Incorporating Transgenerational Epigenetic Inheritance into

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Incorporating transgenerational epigenetic inheritance into ecological risk assessment frameworks. Jennifer L.A. Shaw, Jonathan D Judy, Anupama Kumar, Paul Bertsch, Ming-Bo Wang, and Jason K. Kirby Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01094 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Environmental Science & Technology

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Incorporating transgenerational epigenetic inheritance into ecological risk

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assessment frameworks.

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Jennifer L.A. Shaw1*, Jonathan D. Judy1,3, Anupama Kumar1, Paul Bertsch2, Ming-Bo

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Wang4, Jason, K. Kirby1.

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*Corresponding author email: [email protected], tel: +61 8 8303 8438.

*Commonwealth Scientific and Industrial Research Organisation (CSIRO), Land and Water, Environmental Contaminant Mitigation and Technologies Research Program, Waite Road, Urrbrae, Adelaide, Australia, 5064. 2 Commonwealth Scientific and Industrial Research Organisation (CSIRO), Land and Water, Brisbane, Queensland, Australia, 4001. 3University of Florida, Soil and Water Sciences Department, 1692 McCarthy Drive, Gainesville, Florida, 32611. 4 Commonwealth Scientific and Industrial Research Organisation (CSIRO), Agriculture and Food Unit, Black Mountain, Canberra, ACT, Australia, 2601.

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Abstract

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Chronic exposure to environmental contaminants can induce heritable ‘transgenerational’

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modifications to organisms, potentially affecting future ecosystem health and functionality.

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Incorporating transgenerational epigenetic heritability into risk assessment procedures has

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been previously suggested. However, a critical review of existing literature yielded numerous

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studies claiming transgenerational impacts, with little compelling evidence. Therefore,

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contaminant-induced epigenetic inheritance may be less common than is reported in the

ACS Paragon Plus Environment


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literature. We identified a need for multi-generation epigenetic studies that extend beyond

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what could be deemed ‘direct exposure’ to F1 and F2 gametes, which also include subsequent

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multiple non-exposed generations to adequately evaluate transgenerational recovery times.

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Also, increased experimental replication is required to account for the highly variable nature

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of epigenetic responses and apparent irreproducibility of current studies. Further, epigenetic

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endpoints need to be correlated with observable detrimental organism changes before a need

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for risk management can be properly determined. We suggest that epigenetic-based

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contaminant studies include concentrations lower than current ‘EC10-20’ or ‘Lowest

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Observable Effect Concentrations’ for the organism’s most sensitive phenotypic endpoint, as

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higher concentrations are likely to be already regulated. Finally, we propose a regulatory

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framework and optimal experimental design that enables transgenerational epigenetic effects

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to be assessed and incorporated into conventional ecotoxicological testing.

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Key words: multigenerational | chronic contamination | ecotoxicology | DNA methylation |

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gene expression | environment

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1. Introduction

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Contaminants can enter the environment via multiple pathways such as agricultural

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production, urban runoff, manufacturing, waste disposal and mining. These contaminants can

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have adverse effects on ecologically important organisms1–3 and ecosystem health.4,5 To

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reduce environmental and health impacts, it is critical to understand contaminant-associated

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risks, develop appropriate regulations, and implement appropriate risk management

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strategies. Potential risks are assessed using specific frameworks that incorporate an

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understanding of the biological and functional impacts caused by different exposure

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concentrations and pathways.6–11 Assessments are made by measuring changes to various


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biological and ecological endpoints in response to increasing concentrations of a

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contaminant. Standard methods focus on organismal responses such as decreased biomass,

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growth rates, reproductive fitness, increased mortality, or bioaccumulation of contaminant

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residues, but sometimes also include molecular stress-biomarkers such as increases in

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reactive oxygen species12,13, DNA damage14, and changes to hormonal and enzymatic

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activities.15,16

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More recently, epigenetic biomarkers have been highlighted as a potential future risk-

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assessment tool.

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code, which influence how an organism’s genes are expressed.20 These modifications can

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change in response to a multitude of environmental factors, temporarily or permanently

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altering expression of the DNA code and altering the organism’s phenotype as a result.21

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Most importantly, epigenetic modifications can sometimes become inherited by offspring,22,23

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potentially altering the evolutionary pathway of future generations. Therefore, there is

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potential for transgenerational epigenetic analysis to be used for determining chronic risk to

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future generations.17,24

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We critically evaluate available literature on contaminant-induced transgenerational and

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epigenetic changes, and explore key issues and gaps in current research that hinder our ability

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to utilise these tools in risk assessment. We evaluate whether transgenerational epigenetics

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could add value to existing ecological risk assessment frameworks, and propose a strategy for

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developing useable transgenerational epigenetic endpoints for ecological risk assessment.

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Finally, we propose a tiered assessment framework and experimental design, which

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incorporate transgenerational, chronic risk into current risk assessment frameworks.

17–19

Epigenetics describes chemical modifications that exist along a DNA

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2. Contaminant-induced epigenetic changes



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Numerous environmental contaminants have been shown to induce epigenetic changes to a

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wide range of ecologically relevant organisms (Table 1), such as mammals22,25, plants26–28,

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insects29,30, fish31–33, nematode worms34, molluscs35, and reptiles36,37. Several of these studies

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have cited contaminant-induced hypermethylation of specific genes, suggesting that

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epigenetic alterations vary with contaminant type. For example, studies have demonstrated

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that regions of the rat (Rattus norvegicus) genome are differentially methylated in response to

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exposure from different contaminants including dioxins38, plastics39, and pesticides.40

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Vandegehuchte et al. (2010) found that water fleas (Daphnia magna) exposed to different

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contaminants (7.4 mg/L 5-azacytidine, 4.4 mg/L genistein, and 3.6 mg/L vinclozolin) had

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alternative methylation profiles.41 And in plants, Aina et al., (2004) found that exposure to

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different heavy metals (25 and 100 mg/kg Ni2+, 25 and 100 mg/kg Cd2+, and 25 and 50 mg/kg

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Cr6+) induced site-specific DNA methylation.42 Another study examining plant responses

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found that 100 mg/L concentration of various nanoparticles (ZnO, TiO2, and fullerene soot)

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induced statistically different gene expression profiles 43, and Nair and Chung (2014) found

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that specific genes (mismatch repair MMR and AtPCNA associated genes) were significantly

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up-regulated in plants when exposed to Ag nanoparticles (0.2, 0.5, 1 mg/kg) but not Ag ions

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at equivalent concentrations.44

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Other researchers have argued that these contaminant-induced epigenetic responses are

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simply stochastic in nature. For example, a study by Takiguchi et al. (2003) demonstrated

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that 2.5 µM cadmium reduced genome methylation by interfering with methyltransferase

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molecules.45 Valinuck et al. (2004) similarly demonstrated that metal induced oxidative stress

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interferes with the ability of methyltransferases to interact with DNA46, which could result in

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generalised (stochastic) changes to methylation patterns. These studies confirmed that for

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some contaminants DNA methylation changes occur as a by-product of enzymatic

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inhibition47 and therefore, contaminant-induced methylation effects may not be reproducible.


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Examining the effect of stressors on an organism’s epigenome has become a relatively

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common research subject in recent years. However, the critical question of whether these

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changes can become permanent or semi-permanent across multiple generations is still under-

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studied. In a transgenerational scenario it is hypothesised that an initial exposure to a

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contaminant can result in adverse phenotypic (and epigenetic) outcomes not only for the

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directly exposed parent (P0) and offspring (F1 generation as developing gametes within the

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P0 organism), but also the F2, F3 and later, non-directly exposed generations.22 This

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transgenerational hypothesis assumes that epigenetic modifications can be sustained through

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multiple generations, even in the absence of persistent exposure, and could lead to a chronic

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accumulation of changes to an organisms’ phenotype if exposure does persist. In the next

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section we discuss ecotoxicological studies that have assessed multiple generations.



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Table 1. Studies demonstrating links between epigenetic modifications and environmental contaminants. Organism

Contaminant

Molecular method

Physiological impact

Molecular impact

Length of

identified

identified

exposure (time)

N/A

DNA methylation

45 days

Author, Year

Aquatic vertebrates European Eel

Cadmium (0.4 and 4µg/L-1)

(Anguilla anguilla)

ELISA assay/ methylation sensitive

significantly higher in

PCR

two genes (1.4 – 1.7

Pierron et al. 201348

fold higher). Rainbow trout

Atrazine*

Gene expression

Elevated cortisol,

Gene expression

(Oncorhynchus

(2.3, 18, 59, 555 µg/L)

microarray

decreased lymphocytes.

changes (653 – 845

mykiss) Zebrafish (Danio

4 days

Shelley et al. 201231

genes p < 0.05) BPA* (5, 10, 20µg/L)

rerio)

RT-qPCR, and Chromatin

Promoted apoptosis of

Down-regulated oocyte

ImmunoPrecipitation

reproductive follicles

maturation-promoting

(ChIP) analysis

3 weeks

Santangeli et al. 201632

signals, likely through changes in the chromatin structure mediated by histone modifications.

Invertebrates Water flea (Daphnia magna)

Zinc (388µg/L)

cDNA microarray of gene

N/A

expression

Common genes similarly up or down regulated for two generations (15% of genes).


< 24hrs

Vandegehucht e et al. 201030

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Tiger Mosquito

Phytoestrogen (Genistein*

Global DNA methylation

Decrease in insecticide

Changes to global

(Aedes albopictus)

5mg/L) and fungicide

(UHPLC-MS/MS

sensitivity in unexposed

DNA methylation in P0

(vinclozolin* 3mg/L)

analysis)

offspring.

generation (74%

8 days

Oppold et al. 201529

Genistein and 82% Vinclozolin). Nematode worm

Arsenic (sodium arsenite

Affymetrix expression

Survival assay curves

Dose dependant

(Caenorhabditis

media 0.03 and 0.003%

microarrays

significantly different

differential expression

elegans)

w/v)

(P2-fold gene

7 days

201243

expression difference

(100 mg/L)

Landa et al.

(p=< 0.05) in genes associated with cell organization, biogenesis, translation, nucleosome assembly, stress response genes.

Maize (Zea Mays L.)

Zinc (5, 10, 20, 40 mM)

DNA methylation

Decreases in specific

Dose dependent

7 days (seedling

Erturk et al.,

(restriction enzymes) and

hormones with

increases in

germination)

201526

damage

increasing Zinc

methylation levels with

concentrations (p=
3)

•Determine LC/LD50 •Hours - days

exposure

relationship and ECx

•Applied during gamete

values for sub-acute

development/gestation

•Two generation +

ECx for transgenerational

effects

periods

•Weeks - months

effects

•Days – weeks

•Two generation •Determine ECx values for reproductive impairment •Weeks

•Epigenetic analyses of P0 generation (and F1 generation if modifications are observed at low concentrations)

•Determine dose-response and

•Long term, chronic exposure •Treatments focused around lower concentrations •Determine prevalence of epigenetic memory •Determine recovery time (no. of generations to recover)



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5. Tier 5 experimental design details

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If warranted by lower tier assessment results, then tier 5 risk assessment can be initiated. The

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experimental design for a Tier 5 transgenerational assessment (Table 3) must be adequate to

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determine long-term persistence of any transgenerationally transferred phenotypic alterations

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(Figure 2). First a sufficient number of generations (both exposed and non-exposed) must be

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considered to eliminate the confounding effects of direct exposure to gametes (i.e. the F1

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generation exposed as developing gametes). Further, multiple exposed generations followed

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by multiple non-exposed generations must be assessed, as several studies have suggested that

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multiple generations must be exposed before transgenerational inheritance occurs.51,56 Note

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that careful selection of model organisms with short generation times and well-characterised,

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small genomes would also be critical for minimising time and costs if a tier 5 assessment is

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required.

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Figure 2 depicts a suggested framework for an optimal transgenerational experimental design,

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covering a number of different experimental circumstances. For clarity this figure only

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extends to the F3 generation; however, more generations would likely need to be considered.

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This is a crucial step toward differentiating between true transgenerational modifications and

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repeated direct exposures. However, it is understandable that an experiment of this scale may

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be beyond the spatial and/or financial means of many studies, in which case some sections of

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the design/scheme may be omitted or modified as the experiment progresses. For example,

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section (lineage) A (Figure 3), which assesses continuous re-exposure with zero non-exposed

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progenies, would ideally be omitted in favour of lineage B, D, or H, which includes a number

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of subsequent non-exposed generations after multiple exposed generations. Further,

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epigenetic assessment would not have to occur in every treatment level, instead epigenetic

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testing could be reserved for samples which show a culmination of observable morphological

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detrimental impacts in the F3+ progeny.


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If a transgenerational effect, resulting from multiple-generation chemical exposure, is found

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to persist in subsequent multiple non-exposed generations then it may be necessary to lower

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the current guideline maximum allowable concentrations for that chemical. It would also be

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critical, for research purposes, at this stage to link epigenetic changes with any

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transgenerational morphological or functional changes occurring in later generations. Finally,

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it may also be important to explore the concept of resistance and resilience at this stage (e.g.

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lineage type C, E, F, or G; Figure 3).51,56,105 It may be that the organism has inherited an

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epigenetic profile that enables it to be resilient to the contaminant related stress, in which case

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it must be determined whether these inherited modifications are desirable or not (e.g. in the

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case of economically or functionally important species). If the morphological change does

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not affect the mortality, fecundity or function of the organism or ecosystem, then guidelines

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would not need to be changed.

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It must also be noted that it is critical to adhere to the main principles of sound

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ecotoxicology106 if we are to be able to put transgenerational results into any kind of

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environmental context. These principles include: 1) adequate planning and design of study

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(see figure 2), 2) defining the baseline of what is ‘normal’ for that endpoint in an unexposed

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organism, 3) including an appropriate number of controls, 4) using appropriate exposure

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routes and environmentally realistic exposures, 5) measure the exposure (don’t rely on

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hypothetical/theoretical exposure measurements), 6) minimise variability by having a good

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understanding of the biology and background of the organisms used and also the

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methods/tools used, 7) consider statistical analysis prior to beginning the experiment, 8)

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consider if the number of concentrations will be substantial enough for a dose-response (at

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least three concentrations excluding the controls are needed for reporting dose-responses, and

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unusual patterns need further justification/experimentation), 9) the experiment conclusions

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must be repeatable or validated independently, 10) consider potential confounding factors,



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11) results should be compared with previous studies, 12) do not over-extrapolate or over-

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hype a result with low significance and report negative as well as positive findings.

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Carrying out transgenerational epigenetic assessments in a standardised manner such as this,

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would allow a more robust body of literature to accumulate. This would assist us with a

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clearer understanding of the prevalence of epigenetic transgenerational inheritance in the

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environment. Over time clear pathways or biomarkers of heritable epigenetic alteration may

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become apparent, which may enable risk assessors to predict the likelihood of heritable

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contamination-induced epigenetic changes. If the likelihood of occurrence could be predicted

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with a reasonable level of confidence within the first few generations, then fewer generations

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would need to be tested, reducing both financial and time costs for assessments. However,

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until more robust experimental designs are adopted, any such patterns will remain obscured.


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Figure 2. Schematic of robust experimental design, with lineages including multiple

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generations of exposed organisms, followed by multiple generations of non-exposed

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organisms.

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6. Conclusions

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Contaminant-induced transgenerational epigenetics has received much scientific attention in

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the last decade, but there remains little clarity as to how heritable epigenetic changes might

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be incorporated into ecotoxicological assessments. In part this is due to studies lacking

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sufficient replication, an insufficient number of exposed and non-exposed generations, and

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technological difficulties obtaining appropriate and detailed genetic coverage in organisms

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with complex genomes. For transgenerational epigenetics to be effectively incorporated into

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ecotoxicological risk assessment, a number of issues and gaps must be resolved. Firstly, an

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adequate number of re-exposed and non-exposed progenies are needed to provide evidence of

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transgenerational effects beyond what could be deemed ‘direct exposure’ to gametes. In

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addition to this, greater levels of replication are needed to account for stochastic variability in

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epigenomes across individuals. And also, more relevant dose ranges should be tested, such as

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ranges overlapping the contaminant EC10-20, or LOEC and NOEC concentration levels for the

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organism’s most sensitive phenotypic endpoint. Finally, and perhaps most crucially, more

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attention needs to be paid to the definition of transgenerational inheritance, as some studies

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have reported contaminant-induced transgenerational impacts, when the effect may be caused

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by direct exposure to developing gametes, causing confusion in the body of literature. These

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processes along with the frameworks suggested in this review will assist in developing useful

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transgenerational risk biomarkers for low-level chronic contamination in current risk

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assessment frameworks.

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Funding and image sources: All funding for this work was provided by CSIRO. All images

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were created by- or belong to- the authors on this manuscript.

464


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Figure 2. Schematic of robust experimental design, with lineages including multiple generations of exposed organisms, followed by multiple generations of non-exposed organisms. 218x257mm (300 x 300 DPI)


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Incorporating Transgenerational Epigenetic Inheritance into

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