Center For Food Safety And Applied Nutrition Research Articles

Analyte Extension Method Validation of Elemental Analysis Manual Method 4.7 for Six Additional Elements, Cobalt (Co), Strontium (Sr), Thallium (Tl), Tin (Sn), Uranium (U) and Vanadium (V).

An interlaboratory study was conducted at the U.S. Food and Drug Administration's (FDA) Northeast Food and Feed Laboratory (NFFL) and the Center for Food Safety and Applied Nutrition (CFSAN) with the purpose to expand FDA Elemental Analysis Manual (EAM) method 4.7 (Inductively Coupled Plasma-Mass Spectrometric Determination of Arsenic, Cadmium, Chromium, Lead, Mercury, and Other Elements in Food Using Microwave Assisted Digestion) to include new analytes (1). The goal of the study was to demonstrate the performance of FDA EAM method 4.7 when analyzing new analytes cobalt (Co), strontium (Sr), thallium (Tl), tin (Sn), uranium (U) and vanadium (V). This analyte extension method validation of EAM 4.7 for six additional elements, Co, Sr, Tl, Sn, U and V followed all guidelines for a Level 2 or single laboratory validation and met all acceptance criteria for analyte extensions as per the Guidelines for the Validation of Chemical Methods (3). As per EAM 4.7 (1), this study followed the procedures and used specified equipment operated under recommended conditions. The analyte extension method validation was performed per protocol and with no deviations. All quality control (QC) requirements for this analyte extension method validation of EAM 4.7 passed as evidenced by the analytical data. The results presented demonstrate accuracy, linearity and precision by successful analyses of method blanks, matrix spikes, unfortified test samples and reference materials. The data analyzed met each of the validation requirements for each analyte in all representative matrices. The study showed that the new analytes performed satisfactorily using EAM 4.7 for total acidic extractable elemental analysis of food according to FDA's guidelines (3). The method met or exceeded the performance criteria.

Everyday Evaluation of Herb/Dietary Supplement-Drug Interaction: A Pilot Study.

A lack of reliable information hinders the clinician evaluation of suspected herb-drug interactions. This pilot study was a survey-based study conceived as a descriptive analysis of real-life experiences with herb-drug interaction from the perspective of herbalists, licensed health-care providers, and lay persons. Reported dietary supplement-drug interactions were evaluated against the resources most commonly cited for the evaluation of potential supplement-drug interactions. Disproportionality analyses were performed using tools available to most clinicians using data from the U.S. Federal Adverse Event Reporting System (FAERS) and the US Center for Food Safety and Applied Nutrition (CFSAN) Adverse Event Reporting System (CAERS). Secondary aims of the study included exploration of the reasons for respondent use of dietary supplements and qualitative analysis of respondent's perceptions of dietary supplement-drug interaction. While agreement among reported supplement-drug interactions with commonly cited resources for supplement-drug interaction evaluation and via disproportionality analyses through FAERS was low, agreement using data from CAERS was high.

Characterization and Genetic Diversity of Bacillus cereus Strains Isolated from Baby Wipes.

Bacillus cereus, a ubiquitous environmental microorganism known to cause foodborne illness, was isolated from samples taken from imported baby wipes from two different countries. These strains were characterized using a comprehensive molecular approach involving endpoint PCR, whole genome sequencing (WGS), comparative genomics, and biochemical analyses. A multiplex endpoint PCR assay was used to identify the enterotoxins: hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Phylogenetically, the strains clustered into two major groups according to sequence type (ST) and singleton. We used the Center for Food Safety and Applied Nutrition (CFSAN) GalaxyTrakr BTyper computational tool to characterize the strains further. As an additional means of characterization, we investigated the possible role of carbohydrate transport systems and their role in nutrient uptake by performing a BLAST analysis of the 40 B. cereus genomes recovered from baby wipes. This study outlines a multifaceted workflow that uses the analysis of enterotoxigenic potential, bioinformatics, genomic diversity, genotype, phenotype, and carbohydrate utilization as a comprehensive strategy to characterize these B. cereus strains isolated from baby wipes and further our understanding of the phylogenetic relatedness of strains associated with baby wipe production facilities that could potentially pose an infection risk to a vulnerable infant population.

Integrating the Food and Drug Administration Office of the Coordinated Outbreak Response and Evaluation Network's foodborne illness outbreak surveillance and response activities with principles of the National Incident Management System.

The Food Safety Modernization Act mandates building a national Integrated Food Safety System, which represents a seamless partnership among federal, state, local, territorial, and tribal agencies. During multistate foodborne illness outbreak investigations, local and state partners, the Centers for Disease Control and Prevention, the United States Food and Drug Administration (FDA), or the United States Department of Agriculture Food Safety Inspection Service, depending on the regulated food product, become engaged and assist in coordinating the efforts between partners involved and determine the allocation of resources. The FDA Center for Food Safety and Applied Nutrition (CFSAN) Office of the Coordinated Outbreak Response and Evaluation (CORE) Network coordinates foodborne illness outbreak surveillance, response, and post-response activities related to incidents involving multiple illnesses linked to FDA-regulated human food, dietary supplements, and cosmetic products. FDA has implemented the National Incident Management System (NIMS) Incident Command System (ICS) principles across the agency to coordinate federal response efforts, and CORE has adapted NIMS ICS principles for the emergency management of multistate foodborne illness outbreaks. CORE's implementation of ICS principles has provided several benefits to the operational cycle of foodborne illness outbreak investigations, including establishing a consistent, standardized, and transparent step-by-step approach to outbreak investigations. ICS principles have been instrumental in the development of a national platform for rapid and systematic laboratory, traceback, and epidemiologic information sharing, data analysis, and decision-making. This allows for partners across jurisdictions to reach a consensus regarding outbreak goals and objectives, deploy resources, and take regulatory and public health actions.

GalaxyTrakr: a distributed analysis tool for public health whole genome sequence data accessible to non-bioinformaticians

BackgroundProcessing and analyzing whole genome sequencing (WGS) is computationally intense: a single Illumina MiSeq WGS run produces ~ 1 million 250-base-pair reads for each of 24 samples. This poses significant obstacles for smaller laboratories, or laboratories not affiliated with larger projects, which may not have dedicated bioinformatics staff or computing power to effectively use genomic data to protect public health. Building on the success of the cloud-based Galaxy bioinformatics platform (http://galaxyproject.org), already known for its user-friendliness and powerful WGS analytical tools, the Center for Food Safety and Applied Nutrition (CFSAN) at the U.S. Food and Drug Administration (FDA) created a customized ‘instance’ of the Galaxy environment, called GalaxyTrakr (https://www.galaxytrakr.org), for use by laboratory scientists performing food-safety regulatory research. The goal was to enable laboratories outside of the FDA internal network to (1) perform quality assessments of sequence data, (2) identify links between clinical isolates and positive food/environmental samples, including those at the National Center for Biotechnology Information sequence read archive (https://www.ncbi.nlm.nih.gov/sra/), and (3) explore new methodologies such as metagenomics. GalaxyTrakr hosts a variety of free and adaptable tools and provides the data storage and computing power to run the tools. These tools support coordinated analytic methods and consistent interpretation of results across laboratories. Users can create and share tools for their specific needs and use sequence data generated locally and elsewhere.ResultsIn its first full year (2018), GalaxyTrakr processed over 85,000 jobs and went from 25 to 250 users, representing 53 different public and state health laboratories, academic institutions, international health laboratories, and federal organizations. By mid-2020, it has grown to 600 registered users and processed over 450,000 analytical jobs. To illustrate how laboratories are making use of this resource, we describe how six institutions use GalaxyTrakr to quickly analyze and review their data. Instructions for participating in GalaxyTrakr are provided.ConclusionsGalaxyTrakr advances food safety by providing reliable and harmonized WGS analyses for public health laboratories and promoting collaboration across laboratories with differing resources. Anticipated enhancements to this resource will include workflows for additional foodborne pathogens, viruses, and parasites, as well as new tools and services.

Do topically aplied skin creams containing retinyl palmitate affect the photocarcinogenecity of simulated solar light?

Retinyl palmitate (all-trans-retinyl palmitate; RP) was nominated in 2001 by the U.S. Food and Drug Administration's Center for Food Safety and Applied Nutrition (CFSAN) to the National Toxicology Program (NTP) as a high priority compound for phototoxicity and photocarcinogenicity studies at the National Center for Toxicological Research (NCTR). Studies with SKH-1 hairless mice are required to test whether topical application of RP enhances the phototoxicity and photocarcinogenicity of simulated solar light and UV light. Mechanistic studies are needed to provide insight into the disposition of RP in vitro and on the skin of mice, and to test thoroughly whether genotoxic damage by UV-induced radicals may participate in any toxicity of topically applied RP in the presence of UV light.

Interpreting Whole-Genome Sequence Analyses of Foodborne Bacteria for Regulatory Applications and Outbreak Investigations.

Whole-genome sequence (WGS) analysis has revolutionized the food safety industry by enabling high-resolution typing of foodborne bacteria. Higher resolving power allows investigators to identify origins of contamination during illness outbreaks and regulatory activities quickly and accurately. Government agencies and industry stakeholders worldwide are now analyzing WGS data routinely. Although researchers have published many studies that assess the efficacy of WGS data analysis for source attribution, guidance for interpreting WGS analyses is lacking. Here, we provide the framework for interpreting WGS analyses used by the Food and Drug Administration’s Center for Food Safety and Applied Nutrition (CFSAN). We based this framework on the experiences of CFSAN investigators, collaborations and interactions with government and industry partners, and evaluation of the published literature. A fundamental question for investigators is whether two or more bacteria arose from the same source of contamination. Analysts often count the numbers of nucleotide differences [single-nucleotide polymorphisms (SNPs)] between two or more genome sequences to measure genetic distances. However, using SNP thresholds alone to assess whether bacteria originated from the same source can be misleading. Bacteria that are isolated from food, environmental, or clinical samples are representatives of bacterial populations. These populations are subject to evolutionary forces that can change genome sequences. Therefore, interpreting WGS analyses of foodborne bacteria requires a more sophisticated approach. Here, we present a framework for interpreting WGS analyses that combines SNP counts with phylogenetic tree topologies and bootstrap support. We also clarify the roles of WGS, epidemiological, traceback, and other evidence in forming the conclusions of investigations. Finally, we present examples that illustrate the application of this framework to real-world situations.

Dietary Supplement Adverse Event Report Data From the FDA Center for Food Safety and Applied Nutrition Adverse Event Reporting System (CAERS), 2004-2013.

The Food and Drug Administration (FDA)'s Center for Food Safety and Applied Nutrition (CFSAN) oversees the safety of the nation's foods, dietary supplements, and cosmetic products. To present a descriptive analysis of the 2004-2013 dietary supplement adverse event report (AER) data from CAERS and evaluate the 2006 Dietary Supplements and Nonprescription Drug Consumer Protection Act as pertaining to dietary supplements adverse events reporting. We queried CAERS for data from the 2004-2013 AERs specifying at least 1 suspected dietary supplement product. We extracted the product name(s), the symptom(s) reported, age, sex, and serious adverse event outcomes. We examined time trends for mandatory and voluntary reporting and performed analysis using SAS v9.4 and R v3.3.0 software. Of the total AERs (n = 15 430) received from January 1, 2004, through December 31, 2013, indicating at least 1 suspected dietary supplement product, 66.9% were mandatory, 32.2% were voluntary, and 0.9% were both mandatory and voluntary. Reported serious outcomes included death, life-threatening conditions, hospitalizations, congenital anomalies/birth defects and events requiring interventions to prevent permanent impairments (5.1%). The dietary supplement adverse event reporting rate in the United States was estimated at ~2% based on CAERS data. This study characterizes CAERS dietary supplement adverse event data for the 2004-2013 period and estimates a reporting rate of 2% for dietary supplement adverse events based on CAERS data. The findings show that the 2006 Dietary Supplements and Nonprescription Drug Consumer Protection Act had a substantial impact on the reporting of adverse events.

Welcome Letter

On behalf of the National Center for Natural Products Research, School of Pharmacy, and the University of Mississippi, we would like to welcome you to the “15th International Conference on the Science of Botanicals”. The Conference will be focusing on the progress in the botanical research and development, as well as regulatory and clinical aspects. Further information regarding this conference can also be found at www.oxfordICSB.org. This conference is supported by a cooperative agreement between the NCNPR and the Center for Food Safety and Applied Nutrition (CFSAN) at the U.S. Food and Drug Administration (FDA). It is co-sponsored by the Shanghai Institute of Materia Medica/CAS, China; the Council of Scientific and Industrial Research (CSIR – India); the Ministry of Indigenous Medicine; Sri Lanka; the American Society of Pharmacognosy (ASP); the Society for Medicinal Plant Research (GA); the Korean Society of Pharmacognosy; The Vietnam Academy of Science and Technology (VAST); the Dr. Panjwani Center for Molecular Medicine and Drug Research; International Council on Medicinal and Aromatic.

FDA Said to Miss Adverse Events Caused by Dietary Supplements

Back to table of contents Previous article Next article Professional NewsFull AccessFDA Said to Miss Adverse Events Caused by Dietary SupplementsJun YanJun YanSearch for more papers by this authorPublished Online:19 Apr 2013https://doi.org/10.1176/appi.pn.2013.4b21AbstractContrary to common perception, dietary supplements are not always safe, but the FDA is hampered by a lack of money to collect useful data and monitor safety problems.When it comes to protecting the public from the side effects and toxicities of dietary supplements, the Food and Drug Administration’s (FDA) current adverse-event reporting system is missing a substantial chunk of the complaints that consumers have registered, according to a Government Accountability Office (GAO) report released March 18.Dietary supplements, which include vitamins, minerals, and herbal products, constitute a $30-billion annual business in the United States, and the demand for these products is increasing, according to 2011 estimates published by Nutrition Business Journal. The FDA estimated that about 55,000 dietary supplement products were on the market in 2009. Dietary supplements are primarily regulated by the FDA’s Center for Food Safety and Applied Nutrition (CFSAN), and the agency’s total regulatory activities in this area, ranging from inspections to safety surveillance, had a budget of $18.9 million in 2012, the GAO noted. Unlike prescription drugs, however, dietary supplements can be marketed without the manufacturer having to prove their efficacy and safety. The burden of proof is on the FDA to demonstrate that a product is unsafe or harmful before actions can be taken. To date, one herbal ingredient, ephedrine alkaloids, which was used in weight-loss supplements, has been banned because of its link to several deaths. For less-grave risks, the FDA can issue warning letters, seek court injunctions, block importation, or issue recalls for supplement products that fail inspections, contain adulterated ingredients, or pose other safety risks. In 2011, the agency issued about 90 warning letters related to dietary supplements and 30 voluntary-recall requests. If consumers suffer adverse effects from a dietary supplement, they can report the problem directly to the FDA’s MedWatch program, an online reporting portal (see URL at end of article). These reports are then transferred to CFSAN for evaluation. Consumers can also file a complaint with a local FDA field office or with the supplement’s manufacturer or distributor, whose telephone number is required on the package. Starting in 2008, manufacturers or distributors were required to submit adverse-event reports to the FDA if the event is deemed serious. If the event is not considered serious, the manufacturer is not obligated but can voluntarily report it.(The FDA defines a serious event as one that results “in a death, life-threatening experience, inpatient hospitalization, or birth defect or…require[s] a medical or surgical intervention to prevent the above-cited serious outcomes.”)From 2008 to 2011, the FDA received 6,307 adverse-event reports for dietary supplements, 71 percent of which came from the industry, reported as serious adverse events, according to the GAO audit. The GAO also found that from 2008 to 2010, 57 U.S. poison centers received 4,863 calls about adverse events related to dietary supplements, which was about 1,000 more reports than the FDA received in the same three-year period. These reports were generally not shared with the FDA, the GAO found.Although the GAO recommends that the FDA obtain comprehensive adverse-event data from poison centers, CFSAN officials told the GAO that purchasing poison-center data would be cost prohibitive, as the CFSAN annual budget for adverse-event surveillance is about $400,000, far less than the price poison centers want to charge for the data. Underreporting of adverse events is a constant concern. The GAO report cited reasons for underreporting supplement-related adverse events, in particular that “consumers, health care professionals, and others may not recognize the chronic or cumulative toxic effects of a dietary supplement, or…assume dietary supplements to be safe and not attribute negative effects to them.” ■FDA-issued product recalls are posted at http://www.fda.gov/Safety/MedWatch/default.htm ISSUES NewArchived

Genomic paradigms for food-borne enteric pathogen analysis at the USFDA: case studies highlighting method utility, integration and resolution

Modern risk control and food safety practices involving food-borne bacterial pathogens are benefiting from new genomic technologies for rapid, yet highly specific, strain characterisations. Within the United States Food and Drug Administration (USFDA) Center for Food Safety and Applied Nutrition (CFSAN), optical genome mapping and DNA microarray genotyping have been used for several years to quickly assess genomic architecture and gene content, respectively, for outbreak strain subtyping and to enhance retrospective trace-back analyses. The application and relative utility of each method varies with outbreak scenario and the suspect pathogen, with comparative analytical power enhanced by database scale and depth. Integration of these two technologies allows high-resolution scrutiny of the genomic landscapes of enteric food-borne pathogens with notable examples including Shiga toxin-producing Escherichia coli (STEC) and Salmonella enterica serovars from a variety of food commodities. Moreover, the recent application of whole genome sequencing technologies to food-borne pathogen outbreaks and surveillance has enhanced resolution to the single nucleotide scale. This new wealth of sequence data will support more refined next-generation custom microarray designs, targeted re-sequencing and “genomic signature recognition” approaches involving a combination of genes and single nucleotide polymorphism detection to distil strain-specific fingerprinting to a minimised scale. This paper examines the utility of microarrays and optical mapping in analysing outbreaks, reviews best practices and the limits of these technologies for pathogen differentiation, and it considers future integration with whole genome sequencing efforts.

Best Practices for Use of Historical Control Data of Proliferative Rodent Lesions

CHARLOTTE KEENAN, SUSAN ELMORE, SABINE FRANCKE-CARROLL, RAMON KEMP, ROY KERLIN, SHYAMAL PEDDADA, JOHN PLETCHER, MATTHIAS RINKE, STEPHEN PETER SCHMIDT, IAN TAYLOR, AND DOUGLAS C. WOLF GlaxoSmithKline, King of Prussia, Pennsylvania, USA National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, North Carolina, USA Center for Food Safety and Applied Nutrition (CFSAN), U.S. Food and Drug Administration, College Park, Maryland, USA Merck Research Laboratories, Riom, France Pfizer Inc., Groton, Connecticut, USA NIEHS, Research Triangle Park, North Carolina, USA Charles River, Frederick, Maryland, USA Bayer Schering Pharma AG, Wuppertal, Germany Huntingdon Life Sciences, Eye, United Kingdom U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA

Surrogate endpoints and emerging surrogate endpoints for risk reduction of cardiovascular disease

This article reviews surrogate endpoints and emerging biomarkers that were discussed at the annual "Cardiovascular Biomarkers and Surrogate Endpoints" symposium cosponsored by the US Food and Drug Administration (FDA) and the Montreal Heart Institute. The FDA's Center for Food Safety and Applied Nutrition (CFSAN) uses surrogate endpoints in its scientific review of a substance/disease relationship for a health claim. CFSAN currently recognizes three validated surrogate endpoints: blood pressure, blood total cholesterol, and blood low-density lipoprotein (LDL) concentration in its review of a health claim for cardiovascular disease (CVD). Numerous potential surrogate endpoints of CVD are being evaluated as the pathophysiology of heart disease is becoming better understood. However, these emerging biomarkers need to be validated as surrogate endpoints before they are used by CFSAN in the evaluation of a CVD health claim.

Preparation and characterization of standard reference material 3276, carrot extract in oil

The National Institute of Standards and Technology (NIST), the Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) and Center for Food Safety and Applied Nutrition (CFSAN), and the National Institutes of Health (NIH), Office of Dietary Supplements (ODS) are collaborating to produce a series of standard reference materials (SRMs) for dietary supplements. Standard reference material (SRM) 3276 Carrot Extract in Oil is one in this series, with values assigned for trans-alpha-carotene, trans- and total beta-carotene, delta- and gamma-tocopherol, and twelve fatty acids. Results for carotenoids and tocopherols were obtained by use of combinations of liquid chromatography (LC), on columns of differing selectivity, with absorbance and mass spectrometric (MS) detection. Fluorescence detection also was used for the tocopherols. Results for fatty acids were obtained by use of gas chromatography (GC) with both flame-ionization and mass-spectrometric detection. This material is intended for use as a primary control material when assigning values to in-house (secondary) control materials and for validation of analytical methods for measurement of these analytes in similar matrices.

Histopathology as a predictive biomarker: strengths and limitations.

Cancer is a physical alteration of the relation of cells and their tissues resulting in aberrant social organization. These alterations are detected as masses (tumors) or, in the case of leukemia, as an abnormal number of white blood cells. However, many inflammatory lesions also form masses, and not all neoplasms are malignant. Therefore, histological criteria of malignancy are used for the diagnosis of cancer. Histopathology is the sine qua non of cancer diagnosis. Any other putative marker of cancer must be validated by histopathologic examination. Grading and staging of cancers are used to predict the clinical course and outcome of individual cancers. Grading of cancer is based on the histological criteria of the neoplasm including the degree of deviation from normal of tissue architecture and the differentiation and proliferation of individual cells. Extensive studies have correlated these microscopic characteristics with clinical outcome. Grading of most neoplasms, whether invasive or preinvasive, is useful in prognosis and consequently in therapy decisions. Staging, in most neoplasms, is based on the local extent of tissue involvement and/or the detection of the neoplastic cells in distant sites with microscopic confirmation. The molecular revolution has provided great insight into the genetic alterations leading to cancer and has given hope, in some quarters, that molecular techniques will supplement, or even supplant, microscopic examination. Each biomolecule can now claim its own view of organismic disease states with the suffix ‘‘-omics’’ (genomics, metabolomics, proteomics, glycomics, lipomics, etc.), and each promises to perfect biomarkers as the knowledge base evolves. The discovery and characterization of prostate-specific antigen (PSA) has been one of the great triumphs of this approach. However, some studies indicate that PSA lacks the specificity and sensitivity needed for a biomarker (1–3). The advocates of ‘‘systems biology’’ are diligently using their tools to find the next biomarker. However, their technologies will be validated using the gold standard of cancer diagnosis, microscopic histopathology. Early detection, without doubt, has had a major impact on the successful treatment of cancer. The improved cure rates of diseases such as cervical and breast cancers are indications of the impact of early detection on treatment. Early detection of most solid cancer involves the recognition and understanding of precancers known as carcinoma-in-situ or intraepithelial neoplasia. These foci of atypical cells are considered the precursors to malignancy. Students of specific cancers have identified apparent morphological continua that suggest a sequential acquisition of characteristics leading from normal to malignancy (cancer) (4–14). Although histopathology has successfully detected and characterized these early lesions, these studies also illustrate the limitations of histopathology as a predictive biomarker. Our current concepts of neoplastic progression are largely based on the ‘‘multiple genetic hit’’ hypothesis and the ‘‘linear sequential acquisition’’ models of neoplastic progression. Although somewhat successfully applied to colon cancer, the successive acquisition model has been less predictive in cervical, prostate, and breast cancer, where the relation between ‘‘low-grade’’ intraepithelial neoplasia and invasive cancer has been questioned (15–17). Cervical intraepithelial neoplasia is an excellent example where a specific viral infection may be required to develop a high-grade, progressive in-situ lesion (17). Thus, alternative models of neoplastic evolution have been proposed in which there are several lineages that progress in parallel. These are found to fit observed histological (18) and molecular observations (19) better than a simple linear acquisition model. Mouse models of tumor biology are informative. The insertion or manipulation of genes that are associated with human malignancy has resulted in murine tumors that mimic the 1 Published in a supplement to The Journal of Nutrition. Presented as part of the conference ‘‘The Use and Misuse of Biomarkers as Indicators of Cancer Risk Reduction Following Dietary Manipulation’’ held July 12–13, 2005 in Bethesda, MD. This conference was sponsored by the Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration (FDA), Department of Health and Human Services (DHHS); the Office of Dietary Supplements (ODS), National Institutes of Health, DHHS; and the Division of Cancer Prevention (DCP), National Cancer Institute, National Institutes of Health, DHHS. Guest Editors for the supplement publication were Harold E. Seifried, National Cancer Institute, NIH; and Claudine Kavanaugh, CFSAN, FDA. Guest editor disclosure: H.E. Seifried, no relationships to disclose; C. Kavanaugh, no relationships to disclose. 2 Supported by grants RO1CA089140, U01 CA105490-01 from the National Cancer Institute and U42 RR14905 from the NCRR and 1076-CCR-S0 from the New Jersey Commission on Cancer Research. 3 Author disclosure: no relationships to disclose. * To whom correspondence should be addressed. E-mail: rdcardiff@ucdavis. edu. 7 Abbreviations used: DCIS, ductal carcinoma in situ; GEM, genetically engineered mouse; HPO, hyperplastic outgrowth; MIN, mammary intraepithelial neoplasia; MIN-O, mammary intraepithelial neoplasia outgrowths; PSA, prostatespecific antigen.

Promises and perils of validating biomarkers for cancer risk.

The number of biomarkers potentially relevant to diet and disease has exploded in recent years. Examples include adenomas and intraepithelial neoplasia (tissue); proliferation and apoptosis indices (cell); DNA adducts, strand breaks, genetic polymorphisms (molecular); human papillomavirus (HPV) infection, Helicobacter pylori antibodies (infection); estrogens, PSA (blood analytes); mammographic densities, ovarian ultrasound abnormalities (imaging). Biomarkers can serve three functions in diet and disease research: 1) enhancing the biologic plausibility of diet–disease relations; 2) increasing the strength of diet–disease associations (‘‘relative risk sharpening’’); and 3) serving as surrogate endpoints. With respect to biologic plausibility, an example is the role of blood estrogen levels in explaining the connection between alcohol and breast cancer. Design options for investigating this biologic plausibility include metabolic studies, larger observational epidemiologic studies, and nutrition–gene studies. Nutrition–gene studies can add to biologic plausibility by helping to unravel mixtures (of foods or supplements, for example) or via the Mendelian randomization strategy, which helps rule out bias resulting from confounding and measurement error. Relative risks can be enhanced through the use of specific intake markers (of diet or supplement use—recent studies of serum selenium in relation to colorectal nepolasia, for example) and through investigation of diet–gene interactions (e.g., studies of red meat intake, N-acetyltransferase genotype, and colorectal cancer). Studies with surrogate endpoints can be smaller, faster, and less expensive than those with explicit disease outcomes. This holds for both intervention studies (trials) and observational epidemiologic studies. Surrogate endpoint validity is critical and can be conceived as follows: the study of a dietary factor in relation to the putative surrogate marker gives the correct answer for the relation of that dietary factor to the disease in question. This is a tough requirement. Surrogate validity depends on 3 specific criteria: 1) the marker is associated with disease; 2) the exposure (or intervention) is associated with the marker; and 3) the marker mediates the association between exposure and cancer. Examples of all 3 criteria can be seen in recently published prospective studies of obesity, serum estrogen levels, and breast cancer as well as reports of the interrelations of reproductive factors, HPV infection, and cervical cancer. Standard epidemiologic measures, such as relative risk and attributable proportion, can be used for criteria 1 and 2; regression and stratified analyses can be used to evaluate the third (mediation) criterion. Being on the causal pathway does not in itself guarantee surrogate endpoint validity. A theoretical example of this is given for a well-studied marker, rectal mucosal proliferation assays. Hyperproliferation may not be necessary for colorectal cancer; that is, there may be an alternative pathway bypassing hyperproliferation. The effect of an intervention on the alternative pathway may counterbalance the effect on the hyperproliferation pathway. An agent may reduce proliferation but have no effect on colorectal cancer because it also reduces apoptosis; similarly, an agent may have no effect on proliferation but actually reduce colorectal cancer incidence because it increases apoptosis. The totality of causal connection is key in evaluating surrogate endpoint validity. A high attributable proportion as well as mediation of exposure–disease relations supports the validity of surrogacy. Validity tends to be more assured for surrogates both necessary for and relatively close developmentally to disease (e.g., cervical intraepithelial neoplasia, CIN3, for cervical cancer). A surrogate endpoint may be valid for 1 exposure or intervention in relation to disease but not necessarily valid for another exposure or intervention. That is because, once again, an alternative pathway to the disease may exist. Colorectal adenomas have been studied as surrogates for invasive cancer. The rationale for such use includes the high prevalence of adenomas, the high recurrence rate (2 orders of magnitude greater than that for cancer), the assessment of the endpoint by standard clinical practice (endoscopy), and, reflecting the key biologic rationale, the adenoma–carcinoma 1 Published in a supplement to The Journal of Nutrition. Presented as part of the conference ‘‘The Use and Misuse of Biomarkers as Indicators of Cancer Risk Reduction Following Dietary Manipulation’’ held July 12–13, 2005 in Bethesda, MD. This conference was sponsored by the Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration (FDA), Department of Health and Human Services (DHHS); the Office of Dietary Supplements (ODS), National Institutes of Health, DHHS; and the Division of Cancer Prevention (DCP), National Cancer Institute, National Institutes of Health, DHHS. Guest Editors for the supplement publication were Harold E. Seifried, National Cancer Institute, NIH; and Claudine Kavanaugh, CFSAN, FDA. Guest editor disclosure: H.E. Seifried, no relationships to disclose; C. Kavanaugh, no relationships to disclose. 2 Author disclosure: no relationships to disclose. 3 Abbreviations used: CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; PSA, prostate-specific antigen. * To whom correspondence should be addressed. E-mail: schatzka@mail.nih. gov.

Interaction of genetic and dietary factors in mouse intestinal tumorigenesis.

The profound effect of diet on modulating the development of human intestinal cancer can be effectively studied in the mouse. It has been demonstrated that a Western-style diet (WD) that mimics major risk factors for colon cancer in developed countries (high in fat, low in calcium and vitamin D) accelerates and increases tumor formation in mouse genetic models of cancer, including mice with targeted inactivation of Apc, Apc/ p21, p27, and Apc/p27 (1–3). For example, although there is a gene dosage-dependent increase in tumor formation, and consequent decrease in life span, of Apc1638N mice that are also p21, p21, or p21, the Western diet exerts additive affects on tumor formation for each genotype (3). Further, the targeted inactivation of p27 in mice is sufficient to initiate tumor formation, but not if the animals are fed standard semipurified diet (2; our unpublished data). Tumors form in the large and small intestine of p27 or p27mice if the animals are fed control AIN76A diet, and approximately double if animals of the same genotype are fed the WD diet. The p27 or p27mice also develop intussusception, a pathology seen in patients with intestinal cancer but not yet reported in mouse genetic models, and the incidence of this pathology is markedly increased when the mice are fed the WD (4; our unpublished data). Elevated tumor development in these models is associated with a loss of proper intestinal cell maturation, especially a decrease in differentiated goblet cells (2,3). It has been demonstrated that loss of terminal goblet cell maturation in the intestine of mice with a targeted inactivation of the Muc2 gene is sufficient for tumor development throughout the intestinal tract (4,5) and that goblet cell maturation is more extensively perturbed, and tumor formation enhanced, in Muc2 mice that are also p21 (4). These genetic models of intestinal cancer can therefore be highly useful in dissecting cellular and molecular pathways, and their interactions, that are involved in intestinal cancer. However,.90% of human colon cancer develops in the absence of an inherited mutation in tumor oncogenes or suppressor genes. Similarly, rodent models that utilize DMH and AOM have provided important insight into colon carcinogenesis and its chemo-/nutritional prevention, but these carcinogens are not related to initiators of human tumor development. We therefore developed a new mouse model that mimics the development of ‘‘sporadic’’ colon cancer, the tumors that account for at least 90% of the colon cancer in the United States. In this model, control C57 Bl/6 mice are fed a new western diet (NWD is the original WD, but with low levels of folate, choline, methionine, and fiber in addition to the low levels of calcium and vitamin D (6)) for 2 y, equivalent to about 80% of their normal life span. Although no tumors were seen at 12 mo, tumors appear by 18 mo and increase to ;1 tumor in 25% of the mice. This is similar to the development of single colon tumors found by colonoscopy in 25–50% of individuals after the age of 60 (again, ;80% of the normal life span) in populations that consume a ‘‘higher-risk’’ diet. We therefore consider this a model of common sporadic colon cancer. To gain insight into alterations that elevate risk for tumor development, expression profiling has been carried out utilizing RNA from isolated colonic epithelial cells of these control mice maintained on different diets for 3 mo, well before tumors begin to develop after 1 y. Shifts in expression of over 3000 sequences (of 27,000 screened) were detected in mice fed the NWD compared with those fed control AIN76A. Because prior work had demonstrated that increasing the low levels of calcium and vitamin D in the WD was effective in reversing the increase in tumor formation stimulated by the WD in several mouse genetic models (K. Yang, unpublished data), a third diet in which the NWDwas supplemented with additional calcium and vitamin D was also investigated. In Bl/6 mice fed this supplemented diet, not only was tumor formation initiated by the NWD reversed, but the altered gene expression profile brought about by the NWD was also shifted back toward that seen in the mice fed AIN76A. Further investigation of the sequences whose level of expression closely tracked probabilities of tumor formation as 1 Published in a supplement to The Journal of Nutrition. Presented as part of the conference ‘‘The Use and Misuse of Biomarkers as Indicators of Cancer Risk Reduction Following Dietary Manipulation’’ held July 12–13, 2005 in Bethesda, MD. This conference was sponsored by the Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration (FDA), Department of Health and Human Services (DHHS); the Office of Dietary Supplements (ODS), National Institutes of Health, DHHS; and the Division of Cancer Prevention (DCP), National Cancer Institute, National Institutes of Health, DHHS. Guest Editors for the supplement publication were Harold E. Seifried, National Cancer Institute, NIH; and Claudine Kavanaugh, CFSAN, FDA. Guest editor disclosure: H.E. Seifried, no relationships to disclose; C. Kavanaugh, no relationships to disclose. 2 Author disclosure: no relationships to disclose. 5 Abbreviations used: AOM, azoxymethane; DMH, dimethylhydrazine; NWD, new Western diet; WD, Western diet. * To whom correspondence should be addressed. E-mail: augen@aecom.yu.edu.

What do shifts in indicators of apoptosis indicate about the cancer process?

Overview Apoptosis is genetically programmed cell death (1). It is defined by specific morphological criteria whose hallmark is condensation and fragmentation of the cell nucleus with endonuclease cleavage of DNA, but it is mediated by more than 1 biochemical pathway. The 2 major pathways of apoptotic cell death are the intrinsic or mitochondrial pathway and the extrinsic pathway, which is dependent on death receptor–ligand interactions. These pathways depend on execution caspases to initiate the DNA cleavage and catabolism of the cytoskeleton that results in cell death. There also is a programmed cell death (PCD) pathway that is independent of caspases, and, in addition to these PCD pathways, cancer cells can die through necrosis, mitotic catastrophe, autophagy, and senescence (2,3). Necrosis, which is mediated by processes outside the cell, is unlike apoptosis or PCD because inflammation can result from release of the cell contents. Mitotic catastrophe likely occurs during mitosis and is thought to be secondary to missegregation of chromosomes and/or cell fusion. It can lead to apoptosis. Autophagy is a caspase-independent form of cell death in which cells digest themselves. Senescence occurs whenmetabolically active cells no longer divide and may increase in size. The intrinsic or mitochondrial pathway of PCD is regulated by a family of proteins called the BCL-2 family. The first member of this family to be identified was an antiapoptotic protein named BCL-2 (1). The name was given because it was cloned from a B-cell lymphoma as a chromosomal translocation is which the coding sequence of BCL-2 is placed under transcriptional control of the Ig heavy-chain locus, resulting in significantly increased levels of BCL-2 expression and resistance to apoptosis. Subsequently, it was determined that the human BCL-2 gene was the mammalian counterpart of a gene in the nematode Caenorhabditis elegans that regulated apoptotic cell death. The gene cascade that ultimately governs the intrinsic apoptotic process (that BCL-2 is a member of) was defined in genetic studies performed using the C. elegans developmental model. Unlike C. elegans, in which there is only 1 BCL-2 functional homolog, mammalian species express multiple family members that include both antiapoptotic (such as BCL-2) and proapoptotic members. The relative concentrations of the antiapoptotic and proapoptotic BCL-2 family members act as a rheostat to determine the propensity of the cell to undergo apoptosis by regulating activation of caspases through changes in mitochondrial membrane permeability and release of cytochrome c. The caspases are the executioners of apoptosis and are synthesized as inactive procaspases and subsequently activated by cleavage. Like the BCL-2 family, caspases also represent a family of related proteins with some overlapping functions. Inhibitors of apoptosis (IAPs) are another family of proteins involved in the regulation of apoptosis. They inhibit apoptosis by blocking caspase action. The extrinsic or cell death receptor pathway works independently of the BCL-2 family (3,4). It is triggered by death ligands including tissue necrosis factor-a (TNF-a), Fas ligand (FasL), or TNF-related apoptosis-inducing ligand TRAIL. After ligand-induced activation, the receptors can interact with adapter proteins such as Fas-associated via death domain (FADD), which are homologous to proteins that act in the intrinsic pathway (Apaf-1). Similar to the intrinsic pathway, the extrinsic pathway eventually activates caspase proteins as the ‘‘executioners’’ of apoptosis. p53 is a defined tumor suppressor gene whose loss of function through either mutation or functional inactivation in cancer processes can suppress the apoptotic response triggered by DNA damage and other stimuli (5). Senescence is a state of permanent cell-cycle arrest. It is thought of as a normal physiological process that acts to inhibit the development of cancer by limiting the number of replicative cell cycles that a somatic cell can undergo (6). Most frequently it 1 Published in a supplement to The Journal of Nutrition. Presented as part of the conference ‘‘The Use and Misuse of Biomarkers as Indicators of Cancer Risk Reduction Following Dietary Manipulation’’ held July 12–13, 2005 in Bethesda, MD. This conference was sponsored by the Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration (FDA), Department of Health and Human Services (DHHS); the Office of Dietary Supplements (ODS), National Institutes of Health, DHHS; and the Division of Cancer Prevention (DCP), National Cancer Institute, National Institutes of Health, DHHS. Guest Editors for the supplement publication were Harold E. Seifried, National Cancer Institute, NIH; and Claudine Kavanaugh, CFSAN, FDA. Guest editor disclosure: H.E. Seifried, no relationships to disclose; C. Kavanaugh, no relationships to disclose. 2 This work was supported by grant R01CA89041 from NCI, National Institutes of Health, to Priscilla A. Furth. The contents are solely the responsibility of the authors. 3 Author disclosure: no relationships to disclose. 4 Abbreviations used: FADD, Fas-associated via death domain; IAP, inhibitors of apoptosis; PCD, programmed cell death; TNF-a, tissue necrosis factor-a; TRAIL, TNF-related apoptosis-inducing ligand; TUNEL, terminal deoxynucleotide transferase (TDT)-mediated dUTP nick-end labeling. * To whom correspondence should be addressed. E-mail: paf3@georgetown.edu.

The multifunctional carotenoids: insights into their behavior.

Because it is widely held that damage to DNA by reactive species is a significant contributor to carcinogenesis, it has been postulated that antioxidants in supplements or in foods containing them, which decrease oxidative DNA damage, should have a protective effect against cancer. In antioxidant–cancer risk studies the most common oxidative biomarkers that have been used are: 8-hydroxyguanine in urine, the comet assay in leukocytes, various measures of ‘‘total’’ antioxidant capacity in blood, and serum levels of antioxidant nutrients. It has been shown in many studies that DNA strand breaks can be reduced by antioxidant supplements or by various combinations of fruits and vegetables that are high in antioxidant nutrient content. It is noteworthy, however, that in a study by Duthie et al., the combination of 100 mg of vitamin C, 280 mg of vitamin E, and 25 mg of b-carotene reduced DNA damage, as reflected by the comet assay, only in smokers but not in nonsmokers (1). Mixed results have also been seen when urinary 8-hydroxyguanine was used, and observed effects may not be long lasting. The question remains, therefore, do these biomarker assays of oxidative stress have any predictive value with regard to cancer incidence? The simple answer is that we don’t know. Even if we disregard the technical problems of lack of standardization of the assays in various published studies, no prospective epidemiologic or intervention studies have been done in human populations to show that individuals with lower levels of DNA strand breaks have a lower risk of cancer, or indeed that diets or antioxidant supplements can lower the number of DNA strand breaks over the long run (e.g., years). What is known is that antioxidants can alter short-term oxidation status however it is assayed, but we have no idea of the cancerlowering predictive value of these assays. In this article b-carotene will be used as a model antioxidant in an attempt to answer the question of the clinical significance of diet and changes in oxidative biomarkers with regard to cancer risk. It is now realized that different oxidative effects (antioxidant, pro-oxidant) of b-carotene can be seen, depending on its concentration and other conditions (e.g., the oxygen concentration) (2). Epidemiologic studies have shown an inverse relation between dietary or serum values of b-carotene and the incidence of lung cancer (3). Thus, it was against all expectations that b-carotene given in high doses to smokers resulted in an increased incidence of lung cancer in 2 intervention trials (ATBC, CARET) (4,5). The original interpretation of these trials was that dietary b-carotene in epidemiologic studies was simply acting as a surrogate for some other factor, or that case control studies (the design used in most of the epidemiologic studies on b-carotene) were inherently biased. However, another bothersome explanation was that b-carotene at high dose could be working to promote cancer. It was noted that in the 2 b-carotene intervention trials that resulted in a higher incidence of cancer, the serum b-carotene levels achieved were significantly higher than that in another intervention trial in which there was no increase in lung cancer in response to supplementary b-carotene (Physicians Health Trial) (4–6). These disparate findings could be accounted for by the different concentrations of b-carotene reached in tissue. Although the b-carotene intervention trial results were shocking at the time, other studies have also shown that supplementary antioxidants can increase the incidence of cancer. For example, it has been reported that vitamin E supplementation in patients with treated head and neck cancers resulted in an increased occurrence of secondary primary cancers during the supplementation period (7). This study included 522 patients with stage 1 head and neck cancers who were treated by radiation. These patients were supplemented with 400 international units of vitamin E and 30 mg of b-carotene or placebo, which began on the first day after radiation therapy, and these treatments were continued for 3 years. During the course of the trial, b-carotene was discontinued after 156 patients had been enrolled in the study. The remaining patients received only vitamin E. After a median follow-up of 52 mo, second primary cancers and recurrences of the first tumorwere counted. It was found that patients receiving the vitamin E supplements had a higher rate of second primary cancers during the supplementation 1 Published in a supplement to The Journal of Nutrition. Presented as part of the conference ‘‘The Use and Misuse of Biomarkers as Indicators of Cancer Risk Reduction Following Dietary Manipulation’’ held July 12–13, 2005 in Bethesda, MD. This conference was sponsored by the Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration (FDA), Department of Health and Human Services (DHHS); the Office of Dietary Supplements (ODS), National Institutes of Health, DHHS; and the Division of Cancer Prevention (DCP), National Cancer Institute, National Institutes of Health, DHHS. Guest Editors for the supplement publication were Harold E. Seifried, National Cancer Institute, NIH; and Claudine Kavanaugh, CFSAN, FDA. Guest editor disclosure: H.E. Seifried, no relationships to disclose; C. Kavanaugh, no relationships to disclose. 2 Author disclosure: no relationships to disclose. * To whom correspondence should be addressed. E-mail: rob.russell@tufts.edu.

Oxidative DNA damage and repair: significance and biomarkers.

A range of cellular processes, external factors, and/or disease states can lead to the formation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS), and these themselves have ongoing (and usually detrimental) effects in humans (Fig. 1). For example, chronic inflammatory processes produce an excess of ROS as well as DNA-reactive aldehydes including trans-4hydroxy-2-nonenal and malondialdehyde from lipid peroxidation (LPO) (1). There is evidence that individuals with chronic inflammatory conditions, including chronic pancreatitis, ulcerative colitis, and Crohn’s disease, show elevated levels of oxidative damage including modified DNA bases in various organs, as compared with the normal population (2). They are also at increased risk of cancer. Therefore, dietary modifications that reduce the steady-state levels of these modified bases may be likely to protect against cancer. Not only are there several different ROS and RNS, but these also lead to a range of effects on different macromolecules (Table 1). Protection against ROS primarily occurs through exogenous dietary antioxidants (of which an important class are free radical scavengers) or through enzymatic cellular defense mechanisms. Major mechanisms here are inhibitors of prooxidant enzymes or inducers of endogenous defenses. Important groups of free radical scavengers in the human diet are derived from plants, including ascorbic acid (vitamin C), vitamin E, carotenoids, and certain plant polyphenols (1,3). Potentially more interesting may be molecules such as isothiocyanates that are oxidants themselves but also upregulate the expression of cellular antioxidant proteins and detoxification enzymes (4). Potentially at least, increasing the intake of antioxidants can reduce the levels of ROS or RNS and may affect the efficacy of DNA repair processes. However, proving the efficacy of such measures is not simple, nor are preferred methodologies agreed on. Although a number of studies report on tests of antioxidant activity in vitro or in animal experimental models (e.g., 5), there are a considerable number of uncertainties in extrapolating such data to humans. The most definitive proof that a certain dietary antioxidant is beneficial to humans is to test whether supplementing human diets with the relevant material can decrease their cancer risk. However, the unexpected result that supplementation with b-carotene increased, not decreased, human cancer risk in certain high-risk groups (6) shows the dangers of waiting until cancer develops. Thus, most human trials do not go as far as cancer but utilize a surrogate biomarker that implies (but does not prove) disease risk (7). In considering the ability of a given individual or population group to cope with oxidative stress, one obvious method is to measure levels of free radical scavengers in the plasma. Examples that have been commonly studied thus include all the plantrelated chemicals identified above, at the indicated levels to be expected in plasma: ascorbic acid, 20–80 mM; vitamin E, 50 mM; carotenoids, 0.1–0.4 mM; plant polyphenols, 0.01–0.1 mM; and isothiocyanates, 0.1–1 mM. It is also possible to measure the ability of plasma to scavenge free radicals using, for example, Trolox equivalent antioxidant activity (TEAC) or oxygen radical absorbance activity (ORAC) assays (8). However, this gives only a part of the picture. A considerable number of biomarker studies have assayed excretory products in the urine. For example, etheno-DNA adducts can be measured using an immuno-enriched HPLC fluorescence methodwith a sensitivity of;5 fmol/ml (9). Similarly, 7-hydroxy8-oxo-2#-deoxyguanosine (8-OxodG) (or the corresponding base 8-oxoGua) is a commonly formed modified DNA base product that can be detected in urine after oxidative stress, has been shown to be mutagenic in vivo, and can be measured with high sensitivity (10). Thus, measures of urinary levels of this modified base have been supported as a biomarker of the total extent of oxidative damage to DNA, and a reduction in urinary excretion rates of this molecule interpreted as revealing dietary protection (10). Such methods take advantage of the fact that there is 1 Published in a supplement to The Journal of Nutrition. Presented as part of the conference ‘‘The Use and Misuse of Biomarkers as Indicators of Cancer Risk Reduction Following Dietary Manipulation’’ held July 12–13, 2005 in Bethesda, MD. This conference was sponsored by the Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration (FDA), Department of Health and Human Services (DHHS); the Office of Dietary Supplements (ODS), National Institutes of Health, DHHS; and the Division of Cancer Prevention (DCP), National Cancer Institute, National Institutes of Health, DHHS. Guest Editors for the supplement publication were Harold E. Seifried, National Cancer Institute, NIH; and Claudine Kavanaugh, CFSAN, FDA. Guest editor disclosure: H.E. Seifried, no relationships to disclose; C. Kavanaugh, no relationships to disclose. 2 This work was supported by grants from the Auckland Cancer Society. The contents are solely the responsibility of the authors. 3 Author disclosure: no relationships to disclose. 4 Abbreviations used: FPG, formamidopyrimidine DNA glycosylase; LPO, lipoid peroxidation; ORAC, oxygen radical absorbance activity; RNS, reactive nitrogen species; ROS, reactive oxygen species; TEAC, Trolox equivalent antioxidant activity. * To whom correspondence should be addressed. E-mail: ferguson@auckland.ac.nz.