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Heterocyclic aromatic amines (HAAs) are detrimental substances can develop during the high-temperature cooking of protein-rich foods, such as meat. They are potent mutagens and carcinogens linked to an increased risk of various cancers. HAAs have complex structures with nitrogen-containing aromatic rings and are formed through chemical reactions between amino acids, creatin(in)e, and sugars during cooking. The formation of HAAs is influenced by various factors, such as food type, cooking temperature, time, cooking method, and technique. HAAs exert their toxicity through mechanisms like DNA adduct formation, oxidative stress, and inflammation. The research on HAAs is important for public health and food safety, leading to risk assessment and management strategies. It has also led to innovative approaches for reducing HAAs formation during cooking and minimizing related health risks. Understanding HAAs' chemistry and formation is crucial for developing effective ways to prevent their occurrence and protect human health. The current review presents an overview about HAAs, their formation pathways, and the factors influencing their formation. Additionally, it reviews their adverse health effects, occurrence, and the analytical methods used for measuring them.

SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

The changes occurring in viral quasispecies populations during infection have been monitored using diversity indices, nucleotide diversity, and several other indices to summarize the quasispecies structure in a single value. In this study, we present a method to partition quasispecies haplotypes into four fractions according to their fitness: the master haplotype, rare haplotypes at two levels (those present at <0.1%, and those at 0.1–1%), and a fourth fraction that we term emerging haplotypes, present at frequencies >1%, but less than that of the master haplotype. We propose that by determining the changes occurring in the volume of the four quasispecies fitness fractions together with those of the Hill number profile we will be able to visualize and analyze the molecular changes in the composition of a quasispecies with time. To develop this concept, we used three data sets: a technical clone of the complete SARS-CoV-2 spike gene, a subset of data previously used in a study of rare haplotypes, and data from a clinical follow-up study of a patient chronically infected with HEV and treated with ribavirin. The viral response to ribavirin mutagenic treatment was selection of a rich set of synonymous haplotypes. The mutation spectrum was very complex at the nucleotide level, but at the protein (phenotypic/functional) level the pattern differed, showing a highly prevalent master phenotype. We discuss the putative implications of this observation in relation to mutagenic antiviral treatment.

Cancer is driven by genomic mutations in 'cancer driver' genes, which have essential roles in tumor development. These mutations may be caused by exposure to mutagens in the environment or by endogenous DNA-replication errors in tissue stem cells. Recent observations of abundant mutations, including cancer driver mutations, in histologically normal human tissues suggest that mutations alone are not sufficient for tumor development, thus prompting the question of how single mutant cells give rise to neoplasia. In a concept supported by decades-old data from mouse tumor models, non-mutagenic tumor-promoting agents have been posited to activate the proliferation of dormant mutated cells, thus generating actively growing lesions, with the promotion stage as the rate-limiting step in tumor formation. Non-mutagenic promoting agents, either endogenous or environmental, may therefore have a more important role in human cancer etiology than previously thought.

The risk assessment of chemical carcinogens is one major task in toxicology. Even though exposure has been mitigated effectively during the last decades, low levels of carcinogenic substances in food and at the workplace are still present and often not completely avoidable. The distinction between genotoxic and non-genotoxic carcinogens has traditionally been regarded as particularly relevant for risk assessment, with the assumption of the existence of no-effect concentrations (threshold levels) in case of the latter group. In contrast, genotoxic carcinogens, their metabolic precursors and DNA reactive metabolites are considered to represent risk factors at all concentrations since even one or a few DNA lesions may in principle result in mutations and, thus, increase tumour risk. Within the current document, an updated risk evaluation for genotoxic carcinogens is proposed, based on mechanistic knowledge regarding the substance (group) under investigation, and taking into account recent improvements in analytical techniques used to quantify DNA lesions and mutations as well as “omics” approaches. Furthermore, wherever possible and appropriate, special attention is given to the integration of background levels of the same or comparable DNA lesions. Within part A, fundamental considerations highlight the terms hazard and risk with respect to DNA reactivity of genotoxic agents, as compared to non-genotoxic agents. Also, current methodologies used in genetic toxicology as well as in dosimetry of exposure are described. Special focus is given on the elucidation of modes of action (MOA) and on the relation between DNA damage and cancer risk. Part B addresses specific examples of genotoxic carcinogens, including those humans are exposed to exogenously and endogenously, such as formaldehyde, acetaldehyde and the corresponding alcohols as well as some alkylating agents, ethylene oxide, and acrylamide, but also examples resulting from exogenous sources like aflatoxin B1, allyl

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are ubiquitous environmental contaminants formed during incomplete combustion of organic materials. Our prior work showed that transplacental exposure to BaP depletes ovarian follicles and increases prevalence of epithelial ovarian tumors later in life. We used the MutaMouse transgenic rodent model to address the hypothesis that ovarian mutations play a role in tumorigenesis caused by prenatal exposure to BaP. Pregnant MutaMouse females were treated with 0, 10, 20, or 40 mg/(kg day) BaP orally on gestational days 7-16, covering critical windows of ovarian development. Female offspring were euthanized at 10 weeks of age; some ovaries with oviducts were processed for follicle counting; other ovaries/oviducts and bone marrow were processed for determination of lacZ mutant frequency (MF). Mutant plaques were pooled within dose groups and sequenced to determine the mutation spectrum. BaP exposure caused highly significant dose-related decreases in ovarian follicles and increases in ovarian/oviductal and bone marrow mutant frequencies at all doses. Absence of follicles, cell packets, and epithelial tubular structures were observed with 20 and 40 mg/(kg day) BaP. Depletion of ovarian germ cells was inversely associated with ovarian MF. BaP induced primarily G > T and G > C transversions and deletions in ovaries/oviducts and bone marrow cells and produced a mutation signature highly consistent with that of tobacco smoking in human cancers. Overall, our results show that prenatal BaP exposure significantly depletes ovarian germ cells, causes histopathological abnormalities, and increases the burden of ovarian/oviductal mutations, which may be involved in pathogenesis of epithelial ovarian tumors. Environ. Mol. Mutagen. 60:410-420, 2019. © 2018 Her Majesty the Queen in Right of Canada.

We show that trimethoprim (TMP), an antibiotic in current use, displays a strong synergistic effect on mutagenesis in Escherichia coli when paired with the base analog 2-aminopurine (2AP), resulting in a 35-fold increase in mutation frequencies in the rpoB-Rifr system. Combination therapies are often employed both as antibiotic treatments and in cancer chemotherapy. However, mutagenic effects of these combinations are rarely examined. An analysis of the mutational spectra of TMP, 2AP, and their combination indicates that together they trigger a response via an alteration in deoxynucleoside triphosphate (dNTP) ratios that neither compound alone can trigger. A similar, although less strong, response is seen with the frameshift mutagen ICR191 and 2AP. These results underscore the need for testing the effects on mutagenesis of combinations of antibiotics and chemotherapeutics.

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.

Since its initial sales in the 1970s, the herbicide glyphosate attained widespread use in modern agriculture, becoming the most commercially successful and widely used herbicide of all time as of 2016. Despite a primary mechanism that targets a pathway absent from animal cells and regulatory studies showing safety margins orders of magnitude better than many other, more directly toxic herbicides, the safety status of glyphosate has recently been brought into question by a slow accumulation of studies suggesting more subtle health risks, especially when considered in combination with the surfactants it is usually applied with. Current, official views of respected international regulatory and health bodies remain divided on glyphosate's status as a human carcinogen, but the 2015 International Agency for Research on Cancer decision to reclassify the compound as Category 2A (probably carcinogenic to humans) marked a sea change in the scientific community's consensus view. The goal of this review is to consider the state of science regarding glyphosate's potential as a human carcinogen and genotoxin, with particular focus on studies suggesting mechanisms that would go largely undetected in traditional toxicology studies, such as microbiome disruption and endocrine mimicry at very low concentrations.

ObjectivesElectronic cigarettes (e-cig), which are promoted as safe alternatives to tobacco cigarettes or as aides to smoking cessation, are becoming increasingly popular among adult chronic smokers and adolescents experimenting with tobacco products. Despite the known presence of toxicants and carcinogens in e-cig liquid and vapor, the possible carcinogenic effects of e-cig use in humans are unknown.Materials and methodsWe have utilized two validated in vitro model systems to investigate whether e-cig vapor induces mutation in mouse or human cells. We have exposed transgenic mouse fibroblasts in vitro to e-cig vapor extracts prepared from three popular brands, and determined the induction of mutagenesis in a reporter gene, the cII transgene. Furthermore, we have treated the pSP189 plasmid with e-cig vapor extract, transfected human fibroblast cells with the e-cig-treated plasmid, and screened for the induced mutations in the supF gene.Results and conclusionWe observed no statistically significant increases in relative mutant frequency in the cII transgene or supF gene in the e-cig treated mouse or human cells, respectively. Our data indicate that e-cig vapor extracts from the selected brands and at concentrations tested in this study have limited mutagenicity in both mouse and human cells in vitro.

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

The p53 tumor suppressor protein plays a critical role in orchestrating the genomic response to various stress signals by acting as a master transcriptional regulator. Differential gene activity is controlled by transcription factors but also dependent on the underlying chromatin structure, especially on covalent histone modifications. After screening different histone lysine methyltransferases and demethylases, we identified JMJD2B/KDM4B as a p53-inducible gene in response to DNA damage. p53 directly regulates JMJD2B gene expression by binding to a canonical p53-consensus motif in the JMJD2B promoter. JMJD2B induction attenuates the transcription of key p53 transcriptional targets including p21, PIG3 and PUMA, and this modulation is dependent on the catalytic capacity of JMJD2B. Conversely, JMJD2B silencing led to an enhancement of the DNA-damage driven induction of p21 and PIG3. These findings indicate that JMJD2B acts in an auto-regulatory loop by which p53, through JMJD2B activation, is able to influence its own transcriptional program. Functionally, exogenous expression of JMJD2B enhanced subcutaneous tumor growth of colon cancer cells in a p53-dependent manner, and genetic inhibition of JMJD2B impaired tumor growth in vivo. These studies provide new insights into the regulatory effect exerted by JMJD2B on tumor growth through the modulation of p53 target genes.

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

ObjectivesElectronic cigarettes (e-cig), which are promoted as safe alternatives to tobacco cigarettes or as aides to smoking cessation, are becoming increasingly popular among adult chronic smokers and adolescents experimenting with tobacco products. Despite the known presence of toxicants and carcinogens in e-cig liquid and vapor, the possible carcinogenic effects of e-cig use in humans are unknown.Materials and methodsWe have utilized two validated in vitro model systems to investigate whether e-cig vapor induces mutation in mouse or human cells. We have exposed transgenic mouse fibroblasts in vitro to e-cig vapor extracts prepared from three popular brands, and determined the induction of mutagenesis in a reporter gene, the cII transgene. Furthermore, we have treated the pSP189 plasmid with e-cig vapor extract, transfected human fibroblast cells with the e-cig-treated plasmid, and screened for the induced mutations in the supF gene.Results and conclusionWe observed no statistically significant increases in relative mutant frequency in the cII transgene or supF gene in the e-cig treated mouse or human cells, respectively. Our data indicate that e-cig vapor extracts from the selected brands and at concentrations tested in this study have limited mutagenicity in both mouse and human cells in vitro.

The p53 tumor suppressor protein plays a critical role in orchestrating the genomic response to various stress signals by acting as a master transcriptional regulator. Differential gene activity is controlled by transcription factors but also dependent on the underlying chromatin structure, especially on covalent histone modifications. After screening different histone lysine methyltransferases and demethylases, we identified JMJD2B/KDM4B as a p53-inducible gene in response to DNA damage. p53 directly regulates JMJD2B gene expression by binding to a canonical p53-consensus motif in the JMJD2B promoter. JMJD2B induction attenuates the transcription of key p53 transcriptional targets including p21, PIG3 and PUMA, and this modulation is dependent on the catalytic capacity of JMJD2B. Conversely, JMJD2B silencing led to an enhancement of the DNA-damage driven induction of p21 and PIG3. These findings indicate that JMJD2B acts in an auto-regulatory loop by which p53, through JMJD2B activation, is able to influence its own transcriptional program. Functionally, exogenous expression of JMJD2B enhanced subcutaneous tumor growth of colon cancer cells in a p53-dependent manner, and genetic inhibition of JMJD2B impaired tumor growth in vivo. These studies provide new insights into the regulatory effect exerted by JMJD2B on tumor growth through the modulation of p53 target genes.

The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.

I used classical mutagens in Escherichia coli to study synergies with different classes of antibiotics, test models of antibiotic mechanisms of action, and examine the basis of synergy. The strongest synergies were detected with 4-nitroquinoline 1-oxide (4NQO), which oxidizes guanines and ultimately results in double-strand breaks when paired with the bactericidal antibiotics vancomycin (VAN), ciprofloxacin (CPR), trimethoprim (TMP), gentamicin (GEN), but no synergies with the bacteriostatic antibiotics tetracycline (TET), erythromycin (ERY), and chloramphenicol (CHL). Other mutagens tested display synergies with the bactericidal antibiotics to various degrees, also with some of the other mutagens. The results support models showing that bactericidal antibiotics kill bacteria by generating more double-strand breaks than can be repaired. The synergies represent dose effects of not the proximal target damage but rather the ultimate resulting double-strand breaks. I also used pairwise tests to place the mutagens into functional antibacterial categories within a previously defined drug interaction network.

The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.

I used classical mutagens in Escherichia coli to study synergies with different classes of antibiotics, test models of antibiotic mechanisms of action, and examine the basis of synergy. The strongest synergies were detected with 4-nitroquinoline 1-oxide (4NQO), which oxidizes guanines and ultimately results in double-strand breaks when paired with the bactericidal antibiotics vancomycin (VAN), ciprofloxacin (CPR), trimethoprim (TMP), gentamicin (GEN), but no synergies with the bacteriostatic antibiotics tetracycline (TET), erythromycin (ERY), and chloramphenicol (CHL). Other mutagens tested display synergies with the bactericidal antibiotics to various degrees, also with some of the other mutagens. The results support models showing that bactericidal antibiotics kill bacteria by generating more double-strand breaks than can be repaired. The synergies represent dose effects of not the proximal target damage but rather the ultimate resulting double-strand breaks. I also used pairwise tests to place the mutagens into functional antibacterial categories within a previously defined drug interaction network.

The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.

UnlabelledWe tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 × 10(-8)) in the rpoB gene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T→G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools.ImportanceAlthough mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.

Carbon nanotubes vary greatly in physicochemical properties. We compared cytotoxic and genotoxic response to 15 multiwalled carbon nanotubes (MWCNT) with varying physicochemical properties to identify drivers of toxic responses. The studied MWCNT included OECD Working Party on Manufactured Nanomaterials (WPMN) (NM-401, NM-402, and NM-403), materials (NRCWE-026 and MWCNT-XNRI-7), and three sets of surface-modified MWCNT grouped by physical characteristics (thin, thick, and short I-III, respectively). Each Groups I-III included pristine, hydroxylated and carboxylated MWCNT. Group III also included an amino-functionalized MWCNT. The level of surface functionalization of the MWCNT was low. The level and type of elemental impurities of the MWCNT varied by <2% of the weight, with exceptions. Based on dynamic light scattering data, the MWCNT were well-dispersed in stock dispersion of nanopure water with 2% serum, but agglomerated and sedimented during exposure. FE1-Muta(TM) Mouse lung epithelial cells were exposed for 24 hr. The levels of DNA strand breaks (SB) were evaluated using the comet assay, a screening assay suitable for genotoxicity testing of nanomaterials. Exposure to MWCNT (12.5-200 µg/ml) did not induce significant cytotoxicity (viability above 92%). Cell proliferation was reduced in highest doses of some MWCNT after 24 hr, and was associated with generation of reactive oxygen species and high surface area. Increased levels of DNA SB were only observed for Group II consisting of MWCNT with large diameters and high Fe2 O3 and Ni content. Significantly, increased levels of SB were only observed at 200 µg/ml of MWCNT-042. Overall, the MWCNT were not cytotoxic and weakly genotoxic after 24 hr exposure to doses up to 200 µg/ml.

De laatste decennia wordt UV-straling steeds vaker toegepast in de waterzuivering. Bij lage doses voor desinfectie, bij hoge doses in combinatie met waterstofperoxide (geavanceerde oxidatie) voor verwijdering van organische microverontreinigingen. Bij zulke hoge doses kunnen er echter ook mutagene bijproducten gevormd worden. Twee recente studies uit het bedrijfstakonderzoek (BTO) leverden (i) meer kennis over en inzicht in de vorming van bijproducten, en (ii) een analytisch-chemische tool voor het opsporen van deze bijproducten. Dit type studies kan helpen een verantwoorde toepassing van UV-processen bij diverse watertypen en in diverse zuiveringsschema’s te bewerkstelligen.

Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation. Targeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi. Recombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC)., Published in Parasites & Vectors, v7 n577, 10 Dec 2014. Sponsored in part by USDA.

Traumatic brain injury (TBI) is of great concern both to the general population and to the military. There are at least two phases to brain injury: the first is the actual injury, which may result in locally damaged tissue. The second phase is the response of the body to the injury, which may last days or weeks. This second phase includes inflammation, much of which has been shown to be harmful to the injured brain. Indeed, this inflammation may be the major cause of neuronal damage that leads to many of the negative outcomes associated with brain injury. It has been shown that several cytokines and chemokines (which are key pro-inflammatory proteins) are upregulated upon brain injury. We have studied the structural biology and biochemistry of two proteins, vCCI and vMIP-II, each of which has been shown to have powerful anti-inflammatory properties due to inhibition of chemokines (and each having a separate mechanism of action). This work has included mutagenesis, binding studies, and structural studies by NMR. A greater understanding of these anti-inflammatory proteins can allow a molecular understanding of chemokine inhibition. This could eventually lead to strategies for recovery from traumatic brain injury., The original document contains color images.

The oligomerisation of protein subunits is an area of much research interest, in particular the relationship to protein function. In the last decade, the potential to control the interactions involved in order to design constructs with tuneable oligomeric properties in vitro has been pursued. The subject of this thesis is the quaternary structure of members of the peroxiredoxin family, which have been seen to assume an intriguing array of organisations. Human Peroxiredoxin 3 (HsPrx3) and Mycobacterium tuberculosis alkyl hydroperoxide reductase (MtAhpE) catalyse the detoxification of reactive species, preferentially hydrogen peroxide and peroxynitrite respectively, and form an essential part of the antioxidant defence system. As well as their biomedical interest, the ability of these proteins to form organised supramolecular assemblies makes them of interest in protein nanotechnology. The work described focusses on the elucidation of the quaternary structure of both proteins, resolving previous debates about their oligomeric state. The factors influencing oligomerisation were examined through biophysical characterisation in different conditions, using solution techniques including chromatography, light and X-ray scattering, and electron microscopy. The insight gained, along with analysis of the protein-protein interfaces, was used to alter the quaternary structure through site-directed mutagenesis. This resulted in a level of control over the protein s oligomeric state to be achieved, and novel structures with potential applications in nanotechnology to be generated. The activity of the non-native structures was also assessed, to begin to unravel the relationship between peroxiredoxin quaternary structure to enzyme activity. The formation and structure of very high molecular weight complexes of HsPrx3 were explored using electron microscopy., The original document contains color images.

During this research period, molecular modeling and biochemical studies were conducted to assess the roles of different acidic residues in the binding of the Src homology 2-containing protein tyrosine phosphatase (SHP2) to its substrates. A peptide derived from this substrate was shown to be a selective inhibitor of SHP2. SHP1 was used as a control, and the peptide did not act on this enzyme. Molecular modeling revealed key binding determinants at the atomic level present in SHP2 that could not be recapitulated in SHP1. These modeling observations were tested using mutation of the peptide. Indeed, when the peptide was mutated to abolish these interactions, inhibitory capacity was abolished entirely. Systematic mutation of the peptide in silico further demonstrated that the substrate needed to bind both of the positively-charged amino acid sidechains adjacent to the active site to facilitate the highest binding stability. Mutation of any of the acidic amino acids next to the phosphotyrosine on the substrate produced significant defects in binding as predicted by molecular docking. These findings are now being parlayed into study of the peptide-based inhibitor on cells, which is work that will continue into the coming year., The original document contains color images.

We have identified and characterized a biosensor for auxin and constructed the corresponding biosynthetic cassette. The system is shown in figure 1. Three variants have been made so that we can select on ampicillin, chloramphenicol, or kanamycin. The system also contains a co-transcribed GFP marker for quantitation. After full validation of genotypes we will begin characterizing auxin production and the associated readout provided by the biosensor cassette. Mutagenesis of the host and increased auxin production will follow., The original document contains color images.

Using a benchmark Ames mutagenicity data set, we evaluated the performance of molecular fingerprints as descriptors for developing quantitative structure activity relationship (QSAR) models and defining applicability domains with two machine-learning methods: random forest (RF) and variable nearest neighbor (v-NN). The two methods focus on complementary aspects of chemical mutagenicity and use different characteristics of the molecular fingerprints to achieve high levels of prediction accuracies. Thus, while RF flags mutagenic compounds using the presence or absence of small molecular fragments akin to structural alerts, the v-NN method uses molecular structural similarity as measured by fingerprint-based Tanimoto distances between molecules. We showed that the extended connectivity fingerprints could intuitively be used to define and quantify an applicability domain for either method. The importance of using applicability domains in QSAR modeling cannot be understated; compounds that are outside the applicability domain do not have any close representative in the training set, and therefore, we cannot make reliable predictions. Using either approach, we developed highly robust models that rival the performance of a state-of-the-art proprietary software package. Importantly, based on the complementary approach used by the methods, we showed that by combining the model predictions we raised the applicability domain from roughly 80% to 90%. These results indicated that the proposed QSAR protocol constituted a highly robust chemical mutagenicity prediction model., Published in the Journal of Chemical Education and Modeling, v54 p793-800, 4 Feb 2014. Sponsored in part by DTRA.

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is one of the major environmental pollutants that causes mutagenesis and cancer. BaP has been shown to accumulate in phytoplankton and zooplankton. We have studied the localization and aggregation of BaP in Chlorella sp., a microalga that is one of the primary producers in the food chain, using fluorescence confocal microscopy and fluorescence lifetime imaging microscopy with the phasor approach to characterize the location and the aggregation of BaP in the cell. Our results show that BaP accumulates in the lipid bodies of Chlorella sp. and that there is Förster resonance energy transfer between BaP and photosystems of Chlorella sp., indicating the close proximity of the two molecular systems. The lifetime of BaP fluorescence was measured to be 14 ns in N,N-dimethylformamide, an average of 7 ns in Bold's basal medium, and 8 ns in Chlorella cells. Number and brightness analysis suggests that BaP does not aggregate inside Chlorella sp. (average brightness = 5.330), while it aggregates in the supernatant. In Chlorella grown in sediments spiked with BaP, in 12 h the BaP uptake could be visualized using fluorescence microscopy.

In Central America, the traditional temazcales or wood-fired steam baths, commonly used by many Native American populations, are often heated by wood fires with little ventilation, and this use results in high wood smoke exposure. Urinary mutagenicity has been previously employed as a non-invasive biomarker of human exposure to combustion emissions. This study examined the urinary mutagenicity in 19 indigenous Mayan families from the highlands of Guatemala who regularly use temazcales (N = 32), as well as control (unexposed) individuals from the same population (N = 9). Urine samples collected before and after temazcal exposure were enzymatically deconjugated and extracted using solid-phase extraction. The creatinine-adjusted mutagenic potency of urine extracts was assessed using the plate-incorporation version of the Salmonella mutagenicity assay with strain YG1041 in the presence of exogenous metabolic activation. The post-exposure mutagenic potency of urine extracts were, on average, 1.7-fold higher than pre-exposure samples (P < 0.005) and also significantly more mutagenic than the control samples (P < 0.05). Exhaled carbon monoxide (CO) was ~10 times higher following temazcal use (P < 0.0001), and both CO level and time spent in temazcal were positively associated with urinary mutagenic potency (i.e. P < 0.0001 and P = 0.01, respectively). Thus, the wood smoke exposure associated with temazcal use contributes to increased excretion of conjugated mutagenic metabolites. Moreover, urinary mutagenic potency is correlated with other metrics of exposure (i.e. exhaled CO, duration of exposure). Since urinary mutagenicity is a biomarker associated with genetic damage, temazcal use may therefore be expected to contribute to an increased risk of DNA damage and mutation, effects associated with the initiation of cancer.

The heterogeneous reactions of ambient particulate matter (PM)-bound polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs (NPAHs) with NO3/N2O5, OH radicals, and O3 were studied in a laboratory photochemical chamber. Ambient PM2.5 and PM10 samples were collected from Beijing, China, and Riverside, California, and exposed under simulated atmospheric long-range transport conditions for O3 and OH and NO3 radicals. Changes in the masses of 23 PAHs and 20 NPAHs, as well as the direct and indirect-acting mutagenicity of the PM (determined using the Salmonella mutagenicity assay with TA98 strain), were measured prior to and after exposure to NO3/N2O5, OH radicals, and O3. In general, O3 exposure resulted in the highest relative degradation of PM-bound PAHs with more than four rings (benzo[a]pyrene was degraded equally well by O3 and NO3/N2O5). However, NPAHs were most effectively formed during the Beijing PM exposure to NO3/N2O5. In ambient air, 2-nitrofluoranthene (2-NF) is formed from the gas-phase NO3 radical- and OH radical-initiated reactions of fluoranthene, and 2-nitropyrene (2-NP) is formed from the gas-phase OH radical-initiated reaction of pyrene. There was no formation of 2-NF or 2-NP in any of the heterogeneous exposures, suggesting that gas-phase formation of NPAHs did not play an important role during chamber exposures. Exposure of Beijing PM to NO3/N2O5 resulted in an increase in direct-acting mutagenic activity which was associated with the formation of mutagenic NPAHs. No NPAH formation was observed in any of the exposures of the Riverside PM. This was likely due to the accumulation of atmospheric degradation products from gas-phase reactions of volatile species onto the surface of PM collected in Riverside prior to exposure in the chamber, thus decreasing the availability of PAHs for reaction.

The heterogeneous reactions of ambient particulate matter (PM)-bound polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs (NPAHs) with NO3/N2O5, OH radicals, and O3 were studied in a laboratory photochemical chamber. Ambient PM2.5 and PM10 samples were collected from Beijing, China, and Riverside, California, and exposed under simulated atmospheric long-range transport conditions for O3 and OH and NO3 radicals. Changes in the masses of 23 PAHs and 20 NPAHs, as well as the direct and indirect-acting mutagenicity of the PM (determined using the Salmonella mutagenicity assay with TA98 strain), were measured prior to and after exposure to NO3/N2O5, OH radicals, and O3. In general, O3 exposure resulted in the highest relative degradation of PM-bound PAHs with more than four rings (benzo[a]pyrene was degraded equally well by O3 and NO3/N2O5). However, NPAHs were most effectively formed during the Beijing PM exposure to NO3/N2O5. In ambient air, 2-nitrofluoranthene (2-NF) is formed from the gas-phase NO3 radical- and OH radical-initiated reactions of fluoranthene, and 2-nitropyrene (2-NP) is formed from the gas-phase OH radical-initiated reaction of pyrene. There was no formation of 2-NF or 2-NP in any of the heterogeneous exposures, suggesting that gas-phase formation of NPAHs did not play an important role during chamber exposures. Exposure of Beijing PM to NO3/N2O5 resulted in an increase in direct-acting mutagenic activity which was associated with the formation of mutagenic NPAHs. No NPAH formation was observed in any of the exposures of the Riverside PM. This was likely due to the accumulation of atmospheric degradation products from gas-phase reactions of volatile species onto the surface of PM collected in Riverside prior to exposure in the chamber, thus decreasing the availability of PAHs for reaction.

The heterogeneous reactions of benzo[a]pyrene-d12 (BaP-d12), benzo[k]fluoranthene-d12 (BkF-d12), benzo[ghi]perylene-d12 (BghiP-d12), dibenzo[a,i]pyrene-d14 (DaiP-d14), and dibenzo[a,l]pyrene (DalP) with NO2, NO3/N2O5, and OH radicals were investigated at room temperature and atmospheric pressure in an indoor Teflon chamber and novel mono-NO2-DaiP and mono-NO2-DalP products were identified. Quartz fiber filters (QFF) were used as a reaction surface and the filter extracts were analyzed by GC/MS for nitrated-PAHs (NPAHs) and tested in the Salmonella mutagenicity assay, using Salmonella typhimurium strain TA98 (with and without metabolic activation). In parallel to the laboratory experiments, a theoretical study was conducted to rationalize the formation of NPAH isomers based on the thermodynamic stability of OH-PAH intermediates, formed from OH-radical-initiated reactions. NO2 and NO3/N2O5 were effective oxidizing agents in transforming PAHs to NPAHs, with BaP-d12 being the most readily nitrated. Reaction of BaP-d12, BkF-d12, and BghiP-d12 with NO2 and NO3/N2O5 resulted in the formation of more than one mononitro isomer product, while the reaction of DaiP-d14 and DalP resulted in the formation of only one mononitro isomer product. The direct-acting mutagenicity increased the most after NO3/N2O5 exposure, particularly for BkF-d12 in which di-NO2-BkF-d10 isomers were measured. The deuterium isotope effect study suggested that substitution of deuterium for hydrogen lowered both the direct and indirect acting mutagenicity of NPAHs and may result in an underestimation of the mutagencity of the novel NPAHs identified in this study.

In Central America, the traditional temazcales or wood-fired steam baths, commonly used by many Native American populations, are often heated by wood fires with little ventilation, and this use results in high wood smoke exposure. Urinary mutagenicity has been previously employed as a non-invasive biomarker of human exposure to combustion emissions. This study examined the urinary mutagenicity in 19 indigenous Mayan families from the highlands of Guatemala who regularly use temazcales (N = 32), as well as control (unexposed) individuals from the same population (N = 9). Urine samples collected before and after temazcal exposure were enzymatically deconjugated and extracted using solid-phase extraction. The creatinine-adjusted mutagenic potency of urine extracts was assessed using the plate-incorporation version of the Salmonella mutagenicity assay with strain YG1041 in the presence of exogenous metabolic activation. The post-exposure mutagenic potency of urine extracts were, on average, 1.7-fold higher than pre-exposure samples (P < 0.005) and also significantly more mutagenic than the control samples (P < 0.05). Exhaled carbon monoxide (CO) was ~10 times higher following temazcal use (P < 0.0001), and both CO level and time spent in temazcal were positively associated with urinary mutagenic potency (i.e. P < 0.0001 and P = 0.01, respectively). Thus, the wood smoke exposure associated with temazcal use contributes to increased excretion of conjugated mutagenic metabolites. Moreover, urinary mutagenic potency is correlated with other metrics of exposure (i.e. exhaled CO, duration of exposure). Since urinary mutagenicity is a biomarker associated with genetic damage, temazcal use may therefore be expected to contribute to an increased risk of DNA damage and mutation, effects associated with the initiation of cancer.

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is one of the major environmental pollutants that causes mutagenesis and cancer. BaP has been shown to accumulate in phytoplankton and zooplankton. We have studied the localization and aggregation of BaP in Chlorella sp., a microalga that is one of the primary producers in the food chain, using fluorescence confocal microscopy and fluorescence lifetime imaging microscopy with the phasor approach to characterize the location and the aggregation of BaP in the cell. Our results show that BaP accumulates in the lipid bodies of Chlorella sp. and that there is Förster resonance energy transfer between BaP and photosystems of Chlorella sp., indicating the close proximity of the two molecular systems. The lifetime of BaP fluorescence was measured to be 14 ns in N,N-dimethylformamide, an average of 7 ns in Bold's basal medium, and 8 ns in Chlorella cells. Number and brightness analysis suggests that BaP does not aggregate inside Chlorella sp. (average brightness = 5.330), while it aggregates in the supernatant. In Chlorella grown in sediments spiked with BaP, in 12 h the BaP uptake could be visualized using fluorescence microscopy.

The purpose of this technical report is two-fold. First, this technical report presents the results of a sensory irritation assay conducted in male Swiss-Webster mice exposed to different concentrations of hydroprocessed esters and fatty acids (HEFA) bio-based jet fuels, HEFA-Camelina (HEFA-C) and HEFA-Tallow (HEFA-T). The mice were exposed via a modified nose-only plethysmograph tube to each fuel, administered as an aerosol/vapor combination. For HEFA-C, the predicted RD50 (50 percent reduction in respiratory rate) was greater than 11,755 mg/m3. For HEFA-T, the predicted RD50 was greater than 13,306 mg/m3. This technical report also summarizes toxicity data for HEFA fuels, including HEFA-C, HEFA-T and a HEFA fuel with a feedstock of mixed animal fats and oils (HEFA-F). HEFA toxicity data are then compared with toxicity data from synthetic paraffinic kerosene (SPK) alternative jet fuel and petroleum derived JP-8. The toxicity data are used to develop a comparative occupational exposure limit (OEL) for HEFA fuels that concurs with the current JP-8 OEL (200 mg/m3)., Prepared in collaboration with the Henry M. Jackson Foundation for the Advancement of Military Medicine, Wright-Patterson AFB, OH, the Naval Medical Research Unit-Dayton, Wright-Patterson AFB, OH, and AFRL/RQTF, Wright-Patterson AFB, OH.

Variants of human paraoxonase 1 (PON1) are being developed as catalytic bioscavengers for the organophosphorus chemical warfare agents (OP). It is preferable that the new PON1 variants have broad spectrum hydrolase activities to hydrolyze both G- and V-class OPs. H115W PON1 has shown improvements over wild type PON1 in its capacity to hydrolyze some OP compounds. We improved upon these activities either by substituting a tryptophan (F347W) near the putative active site residues for enhanced substrate binding or by reducing a bulky group (Y71A) at the periphery of the putative enzyme active site. When compared to H115W alone, we found that H115W/Y71A and H115W/F347W maintained VX catalytic efficiency but showed mixed results for the capacity to hydrolyze paraoxon. Testing our double mutants against racemic sarin, we observed reduced values of KM for H115W/F347W that modestly improved catalytic efficiency over wild type and H115W. Contrary to previous reports, we show that H115W can hydrolyze soman, and the double mutant H115W/Y71A is nearly 4-fold more efficient than H115W for paraoxon hydrolysis. We also observed modest stereoselectivity for hydrolysis of the P(-) stereoisomer of tabun by H115W/F347W. These data demonstrate enhancements made in PON1 for the purpose of developing an improved catalytic bioscavenger to protect cholinesterase against chemical warfare agents., The original document contains color images. Prepared in cooperation with the Department of Chemistry and Biochemistry, University of Delaware, Newark. Published in Chemico-Biological Interactions, v203 p181 185, 2013.

Anti-estrogen drugs such as tamoxifen have become widely used to treat patients with estrogen-receptor positive tumors. Although effective in many cases, significant proportions of estrogen-receptor positive tumors are intrinsically unresponsive to anti-estrogens or have acquired resistance over time. Two proteins that have emerged as key players in this resistance are aptly named Breast Cancer Antiestrogen Resistance 3 (BCAR3) and BCAR1 (also known as p130Cas). Not only can BCAR3 and p130Cas confer anti-estrogen resistance individually, but the two have subsequently been found to directly interact with each other. We have solved the crystal structures of the p130Cas interaction domain of BCAR3, and the complex of the close BCAR3 homolog, NSP3, bound to the C-terminal domain of p130Cas. Unbound breast cancer antiestrogen resistance 3 (BCAR3/NSP2) adopts the Cdc25-homology fold characteristic of Ras GTPase exchange factors, but in a unique closed conformation incapable of enzymatic activity. The structure of the BCAR3 relative, NSP3, in complex with p130Cas further reveals that the closed conformation is instrumental for interaction with a focal adhesion-targeting (FAT)- like region in Cas proteins. This novel enzyme-to-adaptor conversion enables high affinity interactions between BCAR3 (or NSP3) and Cas family proteins to link their signaling networks., The original document contains color images.

Vitetta and colleagues identified and characterized a putative vascular leak peptide (VLP) consensus sequence in recombinant ricin toxin A-chain (RTA) that contributed to dose-limiting human toxicity when RTA was administered intravenously in large quantities during chemotherapy. We disrupted this potentially toxic site within the more stable RTA1-33/44-198 vaccine immunogen and determined the impact of these mutations on protein stability, structure and protective immunogenicity using an experimental intranasal ricin challenge model in BALB/c mice to determine if the mutations were compatible. Single amino acid substitutions at the positions corresponding with RTA D75 (to A, or N) and V76 (to I, or M) had minor effects on the apparent protein melting temperature of RTA1-33/44-198 but all four variants retained greater apparent stability than the parent RTA. Moreover, each VLP( ) variant tested provided protection comparable with that of RTA1-33/44-198 against supralethal intranasal ricin challenge as judged by animal survival and several biomarkers. To understand better how VLP substitutions and mutations near the VLP site impact epitope structure, we introduced a previously described thermal stabilizing disulfide bond (R48C/T77C) along with the D75N or V76I substitutions in RTA1-33/44-198. The D75N mutation was compatible with the adjacent stabilizing R48C/T77C disulfide bond and the Tm was unaffected, whereas the V76I mutation was less compatible with the adjacent disulfide bond involving C77. A crystal structure of the RTA1-33/44-198 R48C/T77C/D75N variant showed that the structural integrity of the immunogen was largely conserved and that a stable immunogen could be produced from E. coli. We conclude that it is feasible to disrupt the VLP site in RTA1-33/44-198 with little or no impact on apparent protein stability or protective efficacy in mice and such variants can be stabilized further by introduction of a disulfide bond., Published in Toxins, v5 p224-248, 29 Jan 2013. The original document contains color images. Sponsored in part by DoD and DTRA. Prepared in collaboration with the United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, Walter Reed Army Institute of Research, Silver Spring, MD, and the Naval Research Laboratory, Washington, D.C. Prepared in cooperation with NOVA Research, Inc., Alexandria, VA.

Bladder cancer is a significant public health problem, worldwide. In the United States, bladder cancer is the fourth most common cancer in men, and its recurrence rate is the highest among all malignancies. Tobacco smoking is the leading risk factor for bladder cancer. The risk of bladder cancer is directly related to the intensity and duration of smoking, while quitting smoking reduces this risk. The increased risk of smokers for developing bladder cancer is attributable to their exposure to aromatic amines, which constitute a family of known bladder carcinogens present in tobacco smoke. The underlying mechanism of action of aromatic amines in the genesis of bladder cancer is not, however, fully delineated. Research has identified a genotoxic mode of action, specifically DNA adduction and mutagenicity, for aromatic amines, which may account for their carcinogenicity. The present review summarizes our current knowledge on the DNA adduction and mutagenicity of aromatic amines in relation to smoking-associated bladder cancer. For illustrative purposes, representative results from published research on aromatic amine-induced DNA adduction and mutagenesis are discussed. The direction of future research on the underlying mechanisms of tobacco smoke-associated bladder carcinogenesis is also outlined. Understanding the molecular mechanisms of bladder carcinogenesis is essential for improving future strategies for prevention, early detection, treatment, and prognosis of this malignancy.

Bladder cancer is a significant public health problem, worldwide. In the United States, bladder cancer is the fourth most common cancer in men, and its recurrence rate is the highest among all malignancies. Tobacco smoking is the leading risk factor for bladder cancer. The risk of bladder cancer is directly related to the intensity and duration of smoking, while quitting smoking reduces this risk. The increased risk of smokers for developing bladder cancer is attributable to their exposure to aromatic amines, which constitute a family of known bladder carcinogens present in tobacco smoke. The underlying mechanism of action of aromatic amines in the genesis of bladder cancer is not, however, fully delineated. Research has identified a genotoxic mode of action, specifically DNA adduction and mutagenicity, for aromatic amines, which may account for their carcinogenicity. The present review summarizes our current knowledge on the DNA adduction and mutagenicity of aromatic amines in relation to smoking-associated bladder cancer. For illustrative purposes, representative results from published research on aromatic amine-induced DNA adduction and mutagenesis are discussed. The direction of future research on the underlying mechanisms of tobacco smoke-associated bladder carcinogenesis is also outlined. Understanding the molecular mechanisms of bladder carcinogenesis is essential for improving future strategies for prevention, early detection, treatment, and prognosis of this malignancy.