IN VITRO HEPATIC OXIDATIVE BIOTRANSFORMATION OF TRIMETHOPRIM

Introduction: Trimethoprim-sulfamethoxazole (TMP-SMX) is a commonly used antibiotic often associated with idiosyncratic adverse drug reactions (IADRs). Historically, bioactivation of SMX to a reactive intermediate has been implicated in IADRs. Recently, the bioactivation of TMP has been described as TMP N-acetyl cysteine (NAC) adducts were identified following human liver microsome (HLM) incubations and in the urine of children taking TMP, suggesting cytochrome P450 enzymes (P450s) catalyze TMP bioactivation. In this study, we identified the P450s involved in the formation of six TMP primary metabolites. Methods: A panel of characterized HLMs (n=16), c-DNA expressed P450s, and pooled HLMs in the presence and absence of selective P450 inhibitors were incubated with therapeutic concentrations of TMP. Reactive metabolites were trapped with NAC, and metabolite formation was quantified by UPLC/MS/MS. Correlation coefficients between the rates of metabolite formation and P450 marker reaction rates were determined using least-squares regression analysis and evaluated at α=0.05. Results: 1-NO-TMP, Cα-NAC-TMP and Cα-OH-TMP were formed by CYP3A4 and inhibited by ketoconazole (CYP3A inhibitor). 4'-demethylation was catalyzed by several P450s including CYP3A4, correlated with multiple CYPs, and inhibited primarily with ketoconazole suggesting CYP3A4 contributed to 4'-demethylation. 3-NO-TMP was formed by CYP1A and inhibited by α-naphthoflavone (CYP1A inhibitor). 3'-demethylation was catalyzed by multiple P450s including CYP2C9, correlated with CYP2C9 activity and inhibited by sulphafenazole (CYP2C9 inhibitor). Conclusion: These findings suggest that P450s were responsible for the primary metabolism of TMP with CYPs 2C9 and 3A4 being the most significant contributors to TMP primary metabolism. Factors modulating activity of these enzymes may affect the risk of IADRs

Concluding Commentary: Children in All Cancer Prevention Policy Decisions.

This interesting series of articles on Opportunities for Cancer Prevention During Early Life brings many ideas for the primary prevention of cancer in childhood, or in adults due to early life events. The economic burden not only of cancer mortality but also of lifelong morbidity among cancer survivors, as shown by Guy et al,1 raises the importance of this critical public health issue. The topics of these articles were developed during online seminars with the pioneers in this area, some of whom authored the articles. They reflect the determinants of health diagrammed so eloquently in Healthy People 2020.2 Broadly, the determinants of health outcomes are biology/genetics, the physical environment, individual behavior, the social environment, and health services. The articles have been grouped according to these categories. For example, the article by Terry and Forman3 focuses on interventions at the individual level, and the article by Massetti et al,4 focuses on interventions aimed at social determinants. Classically, mutagenesis was the first basic mechanism clearly identified for carcinogenesis, and either inherited mutations or the mutagenesis of agents, such as tobacco and radiation, were the focus. Viral infections also have long been recognized to be involved with certain cancers. Our current models for carcinogenicity recognize that, for most cancers, multiple stages are required to not only initiate the cancer but also to elude biological mechanisms for repairing DNA, immune surveillance, and other natural defenses against cancer. One carcinogen, diethylstilbestrol (DES), did not neatly fit either the mutagen or viral hypotheses,5,6 and we now understand that in addition to inherited or acquired genetic susceptibilities, a basic mechanism for carcinogenesis due to either physical or social exposures is epigenetics. Epigenetics is the study of how the expression of DNA is modified without a change in the DNA code itself. Such modifications are critical to early developmental cellular differentiation pathways and, for example, explain why, with the same DNA, epidermal cells are so different from neurons. Epigenetic modulation of DNA expression occurs through DNA methylation, histone acetylation, and micro-RNAs. A physical or chemical exposure can interact with an organism leading to a series of signaling events culminating in an epigenetic change.7 This change in DNA expression may occur soon after the exposure, or persist until the particular gene is transcribed when the anomaly in DNA expression is expressed. As these articles demonstrate, to date we have too few answers for how to prevent childhood cancers from arising in the first place; in other words, to do primary prevention. We propose a framework that would consider that in addition to genetic susceptibilities, inherited or acquired, interactions with physical, chemical, microbial, hormonal, and/or nutritional agents are involved in signal cascades that in turn are involved with epigenetic changes contributing to childhood carcinogenesis. Primary prevention would involve understanding the biochemical changes required for the initiation of carcinogenesis so that they can be prevented, blocked, or undone, so that no carcinogenesis would take place. Avoidance of substances that are known to cause mutations is an obvious step that has been taken in many contexts, but we now additionally need to focus on risk factors that cause harmful epigenetic changes. Additionally, several nutrients are being investigated for their ability to reverse or modify epigenetic changes. Finally, as the case of DES suggests, inappropriate hormonal stimulation may be a factor. We know very little about stress (maternal during pregnancy, familial, or in children) and how consequent changes in hormonal expression impact cancer. However, we do know that excessive caloric intake is associated with increased cancer among adults in one study accounting for 14% of all cancer deaths in men and 20% in women.8 This is most certainly mediated via metabolic, and epigenetic, change. This is relevant for prevention during early life, as epigenetic changes are more prominent during fetal and postnatal development. Another current topic in medicine that could play a role here is personalized medicine. Personalized medicine refers to a medical model that proposes the customization of health care, with medical decisions, practices, and/or products being tailored to the individual patient.9 For example, DES exposure in utero to female fetuses results in an elevated risk of clear-cell adenocarcinoma of the vagina (CCAV); however, it is estimated that only 1 of 1000 women who were exposed in utero developed CCAV.5 DES, like estrogen, passes through the plasma and nuclear membranes to bind to various sites on DNA and modify gene expression. The risk factors for developing CCAV are unknown, with the possibility of earlier exposure to DES in utero, or a second exposure to another hormone either through oral contraceptives or pregnancy being one.6 The actual difference in susceptibility may be due to a genetic polymorphism that limits changes in the DES-modulated DNA expression or other unrelated factors like exposures to viruses. Further understanding of why one exposed person develops CCAV and another does not, would help in interpreting what exposure of a hormone to an individual means, which individuals should avoid exposure, and the discovery of cancer promoters that might be important for other cancers in addition to CCAV. Another emerging issue is global climate change and the possibility that cancer may increase as a result. There are many ways that global climate change could cause increases in childhood cancer rates. Heavy rainfalls will increase, and these result in increased toxic runoff into water, including drinking water supplies. Bottle-fed newborn infants are among the heaviest consumers of water, and they may be disproportionately affected. A warmer planet will experience increased air pollution via volatilization of certain environmental chemicals. Because infants and children breathe more air per kilogram than adults, a higher exposure will occur. Other global processes are continuing to deplete the planetary stratospheric ozone layer, the so-called “good” ozone that shields the planet from UV radiation. Sunscreen is typically not recommended for infants younger than 6 months and is not readily available to many children around the world. With the increasing temperature, children are at higher risk of sun exposure and sun burns; such exposures before the age of 20 years are known to increase the risk of melanoma and other skin cancers. This series of articles suggests that several policy changes would substantially reduce the risk of cancer from prenatal and early-life exposures. For prenatal exposures, increased testing of environmentally used chemicals in reproductive toxicity or hormonal activity (including estrogenicity) is warranted. Also, many carcinogens are part of the workplace, such as solvents and heavy metals. There are increased risks not only to the exposed workers, but also to their unborn children. Alcohol, a solvent and known teratogen, causes cancer in adults, and is suspected to lead to cancer in children with fetal alcohol syndrome.10 We do not have evidence about cancer risks for children at lower doses, but we should, given that ethanol is widely used as a fuel and a fuel additive and in that way causes exposures generally. Maternal exposure to industrial solvents is also linked to development of acute lymphoblastic leukemia in their offspring.11These results strongly suggest that occupational exposure limits by the Occupational Safety and Health Administration be set to protect the fetuses of pregnant women. For children, the critical element of time is lacking from the 2020 model of Healthy People. Time is important in many senses of the word: time of exposure, time of biologic development/status, time in the sense of linear progression; what went before influences current health status such that an individual may be more susceptible or more resilient to the carcinogen at every stage of development. Simply put, we as pediatricians know that children are not little adults, but we also know that a fetus is not a little infant, an infant is not a little child, and a child is not a little adolescent. Thus, although we categorize children into stages of human development, it may be that every day is a critical window of susceptibility for yet another molecular event; and the influences on this ever-changing biological organism are complex. Chemical exposures can appear to be straightforward (absorption, metabolism, interaction with target molecule, health outcome) biologically, but there are complex interactions with a number of other factors (stress, hormonal responses, epigenetically altered target molecules, and nutritional state) that add complexity. All of these determinants rely on unique attributes of the organism that occur only during a narrow window of time. If the parts of our DNA that are translated to encode proteins (the exome) are not complicated enough, we now know that we must be concerned about the nontranslated parts and the exponentially complex epigenome. Even with major breakthroughs in mathematics or informatics, we may never be able to precisely understand the factors that contribute to any 1 case of cancer. What we can learn is which factors are increasing risks of cancer. The more relevant model to consider when thinking of cancer prevention during early life may be that of the kaleidoscope presented in the National Academies Press publication, Children’s Health, the Nation’s Wealth.12 Such complexity suggests that a simplified overarching approach to cancer-prevention policies may be to consider the health of fetuses and children as well as the preconception of health of parents in all policy decisions

Neutron Scattering Study of Crystal Field Energy Levels and Field Dependence of the Magnetic Order in Superconducting HoNi2B2C

Elastic and inelastic neutron scattering measurements have been carried out to investigate the magnetic properties of superconducting (Tc~8K) HoNi2B2C. The inelastic measurements reveal that the lowest two crystal field transitions out of the ground state occurat 11.28(3) and 16.00(2) meV, while the transition of 4.70(9) meV between these two levels is observed at elevated temperatures. The temperature dependence of the intensities of these transitions is consistent with both the ground state and these higher levels being magnetic doublets. The system becomes magnetically long range ordered below 8K, and since this ordering energy kTN ~ 0.69meV << 11.28meV the magnetic properties in the ordered phase are dominated by the ground-state spin dynamics only. The low temperature structure, which coexists with superconductivity, consists of ferromagnetic sheets of Ho{3+ moments in the a-b plane, with the sheets coupled antiferromagnetically along the c-axis. The magnetic state that initially forms on cooling, however, is dominated by an incommensurate spiral antiferromagnetic state along the c-axis, with wave vector qc ~0.054 A-1, in which these ferromagnetic sheets are canted from their low temperature antiparallel configuration by ~17 deg. The intensity for this spiral state reaches a maximum near the reentrant superconducting transition at ~5K; the spiral state then collapses at lower temperature in favor of the commensurate antiferromagnetic state. We have investigated the field dependence of the magnetic order at and above this reentrant superconducting transition. Initially the field rotates the powder particles to align the a-b plane along the field direction, demonstrating that the moments strongly prefer to lie within this plane due to the crystal field anisotropy. Upon subsequently increasing the field atComment: RevTex, 7 pages, 11 figures (available upon request); Physica

Office of Regulatory Affairs Strategies for Building an Integrated National Laboratory Network for Food and Feed

An interconnected network of accredited federal, state, local, tribal, and territorial laboratories is critical to ensuring the safety of the U.S. food supply and the development of the Integrated Food Safety System (IFSS). In 2004, as part of a national policy to defend the U.S. food supply against terrorist attacks, major disasters, and other emergencies, the Food Emergency Response Network (FERN) was created to integrate the nation’s multilevel (i.e., federal, state, local, tribal, territorial) food-testing laboratories to detect, identify, respond to, and recover from a bioterrorism act affecting the safety of the food supply, or a public health emergency/outbreak involving the food supply. Since 2004, federal agencies have invested an estimated $200 million in FERN. The majority of this investment has been in the FERN cooperative agreements with FDA and USDA-FSIS investing$95.8 million and $69 million, respectively. FDA has promoted the accreditation of state laboratories through cooperative agreement funding, investing more than$50 million to fund these grants. On November 11, 2014, the Office of Regulatory Affairs (ORA) requested that the FDA Science Board establish a subcommittee to evaluate current investments in: (1) the FERN cooperative agreement funding program (CAP), and (2) funding for state laboratories to achieve International Organization for Standardization (ISO) accreditation. The goal was to ascertain how ORA can advance and establish an effective integrated laboratory network among ORA, FDA Center, and state public health and food- and feed-testing laboratories. In response to this request, the Science Board created the ORA FERN Cooperative Agreement Evaluation Subcommittee on July 1, 2015. This report summarizes the results of the Subcommittee’s review

Key issues for the assessment of the allergenic potential of genetically modified foods: breakout group reports.

On the final afternoon of the workshop "Assessment of the Allergenic Potential of Genetically Modified Foods," held 10-12 December 2001 in Chapel Hill, North Carolina, USA, speakers and participants met in breakout groups to discuss specific questions in the areas of use of human clinical data, animal models to assess food allergy, biomarkers of exposure and effect, sensitive populations, dose-response assessment, and postmarket surveillance. Each group addressed general questions regarding allergenicity of genetically modified foods and specific questions for each subject area. This article is a brief summary of the discussions of each of the six breakout groups regarding our current state of knowledge and what information is needed to advance the field

Assuring access to data for chemical evaluations

Background: A database for studies used for U.S. Environmental Protection Agency (EPA) pesticide and chemical reviews would be an excellent resource for increasing transparency and improving systematic assessments of pesticides and chemicals. There is increased demand for disclosure of raw data from studies used by the U.S. EPA in these reviews. Objectives: Because the Information Quality Act (IQA) of 2001 provides an avenue for request of raw data, we reviewed all IQA requests to the U.S. EPA in 2002–2012 and the U.S. EPA’s responses. We identified other mechanisms to access such data: public access databases, the Freedom of Information Act (FOIA), and reanalysis by a third party. Discussion: Only two IQA requests to the U.S. EPA were for raw data. Both of these were fulfilled under FOIA, not the IQA. Barriers to the U.S. EPA’s proactive collection of all such data include costs to the U.S. EPA and researchers, significant time burdens for researchers, and major regulatory delays. The U.S. EPA regulatory authority in this area is weak, especially for research conducted in the past, not funded by the U.S. government, and/or conducted abroad. The U.S. EPA is also constrained by industry confidential business information (CBI) claims for regulatory testing data under U.S. chemical and pesticide laws. The National Institutes of Health Clinical Trials database systematically collects statistical data about clinical trials but not raw data; this database may be a model for data from studies of chemicals and pesticides. Conclusions: A database that registers studies and obtains systematic sets of parameters and results would be more feasible than a system that attempts to make all raw data available proactively. Such a proposal would not obviate rights under the IQA to obtain raw data at a later point