Lack of hydroxyurea-associated mutagenesis in pediatric sickle cell disease patients.

Hydroxyurea is approved for treating children and adults with sickle cell anemia (SCA). Despite its proven efficacy, concerns remain about its mutagenic and carcinogenic potential that hamper its widespread use. Cell culture- and animal-based investigations indicate that hydroxyurea's genotoxic effects are due to indirect clastogenicity in select cell types when high dose and time thresholds are exceeded (reviewed by Ware & Dertinger, 2021). The current study extends these preclinical observations to pediatric patients receiving hydroxyurea for treatment of SCA. First, proof-of-principle experiments with testicular cancer patients exposed to a cisplatin-based regimen validated the ability of flow cytometric blood-based micronucleated reticulocyte (MN-RET) and PIG-A mutant reticulocyte (MUT RET) assays to detect clastogenicity and gene mutations, respectively. Second, these biomarkers were measured in a cross-sectional study with 26 SCA patients receiving hydroxyurea and 13 SCA patients without exposure. Finally, a prospective study was conducted with 10 SCA patients using pretreatment blood samples and after 6 or 12 months of therapy. Cancer patients exposed to cisplatin exhibited increased MN-RET within days of exposure, while the MUT RET endpoint required more time to reach maximal levels. In SCA patients, hydroxyurea induced MN-RET in both the cross-sectional and prospective studies. However, no evidence of PIG-A gene mutation was found in hydroxyurea-treated children, despite the fact that the two assays use the same rapidly-dividing, highly-exposed cell type. Collectively, these results reinforce the complementary nature of MN-RET and MUT RET biomarkers, and indicate that hydroxyurea can be clastogenic but was not mutagenic in young patients with SCA.

Collaborating With Parks and Recreation Departments to Address Youth Health.

Adolescence is an important time for addressing health, including mental health, obesity, substance use, and food insecurity. As the myriad health and wellness needs of community members expands, it becomes increasingly difficult for one profession alone to address these needs. The park and recreation field was founded to address the health and wellness needs of people, and much of their programming is focused on youth. Thus, municipal park and recreation departments are becoming increasingly involved in collaborative partnerships with other health-serving agencies within their community. This study explored park and recreation directors' experiences in collaborating with health agencies to better address health and wellness factors that affect the youth in their communities. This phenomenological qualitative study employed in-depth interviews with park and recreation directors and used thematic analysis. Nine interviews were conducted from four of the six New England states. The most common health concern seen by the directors was mental health, primarily behavioral health challenges. Parks and recreation collaboration with public health organizations ranged from none to close collaborations; directors regularly spoke about wanting to strengthen their collaborations. Public health and park and recreation staff have similar challenges with staffing and resources; additionally, they have complimentary skills with regard to planning and implementing health-related programs. For that reason, creating purposeful collaborative partnerships between public health agencies and park and recreation departments, focused on measurable outcomes, may increase benefits to the health of youth in a community.

Bruce Nathan Ames - Paradigm shifts inside the cancer research revolution.

Dr. Bruce Ames turned 92 on December 16, 2020. He considers his most recent work linking adequate consumption of 30 known vitamins and minerals with successful aging to be his most important contribution. With the passage of time, it is not uncommon for the accomplishments of a well-known scientist to undergo a parsimonious reductionism in the public mind - Pasteur's vaccine, Mendel's peas, Pavlov's dogs, Ames' test. Those of us in the research generation subsequent to Dr. Ames' are undoubtedly affected by our own unconscious tendencies toward accepting the outstanding achievements of the past as commonplace. In doing so, seminal advances made by earlier investigators are often inadvertently subsumed into common knowledge. But having followed Ames' work since the mid-1970s, we are cognizant that the eponymous Ames Test is but a single chapter in a long and rich narrative. That narrative begins with Ames' classic studies on the histidine operon of Salmonella, for which he was elected to the National Academy of Sciences. A summary of the historical progression of the understanding of chemical carcinogenesis to which Ames and his colleagues contributed is provided. Any summary of a topic as expansive and complex as the ongoing unraveling of the mechanisms underlying chemical carcinogenesis will only touch upon some of the major conceptual advances to which Ames and his colleagues contributed. We hope that scientists of all ages familiar with Ames only through the eponymous Ames Test will further investigate the historical progression of the conceptualization of cancer caused by chemical exposure. As the field of chemical carcinogenesis gradually moves away from primary reliance on animal testing to alternative protocols under the rubric of New Approach Methodologies (NAM) an understanding of where we have been might help to guide where we should go.

A review of mammalian in vivo genotoxicity of hexavalent chromium: implications for oral carcinogenicity risk assessment.

Assessment of genotoxicity is a critical component of mode of action (MOA) analysis and carcinogen risk assessment due to its influence on quantitative risk extrapolation approaches. To date, clear guidance and expert consensus on the determination of a mutagenic MOA remains elusive, resulting in different estimates of carcinogenic risk for the same chemical among different stakeholders. Oral toxicity criteria for hexavalent chromium [Cr(VI)], for example, differ by orders of magnitude due largely to the interpretation of in vivo genotoxicity data. Herein, we review in vivo genotoxicity studies for Cr(VI) to inform the MOA for Cr(VI)-induced tumors observed in a two-year cancer bioassay in mice and rats exposed via drinking water. Overall, genotoxicity results in carcinogenic target tissues (viz., oral cavity and duodenum) are negative. Results in the intestine are consistent with imaging data indicating little to no chromium present in the crypt compartment following oral exposure. Positive genotoxicity results in nontarget tissues have been reported at high doses mostly following nonphysiological routes of exposure. Given the negative genotoxicity results in carcinogenic target organs from oral exposure to Cr(VI), there is scientific justification to support the use of nonlinear low-dose extrapolation methods in the derivation of oral toxicity criteria for Cr(VI). These results highlight important differences between genotoxicity testing for hazard identification purposes and quantitative risk assessment.

Assessment of systemic genetic damage in pediatric inflammatory bowel disease.

The etiology of distal site cancers in inflammatory bowel disease (IBD) is not well understood and requires further study. We investigated whether pediatric IBD patients' blood cells exhibit elevated levels of genomic damage by measuring the frequency of mutant phenotype (CD59-/CD55-) reticulocytes (MUT RET) as a reporter of PIG-A mutation, and the frequency of micronucleated reticulocytes (MN-RET) as an indicator of chromosomal damage. IBD patients (n = 18 new-onset disease, 46 established disease) were compared to age-matched controls (constipation or irritable bowel syndrome patients from the same clinic, n = 30) and young healthy adults age 19-24 (n = 25). IBD patients showed no indication of elevated MUT RET relative to controls (mean ± SD = 3.1 ± 2.3 × 10-6 vs. 3.6 ± 5.6 x 10-6 , respectively). In contrast, 59 IBD patients where %MN-RET measurements were obtained, 10 exceeded the upper bound 90% tolerance interval derived from control subjects (i.e., 0.42%). Furthermore, each of the 10 IBD patients with elevated MN-RET had established disease (10/42), none were new-onset (0/17) (p = .049). Interestingly, each of the subjects with increased chromosomal damage was receiving anti-TNF based monotherapy at the time blood was collected (10/10, 100%), whereas this therapy was less common (20/32, 63%) among patients that exhibited ≤0.42% MN-RET (p = .040). The results clearly indicate the need for further work to understand whether the results presented herein are reproducible and if so, to elucidate the causative factor(s) responsible for elevated MN-RET frequencies in some IBD patients.

Human blood PIG-A mutation and micronucleated reticulocyte flow cytometric assays: Method optimization and evaluation of intra- and inter-subject variation.

We previously described flow cytometry-based methods for scoring the incidence of micronucleated reticulocytes (MN-RET) and PIG-A mutant phenotype reticulocytes (MUT RET) in rodent and human blood samples. The current report describes important methodological improvements for human blood analyses, including immunomagnetic enrichment of CD71-positive reticulocytes prior to MN-RET scoring, and procedures for storing frozen blood for later PIG-A analysis. Technical replicate variability in MN-RET and MUT RET frequencies based on blood specimens from 14 subjects, intra-subject variability based on serial blood draws from 6 subjects, and inter-subject variation based on up to 344 subjects age 0 to 73 years were quantified. Inter-subject variation explained most of the variability observed for both endpoints (≥77%), with much lower intra-subject and technical replicate variability. The relatively large degree of inter-subject variation is apparent from mean and standard deviation values for MN-RET (0.15 ± 0.10%) and MUT RET (4.7 ± 5.0 per million, after omission of two extreme outliers). The influences of age and sex on inter-subject variation were investigated, and neither factor affected MN-RET whereas both influenced MUT RET frequency. The lowest MUT RET values were observed for subjects <11 years old, and males had moderately higher frequencies than females. These results indicate that MN-RET and MUT RET are automation-compatible biomarkers of genotoxicity that bridge species of toxicological interest to include human populations. These data will be useful for appropriately designing future human studies that include these biomarkers of genotoxicity, and highlight the need for additional work aimed at identifying the sources of inter-individual variability reported herein.

3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals.

Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.

In vivo pig-a and micronucleus study of the prototypical aneugen vinblastine sulfate.

The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days -4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen. 59:30-37, 2018. © 2017 Wiley Periodicals, Inc.

A novel Ehrlichia species in blood and Ixodes ornithorhynchi ticks from platypuses (Ornithorhynchus anatinus) in Queensland and Tasmania, Australia.

Worldwide, Ehrlichia spp. are emerging infectious organisms of domestic animals and people, however, most Ehrlichia spp. naturally infect wildlife reservoirs causing mainly asymptomatic infections. Australian ecosystems have been under-explored for these potentially pathogenic organisms, and recent studies have identified a range of novel Ehrlichia, and their sister genera, Anaplasma and 'Candidatus Neoehrlichia' species, from native Australian ticks. We used bacterial 16S rRNA (16S) next-generation sequencing and genus-specific PCR to profile the bacterial communities in platypus (Ornithorhynchus anatinus) blood samples and platypus ticks (Ixodes ornithorhynchi), and identified a high prevalence of Ehrlichia sequences. We also observed Ehrlichia-like intra-neutrophilic inclusions (morulae) in PCR-positive stained platypus blood films that were consistent in morphology with other Ehrlichia spp. Bayesian phylogenetic analysis of 16S (1343 bp), gltA (1004 bp), and groEL (1074 bp) gene sequences group the platypus Ehrlichia with 'Candidatus Ehrlichia khabarensis' from far-eastern Russia, and demonstrate that the platypus Ehrlichia is clearly distinct from all other Ehrlichia spp. Enough genetic divergence exists to delineate this platypus Ehrlichia as a separate species that we propose to designate 'Candidatus Ehrlichia ornithorhynchi'. There is no evidence that 'Candidatus Ehrlichia ornithorhynchi' causes disease in wild platypuses, however, the organism does seem to be widespread in Australia, being found in both Queensland and Tasmania. 'Candidatus Ehrlichia ornithorhynchi' is the second native Australian Ehrlichia described and adds to the rapidly growing diversity of recently described native Australian tick-borne bacteria.

Enabling spontaneous analogy through heuristic change.

Despite analogy playing a central role in theories of problem solving, learning and education, demonstrations of spontaneous analogical transfer are rare. Here, we present a theory of heuristic change for spontaneous analogical transfer, tested in four experiments that manipulated the experience of failure to solve a source problem prior to attempting a target problem. In Experiment 1, participants solved more source problems that contained an additional financial constraint designed to signal the inappropriateness of moves that maximized progress towards the goal. This constraint also led to higher rates of spontaneous analogical transfer to a superficially similar problem. Experiments 2 and 3 showed that the effects of this constraint extend to superficially and structurally different analogs. Experiment 4 generalized the finding to a non-analogous target problem that also benefitted from inhibiting maximizing moves. The results indicate that spontaneous transfer can arise through experience during the solution of a source problem that alters the heuristic chosen for solving both analogical and non-analogical target problems.

A NEED FOR DYNAMIC HEMATOLOGY AND SERUM BIOCHEMISTRY REFERENCE TOOLS: NOVEL USE OF SINE WAVE FUNCTIONS TO PRODUCE SEASONALLY VARYING REFERENCE CURVES IN PLATYPUSES (ORNITHORHYNCHUS ANATINUS).

Seasonal changes in hematology and serum biochemistry results, described by separate reference intervals for different seasons, have been reported in many animals. We developed a novel method to investigate seasonal variation in values and a reference tool (the reference curve) based on sine wave functions that, for suitable variables, represents data more appropriately than a fixed reference interval. We applied these techniques to values observed in blood samples from 126 adult wild platypuses ( Ornithorhynchus anatinus ; 58 females and 68 males). Samples were collected under isoflurane anesthesia from animals captured in the Inglis Catchment in northwest Tasmania. In general, packed cell volume (PCV), red cell count (RCC), and hemoglobin (Hb) values appeared to be lower than those in two studies that previously reported platypus hematology reference intervals. This likely resulted from reduced stress-related splenic contraction or isoflurane-associated splenic sequestration of red blood cells in our study. Reference curves were described for five variables (PCV, RCC, Hb, albumin, and magnesium). We found evidence that this seasonal variation may result from metabolic changes associated with seasonal variations in environmental temperature. These observations suggest that it is important for researchers reporting platypus hematology and serum biochemistry to look for seasonal changes in their data to ensure it is appropriately interpreted.

INVESTIGATION INTO INDIVIDUAL HEALTH AND EXPOSURE TO INFECTIOUS AGENTS OF PLATYPUSES (ORNITHORHYNCHUS ANATINUS) IN TWO RIVER CATCHMENTS IN NORTHWEST TASMANIA.

Changes in the health of individuals within wildlife populations can be a cause or effect of population declines in wildlife species. Aspects of individual platypus ( Ornithorhynchus anatinus ) health have been reported. However, holistic studies investigating potential synergistic effects of both pathogens and environmental factors are needed to expand understanding of platypus individual health. We collected baseline data on the health of platypuses in two Tasmanian river catchments (including evidence of the potentially fatal fungal disease mucormycosis) and on individual, demographic, and geographic patterns associated with health data results. We examined 130 wild platypuses from the Inglis River Catchment and 24 platypuses from the Seabrook Creek Catchment in northwest Tasmania between 29 August 2011 and 31 August 2013. More than 90% of captured platypuses were infected with ticks, Theileria spp., and trypanosomes. Evidence of exposure to other infections, including Salmonella spp., Leptospira spp., and intestinal parasites, was low (<10%). Three platypuses had single fungal granulomas in the webbing of a forefoot, but no evidence of mucormycosis was found in any of the study animals. Possible subclinical hepatopathies or cholangiohepatopathies were found in six platypuses. Exposure to infectious agents did not cluster geographically, demographically, or in individuals, and there was minimal evidence of morbidity resulting from infection. This study has provided important baseline data for monitoring the effects of threatening processes, including mucormycosis, on the health of infected populations.

Dermatophilus congolensis Infection in Platypus ( Ornithorhynchus anatinus ), Tasmania, Australia, 2015.

We report disease due to Dermatophilus congolensis infection in three of 13 (23%) platypuses ( Ornithorhynchus anatinus ) from a catchment in Tasmania, Australia. This pathogen has not previously been reported in platypuses. Two of the three infected platypuses had extensive scab formations, but no substantial hair loss was apparent.

Mouse Pig-a and micronucleus assays respond to N-ethyl-N-nitrosourea, benzo[a]pyrene, and ethyl carbamate, but not pyrene or methyl carbamate.

This laboratory previously described a method for scoring the incidence of peripheral blood Pig-a mutant phenotype rat erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends the method to mouse blood, using the frequency of CD24-negative reticulocytes (RET(CD24-)) and erythrocytes (RBC(CD24-)) as phenotypic reporters of Pig-a gene mutation. Following assay optimization, reconstruction experiments demonstrated the ability of the methodology to return expected values. Subsequently, the responsiveness of the assay to the genotoxic carcinogens N-ethyl-N-nitrosourea, benzo[a]pyrene, and ethyl carbamate was studied in male CD-1 mice exposed for 3 days to several dose levels via oral gavage. Blood samples were collected on Day 4 for micronucleated reticulocyte analyses, and on Days 15 and 30 for determination of RET(CD24-) and RBC(CD24-) frequencies. The same design was used to study pyrene, with benzo[a]pyrene as a concurrent positive control, and methyl carbamate, with ethyl carbamate as a concurrent positive control. The three genotoxicants produced marked dose-related increases in the frequencies of Pig-a mutant phenotype cells and micronucleated reticulocytes. Ethyl carbamate exposure resulted in moderately higher micronucleated reticulocyte frequencies relative to N-ethyl-N-nitrosourea or benzo[a]pyrene (mean ± SEM = 3.0 ± 0.36, 2.3 ± 0.17, and 2.3 ± 0.49%, respectively, vs. an aggregate vehicle control frequency of 0.18 ± 0.01%). However, it was considerably less effective at inducing Pig-a mutant cells (e.g., Day 15 mean no. RET(CD24-) per 1 million reticulocytes = 7.6 ± 3, 150 ± 9, and 152 ± 43 × 10(-6), respectively, vs. an aggregate vehicle control frequency of 0.6 ± 0.13 × 10(-6)). Pyrene and methyl carbamate, tested to maximum tolerated dose or limit dose levels, had no effect on mutant cell or micronucleated reticulocyte frequencies. Collectively, these results demonstrate the utility of the cross-species Pig-a and micronucleated reticulocyte assays, and add further support to the value of studying both endpoints in order to cover two distinct genotoxic modes of action.

First Molecular Characterization of Theileria ornithorhynchi Mackerras, 1959: yet Another Challenge to the Systematics of the Piroplasms.

Piroplasms, tick-transmitted Apicomplexa of the genera Theileria, Babesia and Cytauxzoon, are blood-borne parasites of clinical and veterinary importance. The order Piroplasmida shows a puzzling systematics characterized by multiple clades, soft polytomies and paraphyletic/polyphyletic genera. In the present study, screening of platypuses (Ornithorhynchus anatinus), was performed to infer the parasite molecular phylogeny. DNA was extracted from blood, ectoparasites and tick eggs and the 18S rRNA- hsp70-genes were used for the phylogenetic reconstructions. Microscopic analyses detected pleomorphic intra-erythrocytic organisms and tetrads consistent with previous descriptions of Theileria ornithorhynchi Mackerras, 1959, but observation of possible schizonts could not be confirmed. DNA sequences obtained from blood and ticks allowed resolving the systematics of the first piroplasm infecting a monotreme host. Molecularly, T. ornithorhynchi formed a novel monophyletic group, basal to most known piroplasms' clades. The ancestral position of this clade, isolated from an ancient lineage of mammalian host appears particularly fascinating. The present paper discusses the inadequacies of the current molecular systematics for the Piroplasmida and the consequences of incomplete sampling, morphology-based classification and ambiguous microscopic identifications. Likely when the current sampling bias is rectified and more sequence data is made available, the phylogenetic position of T. ornithorhynchi will be further contextualized without ambiguity.

IWGT report on quantitative approaches to genotoxicity risk assessment I. Methods and metrics for defining exposure-response relationships and points of departure (PoDs).

This report summarizes the discussion, conclusions, and points of consensus of the IWGT Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (QWG) based on a meeting in Foz do Iguaçu, Brazil October 31-November 2, 2013. Topics addressed included (1) the need for quantitative dose-response analysis, (2) methods to analyze exposure-response relationships & derive point of departure (PoD) metrics, (3) points of departure (PoD) and mechanistic threshold considerations, (4) approaches to define exposure-related risks, (5) empirical relationships between genetic damage (mutation) and cancer, and (6) extrapolations across test systems and species. This report discusses the first three of these topics and a companion report discusses the latter three. The working group critically examined methods for determining point of departure metrics (PoDs) that could be used to estimate low-dose risk of genetic damage and from which extrapolation to acceptable exposure levels could be made using appropriate mode of action information and uncertainty factors. These included benchmark doses (BMDs) derived from fitting families of exponential models, the No Observed Genotoxic Effect Level (NOGEL), and "threshold" or breakpoint dose (BPD) levels derived from bilinear models when mechanistic data supported this approach. The QWG recognizes that scientific evidence suggests that thresholds below which genotoxic effects do not occur likely exist for both DNA-reactive and DNA-nonreactive substances, but notes that small increments of the spontaneous level cannot be unequivocally excluded either by experimental measurement or by mathematical modeling. Therefore, rather than debating the theoretical possibility of such low-dose effects, emphasis should be placed on determination of PoDs from which acceptable exposure levels can be determined by extrapolation using available mechanistic information and appropriate uncertainty factors. This approach places the focus on minimization of the genotoxic risk, which protects against the risk of the development of diseases resulting from the genetic damage. Based on analysis of the strengths and weaknesses of each method, the QWG concluded that the order of preference of PoD metrics is the statistical lower bound on the BMD > the NOGEL > a statistical lower bound on the BPD. A companion report discusses the use of these metrics in genotoxicity risk assessment, including scaling and uncertainty factors to be considered when extrapolating below the PoD and/or across test systems and to the human.

IWGT report on quantitative approaches to genotoxicity risk assessment II. Use of point-of-departure (PoD) metrics in defining acceptable exposure limits and assessing human risk.

This is the second of two reports from the International Workshops on Genotoxicity Testing (IWGT) Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (the QWG). The first report summarized the discussions and recommendations of the QWG related to the need for quantitative dose-response analysis of genetic toxicology data, the existence and appropriate evaluation of threshold responses, and methods to analyze exposure-response relationships and derive points of departure (PoDs) from which acceptable exposure levels could be determined. This report summarizes the QWG discussions and recommendations regarding appropriate approaches to evaluate exposure-related risks of genotoxic damage, including extrapolation below identified PoDs and across test systems and species. Recommendations include the selection of appropriate genetic endpoints and target tissues, uncertainty factors and extrapolation methods to be considered, the importance and use of information on mode of action, toxicokinetics, metabolism, and exposure biomarkers when using quantitative exposure-response data to determine acceptable exposure levels in human populations or to assess the risk associated with known or anticipated exposures. The empirical relationship between genetic damage (mutation and chromosomal aberration) and cancer in animal models was also examined. It was concluded that there is a general correlation between cancer induction and mutagenic and/or clastogenic damage for agents thought to act via a genotoxic mechanism, but that the correlation is limited due to an inadequate number of cases in which mutation and cancer can be compared at a sufficient number of doses in the same target tissues of the same species and strain exposed under directly comparable routes and experimental protocols.

Effects of cluster location and cluster distribution on performance on the traveling salesman problem.

Research on human performance in solving traveling salesman problems typically uses point sets as stimuli, and most models have proposed a processing stage at which stimulus dots are clustered. However, few empirical studies have investigated the effects of clustering on performance. In one recent study, researchers compared the effects of clustered, random, and regular stimuli, and concluded that clustering facilitates performance (Dry, Preiss, & Wagemans, 2012). Another study suggested that these results may have been influenced by the location rather than the degree of clustering (MacGregor, 2013). Two experiments are reported that mark an attempt to disentangle these factors. The first experiment tested several combinations of degree of clustering and cluster location, and revealed mixed evidence that clustering influences performance. In a second experiment, both factors were varied independently, showing that they interact. The results are discussed in terms of the importance of clustering effects, in particular, and perceptual factors, in general, during performance of the traveling salesman problem.

Rat Pig-a mutation assay responds to the genotoxic carcinogen ethyl carbamate but not the non-genotoxic carcinogen methyl carbamate.

Determination of the mode of action of carcinogenic agents is an important factor in risk assessment and regulatory practice. To assess the ability of the erythrocyte-based Pig-a mutation assay to discriminate between genotoxic and non-genotoxic modes of action, the mutagenic response of Sprague Dawley rats exposed to methyl carbamate (MC) or ethyl carbamate (EC) was investigated. EC, a potent carcinogen, is believed to induce DNA damage through the formation of a DNA-reactive epoxide group, whereas the closely structurally related compound, MC, cannot form this epoxide and its weaker carcinogenic activity is thought to be secondary to inflammation and promotion of cell proliferation. The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to either MC (doses ranging from 125 to 500 mg/kg/day) or EC (250 mg/kg/day). Significant increases in the frequency of mutant reticulocytes were observed from Days 15 through 43, with a peak mean frequency of 19.9×10(-6) on Day 29 (i.e. 24.9-fold increase relative to mean vehicle control across all four sampling times). As expected, mutant erythrocyte responses lagged behind mutant reticulocyte responses, with a maximal mean frequency of 8.2×10(-6) on Day 43 (i.e. 16.4-fold increase). No mutagenic effects were observed with MC. A second indicator of in vivo genotoxicity, peripheral blood micronucleated reticulocytes, was also studied. This endpoint was responsive to EC (3.3-fold mean increase), but not to MC. These results support the hypothesis that genotoxicity contributes to the carcinogenicity of EC but not of MC, and illustrates the value of the Pig-a assay for discriminating between genotoxic and non-genotoxic modes of action.

Comparison of male versus female responses in the Pig-a mutation assay.

Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, 7- or 14-week old male and female Sprague Dawley rats were exposed to N-ethyl-N-nitrosourea (ENU). In the study with the 7-week old rats, exposure was to 0, 1, 5 or 25mg ENU/kg/day for three consecutive days (study Days 1-3). Pig-a mutant phenotype reticulocyte (RET(CD59-)) and mutant phenotype erythrocyte (RBC(CD59-)) frequencies were determined on study Days -4, 15, 29 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). Additionally, blood samples collected on Day 4 were analysed for micronucleated reticulocyte (MN-RET) frequency (In Vivo MicroFlow®). The percentage of reticulocytes (%RET) was markedly higher in the 7-week old males compared to females through Day 15 (2.39-fold higher on Day -4). At 25mg/kg/day, ENU reduced Day 4 RET frequencies in both sexes, and the two highest dose levels resulted in elevated MN-RET frequencies, with no sex or treatment × sex interaction. The two highest dose levels significantly elevated the frequencies of mean RET(CD59-) and RBC(CD59-) in both sexes from Day 15 onward. RET(CD59-) and RBC(CD59-) frequencies were somewhat lower for females compared to males at the highest dose level studied, and differences in RET(CD59-) resulted in a statistically significant interaction effect of treatment × sex. In the study with 14-week old rats, treatment was for 3 days with 0 or 25mg ENU/kg/day. RET frequencies differed to a lesser degree between the sexes, and in this case there was no evidence of a treatment × sex interaction. These results suggest that the slightly higher response in younger males than in the younger females may be related to differences in erythropoiesis function at that age. In conclusion, while some quantitative differences were noted, there were no qualitative differences in how males and females responded to a prototypical mutagen, and support the contention that both sexes are equally acceptable for Pig-a gene mutation studies.