Polycyclic aromatic hydrocarbons and PAH-related DNA adducts

Investigations on the impact of chemicals on the environment and human health have led to the development of an exposome concept. The exposome refers to the totality of exposures received by a person during life, including exposures to life-style factors, from the prenatal period to death. The exposure to genotoxic chemicals and their reactive metabolites can induce chemical modifications of DNA, such as, for example, DNA adducts, which have been extensively studied and which play a key role in chemically induced carcinogenesis. Development of different methods for the identification of DNA adducts has led to adopting DNA adductomic approaches. The ability to simultaneously detect multiple PAH-derived DNA adducts may allow for the improved assessment of exposure, and offer a mechanistic insight into the carcinogenic process following exposure to PAH mixtures. The major advantage of measuring chemical-specific DNA adducts is the assessment of a biologically effective dose. This review provides information about the occurrence of the polycyclic aromatic hydrocarbons (PAHs) and their influence on human exposure and biological effects, including PAH-derived DNA adduct formation and repair processes. Selected methods used for determination of DNA adducts have been presented.     

Combining environmental exposure and genetic effect measurements in health outcome assessment.

The presence of overwhelming difficulties in assessing the extent or even the presence of a causal association between modern environmental exposures and disease has promoted the use of more complex models in the design of human biomonitoring studies. The concatenation of environmental exposure, genetic effect and individual susceptibility is a key issue in the assessment of risks for populations exposed to environmental pollutants. The use of a biological event laying in the causal pathway from exposure to outcome as surrogate end-point of disease, can potentially anticipate clinical diagnosis, offering a number of possibilities for application of preventive measures. Numerous biomarkers are currently employed to study human populations exposed to environmental carcinogens, among these, the frequency of chromosomal aberration (CA) in peripheral blood lymphocytes has the most abundant literature linking a genetic effect with the occurrence of cancer. Findings from recent epidemiological studies which have followed-up a large group of healthy subjects screened for CAs have lent further support to the use of chromosomal breakage as a relevant biomarker of cancer risk. The applicability of surrogate end-points of cancer on an individual basis thus far seems to be limited to few examples. On the other hand, from a public health outlook, increases in the frequency of surrogate end-points are suggestive of an increased risk of cancer, and for validated biomarkers such as CAs intervention policies and actions in exposed populations showing increased frequency of these end-points should be always recommended.     

Adolescent idiopathic scoliosis (AIS), environment, exposome and epigenetics: a molecular perspective of postnatal normal spinal growth and the etiopathogenesis of AIS with consideration of a network approach and possible implications for medical therapy

ABSTRACT: Genetic factors are believed to play an important role in the etiology of adolescent idiopathic scoliosis (AIS). Discordant findings for monozygotic (MZ) twins with AIS show that environmental factors including different intrauterine environments are important in etiology, but what these environmental factors may be is unknown. Recent evidence for common chronic non-communicable diseases suggests epigenetic differences may underlie MZ twin discordance, and be the link between environmental factors and phenotypic differences. DNA methylation is one important epigenetic mechanism operating at the interface between genome and environment to regulate phenotypic plasticity with a complex regulation across the genome during the first decade of life. The word exposome refers to the totality of environmental exposures from conception onwards, comprising factors in external and internal environments. The word exposome is used here also in relation to physiologic and etiopathogenetic factors that affect normal spinal growth and may induce the deformity of AIS. In normal postnatal spinal growth we propose a new term and concept, physiologic growth-plate exposome for the normal processes particularly of the internal environments that may have epigenetic effects on growth plates of vertebrae. In AIS, we propose a new term and concept pathophysiologic scoliogenic exposome for the abnormal processes in molecular pathways particularly of the internal environment currently expressed as etiopathogenetic hypotheses; these are suggested to have deforming effects on the growth plates of vertebrae at cell, tissue, structure and/or organ levels that are considered to be epigenetic. New research is required for chromatin modifications including DNA methylation in AIS subjects and vertebral growth plates excised at surgery. In addition, consideration is needed for a possible network approach to etiopathogenesis by constructing AIS diseasomes. These approaches may lead through screening, genetic, epigenetic, biochemical, metabolic phenotypes and pharmacogenomic research to identify susceptible individuals at risk and modulate abnormal molecular pathways of AIS. The potential of epigenetic-based medical therapy for AIS cannot be assessed at present, and must await new research derived from the evaluation of epigenetic concepts of spinal growth in health and deformity. The tenets outlined here for AIS are applicable to other musculoskeletal growth disorders including infantile and juvenile idiopathic scoliosis.     

Has the mist been peered through? Revisiting the building blocks of human health risk assessment for electronic cigarette use

Electronic cigarettes, battery-powered nicotine delivery devices, have been increasingly used in the past decade. This critical review provides a qualitative research synthesis of the human health risks associated with E-vapor inhalation in the peer-reviewed literature and our own preliminary experimental results. E-cigarettes may be as efficient as traditional cigarettes in nicotine delivery, especially for experienced users, and studies suggest lower emissions of air toxics from E-cigarette vapor and lower second- and third-hand vapor exposures. Some toxic emissions may however surpass those of traditional cigarettes, especially under high voltage vaping conditions. Experimentally, E-vapor/E-liquid exposures reduce cell viability and promote pro-inflammatory cytokine release. User vulnerability to concomitant environmental agent exposures, such as viruses and bacteria, may potentially be increased. While evidence to date suggests that E-cigarettes release fewer toxins and carcinogens compared to cigarettes, E-vapor is not safe and might adversely affect human immune functions. Major knowledge gaps hinder risk quantification and effective regulation of E-cigarette products including: lack of long-term exposure studies, lack of understanding of biological mechanisms associated with exposure, and lack of integration of exposure and toxicity assessments. Better data are needed to inform human health risk assessments and understand the public health impact of E-vapor exposures.     

High-performance metabolic profiling with dual chromatography-Fourier-transform mass spectrometry (DC-FTMS) for study of the exposome

Studies of gene–environment (G × E) interactions require effective characterization of all environmental exposures from conception to death, termed the exposome. The exposome includes environmental exposures that impact health. Improved metabolic profiling methods are needed to characterize these exposures for use in personalized medicine. In the present study, we compared the analytic capability of dual chromatography-Fourier-transform mass spectrometry (DC-FTMS) to previously used liquid chromatography-FTMS (LC-FTMS) analysis for high-throughput, top-down metabolic profiling. For DC-FTMS, we combined data from sequential LC-FTMS analyses using reverse phase (C18) chromatography and anion exchange (AE) chromatography. Each analysis was performed with electrospray ionization in the positive ion mode and detection from m/z 85 to 850. Run time for each column was 10 min with gradient elution; 10 μl extracts of plasma from humans and common marmosets were used for analysis. In comparison to analysis with the AE column alone, addition of the second LC-FTMS analysis with the C18 column increased m/z feature detection by 23–36%, yielding a total number of features up to 7,000 for individual samples. Approximately 50% of the m/z matched to known chemicals in metabolomic databases, and 23% of the m/z were common to analyses on both columns. Database matches included insecticides, herbicides, flame retardants, and plasticizers. Modularity clustering algorithms applied to MS-data showed the ability to detection clusters and ion interactions. DC-FTMS thus provides improved capability for high-performance metabolic profiling of the exposome and development of personalized medicine.

     

New applications of biological monitoring for environmental exposure and susceptibility monitoring. Report of the 7th International Symposium on Biological Monitoring in Occupational and Environmental Health

Validated biological monitoring methods are used in large-scale monitoring programmes involving determination of ubiquitous environmental pollutants such as metals and pesticides. Some programmes focus on children's exposure, and policies to prevent adverse health effects. Most of these initiatives are aimed at characterizing trends. Some of these programmes are designed to investigate the role of certain exposures in disease. Fewer new biological monitoring methods were presented during the present meeting than in previous meetings. All of these new methods used mass spectrometric-based detection and quantification. There is an increasing use of biomarkers to study genetic polymorphisms of enzyme systems involved in both toxification pathways and metabolite conjugation and DNA repair. At the meeting a discussion was started that could lead to a further harmonization of the scientific fundaments of the use of biological monitoring in occupational health with possible value also for applications in the field of environmental health.     

Ecosystem Functioning and Community Structure as Indicators for Assessing Environmental Impacts: Leaf Processing and Macroinvertebrates in Atlantic Forest Streams

Programs for evaluating environmental impacts are often carried out with chemical analyses that are inadequate for evaluating ecological aspects of rivers. In recent times there has been a strong movement towards biomonitoring using indices based on the structure of communities of organisms. Less attention has been given to functional parameters (ecosystem level processes such as decomposition, primary production, nutrient cycling, etc.), despite recent work that demonstrates their applicability. The breakdown of leaf material in streams provides a system in which an aspect of ecosystem functioning (decomposition) can be measured along with the community structure of the fauna associated with the leaves. We measured rate of leaf processing and the associated macroinvertebrates in 9 streams in 3 categories of environmental impact: Reference (pristine forest), Intermediate (pasture land‐use with intact riparian vegetation) and Impacted (pasture land‐use with degraded banks and stream‐bed). Leaf processing was fastest in the least impacted, “Reference”, streams. The abundance and taxon diversity of the associated fauna were not different among categories of impact. Thus this aspect of ecosystem functioning was more sensitive to perturbation than was the structure of the community in this case. We suggest that leaf processing can be a cheap and indicative parameter for biological assessment and monitoring. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Human Risk Assessment for Nonylphenol

This article presents a risk assessment for human exposure to nonylphenol (NP). We critically reviewed and assessed all relevant full-text publications based on a variety of data quality attributes. Two categories of data, environmental monitoring and biomonitoring from exposed individuals, were used to estimate human exposure to NP. Environmental monitoring data included the measurement of NP in food, water, air, and dust. From these data and estimates of human intake rates for the sources, exposures were estimated from each source and source-specific Margins of Exposure (MOEs) calculated. However, the nature of the populations studied prevented the calculation of aggregate exposure calculations from these data. Rather, the most reliable estimates of aggregate exposure to NP were those derived from biomonitoring studies in exposed individuals. Using the daily absorbed dose estimates for NP, MOEs were calculated for these populations. The MOEs were based on the use of a No-Observed-Adverse-Effect-Level (NOAEL) for sensitive toxicological endpoints of interest, that is, systemic and reproductive toxicity from continuous-feeding more than 3.5 generations (13 mg/kg/day). The MOEs were all greater than 1000 (ranging from 2863 to 8.4 × 10⁷), clearly indicating reasonable certainty of no harm for source-specific and aggregate (based on biomonitoring) exposures to NP.     

Biomonitoring using accessible human cells for exposure and health risk assessment

A major goal for genetic toxicologist is to provide precise information on exposure and health risk assessment for effective prevention of health problems. A frequently used approach for population study has been to utilize readily available blood cells (lymphocytes and red blood cells) as sentinel cell types to detect biological effects from exposure and to provide early warning signals for health risk. However, such approach still cannot be used reliably for developing strategies in risk assessment and disease prevention. It is possible that other available cell types which are more representative of the target cells for disease may be used to overcome the deficiency. In this report, the use of non-blood cells for biomonitoring is briefly reviewed. Their usefulness in certain exposure condition is highlighted and their effectiveness in documenting exposure compared with other cell types such as the traditional blood cells is presented. It is obvious that the decision in using these non-blood cells in biomonitoring is based on the exposure condition and the experimental design. Nevertheless, monitoring studies using non-blood cells should be encouraged with emphasis on providing dose–response information, comparative response with other cell types and effectiveness for health risk assessment.     

A quantitative risk assessment for chronic traumatic encephalopathy (CTE) in football: How public health science evaluates evidence

How should science and policy interpret the recent finding that 110 of 111 former National Football League (NFL) players had brain pathology known as chronic traumatic encephalopathy (CTE) at autopsy? Some physicians view this (and related epidemiologic and mechanistic evidence) skeptically, emphasizing that the association between repeated head trauma (RHT) and CTE may be artifactual, that this “incidence” is biased by self-selection of players with cognitive or emotional symptoms, and that even if RHT causes CTE, the lesions themselves may be inconsequential. Public health scientists look at this emerging evidence quite differently; in particular, they tend not to fall prey to certain illogical arguments justifying inaction. We present a quantitative risk assessment showing that even accounting for the non-representativeness of the 110 cases, the risk of CTE in the NFL workforce amply meets both parts of the test for “a significant risk of material impairment of health” that would permit the U.S. Occupational Safety and Health Administration to intervene to reduce RHT exposure. We further conclude that according to available evidence, CTE is a public health problem, and that lawyers and physicians need to understand that this conclusion is based on standards of evidence at least as long-standing and robust as their own.     

Developmental and transplacental genotoxicology: fluconazole

Over the last 40 years mankind has been facing new types of radiochemical environmental settings with every decade. During the last decade, biomonitoring was additionally focused on assessing associations between environmental exposure(s) and both early and late biological effects in children. Despite efforts to control and avoid child exposure to genotoxic agents the incidence of childhood cancers is increasing. Some cancers in adulthood may be the consequence of a multi-step process which starts with intrauterine and childhood exposure. This highlights the importance of a comprehensive interpretation of multiple health effects, especially considering recent studies suggesting that most health disorders are related to DNA changes. When exposed to genotoxic agents, a developing organism (fetus or child) is constantly being forced to reorganize into new equilibriums in order to adjust to a xenobiotic environment. In addition, the influence of sex hormones on radiochemical sensitivity is still unknown. For this reason special attention should be paid to puberty. The results of recent studies on animal models and follow up studies on children after nuclear accidents show long-lasting cytogenetic damage even after low dose exposures and their transgenerational persistance. To evaluate age-related difference and transplacental genotoxic potency fluconazole (FC) was investigated by in vivo micronucleus (MN) assay in adult mice, young mice and in transplacentally exposed newborn pups. Compared to the baseline values, FC caused no detectable genome damage in adult animals, but there was a significant increase in MN frequency in young animals and in newborn pups. Our study thus exemplifies an age-related chemosensitivity, and argues that cancer-promoting disturbances of complex prenatal developmental mechanisms and maturation during childhood require a new approach using systems biology.     

Assessment of ecological impairment of Arctic streams: Challenges and future directions

As increased growth and development put pressure on freshwater systems in Arctic environments, there is a need to maintain a meaningful and feasible framework for monitoring water quality. A useful tool for monitoring the ecological health of aquatic systems is by means of the analysis and inferences made from benthic invertebrates in a biomonitoring approach. Biomonitoring of rivers and streams within the Arctic has been under‐represented in research efforts. Here, we investigate an approach for monitoring biological impairment in Arctic streams from anthropogenic land use at two streams with different exposure to urban development in Iqaluit, Nunavut, Arctic Canada. Sites upstream of development, at midpoint locations, and at the mouth of each waterbody were sampled during 6 campaigns (2008, 2009, 2014, 2015, 2018, and 2019) to address spatial and temporal variability of the macroinvertebrate community. The influence of taxonomic resolution scaling was also examined in order to understand the sensitivity of macroinvertebrates as indicators in Arctic aquatic systems. We demonstrate that standard biological metrics were effective in indicating biological impairment downstream of sources of point‐source pollutants. A mixed‐design ANOVA for repeated measures also found strong interannual variability; however, we did not detect intra‐annual variation from seasonal factors. When examining metrics at the highest taxonomic resolution possible, the sensitivity of metrics increased. Likewise, when trait‐based metrics (α functional diversity) were applied to indicators identified at high taxonomic resolution, a significant difference was found between reference and impacted sites. Our results show that even though Arctic systems have lower diversity and constrained life‐history characteristics compared to temperate ecosystems, biomonitoring is not only possible, but also equally effective in detecting trends from anthropogenic activities. Thus, biomonitoring approaches in Arctic environments are likely a useful means for providing rapid and cost‐effective means of assessing future environmental impact.     

High-performance metabolic profiling with dual chromatography-Fourier-transform mass spectrometry (DC-FTMS) for study of the exposome

Studies of gene–environment (G × E) interactions require effective characterization of all environmental exposures from conception to death, termed the exposome. The exposome includes environmental exposures that impact health. Improved metabolic profiling methods are needed to characterize these exposures for use in personalized medicine. In the present study, we compared the analytic capability of dual chromatography-Fourier-transform mass spectrometry (DC-FTMS) to previously used liquid chromatography-FTMS (LC-FTMS) analysis for high-throughput, top-down metabolic profiling. For DC-FTMS, we combined data from sequential LC-FTMS analyses using reverse phase (C18) chromatography and anion exchange (AE) chromatography. Each analysis was performed with electrospray ionization in the positive ion mode and detection from m/z 85 to 850. Run time for each column was 10 min with gradient elution; 10 μl extracts of plasma from humans and common marmosets were used for analysis. In comparison to analysis with the AE column alone, addition of the second LC-FTMS analysis with the C18 column increased m/z feature detection by 23–36%, yielding a total number of features up to 7,000 for individual samples. Approximately 50% of the m/z matched to known chemicals in metabolomic databases, and 23% of the m/z were common to analyses on both columns. Database matches included insecticides, herbicides, flame retardants, and plasticizers. Modularity clustering algorithms applied to MS-data showed the ability to detection clusters and ion interactions. DC-FTMS thus provides improved capability for high-performance metabolic profiling of the exposome and development of personalized medicine.     

Comparing Occupational and Environmental Risk Assessment Methodologies Using Pharmacokinetic Modeling

Human health risks from occupational exposures are managed by limiting exposures to acceptable levels established by the American Conference of Governmental Industrial Hygienists or another similar body. Acceptable environmental exposures are benchmarked by values such as U.S. Environmental Protection Agency's Reference Doses and Reference Concentrations. The approaches to establishing these values are different, as are the groups they are intended to protect, complicating direct comparisons. Occupational limits are based on a healthy workforce in a narrow age range and do not generally consider sensitive populations. Limits for environmental exposures consider sensitive populations. In this evaluation, physiologically based pharmacokinetic modeling was used to predict tissue doses from acceptable/safe exposures as established by different organizations and agencies. Internal doses calculated for an agency's acceptable/safe exposures via oral and inhalation routes may differ substantially, but are sometimes in excellent agreement. The finding that internal doses resulting from occupational exposures are almost uniformly greater than those from environmental exposures suggests different mindsets among these groups regarding how safe is “safe.”     

Mendelian randomization in health research: Using appropriate genetic variants and avoiding biased estimates

Mendelian randomization methods, which use genetic variants as instrumental variables for exposures of interest to overcome problems of confounding and reverse causality, are becoming widespread for assessing causal relationships in epidemiological studies. The main purpose of this paper is to demonstrate how results can be biased if researchers select genetic variants on the basis of their association with the exposure in their own dataset, as often happens in candidate gene analyses. This can lead to estimates that indicate apparent “causal” relationships, despite there being no true effect of the exposure. In addition, we discuss the potential bias in estimates of magnitudes of effect from Mendelian randomization analyses when the measured exposure is a poor proxy for the true underlying exposure. We illustrate these points with specific reference to tobacco research.     

A lifestage approach to assessing children's exposure

Understanding and characterizing risks to children has been the focus of considerable research efforts at the U.S. Environmental Protection Agency (EPA). Potential health risks resulting from environmental exposures before conception and during pre‐ and postnatal development are often difficult to recognize and assess because of a potential time lag between the relevant periods of exposure during development and associated outcomes that may be expressed at later lifestages. Recognizing this challenge, a lifestage approach for assessing exposure and risk is presented in the recent EPA report titled A Framework for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA, 2006 ). This EPA report emphasizes the need to account for the potential exposures to environmental agents during all stages of development, and consideration of the relevant adverse health outcomes that may occur as a result of such exposures. It identifies lifestage‐specific issues associated with exposure characterization for regulatory risk assessment, summarizes the lifestage‐specific approach to exposure characterization presented in the Framework, and discusses emerging research needs for exposure characterization in the larger public‐health context. This lifestage approach for characterizing children's exposures to environmental contaminants ensures a more complete evaluation of the potential for vulnerability and exposure of sensitive populations throughout the life cycle. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

On the road to synthetic life: the minimal cell and genome-scale engineering

Synthetic biology employs rational engineering principles to build biological systems from the libraries of standard, well characterized biological parts. Biological systems designed and built by synthetic biologists fulfill a plethora of useful purposes, ranging from better healthcare and energy production to biomanufacturing. Recent advancements in the synthesis, assembly and “booting-up” of synthetic genomes and in low and high-throughput genome engineering have paved the way for engineering on the genome-wide scale. One of the key goals of genome engineering is the construction of minimal genomes consisting solely of essential genes (genes indispensable for survival of living organisms). Besides serving as a toolbox to understand the universal principles of life, the cell encoded by minimal genome could be used to build a stringently controlled “cell factory” with a desired phenotype. This review provides an update on recent advances in the genome-scale engineering with particular emphasis on the engineering of minimal genomes. Furthermore, it presents an ongoing discussion to the scientific community for better suitability of minimal or robust cells for industrial applications.     

Defining and measuring river health

1. Society benefits immeasurably from rivers. Yet over the past century, humans have changed rivers dramatically, threatening river health. As a result, societal well-being is also threatened because goods and services critical to human society are being depleted. 2. ‘Health’— shorthand for good condition (e.g. healthy economy, healthy communities) — is grounded in science yet speaks to citizens. 3. Applying the concept of health to rivers is a logical outgrowth of scientific principles, legal mandates, and changing societal values. 4. Success in protecting the condition, or health, of rivers depends on realistic models of the interactions of landscapes, rivers, and human actions. 5. Biological monitoring and biological endpoints provide the most integrative view of river condition, or river health. Multimetric biological indices are an important and relatively new approach to measuring river condition. 6. Effective multimetric indices depend on an appropriate classification system, the selection of metrics that give reliable signals of river condition, systematic sampling protocols that measure those biological signals, and analytical procedures that extract relevant biological patterns. 7. Communicating results of biological monitoring to citizens and political leaders is critical if biological monitoring is to influence environmental policies. 8. Biological monitoring is essential to identify biological responses to human actions. By using the results to describe the condition, or health, of rivers and their adjacent landscapes and to diagnose causes of degradation, we can develop restoration plans, estimate the ecological risks associated with land use plans in a watershed, or select among alternative development options to minimize river degradation.     

Climate change and health with an emphasis on interactions with ultraviolet radiation: a review

Climate change is increasingly recognized as a major risk to human health, and health concerns are assuming more importance in international debates on mitigation and adaptation strategies. Health consequences of climate change will occur through direct and indirect routes, and as a result of interactions with other environmental exposures. Heatwaves will become more common and are associated with higher mortality particularly in the elderly and those with pre‐existing cardiovascular and respiratory illnesses. Warmer ambient temperatures will result in more dehydration episodes and increased risks of renal disease and, through effects on pollen seasons, there may be an increase in allergic disease such as asthma and hayfever. Other adverse effects including on air quality, food safety and security and an expanding distribution of some infectious diseases, including vector‐borne diseases, are postulated. A related but separate environmental exposure is that of ultraviolet radiation (UVR). Interactions between climate change and stratospheric ozone (and the causes of ozone depletion) will cause changes to levels of ambient UVR in the future and warmer temperatures are likely to change sun exposure behaviour. Co‐occurring effects on aquatic and terrestrial ecosystems have potential consequences for food safety, quality and supply. Climate change‐related exposures are likely to affect the incidence and distribution of diseases usually considered as caused by UVR exposure; and changes in UVR exposure will modulate the climate change effects on human health. For example, in some regions warmer temperatures due to climate change will encourage more outdoor behaviour, with likely consequences for increasing skin cancer incidence. Although many of the health outcomes of both climate change and the interaction of climate change and UVR exposure are somewhat speculative, there are risks to over‐ or under‐estimations of health risks if synergistic and antagonistic effects of co‐occurring environmental changes are not considered.     

Methods for evaluating variability in human health dose–response characterization

Abstract

The Reference Dose (RfD) and Reference Concentration (RfC) are human health reference values (RfVs) representing exposure concentrations at or below which there is presumed to be little risk of adverse effects in the general human population. The 2009 National Research Council report Science and Decisions recommended redefining RfVs as “a risk-specific dose (for example, the dose associated with a 1 in 100,000 risk of a particular end point).” Distributions representing variability in human response to environmental contaminant exposures are critical for deriving risk-specific doses. Existing distributions estimating the extent of human toxicokinetic and toxicodynamic variability are based largely on controlled human exposure studies of pharmaceuticals. New data and methods have been developed that are designed to improve estimation of the quantitative variability in human response to environmental chemical exposures. Categories of research with potential to provide new data useful for developing updated human variability distributions include controlled human experiments, human epidemiology, animal models of genetic variability, in vitro estimates of toxicodynamic variability, and in vitro-based models of toxicokinetic variability. In vitro approaches, with further development including studies of different cell types and endpoints, and approaches to incorporate non-genetic sources of variability, appear to provide the greatest opportunity for substantial near-term advances.