Linking Pharmacokinetics and Biomarker Data to Mechanism of Action in Risk Assessment

Incorporating information on metabolism, pharmacokinetics, and DNA and protein biomarkers provides a means to integrate these important factors into the risk assessment process. Such data are useful for species to species extrapolation, high- to low-dose extrapolation and PBPK modeling. In addition, these data are critical for understanding the mode of action for chemical carcinogens. Through the use of mass spectrometry, stable isotopes can be used to unequivocally demonstrate pathways of formation of biomarkers and relationships between exogenous and endogenous processes. This paper reviews what has been learned for two carcinogens, vinyl chloride and butadiene. It is clear that such data play major roles in improving the understanding of how chemicals cause cancer, extending the range of data on exposure-response relationships, and examining interspecies differences and inter-individual differences that may affect susceptibility. As such, it is also clear that these data play a critical role in improving the accuracy of risk assessments.     

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.     

Interspecies extrapolation based on mechanistic determinants of chemical disposition

Uncertainty factors are applied in methods developed by the Environmental Protection Agency (EPA) to derive dose‐response estimates. The uncertainty factors are applied to account for uncertainties in defined extrapolations from the laboratory animal experimental data conditions to a dose‐response estimate appropriate for the assumed human scenario. The conceptual difference between these uncertainty factors and safety factors is best illustrated by how uncertainty factors can be modified as scientific data inform our understanding of the key factors that influence chemical disposition and toxicity. Mechanistic data help describe the major factors influencing chemical disposition and toxicant‐target tissue interactions, and should increase the accuracy of exposure‐dose‐response assessment. Mechanistic data on the determinants of inhaled chemical disposition were used to construct default dosimetry adjustments applied by the EPA in its inhalation Reference Concentration (RfC) methods. Because these adjustments account for interspecies dosimetric differences to some degree, the uncertainty factor for interspecies extrapolation was modified. A framework is presented that allows for incorporation of mechanistic data in order to ensure that required extrapolations are commensurate with the state‐of‐the‐science. Future applications of mechanistic data to modify additional uncertainty factors are outlined.     

Mutagenic activity of carcinogens detected in transgenic rodent mutagenicity assays at dose levels used in chronic rodent cancer bioassays

Data on transgenic rodent mutagenicity of five human carcinogens were summarised and compared with the results from rodent carcinogenicity studies. Four out of five carcinogens showed mutagenic activity already at daily dose levels which induced cancer in long-term rodent bioassays in at least one target tissue of carcinogenesis. In several of these studies, even single dose applications were sufficient to significantly increase the mutation frequency in vivo. Other genotoxic carcinogens required application of multiple dosing at dose-levels used in rodent cancer bioassays to show their in vivo mutagenicity. A rodent respiratory tract carcinogen, 1,2-dibromoethane (DBE), following inhalation exposure, displayed no mutagenic activity, neither in lung nor in nasal mucosa, at a single 2-h exposure to 30 ppm, which is below the highest concentration used in a NTP cancer bioassay. In contrast, after multiple treatment for 10 days at the same daily doses, a significant increase of the mutation frequency in nasal mucosa was apparent. We conclude, that especially when studying new chemicals in these transgenic rodent mutation assays, a multiple dosing protocol should be preferred. For dose selection, the same criteria could be applied as for chronic rodent bioassays.     

The Causes and Prevention of Cancer: The Role of Environment

The idea that synthetic chemicals such as DDT are major contributors to human cancer has been inspired, in part, by Rachel Carson's passionate book, Silent Spring. This chapter discusses evidence showing why this is not true. We also review research on the causes of cancer, and show why much cancer is preventable.Epidemiological evidence indicates several factors likely to have a major effect on reducing rates of cancer: reduction of smoking, increased consumption of fruits and vegetables, and control of infections. Other factors are avoidance of intense sun exposure, increases in physical activity, and reduction of alcohol consumption and possibly red meat. Already, risks of many forms of cancer can be reduced and the potential for further reductions is great. If lung cancer (which is primarily due to smoking) is excluded, cancer death rates are decreasing in the United States for all other cancers combined.Pollution appears to account for less than 1% of human cancer; yet public concern and resource allocation for chemical pollution are very high, in good part because of the use of animal cancer tests in cancer risk assessment. Animal cancer tests, which are done at the maximum tolerated dose (MTD), are being misinterpreted to mean that low doses of synthetic chemicals and industrial pollutants are relevant to human cancer. About half of the chemicals tested, whether synthetic or natural, are carcinogenic to rodents at these high doses. A plausible explanation for the high frequency of positive results is that testing at the MTD frequently can cause chronic cell killing and consequent cell replacement, a risk factor for cancer that can be limited to high doses. Ignoring this greatly exaggerates risks. Scientists must determine mechanisms of carcinogenesis for each substance and revise acceptable dose levels as understanding advances.The vast bulk of chemicals ingested by humans is natural. For example, 99.99% of the pesticides we eat are naturally present in plants to ward off insects and other predators. Half of these natural pesticides tested at the MTD are rodent carcinogens. Reducing exposure to the 0.01% that are synthetic will not reduce cancer rates. On the contrary, although fruits and vegetables contain a wide variety of naturally-occurring chemicals that are rodent carcinogens, inadequate consumption of fruits and vegetables doubles the human cancer risk for most types of cancer. Making them more expensive by reducing synthetic pesticide use will increase cancer. Humans also ingest large numbers of natural chemicals from cooking food. Over a thousand chemicals have been reported in roasted coffee: more than half of those tested (19/28) are rodent carcinogens. There are more rodent carcinogens in a single cup of coffee than potentially carcinogenic pesticide residues in the average American diet in a year, and there are still a thousand chemicals left to test in roasted coffee. This does not mean that coffee is dangerous but rather that animal cancer tests and worst-case risk assessment, build in enormous safety factors and should not be considered true risks.The reason humans can eat the tremendous variety of natural chemical "rodent carcinogens" is that humans, like other animals, are extremely well protected by many general defense enzymes, most of which are inducible (i.e., whenever a defense enzyme is in use, more of it is made). Since the defense enzymes are equally effective against natural and synthetic chemicals one does not expect, nor does one find, a general difference between synthetic and natural chemicals in ability to cause cancer in high-dose rodent tests.The idea that there is an epidemic of human cancer caused by synthetic industrial chemicals is false. In addition, there is a steady rise in life expectancy in the developed countries. Linear extrapolation from the maximum tolerated dose in rodents to low level exposure in humans has led to grossly exaggerated mortality forecasts.Such extrapolations can not be verified by epidemiology. Furthermore, relying on such extrapolations for synthetic chemicals while ignoring the enormous natural background, leads to an imbalanced perception of hazard and allocation of resources. It is the progress of scientific research and technology that will continue to lengthen human life expectancy.Zero exposure to rodent carcinogens cannot be achieved. Low levels of rodent carcinogens of natural origin are ubiquitous in the environment. It is thus impossible to obtain conditions totally free of exposure to rodent carcinogens or to background radiation. Major advances in analytical techniques enable the detection of extremely low concentrations of all substances, whether natural or synthetic, often thousands of times lower than could be detected 30 years ago.Risks compete with risks: society must distinguish between significant and trivial risks. Regulating trivial risks or exposure to substances erroneously inferred to cause cancer at low-doses, can harm health by diverting resources from programs that could be effective in protecting the health of the public. Moreover, wealth creates health: poor people have shorter life expectancy than wealthy people. When money and resources are wasted on trivial problems, society's wealth and hence health is harmed.     

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.     

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.     

How many high production chemicals are rodent carcinogens? Why should we care? What do we need to do about it?

High production volume (HPV) chemicals are produced in or imported to the US in amounts greater than 1 million pounds per chemical per year. The EPA has identified thousands of HPVs. Due to their abundance, such chemicals bring a substantial risk for human exposure, and as a result some level of adverse consequences to health are to be expected. In order to examine the potential for cancer risk associated with HPVs, this paper examines HPVs that have been tested in the National Toxicology Program's rodent cancer bioassay. The chemicals tested in the bioassay represent a small sample of the universe of environmental chemicals to which people are exposed. Unexpectedly, 60% of the 128 HPVs evaluated in the bioassay proved to be rodent carcinogens. This value compares to a predicted prevalence of only 16.5% carcinogens in a previous study. The previous study concluded that HPVs were less likely to be toxic by a variety of other test criteria as well. However, the approach involved identifying putative carcinogens using quantitative chemical structure-activity relationships (QSAR) in contrast to the direct tabulation of bioassay test results performed here. Detailed examination of bioassay results reveals that test outcomes depend heavily on route of administration as well as on dose level, sex, strain, and species used. Since most of these factors have little or no apparent relationship to chemical structure, results of this study suggest that QSAR, as well as virtually all other alternative methods, may not generally provide accurate predictions of carcinogenic potential. As we wait for efficient and effective methods for predicting carcinogens to be developed, people continue to be exposed to environmental carcinogens. Progress on regulating environmental carcinogens as well as on developing more effective test methods has been minimal since "war on cancer" began approximately 30 years ago. The present study questions whether the current strategy for dealing with environmental carcinogens will ever be successful. Close examination of rodent cancer test results seems to suggest that almost all chemicals may have carcinogenic potential in some genotypes under some exposure circumstances. If this hypothesis is correct, it would explain the general lack of progress in developing methods to differentiate carcinogens from noncarcinogens. A completely new strategy for dealing with cancer caused by exposures to environmental chemicals seems to be needed.     

Hemoglobin promotes Staphylococcus aureus nasal colonization

Staphylococcus aureus nasal colonization is an important risk factor for community and nosocomial infection. Despite the importance of S. aureus to human health, molecular mechanisms and host factors influencing nasal colonization are not well understood. To identify host factors contributing to nasal colonization, we collected human nasal secretions and analyzed their ability to promote S. aureus surface colonization. Some individuals produced secretions possessing the ability to significantly promote S. aureus surface colonization. Nasal secretions pretreated with protease no longer promoted S. aureus surface colonization, suggesting the involvement of protein factors. The major protein components of secretions were identified and subsequent analysis revealed that hemoglobin possessed the ability to promote S. aureus surface colonization. Immunoprecipitation of hemoglobin from nasal secretions resulted in reduced S. aureus surface colonization. Furthermore, exogenously added hemoglobin significantly decreased the inoculum necessary for nasal colonization in a rodent model. Finally, we found that hemoglobin prevented expression of the agr quorum sensing system and that aberrant constitutive expression of the agr effector molecule, RNAIII, resulted in reduced nasal colonization of S. aureus. Collectively our results suggest that the presence of hemoglobin in nasal secretions contributes to S. aureus nasal colonization.     

Machine learning methods without tears: a primer for ecologists

Machine learning methods, a family of statistical techniques with origins in the field of artificial intelligence, are recognized as holding great promise for the advancement of understanding and prediction about ecological phenomena. These modeling techniques are flexible enough to handle complex problems with multiple interacting elements and typically outcompete traditional approaches (e.g., generalized linear models), making them ideal for modeling ecological systems. Despite their inherent advantages, a review of the literature reveals only a modest use of these approaches in ecology as compared to other disciplines. One potential explanation for this lack of interest is that machine learning techniques do not fall neatly into the class of statistical modeling approaches with which most ecologists are familiar. In this paper, we provide an introduction to three machine learning approaches that can be broadly used by ecologists: classification and regression trees, artificial neural networks, and evolutionary computation. For each approach, we provide a brief background to the methodology, give examples of its application in ecology, describe model development and implementation, discuss strengths and weaknesses, explore the availability of statistical software, and provide an illustrative example. Although the ecological application of machine learning approaches has increased, there remains considerable skepticism with respect to the role of these techniques in ecology. Our review encourages a greater understanding of machin learning approaches and promotes their future application and utilization, while also providing a basis from which ecologists can make informed decisions about whether to select or avoid these approaches in their future modeling endeavors.     

In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH>13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague-Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3-6 and 22-26h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology. Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.     

Metabolism-dependent activation and toxicity of chemicals in nasal glands

The mucosae of the nasal passages contain a large amount of glands which express secretory proteins as well as phase I and phase II biotransformation enzymes. In this review the metabolic activation, covalent binding and toxicity of chemicals in the Bowman's glands in the olfactory mucosa, in the sero-mucous glands in the nasal septum and in the lateral nasal glands and maxillary glands around the maxillary sinuses are discussed. Light microscopic autoradiographic studies have demonstrated a selective covalent binding of nasal toxicants and carcinogens such as halogenated hydrocarbons and N-nitrosamines, especially in the Bowman's glands following a single systemic exposure, suggesting a high rate of metabolic activation of chemicals in these glands. Special attention is put on the herbicide dichlobenil which induces necrosis in the olfactory mucosa following a cytochrome-P450-mediated metabolic activation and covalent binding in the Bowman's glands.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC publication No. 13. The need for biological risk assessment in reaching decisions about carcinogens

The prudent assumption that carcinogen bioassays in rodents predict for human carcinogenicity is examined. It is suggested that in certain cases, as for example the induction of tumors against a high incidence in controls, or in situations in which high dose toxicity may be a critical factor in the induction of cancer, the probability that animal bioassays predict for humans may be low. The term 'biological risk assessment' is introduced to describe that part of risk assessment concerned with the relevance of specific animal results to the induction of human cancer. Biological risk assessment, which is almost entirely dependent on an understanding of carcinogenesis mechanisms, is an important addition to present mathematical modeling used to predict the effects of animal carcinogens that have been demonstrated after high dose exposure, to the effects of the much smaller doses to which humans are perceived to be exposed. Evidence for the conclusions reached by biological risk assessment may sometimes be supported by a careful review of human epidemiological data.     

Acrylamide: a review of its genotoxicity and an assessment of heritable genetic risk

An updated review of the genotoxicity studies with acrylamide is provided. Then, using data from the studies generating quantitative information concerning heritability of genetic effects, an assessment of the heritable genetic risk presented by acrylamide is presented. The review offers a discussion of the reactions and possible mechanisms of genotoxic action by acrylamide and its epoxide metabolite glycidamide. Several genetic risk approaches are discussed, including the parallelogram, direct (actually a modified direct), and doubling dose approaches. Using data from the specific-locus and heritable translocation assays, the modified direct and doubling dose approaches are utilized to quantitate genetic risk. Exposures of male parents to acrylamide via inhalation, ingestion, and dermal routes are also quantitated. With these approaches and measurements and their underlying assumptions concerning extrapolation factors (including germ cell stage specificity, DNA repair variability, locus specificity), number of human loci associated with dominant disease alleles, and spontaneous mutation rates, an assessment of heritable genetic risk for humans is calculated for the three exposure scenarios. The calculated estimates for offspring from fathers exposed to acrylamide via drinking water are up to three offspring potentially affected with induced genetic disease per 10⁸ offspring. Estimates for inhalation or dermal exposures suggest higher risks for induced genetic disease in offspring from fathers exposed in occupational settings.     

Study of genetic control of radiosensitivity

The results of radiation genetics studies are reviewed. The first series of studies concerned the role of heterogeneity of the human population for radiosensitivity of chromosomes in determining the pattern of dose-response relationships; correctness of extrapolation of averaged experimental data to low doses was demonstrated. In the second series of experiments, the radiation-induced adaptive response and the contribution of different factors, including genetic ones, to its formation in human cells were studied. A conclusion was made about impossibility of extrapolating data obtained for cell cultures to an organism as a whole or to a population. The third part of the study was of applied character: cytogenetic methods of biological dosimetry were used to estimate the doses of internal and external irradiation of children living on the territory of the Bryansk oblast contaminated after the Chernobyl accident. The results are discussed in the context of the present-day concepts of genetic control of sensitivity to environmental factors.     

Health Risk Assessment and Vapor Intrusion: A Review and Australian Perspective

Soil contamination by volatile hydrocarbons is of public health importance due to vapor intrusion and indoor inhalation exposures. These are assessed using measurement or predictive modeling and need to consider the key areas of subsurface partitioning and transport, dwelling ventilation, and receptor inhalation dosimetry. While subsurface partitioning and transport have been subject to intensive international investigation, limited consideration has been given to the latter. Building ventilation research has developed multi-zone airflow and contaminant dispersal models including AccuRate, an Australian model that examines natural ventilation modeling, roof and sub-floor ventilation, and identifies the importance of geometry and thermal factors on ventilation (the most sensitive variable) and indoor pollutant concentrations. Inhalation dosimetry has received recent attention due to concerns over child inhalation susceptibility and dose metrics. Research using coupled computational fluid dynamics (CFD) and physiologically based pharmaco-kinetic (PBPK) models has reported variance from previous animal models’ extrapolation while CFD modeling of transient lung vapor absorption suggests the significance of transient versus steady-state evaluation of volatiles absorption into tissue and blood. The transient nature of sub-surface fate and transport, ventilation, and inhalation uptake thus warrants integrated exploration and application in order to realize improvements in vapor intrusion assessments. These perspectives and Australian modeling initiatives are presented in this article.     

Cancer risk assessment for 1,3-butadiene: data integration opportunities

The US Environmental Protection Agency recently released its new guidelines for carcinogen risk assessment together with supplemental guidance for assessing susceptibility from early-life exposure to carcinogens. In particular, these guidelines encourage the use of mechanistic data in support of dose-response characterization at doses below those at which an increase in tumor frequency over background levels might be detected. In this context of the utility of mechanistic data for human cancer risk assessment, the International Life Sciences Institute (ILSI) has developed a human relevance framework (HRF) that can be used to assess the plausibility of a mode of action (MoA) described for animal models operating in humans. The MoA is described as a sequence of key events and processes that result in an adverse outcome. A key event is a measurable precursor step that is in itself a necessary element of the MoA or is a bioindicator for such an element. A number of cellular and molecular perturbations have been identified as key events whereby DNA-reactive chemicals can produce tumors. These include DNA adducts in target tissues, gene mutations and/or chromosomal alterations in target tissues and enhanced cell proliferation in target tissues. This type of data integration approach to quantitative cancer risk assessment can be applied to 1,3-butadiene, for example, using data on biomarkers in exposed Czech workers [1]. For this study, an extensive range of biomarkers of exposure and response was assessed, including: polymorphisms in metabolizing enzymes; urinary concentrations of several metabolites of 1,3-butadiene; hemoglobin adducts; HPRT mutations in T-lymphocytes; chromosomal aberrations by FISH and conventional staining procedures; sister chromatid exchanges. Exposure levels were monitored in a comprehensive fashion. For risk assessment purposes, these data need to be considered in the context of how they inform the MoA for leukemia, the tumor type reported to be increased in synthetic rubber workers exposed to 1,3-butadiene. Also, for the HRF it is necessary to establish key events for a MoA in rodents for the induction of tumors by 1,3-butadiene. There is clearly a species difference in sensitivity to tumor induction, with mice being much more sensitive than rats; key events need to explain this difference. For butadiene, the MoA is DNA-reactivity and subsequent mutagenicity and so following the EPA's cancer guidelines, a linear extrapolation is used from the point of departure (POD), unless additional data support a non-linear extrapolation. For the present case, the human bioindicator data are not informative as far as dose-response characterization is concerned. Mouse chromosome aberration data for in vivo exposures might be used for establishing a POD, with linear extrapolation from this POD. The available cytogenetic data from rodent studies appear to be sufficiently extensive and consistent for this to be a viable approach. This approach of using MoA and key events to establish the human relevance can lead to the development of specific informative bioindicators of response that can be used as surrogates to predict the shape of the tumor dose response curve at low doses. Truly informative predictors of tumor responses should be able to provide estimates of human tumor frequencies at low, environmental exposures to 1,3-butadiene.     

In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH > 13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague–Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3–6 and 22–26 h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20 mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology.Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40 min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.