An adverse outcome pathway (AOP) is a conceptual framework that organizes existing knowledge concerning biologically plausible, and empirically supported, links between molecular-level perturbation of a biological system and an adverse outcome at a level of biological organization of regulatory relevance. Systematic organization of information into AOP frameworks has potential to improve regulatory decision-making through greater integration and more meaningful use of mechanistic data. However, for the scientific community to collectively develop a useful AOP knowledgebase that encompasses toxicological contexts of concern to human health and ecological risk assessment, it is critical that AOPs be developed in accordance with a consistent set of core principles. Based on the experiences and scientific discourse among a group of AOP practitioners, we propose a set of five fundamental principles that guide AOP development: (1) AOPs are not chemical specific; (2) AOPs are modular and composed of reusable components-notably key events (KEs) and key event relationships (KERs); (3) an individual AOP, composed of a single sequence of KEs and KERs, is a pragmatic unit of AOP development and evaluation; (4) networks composed of multiple AOPs that share common KEs and KERs are likely to be the functional unit of prediction for most real-world scenarios; and (5) AOPs are living documents that will evolve over time as new knowledge is generated. The goal of the present article was to introduce some strategies for AOP development and detail the rationale behind these 5 key principles. Consideration of these principles addresses many of the current uncertainties regarding the AOP framework and its application and is intended to foster greater consistency in AOP development.

a b s t r a c tInhaled aerosol dose models play critical roles in medicine, the regulation of air pollutants and basic research. The models fall into several categories: traditional, computational fluid dynamical (CFD), physiologically based pharmacokinetic (PBPK), empirical, semi-empirical, and "reference". Each type of model has its strengths and weaknesses, so multiple models are commonly used for practical applications. Aerosol dose models combine information on aerosol behavior and the anatomy and physiology of exposed human and laboratory animal subjects. Similar models are used for in-vitro studies. Several notable advances have been made in aerosol dose modeling in the past 80 years. The pioneers include Walter Findeisen, who in 1935 published the first traditional model and established the structure of modern models. His model combined aerosol behavior with simplified respiratory tract structures. Ewald Weibel established morphometric techniques for the lung in 1963 that are still used to develop data for modeling today. Advances in scanning techniques have similarly contributed to the knowledge of respiratory tract structure and its use in aerosol dose modeling. Several scientists and research groups have developed and advanced traditional, CFD, and PBPK models. Current issues under study include understanding individual and species differences; examining localized particle deposition; modeling non-ideal aerosols and nanoparticle behavior; linking the regions of the respiratory tract airways from nasal-oral to alveolar; and developing sophisticated supporting software. Although a complete history of inhaled aerosol dose modeling is far too extensive to cover here, selected highlights are described in this paper.

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Thymine DNA glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily of DNA repair enzymes. Owing to its ability to excise thymine when mispaired with guanine, it was proposed to act against the mutability of 5-methylcytosine (5-mC) deamination in mammalian DNA. However, TDG was also found to interact with transcription factors, histone acetyltransferases and de novo DNA methyltransferases, and it has been associated with DNA demethylation in gene promoters following activation of transcription, altogether implicating an engagement in gene regulation rather than DNA repair. Here we use a mouse genetic approach to determine the biological function of this multifaceted DNA repair enzyme. We find that, unlike other DNA glycosylases, TDG is essential for embryonic development, and that this phenotype is associated with epigenetic aberrations affecting the expression of developmental genes. Fibroblasts derived from Tdg null embryos (mouse embryonic fibroblasts, MEFs) show impaired gene regulation, coincident with imbalanced histone modification and CpG methylation at promoters of affected genes. TDG associates with the promoters of such genes both in fibroblasts and in embryonic stem cells (ESCs), but epigenetic aberrations only appear upon cell lineage commitment. We show that TDG contributes to the maintenance of active and bivalent chromatin throughout cell differentiation, facilitating a proper assembly of chromatin-modifying complexes and initiating base excision repair to counter aberrant de novo methylation. We thus conclude that TDG-dependent DNA repair has evolved to provide epigenetic stability in lineage committed cells.

DNA mismatch recognition and binding in human cells has been thought to be mediated by the hMSH2 protein. Here it is shown that the mismatch-binding factor consists of two distinct proteins, the 100-kilodalton hMSH2 and a 160-kilodalton polypeptide, GTBP (for G/T binding protein). Sequence analysis identified GTBP as a new member of the MutS homolog family. Both proteins are required for mismatch-specific binding, a result consistent with the finding that tumor-derived cell lines devoid of either protein are also devoid of mismatch-binding activity.

Proliferating cell nuclear antigen (PCNA) has been implicated in eukaryotic postreplicative mismatch correction, but the nature of its interaction with the repair machinery remained enigmatic. We now show that PCNA binds to the human mismatch binding factors hMutS␣ and hMutS␤ via their hMSH6 and hMSH3 subunits, respectively. The N-terminal domains of both proteins contain the highly conserved PCNA-binding motif Qxx[LI]xx [FF]. A variant of hMutS␣, lacking this motif because of deletion of 77 N-terminal residues of the hMSH6 subunit, no longer was able to interact with PCNA in vitro and failed to restore mismatch repair in hMSH6-deficient cells. Colocalization of PCNA and hMSH6 or hMSH3 to replication foci implies an intimate link between replication and mismatch correction. We postulate that PCNA plays a role in repair initiation by guiding the mismatch repair proteins to free termini in the newly replicated DNA strands. Mismatches introduced into DNA during replication are addressed by the postreplicative mismatch repair (MMR) system. In Escherichia coli, binding of the mismatch by the MutS protein, which is able to recognize both basebase mismatches (Jiricny et al. 1988;Su et al. 1988) and insertion-deletion loops (IDLs) containing up to four extrahelical nucleotides (Parker and Marinus 1992), triggers an ATP-driven assembly of the MMR repairosome. This contains, in addition to the homodimeric MutS protein, also the strand-discrimination endonuclease MutH and the MutL homodimer, thought to play a bridging role between MutS and MutH. The process also requires the DNA helicase UvrD, single-strand DNA-binding protein Ssb, one of several exonucleases, DNA polymerase III holoenzyme, and DNA ligase. The Dam methylase, which modifies GATC sites in E. coli DNA, plays a key accessory role in the MMR process. Because this enzyme lags behind the replication fork by ∼2 min (Barras and Marinus 1989), the newly synthesized strand remains unmethylated during this time and therefore can be distinguished from the methylated, template DNA. This property is used by the MutH endonuclease, which initiates the repair process by incising the newly synthesized, unmethylated strand of the DNA heteroduplex 5Ј from an unmethylated GATC sequence. The MutL protein then loads the helicase-exonuclease complex at this site (Dao and Modrich 1998), and the error-containing strand is degraded until the mispair is eliminated. The repair tract, stabilized by the Ssb protein, is filled in by DNA polymerase III, and ligation of the remaining nick and modification of the hemimethylated DNA by Dam methylase completes the repair process (for review, see Modrich 1989Modrich , 1991Modrich and Lahue 1996).The MMR process is highly conserved throughout evolution. In eukaryotes, the mismatch recognition function is fulfilled by two heterodimeric factors composed of MutS homologs MSH2 and MSH6 (MutS␣) or MSH2

We tested the ability of recombinant hMutS␣ (hMSH2͞hMSH6) and hMutS␤ (hMSH2͞hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base͞base mispairs and insertion-deletion loops was restored by hMutS␣, only the latter substrates were addressed in extracts supplemented with hMutS␤. hMutS␣ was also able to complement a defect in the repair of base͞base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutS␣ and MutS␤. As a rule, MSH2 is primarily complexed with MSH6. MutS␣ is thus relatively abundant in mammalian cell extracts, whereas MutS␤ levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutS␤. This leads to degradation of the partnerless MSH6 and depletion of MutS␣. CHO R and HL60R cells therefore lack correction of base͞base mispairs, whereas loop repair is maintained by MutS␤. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al.

Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of baseexcision repair enzymes.