it contains three chains, two of which, alpha and beta, display high sequence homology to other complement and CTL pore-forming proteins. The third chain, C8 gamma, is covalently linked to C8 alpha by a disulfide linkage; it is demonstrated that Cys40 of C8 gamma is linked to Cys164 of C8 alpha, a unique cysteine located in a loop located between the cysteine-rich LDL-receptor class A module and the membrane-inserting region of C8 alpha. C8 gamma was recently identified as a member of the lipocalin protein family, in which all proteins were either shown to, or are believed to bind small hydrophobic ligands. The present results now demonstrate that C8 gamma incorporates retinol and retinoic acid in the presence of 2 M NaCl. Molecular modeling of C8 gamma, based on the crystal structure of the homologous beta-lactoglobulin, reveals a structure of eight antiparallel beta-strands, bearing a highly hydrophobic binding pocket. The residues participating in the pocket formation are highly conserved when compared with the structures of beta-lactoglobulin and retinol-binding protein, both of which are known to interact with retinol. It is therefore proposed that C8 gamma may act as a retinol transporting protein in plasma.

Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and perforin.

Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.

Mutations in the putative lipid-interaction domain of complement C9 result in defective secretion of the functional protein.

Complement protein C9 assembles with C5, C6, C7, C8 on the surface of target cells to form the lytic membrane attack complex (MAC). During MAC assembly and insertion into the target membrane, the hydrophilic, globular C9 partially unfolds to expose a hydrophobic lipid interaction domain. Several copies of amphiphilic C9 subsequently polymerize to form the characteristic ring-like MAC. Using a combined photoaffinity label and computer modeling approach, two amphipathic helices in a segment encompassing the amino acids 293-334 have been predicted to interact with membrane lipids. To elucidate the mechanism of C9 lipid binding and insertion, site-directed mutagenesis was used to change the amphipathic character of the helices. While some conservative amino acid replacements such as Thr307 by a Leu were tolerated and yielded fully active C9 when expressed in COS cells, successive changes of Leu305 into Val, Ala, and Glu on the hydrophobic site of the first helix gave rise to only partly or not secreted C9. All non-conservative amino acid replacements introduced on either side of the helices resulted in non-secreted C9 that was subsequently degraded intracellularly, indicating the importance of the correct folding of the presumptive transmembrane domain during biosynthesis. A natural secretion-incompetent mutant was found in which Val293, located in the proposed lipid-binding region, was lacking. Taken together, these findings suggest that the high incidence of homozygous C9 deficiencies may be due to a blockage in intracellular transport and secretion due to point mutations in this 'hot spot' region of the molecule.

Identification of gene expression signature for cigarette smoke exposure response--from man to mouse.

Gene expression profiling data can be used in toxicology to assess both the level and impact of toxicant exposure, aligned with a vision of 21st century toxicology. Here, we present a whole blood-derived gene signature that can distinguish current smokers from either nonsmokers or former smokers with high specificity and sensitivity. Such a signature that can be measured in a surrogate tissue (whole blood) may help in monitoring smoking exposure as well as discontinuation of exposure when the primarily impacted tissue (e.g., lung) is not readily accessible. The signature consisted of LRRN3, SASH1, PALLD, RGL1, TNFRSF17, CDKN1C, IGJ, RRM2, ID3, SERPING1, and FUCA1. Several members of this signature have been previously described in the context of smoking. The signature translated well across species and could distinguish mice that were exposed to cigarette smoke from ones exposed to air only or had been withdrawn from cigarette smoke exposure. Finally, the small signature of only 11 genes could be converted into a polymerase chain reaction-based assay that could serve as a marker to monitor compliance with a smoking abstinence protocol.

Biological impact of cigarette smoke compared to an aerosol produced from a prototypic modified risk tobacco product on normal human bronchial epithelial cells.

Cigarette smoking causes serious and fatal diseases. The best way for smokers to avoid health risks is to quit smoking. Using modified risk tobacco products (MRTPs) may be an alternative to reduce the harm caused for those who are unwilling to quit smoking, but little is known about the toxic effects of MRTPs, nor were the molecular mechanisms of toxicity investigated in detail. The toxicity of an MRTP and the potential molecular mechanisms involved were investigated in high-content screening tests and whole genome transcriptomics analyses using human bronchial epithelial cells. The prototypic (p)MRTP that was tested had less impact than reference cigarette 3R4F on the cellular oxidative stress response and cell death pathways. Higher pMRTP aerosol extract concentrations had impact on pathways associated with the detoxification of xenobiotics and the reduction of oxidative damage. A pMRTP aerosol concentration up to 18 times higher than the 3R4F caused similar perturbation effects in biological networks and led to the perturbation of networks related to cell stress, and proliferation biology. These results may further facilitate the development of a systems toxicology-based impact assessment for use in future risk assessments in line with the 21st century toxicology paradigm, as shown here for an MRTP.

Increased p34cdc2-dependent kinase activity during apoptosis: a possible activation mechanism of DNase I leading to DNA breakdown.

Cells undergoing apoptosis typically exhibit distinctive morphological characteristics. Early events include the rounding up of the cell, chromatin condensation, nuclear membrane breakdown and blebbing of the cellular membrane. Strikingly similar changes take place in the cell cycle progression, at the entry into mitosis, suggesting a link between mitosis and apoptosis. Here we show that expression of active p34cdc2 at inappropriate phases during the cell cycle leads to morphological changes reminiscent of apoptosis, including DNA degradation. Cells cotransfected with the active mutant of p34cdc2 and DNase I displayed degraded DNA, which was absent in p34cdc2 wild-type and DNase I-transfected cells, in spite of similar DNase activities. Upon induction of apoptosis in thymocytes, transient p34cdc2 activation was detected prior to lamina breakdown and nuclease activation. P34cdc2 activation was also observed during APO-1 (Fas/CD95)-induced apoptosis in a B lymphoblastoma cell line. Our results suggest that unscheduled activation of p34cdc2 may participate in the initiation of the typical apoptotic phenotype.

Overexpression of deoxyribonuclease I (DNase I) transfected into COS-cells: its distribution during apoptotic cell death.

COS-cells were transiently transfected with the pSG5 plasmid containing the cDNA of rat parotid deoxyribonuclease I (DNase I) either in right or inverse orientation. Expression of DNase I in transfected cells was only observed when the plasmid contained the cDNA in the right orientation. Expression of DNase I was monitored by measuring the DNase I specific DNA-degrading activity present in the conditioned cell culture medium and in cell homogenates. The expressed DNase I activity could be inhibited by monospecific polyclonal antibodies and by G-actin. Immunofluorescence indicated that approximately 20% of the COS-cells transfected with the DNase I-cDNA in right orientation expressed DNase I. These transfected cells contained large amounts of DNase I, which was found to be localized within the rough endoplasmic reticulum, the Golgi-complex and finally concentrated in a perinuclear location. Occasionally cells were observed which contained the DNase I in small apparently secretory transport vesicles. Transfected cells with perinuclear concentration of DNase I exhibited progressive nuclear destruction, i.e., pyknosis and cytoplasmic shrinkage. Solely the DNA extracted from isolated nuclei of cells transfected with the DNase I-cDNA in correct orientation revealed an internucleosomal DNA-degradation (ladder formation) typical for apoptosis after incubation in the presence of CaCl2 and MgCl2. Only the conditioned medium of COS-cells transfected with the right-oriented DNase I-cDNA contained the nucleolytic activity able to internucleosomally degrade the chromatin of substrate nuclei. Thus, these results indicate that overexpression of DNase I alone is sufficient to induce the morphological and biochemical changes observed during apoptosis.

Distribution of deoxyribonuclease I in rat tissues and its correlation to cellular turnover and apoptosis (programmed cell death).

The expression of deoxyribonuclease I (DNase I) in various rat tissues was screened by use of a cDNA-probe of rat parotid DNase I and monospecific polyclonal antibodies. High amounts of DNase I-specific mRNA were found in the parotid gland, kidney and small intestine. Homogenates of these organs also contain elevated levels of DNase I-specific DNA-degrading activity as verified by the zymogram technique and immunoblots. Affinity-purified polyclonal antibodies against rat parotid DNase I were employed in an immunohistochemical study of the cellular distribution of DNase I antigen in rat parotid gland, kidney, small intestine, and a number of stratified epithelia. In the parotid gland the DNase I antigenicity was found to be confined to the secretory cells. Within these cells the secretory granules exhibit the highest immunoreactivity. In contrast, within the small intestine and stratified epithelia we found a preferential localization and concentration of DNase I in cells prone to undergo apoptosis (programmed cell death), i.e., within the migrating enterocytes present at the villar tips and the keratinocytes above the basal cell layer. Within the kidney, the cells lining the convoluted distal tubules and collecting ducts exhibit strong DNase I immunoreactivity which was found to often localize perinuclearly. The cells exhibiting chromatin fragmentation were identified on paraffin-embedded sections by in situ end-labeling of free 3'-OH-ends of cleaved DNA using fluorescent dATP or dUTP and terminal transferase. It was found that only a small fraction of the DNase I positive cells showed signs of apoptotic chromatin degradation. Thus only a few enterocytes at the uppermost villar tips and very few keratinocytes underneath the keratinized layer were in situ end-labeled, i.e., exhibited a high concentration of fragmented DNA. This result is taken as evidence that these cells express DNase I in advance of their apoptotic death and furthermore that the actual apoptosis is a rapid process only detectable in a few cells. In contrast, no in situ end-labeled apoptotic nuclei were detected in rat kidney provided that care was taken to rapidly excise and fix this organ.