Historical perspective on the use of animal bioassays to predict carcinogenicity: evolution in design and recognition of utility.

The animal testing protocols used today to evaluate the carcinogenicity of chemicals are very different from those used in the earlier part of the 20th century. To explore how cancer bioassays have changed over time, we surveyed the literature discussing test design and interpretation from the 1930s to the present. We also analyzed compendia of bioassays published by the US Public Health Service (US PHS) from 1938 to 1978, and evaluated the data to understand the evolution of testing methodology (e.g., animals used, test duration) and the types of chemicals being studied. The cancer bioassay evolved in several stages. At the beginning of the 20th century, animal bioassays were primarily used to re-create known human diseases, whereas in the 1940s to 1960s, animal bioassays were largely used to evaluate the safety of chemicals in foods, drugs, and cosmetics. Beginning in the late 1960s and 1970s, chemicals primarily associated with occupational or environmental exposures were also evaluated. Testing strategies now emphasize a suite of tests including multiple in vitro tests and both short-term and long-term animal tests. The objectives of testing are broader, too, with test goals encompassing information regarding mode of action and other parameters aimed at evaluating potential species differences (e.g., in toxicokinetics) and their relevance for evaluating human risks. It is important to consider this evolution when evaluating the testing methodology and scientific conclusions in earlier eras. As toxicology continues to develop, testing methods will continue to change in concert with increased knowledge and understanding.

Music therapy was noninferior to verbal standard treatment of traumatized refugees in mental health care: Results from a randomized clinical trial.

Background: Many people with refugee backgrounds suffer from trauma-related complex social and psychological problems, and compliance with standard psychological treatment tends to be low. More culturally adaptable treatment options seem to be needed. Objective: We aimed to investigate whether the music therapy method: 'trauma-focused music and imagery' (tr-MI), characterized by a particular focus on arousal and affect regulation, would be equally effective as the standard psychological talk therapies for ameliorating trauma symptoms in Danish refugees. Methods: A pragmatic, noninferiority, parallel, randomized controlled trial with six-month follow-up was carried out at three clinics for refugees in the public mental health services of the Psychiatry (DK). Seventy-four adults diagnosed with posttraumatic stress disorder (PTSD) were allocated to either music therapy sessions (tr-MI, N = 39) or psychological treatment as usual (TAU, N = 35). Western classical music, new age music, and music from the participants' own national culture were used to generate inner imagery, following a phased treatment protocol. Homework entailed listening to music. The primary outcome was the measurement of trauma symptoms by the Harvard Trauma Questionnaire, section IV (HTQ-IV); secondary measures were somatoform and psychoform dissociation (DSS-20), SDQ-20), attachment (RAAS), and well-being (WHO-5). Treatment effects reflected by primary and secondary measures were estimated using linear mixed models. Results: Tr-MI was noninferior to TAU (mean difference at follow-up HTQ-IV: 0.14, CI (-0.10; 0.38), with a - 0.3 noninferiority margin). A high dropout rate of 40% occurred in the TAU group, compared to 5% in the music therapy group. Secondary measures generated small to medium effect sizes in both groups, with significant medium effect sizes for well-being and psychoform dissociation at follow-up in tr-MI. Conclusions: Tr-MI is an innovative form of psychological treatment in refugee mental health services. Trials comparing music therapy to standardized therapy are needed to substantiate the evidence base for tr-MI therapy.

Antecedentes: Muchas personas con antecedente de condición de refugiadas sufren complejos problemas psicológicos y sociales relacionados con el trauma, y el cumplimiento con el tratamiento psicológico estándar tiende a ser bajo. Parecen ser necesarias opciones de tratamiento más culturalmente adaptadas.Objetivo: Apuntamos a investigar si el método de músico-terapia: 'música e imaginería focalizada en el trauma' (tr-MI), caracterizado por un enfoque particular en el nivel de alerta y regulación emocional, podría ser igualmente efectivo como las terapias psicológicas de conversación para aliviar síntomas de trauma en refugiados daneses.Métodos: Se llevó a cabo un ensayo clínico aleatorizado controlado, pragmático, de no inferioridad, paralelo, con seis meses de seguimiento en tres clínicas para refugiados en servicios públicos de salud mental de Psiquiatría en Dinamarca. Setenta y cuatro adultos diagnosticados con Trastorno de estrés postraumático (TEPT) fueron asignados a sesiones de músico-terapia (tr-MI, N = 39) o al tratamiento psicológico de costumbre (TAU, N = 35). Se utilizó música clásica occidental, música new age, y música de la propia cultura nacional de los participantes, para generar imaginería interior, siguiendo un protocolo de tratamiento por fases. La tarea implicaba escuchar música. El resultado principal fue la medición de síntomas de trauma a través del Cuestionario de Trauma de Harvard, sección IV (HTQ-IV); las mediciones secundarias fueron disociación somatomorfa y psicomorfa (DSS-20, SDQ-20), apego (RAAS), y bienestar (WHO-5). Los efectos del tratamiento reflejados por mediciones primarias y secundarias fueron estimados usando modelos lineales mixtos.Resultados: Tr-MI no fue inferior a TAU (diferencia promedio al seguimiento HTQ-IV: 0.14, IC −0.10; 0.38), con un margen de no-inferioridad de −0.3). Una alta tasa de deserción de un 40% ocurrió en el grupo TAU, comparado con un 5% en el grupo de músico-terapia. Las mediciones secundarias generaron tamaños de efecto pequeños a medianos en ambos grupos, con un tamaño del efecto mediano significativo para bienestar y disociación psicomorfa al seguimiento en tr-MI.Conclusiones: Tr-MI es una forma innovadora de tratamiento psicológico en servicios de salud mental para refugiados. Se necesitan ensayos comparando músico-terapia con terapia estandarizada para probar la base de evidencia para la terapia tr-MI.

An evaluation of the US Environmental Protection Agency definition of a risk assessment.

A full presentation of relevant information, including both non-adverse and beneficial effects, of chemicals is important to developing sound and balanced risk assessments. Such considerations are not new. For example, the American Thoracic Society has developed criteria for defining adverse and non-adverse pulmonary effects. Failing to allow risk assessors to even consider non-adverse and beneficial effects will discourage the use of information from developing technologies, such as genomics, and from new understandings of dose-response relationships, as reflected in the hormetic model. Failing to provide such information to risk managers potentially provides a biased perspective on risk.

Effect of BRCA mutations on the length of survival in epithelial ovarian tumors.

PURPOSE: To study the role of BRCA mutations in ovarian cancer survival. PATIENTS AND METHODS: Blood samples and specimens of ovarian tumors (whenever blood samples were not available) at the time of the primary surgery were obtained in the course of a nationwide case-control study of women with ovarian cancer in Israel. The three common BRCA mutations in Israel (185delAG, 5382insC, and 6174delT) were analyzed with a multiplex polymerase chain reaction to amplify the exons containing the three mutations using fluor-labeled primers in a single reaction. Because each mutation is a small insertion or deletion, they can be detected as length polymorphisms. Patients were followed for up to 5 years (range, 20 to 64 months). Statistical analysis was performed using the Kaplan-Meier method and the log-rank test. Stepwise Cox regression analysis was used for determination of independent prognostic factors. RESULTS: This report is based on 896 blood or tumor specimens analyzed for the presence of the BRCA mutations. Of these, 234 women (26.1%) were found to be positive. A significant difference in survival pattern was found between BRCA1/BRCA2 carriers and noncarriers among the women with invasive ovarian cancer (median survival, 53.4 months v. 37.8 months; 3-year survival, 65.8% v. 51.9%, respectively). These differences were independent of age at diagnosis or stage of the disease. CONCLUSION: Our data indicate that the survival of patients with ovarian cancer is affected by BRCA germline mutation, at least in the early years after diagnosis.

Risk evaluation of occupational exposure to methylene dianiline and toluene diamine in polyurethane foam.

Methylene diphenylisocyanate (MDI) and toluene diisocyanate (TDI) are widely used in industry to produce polyurethane foam products. Small amounts of methylenedianiline (MDA) and toluene diamine (TDA) are released during MDI and TDI polymerization and may be present in newly finished polyurethane foam parts. MDA and TDA concentrations in foam decline exponentially within several hours of demolding. MDA and the 2,4-isomer of TDA are known animal carcinogens and, in addition, have significant non-carcinogenic health effects. Our goal was to determine whether worker exposure to MDA or TDA in freshly produced polyurethane foams was associated with unacceptable health risks. Sampling and analysis of the fresh foam indicated that MDA and TDA concentrations varied considerably among products, but concentrations in all materials evaluated declined rapidly over time. We found that, under a worst-case exposure scenario, cancer risks from TDA exposure were approximately 5 x 10(-6), whereas cancer risks from MDA exposure resulted in a tumorigenic margin of exposure (MOE) of 85 000. Non-cancer chronic hazard indices were well below 1.0. Therefore, the potential cancer and non-cancer health risks from MDA or TDA exposure to newly manufactured foam parts appear to fall well within acceptable health risk criteria.

Arsenic risk assessment.

We review recent publications by Hopenhayn-Rich et al. and Smith et al. regarding two critical issues in arsenic risk assessment: the role of methylation in the dose-response relationship and the role of internal cancers. Hopenhayn-Rich et al. applied simple linear regression to data from several studies to determine whether the percentage of inorganic arsenic in urine increases with increasing dose. Although their results failed to show a correlation between percent inorganic arsenic and urinary arsenic concentration, their evaluation does not demonstrate the absence of a methylation threshold because of the relatively low level of arsenic in urine and the use of grab samples in evaluating methylating capacity. Using data from an epidemiological study in Taiwan, Smith et al. have indicated that arsenic could be an important risk factor not only for skin cancer (the basis of the current EPA cancer slope factor), but also for several internal cancers including lung, liver, bladder, and kidney. We note the following deficiencies in the analysis of Smith et al: 1) the likely underestimated exposure estimate due to lack of consideration on nonwater sources of arsenic and the underestimate of water consumption, 2) lack of consideration of detoxification in estimating potential risks from low-level exposures typical of the U.S. population, and 3) lack of consideration of key differences, particularly nutritional differences, between the Taiwanese and U.S. populations that could affect potential risks.

Inorganic arsenic: a need and an opportunity to improve risk assessment.

This paper presents views on the current status of (inorganic) arsenic risk assessment in the United States and recommends research needed to set standards for drinking water. The opinions are those of the Arsenic Task Force of the Society for Environmental Geochemistry and Health, which has met periodically since 1991 to study issues related to arsenic risk assessment and has held workshops and international conferences on arsenic. The topic of this paper is made timely by current scientific interest in exposure to and adverse health effects of arsenic in the United States and passage of the Safe Drinking Water Act Amendment of 1996, which has provisions for a research program on arsenic and a schedule mandating the EPA to revise the maximum contaminant level of arsenic in drinking water by the year 2001. Our central premise and recommendations are straightforward: the risk of adverse health effects associated with arsenic in drinking water is unknown for low arsenic concentrations found in the United States, such as at the current interim maximum contaminant level of 50 microg/l and below. Arsenic-related research should be directed at answering that question. New epidemiological studies are needed to provide data for reliable dose-response assessments of arsenic and for skin cancer, bladder cancer, or other endpoints to be used by the EPA for regulation. Further toxicological research, along with the observational data from epidemiology, is needed to determine if the dose-response relationship at low levels is more consistent with the current assumption of low-dose linearity or the existence of a practical threshold. Other recommendations include adding foodborne arsenic to the calculation of total arsenic intake, calculation of total arsenic intake, and encouraging cooperative research within the United States and between the United States and affected countries.

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells.

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.

Translocation of MRE11 from the nucleus to the cytoplasm as a mechanism of radiosensitization by heat.

Zhu, W-G., Seno, J. D., Beck, B. D. and Dynlacht, J. R. Translocation of MRE11 from the Nucleus to the Cytoplasm as a Mechanism of Radiosensitization by Heat. Radiat. Res. 156, 95-102 (2001).Hyperthermia sensitizes mammalian cells to ionizing radiation, presumably by inhibiting the repair of radiation-induced double-strand breaks (DSBs). However, the mechanism by which heat inhibits DSB repair is unclear. The nuclear protein MRE11 is a component of a multi-protein complex involved in nonhomologous end joining (NHEJ) of radiation-induced DSBs. Using one-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blotting, we found that MRE11 is translocated from the nucleus to the cytoplasm when human U-1 melanoma or HeLa cells are heated for 15 min at 45.5 degrees C or when cells are heated after irradiation with 12 Gy of X rays. No such translocation is observed in unheated irradiated cells. The kinetics of migration of MRE11 to the cytoplasm was dependent upon whether the heated cells were irradiated, while the magnitude of redistribution of MRE11 was dependent upon post-treatment incubation time at 37 degrees C. Cytoplasmic MRE11 content reached a maximum 2-4 h after heating; the increase was not due to new protein synthesis. Partial recovery of nuclear MRE11 content was observed when heated cells or heated irradiated cells were incubated for up to 7 h at 37 degrees C after treatment. Western blotting results showing translocation of MRE11 from the nucleus to the cytoplasm after heating and irradiation were confirmed using confocal microscopy and immunofluorescence staining of fixed cells. Our data suggest that radiosensitization by heat may be caused, at least in part, by translocation of the DNA repair protein MRE11 from the nucleus to the cytoplasm.

The enigma of arsenic carcinogenesis: role of metabolism.

Inorganic arsenic is considered a high-priority hazard, particularly because of its potential to be a human carcinogen. In exposed human populations, arsenic is associated with tumors of the lung, skin, bladder, and liver. While it is known to be a human carcinogen, carcinogenesis in laboratory animals by this metalloid has never been convincingly demonstrated. Therefore, no animal models exist for studying molecular mechanisms of arsenic carcinogenesis. The apparent human sensitivity, combined with our incomplete understanding about mechanisms of carcinogenic action, create important public health concerns and challenges in risk assessment, which could be met by understanding the role of metabolism in arsenic toxicity and carcinogenesis. This symposium summary covers three critical major areas involving arsenic metabolism: its biodiversity, the role of arsenic metabolism in molecular mechanisms of carcinogenesis, and the impact of arsenic metabolism on human risk assessment. In mammals, arsenic is metabolized to mono- and dimethylated species by methyltransferase enzymes in reactions that require S-adenosyl-methionine (SAM) as the methyl donating cofactor. A remarkable species diversity in arsenic methyltransferase activity may account for the wide variability in sensitivity of humans and animals to arsenic toxicity. Arsenic interferes with DNA methyltransferases, resulting in inactivation of tumor suppressor genes through DNA hypermethylation. Other studies suggest that arsenic-induced malignant transformation is linked to DNA hypomethylation subsequent to depletion of SAM, which results in aberrant gene activation, including oncogenes. Urinary profiles of arsenic metabolites may be a valuable tool for assessing human susceptibility to arsenic carcinogenesis. While controversial, the idea that unique arsenic metabolic properties may explain the apparent non-linear threshold response for arsenic carcinogenesis in humans. In order to address these outstanding issues, further efforts are required to identify an appropriate animal model to elucidate carcinogenic mechanisms of action, and to define dose-response relationships.

Cognitive tests: interpretation for neurotoxicity? (Workshop summary).

The appropriate use and interpretation of cognitive tests presents important challenges to the toxicologist and to the risk assessor. For example, intelligence cannot be measured directly; rather intelligence is quantified indirectly by scoring responses (i.e., behaviors) to specific situations (problems). This workshop, "Cognitive Tests: Interpretation for Neurotoxicity?" provided an overview on the types of cognitive tests available and described approaches by which the validity of such tests can be assessed. Unlike many tools available to the toxicologist, cognitive tests have a particular advantage. Being noninvasive and species-neutral, the same test can be performed in different mammalian species. This enhances one's ability to assess the validity of test results. Criteria for test validity include comparable responses across species as well as similar disruption by the same neurotoxicant across species. Test batteries, such as the Operant Test Battery, have indicated remarkable similarity between monkeys and children with respect to performance of certain tasks involving, for example, short-term memory. Still, there is a need for caution in interpretation of such tests. In particular, cognitive tests, especially when performed in humans, are subject to confounding by a range of factors, including age, gender, and, in particular, education. Moreover, the ability of such tests to reflect intelligence must be considered. Certain aspects of intelligence, such as the ability to plan or carry out specific tasks, are not well reflected by many of the standard tests of cognition. Nonetheless, although still under development, cognitive tests do hold promise for reliably predicting neurotoxicity in humans.

Chemical mixtures from a public health perspective: the importance of research for informed decision making.

When considered from a public health perspective, the central question regarding chemical mixtures is deceptively simple: Are current approaches to risk assessment for chemical mixtures affording effective (adequate) and efficient (cost-effective) protection for members of our society? Answering this question realistically depends on an understanding of the hierarchical goals of public health (i.e. prevention, intervention, treatment) and an accurate evaluation of the extent to which these goals are being achieved. To allow decision makers to make informed judgments about the health risks of chemical mixtures, adequate scientific knowledge and understanding must be available to support risk assessment activities, which are an integral part of the regulatory decision making process. Designing and implementing relevant research depends on the existence of a feedback loop between researchers and regulators, where the information needs of regulators influence the nature and direction of research and the information and understanding generated by researchers improves the scientific basis for public health decisions. A clear, consistent, commonly accepted taxonomy for describing important mixture-related phenomena is a key factor in creating and maintaining the necessary feedback loop. Ultimately, both researchers and regulators share a common goal with regard to chemical mixtures; improving the state-of-the-science so that we can make informed decisions about protecting public health. A survey of research issues and needs that are crucial to attaining this goal is presented.

Cytoplasmic enzyme patterns in isolated hamster pulmonary alveolar type II cells.

Three cytoplasmic enzyme patterns were studied in pulmonary alveolar type II cells isolated from normal adult hamster lung: lactate dehydrogenase (total and isoenzymes), peroxidase, and beta-N-acetylglucosaminidase. Enzyme patterns of freshly-isolated type II cells were found to be different from those of freshly-isolated pulmonary hamster fibroblasts. After both types of cells had been cultured for seven days, no difference in cytoplasmic enzyme patterns remained. Lactate dehydrogenase isoenzyme patterns for type II cells were different from those obtained from polymorphonuclear leukocytes and alveolar macrophages. These data may be useful in detecting sources of lung injury by assessment of enzyme patterns in bronchoalveolar lavage fluid.

The development of a stochastic physiologically-based pharmacokinetic model for lead.

This presentation describes the development of a prototype Monte Carlo module for the physiologically-based pharmacokinetic (PBPK) model for lead, created by Dr Ellen O'Flaherty. The module uses distributions for the following: exposure parameters (soil and dust concentrations, daily soil and ingestion rate, water lead concentration, water ingestion rate, air lead concentration, inhalation rate and dietary lead intake); absoption parameters; and key pharmacokinetic parameters (red blood binding capacity and half saturation concentration). Distributions can be specified as time-invariant or can change with age. Monte Carlo model predicted blood levels were calibrated to empirically measured blood lead levels for children living in Midvale, Utah (a milling/smelting community). The calibrated model was then evaluated using blood lead data from Palmerton, Pennsylvania (a town with a former smelter) and Sandy, Utah, (a town with a former smelter and slag piles). Our initial evaluation using distributions for exposure parameters showed that the model accurately predicted geometric (GM) blood lead levels of Palmerton and Sandy and slightly over predicted the GSD. Consideration of uncertainty in red blood cell parameters substantially inflated the GM. Future model development needs to address the correlation among parameters and the use of parameters for long-term exposure derived from short-term studies.

Systemic uptake of inhaled arsenic in rabbits.

Human occupational exposure to sufficiently high levels of arsenic in air has been associated with lung cancer, but generally not other types of cancer. Thus, a better understanding of the relationship between airborne arsenic exposures and systemic uptake is essential. In this study, rabbits were exposed to one of four levels of arsenic trioxide in air for 8 h/day, 7 days/week, for 8 weeks (0.05, 0.1, 0.22, or 1.1 mg/m3). Plasma levels of inorganic arsenic, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were measured following the last exposure. Although there was a dose-related increase in plasma levels of methylated arsenic metabolites, statistically significant increases in mean inorganic arsenic levels in plasma were observed only in male rabbits exposed to 0.22 mg/m3, and in both males and females exposed to 1.1 mg/m3. Mean inorganic arsenic levels in plasma in males and females exposed to 0.05 and 0.1 mg/m3, and females exposed to 0.22 mg/m3, were not significantly elevated compared to controls. These results suggest that arsenic inhalation has a negligible impact on body burden of inorganic arsenic until air levels are significantly elevated. Based on plasma measurements of inorganic arsenic, the two lowest exposure levels in this study (0.05 and 0.1 mg/m3) are indistinguishable from background.

The use of information on susceptibility in risk assessment: state of the science and potential for improvement.

The aim of this paper is to evaluate the use of information on susceptibility or variability in risk assessment. Are we using it well? Are we not using it well or not at all? And finally, what can we do to improve the use of information on variability in risk assessment? The presentation is organized using the risk assessment paradigm developed by the National Research Council in 1983 (National Research Council, 1983. Risk Assessment in the Federal Government: Managing the Process. National Academy Press, Washington, DC.) which conceptualized risk assessment as consisting of four phases: hazard identification, dose response assessment, exposure assessment, and risk characterization. Because risk assessment procedures differentiate cancer risk from risk for systemic toxicity (i.e. non-cancer), cancer and noncancer risk assessment are discussed separately.

Recent trends in childhood blood lead levels.

Blood lead levels in children in the United States have declined through 1994, the date of the most recent National Health and Nutrition Examination Survey. In this investigation, the authors analyzed whether blood lead levels have changed since 1994 and quantified the magnitude of any change. The authors evaluated blood lead levels from 12 longitudinal data sets from 11 states and 1 city. Geometric mean blood lead levels declined between 4%/year and 14%/year in 8 of the data sets. No differences in decline rates were observed between data sets from states that had universal screening as a goal or that included repeat measures for an individual child and those data sets that did not. The authors' best estimate for these populations was a decline rate of 4-7%/year, which was comparable to the decline rate prior to 1994.

Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart.

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart.