Critical factors in assessing risk from exposure to nasal carcinogens

Anatomical, physiological, biochemical and molecular factors that contribute to chemical-induced nasal carcinogenesis are either largely divergent between test species and humans, or we know very little of them. These factors, let alone the uncertainty associated with our knowledge gap, present a risk assessor with the formidable task of making judgments about risks to human health from exposure to chemicals that have been identified in rodent studies to be nasal carcinogens. This paper summarizes some of the critical attributes of the hazard identification and dose–response aspects of risk assessments for nasal carcinogens that must be accounted for by risk assessors in order to make informed decisions. Data on two example compounds, dimethyl sulfate and hexamethylphosphoramide, are discussed to illustrate the diversity of information that can be used to develop informed hypotheses about mode of action and decisions on appropriate dosimeters for interspecies extrapolation. Default approaches to interspecies dosimetry extrapolation are described briefly and are followed by a discussion of a generalized physiologically based pharmacokinetic model that, unlike default approaches, is flexible and capable of incorporating many of the critical species-specific factors. Recent advancements in interspecies nasal dosimetry modeling are remarkable. However, it is concluded that without the development of research programs aimed at understanding carcinogenic susceptibility factors in human and rodent nasal tissues, development of plausible modes of action will lag behind the advancements made in dosimetry modeling.     

Differences in Metabolism of Chemical Carcinogens in Cultured Human Epithelial Tissues and Cells

The metabolism of chemical carcinogens has been studied in cultured human bronchus, colon, duodenum, pancreatic duct, and esophagus. Metabolite patterns and carcinogen-DNA adducts are generally qualitatively similar among animal species, individuals within a species, and tissues within an individual. However, wide quantitative differences are observed between individuals in out-bred animal species, including humans. These interindividual differences in amounts of carcinogen-DNA adducts and in activities of enzymes that are important in the metabolism of chemical carcinogens are similar in magnitude (10-to 150-fold) to those observed in pharmacogenetic studies of drug metabolism. The role of these differences as risk factors in human cancer is being investigated.     

Who's related to whom? Recent results from molecular systematic studies

Similarities among model systems can lead to generalizations about plants, but understanding the differences requires systematic data. Molecular phylogenetic analyses produce results similar to traditional classifications in the grasses (Poaceae), and relationships among the cereal crops are quite clear. Chloroplast-based phylogenies for the Solanaceae show that tomato is best considered as a species of Solanum, closely related to potatoes. Traditional classifications in the Brassicaceae are misleading with regard to true phylogenetic relationships and data are only now beginning to clarify the situation. Molecular data are also being used to revise our view of relationships among flowering plant families. Phylogenetic data are critical for interpreting hypotheses of the evolution of development.     

Flavonoids and alkenylbenzenes: mechanisms of mutagenic action and carcinogenic risk

The present review focuses on the mechanisms of mutagenic action and the carcinogenic risk of two categories of botanical ingredients, namely the flavonoids with quercetin as an important bioactive representative, and the alkenylbenzenes, namely safrole, methyleugenol and estragole. For quercetin a metabolic pathway for activation to DNA-reactive species may include enzymatic and/or chemical oxidation of quercetin to quercetin ortho-quinone, followed by isomerisation of the ortho-quinone to quinone methides. These quinone methides are suggested to be the active alkylating DNA-reactive intermediates. Recent results have demonstrated the formation of quercetin DNA adducts in exposed cells in vitro. The question that remains to be answered is why these genotoxic characteristics of quercetin are not reflected by carcinogenicity. This might in part be related to the transient nature of quercetin quinone methide adducts, and suggests that stability and/or repair of DNA adducts may need increased attention in in vitro genotoxicity studies. Thus, in vitro mutagenicity studies should put more emphasis on the transient nature of the DNA adducts responsible for the mutagenicity in vitro, since this transient nature of the formed DNA adducts may play an essential role in whether the genotoxicity observed in vitro will have any impact in vivo. For alkenylbenzenes the ultimate electrophilic and carcinogenic metabolites are the carbocations formed upon degradation of their 1'-sulfooxy derivatives, so bioactivation of the alkenylbenzenes to their ultimate carcinogens requires the involvement of cytochromes P450 and sulfotransferases. Identification of the cytochrome P450 isoenzymes involved in bioactivation of the alkenylbenzenes identifies the groups within the population possibly at increased risk, due to life style factors or genetic polymorphisms leading to rapid metaboliser phenotypes. Furthermore, toxicokinetics for conversion of the alkenylbenzenes to their carcinogenic metabolites and kinetics for repair of the DNA adducts formed provide other important aspects that have to be taken into account in the high to low dose risk extrapolation in the risk assessment on alkenylbenzenes. Altogether the present review stresses that species differences and mechanistic data have to be taken into account and that new mechanism- and toxicokinetic-based methods and models are required for cancer risk extrapolation from high dose experimental animal data to low dose carcinogenic risks for man.     

Understanding the role of xenobiotic-metabolism in chemical carcinogenesis using gene knockout mice

Most chemical carcinogens require metabolic activation to electrophilic metabolites that are capable of binding to DNA and causing gene mutations. Carcinogen metabolism is carried out by large groups of xenobiotic-metabolizing enzymes that include the phase I cytochromes P450 (P450) and microsomal epoxide hydrolase, and various phase II transferase enzymes. It is extremely important to determine the role P450s play in the carcinogenesis and to establish if they are the rate limiting and critical interface between the chemical and its biological activities. The latter is essential in order to validate the use of rodent models to test safety of chemicals in humans. Since there are marked species differences in expressions and catalytic activities of the multiple P450 forms that activate carcinogens, this validation process becomes especially difficult. To address the role of P450s in whole animal carcinogenesis, mice were produced that lack the P450s known to catalyze carcinogen activation. Mouse lines having disrupted genes encoding the P450s CYP1A2, CYP2E1, and CYP1B1 were developed. Mice lacking expression of microsomal epoxide hydrolase (mEH) and NADPH-quinone oxidoreductase (NQO1) were also made. All of these mice exhibit no gross abnormal phenotypes, suggesting that the xenobiotic-metabolizing enzymes have no critical roles in mammalian development and physiological homeostasis. This explains the occurrence of polymorphisms in xenobiotic-metabolizing enzymes among humans and other mammalian species. However, these null mice do show differences in sensitivities to acute chemical toxicities, thus establishing the importance of xenobiotic metabolism in activation pathways that lead to cell death. Rodent bioassays using null mice and known genotoxic carcinogens should establish whether these enzymes are required for carcinogenesis in an intact animal model. These studies will also provide a framework for the production of transgenic mice and carcinogen bioassay protocols that may be more predictive for identifying the human carcinogens and validate the molecular epidemiological studies ongoing in humans that seek to establish a role for polymorphisms in cancer risk.     

Consideration of both genotoxic and nongenotoxic mechanisms in predicting carcinogenic potential

Bacterial and cell culture genotoxicity assays have proven to be valuable in the identification of DNA reactive carcinogens because mutational events that alter the activity or expression of growth control genes are a key step in carcinogenesis. The addition of metabolizing enzymes to these assays have expanded the ability to identify agents that require metabolic activation. However, chemical carcinogenesis is a complex process dependent on toxicokinetics and involving at least steps of initiation, promotion and progression. Identification of those carcinogens that are activated in a manner unique to the whole animal, such as 2,6-dinitrotoluene, require in vivo genotoxicity assays. There are many different classes of non-DNA reactive carcinogens ranging from the potent promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that acts through a specific receptor, to compounds that alter growth control, such as phenobarbital. Many compounds, such as saccharin, appear to exhibit initiating, promotional and/or carcinogenic activity as events secondary to induced cytotoxicity and cell proliferation seen only at the chronic lifetime maximum tolerated doses mandated in rodent bioassays. Simple plus/minus vs. carcinogen/noncarcinogen comparisons used to validate the predictivity of bacterial and cell culture genotoxicity assays have revealed that a more comprehensive analysis will be required to account for the carcinogenicity of so many diverse chemical agents. Predictive assays and risk assessments for the numerous types of nongenotoxic carcinogens will require understanding of their mechanism of action, reasons for target organ and species specificity, and the quantitative dose-response relationships between endpoints such as induced cell proliferation and carcinogenic potential.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper No. 5. Genotoxicity tests as predictors of carcinogens: an analysis

Differences between the results of numerical validation studies comparing in vitro and in vivo genotoxicity tests with the rodent cancer bioassay are leading to the perception that short-term tests predict carcinogenicity only with uncertainty. Consideration of factors such as the pharmacokinetic distribution of chemicals, the systems available for metabolic activation and detoxification, the ability of the active metabolite to move from the site of production to the target DNA, and the potential for expression of the induced lesions, strongly suggests that the disparate sensitivity of the different test systems is a major reason why numerical validation is not more successful. Furthermore, genotoxicity tests should be expected to detect only a subset of carcinogens, namely genotoxic carcinogens, rather than those carcinogens that appear to act by non-genetic mechanisms. Instead of relying primarily on short-term in vitro genotoxicity tests to predict carcinogenic activity, these tests should be used in a manner that emphasizes the accurate determination of mutagenicity or clastogenicity. It must then be determined whether the mutagenic activity is further expressed as carcinogenicity in the appropriate studies using test animals. The prospects for quantitative extrapolation of in vitro or in vivo genotoxicity test results to carcinogenicity requires a much more precise understanding of the critical molecular events in both processes.     

Prospects for applying genotypic selection of somatic oncomutation to chemical risk assessment.

Genotypic selection methods detect rare sequence changes in populations of DNA molecules. These methods have been used to investigate the chemical induction of mutation and for the detection and diagnosis of cancer. The possible use of genotypic selection for improving current risk assessment practices is based on the premise that the frequency of somatic mutation is of critical importance in understanding and modeling carcinogenesis. If genotypic selection can measure the induction of specific mutations that disrupt normal cell/tissue homeostasis, then it could provide key mechanistic information for cancer risk assessment. For example, genotypic selection data might support a particular low-dose extrapolation method or characterize the relationship between rodent and human cancer risk. Strategies for evaluating the use of genotypic selection in cancer risk assessment include the concept of developing a battery of targets that detect a range of agent-specific effects. Ideal targets to examine by genotypic selection are the oncogene and tumor suppressor gene mutations frequently detected in human tumors because these are thought to represent tumor-initiating events. The most commonly occurring basepair (bp) substitutions within the ras and p53 genes are identified. Also, the battery of genotypic selection methods is defined in terms of the most important mutational specificities to include. In theory, the major basepair substitution mutations induced by 29 of 31 chemical carcinogens could be detected by analyzing three different mutations: G:C-->T:A, G:C-->A:T, and A:T-->T:A. Genotypic selection will have the greatest impact on risk assessment if measurement of spontaneous mutation is possible. Data from phenotypic selection assays suggest this corresponds to detection of mutant fractions of approximately 10(-7), and this would necessitate examining DNA samples containing >10(7) target molecules. Despite its apparent potential, considerable development and validation is needed before genotypic selection data can be applied to cancer risk assessment.     

基于Rag1基因序列变异的鲤形目鱼类系统发育重建:采样策略对生命之树的影响

The phylogenetic relationships of species are fundamental to any biological investigation, including all evolutionary studies. Accurate inferences of sister group relationships provide the researcher with an historical framework within which the attributes or geographic origin of species (or supraspecific groups) evolved. Taken out of this phylogenetic context, interpretations of evolutionary processes or origins, geographic distributions, or speciation rates and mechanisms, are subject to nothing less than a biological experiment without controls. Cypriniformes is the most diverse clade of freshwater fishes with estimates of diversity of nearly 3,500 species. These fishes display an amazing array of morphological, ecological, behavioral, and geographic diversity and offer a tremendous opportunity to enhance our understanding of the biotic and abiotic factors associated with diversification and adaptation to environments. Given the nearly global distribution of these fishes, they serve as an important model group for a plethora of biological investigations, including indicator species for future climatic changes. The occurrence of the zebrafish, Danio rerio, in this order makes this clade a critical component in understanding and predicting the relationship between mutagenesis and phenotypic expressions in vertebrates, including humans. With the tremendous diversity in Cypriniformes, our understanding of their phylogenetic relationships has not proceeded at an acceptable rate, despite a plethora of morphological and more recent molecular studies. Most studies are pre-Hennigian in origin or include relatively small numbers of taxa. Given that analyses of small numbers of taxa for molecular characters can be compromised by peculiarities of long-branch attraction and nodal-density effect, it is critical that significant progress in our understanding of the relationships of these important fishes occurs with increasing sampling of species to mitigate these potential problems. The recent Cypriniformes Tree of Life initiative is an effort to achieve this goal with morphological and molecular (mitochondrial and nuclear) data. In this early synthesis of our understanding of the phylogenetic relationships of these fishes, all types of data have contributed historically to improving our understanding, but not all analyses are complementary in taxon sampling, thus precluding direct understanding of the impact of taxon sampling on achieving accurate phylogenetic inferences. However, recent molecular studies do provide some insight and in some instances taxon sampling can be implicated as a variable that can influence sister group relationships. Other instances may also exist but without inclusion of more taxa for both mitochondrial and nuclear genes, one cannot distinguish between inferences being dictated by taxon sampling or the origins of the molecular data.     

Tropical terrestrial invertebrates—Where to from here?

There are over one million described invertebrate species on Earth, the majority of which are likely to inhabit the highly biodiverse rain forests around the equator. These are some of the most vulnerable ecosystems on Earth due to the pressures of deforestation and climate change with many of their inhabitants at risk of extinction. Invertebrates play a major role in ecosystem functioning from decomposition and nutrient cycling to herbivory and pollination; however, while our understanding of these roles is improving, we are far from being able to predict the consequences of further deforestation, climate change, and biodiversity loss due to the lack of comparative data and the high proportion of species which remain to be discovered. As we move into an era of increased pressure on old-growth habitats and biodiversity, it is imperative that we understand how changes to invertebrate communities, and the extinction of species, affect ecosystems. Innovative and comprehensive methods that approach these issues are needed. Here, we highlight priorities for future tropical terrestrial invertebrate research such as the efficiency of sustainable land management, exploration of innovative methods for better understanding of invertebrate ecology and behavior, and quantifying the role of invertebrates in ecosystem functioning.     

Formation and analysis of heterocyclic aromatic amine-DNA adducts in vitro and in vivo

The detection and quantification of heterocyclic aromatic amine (HAA)-DNA adducts, critical biomarkers in interspecies extrapolation of toxicity data for human risk assessment, remains a challenging analytical problem. The two main analytical methods currently in use to screen for HAA-DNA adducts are the 32P-postlabeling assay and mass spectrometry, using either accelerated mass spectrometry (AMS) or liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS). In this review, the principal methods to synthesize and characterize DNA adducts, and the methods applied to measure HAA-DNA adduct in vitro and vivo are discussed.     

Carcinogen DNA adducts and the risk of colon cancer: case-control study

Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case-control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by ³²P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 10¹⁰ nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 10¹⁰ nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individual's internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833-15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.     

Setting Risk-Based Occupational Exposure Limits for No-Threshold Carcinogens

Several regulators have recently issued so-called risk-based occupational exposure limits for carcinogenic substances, and also reported estimates of the risk of fatality that exposure to the limit value would give rise to. This practice provides an opportunity to study how differences in the exposure limits set by different regulators are influenced by differences in the scientific judgment (what is the risk at different levels?) and in the policy judgment (how should large risks be accepted?). Based on a broad search, a list was compiled of exposure limits for carcinogens that the respective regulator associates with a numerical risk estimate. For benzene, such data was available from six regulators. The differences in estimates of the risk/exposure relationship and in risk tolerance were about equal in size for benzene, while the range for acceptability was somewhat wider. A similar pattern was observed, although less clearly, for substances with data from only two or three regulators. It is concluded that the science factor and the policy factor both contribute to differences in exposure limits for carcinogens. It was not possible to judge which of these two factors has the larger influence.     

What do we gain from simplicity versus complexity in species distribution models?

Species distribution models (SDMs) are widely used to explain and predict species ranges and environmental niches. They are most commonly constructed by inferring species' occurrence–environment relationships using statistical and machine‐learning methods. The variety of methods that can be used to construct SDMs (e.g. generalized linear/additive models, tree‐based models, maximum entropy, etc.), and the variety of ways that such models can be implemented, permits substantial flexibility in SDM complexity. Building models with an appropriate amount of complexity for the study objectives is critical for robust inference. We characterize complexity as the shape of the inferred occurrence–environment relationships and the number of parameters used to describe them, and search for insights into whether additional complexity is informative or superfluous. By building ‘under fit’ models, having insufficient flexibility to describe observed occurrence–environment relationships, we risk misunderstanding the factors shaping species distributions. By building ‘over fit’ models, with excessive flexibility, we risk inadvertently ascribing pattern to noise or building opaque models. However, model selection can be challenging, especially when comparing models constructed under different modeling approaches. Here we argue for a more pragmatic approach: researchers should constrain the complexity of their models based on study objective, attributes of the data, and an understanding of how these interact with the underlying biological processes. We discuss guidelines for balancing under fitting with over fitting and consequently how complexity affects decisions made during model building. Although some generalities are possible, our discussion reflects differences in opinions that favor simpler versus more complex models. We conclude that combining insights from both simple and complex SDM building approaches best advances our knowledge of current and future species ranges.     

Extrapolations are the Achilles heel of risk assessment

This Reflections article considers the problems associated with the various extrapolations that are required for the estimation of human cancer risks from exposure to environmental carcinogens at low doses. These include extrapolation between species (particularly rodent to human), from responses at high doses to those at low doses, and among different stages of life. Reductions in uncertainty in risk estimates are closely coupled to the ability to conduct reliable extrapolations. The best way forward appears to be the use of data on mechanisms of carcinogenesis to develop bioindicators of responses related to the pathway to tumor formation. Such an approach is proposed based on the phenotypes represented by the six acquired characteristics forming the Hanahan-Weinberg model for carcinogenesis (The Hallmarks of Cancer). In addition, approaches can be established that use the Hanahan-Weinberg model as the basis for the collection and/or analysis of microarray or similar data. The reduction in reliance on default options and safety factors in the risk assessment process is a real possibility.     

Yaks and sheep trigger different changes in the grasshopper assemblages of the Qilian Mountains via differentially altering plant assemblages

1. The Qilian Mountains represent one of the key livestock‐raising grasslands in China. The two main herbivore species raised in this area – yaks and sheep – are of critical economical value. Grasshoppers compete with these animals for available nutrients, creating multifaceted relationships between livestock, grasshoppers and plants. A clear understanding of such relationships is lacking and is urgently needed to guide conservation efforts. 2. This study aims to document the effects of yak and sheep grazing on grasshopper assemblages and to elucidate the underlying mechanisms of such effects. 3. It is shown here that yaks and sheep impact grasshopper assemblages differently. Grasshopper assemblages exhibited lower density, biodiversity, richness, and evenness of distribution in yak‐grazed pastures than in grazing‐free grasslands. Sheep‐grazed pastures exhibited a dramatically divergent picture, with elevated density, biodiversity and richness, and a slightly decreased evenness of distribution. Grasshoppers were generally larger in grazed pastures than in grazing‐free grasslands, especially in yak‐grazed plots. 4. The present study suggests that differences between yak and sheep pastures in plant assemblage structure and plant traits are probably the underlying forces driving the differences in grasshopper assemblage structure and grasshopper traits, respectively. 5. The study shows that the grasshopper habitat indicator species differ between yak and sheep pastures, raising the possibility that such indicators can be used to monitor grassland usage and degradation in the Qilian Mountains. 6. These results provide novel insights into the dynamic interactions of common domesticated herbivore species, grasshoppers and plants in Qilian Mountains, which augment current knowledge and may ultimately lead to better conservation practices.     

The effects of phenological mismatches on demography

Climate change is altering the phenology of species across the world, but what are the consequences of these phenological changes for the demography and population dynamics of species? Time-sensitive relationships, such as migration, breeding and predation, may be disrupted or altered, which may in turn alter the rates of reproduction and survival, leading some populations to decline and others to increase in abundance. However, finding evidence for disrupted relationships, or lack thereof, and their demographic effects, is difficult because the necessary detailed observational data are rare. Moreover, we do not know how sensitive species will generally be to phenological mismatches when they occur. Existing long-term studies provide preliminary data for analysing the phenology and demography of species in several locations. In many instances, though, observational protocols may need to be optimized to characterize timing-based multi-trophic interactions. As a basis for future research, we outline some of the key questions and approaches to improving our understanding of the relationships among phenology, demography and climate in a multi-trophic context. There are many challenges associated with this line of research, not the least of which is the need for detailed, long-term data on many organisms in a single system. However, we identify key questions that can be addressed with data that already exist and propose approaches that could guide future research.     

Epidemiological studies on radiation carcinogenesis in human populations following acute exposure: nuclear explosions and medical radiation.

The present review provides an understanding of our current knowledge of the carcinogenic effect of low-dose radiation in man, and surveys the epidemiological studies of human populations exposed to nuclear explosions and medical radiation. Discussion centers on the contributions of quantitative epidemiology to present knowledge, the reliability of the dose-incidence data, and those relevant epidemiological studies that provide the most useful information for risk estimation of cancer induction in man. Reference is made to dose-incidence relationships from laboratory animal experiments where they may obtain, for problems and difficulties in extrapolation from data obtained at high doses to low doses, and from animal data to the human situation. The paper describes the methods of application of such epidemiological data for estimation of excess risk of radiation-induced cancer in exposed human populations and discusses the strengths and limitations of epidemiology in guiding radiation protection philosophy and public health policy.     

Nutrition and carcinogenesis

Traditional views of nutritional carcinogenesis depend on the identification of exogenous carcinogens as major risk factors. As our understanding evolves, it is clear that the pattern of events involves not only exogenous carcinogens, but also metabolic processes and endogenous and exogenous anticarcinogens. The process is modulated by the immune system, and genetics plays a significant role. New monitoring methods provide much-needed tools for providing proof of involvement of various factors at the level of human populations.     

DNA adducts in humans as dosimeters of exposure to environmental, occupational, or dietary genotoxins.

The quantitation of adducts of genotoxins with DNA is probably one of the best indicators of genetic damage due to exposure to toxins or carcinogens. It is generally believed that such adducts can lead to mutations, which in turn can trigger the initiation of the carcinogenic process. DNA adducts have been quantitated in white blood cells and in various tissues of smokers, persons in certain high-exposure occupations, and persons consuming foods contaminated with certain carcinogens. The feasibility of this approach for biochemical epidemiologic studies has been demonstrated using methods such as 32P-postlabeling, enzyme-linked immunosorbent assay, and synchronous fluorescence spectrophotometry. Relatively large interindividual differences in DNA adducts have been observed in both exposed and nonexposed persons. As a result, there are only a few studies in which clear quantitative and qualitative differences between these two groups have been observed. In addition, it appears that in some studies the 32P-postlabeling method does not detect the presence of the polycyclic aromatic hydrocarbon DNA adducts that are detectable by immunoassays. More extensive studies in additional populations at risk should shed further light on the utility of DNA adduct analysis in biochemical monitoring, especially if further refinements in methodology would result in increased sensitivity and specificity.