Toxicology 391 (2017) 64–74

Contents lists available at ScienceDirect

Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Ecological significance of mitochondrial toxicants MARK

Nishad Jayasundara
University of Maine, Orono, ME, 04469, United States

A R T I C L E I N F O A B S T R A C T

Keywords: Mitochondrial dysfunction with exposure to anthropogenic contaminants is a critical concern in biomedical and
Mitochondria ecological health. Importantly, understanding the role of mitotoxicants in modulating organismal fitness and
Pollutants survival, particularly in the context of species’ resilience to shifts in abiotic factors with climate change, is
Climate change emerging as a key consideration. To date, only a few studies have focused on effects of mitotoxicants on or-
Stress responses
ganismal responses to additional abiotic stressors (e.g., shifts in temperature, levels of oxygen, and salinity);
Ecotoxicology
however, these serve to demonstrate that chemical exposure could alter stress responses. Here a theoretical
framework is presented to integrate mitochondrial dysfunction into organismal response to additional stressors,
highlighting the importance of sustaining the capacity to conserve or regenerate a healthy population of mi-
tochondria, i.e., maintaining mitochondrial plasticity, in a multi-stressor environment. The complexity of mi-
tochondria-associated processes and impact of mitotoxicants in the context of shifts in fate and transport of
anthropogenic chemicals with climate change are also considered. Considering the crucial role of mitochondria
in multi-stressor environments and their vulnerability to physical and chemical stressors, this review proposes
the use of mitochondrial function as a physiological marker of species resilience to environmental change.
Finally, impacts of contaminants on mitochondrial health of wildlife are important in their own right, but the
potential use of these studies, especially mitochondrial DNA analyses, to inform long-term impacts of chemicals
on mitochondria in a human health context is also discussed.

1. Introduction mitochondrial function and integrity with exposure to multiple abiotic

Mitochondria are key cellular organelles involved in a number of
biological processes and mitochondrial health is critical to organismal
survival and fitness. In addition to serving as the energy powerhouse,
mitochondria are associated with apoptotic signaling including initia-
tion and amplification of apoptosis (Raimundo et al., 2012; Tann et al.,
2011), calcium signaling and calcium homeostasis (Clapham, 2007),
copper and iron homeostasis (Askwith and Kaplan, 1998), non-shi-
vering thermogenesis (Nedergaard et al., 2001), protein activating iron-
sulfur cluster assembly (Gakh et al., 2016), and synthesis of macro-
molecular structures (Ahn and Metallo, 2015), ion exchange (Shoshan-
Barmatz et al., 2015), and metabolite transport (Palmieri and Pierri,
2010). Mitochondria also communicate with the nucleus and cytoplasm
via reactive oxygen and nitric oxide species signaling (ROS and NOS,
respectively) (Schulz et al., 2014; Storz, 2006) and cell cycle arrest
signaling (Koczor et al., 2009). Considering the complex and important
roles of these organelles, maintaining a healthy population of mi-
tochondria is crucial for organismal survival and fitness. In particular,
mitochondrial plasticity, the capacity to maintain optimum
stressors, is critical when exposed to environmental perturbations.
The increasing recognition of mitochondrial etiology of a range of
human health disorders has led to an emergence of studies focusing on
mitochondrial toxicants (Meyer et al., 2013) and remain a critical area
of research as discussed in detail in this issue. In addition to research on
human health implications of mitotoxicants, a number of studies de-
monstrate that environmental contaminants have significant effects on
mitochondrial structure and function in organisms across taxa. For
example, studies in amphibians (Stabenau et al., 2008; Swann et al.,
1996), aquatic and terrestrial invertebrates (de Quadros et al., 2016;
Xie et al., 2006; Mommaerts et al., 2011; Cutkomp et al., 1971), and
fish from the tropics to the polar regions (Cutkomp et al., 1971;
Dussauze et al., 2014; Tissier et al., 2015; Padmini et al., 2009; Knecht
et al., 2013; Cambier et al., 2009; Smolders et al., 2003; Pandey et al.,
2008), show that exposure to heavy metals, pharmaceuticals, pesti-
cides, complex hydrocarbon mixtures, nanomaterials, UV radiation and
various other organic and inorganic contaminants can disrupt mi-
tochondrial structure and function. Examples of pollution effects on
plant mitochondria also exist (Schulte Hostede et al., 1987). However,

Abbreviations: mtDNA, mitochondrial DNA; NOS, nitric oxide species; OXPHOS, oxidative phosphorylation; PAH, polycyclic aromatic hydrocarbons; PCB, polychlorinated biphenyls;
POP, persistent organic pollutant; ROS, reactive oxygen species; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin
E-mail address: nishad.jayasundra@maine.edu.

http://dx.doi.org/10.1016/j.tox.2017.07.015
Received 21 April 2017; Received in revised form 19 July 2017; Accepted 24 July 2017
Available online 01 August 2017
0300-483X/ © 2017 Elsevier B.V. All rights reserved.
N. Jayasundara
effects of anthropogenic chemicals on mitochondrial health of wildlife
in the context of organismal responses to multiple abiotic stressors have
received less attention. This remains a key area of research considering
rapid environmental change due to anthropogenic activities. In parti-
cular, sub-lethal concentrations of mitotoxicants could affect orga-
nismal aerobic performance and perturb food-web dynamics, re-
productive outcomes, and response to other abiotic stressors, leading to
significant ecosystem level impacts (Seiler, 2002; Di Giulio and Clark,
2015). Given the importance of mitochondrial plasticity in organismal
fitness and survival, especially when exposure to multiple stressors,
understanding the ecological impact of mitochondrial toxicants remain
a crucial consideration and warrants further analysis on impacts of
mitochondrial dysfunction at the organismal, population, and eco-
system level. As discussed later, these studies may also provide critical
insights into effects of mitotoxicants on human health. Accordingly, this
review considers the implications of mitochondrial toxicants with a
specific focus on their effects on organismal response to shifts in their
abiotic environment (Løkke et al., 2013) and briefly discusses the po-
tential implications of these studies on human mitochondrial health
research.
2. Complexity of mitochondria
Mitochondria contain their own genome and each organelle may
consist of several copies of mitochondrial DNA (mtDNA). mtDNA is
transcribed at a high rate, especially in highly energetically active tis-
sues such as hearts (Mercer et al., 2011), and encodes for ∼13 proteins
that contribute to the enzyme complexes of the oxidative phosphor-
ylation (OXPHOS) system (Friedman and Nunnari, 2014). Since some
proteins of the electron transport chain complexes are encoded in the
nuclear genome, a tight regulation of mitochondrial function by the
nucleus and cytoplasm is required to maintain optimal mitochondrial
function (Rand et al., 2004).
Additionally, many gene products (∼1000–1500 proteins) (Calvo
and Mootha, 2010) that are encoded in the nuclear genome are im-
ported into the mitochondria to perform various functions. Mitochon-
drial interactions and biogenesis (growth and division of pre-existing
mitochondria) are highly tissue dependent (Jornayvaz and Shulman,
2010) and this specificity is reflected in mitochondrial morphology
(spherical vs. elongated) and organization (discrete organelles vs.
highly interconnected dynamic networks), as well as the total mi-
tochondrial amount, content, and transcriptome of these organelles
(Okamoto and Shaw, 2005; Vafai and Mootha, 2012; Benard et al.,
2007). Ontogenetic shifts and environmental factors can also affect
mitochondrial morphology, content, and copy number (Jendrach et al.,
2008). Mitochondrial biogenesis is a highly coordinated process with
mtDNA replication, fusion and fission, and nuclear-cytoplasmic sig-
naling and is heavily influenced by environmental stressors. Precise
regulation of biogenesis, degradation, fission and fusion, as well as
maintenance of nuclear-cytoplasmic signaling are critical for optimum
mitochondrial function (Meyer et al., 2013; Green and Van Houten,
2011; Piantadosi and Suliman, 2012; Youle and Van Der Bliek, 2012;
Blajszczak and Bonini, 2017; Roubicek and de Souza Pinto, 2017; Chan,
2017; Meyer et al., 2017).
The aforementioned mitochondria-associated cellular processes,
including OXPHOS, are highly sensitive to changes in the chemical and
physical properties of the cellular milieu. In addition, anthropogenic
chemicals and other stressors could impact macromolecular mi-
tochondrial structures via molecular interference or mechanical da-
mage. For example, the ion impermeability of the inner mitochondrial
membrane is critical for the generation of the electrochemical gradient
for oxidative phosphorylation. However, it is a likely target of xeno-
biotic elements (Meyer et al., 2013; Smith et al., 2003) and physical
stressors (as reviewed by Somero et al., 2017) as they can damage
membrane structure and/or alter membrane potential.
mtDNA is also highly susceptible to changes in physical and
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Toxicology 391 (2017) 64–74
chemical environment of the cell. In contrast to nuclear DNA, mtDNA is
not protected by histone proteins and is thought to be damaged at a
higher rate by ROS that are routinely generated in the mitochondria
(Yakes et al., 1997) (e.g., loss of electrons during electron transport
chain can lead to transformation of some oxygen (∼0.1–0.4%) into
superoxide anion radicals (Hermes-Lima, 2004)). Furthermore, com-
pared to nuclear DNA, mtDNA is susceptible to higher mutation rates
(∼10–20 times) and the rates of mutation accumulation depends on the
region of mtDNA that is being tested (Neiman and Taylor, 2009) and
may also depend on the type of mutagen (Valente et al., 2016). Ad-
ditionally, protein products of altered mtDNA and mitochondrial
structural damage can lead to mitochondrial dysfunction resulting in
increased production of ROS (e.g., H2O2) (∼10× more than normal
mitochondria), and in some conditions may potentially lead to systemic
mitochondrial oxidative damage and dysfunction (Yakes et al., 1997;
Grivennikova et al., 2010). It is noteworthy that the extent of mi-
tochondrial perturbation is influenced by the tissue type, since tissues
differ in their bioenergetic capacity and ability to remove ROS and
repair damaged mtDNA (Szczesny et al., 2010).
Despite decades of research, large gaps in knowledge exist in our
understanding of the complex nature of mitochondria, including their
origin, structure, function, and repair and removal processes (e.g., mi-
tophagy—selective removal of damaged organelles (Kim and Lemasters,
2011)). Nonetheless, it is clear that these vital organelles are vulnerable
to changes in the surrounding physical and chemical environment,
implying that mitotoxicants are an important consideration in assessing
organismal fitness and survival in their natural environment.
3. Mitochondrial toxicants in the environment
Mitochondria are vulnerable to certain anthropogenic chemicals
due in part to the membranous high lipid content that facilitates ac-
cumulation of lipophilic compounds, as well as the negative charge of
the matrix that facilitates accumulation of cationic metals (Meyer et al.,
2013). Various classes of anthropogenic contaminants have emerged as
potent mitochondrial toxicants and Meyer et al. (2013) provide an
excellent review of these chemicals and their mechanisms of toxicity.
These compounds include persistent organic pollutants (POP) (e.g.,
polycyclic aromatic hydrocarbons (PAH), 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD), and bisphenol A (BPA)) and various metals (e.g., Cd, Cu,
Pb, Hg, As, Methyl-Hg, Mn, Se, and Zn) as well as chemicals designed to
target mitochondrial complexes (e.g., rotenone). Furthermore, a com-
prehensive analysis of Comparative Toxicogenomics Database that
contains manually curated information about chemical–gene/protein
interactions, chemical–disease and gene–disease relationships
(Mattingly et al., 2006), indicated that over 6000 chemicals from many
classes of compounds affect mitochondrial oxidative phosphorylation
(Supplemental Fig. S1).
Certain mitotoxicants could present a higher risk for wildlife com-
pared to human health due to elevated bioavailability in the environ-
ment, especially in aquatic ecosystems. Notably, selenium toxicity has
gained recent attention as a coal combustion waste product released
into the aquatic environment and is shown to affect fish mitochondrial
membrane potential leading to apoptosis (Selvaraj et al., 2013). BPA
and phthalates, commonly known for their endocrine disrupting prop-
erties, are ubiquitous environmental contaminants, especially in the
aquatic environment (Kang et al., 2007). Mounting evidence suggests
that these chemicals affect mitochondrial membrane potential, mtDNA
copy number, mitochondrial mass, mtDNA mutations, and ATP content
(Lin et al., 2013; Kaur et al., 2014). Furthermore, interactive effects of
chemical mixtures and other environmental factors are a significant
concern in an ecological context. For instance, exposure to UV radiation
(de Quadros et al., 2016) induces mitochondrial toxicity and is likely to
be further exacerbated by synergistic effects with other mitoxicants
such as PAHs (de Quadros et al., 2016).
In addition to chemicals released from various industrial and
N. Jayasundara
agricultural activities, pharmaceuticals are an environmentally im-
portant category of mitotoxicants. Evidence of drug-induced mi-
tochondrial dysfunction (e.g., effects on electron transport chain, mi-
tochondrial transport pathways, and mtDNA replication etc.) is rapidly
accumulating (Begriche et al., 2011) and specific examples are re-
viewed by Meyer et al. (2013). Certain bactericidal antibiotics are of
particular concern, as they can cause mitochondrial dysfunction and
ROS overproduction, potentially via disruption of mitochondrial pro-
tein synthesis—given the common evolutionary origins of mitochondria
and bacteria (Schägger, 2002), antibiotics that are designed to target
bacterial ribosomes could also target mitochondrial ribosomes (Meyer
et al., 2013). Antibiotics, other pharmaceuticals and their metabolites
are often detected in the environment, especially in aquatic habitats,
sometimes at concentrations that are orders of magnitude higher than
prescribed for human consumption (Bernhardt et al., 2017). Pharma-
ceuticals, especially the lipophilic and persistent ones, have the po-
tential to accumulate in the mitochondria and biomagnify in non-target
aquatic species. In addition, as reviewed previously (Seiler, 2002) cer-
tain pharmaceuticals, potentially including compounds that target mi-
tochondria, that have negligible biological effects at prescribed con-
centrations in human health contexts may have unintended chemical
and physical interactions in the environment. They may potentially
lead to organismal level consequences.
Risk of mitoxicity can also increase with shifts in fate and transport
of chemicals in ecosystems due to natural and anthropogenic activities.
For example, toxic and trace metal exposures can vary rapidly as a
result of predicted shifts in fate and transport of chemicals in the abiotic
and biotic environment with climate change (Gambrell, 1994). Several
reviews have described mechanisms and predicted consequences of
altered fate and transport of chemicals due to changes in temperature,
salinity, and pH as well changes in frequency of weather events such as
precipitation and floods, cloud cover, and forest fires (Noyes et al.,
2009; Hooper et al., 2013; Noyes and Lema, 2015). For instance, metal
distribution, speciation, and biotransformation will be affected with
shifts in geophysical and chemical properties (e.g., temperature), and
bioavailability and accumulation of these chemicals is likely to change
as a result (Noyes and Lema, 2015; Anawar, 2013). An indirect con-
sequence of warming is elevated release and bioavailability of seques-
tered mitochondrial toxicants such as polychlorinated biphenyls (PCBs)
and methyl mercury with melting sea ice and snow, especially in the
polar regions (Noyes et al., 2009). Furthermore, climate-induced shifts
in behavior and physiology in polar organisms results in altered bio-
logical toxicokinetics within an organism as well as across trophic
cascades with rapid environmental change (Noyes et al., 2009) (ex-
amples are discussed later). Overall, it is evident that changes in abiotic
factors have significant effects on fate and transport of chemicals in-
cluding mitotoxicants in the abiotic and biotic environment remains an
important, yet highly complex and challenging consideration when
determining ecological relevance of these chemicals.
4. Consequences of mitotoxicant exposure on organismal
response to effects of climate change
As highlighted previously, a number of studies demonstrate sig-
nificant effects of anthropogenic chemicals on mitochondrial function
and structure, especially in aquatic organisms. A key area of emerging
research is beginning to focus on effects of mitochondrial toxicants on
organismal response to additional abiotic stressors. Here, the existing
research examining effects of mitotoxicants on organismal response to
multiple stressors are presented with a discussion on five broad me-
chanisms potentially underlying ecologically relevant effects of mito-
toxicants.
Previous studies show that, while certain compounds such as rote-
none directly (and on some cases are designed to) target mitochondria,
other chemicals affect mitochondrial function via various indirect me-
chanisms. Therefore, based on previous research, ecologically relevant
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Toxicology 391 (2017) 64–74
mitochondrial dysfunction with anthropogenic chemicals can be at-
tributed to five processes: (i) direct inhibition of mitochondrial bio-
chemical processes; (ii) structural perturbation (e.g., membrane per-
turbation and mitochondrial DNA damage and mutation) from
exposure to parent compounds and metabolites (e.g., free radicals); (iii)
direct effects on nuclear-cytoplasmic processes involved in regulation of
mitochondrial function (e.g., hormonal activity, oxygen availability,
and ion regulation etc.) and signaling (e.g., ROS, apoptotic, and Ca2+
signaling); (iv) compensatory mitochondrial changes to meet the in-
creased energy demand following a chemical insult to induce an ade-
quate stress response; and (v) compensatory mitochondrial changes
with altered organismal energy metabolic phenotype due to shifts in
substrate (e.g., fatty acids) availability as a result of pollutant effects on
predator-prey interactions and food availability. A combination of these
direct and indirect effects of exposure to an individual chemical or a
mixture can affect mitochondrial function in a life-history dependent
manner. Of particular importance are the potential for effects of ex-
posure to mitotoxicants during development that may have long-term
consequences across generations (Kozal et al., 2017; Stapleton et al.,
2015).
Regardless of the mechanism, mitochondrial impairment via the
aforementioned processes is likely to have a significant impact on or-
ganismal fitness and survival. Mitochondrial function and integrity are
common targets of both chemical (Meyer et al., 2013) and physical
(Somero et al., 2017) stressors. For example, as reviewed by Somero
et al. (2017), temperature stress alters mitochondrial reaction rates,
affects mitochondrial membrane fluidity, increases ROS production,
induces mtDNA mutations, and alters other macromolecular structures
and functions that are important for peak mitochondrial performance.
Consequently, optimum mitochondrial capacity is critical in inducing
acute, acclimatory, and adaptive responses to enable organisms mini-
mize the impact of climate change. Here these effects are discussed with
a focus on the impact on aquatic organisms, since water bodies often
serve as ‘sinks’ for anthropogenic waste products and are exceedingly
vulnerable to impacts of climate change. Aquatic organisms are not
only exposed to pollutants due to agricultural runoff, industrial ef-
fluents, and other processes, they can experience daily and seasonal
fluctuations in abiotic factors such as temperature, salinity, oxygen, and
pH (Harley et al., 2006; Schulte, 2007).
Several recent studies have considered the role of mitochondrial
toxicants in multi-stressor experiments. Ivanina et al. (2012) demon-
strated that exposure to Cd and acute change in temperature affects
mitochondrial capacity to maintain function with changes in environ-
mental oxygen concentrations in oysters (Crassostrea virginica). Studies
in oysters exposed to Cd also indicate that mitochondrial vulnerability
to chemical insults with thermal stress may depend on the latitudinal
positioning (i.e., prior thermal history and thermal adaption) of a po-
pulation (Cherkasov et al., 2010). Furthermore, Cd and temperature co-
exposure studies showed higher sensitivity of mitochondrial enzymes to
the inhibitory effects of Cd, a decrease in activation energy of mi-
tochondrial enzymes (Ivanina et al., 2008), and increased degradation
of oxidatively damaged protein as a protective mechanism against Cd
mitotoxicity (Sanni et al., 2008).
Similar studies in rainbow trout (Oncorhynchus mykiss) indicated
that electron transport chain, tricarboxylic acid cycle, and proton leak
are common targets of metals such as Cd and Cu and exposure to these
chemicals affect responses to temperature changes and hypoxia-reox-
ygenation. In these fish, warm acclimation induced electron transport
chain shifts to compensate for acute thermal stress, but resulted in
mitochondria more sensitized to Cu and Cd toxicity. However, mi-
tochondrial hormesis—mild mitochondrial stress leaving the mi-
tochondria less susceptible to subsequent perturbations—was detected
with low dose Cd exposure, such that the effects of temperature and
hypoxia-reoxygenation on proton leak and complex 1 were rescued
(Sappal et al., 2015a, 2016, 2015b, 2014; Onukwufor et al., 2016a,
2016b). In addition to metals, on going studies suggest that
N. Jayasundara
developmental and adult exposure to PAHs can affect mitochondrial
response to temperature response later in life and across several gen-
erations in zebrafish (Danio rerio) (Kozal et al., 2017) and killifish
(Fundulus heteroclitus) (Jayasundara et al., 2017). Collectively, these
studies provide evidence for a direct link between mitochondrial toxi-
city and thermal, hypoxia, and other stress tolerance and limits. Im-
portantly, it is evident that mitochondrial effects of chemical and
physical stressors were dependent on magnitude and duration of the
chemical exposure and the history of exposure and adaptation to other
stressors.
5. Potential mechanisms that mediate ecological impact of
mitotoxicants
Based on aforementioned studies and previous studies on orga-
nismal response to abiotic stressors (Somero et al., 2017; Schulte, 2007;
Ivanina and Sokolova, 2016; Haider et al., 2016), three key con-
siderations emerge as likely mediators of mitochondrial toxicity in a
multi-stressor environment. These, addressed in more detail below, are
(i) bioenergetic imbalance; (ii) oxidative stress; and (iii) common mo-
lecular pathways (e.g. apoptotic response) that are targets of mitotox-
icants and other abiotic stressors (Fig. 1).
5.1. Bioenergetic imbalance
A direct consequence of mitochondrial dysfunction is altered energy
homeostasis (Sokolova et al., 2012, 2011) and this is a critical para-
meter in determining ecological significance of mitotoxicants. Previous
studies have demonstrated whole organismal bioenergetic effects of
chemical exposures across taxa. For instance, developmental and acute
exposure to individual PAH compounds and complex PAH mixtures
affects routine (or standard) and maximum metabolic rates, leading to
reduced aerobic scope in adult and juvenile teleosts (Davoodi and
Claireaux, 2007; Gerger et al., 2015; Gerger and Weber, 2015; Mager
et al., 2014). Ongoing studies also show that dietary exposure to PAHs
has transgenerational effects on fish metabolic rates (Kozal et al.,
2017). PCB exposure also resulted in reduced standard metabolic rates
in European eel (Anguilla anguilla) (van Ginneken et al., 2009) and al-
tered energy metabolic enzymes in killifish (F. heteroclitus) (Kraemer
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Toxicology 391 (2017) 64–74
Fig. 1. Conceptual figure demonstrating mitochon-
dria as a target of chemical and physical stressors,
consequences of exposure to these stressors and the
use of mitochondrial health index (MHI) as a marker
of optimum mitochondrial function.
and Schulte, 2004). Furthermore, as discussed by Noyes and Lema
(2015) exposure to metals and organochlorine pesticides can alter
metabolic rates in fish, crustaceans, and mollusks. It is possible that
these observed changes are independent of mitochondrial toxicity and
are due to altered metabolic demand and signaling (Casals-Casas and
Desvergne, 2011; Grun and Blumberg, 2006) or substrate (e.g., fatty
acids, glucose) composition shifts as a result of behavioral and phy-
siological changes with chemical exposure (De Smet and Blust, 2001;
Fabbri et al., 2003). For example, as discussed by Sokolova et al.
(2012), elevated basal metabolic function observed in aquatic in-
vertebrates with metal exposure is thought to result from increased ATP
demand to maintain cellular integrity. In contrast, higher levels of ex-
posure to metals are thought to reduce aerobic scope via direct effects
on mitochondrial function. Overall, it is evident that chemical exposure
can alter organismal energy balance that may or may not depend on
direct mitochondrial toxicity of pollutants. Nonetheless, the consequent
shifts in ATP demand and synthesis may affect mitochondrial function
and the capacity to meet this demand is considered critical in ecological
success across eukaryotic taxa (Sokolova et al., 2012).
Shifts in ATP demand and synthesis with chemical exposure are
likely to reduce energy allocation for somatic maintenance, cellular
stress responses, growth and reproduction, and aerobic performance,
and the magnitude of this effect depends on a number of factors
(Sokolova et al., 2012). Perturbed ATP allocation is further exacerbated
by direct and indirect effects on bioenergetics and mitochondrial
function and integrity via shifts in physical abiotic factors such as
temperature, pH, oxygen, and salinity that are anticipated with climate
change (Somero et al., 2017). This energy imbalance with exposure to
multiple abiotic stressors may have significant population level fitness
consequences and ecosystem impacts (Calow and Forbes, 1998).
To this end, several useful models have incorporated altered orga-
nismal bioenergetics to evaluate ecosystem level impacts of anthro-
pogenic stressors, particularly focusing on thermal stress. As summar-
ized by Noyes and Lema (2015), a number of studies indicate that
chemical exposure can alter thermal limits in ectotherms, though the
underlying mechanisms are not always clear. As described by the
oxygen- and capacity-limited thermal tolerance framework, the capa-
city to maintain an adequate oxygen supply to meet the increased
oxygen demand at different temperatures is thought to define thermal
N. Jayasundara Toxicology 391 (2017) 64–74

limits in aquatic ectotherms. Accordingly, further studies suggest that
aerobic metabolism at a given temperature and the thermal acclimatory
capacity of aerobic metabolic processes are directly associated with
thermal limits and tolerance in ectotherms (Pörtner and Farrell, 2008;
Jayasundara and Somero, 2013). Studies in mollusks indicate that
thermal tolerance limits are indeed reduced by decreased aerobic scope
with metal exposure in aquatic invertebrates (Lannig et al., 2008;
Bagwe et al., 2015; Sokolova and Lannig, 2008). Notably, mitochondria
play an important role in defining these limits, where, in teleost fish,
cardiac bioenergetics and mitochondria are linked with thermal
boundaries and optimum performance (Jayasundara and Somero, 2013;
Iftikar and Hickey, 2013).
Noyes and Lema (2015) present a synthesis of incorporating pollu-
tant effects on organismal energetics and temperature tolerance, by
focusing on thermal performance curves (TPC) (Pörtner and Farrell,
2008; Huey et al., 2012)—TPC presents a mechanistic framework to
examine the temperature vs fitness (e.g., aerobic scope, reproductive
capacity, swimming performance) relationship. TPC of an ectothermic
organism characterizes the upper and lower thermal limits and the
temperature at which an organism maintains optimal performance.
Extending this analysis beyond thermal stress, Sokolova et al. (2012)
present an excellent analysis integrating organismal level bioenergetic
effects of multiple stressors with long-term, population-level con-
sequences accounting for magnitude and duration of stress exposure. In
the context of mitotoxicants, this approach provides a framework to
incorporate mitochondrial dysfunction in assessment of population
level impact of anthropogenic stressors (Fig. 2) and is discussed later.
5.2. Oxidative stress
In addition to bioenergetic constraints, ROS production, oxidative
stress response and oxidative damage derived from mitochondrial
dysfunction may play an important role organismal fitness in multi-
stressor environments (Abele and Puntarulo, 2004; Lesser, 2006). Di-
rect inhibition of mitochondrial function (e.g., rotenone inhibition of
complex I, which is a major source of intercellular ROS) and indirect
effects on mitochondria as well as pollutant-specific biotransformation
and detoxification processes can lead to increased ROS production. A
number of studies show that both chemical (e.g., metals and POPs) and
physical (e.g., temperature and UV radiation) environmental stressors
are ROS inducers, eliciting oxidative stress responses. In Antarctic
scallop (Adamussium colbecki), exposure to temperature, pH, and Cd
stress showed tissue specific oxidative stress response and damage de-
pending on the magnitude of the stressors (Benedetti et al., 2016).
Studies in oysters (Crassostrea angulate) showed that As effects on an-
tioxidant capacity are increased at higher salinities (Moreira et al.,
2016). Differential effects on oxidative stress response markers were
detected in coral (Porites astreoides) larvae with exposure to pesticides
and thermal stress (Ross et al., 2015). Falfushynska et al. (2015) de-
monstrated that antioxidant response pathways in freshwater mussels
(Unio tumidus) are overwhelmed by combined exposure to temperature
stress, organics pollutants, and ZnO nanoparticles and suggested that
toxicity of ZnO particles was modulated by the presence of other
stressors. In European bullhead (Cottus gobio), a teleost fish, oxidative
stress responses were tissue and stressor specific and were influenced by
elevated temperature and Cd (Dorts et al., 2014).
As exemplified in these studies, evaluating ROS levels, oxidative
stress responses, and oxidative damage remains important, but is likely
to be challenging. For example, mitochondria are both a source and
target of ROS. Thus, changes in oxidative stress response and oxidative
damage could be a result of direct effects of pollutants on mitochondria
leading to ROS generation, but also due to ROS generated via bio-
transformation of xenobiotic elements, which may in turn lead to mi-
tochondrial damage (Banerjee et al., 2016). Another major challenge is
distinguishing whether ROS production is an effect of stress exposure or
a secondary stress response including a cell death response110. In ad-
dition, ROS signaling is crucial in physiological responses to regulate
cellular homeostasis. Thus, changes in ROS levels and antioxidant re-
sponses may reflect adaptive changes following exposure to con-
taminants (Shadel and Horvath, 2015). These aspects underscore the
complexity in uncovering specific effects of chemical and physical
stressors on ROS production and oxidative stress responses. None-
theless, ROS-induced damage, especially mtDNA and membrane da-
mage, can be important in assessing the impact of mitochondrial tox-
icants on organismal response to multi-stressor environments (Lesser,
2006).
5.3. Common targets of cellular stress response and cell death
There are number of common mitochondria-associated pathways

Fig. 2. Impacts of mitochondrial toxicants on aerobic activity of organisms and its relationship with organismal fitness. (a) A theoretical framework for integrating bioenergetic analysis
with thermal tolerance based on thermal performance curves [adapted from Huey (1982) and Pörtner and Farrell (2008)]. Solid blue line depicts the natural thermal range of an
ectotherm defined based on organismal aerobic fitness. Solid green lines illustrate effects of thermal acclimation shifting thermal range towards cold or warm tolerance. Dotted blue line
represents narrowed range for thermal tolerance with exposure to mitotoxicants due to direct and indirect effects on energy metabolism as discussed in the text. This suppression can
affect cold or warm tolerance (solid arrow 1 and 3 respectively), thermal optimum (solid arrow 2) or the overall thermal tolerance (dotted blue line) (b) Mitochondrial function may serve
as a marker of organismal fitness in multi-stressor environment. As discussed in ovo and ex vivo functional studies (e.g., XFe24 Extracellular Flux Analyzer assays) will be useful in these
analyses and utilized for developing a mitochondrial health index (MHI). MHI may vary depending on the duration and the magnitude of the stressor (Δ stressor) e.g., temperature, pH,
salinity (solid blue line). Exposure to mitotoxicants is expected to shift MHI affecting the response to Δ stressor (dotted blue line). The direction of this suppression is toxicant dependent
and is depicted by solid arrows. (c) Mitochondrial plasticity can be measured as the capacity to maintain MHI in a multi-stressor environment. The duration and the magnitude of a given
stressor (Δ stressor) are likely to determine mitochondrial plasticity. Identification of the flexion point of this relationship, where mitochondria fails to maintain plasticity can reveal
vulnerability of an organism to environmental stressors. The flexion point is likely to be right shifted with exposure to mitotoxicants, except for potentially in mitohormesis scenarios
(dotted orange line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

68
N. Jayasundara
(e.g., induction of apoptosis) that are involved in xenobiotic response
and cellular response to physical stressors. For example, an important
consequence of mitochondrial ROS is apoptotic and necrotic cell death.
Mitochondria-generated ROS lead to release of cytochrome C and other
pro-apoptotic proteins that elicit cellular apoptotic response (e.g., Ca2+
dependent mitochondrial permeability transition (MPT) and caspase
activation) (Ott et al., 2007). Induction of apoptotic pathways is also a
key element of abiotic stress responses including temperature and hy-
poxia/reoxygenation response (Kültz, 2005). Acute and chronic ex-
posure to warm temperature leads to apoptotic gene and protein ex-
pression level changes in fish hearts and gills (Logan and Buckley, 2015;
Jayasundara et al., 2015a). These responses include protein expression
changes in voltage-dependent anion channel (VDAC) proteins that are
involved in apoptotic signaling, as well as global regulation and sup-
pression of mitochondria (Jayasundara et al., 2015a). Hypoxic and
anoxic conditions also activate similar cascades of apoptotic pathways,
including regulators of VDAC via induction of ROS and NOS, cytokines,
kinases, and increased levels of NADH (Lemasters and Holmuhamedov,
2006). Importantly, VDAC forms a multimeric complex with adenine
nucleotide translocase and cyclophilin D that forms the MPT pore, that
is a target of various pharmaceuticals (e.g., doxorubicin and arsenic
trioxide), as well as ROS that are generated from pollutant exposure and
metabolism (Oliveira and Wallace, 2006; Andreau et al., 2012). The
precise composition of the MPT pore complex and the role of its com-
ponents are yet to be fully resolved. However, the ROS generated from
chemical and physical factors are likely to affect the MPT pore complex
leading to cell death and presents an example of a common mi-
tochondrial target in a multi-stressor environment.
Besides ROS, interactions among environmental sensor molecules
may play an important role in mediating mitochondrial dysfunction in
multi-stressor environments. Notably, the crosstalk between the aryl
hydrocarbon receptor (AHR) and hypoxia inducible factor-1α (HIF-1α),
and their common binding partner heat shock protein 90 are targets of
exposure to xenobiotics, hypoxia stress and temperature changes
(Gabriely et al., 2017; Ramsay and oreen Cantrell, 2015). Importantly,
the recent discovery of the presence of the (AHR) in the inner mi-
tochondrial membrane space suggests a unique role for this protein in
mitochondrial regulation. AHR is well known for xenobiotic metabo-
lism of its agonists (e.g., TCDD and certain PAHs). Studies show that
exposure to AHR agonists have profound effects on mitochondrial
membrane potential and retrograde signaling (Hwang et al., 2016a,
2016b; Tappenden et al., 2011) and that fish resistant to PAHs, an AHR
agonist, have evolved specialized mitochondria (Du et al., 2016).
Considering the detection of AHR in the mitochondria and its interac-
tion with HSP90 and HIF-1α, it is possible that AHR is an important
mitochondria-associated common target in a multi stressor environ-
ment.
Collectively, it is evident that anthropogenic chemicals have direct
and indirect effects on mitochondrial function that may impair orga-
nismal capacity to respond to additional abiotic stressors.
6. Mitotoxicity relevance to mammalian and avian species
Compared to lower vertebrates, especially in the aquatic environ-
ment, impacts of mitotoxicants on mammalian and bird species are less
well studied in ecological contexts. Nevertheless, rodent studies have
significantly contributed to the current understanding of mitochondrial
toxicants. Considering the taxonomic similarity, it is likely that the
response to mitotoxicants is shared among rodents, humans, and other
mammals, potentially extending to bird species, and exposure may lead
to numerous health disorders (e.g., neurodegenerative diseases and
metabolic syndromes). Furthermore, mitochondrial toxicity during ge-
station is an important consideration in mammals and several studies
indicate that mitochondrial function in the placenta is disrupted with
exposure to air pollutants, lead and endocrine disruptors, and may have
multi generational consequences (Brunst et al., 2015).
69
Toxicology 391 (2017) 64–74
While mitochondrial toxicity is likely an important concern for
health and fitness of mammals and birds, they may pose a greater risk
for organisms that have evolved specialized mitochondrial functions to
inhabit challenging habitats. For example, organisms have evolved
various energetic adaptations to inhabit higher altitudes, polar-regions,
and cold ocean depths. With changes in abiotic environmental factors
(e.g., temperature), mitochondrial dysfunction could manifest via (i)
increased mitotoxicant potency with altered biological and environ-
mental toxicokinetics and (ii) increased mitochondrial demand to
maintain homeostasis during dramatic seasonal or thermocline fluc-
tuations. For example, higher levels of organohalogen and PCBs, known
mitotoxicants, are detected in polar bears (Ursus maritimus) and were
particularly prominent in females during their reproductive and cub
rearing period. Elevated contaminant levels appear to be associated
with shifts in diet, mating, feeding, and birth and survival rates that are
linked to direct impacts of climate change (e.g., levels of sea ice) (Noyes
et al., 2009). The capacity to maintain bioenergetic homeostasis for
periods of fasting is critical to the survival of adult and juvenile polar
bears and may lead females to abandon reproductive efforts (Derocher
and Stirling, 1996; Robbins et al., 2012).
Studies show that extreme cold-tolerant organisms, such as polar
bears, have evolved specific mitochondrial adaptations to arctic habi-
tats (Welch et al., 2014). Thus, even subtle changes in mitochondrial
function may have significant energetic consequences, especially
during hibernation. In addition to bioenergetics, mitochondria play an
important role in thermal regulation of polar and high-altitude organ-
isms. Notably, heat production in hibernators as well as in mammalian
neonates is dependent on non-shivering thermogenesis that occurs in
large deposits of brown adipose tissue (BAT) (Stephens et al., 2011).
BAT has high mitochondrial content and activity, and they produce
heat via uncoupling of oxidative phosphorylation mediated by un-
coupling protein 1 (UCP1) (Stephens et al., 2011). Perturbed mi-
tochondrial function may significantly alter BAT activity, thus ther-
mogenesis. In addition, organisms such as hummingbirds that maintain
remarkably high metabolic rates (Suarez et al., 1991), flying bats (Shen
et al., 2010), and Antarctic killer whales (Foote et al., 2011), have
evolved specialized mitochondria to maintain unique life-history stra-
tegies or inhabit challenging environments are likely to be directly
impacted by mitochondrial toxicants. Besides mammals, partially en-
dothermic (mesothermic), apex oceanic predators (e.g., bluefin tunas
and lamnid sharks) also maintain high metabolic rates and specialized
cardiac Ca2+ homeostatic processes to maintain a highly aerobic life
style. Thus, optimal mitochondrial function is critical for their success
as apex predators (Jayasundara et al., 2013). Due to their positions on
the trophic chain, these organisms contain high levels of mitotoxicants
such as methy-Hg and POPs (Hisamichi et al., 2010) and are likely to be
susceptible to mitochondrial damage from exposure. These examples
indicate the impact of mitotoxicants across taxa and food webs, po-
tentially leading to significant ecological consequences.
7. Mitochondria as markers of species’ resilience to global change
Though characterizing the effects of mitotoxicants on organismal
health is complex and warrants further studies, mitochondrial function
may serve as a marker of impact of mitotoxicant exposure on orga-
nismal response to multiple stressors.
Most studies have focused on in vitro model systems to quantify
function in isolated mitochondria or primary cell-culture. While these
approaches provide important insights under varying substrate and
ADP levels, particularly into altered mitochondrial membrane poten-
tial, they are often cumbersome and low throughput, and the results are
likely to be confounded by perturbed cellular conditions (Kuznetsov
et al., 2008). Several recent studies have utilized XFe Extracellular Flux
Analyzer as a semi- and high-throughput method for mitochondrial
function analysis in ovo (Stackley et al., 2011), ex vivo (Jayasundara
et al., 2015b) and in vivo (whole larvae) (Raftery et al., 2017). In these
N. Jayasundara
Fig. 3. Parameters of mitochondrial health index. Ex vivo and in ovo mitochondrial studies
utilizing pharmacological inhibitors can indicate oxygen consumption associated with
mitochondrial and non-mitochondrial respiration, ATP-linked respiration, reserve capa-
city and proton leak. Oligomycin inhibits complex V; FCCP (carbonyl cyanide 4-(tri-
fluoromethoxy) phenylhydrazone) is an uncoupler; Antimycin A and rotenone mixture
inhibits oxidative phosphorylation via inhibiting complex III and complex I respectively.
Sodium azide can be used as a substitute for mitochondrial inhibition. Figure adapted
from Jayasundara et al. (2015a,b).
studies, different pharmacological agents were used to quantify total
mitochondrial and non-mitochondrial respiration, maximal respiration,
and reserve mitochondrial capacity (Fig. 3). The ex vivo analysis on
whole heart, brain and liver (Jayasundara et al., 2015b) tissues of tel-
eosts is particularly useful in an ecological context, considering the
tissue-specificity of mitochondrial function, structure and susceptibility
to chemical exposure. The capability to examine extra cellular acid-
ification rates (an index for glycolysis) in developing embryos and
whole tissues is an added advantage of this method and can be a useful
metric in assessing environmental conditions that trigger anaerobic
metabolism due to or independent of mitochondrial impairment.
To this end, focusing on mitochondrial plasticity may provide im-
portant insights into overall organismal fitness in changing environ-
ments. The capacity for mitochondria to repair damage and alter mi-
tochondrial structure, content, and copy number (i.e., conserving a
health population of mitochondria by regenerative biogenesis) is cri-
tical in maintaining mitochondrial plasticity. This can be assessed based
on the following parameters (Szendroedi et al., 2012; Jelenik and
Roden, 2013): (i) In vivo resting oxidative phosphorylation at low ADP
concentrations; (ii) In vivo submaximal ADP-stimulated oxidative
phosphorylation; (iii) Ex vivo maximal ADP-stimulated oxidative
phosphorylation in isolated mitochondria or permeabilized fibers, or
whole tissues assessed based on oxygen consumption rate measure-
ments; (iv) Ex vivo mitochondrial reserve capacity—the difference be-
tween ATP coupled oxygen consumption rate and uncoupled oxygen
consumption rates. Specifically, ex vivo and in ovo measurements using
the XFe Flux Analyzer will be highly useful in evaluating the mi-
tochondrial plasticity of wild-caught fish species and potentially others
organisms, thereby enabling a population level multi-stress response
assessment.
Mitochondrial health index (MHI)—a concept introduced by Chacko
et al. (2014) to describe a bioenergetic health index (BHI) in human
populations—derived from bioenergetic reserve capacity, ATP-linked
respiration, proton leak (Fig. 3) and non-mitochondrial respiration can
potentially serve as an indicator of mitochondrial health and plasticity
of an organism. Similar to BHI (Chacko et al., 2014), MHI is calculated
(reserve capacity )a × (ATP linked)b
as following: MHI = log , where a, b, c, and d
(non-mitochindrial)c × (proto leak )d
are predefined parameters based on control groups. In fact a major
limitation in this approach is that MHI calculation is highly dependent
on parameters a,b,c, and d. Thus, the application of MHI approach at
70
Toxicology 391 (2017) 64–74
the population level warrants careful control studies to define these
parameters. Another limitation is that altered substrate availability due
to an environmental perturbation may affect ATP linked respiration. In
addition, certain organisms are facultative anaerobes (e.g., blue mussel,
Mytilus edulis when under hypoxia) (Müller et al., 2012), limiting the
potential use of MHI as an appropriate indicator of organismal level
bioenergetic effects of exposure to an environmental perturbation.
Despite these challenges, this approach provides a comparative analysis
in multi-stressor contexts to assess mitochondrial health. Accordingly, a
theoretical framework based on TPCs (Fig. 3a) for utilizing MHI as an
indicator for mitochondrial function and plasticity, thus organismal
fitness, in the context of other stressors is presented in Fig. 2b and c. A
similar framework can be utilized for other physical stressors (e.g.,
salinity, pH, and hypoxia changes) that are likely to alter organismal
metabolic rates via direct effects on mitochondrial function and indirect
effects on metabolic signaling. Integrating mitochondrial plasticity
under multiple abiotic stressors will be crucial for assessing population
vulnerability and may further contribute to various quantitative bioe-
nergetic modeling approaches (e.g., dynamic energy budget modeling).
One disadvantage of functional analyses, especially the methods
that are based on oxygen consumption rate measurements, is that
chronic toxicant exposure effects may be masked by compensatory re-
sponses by the organism. Integrating functional analyses with changes
in protein, lipid and nucleic acid molecules associated with mitochon-
dria may better indicate altered cellular metabolic status and mi-
tochondrial dysfunction, though they may reflect secondary effects.
Furthermore, there may be masking effects in multi-stressor contexts.
For example, studies in oysters indicated that hypoxia induced com-
pensatory responses in mitochondrial enzymes were suppressed by Cd
exposure (Ivanina et al., 2011). Similarly, while markers of ROS pro-
duction and oxidative stress responses are typical indicators of mi-
tochondrial stress, they may not be useful in the context of assessing
mitochondrial health in multi-stressor contexts, since both chemical
and physical stressors are known to disrupt the balance in ROS gen-
eration and removal. For example, Cd exposure masked the tempera-
ture effect on mitochondrial aconitase, a marker of oxidative stress in
oysters (Sanni et al., 2008). However, a battery of oxidative stress re-
sponse enzymes (e.g., manganese superoxide dismutase, catalase, glu-
tathione peroxidase etc.) and markers of oxidative damage (e.g., lipid
peroxidation) are often used and may serve as indicators of mi-
tochondrial vulnerability to ROS production and ROS-induced damage
(Massarsky et al., 2017).
Although molecular changes, including mtDNA sequence changes as
discussed next, are excellent indicators of mitochondrial health, cou-
pling such markers with functional assays will be crucial in assessing
mitochondrial function at a population level. The in ovo and ex vivo
functional assays provide a rapid and relatively simple approach to
estimate mitochondrial bioenergetics. An important consideration in
functional studies is life history stages, since periods of gametogenesis
and embryonic development are critical windows for exposure to mi-
totoxicants (Stackley et al., 2011). Species’ vulnerability to mitotox-
icants is likely to be highest during the developmental window of surge
in mitochondrial biogenesis, where a profound increase in dynamics
(mitophagy, fusion, and fission), and consequent shifts in distribution
and density of mitochondria occurs (Dorn et al., 2015). In fact in heart
tissues, mitochondrial function and architecture are defined during
developmental mitochondrial biogenesis (Dorn et al., 2015). Thus,
considering the role of hearts in setting stress tolerance limits as dis-
cussed previously, cardiac mitochondria and its plasticity (the capacity
to maintain function and integrity with exposure to stressors) may serve
as a useful ecologically important marker of mitochondrial toxicity, at
least in teleost fish. Collectively, in ovo and ex vivo bioenergetic studies
are a powerful tool in assessment of population level mitochondrial
fitness across temporal and spatial scales, i.e., landscape bioenergetic
analyses.
N. Jayasundara
8. Canneries in the coalmine: human health implications of
ecotoxicology of mitotoxicants
As discussed in the previous section, certain mitochondrial bio-
markers can help to evaluate population level impacts of mitochondrial
toxicants in wildlife, but can also be informative in human health
contexts. Especially, mtDNA sequence analyses can provide important
insights into transgenerational effects of exposure and later life con-
sequences of developmental exposure to mitotoxicants in wildlife and
human health studies, though may present some caveats. For example,
in evolutionary biology mtDNA is often used as a genetic marker due to
their maternal inheritance, absence or very low levels (Neiman and
Taylor, 2009) of recombination, the presence of highly conserved as
well as less conserved regions within the same circular DNA strand, and
the relatively higher mutation rates compared to the nuclear DNA
(Ladoukakis and Zouros, 2017). However, these fundamental premises
are complicated by emerging evidence on paternal transfer (or leakage)
of mtDNA (Schwartz and Vissing, 2002) and occurrence of re-
combination (Neiman and Taylor, 2009; Kraytsberg et al., 2004) across
various taxa including in humans. Despite these challenges, evolu-
tionary analyses based on mtDNA in organisms exposed to anthro-
pogenic chemicals over multiple generations may contribute to ex-
amining effects of contaminants on mtDNA. Additionally, with greater
understanding of a population baseline, these studies can serve as in-
dicators of historical exposure to mitotoxicants and other stressors.
Furthermore, mtDNA analyses provide important insights into popula-
tion connectivity and migration. Collectively, in addition to informing
population dynamics within a species, including in the contexts of or-
ganisms inhabiting contaminated sites (Bickham, 2011), these analyses
may have important implications in mitochondrial mutagenesis re-
search.
More specifically, these studies may help understand sources of
mitochondrial mutations and mutation accumulation rates with con-
taminant exposure—areas of research that are particularly con-
troversial in human mitochondrial health studies as highlighted in this
issue (Meyer et al., 2017; Chan, 2017) and elsewhere (Valente et al.,
2016). However, several challenges are associated with these ap-
proaches. Naturally occurring mutations, especially at a higher rate in
mitochondria, migration or mixing of populations, and diversifying
selection confound these analyses adding a layer of complexity in de-
termination of new mutations resulting from pollution exposures
(Bickham, 2011). For example, mitochondrial polymorphisms, a key
evolutionary marker indicating genetic diversity (haplotype diversity)
of a population (Moore, 1995) can reveal increased mutation rates due
to mitotoxicant exposure and may show greater genetic diversity, but
also may mask loss of genetic diversity (Bickham, 2011). Thus, asses-
sing both nucleotide and haplotype diversity in these contexts is im-
portant in providing further insights into shifts in overall genetic di-
versity. In addition to mtDNA polymorphisms, mtDNA damage levels,
changes in copy number, and shifts in mtDNA heterogeneity, also
known as mitochondrial heteroplasmy, could be used in these studies as
markers of altered mitochondria. However, it is thought that as a result
of increased mutation rates and limited protection against oxidative
stress, a baseline level of mitochondrial mutations and heteroplasmy
are present within a cell or a tissue (Chinnery et al., 2012). Thus, to
evaluate effects of mitotoxicants on mtDNA, well-controlled studies are
necessary to determine baseline mtDNA mutation rates, damage, copy
number, and heteroplasmy. Furthermore, a potential increase in mu-
tation rates with chemical exposure may generate a mixed population
of mtDNAs, resulting in increased heteroplasmy. As discussed pre-
viously by Brunst et al. (2015), despite the advantages mitochondrial
heterogeneity within an organism, beyond a certain threshold, in-
creased damage and heteroplasmy affect optimal function; a trait that
might be selected against in a population which may lead to under-
estimations of mtDNA mutations in a population.
Despite these challenges, mtDNA sequence comparisons between
71
Toxicology 391 (2017) 64–74
populations or subpopulations of species inhabiting contaminated sites
with clean conditions present unique natural experimental setups for
examining mutagenesis with pollution exposure. For example, a mtDNA
analysis showed an increase in haplotypes and polymorphic loci, ge-
netic diversity, and base pair substitutions in rodents exposed to low
levels of radiation over 50 generations (Baker et al., 2017). Similar
studies showed altered mitochondrial genome diversity in marsh frogs
(Rana ridibunda) in an industrial waste site (Matson et al., 2006) and in
kangaroo rats (Dipodomys merriami) inhabiting an atomic blast site
(Theodorakis and Bickham, 2004). With the advent of next generation
sequencing capabilities, these analyses are likely to become more ac-
curate and informative, and will enable studies in non-model organisms
to better inform effects of contaminants on mtDNA. Thus, in accordance
with the “One Health” and “One Toxicology” concepts that are cur-
rently being discussed to integrate human and wildlife health impacts
of chemical exposures(Beasley, 2009; Buttke, 2011), assessment of
mtDNA as well as other mitochondrial markers in animal populations
can potentially serve as key indicators of long-term human health ef-
fects of exposure to anthropogenic chemicals.
9. Future directions
Two emerging aspects of mitochondrial biology that warrant sig-
nificant attention in the context of mitotoxicants are mitochondrial
epigenetics and cross talk between the commensal microbiome and
mitochondria. The latter may have profound ecological implications
considering the rapid shifts in microbial communities that may occur
with physical and chemical stressors including increased release of anti-
microbial compounds (e.g. antibiotics and nano materials) to the en-
vironment. Mitochondrial epigenetics, includes transcriptional regula-
tion of mtDNA via differential methylation, and the mitochondrial-
epigenome cross talk. Mitochondrial epigenetics is likely to be a central
consideration in determining the role of mitochondria and their dys-
function in modulation of acute and acclimatization responses to
abiotic stressors.
Methylation of mitochondrial DNA remains contentious with studies
showing contradictory evidence (Liu et al., 2016; van der Wijst and
Rots, 2015) and is discussed in detail in this issue (Weinhouse, 2017).
Nonetheless, the discovery of the DNA methyltransferase 1 in mi-
tochondria (Shock et al., 2011) and that ROS can alter DNA methyla-
tion, suggest that epigenetic changes of mtDNA are likely and could be
altered with chemical exposures. Furthermore, role of mitotoxicants on
the mitochondrial modulation of nuclear DNA methylation is of further
significance (Guha and Avadhani, 2013). As discussed in detail by
Weinhouse (2017), mitochondria-epigenome crosstalk is an important
target of mitotoxicants, and the ecological implications of such changes
are yet to be studied. Via mitochondria-epigenome crosstalk, it is pos-
sible that a diverse mitochondrial population may lead to differential
organismal gene expression patterns (Hill, 2015; Hoekstra et al., 2013),
although a recent study in killifish Fundulus heteroclitus indicated only
modest changes in gene expression patterns with cold temperature
exposure between populations with different mitochondrial genotypes
(Healy et al., 2017). Nonetheless, a greater understanding of environ-
mental regulation of mitochondrial epigenetics will be critical in de-
termining organismal response to multiple anthropogenic stressors.
10. Conclusions
Mitochondria are key organelles involved in maintaining cellular
homeostasis and also are targets of physical and chemical abiotic
stressors. Emergence of mitochondrial toxicants as important category
of anthropogenic chemicals indicate the need to better understand the
effects of these compounds on organismal response to global climate
change. To that end, further research is necessary to gain mechanistic
insights into differential effects of mitotoxicants based on the magni-
tude and duration of the exposure and especially history of exposure
N. Jayasundara
(e.g., the protective role of mitohormesis) and methods to incorporate
such findings into population level analyses. Importantly, a greater
understanding of mitochondrial epigenetics and impact of mitotox-
icants on this process may provide crucial insights into mitochondrial,
cellular, and organismal responses to biotic and abiotic stressors.
Accordingly, the discussion presented here indicates that mitochondria
may in fact serve as a potential marker of physiological resilience of
organisms to environmental shifts and may help to portend species’
fitness and survival with climate change. Additionally, these analyses
can be important in predicting human health impacts of mitotoxicants
and lends to the “One Health” concept that is being widely discussed as
an important approach in toxicology and human health (Beasley, 2009;
Buttke, 2011).
Acknowledgments
The author would like to thank the editors, the reviewers and Dr.
Andrey Massarsky, Duke University, NC for their critical insights and
feedback.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.tox.2017.07.015.
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