Dose and litter allocations in the design of teratological studies for detecting hormesis

BACKGROUND: Hormesis is being recognized in the field of toxicology due to the stimulating effects of some toxic compounds at low exposure levels. Therefore, it is desirable that experimental designs for toxicological studies be flexible enough to aid in the detection of hormetic effects. Current designs may still not have enough power to do this. METHODS: A simulation study was conducted to determine teratological study designs that would yield more power over standard designs in detecting hormesis. Developmental toxicity endpoints of interest are the number of dead/resorbed or malformed fetuses in a litter. The simulation designs mimic teratological experiments in terms of sample size and number of dose levels. Modified designs with even dose spacing at low levels and reallocated litters are investigated to determine the power of hormetic detection. RESULTS: Designs with reallocated litters (with more litters at low exposure levels than at high levels) and even dose spacing have more power than those with equal litters per group and uneven dose spacing. CONCLUSIONS: Through appropriate modifications of current experimental designs, such as reallocation of litters and even dose spacing, we can better detect hormetic effects.     

Hormetic Effect of Berberine Attenuates the Anticancer Activity of Chemotherapeutic Agents

Hormesis is a phenomenon of biphasic dose response characterized by exhibiting stimulatory or beneficial effects at low doses and inhibitory or toxic effects at high doses. Increasing numbers of chemicals of various types have been shown to induce apparent hormetic effect on cancer cells. However, the underlying significance and mechanisms remain to be elucidated. Berberine, one of the major active components of Rhizoma coptidis, has been manifested with notable anticancer activities. This study aims to investigate the hormetic effect of berberine and its influence on the anticancer activities of chemotherapeutic agents. Our results demonstrated that berberine at low dose range (1.25 ~ 5 μM) promoted cell proliferation to 112% ~170% of the untreated control in various cancer cells, while berberine at high dose rage (10 ~ 80 μM) inhibited cell proliferation. Further, we observed that co-treatment with low dose berberine could significantly attenuate the anticancer activity of chemotherapeutic agents, including fluorouracil (5-FU), camptothecin (CPT), and paclitaxel (TAX). The hormetic effect and thereby the attenuated anticancer activity of chemotherapeutic drugs by berberine may attributable to the activated protective stress response in cancer cells triggered by berberine, as evidenced by up-regulated MAPK/ERK1/2 and PI3K/AKT signaling pathways. These results provided important information to understand the potential side effects of hormesis, and suggested cautious application of natural compounds and relevant herbs in adjuvant treatment of cancer.     

Hazards from chemicals: scientific questions and conflicts of interest.

All substances are toxic when the dose is large enough. In order to regulate the use of chemicals, we need to measure the level at which toxic effects are found. Epidemiological evidence suggests that present levels of chemical use do not lead to widespread harmful contamination of the human environment. For chemicals, most of the problems of toxicity are found in the workplace, while the population at large gets most of its toxic effects from voluntary exposure to substances such as tobacco smoke and ethanol. The prevention and control of toxic effects depends on a series of steps. This begins with measurement of toxicity in model systems, such as laboratory animals, and the estimation of the likely exposure of workers or consumers. Reliable extrapolation of information gathered from animals to the diverse and biochemically differing human population depends on understanding mechanisms of toxic effects. The toxic effect and mechanisms of action of substances such as carbon tetrachloride or paracetamol have been extensively investigated, and our ability to predict toxicity or develop antidotes to poisoning has had some success, but epidemiology is still an essential part of assessment of toxic effects of new chemicals. The example of phenobarbitone shows how animal experiments may well lead to conclusions which do not apply to man. After measurement of toxicity and assessment of likely hazards in use comes the final evaluation of the use of a chemical. This depends not only on its toxicity, but also on its usefulness. The direct effects on health may be small in comparison with the indirect advantageous effects which a useful substance such as vinyl chloride may bring. The assessment of risks and benefits of new chemicals can be partly removed from a political style of discourse, but the evaluation of the relative weight to be attached to these risks and benefits is inescapably political. The scientific contribution must be to allow the debate to take place in the light of maximum clarity of information about the consequences of use of chemicals.     

Testing threshold and hormesis in a random effects dose-response model applied to developmental toxicity data

Here we describe a random effects threshold dose-response model for clustered binary-response data from developmental toxicity studies. For our model we assume that a hormetic effect occurs in addition to a threshold effect. Therefore, the dose-response curve is based on two components: relationships below the threshold (hormetic u-shaped model) and those above the threshold (logistic model). In the absence of hormesis and threshold effects, the estimation procedure is straightforward. We introduce score tests that are derived from a random effects hormetic-threshold dose-response model. The model and tests are applied to clustered binary data from developmental toxicity studies of animals to test for hormesis and threshold effects. We also compare the score test and likelihood ratio test to test for hormesis and threshold effects in a simulated study.     

Transcription‐blocking DNA damage in aging: a mechanism for hormesis

Recent evidence from studies on DNA repair systems that are implicated in accelerated aging syndromes, have revealed a mechanism through which low levels of persistent damage might exert beneficial effects for both cancer prevention and longevity assurance. Beneficial effects of adaptive responses to low doses of insults that in higher concentrations show adverse effects are generally referred to as hormesis. There are numerous examples of hormetic effects ranging from mild stresses of irradiation to heat stress, hypergravity, pro‐oxidants, or food restriction. Although the notion of hormesis is supported by many observations in various organisms, at least two major caveats have thus far prevented the application of hormesis for disease prevention in humans. First, the very nature of hormesis using toxins as a treatment regimen harbors the inherent danger of detrimental consequences. Second, the molecular mechanisms through which insults might exert beneficial effects have thus far remained elusive. Here, I discuss a mechanistic basis for hormesis and its implications for cancer prevention and healthy aging.     

Maternal toxicity: a possible etiological factor in embryo-fetal deaths and fetal malformations of rodent-rabbit species

Data from animal teratology studies were surveyed to determine whether embryo-fetal mortality and fetal malformations result from a primary action of the agent on the conceptus or if they are secondary to maternal toxicity--a consequence of administration with high dose levels of test chemicals. A fairly strong association between embryo-fetal mortality and maternal toxicity was revealed by analysis of data from hamsters, mice, rats, and rabbits in 234 studies of chemical and physical agents, of which 83 were conducted at both maternotoxic and nonmaternotoxic doses, 94 only at maternotoxic doses, and 49 at nonmaternotoxic doses. In the above studies, only nine chemicals (four each in hamsters and rabbits and one in rats) were reported to induce embryo-fetal deaths at apparently nonmaternotoxic doses. These findings tend to suggest a contributory role for maternal toxicity in the induction of embryo-fetal deaths. The previously reported hypothesis that certain fetal defects in mice may perhaps be caused by maternal toxicity was also found to be true in a review of data on hamsters, rats, and rabbits. Salient maternal toxicity-associated fetal malformations were exencephaly, encephalocele, micro- or anophalmia, and fused ribs in hamsters and defective (fused, missing, or extra) ribs, vertebrae, and sternebrae, ex-, an-, or microphthalmia, and cleft palate in rats and rabbits. These malformations occurred at low frequencies, generally with no readily apparent dose-response relationship. Presumptive evidence indicates that embryo-fetal deaths, and the above-mentioned fetal malformations in experimental animals, which in published literature are presently attributed to chemical induction for a large number of chemicals, may be a consequence of maternal toxicity per se.     

The nutrient-toxin dosage continuum in human evolution and modern health.

Recent findings support the long-recognized principle that nutritive and toxic effects of an ingested material depend not only on its nature but very much on its quantity. The well known observation that essential nutrients can be toxic at high dosages suggests that the same reversal of effect may be true of many substances that could be beneficial but not essential at low dosages (the phenomenon of hormesis). This has been demonstrated for many well known toxins. We suggest a mathematical model that describes these dosage effects as an expected result of the evolution of human metabolic and dietary adaptations for maximizing benefits and minimizing costs of the ingestion or other intake of any substance. Evolved mechanisms for achieving benefits may be unrelated to those for reducing costs. These evolutionary considerations suggest important consequences demonstrable by experimental or epidemiological studies. They also suggest ways in which our evolved dietary adaptations may be currently maladaptive, and individual development of taste preferences poorly calibrated by early experience in modern environments. The apparent reality of hormesis raises the possibility of counterproductive effects of current dosage recommendations and limits for nutrients and pollutants. We propose that some conceptual and factual problems are urgently in need of resolution. Fundamental to evolutionary biology is the tendency for organisms to become increasingly adapted to those environments to which they are most commonly exposed (Parsons 1990).     

Human Health Impact as a Boundary Selection Criterion in the Life Cycle Assessment of Pultruded Fiber Reinforced Polymer Composite Materials

The human health impact of fiber reinforced polymer (FRP) composite materials manufactured by the pultrusion industry is not fully understood. In particular, it is unclear whether the human health impact of toxic chemicals present in low concentrations in fire retardant pultruded FRP materials is disproportionately high. This impact may be an important criterion when making boundary selection decisions in the life cycle assessment (LCA) of these materials. The North American pultrusion industry was surveyed to determine resin mix concentration levels and workplace inhalation toxicity exposure levels. LCAs were then conducted on three building panel resin mixes to determine whether the human health impact of toxic chemicals used in the mixes was low enough to exclude the chemicals from the life cycle inventory (LCI) boundary. The first resin mix represented a typical pultruded product, the second mix removed toxic chemicals present in small concentrations, and the third mix replaced toxic chemicals present in small concentrations with a nontoxic chemical. Results showed that toxicity levels fell below exposure limits and no significant difference in human health impact existed among the LCAs. The research concludes that human health impact is a useful criterion when defining an LCI boundary. Toxic chemicals present in small concentrations in pultruded FRP materials may be excluded from the LCI boundary, as their human health impacts are low. Because these levels are marginal in North American pultrusion factories, no changes in resin mixes are recommended for the pultrusion industry.     

A Proposed Working Definition for the Novel Concept of Neurobehavioral Hormesis

It is proposed that a novel concept, neurobehavioral hormesis, be considered for integration into the field of toxicology. Hormesis results in a non-linear dose response where low dose exposures to toxicants cause beneficial effects, and detrimental effects at higher doses. Hormesis has not been systematically incorporated into traditional risk assessment methodologies, yet there is recent evidence that this pattern of results is relatively prevalent. In this paper, hormesis is applied to neurobehavioral toxicology, and an operational definition is proposed for application to putative examples of neurobehavioral hormesis. The two primary criteria used for the operational definition are: (1) performance is enhanced with low dose exposure and denigrated at higher doses, and (2) the change in behavior persists following a recovery period. In recent research from our laboratory it was reported that rats exposed to JP-8 jet fuel vapor demonstrated such a pattern of neurobehavioral performance on tests of learning and memory. Specifically, animals with long-term exposure to low concentrations of jet fuel demonstrated enhanced performance on specific operant tasks as compared both to controls and to animals exposed to higher concentrations. The effect was most apparent during complex versus simple operant tests, and was observed months following the last exposure to jet fuel. The effects meet both criteria for the proposed working definition of neurobehavioral hormesis, and thus provide evidence of the validity for considering neurobehavioral hormesis in published and future research, and suggests a more systematic investigation of existing literature may be warranted. Also, it provides additional support for the overall proposal to include hormetic effects in formal risk assessment paradigms.     

Hormesis and ecological risk assessment: Fact or fantasy?

Hormesis is a widespread phenomenon across occurring many taxa and chemicals, and, at the single species level, issues regarding the application of hormesis to human health and ecological risk assessment are similar. However, interpreting the significance of hormesis for even a single species in an ecological risk assessment can be complicated by competition with other species, predation effects, etc. In addition, ecological risk assessments may involve communities of hundreds or thousands of species as well as a range of ecological processes. Applying hormetic adjustments to threshold effect levels for chemicals derived from sensitivity distributions for a large number of species is impractical. For ecological risks, chemical stressors are frequently of lessor concern than physical stressors (e.g., habitat alteration) or biological stressors (e.g., introduced species), but the relevance of hormesis to non‐chemical stressors is unclear. Although ecological theories such as the intermediate disturbance hypothesis offer some intriguing similarities between chemical hormesis and hormetic‐like responses resulting from physical disturbances, mechanistic explanations are lacking. While further exploration of the relevance of hormesis to ecological risk assessment is desirable, it is unlikely that hormesis is a critical factor in most ecological risk assessments, given the magnitude of other uncertainties inherent in the process.     

Detecting and Estimating Hormesis Using a Model-Based Approach

Hormesis is defined as a dose-response relationship that is stimulatory at low doses, but is inhibitory at higher doses. In a given experiment, it is not unusual to observe enhanced responses at low doses, however, such enhanced responses may not imply hormesis, but the random fluctuation of the data. Statistical tests can be developed to detect hormesis when enhanced responses at low concentrations are observed. We propose the use of a model-based approach to detect the presence of, and estimate the extent of, hormesis. This approach includes two steps: detection and estimation. In the detection step, we compare the full and the reduced models. The full model describes the dose-response relationship incorporating the hormetic effect; the reduced model describes the dose-response relationship without the hormetic effect. The full model is an extension of the reduced model and has an extra parameter that measures the amount of increase in response at low doses. A test of statistical significance of this extra parameter can essentially be a test for detecting hormesis. In the estimation step, we obtain the area under the best-fitted dose-response curve falling within the hormetic zone. Considering both the number of concentrations within the hormetic zone and the magnitude of the stimulatory response, we propose using the ratio of the area under the hormetic zone (AUC_H) and the area under the best-fitted curve from zero to zero equivalent point (AUC_ZEP) as an estimate of magnitude of the hormetic effect. Two numerical examples are used to illustrate the use of this model-based approach.     

Test of six chemicals for embryotoxicity using fetal mouse salivary glands in culture

Many new chemicals come into use each year, and the need for rapid and cost-effective methods for testing developmental toxicity is apparent. Establishing reliable in vitro techniques is important to a tier approach to testing for developmental toxicity. The fetal mouse salivary gland was selected as a possible test system because several interacting developmental processes occur in gland growth, and development is quantifiable by counting lobes. For each chemical tested, 20 glands from 13-day embryos were treated in a control media and in three concentrations of the test chemicals. The number of lobes present after 48 hours is dependent on the number of lobes at explantation. Glands with different numbers of lobes at explantation were compared by dividing the number of lobes present after 48 hours by the number present at explantation to determine a growth ratio. Mean growth ratios were used to construct dose-response curves, and from these curves the concentration that reduced growth by 50% (TP50) was determined. Comparisons with in vivo data were made by calculating three ratios; the TP50 was divided into the lowest teratogenic dose, the lowest maternal toxic dose, and the dose that was lethal to 50%. Four in vivo teratogens, 6-aminonicotinamide, cytochalasin B, hydroxyurea, and 3-acetylpyridine, all had ratios much higher than 1, indicating a very sensitive response by the glands. One in vivo teratogen, dexamethasone, had much lower ratios, indicating less sensitivity. Acetaminophen, a nonteratogen in vivo, actually stimulated growth of the glands at 10(-5) M and had very low ratios indicating a minimal response by the glands.     

New considerations on hormetic response against oxidative stress

In order to survive living organisms have developed multiple mechanisms to deal with tough environmental conditions. Hormesis is defined as a process in which exposure to a low dose of a chemical agent or environmental factor that is damaging at higher doses induces an adaptive beneficial effect on the cell or organism. In this paper, we examine several ideas that might be taken into consideration before using hormesis as a therapeutic tool to improve health and life span, and hopefully will open the discussion for new and interesting debates regard hormesis. The first one is to understand that the same stressor or inductor can activate different pathways in a parallel or dual response, which might lead to diverse outcomes. Another idea is related to the mechanisms involved in activating Nrf2, which might be different and have diverse hormetic effects.Last, we discuss mild oxidative stress in association to low-grade chronic inflammation as a stimulating avenue to be explored and the unexpected effects proposed by the obesity paradox theory. All the previous might help to clarify the reasons why centenarians are able to reach the extreme limits of human life span, which could probably be related to the way they deal with homeostasis maintenance, providing an opportunity for hormesis to intervene significantly.Keyword: Aging, Hormesis, Inflammation, Nrf2, Obesity, Oxidative stress     

Embryos as targets of endocrine disrupting contaminants in wildlife

Environmental contaminants are now a ubiquitous part of the ecological landscape, and a growing literature describes the ability of many of these chemicals to alter the developmental trajectory of the embryo. Because many environmental pollutants readily bioaccumulate in lipid rich tissues, wildlife can attain considerable body burdens. Embryos are often exposed to these pollutants through maternal transfer, and a growing number of studies report long-term or permanent developmental consequences. Many biological mechanisms are reportedly affected by environmental contaminants in the developing embryo and fetus, including neurodevelopment, steroidogenesis, gonadal differentiation, and liver function. Embryos are not exposed to one chemical at a time, but are chronically exposed to many chemicals simultaneously. Mixture studies show that for some developmental disorders, mixtures of chemicals cause a more deleterious response than would be predicted from their individual toxicities. Synergistic responses to low dose mixtures make it difficult to estimate developmental outcomes, and as such, traditional toxicity testing often results in an underestimate of exposure risks. In addition, the knowledge that biological systems do not necessarily respond in a dose-dependent fashion, and that very low doses of a chemical can prove more harmful than higher doses, has created a paradigm shift in studies of environmental contaminant-induced dysfunction. Although laboratory studies are critical for providing dose-response relationships and determining specific mechanisms involved in disease etiology, wildlife sentinels more accurately reflect the genetic diversity of real world exposure conditions, and continue to alert scientists and health professionals alike of the consequences of developmental exposures to environmental pollutants.     

Fluctuating Asymmetry as Risk Marker for Stress and Structural Defects in a Toxicologic Experiment

Fluctuating asymmetry (the directionally random asymmetry of bilateral structures, FA) is commonly used as a measure of developmental instability, and may increase with stress. As several studies reported a relation between FA and developmental abnormalities, we investigate whether FA could be an additional perhaps more sensitive marker of developmental toxicity. The aim of this work is analyzing patterns of FA in multiple traits in a large dataset of rabbit fetuses, which were prenatally exposed to a toxic compound and sacrificed just before natural delivery. Gravid females were exposed to three doses of this compound, inducing abnormalities in the fetuses at the high dose only. The average FA, however, was already higher than control in rabbit fetuses of the low‐dose group but did not further increase with higher concentrations. Moreover, the increase in FA differed between traits, with the hindlimbs showing the strongest response. In addition, we did not find any association between FA and the presence of fetal abnormalities at the individual level. Although these results suggest that FA may act as “an early warning system,” we did not find a dose–response relationship with increasing stress and effects were trait‐specific. Further testing is needed before FA may be considered as a sensitive marker in developmental toxicity studies.     

Acetaldehyde utilization in Drosophila: an example of hormesis

For survival measured by longevity, acetaldehyde is beneficial at low and stressful at high concentrations in Drosophila utilizing ethanol as a major resource. This is an example of the increasingly common observation of hormesis which is the stimulatory effect by toxic agents at subinhibitory levels. Organic metabolites such as acetaldehyde and organisms such as Drosophila offer the possibility of model genetical and molecular studies of hormesis. This may assist our understanding of suggestions of enhanced fitness at natural background radiation levels and in habitats simulating these levels.     

Insecticide‐induced hormesis in an insecticide‐resistant strain of the maize weevil,Sitophilus zeamais

Sublethal responses to insecticides are frequently neglected in studies of insecticide resistance, although stimulatory effects associated with low doses of compounds toxic at higher doses, such as insecticides, have been recognized as a general toxicological phenomenon. Evidence for this biphasic dose–response relationship, or hormesis, was recognized as one of the potential causes underlying pest resurgence and secondary pest outbreaks. Hormesis has also potentially important implications for managing insecticide‐resistant populations of insect‐pest species, but evidence of its occurrence in such context is lacking and fitness parameters are seldom considered in these studies. Here, we reported the stimulatory effect of sublethal doses of the pyrethroid insecticide deltamethrin sprayed on maize grains infested with a pyrethroid‐resistant strain of the maize weevil (Sitophilus zeamais) (Coleoptera: Curculionidae). The parameters estimated from the fertility tables of resistant insects exposed to deltamethrin indicated a peak in the net reproductive rate at 0.05 ppm consequently leading to a peak in the intrinsic rate of population growth at this dose. The phenomenon is consistent with insecticide‐induced hormesis and its potential management implications are discussed.     

Selection of test chemicals for the ECVAM international validation study on in vitro embryotoxicity tests. European Centre for the Validation of Alternative Methods

The European Centre for the Validation of Alternative Methods (ECVAM) has sponsored a large international prevalidation and validation study of three embryotoxicity tests, involving embryonic stem cells, limb bud micromass cultures, and post-implantation whole-embryo cultures. The main objective of the study was to assess the performance of these in vitro tests in discriminating between non-embryotoxic, weakly embryotoxic and strongly embryotoxic compounds. An initial part of the study was to select 20 test substances for the formal validation trial, conducted under blind conditions. A database of in vivo and in vitro developmental toxicity test results was complied on 310 chemicals that had been used in previous validation studies, or suggested for such use, or that had good quality "segment II"-type in vivo data, or for which there were human data. From this database, a shortlist of about 30 candidates was constructed. Because the ECVAM study would not include metabolic activation, chemicals known to require activation for their developmental effects were excluded as candidates, although some known stable metabolites were included. Attempts were made: to include substances of diverse mechanism; to avoid overemphasis on pharmaceuticals; to avoid biologically inert substances as non-embryotoxicants; and to make the list different from those used previously. The candidates were of three categories: Class 3, strongly embryotoxic, was defined as developmentally toxic in all species tested, inducing multiple developmental effects, and with a high A/D ratio. Class 1, non-embryotoxic, was defined as not developmentally toxic at maternally toxic exposures, but which may show some minor embryo/fetal toxicity, which cannot be separated from maternal toxicity. Class 2, weakly embryotoxic, were chemicals of intermediate activity. From this candidate list, chemicals of known receptor (androgen, oestrogen, glucocorticoid, aryl hydrocarbon) mechanisms were excluded, on the basis that simple tests for such activity are already available. In addition, chemicals not freely available were excluded, and an emphasis on human data was applied. The final list of 20 chemicals was: Class 3--6-aminonicotinamide, 5-bromo- 2'-deoxyuridine, hydroxyurea, methylmercury chloride, methotrexate, all-trans-retinoic acid; Class 2--boric acid, dimethadione, lithium chloride, methoxyacetic acid, valproic acid (VPA), 2-propyl-4-pentynoic acid (4-yn-VPA), salicylic acid sodium salt; and Class 1--acrylamide, D-(+)-camphor, dimethyl phthalate, diphenhydramine hydrochloride, 2-ethyl-4- methylpentanoic acid (isobutyl-ethyl-VPA), Penicillin G sodium salt, saccharin sodium hydrate.     

NTP carcinogens--interpretational problems

The National Toxicology Program (NTP) was established in 1978 with the broad goal of strengthening the science base of chemical toxicity, thus providing better information to regulatory and research agencies. Since that time the NTP has conducted in-depth toxicity/carcinogenesis studies on over 200 chemicals of importance to industry, the public at large and the general environment; clearly the largest such database in the world. This database is unique in that it represents an objective fairly standard accumulation of peer-reviewed information on a myriad of chemicals composed of various chemical classes, non-carcinogens as well as carcinogens. The results of these studies are reported as "no evidence, equivocal evidence, some evidence or clear evidence of carcinogenic activity" in a single sex/species. There is also an "inadequate" category for studies that have major limitations. Although noted, no attempt is made to give added weight to chemicals which cause neoplasms at multiple sites, at rare versus common sites, in both species/sexes, which occur early in the study, at low as well as high doses, or those observed in the presence or absence of toxicity (necrosis, degeneration, etc.) in the same organ. Such observational data may serve as "markers" or "alerts" for whether a chemical's in vivo carcinogenic activity is the result of mutagenic or non-mutagenic activity.     

Phenomena leading to cell survival values which deviate from linear-quadratic models

For several decades, the prevailing paradigm for modeling the effects of ionizing radiation (IR) on living systems was the target model with its inherent assumptions--that only those cells in the radiation path whose molecules sustained collisions with high energy particles and rays were damaged, that the damage was proportional to the energy absorbed by each cell and to the number of cells absorbing energy, and that all cells had identical sensitivities to radiation. However, evidence has accumulated that cells exhibit phenomena at low radiation exposures that appear to contradict at least one of these assumptions. Some of these phenomena currently under active study include low-dose hypersensitivity (HRS), increased radiation radioresistance (IRR), the adaptive response (AR), the bystander effect (BE), and death-inducing factor (DIE). These effects may interact to give rise to other phenomena such as hormesis, in which small amounts of otherwise toxic agent appear to be beneficial. Elucidating the cellular and molecular bases for these phenomena will lead to greater understanding of the relationships of these processes, including hormesis, to human health.