Quantitative assessment of cell population diversity in single-cell landscapes.

Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and definition of altered cell infiltrates in tumorigenesis, and benchmarking batch-correction tools. sc-UniFrac provides a framework for quantifying diversity or alterations in cell populations across conditions and has broad utility for gaining insight into tissue-level perturbations at the single-cell resolution.

Cultural adaptation to Brazilian Portuguese of the Face, Legs, Activity, Cry, Consolability revised (FLACCr) scale of pain assessment / Adaptação cultural para o português do Brasil da escala de avaliação de dor Face, Legs, Activity, Cry, Consolability revised(FLACCr) / Adaptación cultural para el portugués de Brasil de la escala de evaluación del dolor Face, Legs, Activity, Cry, Consolability revised(FLACCr)

AbstractObjective: to perform the translation into Brazilian Portuguese and cultural adaptation of the Face, Legs, Activity, Cry, Consolability revised (FLACCr) scale, with children under 18 years old, affected by cerebral palsy, presenting or not cognitive impairment and unable to report their pain.Method: methodological development study of translation into Portuguese and cultural adaptation of the FLACCr. After approval by the ethics committee, the process aimed at translation and back-translation, evaluation of translation and back-translation using the Delphi technique and assessment of cultural equivalence. The process included the five categories of the scale and the four application instructions, considering levels of agreement equal to or greater than 80%.Results: it was necessary three rounds of the Delphi technique to achieve consensus among experts. The agreement achieved for the five categories was: Face 95.5%, Legs 90%, Activity 94.4%, Cry 94.4% and Consolability 99.4%. The four instructions achieved the following consensus levels: 1st 99.1%, 2nd 99.2%, 3rd 99.1% and 4th 98.3%.Conclusion: the method enabled the translation and cultural adaptation of the FLACCr. This is a study able to expand the knowledge of Brazilian professionals on pain assessment in children with CP.

ResumoObjetivo:realizar a tradução para a língua portuguesa do Brasil e adaptação cultural da escala Face, Legs, Activity, Cry, Consolability revised(FLACCr), com crianças de até 18 anos de idade, acometidas por paralisia cerebral, apresentando ou não comprometimento cognitivo e impossibilitadas de relatar sua dor.Método:estudo de desenvolvimento metodológico de tradução para o português e adaptação cultural da FLACCr. Após aprovação do comitê de ética, o processo contemplou tradução e retrotradução, avaliação da tradução e da retrotradução utilizando a técnica de Delphi e avaliação da equivalência cultural. O processo incluiu as cinco categorias da escala e as quatro orientações de aplicação, considerando nível de concordância igual ou maior a 80%.Resultados:foram necessários três ciclos da técnica de Delphi para consenso entre os juízes. A concordância obtida para as cinco categorias foi: Face 95,5%, Pernas 90%, Atividade 94,4%, Choro 94,4% e Consolabilidade 99,4%. As quatro orientações alcançaram os seguintes níveis de consenso: 1ª 99,1%, 2ª 99,2%, 3ª 99,1% e 4ª 98,3%.Conclusão:o método possibilitou o desenvolvimento da tradução e adaptação cultural da FLACCr. Sendo um estudo capaz de ampliar o conhecimento de profissionais brasileiros sobre a avaliação da dor em crianças com PC.

ResumenObjetivo:realizar la traducción para el portugués de Brasil y la adaptación cultural de la escala, Face, Legs, Activity, Cry, Consolability revised(FLACCr), con niños menores de 18 años de edad, afectados por la parálisis cerebral, presentando o no deterioro cognitivo y que no pueden comunicar su dolor.Método:estudio de desarrollo metodológico de traducción al portugués y adaptación cultural de la FLACCr. Después de la aprobación por el comité de ética, el proceso incluyó la traducción y retrotraducción, evaluación de la traducción y retrotraducción utilizando la técnica Delphi y evaluación de la equivalencia cultural. El proceso incluyó las cinco categorías de la escala y las cuatro orientaciones de aplicación, teniendo en cuenta nivel de concordancia igual o superior al 80%.Resultados:fueron necesarios tres ciclos de la técnica Delphi para el consenso entre los jueces. La concordancia obtenida para las cinco categorías fue: Cara 95,5%, Piernas 90%, Actividad 94,4%, Llanto 94,4% y Capacidad de Consuelo 99,4%. Las cuatro orientaciones alcanzaron los siguientes niveles de consenso: 1ª 99,1%, 2ª 99,2%, 3ª 99,1% y 4ª 98,3%.Conclusión:el método permitió el desarrollo de la traducción y adaptación cultural de la FLACCr. Este estudio fue capaz de aumentar el conocimiento de los profesionales brasileños en la evaluación del dolor en niños con PC.

Temporal concordance between apical and transcriptional points of departure for chemical risk assessment.

The number of legacy chemicals without toxicity reference values combined with the rate of new chemical development is overwhelming the capacity of the traditional risk assessment paradigm. More efficient approaches are needed to quantitatively estimate chemical risks. In this study, rats were dosed orally with multiple doses of six chemicals for 5 days and 2, 4, and 13 weeks. Target organs were analyzed for traditional histological and organ weight changes and transcriptional changes using microarrays. Histological and organ weight changes in this study and the tumor incidences in the original cancer bioassays were analyzed using benchmark dose (BMD) methods to identify noncancer and cancer points of departure. The dose-response changes in gene expression were also analyzed using BMD methods and the responses grouped based on signaling pathways. A comparison of transcriptional BMD values for the most sensitive pathway with BMD values for the noncancer and cancer apical endpoints showed a high degree of correlation at all time points. When the analysis included data from an earlier study with eight additional chemicals, transcriptional BMD values for the most sensitive pathway were significantly correlated with noncancer (r = 0.827, p = 0.0031) and cancer-related (r = 0.940, p = 0.0002) BMD values at 13 weeks. The average ratio of apical-to-transcriptional BMD values was less than two, suggesting that for the current chemicals, transcriptional perturbation did not occur at significantly lower doses than apical responses. Based on our results, we propose a practical framework for application of transcriptomic data to chemical risk assessment.

In vivo identification and specificity assessment of mRNA markers of hypoxia in human and mouse tumors.

BACKGROUND: Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers. METHODS: Mice carrying human (FaDudd) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared. RESULTS: In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDudd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker. CONCLUSIONS: The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.

In vivo functional and anatomic imaging for assessment of in vivo gene transfer.

PURPOSE: To assess whether a combination of in vivo anatomic and functional imaging can help quantify expression of somatostatin receptor type 2 (SSTR2)-based reporters after in vivo gene transfer. MATERIALS AND METHODS: All animal experiments were approved by an institutional animal care and use committee. Six nude mice bearing two subcutaneous L3.6pl (human pancreatic cancer) tumors were injected intratumorally with an adenovirus containing a human somatostatin receptor type 2 gene chimera (Ad-HA-SSTR2) or a control adenovirus containing green fluorescent protein (Ad-GFP). Two days later, magnetic resonance (MR) imaging was performed to derive tumor weight and analyze morphology. Intravenous injection of Food and Drug Administration-approved indium 111 octreotide was followed by gamma camera imaging (planar imaging and single photon emission computed tomography [SPECT]) the next day. Region-of-interest analysis followed. The procedure was also performed in six nude mice with slow-growing MDA-MB-435 (human breast carcinoma) tumors, which allowed serial imaging 3 days and 2 weeks after adenovirus injection. After imaging, excised tumor weight and biodistribution were assessed. Statistical analyses included a Student t test and linear regression. RESULTS: With both tumor types, ex vivo and image-based in vivo biodistribution demonstrated greater uptake (percentage of injected dose per gram) in tumors infected with Ad-HA-SSTR2 than in those infected with Ad-GFP (P < .05). Furthermore, in vivo and ex vivo biodistribution analysis correlated (ex vivo vs planar and MR imaging: r = 0.87, P < .05, n = 24; ex vivo vs SPECT and MR imaging: r = 0.84, P < .05, n = 24). Moreover, in vivo biodistribution distinguished greater expression at the earlier time point in MDA-MB-435 tumors infected with Ad-HA-SSTR2 from waning expression at the later time point (P < .05). CONCLUSION: A combination of in vivo functional and anatomic imaging methods can help quantify gene expression after in vivo gene transfer.

Graph models for phenotype and genotype association between oral mucosa and submandibular gland tumorigenesis in rat.

BACKGROUND: In recent years, success of statistics in field of genetics has been the identification of genes that affect the process of disease. Experimental models using animals enable early stages of tumor development to be studied. The aim of this study was to apply graph models to assess the association between the observed phenotypic changes in rat oral mucosa and induced tumorigenesis in the submandibular gland (SMG). MATERIALS AND METHODS: We studied changes in oncogenes TP53 and bcl-2, histopathological and immunomarker variables in samples of oral mucosa and SMG of Wistar male rats, 60 days old and 180 g in weight, in which tumorigenesis was induced in their SMG by a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene in acetone. A set of linear structural equations were defined, with each formula indicating the response variables and the direct influences. In graph models, saliva was considered as a latent variable. The association was analyzed using Graphical Gaussian Markov models and odd ratios. RESULTS: About 40% of animals treated with 9, 10-dimethyl-1, 2-benzanthracene showed histological alterations in the epithelial basal strata of their oral mucosa only at 150 days. Statistical models indicated a relationship between gene alteration in gene bcl-2 in the SMG and histological changes observed in the oral mucosa (P = 0.04). CONCLUSION: Graph statistical model with one latent variable allows to conclude that these results associated with other clinical parameters may be useful in detecting early changes in SMG tumorigenesis. Furthermore, the design of randomized sampling of oral mucosa allows to validate these results and establish a reliable methodology for presumptive diagnosis or screening in the future.

Genetic assessment of the importance of galectin-3 in cancer initiation, progression, and dissemination in mice.

The galectin family of beta-galactoside binding lectins is involved in normal and pathological processes. Altered expression of galectin-3 has been described in many cancers, and studies of cancer cell lines have implicated this lectin in various aspects of the tumorigenic cascade. The goal of this report was to directly assess the importance of galectin-3 in tumor biology by introducing the galectin-3 null mutation (galectin-3(-/-)) into mouse lines genetically programmed to develop cancers. We used two mouse models of human intestinal cancer, the Apc(Min) and Apc(1638N) lines, to study tumor initiation and tumor progression. We also crossed the galectin-3(-/-) mice with PyMT transgenic animals, a model in which primary mammary gland tumors give rise to lung metastases at high frequency. Unexpectedly, we show that the absence of galectin-3 does not affect the evolution of the disease in any of these three situations.

Approaches to the risk assessment of genotoxic carcinogens in food: a critical appraisal.

The present paper examines the particular difficulties presented by low levels of food-borne DNA-reactive genotoxic carcinogens, some of which may be difficult to eliminate completely from the diet, and proposes a structured approach for the evaluation of such compounds. While the ALARA approach is widely applicable to all substances in food that are both carcinogenic and genotoxic, it does not take carcinogenic potency into account and, therefore, does not permit prioritisation based on potential risk or concern. In the absence of carcinogenicity dose-response data, an assessment based on comparison with an appropriate threshold of toxicological concern may be possible. When carcinogenicity data from animal bioassays are available, a useful analysis is achieved by the calculation of margins of exposure (MOEs), which can be used to compare animal potency data with human exposure scenarios. Two reference points on the dose-response relationship that can be used for MOE calculation were examined; the T25 value, which is derived from linear extrapolation, and the BMDL10, which is derived from mathematical modelling of the dose-response data. The above approaches were applied to selected food-borne genotoxic carcinogens. The proposed approach is applicable to all substances in food that are DNA-reactive genotoxic carcinogens and enables the formulation of appropriate semi-quantitative advice to risk managers.

Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia.

DNA methylation is associated with malignant transformation, but limitations imposed by genetic variability, tumor heterogeneity, availability of paired normal tissues and methodologies for global assessment of DNA methylation have limited progress in understanding the extent of epigenetic events in the initiation and progression of human cancer and in identifying genes that undergo methylation during cancer. We developed a mouse model of T/natural killer acute lymphoblastic leukemia that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We used restriction landmark genomic scanning with this mouse model of preleukemia reproducibly progressing to leukemia to show that specific genomic methylation is associated with only the leukemic phase and is not random. We also identified Idb4 as a putative tumor-suppressor gene that is methylated in most mouse and human leukemias but in only a minority of other human cancers.

Messenger RNA-based vaccines.

RNA is the only molecule known to recapitulate all biochemical functions of life: definition, control and transmission of genetic information, creation of defined three-dimensional structures, enzymatic activities and storage of energy. Because of its versatility and thanks to several recent scientific breakthroughs, RNA became the focus of intense research in molecular medicine at the beginning of the millennium. In particular, mRNA can be seen as a safe and efficient alternative to protein-, recombinant virus- or DNA-based therapies in the field of vaccination. This review summarises the most remarkable advances in this area and presents the advantages and limits of the five different mRNA-based vaccination methods. The paper will present the official, industrial and financial aspects of mRNA-based vaccination that are paving the way for therapeutic and prophylactic drugs with mRNA as the active component.

Use of transgenic mice in carcinogenicity hazard assessment.

Determining the carcinogenic potential of materials to which humans have significant exposure is an important, complex and imperfect exercise. Not only are the methods for such determinations protracted, expensive and utilize large numbers of animals, extrapolation of data from such studies to human risk is imprecise. Toxicologists have long recognized these shortcomings but the 2-year chronic rodent study has remained the gold standard. Recent developments in the field of molecular oncology and development of methods to insert or inactivate specific genes in animals have provided the tools with which to develop the next generation of carcinogenicity assays. With improved understanding of oncogene activation and tumor suppressor gene inactivation a number of animal models have been developed to dramatically reduce latency for chemically induced cancers. This has led to the development of shorter carcinogenicity assays. Also, because the spontaneous tumor frequencies in these animals are low during the in-life portion of the study, and studies are terminated well before the health complications of advanced aging are observed, it has been possible to reduce the group sizes and reduce animal usage. FDA's adoption of ICH S1B in 1997, (ICH, 1997) "Testing for the Carcinogenicity of Pharmaceuticals," opened the door for the use of such transgenic models in regulatory toxicology. This presentation reviews the current state of the science and its application to regulatory issues.

Background and framework for ILSI's collaborative evaluation program on alternative models for carcinogenicity assessment. International Life Sciences Institute.

The willingness of the agencies involved in the regulation of pharmaceuticals to accept data from newly proposed models for carcinogenicity testing (eg, transgenic animals, neonatal rodent models, initiation-promotion models) has stimulated international interest in gaining experience and a greater understanding of the strengths and limitations of the specific models. Over a 4-year period, the International Life Sciences Institute (ILSI) Health and Environmental Science Institute (HESI) has coordinated a large-scale collaborative research program to help to better characterize the responsiveness of several models proposed for use in carcinogenicity assessment. The overall objective of this partnership among industry, government, and academic scientists was to evaluate the ability of these new models to provide useful information for human cancer risk assessment. This research program reflected a commitment of nearly US$35 million by over 50 industrial, govemment, and academic laboratories from the United States, Europe, and Japan. Evaluation of the models required the development of standardized protocols to allow reproducibility and comparability of data obtained across multiple laboratories. Test compounds were selected on the basis of mechanistically meaningful carcinogenic activity or noncarcinogenicity in the rodent bioassay as well as humans. Criteria were established for dose selection, pathology review, quality control, and for evaluation of study outcome. The database from these studies represents an important contribution to the future application of new models for human cancer risk assessment. Beyond the data, the collaborative process by which the models were evaluated may also represent a prototype for assessing new methods in the future.

Panel discussion on the application of alternative models to cancer risk assessment.

The International Life Sciences Institute Alternative Carcinogenicity Testing (ILSI ACT) Workshop concluded with a panel discussion that addressed the framework issue of the appropriate application of alternative models to human cancer risk assessment. This discussion encompassed both technical issues relating to the level of understanding of these models and their output as well as issues relating to the regulatory acceptance of these data. Although there were many different perspectives represented by the panelists, there was also significant consensus on many broad issues. This article focuses on several key areas of emphasis that were addressed by panelists and are considered critical issues for future discussions and evaluations.

Use of transgenic animals for carcinogenicity testing: considerations and implications for risk assessment.

Advances in genetic engineering have created opportunities for improved understanding of the molecular basis of carcinogenesis. Through selective introduction, activation, and inactivation of specific genes, investigators can produce mice of unique genotypes and phenotypes that afford insights into the events and mechanisms responsible for tumor formation. It has been suggested that such animals might be used for routine testing of chemicals to determine their carcinogenic potential because the animals may be mechanistically relevant for understanding and predicting the human response to exposure to the chemical being tested. Before transgenic and knockout mice can be used as an adjunct or alternative to the conventional 2-year rodent bioassay, information related to the animal line to be used, study design, and data analysis and interpretation must be carefully considered. Here, we identify and review such information relative to Tg.AC and rasH2 transgenic mice and p53+/- and XPA-/- knockout mice, all of which have been proposed for use in chemical carcinogenicity testing. In addition, the implications of findings of tumors in transgenic and knockout animals when exposed to chemicals is discussed in the context of human health risk assessment.

Current status and use of short/medium term models for carcinogenicity testing of pharmaceuticals--scientific perspective.

Short- and medium-term rodent bioassays have been proposed under ICH guidelines for use in testing for the carcinogenic potential of pharmaceuticals. Further evaluation of these models is needed urgently and coordinated efforts are in progress worldwide to expand the available database. Models currently being investigated include transgenic mice (Tg-rasH2, Tg.AC, p53(+/-), XPA(-/-)) and neonatal mice. As more data become available on the performance of these assays, regulatory and industry scientists will be faced with the difficult challenge of determining how the performance (accuracy) of each assay will be measured and deciding which assays have value in the risk assessment process.

Effects of streptococcal preparation OK-432 on cytokine induction in spleen and tumour tissues of mice bearing MH-134 tumour cells.

Streptococcal preparation OK-432 is a bacterial immunopotentiator extensively used in Japan for adjuvant cancer therapy. Using C3H/He mice bearing MH-134 tumour cells, cytokine inductions of tumour necrosis factor-alpha, interleukin 1 beta, interleukin 6 and interferon-gamma were determined in spleen and tumour tissues by reverse transcriptase-polymerase chain reaction analysis. No significant induction of cytokine mRNA was observed after subcutaneous administration of OK-432 (OK-432, s.c.) or after intratumoural injection of IFN-gamma (IFN-gamma, i.t.), compared with controls, either in spleen or tumour tissues. In contrast, subcutaneous administration of OK-432 followed by intratumoural OK-432 injection (OK-432, s.c. + i.t.) was found to induce some cytokine mRNAs significantly. The mRNA levels of tumour necrosis factor alpha and interferon-gamma in spleen tissue and those of interleukin 1 beta and interferon-gamma in tumour tissues were significantly elevated in mice with OK-432, s.c. + i.t. treatment compared with controls. These results suggest that treatment with OK-432, s.c. + i.t. effectively induced splenic antitumour immunity as well as local immunity against tumour cells.

What is the significance of increases in background levels of carcinogen-derived protein and DNA adducts? Some considerations for incremental risk assessment.

Improvements in analytical methodology have led to the detection and quantification of 'background' levels of a number of DNA and protein adducts. Many of these adducts are derived from 'low molecular weight' reactive species which may be generated during normal physiological processes, metabolic pathways or inflammatory processes. The adducts have been detected using gas chromatography-mass spectrometry, HPLC in combination with various detection systems, 32P-postlabelling and immunoassay methods. The reliability and accuracy of many widely used methods for adduct measurements are discussed with reference to several examples where human data is available, namely 4-aminobiphenyl, malondialdehyde, methylating agents, ethylene oxide and hydroxyl radical damage. The accurate and specific quantitation of 'background' levels of damage is essential if reliable estimates of increases in risk associated with incremental increases in exposure to exogenous agents are to be calculated. In experimental studies using low dose exposures to carcinogens, such as N-nitrosodimethylamine, adduct levels in liver correlate closely with tumour incidence. In all likelihood, such relationships need to be established for each exposure and, in order to be relevant to human risk assessment, need to take into account factors such as DNA repair and mutagenic efficiency. Finally, in order to estimate the increase in cancer attributable to a given level of external exposure, it is clearly important to establish background levels of corresponding DNA damage so that the scale of the incremental increase can be calculated.

Gene knockout and transgenic technologies in risk assessment: the next generation.

Transgenic and knockout mice have been proposed as substitutes for one of the standard 2-yr rodent assays. The advantages of using genetically engineered mouse models is that fewer mice are needed, the time to develop disease is greatly reduced, and the mice are predisposed to developing cancer by virtue of gain or loss of functions. The models currently being used have yielded a large amount of data and have proved to be informative for risk assessment; however, they are still far from ideal. In fact, they inherently do not reflect the complexity of mutation and carcinogenesis in humans. Recent advances in technology and the creation of new knockout mice may produce more useful and more sensitive models. This review covers two recent advances in technology--inducible and regulatable gene expression and targeted genetic modifications in the genome--that will allow us to make better models. I also discuss new gene deletion and transgenic mouse models and their potential impact on risk-assessment assays. These models are presented in the context of four basic components or events that occur in the multistep process leading to cancer: maintenance of gene expression patterns, genome stability and DNA repair, cell-cell communication and signaling, and cell-cycle regulation. Finally, surrogate markers and utility in risk assessment are also discussed. This review is meant to stimulate further discussion in the field and to generate excitement about working toward the next generation of risk-assessment models.

Epigenetic carcinogens: evaluation and risk assessment.

Regulatory policies in the U.S. have been developed based upon a single model of cancer causation, which assumes chemical-induced genetic alterations. Such a model predicts some degree of cancer risk even at extremely low exposure levels. Many chemicals that produce tumors in experimental animals have been shown to act by epigenetic mechanisms that do not involve an attack by the chemical on DNA leading to subsequent genetic alteration. Such indirect mechanisms require prolonged exposures to high levels of chemicals for the production of tumors. For chemicals that are carcinogenic in this manner, the cancer mechanism would not be operative at exposures below a threshold at which the relevant cellular effect does not occur. Also, in contrast to DNA-reactive mechanisms, epigenetic effects may be unique to the rodent species used for testing. Certain chemical tumorigens have been well studied and provide examples for the use of mechanistic information in risk assessment. Butylated hydroxyanisole and saccharin are nongenotoxic food additives for which no risk to humans is predicted based upon low exposure levels and the likelihood that humans are either insensitive or much less sensitive to the tumorigenic effects found in rodent test species. For another non-genotoxic food additive d-limonene, the mechanism that underlies kidney tumor development in male rats is not expected to be operative in humans at all. The pharmaceutical phenobarbital represents a large group of non-genotoxic liver microsome enzyme inducers, which produce liver cancer in mice at levels that are near to therapeutic doses in humans. Epidemiology studies have not shown phenobarbital-related tumors in humans, indicating that humans may be less sensitive to the effects of phenobarbital. The mechanistic considerations involved in the risk assessment of these agents demonstrate that humans are not at risk from current exposure levels of many epigenetic carcinogens.