Hormesis is a toxicological concept characterized by low-dose stimulation and high-dose inhibition (1, 2, 3, 4, 5). Extensive examinations of scientific literature by Calabrese and his collaborators reported that hormetic dose-responses are common across biological systems and stressors (4, 6, 7, 8). Scientific literature provides evidence that hormesis can be caused by multiple stimuli (9), such as chemicals (4, 10, 11), radiation (12; 29), heat (13), stress (14), and even exercise (15). Dose-response curves displaying hormesis are characterized by a biphasic behavior (Fig.1). The hormetic zone includes a range of subinhibitory doses that are stimulant, with a peak at the maximum stimulation dose (MSD), and ends at the no observable adverse level (NOAEL), that typically precedes the inhibitory doses (Fig. 1). Our interest in hormesis pertains to the effects of fungicides at subinhibitory doses on fungal and oomycete growth and pathogenicity. Thereafter, for the purposes of this book, we will focus on chemical hormesis alone. Some of the most familiar examples of biphasic dose-responses include vitamins, alcohol, essential minerals, and many drugs (16, 17, 18, 19, 20). Hormesis has been measured using diverse endpoints in multiple biological systems (8). One of the most common endpoints used in hormesis research is growth, but several others have been studied including CO2 production (1; 21), longevity (14), other metabolic processes, and cellular functions (22). Southam and Ehrlich (2) coined the term hormesis to describe the biphasic dose-response because its Greek etymological root
2. Historic background
The first references to dose-responses have been attributed to Paracelsus, who allegedly said “all things are poison and nothing is without poison, only the dose permits something not to be poisonous” (24). However, the first scientific reports of chemical stimulation at low doses and inhibition at high concentrations go back to the 1800s. In 1854, Virchow (22) reported that low concentrations of sodium hydroxide increased the frequency and intensity of beating of ciliae of human tracheal ciliated epithelia; while at higher concentrations or longer exposures the same compound paralyzed ciliae and caused cell death. In 1865, Reveil (25) reported that sodium hypochlorite stimulated seed germination at low concentrations (0.1% solution) but it was phytotoxic at high concentrations. According to Calabrese and Baldwin, in their review of the historical foundations of chemical hormesis as a biological hypothesis (5), two researchers are considered as the founders of modern hormesis, Rudolf Arndt and Hugo Schulz. In 1887, Schulz (1) observed experimentally that several chemicals caused low-dose stimulation and high-dose stimulation on yeast fermentation. Because Schulz’s views supported those of Arndt, a homeopathic physician, soon they were merged in to what was known as the “Arndt-Schulz law”. The law stated that “for every substance, small doses stimulate, moderate doses inhibit and large doses kill” and that their findings could be generalized to all organisms and all toxic agents. Mainly because of the difficulty at the time to demonstrate the universality of this law without offering explanation of its biological causes, and probably also because at the time it was conceptually associated to homeopathy, over time it fell out of use. However, a few years later Ferdinand Hueppe (1896), a distinguished bacteriologist, made similar observations in bacteria and described the phenomenon in his books and scientific publications (26). Hueppe recognized the validity of Schulz’s scientific research but stated certain limitations and exceptions to the Arndt-Schulz law. Soon the notion that “substances which inhibit biological processes at sublethal doses may be expected to stimulate them at lower levels” became known as the “Hueppe rule” and was broadly adopted in international literature (3, 5).
Years later, in 1929, Branham (21) confirmed Schulz’s observations using a series of different chemicals and an improved apparatus to detect CO2 production, and demonstrated that very small doses of inhibitor compounds had an apparent stimulatory effect on carbon dioxide production by yeasts. One of her experiments assessed the effect of adding crystals of 1,2,5,6-dibenzanthracene to yeast suspensions, finding that at a concentration of 9 x 10-4 molar yeast proliferation increased. The term hormesis was coined by Southam and Ehrlich in their 1943 report of growth stimulation of a wood-decaying fungus (
Research focused on chemical subinhibitory dose-responses continued through the early decades of the 1900s, but scientific attention faded away as the science of Toxicology became established and few hormesis studies were published until the 1980s (3, 27, 28). Toxicology is the study of the adverse effects of chemical, physical, or biological agents on biological systems, their prevention and amelioration. Hence, by definition, Toxicology deals with the negative effects of such agents at doses above the no observed adverse effect level (NOAEL), and essentially ignores the effects of subinhibitory doses. Stebbing (3) reexamined the hormesis concept and provided the first update on the validity of this concept based on the abundance of scientific reports of data with biphasic distributions. Stebbing reported his conclusions after searching the scientific literature for an explanation to his own observations of growth stimulation of the colonial hydroid
Calabrese first report related to hormesis described stimulation of
3. Hormesis as a general phenomenon
Based on their extensive literature review, Calabrese and Baldwin stated that hormetic responses often, but not always, display the following characteristics: i) Stimulation zone of the dose-response could be found within a 10-fold range; ii) Stimulatory responses were 30-60% greater than the controls; and iii) the NOAEL three to six-fold greater than the MSD (4). Using these criteria they identified hundreds of toxicological studies that potentially displayed hormetic responses (48). In recent years researchers from many different disciplines have been inspired by Dr. Calabrese’s work and have studied hormesis in their biological systems of interest. There are numerous recent reports of biphasic dose-responses in plants, animals, as well as eukaryotic and prokaryotic microorganisms in the scientific literature. A few examples of studies reporting chemical hormesis are presented below.
The better documented example of hormesis in animals is lifespan increase as a result of restricted caloric intake in diet (24). While high calorie diets have been associated with increased risk of several age-related diseases in animal systems (cardiovascular disease, type 2 diabetes, stroke, among others), dietary energy restriction (i.e. controlled caloric restriction or intermittent fasting) has been reported to have anti-oxidative effects, increasing the cells’ tolerance to several types of stresses. For example, restricted calorie diets protected rodents against several types of cancers (51); furthermore, alternate day calorie restricted diet in humans seems to improve inflammatory symptoms in asthmatic patients (52). A now classic example of chemical hormesis are vitamins in human diet, since small amounts of them are necessary and beneficial, but large amounts are toxic and can cause hypervitaminosis, tissue mineralization, and chemical imbalances (45). Other examples of hormesis in animal systems include inhibition of N-diethylnitrosamine (DEN)- initiated pre-neoplastic lesions by phenobarbital at low-doses, while higher doses promote activity (53), and survival and fertility enhancement in
There are abundant examples of hormesis in prokaryotic and eukaryotic microbial systems. As related in the historical review, the first reports of biphasic dose-responses were on bacteria and fungi (1, 2, 26, 21) and several more studies have been published in more recent years. Hotchkiss (55) found that TiCl2, MgCl2 and, NaCl had hormetic effects on the growth of
4. Evidence of chemical hormesis in phytopathological literature
Our review of mycological and phytopathological literature found several studies of fungicide effects on fungi and oomycetes with results that reflect potential hormetic responses. We present some interesting examples below, while an exhaustive literature review will be reported elsewhere. Southam and Ehrlich (2) observed that extracts of western red-cedar heartwood were stimulatory at low doses on the growth of a wood-decaying fungus (
Similar references can be found on oomycetes literature. Fenn and Coffey (70) observed that 69 µg/ml of phosphorous acid (H3PO3) was stimulatory on the growth of
5. Fungicides hormesis and its impact on fungal plant pathogens
Recent research on chemical hormesis on fungal pathogens has focused on the effects of subinhibitory doses of fungicides on radial growth and pathogenicity of fungi and oomycetes (66, 74). Garzon
6. The biological basis of hormesis
Hormesis can result from overcompensation after a disruption of homeostasis by stressors, by direct stimulation, or as a response to an adaptating dose followed by a larger dose (3, 75, 76). The research by Branham (21) on the effect of 16 chemicals on CO2 production by Baker’s yeast, provided clear evidence, in 12 of the chemicals, of an initial mild inhibition followed by significant stimulation. These results supported the hypothesis of stimulation due to overcompensation, being most evident for formaldehyde, phenol, iodine, and metaphen. Other examples of over-compensatory responses include ethanol stimulation of locomotion in mice (77), increased serotonin levels in rat neurons after treatment with below toxic doses of 5,6-dihydroxytryptamine (78); and growth stimulation in peppermint following an initial decrease after treatment with phosfon, a plant growth regulator (39), among others. Stebbing (79) provided an “improved” explanation for hormesis due to overcompensation by describing a model using two overlapping curves, an
The underlying mechanisms that generate hormetic responses have not yet been fully understood. Conolly and Lutz (80) hypothesized that hormetic responses may occur due the superimposition of two monotonic dose-responses, one that takes effect at low doses and other that overtakes at higher doses undermining the first one. They demonstrated by computational modeling that four different cellular models could generate biphasic dose-responses: i) Membrane receptor subtypes with opposite downstream effect; ii) Androgen receptor mediated gene expression; iii) Induction of DNA repair and “co-repair” of background DNA damage; and, iv) Modulation of the cell cycle and effect on rate of mutation (80). Subsequent studies have found empirical evidence of hormesis attributable to the presence of antagonistic membrane receptors (81) or to the induction of DNA repair (82). Bae
7. Studying hormesis
Detection of hormesis is often challenging due to the multiple factors that can affect metabolic responses of the target organisms. For example, when studying fungicide hormesis in oomycetes using radial growth as endpoint it is fundamental to standardize every experimental factor involved; in addition to growing media type and concentration, fungicide treatments, and incubation temperature, other factors are also relevant, including light, growing media depth, inoculum age and developmental stage, mixing time when preparing fungicide dilutions, using fungicide stock solutions prepared on the same day of the experiments, etc. Variation in any of these parameters can influence mycelial growth significantly, hence introducing experimental variation that could affect the reproducibility of results (66).
When trying to prove the existence of hormesis there are some requirements that the experimental design should fulfill: i) The NOAEL should be determined; ii) doses below the NOAEL need to be tested with five equally spaced doses providing enough data to detect hormesis; and iii) the separation between doses should generally be smaller than one order of magnitude since the hormetic zone is usually within a ten-fold range (42). To test for hormesis researchers must compare the effect of small doses with the response of the non-treated control. Therefore, there should always be background incidence in the control, without background incidence there is no way to detect a stimulus (80). Evaluation of data is very important when proving hormesis. Crump suggests the criteria for evaluating hormesis as follows: strength of evidence, soundness of data, consistency and biological plausibility (86). Statistical analyses should be performed in order to differentiate a small stimulus from background occurrence.
Different methods have been used throughout the years for the detection and estimation of hormesis including parametric, non-parametric, and model-based approaches. The hormetic zone of a dose-response curve follows a non-monotonic relationship between two variables, similar to what is known as umbrella alternatives. Umbrella alternatives are important in many fields of science; a classical example is the ability of learning as a function of age in humans (87). As we grow older our ability to learn new things reaches a peak and later declines. Tests for umbrella alternatives can be used to detect if a dose-response curve follows a non-monotonic trend compared to a monotonic one where hormesis would not be present. The firsts to describe a test for umbrella alternatives were Mack and Wolfe (87) who used a non-parametric method where the distribution of the data is not assumed a-priori. In the Mack and Wolfe test (87) the maximum stimulation detected experimentally is compared to the response at all the other doses using Mann-Whitney counts, a test statistic is calculated and compared with simulated critical values to determine if the dose-response is biphasic. Buning and Kossler (88) demonstrated that the Mack and Wolfe (87) test with Mann-Whitney counts is appropriate for testing data with symmetric and medium-up to long tailed distributions but they suggested the use of different two-sample statistics, i.e. Hogg
For the detection of hormesis there are also parametric analyses which assume a normal distribution of the data and can have more statistical power than non-parametric analyses if such assumptions are correct. Among the parametrical tests we can highlight the one proposed by Buning and Kossler (88), a modification for umbrella alternatives of the test by Barlow
When testing for the stimulation at low doses of chemicals on fungal plant pathogens, Flores and Garzon (74) used a model-based approach. The Brain and Cousens model was appropriate for this case where most of the dose-response datasets analyzed yielded a β higher than 1. Because of the relevance of EC50, NOAEL, and MSD for disease management, the modified model by Schabenberger
8. Why is chemical hormesis relevant for crop management?
Multiple chemicals with distinct modes of action are available for management of fungal and oomycete diseases. Although integrated disease management is practiced extensively, the productivity of many agricultural systems relies strongly on chemical control. The limited access to registered products for certain agricultural environments, such as greenhouses, as well as inappropriate use has led to the emergence of fungicide resistant strains in multiple species (100, 101, 102, 103, 104, 105, 106). Currently, the effects of subinhibitory doses of fungicides on fungal plant pathogens are unknown. The evidence gathered from literature indicates that stimulation of fungi and oomycetes by sub-inhibitory doses of fungicides has been observed in ascomycetes (64, 65, 67, 68), basidiomycetes (2, 69, 74), as well as in oomycetes (66, 70, 71, 72, 73, 74). Several fitness factors could be affected for the benefit of pathogens, including mycelial growth, spore germination, toxin production and pathogenicity (66, 67, 68, 74). Exposure to subinhibitory doses can occur accidentally in agricultural fields, orchards, nurseries and greenhouses, under diverse circumstances, such as inappropriate fungicide application, low-dose applications to reduce costs, presence of fungicide resistant strains, etc. Thereafter, the possibility of fungal pathogen stimulation due to fungicide hormesis in actual agricultural scenarios is real. The potential effects of fungicide hormesis are highly detrimental, since it could result in larger crop losses, reduced seed and crop quality, higher mycotoxin levels in grain, and wasteful use of fungicides.
In spite of the potential detrimental effects of fungicide hormesis on fungal plant pathogens to agricultural productivity, a complete lack of awareness has meant the exclusion of this important concept from the design of disease chemical management strategies. In some fungicide-pathogen systems, the value of the EC50 can be different when hormesis is included in the analysis, hence it is important to consider this concept to avoid bias in EC50 calculations. Awareness of the risk taken by growers by the inappropriate use of reduced-dose fungicides (reduced-dose fungicides can be used in combination with two or more other formulations, with different active ingredients ) and careless chemical application will help to promote the use of best-management practices and responsible use of fungicides.
More research is needed to understand the processes involved in fungicide hormesis, the prevalence of hormesis in fungi and oomycete species and populations, fungicide class risks, whether mixtures can prevent stimulation, etc. Hormesis is not a new concept but its use in plant pathology is recent, and its application to disease management may open new opportunities to improve plan health and crop productivity.