Cross-species Extrapolation Research Articles

Inducibility of cytochromes P-450 by dioxin in liver and extrahepatic tissues of the marmoset monkey ( Callithrix jacchus)

Cytochrome P-450 induction was investigated in the marmoset monkey, a non-human primate, using dioxins as inducing agents. Animals received a single subcutaneous dose of 1.6 nmol tetrachlorodibenzo- p-dioxin or tetrabromodibenzo- p-dioxin/kg body weight. Microsomal fractions were prepared from liver, lung and kidney, and homogenates were prepared from gut and adrenal glands. Anti-peptide antibodies which bind to CYPIA1, CYPIA2, CYP2B6 and CYP3A4 in human were used to identify related forms in the marmoset. The results indicate that CYPIA2 is constitutively expressed in liver, but not in lung, kidney, gut or adrenal gland and that CYPIAI is not expressed in any of these tissues in untreated animals. Treatment with dioxin induced both CYPIAI and CYPIA2 in liver, but only CYPIA1 in lung. No induction of CYPIA1 or CYPIA2 was found in kidney, small intestine or adrenal glands. Methoxy-, ethoxy-, pentoxy- and benzoyloxyresorufin O-dealkylases and high affinity phenacetin O-deethylase activities were induced in the liver, whereas ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities were not affected by dioxin treatment. High-affinity phenacetin O-deethylase and CYPIA2 apoprotein were detected only in liver, consistent with this activity being specifically catalysed by CYPIA2. Furafylline was found to be a competitive inhibitor of methoxyresorufin O-demethylase activity with a K i of 10 μM. In the lung the induction of CYPIAI was accompanied by 15- and 23-fold increases in ethoxyresorufin O-deethylase and methoxyresorufm O-demethylase activities, respectively, suggesting that both activities are catalysed by CYP1A1. In contrast, there was no induction of aryl hydrocarbon hydroxylase activity in lung or liver showing that, unlike in many other species, marmoset CYPIAI does not catalyse this reaction efficiently. The expression, distribution, induction and substrate specificities of marmoset monkey P-450 enzymes differ from the situation found in rodents and other species, demonstrating that caution has to be exercised when making cross-species extrapolations.

DNA damage, and repair in mutagenesis and carcinogenesis: implications of structure-activity relationships for cross-species extrapolation

Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TD 50 estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TD 50 values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA; (2) genotoxins capable of forming DNA etheno-adducts; and (3) N-substituted aryl compounds forming covalent adducts at the C8 position of guanine in DNA. In general, clear SARs and AARs (activity-activity relationships) between physiochemical parameters ( s-values, 0 6/ N7-alkylguanine ratios, CBI), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (mouse, Drosophila) became apparent when the following descriptors were used: TD 50 estimates (lifetime doses expressed in mg/kg b.wt. or mmol/kg b.wt.) from cancer bioassays in rodents; the degree of germ-cell specificity, i.e., the ability of a genotoxic agent to induce mutations in practically all cell stages of the male germ-cell cycle of Drosophila (this project) and the mouse (literature search), as opposed to a more specific response in postmeiotic stages of both species; the M exr−/M exr+ hypermutability ratio, determined in a repair assay utilizing Drosophila germ cells; mutation spectra induced at single loci (the 7 loci used in the specific-locus test of the mouse (published data), and the vermilion gene of Drosophila); and doubling doses (DD) in mg/kg (mmol/kg) for specific locus test results on mice. By and large, the TD 50 values, the inverse of which can be considered as measures of carcinogenic potency, were shown to be predictable from knowledge of the in vivo doses associated with the absorbed amounts of the investigated alkylators and with the second-order constant, k c, reaction at a critical nucleophilic strength, n c. For alkylating agents k c can be expressed as the second-order rate constant for hydrolysis, k H 2O , and the substrate constant s: k H 2O TD 50 is a function of a certain accumulated degree of alkylation, here given as the (average) daily increment, a c, for 2 years exposure of the rodents. The TD 50 in mmol/kg × day) could then be written: T−fsD 50 ∗= a c×λ k H 2O ×10 nc s . This expression would be valid for monofunctional alkylators provided the reactive species are uncharged. This is the case for most S N2 reagents. Although it appears possible to predict carcinogenic potency from measured in vivo doses and from detailed knowledge of reaction-kinetic parameter values, it is at present not possible to quantify the uncertainty of such predictions. One main reason for this is the complication due to uneven distribution in the body, with effects on the dose in target tissues. The estimation can be improved by considering the distribution of dose in the body. In the present analysis, direct-acting alkylators were studied. It is evident that dosimetry and reaction-kinetic characterization of genotoxic metabolites would render it possible to predict the potency of precarcinogens as well.

Biomarkers for Great Lakes priority contaminants: halogenated aromatic hydrocarbons.

One of the major goals of the Great Lakes Action Plan is to actively accumulate and assess toxicological information on persistent toxic substances found in the Great Lakes basin. As part of Health Canada's commitment to this plan, a review of biomarkers for the environmental contaminants polychlorinated biphenyls (PCBs) and polychlorinated dibenzodioxins/dibenzofurans (PCDDs/PCDFs) was conducted. In general, while food consumption was identified as the major source of human exposure to both contaminant groups, certain commodities, such as fish, milk and dairy products, and meat, were found to predominate. Due to the ubiquitous nature of these environmental contaminants and their propensity to bioaccumulate, all humans will have detectable body burdens, which in certain cases can be positively associated with the consumption of particular foods (i.e., PCBs and freshwater fish from the Great Lakes). When dealing with environmental exposure only, relating specific effect biomarkers to contaminant exposure or tissue levels was difficult, due in part to the complex nature of the exposure and the nonspecific nature of the effect. For PCBs, the most likely biomarkers of effect included some form of alteration in lipid metabolism (serum triglyceride/cholesterol levels) and elevation of hepatic-related enzymes, aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT). Cross-species extrapolation also indicates the potential for neurotoxicologic effects to occur in humans. For PCDDs/PCDFs, dermatologic lesions (chloracne) and indications of hepatic enzyme induction have been documented, but primarily due to occupational or high acute accidental exposures. Recent evidence suggests that neonates may represent a potential at-risk population due to relatively high exposure to PCDDs/PCDFs, as with PCBs, during breast feeding as compared to standard adult dietary intake. Future areas of potential benefit for biomarker development include immunologic and endocrine effects, primarily based on biologic plausibility from experimental animal research.

Use of quantitative modelling in methylene chloride risk assessment

The benefits of basing quantitative risk assessment on measures of ‘internal dose’, i.e. target organ exposures as estimated, for instance, by pharmacokinetic models, have been extensively discussed. Recasting risk assessment methods at the level of internal dose raises novel issues, however, some of which are explored by examining the 1987 revision by the U.S. Environmental Protection Agency (EPA) of its cancer risk assessment for inhaled methylene chloride, which was based on the 1987 pharmacokinetic model results of Andersen and coworkers. The internal dose measure was the daily amount of methylene chloride metabolized by a glutathione- S-transferase pathway per 1 of target organ (liver and lung). Owing to high-dose saturation of a competing detoxification reaction, this metabolic activation is less-than-proportionally active at low exposure levels. For a given inhalation exposure, humans have relatively less metabolic activation than do mice, but this is shown to be a foreseeable consequence of their relatively lower breathing rate, a cross-species difference already accounted for in standard EPA methodology. Indeed, many species differences in the rates and tempos of physiological processes evince regular ‘scaling’ relationships across differently sized mammals. EPA's practice of scaling carcinogen doses by body surface area for cross-species extrapolation, often viewed as a correction for metabolic activation, is shown to be more reasonably regarded as an accommodation for the more general species variation in the pace of physiological processes underlying both pharmacokinetics and the carcinogenic response to internal doses. Under this view, the issue of cross-species dose scaling is not obviated by the use of pharmacokinetics.

Risk assessment and biological mechanisms: lessons learned, future opportunities

During the past decade, texicological research has been dominated by two themes: investigations to elucidate the mechanisms of action of toxicants and studies to provide information to support improved assessments of human health risks. The conduct of mechanistic investigations was given an early impetus by advances in biochemistry and cell biology and, more recently, by related advances in molecular biology. Research to provide information for improved human health risk assessments was stimulated by the 1983 NAS/NRC report that provided a codified structure for conducting risk assessments. At first glance, it would appear that the two themes are closely related and, indeed, should represent parts of a joined theme. However, examination of the toxicology/risk assessment literature of the past decade indicates that this has not been the case. Reports of mechanistic studies infrequently indicate how the information can be used to provide improved estimates of human risk from exposure to toxicants. If reference is made, it is usually qualitative in nature. Neither is examination of the risk assessment literature reassuring. Mechanistic studies may be cited; however, the final step of the process, risk characterization, is usually dominated by the use of default options grounded in conservative interpretations of generic scientific knowledge. Two examples are reviewed that stand out as illustrations of how mechanistic information can be used to make a difference in risk assessments: (1) consideration of the α 2 u -globulin-mediated mechanism for evaluating male rat data for relevance in assessing human risks of renal cancer and (2) the use of DNA-protein cross-links as an internal dose metric in cross-species extrapolation of nasal cancer risks from inhaled formaldehyde. This paper reviews past experience on these topics and suggests a strategy for increasing the use of mechanistic information in risk assessments. A key component of the strategy is to use the risk assessment process to identify research needs/opportunities that, if addressed, will reduce the use of default options, thereby reducing the uncertainties in risk assessments. Another component of the strategy is to identify a few chemicals anticipated to exert their effect via different mechanisms and whose mechanisms of dosimetry and disease pathogenesis can be investigated in-depth within a risk assessment framework; this identification will create prototype approaches as alternatives to the use of default options that have major impact on the outcome of the risk assessment process.

In Vivo Tissue Dosimetry as a Basis for Cross-Species Extrapolation in Cancer Risk Assessment of Propylene Oxide

The potential for causing carcinogenic and mutagenic effects has been the main concern when assessing the risks associated with low-level exposures of humans to the industrially important epoxide, propylene oxide (PrO). For regulatory purposes, surface-based extrapolation has been used to determine the human equivalent dose from cancer data obtained in rodents. In this context the tissue dose will more adequately reflect inter- and intraspecies differences with respect to pharmacokinetic parameters than is the case for conventional representations of exposure. The formation of adducts in nucleophilic molecular targets by directly acting electrophilic agents, like epoxides, is thought to be closely linked to the process of cancer initiation. To investigate whether tissue dose is correlated to surface area of the exposed organism, the in vivo adduct levels in hemoglobin and DNA have been determined in mice, rats, and dogs after exposure to PrO by injection as well as by inhalation. The results obtained indicate that the dose in blood is virtually the same in the three investigated animal species, whereas surface-area based extrapolation predicts a difference by a factor of about seven between the mouse and the dog. Although the data base is more limited, this conclusion is also supported by measurements of DNA alkylation in selected tissues. The variations actually observed are not related to the surface area of the animal. No significant differences could be found between administration of PrO by injection or by inhalation. For this reason, the surface-based extrapolation model for estimation of the human equivalent dose is not appropriate, and the carcinogenic potency factors for PrO as previously derived by the U.S. EPA should probably be revised downward by a factor of 10 to 13.

In vivo tissue dosimetry as a basis for cross-species extrapolation in cancer risk assessment of propylene oxide

The potential for causing carcinogenic and mutagenic effects has been the main concern when assessing the risks associated with low-level exposures of humans to the industrially important epoxide, propylene oxide (PrO). For regulatory purposes, surface-based extrapolation has been used to determine the human equivalent dose from cancer data obtained in rodents. In this context the tissue dose will more adequately reflect inter- and intraspecies differences with respect to pharmacokinetic parameters than is the case for conventional representations of exposure. The formation of adducts in nucleophilic molecular targets by directly acting electrophilic agents, like epoxides, is thought to be closely linked to the process of cancer initiation. To investigate whether tissue dose is correlated to surface area of the exposed organism, the in vivo adduct levels in hemoglobin and DNA have been determined in mice, rats, and dogs after exposure to PrO by injection as well as by inhalation. The results obtained indicate that the dose in blood is virtually the same in the three investigated animal species, whereas surface-area based extrapolation predicts a difference by a factor of about seven between the mouse and the dog. Although the data base is more limited, this conclusion is also supported by measurements of DNA alkylation in selected tissues. The variations actually observed are not related to the surface area of the animal. No significant differences could be found between administration of PrO by injection or by inhalation. For this reason, the surface-based extrapolation model for estimation of the human equivalent dose is not appropriate, and the carcinogenic potency factors for PrO as previously derived by the U.S. EPA should probably be revised downward by a factor of 10 to 13.

The Use of Visual and Chemosensory Evoked Potentials in Environmental and Occupational Health

The application of visual (VEP) and chemosensory evoked potentials (CSEP) in occupational and environmental health is briefly reviewed. VEPs have been used extensively in experimental neurotoxicology and play an increasingly important role in human neurotoxicity testing. The similarity of VEP waveforms in different species renders them useful for cross-species extrapolation. CSEPs, used in conjunction with traditional psychophysical tests and rating scales, offer a promising new approach to the study of indoor air pollution.

Extrapolation from animals to humans: scientific and regulatory aspects

The necessity to protect humans from the adverse effects of chemicals on structure and function of the developing and/or mature nervous system is increasingly recognized among regulatory bodies throughout the world. Whereas structural changes dominated much of neurotoxicological research in the past, functional markers of neurotoxicity are gaining acceptance as early signs of insult. Among the more ambitious protective efforts are legal requirements of premarket testing for “behaviour-disrupting properties” of chemicals. Assumed or proven validity of cross species extrapolation underlies the use of animals in primary or secondary screening schemes. Two steps must be distinguished here. The first step is endpoint-base or qualitative, whereas the second one is dose-based or quantitative. Species comparisons in terms of endpoints is typically done within a framework of broad functional categories, such as sensory, motivational, cognitive, motor or social functions. Dose-based extrapolation requires knowledge about species differences in terms of toxicokinetics or metabolism in order to arrive at valid translations of dose-response contingencies. Principles of cross-species extrapolation in neurotoxicology will be exemplified by means of representative neurobehavioural and neurophysiological findings for neurotoxic chemicals of environmental concern, for which an adequate data base is available for comparative purposes, such as inorganic lead and polychlorinated biphenyls (PCB).

Cross-species extrapolation in the assessment of neurotoxicity: David A. Eckerman. University of North Carolina, Chapel Hill, NC

Cross-species extrapolation in the assessment of neurotoxicity: David A. Eckerman. University of North Carolina, Chapel Hill, NC

Quantitative methods for cross-species mapping (CSM)

Cross-species extrapolation will be defined as prediction from one species to another without empirical vetification. Cross-species mapping (CSM) is the same except empirical vetification is performed. CSM may be viewed as validation of methods for extrapolation. Algorithms for CSM may originate from theory, from empirical observations or a combination of the two. Regardless of their origins, CSM algorithms must be explicated and confidence intervals given around their predictions. This paper offers a quantitative method for constructing CSM equations which is useful in evaluation of the CSM and as an aid in the design of new experiments in CSM and extrapolation. The method requires fitting mathematical models for the physiological or behavioral phenomena to be mapped across species. A CSM equation can then be derived from the models in each species and approximate confidence limits may be obtained for predictions from the equation. The method is useful even when the models in the two species differ in form, implying differences in physiology or behavioral principles between species. The method proposed has a number of remaining uncertainties and possible problems.

Physiological alterations during pregnancy: Impact on toxicokinetics

The physiological changes that occur in the alimentary, cardiovascular, pulmonary, and renal organ systems during pregnancy are designed to increase availability of nutrients to and remove wastes from the fetus. Although this is a general requirement, not all animals use the same strategies to meet these goals. These physiological adaptations will impact on toxicokinetics and may alter toxicodynamics. Absorption, distribution, metabolism, transfer between maternal and fetal compartments, and elimination will change for many xenobiotics during pregnancy. The changes in body weight, total body water, plasma proteins, body fat, and cardiac output will alter the distribution of many xenobiotics (Hytten and Leitch, 1971; Hytten and Chamberlain, 1980; Mattison, 1986). As the toxicokinetic parameters change across species, it is important to understand their impact on chemicals associated with maternal, placental, and fetal toxicity for appropriate cross-species extrapolation.

A selective critique of animal experiments in human-orientated biological research

The advantages and justifications for using small animals in human-orientated research are briefly over-viewed. Some of the potential pitfalls of extrapolating animal-derived data to humans are then discussed more fully, using examples biased towards nutrition. A number of particular problems of animal experimentation are highlighted: the applicability of the results of controlled, deliberately reductionist, laboratory experiments to arguments about free-living animals and humans; species differences in nutrient requirements; differences in pattern of feeding and type of diet of mammalian species; and various facets of the problem of scaling from small to large species. The enormous contribution of animal experimentation to the advancement of human biological understanding is accepted, but by focusing attention of biological educators on examples, perceived by the author to have involved inappropriate cross-species extrapolation, it is hoped that this paper may help to increase the future effectiveness and reduce waste of experimental animals.

Model Selection in Toxicology: Principles and Practice

The key assumptions underlying modern toxicology are that (1) animals can serve as accurate predictive models of toxicity in humans, (2) that selection of an appropriate model to use is the key to accurate prediction in humans, and (3) that understanding the strengths and weaknesses of any particular model is essential to understanding the relevance of specific findings to humans. It must first be made clear, however, that the term model encompasses more than a test organism. Then the basic principles underlying these assumptions and the general limitations of models (both in vivo and in vitro) can be critically examined. The actual practice of model selection and why it deviates from the generally accepted principles are overviewed, along with the issue of errors induced by the “high-class model” problem. Historical results as to the accuracy of prediction of human effects are summarized. Finally, the reasons that models fail to predict effects in humans in some cases are examined, along with methods and problems in cross-species extrapolation and the issue of species peculiarities.

Electrophysiological measures of visual and auditory function as indices of neurotoxicity

The application of auditory and visual evoked potentials (VEP) to neurotoxicity testing of humans and animals is reviewed. VEPs elicited by flash, reversing-checkerboard patterns, and sine wave grating are described. The flask evoked potential in rats is altered by exposure to many heavy metals, pesticides and solvents. The brainstem auditory evoked potential also appears to be sensitive to neurotoxic chemicals, but the evidence available is limited. The homology of auditory and visual evoked potentials in rats and humans is useful for cross-species extrapolation in neurotoxicology research.

Cross-species extrapolations and the biologic basis for safety factor determinations in developmental toxicology

Designations of agents as teratogenic or nonteratogenic often are inaccurate, as adverse effects are more a reflection of the timing and severity of treatment during pregnancy than agent nature. Careful consideration of both the similarities and the differences between developmental effects in animals and humans and the extent and nature of the data available are essential for protection of the human conceptus. Animal surrogates prove reliable predictors of human developmental effect levels. When the data are evaluated consistent with contemporary concepts of developmental toxicity, for example, where the effect in the embryo is only seen at maternally toxic doses and exposure is below the adult toxic level, relatively modest safety factors are sufficient for safe cross-species extrapolation. Developmental toxicity safety factor magnitude is predicated on data quality and the fact that thresholds of effect exist in mammalian pregnancy. Safety of human concepti is achiaved by considering both the developmental hazard index of the chemicals in question and the severity of exposure.