Non-monotonic Dose-response Curves Research Articles

The ENDOMIX perspective: how everyday chemical mixtures impact human health and reproduction by targeting the immune system†.

Endocrine disruptor chemicals (EDCs) are natural and synthetic compounds found ubiquitously in the environment that interfere with the hormonal-immune axis, potentially impacting human health and reproduction. Exposure to EDCs has been associated with numerous health risks, such as neurodevelopmental disorders, metabolic syndrome, thyroid dysfunction, infertility, and cancers. Nevertheless, the current approach to establishing causality between EDCs and disease outcomes has limitations. Epidemiological and experimental research on EDCs faces challenges in accurately assessing chemical exposure and interpreting non-monotonic dose response curves. In addition, most studies have focused on single chemicals or simple mixtures, overlooking complex real-life exposures and EDC mechanistic insights, in particular regarding their impact on the immune system. The ENDOMIX project, funded by the EU's Horizon Health Program, addresses these challenges by integrating epidemiological, risk assessment, and immunotoxicology methodologies. This systemic approach comprises the triangulation of human cohort, in vitro, and in vivo data to determine the combined effects of EDC mixtures. The present review presents and discusses current literature regarding human reproduction in the context of immunotolerance and EDC mode of action. It further underscores the ENDOMIX perspective to elucidate the impact of EDCs on immune-reproductive health.

Associations between per- and polyfluoroalkyl substances (PFAS) and diabetes in two population-based cohort studies from Sweden

BackgroundPer- and polyfluoroalkyl substances (PFAS) have been suggested to contribute to the development of metabolic diseases such as obesity, diabetes and non-alcoholic fatty liver disease (NAFLD). However, evidence from epidemiological studies remain divergent. The aim of the present study was to evaluate associations between PFAS exposure and prevalent diabetes in a cross-sectional analysis and fasting glucose in a longitudinal analysis.MethodsIn 2373 subjects aged 45–75 years from the EpiHealth study, three PFAS; perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) were analyzed in plasma together with information on prevalent diabetes. Participants in the PIVUS study (n = 1016 at baseline, all aged 70 years) were followed over 10 years regarding changes in plasma levels of six PFAS; PFHxS, PFOA, PFOS, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUnDA), and changes in plasma levels of fasting glucose.ResultsIn the EpiHealth study, no overall associations could be observed between the levels of PFOA, PFOS or PFHxS and prevalent diabetes. However, there was a significant sex-interaction for PFOA (p = 0.02), and an inverse association could be seen between PFOA (on a SD-scale) and prevalent diabetes in women only (OR: 0.71, 95% CI: 0.52, 0.96, p-value: 0.02). This association showed a non-monotonic dose-response curve. In the PIVUS study, inverse relationships could be observed between the changes in levels (ln-transformed) of PFOA and PFUnDA vs the change in fasting glucose levels (ln-transformed) over 10 years (p = 0.04 and p = 0.02, respectively). As in EpiHealth, these inverse associations were significant only in women (PFOA: β: −0.03, p = 0.02, PFUnDA: β: −0.03, p = 0.03).ImpactExposure to per- and polyfluoroalkyl substances (PFAS) has been linked to unfavorable human health, including metabolic disorders such as obesity, diabetes and non-alcoholic fatty liver disease. However, results from in vivo, in vitro and epidemiological studies are incoherent. The aim of the present study was therefore to investigate associations between PFAS and diabetes in a cross-sectional study and glucose levels in a longitudinal study. Results show inverse associations in women only. Results also display non-monotonic dose response curves (i.e., that only low levels of PFOA are related to higher probability of prevalent diabetes). This suggests that sex differences and complex molecular mechanisms may underlie the observed findings. A better understanding of the factors and molecular mechanisms contributing to such differences is recognized as an important direction for future research.ConclusionsPFOA was found to be inversely related to both prevalent diabetes and changes in plasma glucose levels among women only. Thus, our findings suggest there are sex differences in the inverse relationship of PFOA and type 2 diabetes and glucose levels.

Contrasting dose response relationships of neuroactive antidepressants on the behavior of C. elegans

Antidepressant prescriptions are on a rise worldwide and this increases the concerns for the impacts of these pharmaceuticals on nontarget organisms. Antidepressants are neuroactive compounds that can affect organism’s behavior. Behavior is a sensitive endpoint that may also propagate effects at a population level. Another interesting aspect of antidepressants is that they have shown to induce non-monotonic dose-response (NMDR) curves. While such NMDR relationships may have clear implications for the environmental risk, the resolution of current studies is often too coarse to be able to detect relevant NMDR. Therefore, the current study was performed into the behavioral effects (activity, feeding and chemotaxis) in Caenorhabditis elegans as the model organism of the selective serotonin reuptake inhibitors fluoxetine and sertraline and the acetylcholinesterase inhibiting pesticide chlorpyrifos, using a wide range of concentrations (ng/l to mg/l). In order to statistically examine the non-monotonicity, nonlinear regression models were applied to the results. The results showed a triphasic dose-response relationship for activity and chemotaxis after exposure to fluoxetine, but not to sertraline or chlorpyrifos. Effects of fluoxetine already occurred at low concentrations in the range of ng/l while sertraline only showed effects at concentrations in the μg/l range, similar to chlorpyrifos. The different responses between fluoxetine and sertraline, both SSRIs, indicate that response patterns may not always be extrapolated from chemicals with the same primary mode of action. The effects of fluoxetine at low concentrations, in a non-monotonic manner, confirm the relevance of examining such responses at low concentrations.

Metformin disrupts Danio rerio metabolism at environmentally relevant concentrations: A full life-cycle study

Metformin (MET), an anti-diabetic pharmaceutical of large-scale consumption, is increasingly detected in surface waters. However, current knowledge on the long-term effects of MET on non-target organisms is limited. The present study aimed to investigate the effects of MET in the model freshwater teleost Danio rerio, following a full life-cycle exposure to environmentally relevant concentrations (390 to 14 423 ng/L). Considering that the mode of action (MoA) of MET on non-target organisms remains underexplored and that MET may act through similar human pathways, i.e., lipid and energy metabolisms, biochemical markers were used to determine cholesterol and triglycerides levels, as well as mitochondrial complex I activity in zebrafish liver. Also, the hepatosomatic index as an indication of metabolic disruption, and the expression levels of genes involved in MET's putative MoA, i.e. acaca, acadm, cox5aa, idh3a, hmgcra, prkaa1, were determined, the last by qRT-PCR. A screening of mRNA transcripts, associated with lipid and energy metabolisms, and other signaling pathways potentially involved in MET-induced toxicity were also assessed using an exploratory RNA-seq analysis. The findings here reported indicate that MET significantly disrupted critical biochemical and molecular processes involved in zebrafish metabolism, such as cholesterol and fatty acid biosynthesis, mitochondrial electron transport chain and tricarboxylic acid cycle, concomitantly to changes on the hepatosomatic index. Likewise, MET impacted other relevant pathways mainly associated with cell cycle, DNA repair and steroid hormone biosynthesis, here reported for the first time in a non-target aquatic organism. Non-monotonic dose response curves were frequently detected in biochemical and qRT-PCR data, with higher effects observed at 390 and 2 929 ng/L MET treatments. Collectively, the results suggest that environmentally relevant concentrations of MET severely disrupt D. rerio metabolism and other important biological processes, supporting the need to revise the proposed environmental quality standard (EQS) and predicted no-effect concentration (PNEC) for MET.

The Joint i3+3 (Ji3+3) design for phase I/II adoptive cell therapy clinical trials

ABSTRACT Adoptive cell therapy (ACT), such as chimeric antigen receptor (CAR) T-cell therapy, has demonstrated promising therapeutic effects with potentially non-monotonic dose–response curves. Building upon the i3 + 3 design for cytotoxic agents [1], we propose a new method – joint i3 + 3 (Ji3 + 3) that takes into account of both safety and efficacy outcomes in making dosing recommendations. This allows for efficient dose escalation and identification of biological optimal dose of ACTs which may not be cytotoxic. The Ji3 + 3 design is rule based, easy to understand for clinicians, and is simple to implement. Simulation results show that Ji3 + 3 outperforms existing designs when monotonic dose–response assumption is violated, and still achieves comparable performance when the assumption holds. The simplicity and superior operating characteristics make Ji3 + 3 a good candidate for phase I/II ACT dose-finding trials in the clinical community when toxicity and efficacy are both considered as binary endpoints.

Early-life exposure to bisphenol A and reproductive-related outcomes in rodent models: a systematic review and meta-analysis.

We performed this meta-analysis to elucidate the associations between early-life BPA exposure and reproductive-related outcome indicators. The standardized mean differences (SMDs) and its 95% confidence intervals (CIs) were measured by fixed-effects or random-effects models. The results revealed that BPA exposure at extremely-high dose (>50mg/kg/day) was significantly associated with negative reproductive-related outcomes (Prostate weight: SMD: -4.21; 95% Cl: -5.97, -2.44; Testis weight: SMD: -1.92; 95% Cl: -2.61, -1.23; Epididymis weight: SMD: -2.16; 95% Cl: -3.47, -0.86; Daily sperm production; SMD: -1.90; 95% Cl: -3.27, -0.53; Epididymal sperm count; SMD: -3.42; 95% Cl: -3.87, -2.97). Meanwhile, regardless of the dose, early-life BPA exposure could result in an adverse effect on sperm parameters of F1 generation male rodents at any period. Also, we found the non-monotonic dose response curves of BPA in specific tissues or organs, which may challenge the traditional mindset of "safe dose". This study demonstrated that bisphenol A exposure was relevant to adverse reproductive-related outcomes at specially appointed dose and period of life. Yet the assumption that no adverse effects can occur below the "safe" dose is suspected.

A Combined Morphometric and Statistical Approach to Assess Nonmonotonicity in the Developing Mammary Gland of Rats in the CLARITY-BPA Study.

Background:The Consortium Linking Academic and Regulatory Insights on Bisphenol-A (CLARITY-BPA) is a rare collaboration of guideline-compliant (core) studies and academic hypothesis-based studies to assess the effects of bisphenol A (BPA).Objectives:We aimed to a) determine whether BPA showed effects on the developing rat mammary gland using new quantitative and established semiquantitative methods in two laboratories, b) develop a software tool for automatic evaluation of quantifiable aspects of the mammary ductal tree, and c) compare those methods.Methods:Sprague-Dawley rats were exposed to BPA, vehicle, or positive control [ethinyl estradiol (EE2)] by oral gavage beginning on gestational day (GD)6 and continuing with direct dosing of the pups after birth. There were two studies: subchronic and chronic. The latter used two exposure regimes, one stopping at postnatal day (PND)21 (stop-dose) the other continuing until tissue harvest (continuous). Glands were harvested at multiple time points; whole mounts and histological specimens were analyzed blinded to treatment.Results:The subchronic study’s semiquantitative analysis revealed no significant differences between control and BPA dose groups at PND21, whereas at PND90 there were significant differences between control and the lowest BPA dose and between control and the lowest EE2 dose in animals in estrus. Quantitative, automatized analysis of the chronic PND21 specimens displayed nonmonotonic BPA effects, with a breaking point between the 25 and body weight (BW) per day doses. This breaking point was confirmed by a global statistical analysis of chronic study animals at PND90 and 6 months analyzed by the quantitative method. The BPA response was different from the EE2 effect for many features.Conclusions:Both the semiquantitative and the quantitative methods revealed nonmonotonic effects of BPA. The quantitative unsupervised analysis used 91 measurements and produced the most striking nonmonotonic dose–response curves. At all time points, lower doses resulted in larger effects, consistent with the core study, which revealed a significant increase of mammary adenocarcinoma incidence in the stop-dose animals at the lowest BPA dose tested. https://doi.org/10.1289/EHP6301

Annealing Kinetics of Ti- and Ge-Related Centers in Quartz

Radiation defects in quartz are widely used in trapped charge chronometry. Analytical function which simulates dose–response curve is a very important for the correct measurement of equivalent dose. Ge- and Ti-related centers in quartz which may be used in electron paramagnetic resonance (EPR) dating have non-monotonic dose–response curves. In addition to accumulation, very significant parameter of radiation centers is their thermal stability. Obtained in this work results show that increase of concentration of paramagnetic Ge- and Ti-centers in quartz at the beginning of annealing may be explained by the existence of electron centers with two captured electrons which are not detected with used EPR spectrometer. Proposed model of formation and recombination processes satisfactorily simulates the experimental data—the dose dependencies and isothermal annealing data. It also explains why first- or second-order kinetics used in previous investigations cannot simulate annealing dependences correctly.

Multi-scale impact of chronic exposure to environmental concentrations of chlordecone in freshwater cnidarian, Hydra circumcincta.

Chlordecone (CLD) is an organochlorine pesticide widely used in the past to control pest insects in banana plantations in the French West Indies. Due to its persistence in the environment, CLD has contaminated the soils where it has been spread, as well as the waters, and is still present in them. The objective of our study was to evaluate the effects of chronic exposure to environmentally relevant CLD concentrations in an animal model, the freshwater hydra (Hydra circumcincta). In a multi-marker approach, we have studied the expression of some target stress genes, the morphology, and the asexual reproduction rates. Our data showed that exposure to low concentrations of chlordecone leads to (i) a modulation of the expression of target genes involved in oxidative stress, detoxification, and neurobiological processes, and (ii) morphological damages and asexual reproduction impairment. We have observed non-monotonic dose-response curves, which agree with endocrine-disrupting chemical effects. Thus, "U-shaped" dose-response curves were observed for SOD, GRed, Hym355, and potentially GST gene expressions; inverted "U-shaped" curves for GPx and CYP1A gene expressions and reproductive rates; and a biphasic dose-response curve for morphological damages. Therefore, in the range of environmental concentrations tested, very low concentrations of CLD can produce equally or more important deleterious effects than higher ones. Finally, to our knowledge, this study is the first one to fill the lack of knowledge concerning the effects of CLD in Hydra circumcincta and confirms that this diploblastic organism is a pertinent freshwater model in the risk assessment.

Nonmonotonic Dose-Response Curves Occur in Dose Ranges That Are Relevant to Regulatory Decision-Making.

Non-monotonic dose response curves (NMDRCs) occur in cells, tissues, animals and human populations in response to nutrients, vitamins, pharmacological compounds, hormones and endocrine disrupting chemicals (EDCs). Yet, regulatory agencies have argued that NMDRCs are not common, are not found for adverse outcomes, and are not relevant for regulation of EDCs. Under the linear dose response model, high dose testing is used to extrapolate to lower doses that are anticipated to be ‘safe’ for human exposures. NMDRCs that occur below the toxicological no-observed-adverse-effect level (NOAEL) would falsify a fundamental assumption, that high dose hazards can be used to predict low dose safety. In this commentary, we provide examples of NMDRCs and discuss how their presence in different portions of the dose response curve might affect regulatory decisions. We provide evidence that NMDRCs do occur below the NOAEL dose, and even below the ‘safe’ reference dose, for chemicals such as resveratrol, permethrin, chlorothalonil, and phthalates such as DEHP. We also briefly discuss the recent CLARITY-BPA study, which reported mammary adenocarcinomas only in rats exposed to the lowest BPA dose. We conclude our commentary with suggestions for how NMDRCs should be acknowledged and utilized to improve regulatory toxicity testing and in the calculation of reference doses that are public health protective.

Probabilistic assessment method of the non-monotonic dose-responses-Part II: Robustness assessment

In a previous study, we presented a new method that uses a large-scale sampling system to probabilistically assess non-monotonic dose-response curves. The statistical plausibility of the characterization was governed by the probability of the dominant category, but gave no information about the specific robustness of the curve.In this paper we propose an improvement to the method by integrating a scoring system based on 4 criteria which can be used to assess the slope robustness of each of the 10,000 sampled curves. The distribution criterion which assesses the number of doses forming a slope, the intensity criterion which assesses the amplitude of the response, and the minimum and maximum confirmation criteria which increase the certainty that the response is present. The probabilistic method was tested on 294 dose-response curves taken from 2 databases and 2 other methodologies currently proposed. A total of 544 dose-response curves have been processed.The developed method offers a concrete and probabilistic characterization of the type of curve analyzed. It evaluates its statistical plausibility and its robustness according to its sampling curves. This method is applicable to all types of data (continuous and discrete) and all experimental curves starting from theoretically 3 doses at least.

Linking in vivo toxicity data to ToxCast/Tox21 in vitro assay data

More and more studies aim to characterize non-monotonic dose response curves (NMDRCs). The greatest difficulty is to assess the statistical plausibility of NMDRCs from previously conducted dose response studies. This difficulty is linked to the fact that these studies present (i) few doses tested, (ii) a low sample size per dose, and (iii) the absence of any raw data.In this study, we propose a new methodological approach to probabilistically characterize NMDRCs. The methodology is composed of three main steps: (i) sampling from summary data to cover all the possibilities that may be presented by the responses measured by dose and to obtain a new raw database, (ii) statistical analysis of each sampled dose-response curve to characterize the slopes and their signs, and (iii) characterization of these dose-response curves according to the variation of the sign in the slope.This method allows characterizing all types of dose-response curves and can be applied both to continuous data and to discrete data.The aim of this study is to present the general principle of this probabilistic method which allows to assess the non-monotonic dose responses curves, and to present some results.

Probabilistic assessment method of the non-monotonic dose-responses-Part I: Methodological approach

More and more studies aim to characterize non-monotonic dose response curves (NMDRCs). The greatest difficulty is to assess the statistical plausibility of NMDRCs from previously conducted dose response studies. This difficulty is linked to the fact that these studies present (i) few doses tested, (ii) a low sample size per dose, and (iii) the absence of any raw data.In this study, we propose a new methodological approach to probabilistically characterize NMDRCs. The methodology is composed of three main steps: (i) sampling from summary data to cover all the possibilities that may be presented by the responses measured by dose and to obtain a new raw database, (ii) statistical analysis of each sampled dose-response curve to characterize the slopes and their signs, and (iii) characterization of these dose-response curves according to the variation of the sign in the slope.This method allows characterizing all types of dose-response curves and can be applied both to continuous data and to discrete data.The aim of this study is to present the general principle of this probabilistic method which allows to assess the non-monotonic dose responses curves, and to present some results.

Atrazine exposure affects longevity, development time and body size in Drosophila melanogaster

Atrazine is the one of the most widely used herbicides in the United States and non-target organisms may encounter it in the environment. Atrazine is known to affect male reproduction in both vertebrates and invertebrates but less is known about its effects on other fitness traits. Here we assessed the effects of five different chronic exposure levels on a variety of fitness traits in Drosophila melanogaster. We measured male and female longevity, development time, proportion pupated, proportion emerged, body size, female mating rate, fertility and fecundity. Atrazine exposure decreased the proportion pupated, the proportion emerged and adult survival. Development time was also affected by atrazine and exposed flies pupated and emerged earlier than controls. Although development time was accelerated, body size was actually larger in some of the exposures. Atrazine exposure had no effect on female mating rate and the effects on female fertility and fecundity were only observed in one of the two independent experimental blocks. Many of the traits showed non-monotonic dose response curves, where the intermediate concentrations showed the largest effects. Overall this study shows that atrazine influences a variety of life history traits in the model genetic system, D. melanogaster, and future studies should aim to identify the molecular mechanisms of toxicity.

Regulatory Perspective on Non-Monotonic Dose-Response Curves and “Low dose effects”

Regulatory Perspective on Non-Monotonic Dose-Response Curves and “Low dose effects”

Complex, non-monotonic dose-response curves with multiple maxima: Do we (ever) sample densely enough?

We usually expect the dose-response curves of biological responses to quantifiable stimuli to be simple, either monotonic or exhibiting a single maximum or minimum. Deviations are often viewed as experimental noise. However, detailed measurements in plant primary tissue cultures (stem pith explants of kale and tobacco) exposed to varying doses of sucrose, cytokinins (BA or kinetin) or auxins (IAA or NAA) revealed that growth and several biochemical parameters exhibit multiple reproducible, statistically significant maxima over a wide range of exogenous substance concentrations. This results in complex, non-monotonic dose-response curves, reminiscent of previous reports of analogous observations in both metazoan and plant systems responding to diverse pharmacological treatments. These findings suggest the existence of a hitherto neglected class of biological phenomena resulting in dose-response curves exhibiting periodic patterns of maxima and minima, whose causes remain so far uncharacterized, partly due to insufficient sampling frequency used in many studies.

A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators.

A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and subsequent binding the activated AHR to xenobiotic response elements (XREs) on DNA. The model was based on effects observed in cells lines commonly used as in vitro experimental systems. Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT). In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs) at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism’s ability to respond on a phenotypic level to various stimuli within an inconstant environment.

Non-monotonic dose responses in EDSP Tier 1 guideline assays

In the fields of endocrinology and toxicology, there are ongoing debates about whether endocrine disrupting chemicals (EDCs) produce non-monotonic dose responses. This type of response is typically characterized by a U- or inverted U-shaped relationship between dose and effect. In a recent report, a US EPA panel concluded that non-monotonicity is observed for EDCs, but that these responses are not expected in vivo, and are not typically observed in apical endpoints, i.e. endpoints that are indicators of adverse effects. Here, we have analyzed the shapes of the dose response curves in an EPA report analyzing the effectiveness of the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays. This report included the results of 11 guideline assays for each of 4 chemicals. We found indications of non-monotonic dose response curves (iNMDRCs) for 3 of the 4 coded chemicals. In total, 27% of assays with dose response data included at least one iNMDRC. When the endpoints from the 4 test chemicals with dose respo...

Non-monotonic dose responses in studies of endocrine disrupting chemicals: bisphenol a as a case study.

Non-monotonic dose response curves (NMDRCs) have been demonstrated for natural hormones and endocrine disrupting chemicals (EDCs) in a variety of biological systems including cultured cells, whole organ cultures, laboratory animals and human populations. The mechanisms responsible for these NMDRCs are well known, typically related to the interactions between the ligand (hormone or EDC) and a hormone receptor. Although there are hundreds of examples of NMDRCs in the EDC literature, there are claims that they are not 'common enough' to influence the use of high-to-low dose extrapolations in risk assessments. Here, we chose bisphenol A (BPA), a well-studied EDC, to assess the frequency of non-monotonic responses. Our results indicate that NMDRCs are common in the BPA literature, occurring in greater than 20% of all experiments and in at least one endpoint in more than 30% of all studies we examined. We also analyzed the types of endpoints that produce NMDRCs in vitro and factors related to study design that influence the ability to detect these kinds of responses. Taken together, these results provide strong evidence for NMDRCs in the EDC literature, specifically for BPA, and question the current risk assessment practice where 'safe' low doses are predicted from high dose exposures.

A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays

In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic ‘switching’. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.