An illusion of hormesis in the Ames test: statistical significance is not equivalent to biological significance

A recent report (Calabrese et al., Mutat. Res. 726 (2011) 91-97) concluded that an analysis of Ames test mutagenicity data provides evidence of hormesis in mutagenicity dose-response relationships. An examination of the data used in this study and the conclusions regarding hormesis reveal a number of concerns regarding the analyses and possible misinterpretations of the Salmonella data. The claim of hormesis is based on test data from the National Toxicology Program using Salmonella strain TA100. Approximately half of the chemicals regarded as hormetic, and the majority of the specific dose-responses identified as hormetic, were actually nonmutagenic. We conclude that the data provide no evidence of hormetic effects. The Ames test is an excellent measure of bacterial mutagenicity, but the numbers of revertant (mutant) colonies on the plate are the result of a complex interaction between mutagenicity and toxicity, which renders the test inappropriate for demonstrating hormesis in bacterial mutagenicity experiments.     

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.     

Response of the L-arabinose forward mutation assay of Salmonella typhimurium to frameshift-type mutagens

The present study was designed to evaluate the capacity of the L-arabinose resistance test of Salmonella typhimurium in the detection of frameshift-type mutagens. To this end the response of the Ara test was examined with respect to 15 chemicals which had been previously described as able to revert the Ames tester strain TA97. The mutagenicity of each compound was determined by the liquid test under experimental conditions which optimize the mutagenic response of the Ara test with the tester strain BA9. Strain TA97 was used simultaneously with BA9. The Ara forward-mutation assay efficiently detected the mutagenic activity of 14 out of the 15 chemicals assayed. PR toxin was the only compound which gave a weak dose response without doubling the spontaneous mutant level. In comparison with the Ara test, a total of 3 chemicals (HZ, PE and PR toxin) were not found to be mutagenic with strain TA97. In most cases (11/15) the mutagenic response of the Ara test was comparatively greater than that of strain TA97. Three chemicals (DEO, PRF and 9-AA) were detected with quite similar degrees of sensitivity by both mutation assays. ICR-191, which seems highly specific in reverting frameshift mutations with added cytosines in a run of cytosines, was the only chemical with a lower mutagenic activity in the Ara test than in strain TA97. The results enhance the interest of the L-arabinose forward-mutation assay as an alternative to the set of specific tester strains used by the histidine reverse-mutation assay in massive, general and primary screening for genotoxic agents.     

Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.     

Examination of the mutagenicity of RDX and its N-nitroso metabolites using the Salmonella reverse mutation assay

The mutagenicity of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its N-nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) were evaluated using the Salmonella tryphimurium reverse mutation assay (Ames assay) with strains TA97a, TA98, TA100, and TA102. Using a preincubation procedure and high S9 activation (9%), RDX was observed to induce weak mutagenesis to strain TA97a with a mutagenicity index (MI) of 1.5-2.0 at a dose range of 32.7-1090microg/plate. MNX induced moderate mutagenesis to strain TA97a with an MI of 1.6-2.8 at a dose range of 21.7-878microg/plate. TNX also induced moderate mutagenesis in strain TA97a with an MI of 2.0-3.5 to TA97a at a dose range of 22.7-1120microg/plate. TNX also caused weak mutagenesis to strain TA100 with S9 activation at the dose of 1200microg/plate. MNX and TNX induced weak to moderate mutagenesis to strain TA102. Strain TA97a was found to be the most sensitive strain among these four strains. No cytotoxicity of RDX, MNX, and TNX was observed at the concentrations used in this study. Doses were verified by HPLC.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Epidemiological Assessment of Hormesis in Studies with Low-Level Exposure

Despite its resurgence within toxicology and, specifically, risk assessment, the concept of hormesis remains peripheral to current epidemiological practice. In this paper we examine some reasons for this, focusing on applications within occupational and environmental epidemiology. Unclear in the existing literature is whether hormesis pertains to a single biological mechanism or response, or the aggregate effect of all correlates of exposure. Although J-shaped and U-shaped relationships between risk factors and disease endpoints have been identified epidemiologically, it is unclear whether such patterns reflect biological hormesis or a combination of factors resulting in a hormetic-looking relationship. Given the potential importance of assessing hormetic responses in epidemiological studies, we identify and discuss key limitations of epidemiology in validly detecting and interpreting hormesis. For example, most observational occupational and environmental studies lack the ability to determine the dose received by each individual, and therefore poor surrogates of exposure are frequently used, potentially introducing considerable systematic and random error. Further, because exposure is not randomly assigned to humans, the potential for confounding is great. Finally, using a simple simulation to assess the impact of ignoring hormesis in the analysis of epidemiological data containing mild hormesis, we demonstrate a resulting “hormetic bias,” in which relative risks at exposure levels above the hormetic region are systematically overestimated.     

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.     

Are ecosystems hormetic?

The phenomenon of hormesis has been observed mainly for the response of individual organisms to stress. A reasonable line of inquiry might explore the possibility of observing hormesis at other levels of ecological organization. This initial examination focuses on ecosystem hormesis. Explorations of hormetic responses of ecosystems to stress cannot be made independently of a fundamental concept of ecosystem. The scale‐dependence of ecosystem dynamics also influences whether an ecological disturbance is in reality a stressor. Ecosystem hormesis might be claimed if one or more components of an ecosystem exhibit hormesis. By this definition, ecosystem hormesis would be a trivial extension of hormesis observed for individual organisms. A non‐trivial extension of ecosystem hormesis would include the observation that integrated (i.e., holistic) measures of ecosystem structure or function displayed an hormetic response to an ecological stressor. Several such examples of ecosystem structural and functional hormesis are presented.     

Hormesis provides a generalized quantitative estimate of biological plasticity

Phenotypic plasticity represents an environmentally-based change in an organism’s observable properties. Since biological plasticity is a fundamental adaptive feature, it has been extensively assessed with respect to its quantitative features and genetic foundations, especially within an ecological evolutionary framework. Toxicological investigations on the dose-response continuum (i.e., very broad dose range) that include documented evidence of the hormetic dose response zone (i.e., responses to doses below the toxicological threshold) can be employed to provide a quantitative estimate of phenotypic plasticity. The low dose hormetic stimulation is an adaptive response that reflects an environmentally-induced altered phenotype and provides a quantitative estimate of biological plasticity. Analysis of nearly 8,000 dose responses within the hormesis database indicates that quantitative features of phenotypic plasticity are highly generalizable, being independent of biological model, endpoint measured and chemical/physical stress inducing agent. The magnitude of phenotype changes indicative of plasticity is modest with maximum responses typically being approximately 30–60% greater than control values. The present findings provide the first quantitative estimates of biological plasticity and its capacity for generalization. Summary This article provides the first quantitative estimate of biological plasticity that may be generalized across plant, microbial, animal systems, and across all levels of biological organization. The quantitative features of plasticity are described by the hormesis dose response model. These findings have important biological, biomedical and evolutionary implications.     

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.     

Genotoxicity tests with 6-acetyl-1,1,2,4,4,7-hexamethyltetraline and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyr an

6-Acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB), synthetic fragrance ingredients, were evaluated for potential genotoxicity in a battery of short-term tests. Salmonella typhimurium/Escherichia coli plate incorporation and liquid preincubation assays were conducted on AHTN using tester strains TA97, TA98, TA100, TA102, TA1535, TA1537 and WP2 uvrA +/- S9 activation at doses from 8 to 5000 micrograms/plate. The plate incorporation mutagenicity assay was conducted on HHCB using tester strains TA98, TA100, TA1535, TA1537, TA1538 and WP2 uvrA +/- S9 activation at doses from 10 to 5000 micrograms/plate. An in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells was conducted with AHTN and HHCB at three concentrations each with +/- S9 activation. In the non-activated study, the exposure/harvest periods were 4/20-, 20/20- and 44/44-h. In the S9 activated study, the exposure/harvest periods were 4/20- and 4/44-h. In vitro unscheduled DNA synthesis (UDS) assays were conducted in primary rat hepatocytes at concentrations between 0.15 and 50 micrograms/ml for AHTN and HHCB. In vivo mouse micronucleus assays were conducted with high doses of 1600 mg AHTN/kg and of 1500 mg HHCB/kg in corn oil. No positive responses were observed in any of the tests with HHCB. With AHTN, no positive responses were observed except for cells with structural aberrations in the in vitro cytogenetics assay in CHO cells with S9 activation at the treatment/harvest time of 4/20 h. In initial studies with AHTN, the high dose of 7.8 micrograms/ml showed 0.5% aberrant cells, with the mitotic index at 41% relative to vehicle control and cell growth inhibition in the range of 25-50%. Thus the genotoxicity findings with AHTN were limited to this one positive response; all other genotoxicity tests with AHTN were considered as negative. In particular, the negative finding in the in vivo assay supports AHTN as not likely to be mutagenic in mammalian systems. These considerations, along with other negative published data, lead to the conclusion that both AHTN and HHCB do not have significant potential to act as genotoxic carcinogens.     

Interspecies Variability of Plant Hormesis by the Antiauxin PCIB in a Laboratory Bioassay

Chemical hormesis constitutes an alternative possible use of herbicidal agents for crop enhancement that is, however, compromised by the apparent variability of this low-dose stimulation phenomenon. Studies demonstrating the variability are rare and, therefore, this study investigated the interspecies variability of growth stimulation induced by the auxin-inhibitor PCIB [2-(p-chlorophenoxy)-2-methylpropionic acid] to determine if hormesis is generalizable enough and sufficiently stable between species/cultivars for practical use or which implications may have to be taken into account. In 85 complete dose–response bioassays with 23 cultivars of five species, the variability of PCIB effects was evaluated. The expression of PCIB hormesis proved to depend on the species/cultivar tested, ranging from a cultivar-dependent hormetic efficacy and an occasional lack of hormesis, to a complete lack of hormetic effectiveness in certain species/cultivars. Therefore, frequency estimations, as well as the pattern of dose-dependent variability of dose–response quantities, may inevitably depend on the biological model(s) used and, thus, apply only to the specific conditions for characterization. Comparing the frequency distribution of effective doses demonstrated a risk of a previously hormetic dose causing a loss of hormesis or inhibitory effects in another species/cultivar. Therefore, selecting a dose that will induce hormesis in every species/cultivar is unrealistic. This may limit the window for practical applications to stimulants with negligible varietal differences, to cultivar selective treatments, and/or to cultivars that enable a beneficial long-term use. Hence, efficient crop enhancement by chemical hormesis needs not only a good stimulant, but also a species/cultivar able to convert a specific low-dose treatment into an economic benefit.     

Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium in vitro and in rodent host-mediated assays.

The fungicide benomyl and its commercial preparations Fundazol 50WP and Benlate 50WP and the benomyl metabolite methyl-2-benzimidazole carbamate and its commercial preparation MBC 50WP were tested for mutagenicity in in vitro spot tests, in microsomal plate assay, in liquid-culture treatments, or in rodent host-mediated assay. The base-pair substitution Salmonella typhimurium mutant hisG46 and the hisG46-bearing uvrB excision-repair-deficient mutants TA100, TA1530, TA1535 or TA1950 were used as test organisms. Complete genotypic information of these mutants is given in Ames et al. [2]. Captain 50WP, streptozotocin (SZN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminopurine and N-acetylaminofluorene were used as positive control compounds. In nonoverlay spot tests Benlate 50WP was not mutagenic over a dose range of 50-5000 microgram/spot in hisG46 and TA1535. In overlay spot tests 50 or 100 microgram/spot Benomyl, MBC, Fundazol 50WP, Benlate 50WP and MBC 50WP were tested in hisG46, TA1530 or TA1950. Only a non-commercial MBC sample at 100 microgram/spot showed weak mutagenic activity in hisG46. In microsomal activation plate assay MBC, benomyl, Fundazol 50WP and Benlate 50WP were tested in TA100 over a dose range of 50-2000 microgram/plate. None of the compounds showed mutagenicity. In a 20-h liquid-culture treatment 10, 100, 1000 and 10 000 microgram/ml Fundazol 50WP were not mutagenic in TA 30. In 1-h liquid-culture treatments benomyl, Benlate 50WP or Fundazol 50WP failed to induce mutations in hisG46, TA100 or TA1950 over a dose range of 0.25-1000 microgram/ml. Appropriate positive controls were mutagenic in each experiment. The consistently negative results in this study with commercial MBC and benomyl preparations are contrary to positive results reported earlier with similar methods and similar commercial preparations. Possible reasons to explain the different results are presented. The alkylating agents SZN and MNNG induced fewer mutations in TA1530 and TA1950 uvrB excision-repair-deficient strains than in the hisG46 excision-proficient strain, indicating that with these mutagens excision-repair is also a mutation-prone process. In rodent host-mediated assays with Fundazol 50WP in mice 3 consecutive subcutaneous hourly doses of 500 mg/kg in hisG46 and TA1950 and in rats or mice an oral dose of 4000 mg/kg in TA1950 were not mutagenic. The positive control SZN was mutagenic.     

Evaluation of the genetic and embryotoxic effects of bis(tri-n-butyltin)oxide (TBTO), a broad-spectrum pesticide, in multiple in vivo and in vitro short-term tests

The genetic and embryotoxic effects of bis(tri-n-butyltin)oxide (TBTO) were evaluated in multiple in vivo and in vitro short-term tests preparatory to its potential wide use as a molluscicide in control of schistosomiasis. When tested in the rec assay in Bacillus subtilis, TBTO was not mutagenic and it did not induce reverse mutations in Klebsiella pneumoniae. Neither in the presence nor in the absecne of rat liver activation system did TBTO produce point mutations in Salmonella typhimurium strains TA1530, TA1535, TA1538, TA97, TA98 or TA100. TBTO was matagenic in strain TA100 in a fluctuation test, but only in the presence of rat liver S9 (Aroclor-induced). TBTO did not induce gene mutations in the yeast Schizosaccharomyces pombe, mitotic gene conversions in the yeast Saccharomyces cerevisiae, nor sister-chromatid exchange in Chinese hamster ovary cells in the presence or absence of rat or mouse liver S9. In the latter cells, structural chromosomal aberrations, endoreduplicated and polyploid cells were induced. TBTO did not induce gene mutations in V79 Chinese hamster cells (to 8-azaguanine-, ouabain- or 6-thioguanine-resistance) in the presence of a rat liver postmitochondrial fraction or in cell (hamster embryo cells and human and mouse epidermal keratinocyte)-mediated assays. In mouse lymphoma cells, TBTO did not induce 6-thioguanine- or BUdR-resistant mutations. As many tumour promoters inhibit metabolic cooperation between V79 Chinese hamster 6-thioguanine-resistant/-sensitive cells, TBTO was tested but showed no such activity. TBTO was examined for the induction of recessive lethal mutations in adult Berlin K male Drosophila melanogaster, either by feeding or by injection. Doses of 0.37 or 0.74 mM did not increase the number of X-linked recessive lethal mutations. An increased number of micronuclei was observed in the polychromatic erythrocytes of male BALB/c mice 48 h after a single oral dose of TBTO (60 mg/kg bw), while a lower dose (30 mg/kg bw) was ineffective. Neither of the two doses had induced micronuclei 30 h after treatment. The reproductive toxicity of TBTO was studied in NMRI mice. In a 10-day toxicity study, the LD50 and LD10 were 74 and 34 mg/kg bw, respectively. An increased frequency of cleft palates was seen in the fetuses of mice (compared with controls, 0.7%) treated orally during pregnancy with 11.7 mg/kg TBTO (7%), 23.4 mg/kg (24%) or 35 mg/kg (48%).(ABSTRACT TRUNCATED AT 400 WORDS)     

Statistical Dose-Response Models with Hormetic Effects

Monotonically increasing or decreasing functions are often used to model the relationship between the response of an experimental unit and the dose of a given substance. Of late, there has been an increased interest in dose-response relationships that exhibit hormetic effects. These effects may be characterized by an increase in response at low doses instead of the expected decrease in response that is observed at higher doses. Herein, we study the statistical implications of hormesis in several ways. First, we present a broad class of parametric mathematical-statistical models, constructed from standard dose-response models, that allow the incorporation of hormetic effects in such a way that the presence of hormesis can be tested statistically. Second, we consider the impact of model misspecification on effective dose estimation, such as the ED50 and the limiting dose for stimulation, when the hormetic effect is present but ignored in the dose-response model by the researcher (model underspecification) and when an hormetic effect is not present but incorporated into the dose-response model (model overspecification). Our simulation study reveals that it is more damaging to the estimation of effective dose to ignore the hormetic effect through model underspecification than to include the hormetic effect in the model through model overspecification. Third, we develop a nonpara-metric regression technique useful as an exploratory procedure to indicate hormetic effects when present. Finally, both parametric and nonparametric methods are illustrated with an example.     

Implementing Hormetic Effects in the Risk Assessment Process: Differentiating Beneficial and Adverse Hormetic Effects in the RfD Derivation Process

Hormetic effects have been purported to be both beneficial and adverse. The decision of whether an effect is adverse or beneficial can have important implications for public health and risk assessment. A functional approach is proposed for determining the types of responses available for the incorporation of hormesis in the RfD process. These response categories include: (1) beneficial; (2) adverse; and (3) either beneficial or adverse depending on specific circumstances. Examples of endpoints in each category are presented and discussed. Other issues affecting the RfD process include: (1) the integration of multiple hormetic responses; (2) the importance of endpoint selection in determining the type of hormetic response; and (3) the assumption of hormesis when responses are unobservable due to low background response in controls.     

Evaluation of the mutagenic potential of endosulfan using the Salmonella/mammalian microsome assay

The mutagenic potential of endosulfan, a polychlorinated insecticide, was assessed using the highly sensitive Salmonella tester strains TA97(a), TA98, TA100 and TA102. It exhibited a toxic effect at dose levels of 50 micrograms/plate and higher. Plate incorporation studies did not show mutagenic response with any of the tester strains used. A modification of the assay using a preincubation procedure showed mutagenic activity with and without metabolic activation with TA97(a) only. Increased toxicity was observed after addition of phenobarbital-induced S9 mix.     

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.     

Comparative direct-acting mutagenicity of 1- and 2-nitropyrene: Evidence for 2-nitropyrene mutagenesis by both guanine and adenine adducts

The mutations and DNA adducts produced by the environmental pollutant 2-nitropyrene were examined in Salmonella typhimurium tester strains. 2-Nitropyrene was a stronger mutagen than its extensively studied structural isomer 1-nitropyrene in strains TA96, TA97, TA98, TA100, TA102, TA104 and TA1538. Both 1- and 2-nitropyrene were essentially inactive in TA1535. The mutagenicity of 1- and 2-nitropyrene in TA98 was much higher than in TA98NR and the activity of these compounds in TA100 was much higher than in TA100NR. While 1-nitropyrene exhibited similar mutagenicity in strains TA98 and TA98/1,8-DNP₆, the mutagenicity of 2-nitropyrene in TA98/1,8-DNP₆ was much lower than in TA98. Analysis of DNA from TA96 and TA104 incubated with 2-nitropyrene indicated the presence of two adducts, N-(deoxyguanosin-8-yl)-2-aminopyrene and N-deoxyadenosin-8-yl)-2-aminopyrene. The results suggest that 2-nitropyrene is metabolized by bacterial nitroreductase(s) to N-hydroxy-2-aminopyrene, and possibly by activation to a highly mutagenic O-acetoxy ester. DNA adduct formation with deoxyguanosine and deoxyadenosine correlates with the mutagenicity of 2-nitropyrene in tester strains possessing both G:C and A:T mutational targets.