\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[greek,ngerman,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Impacts of di(2-ethylhexyl) terephthalate on multi-generational fitness
of Caenorhabditis elegans via lipid metabolism and neural regulation}



\author[1]{Zhenyang Yu}%
\author[2]{Jing Zhang}%
\author[2]{Lei Wang}%
\author[3]{Evgenios Agathokleous}%
\author[2]{Shijin Ma}%
\affil[1]{Jiaxing Tongji Institute for Environment}%
\affil[2]{Shanghai Institute of Technology}%
\affil[3]{Nanjing University of Information Science and Technology}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Di(2-ethylhexyl) terephthalate (DEHTP) is a substitutive plasticizer
with various industrial applications. However, its wide occurrence in
environmental matrices and human tissues is urging concerns on its
toxicities. Presently, the effects of DEHTP on the fitness of
\emph{Caenorhabditis elegans} were explored with a consecutive exposure
from F1 to F4 generations. The fitness was represented by reproduction,
lifespan and behavior. Effects of DEHTP on reproduction showed an
oscillation pattern with alteration from stimulation to inhibition and
backwards. Influences of DEHTP on reproduction and lifespan showed
trade-off relationships. Regarding behavior, DEHTP inhibited satiety
quiescence duration, body bending and head swing, while stimulated
reverse and Omega turns. At the biochemical level, DEHTP disturbed the
lipid metabolites and lipid metabolism enzymes. Moreover, positive
correlations were found between the effects on reproduction and with
those on fatty acid synthase (FAS) and acyl-CoA (FA-CoA). DEHTP also
disturbed the neural regulations including neural transmitters and the
expressions of related genes. Out of expectation, the effects on neural
regulations were positively correlated with those on lipid metabolism,
but not with locomotion behavior. Hierarchical clustering analysis (HCA)
results also indicated oscillatory changes underlying multi-generational
effects of DEHTP, e.g., serotonin was more connected with other neural
regulations than other indicators in F1 and F3, while it was more
connected with behavior than others in F2 and F4.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

Phthalates, e.g., di-2-ethylhexyl phthalate (DEHP), are widely used as
plasticizers and they were detected in environments and also human
tissues with various health impacts (Ma et al., 2022; Wang et al.,
2022b; Zhao et al., 2022). Di(2-ethylhexyl) terephthalate (DEHTP), as a
substitutive plasticizer, is increasingly used in toys, food packing
materials and even medical devices (Bernard et al., 2023; Lessmann et
al., 2019). Consequently, DEHTP and its metabolites were detected in
environmental matrices such as sediments at \selectlanguage{greek}μ\selectlanguage{english}g/kg levels (Bulbul et al.,
2022) and also in human urine samples at \selectlanguage{greek}μ\selectlanguage{english}g/L levels (Bernard et al.,
2023; Lessmann et al., 2019; Schwedler et al., 2020). Accordingly, the
effects of DEHTP on living organisms need further investigation to fully
demonstrate its hazards.

Reproductive toxicities of phthalates had been widely reported on both
humans and model organisms (Sedha et al., 2021; Sree et al., 2023).
Notably, the reproduction processes involve embryo and germline
development to connect adjacent generations (Wang et al., 2023).
Accordingly, the long-term influences of phthalates earned special
attentions. On one hand, the long-term influences of DEHP (one
traditional phthalate) were recently explored with particular attentions
on the hazards over generations. For example, parental or maternal
exposure to DEHP resulted in obesogenic outcomes in offspring of
drosophila (Chen et al., 2019) and behavior disorder and reproductive
dysfunction in mice (Quinnies et al., 2015; Rattan et al., 2018).
Moreover, the disturbances of DEHP on liver functions lasted over three
generations in mice (Wen et al., 2020). On the other hand, the
substitutive DEHTP can transfer over generations (Pacyga et al., 2023).
Both the reproductive and generational effects demonstrated the impacts
of plasticizers on the fitness which is important in sustaining
ecological stability (Yue et al., 2021). Accordingly, the long-term
effects of DEHTP are urging comprehensive studies and mechanism
exploration.

Lipid metabolism provides energy for reproduction that connects
generations. As expected, lipid metabolism disorder was employed to
explain the multigenerational reproductive toxicities of environmental
pollutants such as ionic liquids (Zhang et al., 2022). It was reported
that phthalates disturbed lipid metabolism in \emph{Daphnia magna,} with
impacts on reproduction, development and lifespan (Seyourm and Pradhan,
2019) and they also impacted lipid metabolism in the dolphin (Xie et
al., 2023). Therefore, DEHTP is assumed to provoke long-term exposure
over generations, including lipid metabolism disorder. However, such an
assumption needs support by empirical evidences.

Neural signals, e.g., dopamine (DA), acetylcholine (ACh),
acetylcholinesterase (AChE), \selectlanguage{greek}γ-\selectlanguage{english}aminobutyric acid (GABA) and serotonin
(5-HT), are also essential for the behavior and reproduction (Liu et
al., 2013). Moreover, it is also well connected with changes in lipid
metabolism (Luo et al., 2022; Magnan et al., 2015). The stimulation on
DA and inhibition on 5-HT reduced fat accumulation and metabolic
dysfunction (Ali and Mirza, 2021; Crane et al., 2015). Meanwhile, the
disturbance of antibiotics on the neural signals significantly inhibited
satiety to induce fat accumulation (Luo et al., 2022). Lipid metabolism
signals can also trigger neural regulations (Hamilton et al., 2015;
Magnan et al., 2015). Therefore, investigations combining both neural
signals and lipid metabolism are expected to provide more information
regarding the toxicity mechanisms of DEHTP.

The purpose of the present study was to analyze the multi-generational
toxicities of DEHTP on \emph{Caenorhabditis elegans} . Moreover, lipid
metabolism and neural regulations were explored to explain the
multi-generational toxicities. We hypothesized that the
multigenerational effects of DEHTP involved with both lipid metabolism
and neural regulations. The findings not only confirmed such hypothesis
and also showed oscillatory patterns in the effects over generations.
The environmental hazards of plasticizer substitutes are indeed of
concern, and further investigation is still needed to explore both
impacts and underlying mechanisms.

\textbf{2. Materials and Methods}

\textbf{2.1. Chemicals}

Di(2-ethylhexyl) terephthalate (DEHTP, CAS: 6422-86-2, Purity
\textgreater{} 98\%) was purchased from Adamas (Swiss). The stock
solutions were prepared with 1\% dimethyl sulfoxide (DMSO, Amresco, USA)
in sterile K-medium (0.051 M NaCl and 0.032 M KCl) (Yu et al., 2022).
The stock solutions were maintained at 4 \selectlanguage{ngerman}°C until use.

\textbf{2.2. Nematode preparation}

\emph{Escherichia coli} (\emph{E. coli} ) OP50 was cultured in lysogeny
broth (LB) medium at 37 ºC and served as the food for subsequent
nematode culture. \emph{Caenorhabditis elegans} (\emph{C. elegans} ,
wild-type N2) nematodes were cultured at 20 ºC on nematode growth medium
(NGM) agar. Gravid hermaphrodite nematodes were lyzed by fresh Clorox
solution (1\% NaClO and 0.5 mol/L NaOH), and the eggs were resistant to
such lysis and therefore were age-synchronized for subsequent
experiments (Li et al., 2019).

\textbf{2.3. Multi-generational experiment and measurement on lifespan
and reproduction}

In preliminary experiments, DEHTP at 8.0 mg/L provoked lethality smaller
than 5\% (LC5). Accordingly, the nominal exposure concentrations were
arranged as 0.0008, 0.008, 0.08, 0.8, and 8.0 mg/L, three of which
represented realistic \selectlanguage{greek}μ\selectlanguage{english}g/L levels (Bulbul et al., 2022). Briefly, warm
NGM were mixed with DEHTP stock solutions or sterile K-medium solutions
containing 1\% DMSO (as the control) with a volume ration of 99:1 to
reach the nominal concentrations. Then, they were aliquoted into Petri
dishes. After cooling down to form agar, they received the same amounts
of \emph{E. coli} OP50 suspensions with the same bacterial density.
Then, they were kept overnight for mild evaporation to form a bacterial
lawn. At last, the age-synchronization eggs were added on the NGM agars
to start the first-generation exposure (marked as F1) (Li et al., 2019;
Yu et al., 2017; Yue et al., 2021). Each experimental group including
the control had at least ten replicates of NGM agars.

On the 3\textsuperscript{rd} day of the F1 exposure, in each
experimental group the nematodes were collected and randomly allocated
to four experimental manipulations: (1) 48 adults were transferred onto
48 NGM agars with continuous treatment of DEHTP or K-medium solutions to
measure lifespan and reproduction where disappeared nematodes were not
calculated (Liu et al., 2020; Yu et al., 2017); (2) approximately 1000
adults were transferred onto clean NGM agars for behavior measurement;
(3) 200 adults were transferred onto new NGM agars with continuous DEHTP
or K-medium treatment to reproduce eggs for 24 h. Then, the adults were
discarded, and the age-synchronized eggs started next generation
exposure (i.e., F2) (Yue et al., 2021); (4) the remaining nematodes in
each group were collected to 20 centrifuge tubes and stored at -80 \selectlanguage{ngerman}ºC
until assayed. The procedure was repeated to finish a multi-generational
exposure with 4 consecutive generations from F1 to F4.

\textbf{2.4. Behavior measurement}

Briefly, nematodes were captured for videos by dissecting microscope
(XTL-BM-9TD, Shanghai BM optical instruments manufacture co., Ltd)
equipped with an industrial digital camera (UCMOS05100KPA). The head
swing, body bending frequency, reversal movement and Omega turns in
videos were counted for each individual for one minute (Yu et al.,
2011). A slow-playing mode was employed to facilitate the counting and
recording of the pharyngeal pumping times within 10 s (Luo et al.,
2022). The satiety quiescence referred to the time when nematodes did
not move for more than 5 s within 1 min, and the satiety quiescence
duration was also recorded (Luo et al., 2022). Each group was consisted
of at least 6 replicates.

\textbf{2.5. Biochemical assays}

Briefly, nematodes were homogenized followed by a centrifugation at 4
ºC, and the supernatants were aliquoted for subsequent assays. Regarding
the neural signals, the contents of 5-HT, DA, ACh and GABA and the
activities of AChE were determined using the commercial enzyme-linked
immune-sorbent assay (ELISA) kits (Shanghai Ketao Biotechnology Co. LTD)
according to the instructions (Liu et al., 2020; Yue et al., 2021).
Regarding the lipid metabolism, the contents of triacylglycerol (TG),
non-esterified fatty acid (NEFA) and the metabolites acetyl-CoA (ACA)
and acyl-CoA (FA-CoA) that combine both lipogenesis and lipolysis were
measured. In addition, the activities of fatty acid synthase (FAS),
acetyl-CoA carboxylase (ACC) and glycerol-3-phosphateacyl transferases
(GPAT), which facilitate lipogenesis, and lipase, acyl-CoA synthetase
(ACS) and carnitine palmitoyl transferase (CPT), were analyzed. Finally,
adipose triglyceride lipase (ATGL) that facilitates lipolysis was also
measured. Measurements were conducted with the ELISA method (Zhang et
al., 2022). Briefly, the ELISA kits provided 96-well plates, substrates,
buffer and standard enzyme solutions. Sufficient substrates were added
with a series of enzyme solutions (diluted with the buffer) whose
concentrations were known, and the results were used to establish
standard curves which were used to calculate the results of the samples.
In each group, three were at least six replicates with 500 nematodes in
each to ensure the methodological accuracy (Zhang et al., 2017). The
biomass among samples was balanced by the normalization where each
biochemical was represented as its proportion against the total protein
(TP) in each sample. Each group was composed of at least three
replicates.

\textbf{2.6. Gene expression analysis}

Genes including \emph{ser-1} , \emph{mod-1} , \emph{ser-6} ,
\emph{acs-4} ,\emph{dop-3} and \emph{cpt-1} are directly related with
neural signals and lipid metabolism (Lemieux and Ashrafi, 2016;
Srinivasan et al., 2008; Watts, 2009). Their expression levels were
determined with real-time polymerase chain reaction (RT-PCR) (Li et al.,
2020). Briefly, TRIzol reagents were used to extract the total RNA which
was further synthesized to cDNA for subsequent RT-PCR with SRBR Premix
Ex Taq\selectlanguage{english} (Takara Bio Inc., Japan) in FTC-3000 (Funglyn Biotech Inc.,
Canada). The expression levels of chosen genes were quantified by the
2\selectlanguage{greek}\textsuperscript{-ΔΔCT}\selectlanguage{english} method with \emph{gpd-2} as the endogenous
reference gene. The primers are listed in Table S1 in Supporting
Information.

\textbf{2.7. Data presentation and statistical analysis}

Briefly, all data were presented as fold-change against the concurrent
control in each generation (Liu et al., 2020; Yue et al., 2021). The
values in the concurrent control in each indicator were normalized to
1.0. Therefore, fold-change values lower than 1.0 indicate inhibition
and those higher than 1.0 indicate stimulation. All data passed
normality test and went through two-way ANOVA with a Tukey \emph{post
hoc} test at \selectlanguage{greek}\emph{α} \selectlanguage{english}= 0.05 (\emph{p}\textless{} 0.05) to determine
significant differences among treatments (control and two
concentrations) and among generations. Parallel analysis presented the
individual data of each sample in each indicator with a purpose to check
the changing patterns between indicators in corresponding samples. At
the same time, the mean values that were calculated from the individual
data of each sample went through Spearman's rank correlation and
hierarchical clustering analysis (HCA) to further analyze connections
among the indicators (Yue et al., 2021).

\textbf{3. Results}

\textbf{3.1. Multi-generational effects on fitness}

Reproductive influences represent the first aspect in fitness
evaluation. The effects of DEHTP on the reproduction are shown in Figure
1(A). Collectively, DEHTP caused oscillatory effects over generations.
For example, at 0.8 mg/L, DEHTP showed stimulation in F1, inhibition in
F2 and stimulation again in F3, and the stimulation was much less in F4.
Generally, the stimulations were commonly greater in F3 than in F1, and
the inhibitions were usually less severe in F4 than in F2. Moreover, the
results of reproduction even showed a regular oscillation pattern, with
stimulation in F1 and F3 and inhibition in F2 and F4.

(Figure 1 around here)

The influences on the lifespan represent the second aspect in fitness
evaluation. The effects on lifespan (Figure 1(B)) also showed
alterations between inhibition and stimulation, but there were no
particular concentration- or generation-dependent patterns. Notably, the
stimulation of DEHTP on reproduction (Figure 1(A)) was accompanied with
inhibition of lifespan in F1 and F3 at most concentrations (Figure
1(B)). Meanwhile, the inhibition on reproduction in F2 and F4 was
accompanied with the stimulation on lifespan in the corresponding
generations. The opposite relationship between effects on reproduction
and those on lifespan showed potential trade-off relationships. Further
parallel analysis was performed with the data of reproduction and
lifespan in each individual. Results showed that the individual with the
greatest stimulation on reproduction showed the greatest inhibition on
lifespan, and that with the greatest inhibition on reproduction showed
stimulation on lifespan (Figure 1(C)). Moreover, such relationship
covered 65\% of the total cases, and confirmed the trade-off
relationships.

The impacts on the behavior represent the third aspect in fitness
evaluation. Nematodes have more quiescence duration at satiety with
reverse and Omega turns or no locomotion. In F1, DEHTP inhibited the
satiety quiescence duration at 0.8 and 8.0 mg/L (Figure 2(A) and Figure
S1). The shorter satiety quiescence duration should spare more time for
foraging via body bending and head swing. Out of expectation, the
effects of DEHTP in F1 showed inhibitions on body bending at 8.0 mg/L
(Figure 2(A) and Figure S2), with stimulations on reverse and Omega
turns. In other generations, the effects showed similarities, i.e.,
inhibition on the satiety quiescence duration and body bending, and
stimulation on reverse and Omega turns (Figure 2(B-D)). Such
similarities were supported by Spearman's correlation analysis where the
satiety quiescence duration was positively correlated with body bending
and head swing but negatively correlated with reverse locomotion (Figure
2(E)). Collectively, the results showed that DEHTP dispatched less time
in foraging but more time in backward movement. Moreover, DEHTP also
inhibited the pharyngeal pumping (Figure S2), which further aggravated
the lack of food supply.

(Figure 2 around here)

\textbf{3.2. Multi-generational effects on lipid metabolism}

The multigenerational effects of DEHTP on the lipid metabolites,
including TG, NEFA, ACA and FA-CoA, are shown in Figure S2. In F1, at
0.8 mg/L DEHTP inhibited TG, ACA, lipase, ATGL, ACS, CPT and ACC but
stimulated NEFA, FA-CoA, GPAT and FAS. In F2 to F4, the effects showed
alterations between inhibition and stimulation without specific
tendencies or relationships (Figure S2).

Notably, there were opposite effects on TG and NEFA, with 60\% of the
cases where the stimulations on TG were usually accompanied with
inhibitions on NEFA and vice versa (Figure 3(A)). The opposite effects
were also found between lipase and ATGL (40\% of the total cases), and
also between the effects on ATGL and GPAT (55\% of the total cases)
(Figure 3(B)).

The Spearman's correlation analysis demonstrated the connection between
the metabolites and the enzymes (Figure 3(C)). The significant
stimulations of DEHTP on ACA were accompanied with positively correlated
stimulation on a series of enzymes in ACA metabolism (ACS, CPT and ACC)
and also in the balance between TG and NEFA (ATGL and GPAT). Combining
the apical and biochemical effects, there were positive correlations
between the effects on reproduction and with those on FAS and FA-CoA,
and negative ones between the influences on behavior and those on
FA-CoA, ACA and GPAT (Figure 3(E)).

\textbf{3.3. Multi-generational effects on neural regulation}

In F1, at 0.8 mg/L DEHTP stimulated 5-HT, DA, AChE and GABA and
inhibited ACH, with down-regulations on the expressions of \emph{ser-1}
,\emph{ser-6} , \emph{mod-1} and \emph{dop-3} (Figure S3). In F2 to F4,
DEHTP stimulated 5-HT, DA, AChE, ACH, GABA, with both up- and
down-regulations on the gene expressions. Moreover, DEHTP caused mostly
stimulations of 5-HT, DA, AChE and GABA, while it provoked mostly
inhibition of ACH. The results showed the opposite effects on AChE and
ACH, where stimulations on AChE with inhibition on ACH (and vice versa)
covered 55\% of the total cases (Figure 4). Moreover, the effects of
DEHTP on 5-HT and DA showed positive Spearman's correlation with each
other (0.5534, \emph{p} \textless{} 0.05). However, there were no
correlations between the effects on neural regulations and those on
locomotion (Figure 4).

\textbf{3.4. Overall multi-generational influences of DEHTP combining
various aspects}

Further analysis combined the aforementioned aspects to explore the
underlying connections. The correlation analysis demonstrated positive
correlations (i) between the effects on 5-HT and those on GPAT and ACS,
(ii) between DA and GPAT, FAS and FA-CoA, (iii) between ACH and ATGL,
GPAT, ACS, FAS and ACA, (iv) between AChE and NEFA, and (v) between GABA
and NEFA (Figure 4).

The HCA further assessed the potential strategies in the nematode
response to DEHTP over generations (Figure 5). The indicators in F1 had
shorter distance (with the longest distance \textless{} 2 in x axis)
than those in F2-F4. Such results implied closer connections among
neural regulation, lipid metabolism, reproduction and locomotion in F1
than subsequent generations. Notably, 5-HT was connected firstly with
other neural indicators and secondly with behavior and lipid metabolism
in F1, while it was connected firstly with behavior and later with other
neural indicators. Interestingly, 5-HT showed closer connection with
other neural indicators in F3 again, which was similar to the results in
F1, while it had later connection in F4 which was similar to those in
F2.

\textbf{4. Results and Discussion}

\textbf{4.1. Multi-generational influences of DEHTP on fitness}

The fitness evaluation was performed in reproduction, lifespan and
behavior. In the aspect of reproduction, the oscillation between
stimulation and inhibition over generations was also reported in the
multi-generational effects of ionic liquids and antibiotics (Zhang and
Feng, 2022; Zheng et al., 2022). On one hand, the alteration from
inhibition in earlier generation (e.g., in F2) to stimulation in later
one (F3) showed generation-dependent hormesis, which was also observed
in the reproductive effects of antibiotics (Zheng et al., 2022), ionic
liquids (Shi et al., 2021; Zhang and Feng, 2022) on \emph{C. elegans}
and those of graphene oxide on \emph{Acheta domesticus} (Dziewi\selectlanguage{polish}ę\selectlanguage{english}cka et
al., 2020). Hormesis over generations (also referred as trans-hormesis)
were found in various organisms (Agathokleous et al., 2022; Costantini,
2022). On the other hand, the alteration in effects across generations
indicated that the organisms actively adjusted fetal response to the
maternal challenges, which is essential to ensure the fitness or
adaption to sustain the population survival (Yue et al., 2021). Notably,
the responses in the offspring did not always match the stresses that
the offspring actually experience. From an ecological perspective,
pre-conditioning hormesis can improve resilience to subsequent exposure
(Costantini et al., 2010; Costantini et al., 2014). From a health
perspective, it may even strengthen therapeutic benefit (Dhawan et al.,
2020). However, pre-conditioning hormesis is costly under the mismatch
between the actual environment in offspring and the predicted
environment by parents. Therefore, the long-term effects of DEHTP
deserve ecological concerns.

The trade-off effects between lifespan and reproduction by DEHTP were
also observed in the toxicities of other pollutants, e.g., ionic liquids
(Shi et al., 2021; Zhang and Feng, 2022) and antibiotics (Yu et al.,
2017). The trade-off relationship showed a common strategy to balance
energy investment in adapting environments for better fitness. On one
hand, more reproduction at the expense of lifespan is essential to the
population's survival and continuation (Yu et al., 2017). On the other
hand, lower reproduction with an extended lifespan aids individual
organisms to escape from environmental stress. Both aspects were
observed in the effects of DEHTP but in different generations. Again,
the trade-off effects over generations also supported the active
adjustment of organisms to environmental stresses. The results
collectively indicated that phthalate might potentially disturb
adaptation and evolutionary changes.

In the aspect of behavior, the results demonstrated that DEHTP caused
avoidant behavior in nematodes, which allows the individuals to escape
from environmental stresses. Notably, the influences of DEHTP on
reproduction, lifespan, growth and behavior were all observable at 0.8
mg/L and even lower concentrations that are environmental realistic
(e.g., 0.07-6.1 mg/kg as the arithmetic mean values in dust) (Nagorka et
al., 2011). The results also brought up an assumption that DEHTP
resulted in caloric restriction by less foraging behavior and less
efficient food uptake. The caloric restriction decreased the overall
available energy, which subsequently resulted in energy allocation among
life traits according to the energy budget theory (Matyja et al., 2020;
Yu et al., 2018). The energy allocation also influenced the trade-off
relationships over generations (Wang et al., 2022a). Moreover, the
caloric restriction is important in the evolutionary responses with more
resilient to other stressors, and therefore partially explained the
pre-conditioning hormesis (Speakman, 2020). Therefore, further studies
on the caloric restriction would explain the aforementioned trade-off
relationships between reproduction and lifespan, and also the
generational effects.

\textbf{4.2. Lipid metabolism underlying the multi-generational effects}

Lipid metabolism plays crucial roles in supporting reproduction and
growth and regulating lifespan (Hansen et al., 2013). It is known that
TG is hydrolyzed to NEFA by lipase and ATGL, and NEFA can be synthesized
back to TG by GPAT (Figure 3(D)) (Pradhan et al., 2018; Srinivasan et
al., 2008). Therefore, the opposite effects on the metabolites (between
TG and NEFA) and enzymes (between lipase and ATGL, between ATGL and
GPAT) demonstrated that DEHTP disturbed the overall lipid metabolism
with potential biased directions. Moreover, NEFA can form FA-CoA by ACS,
and FA-CoA can react to ACA by CPT, while ACA can re-form NEFA by ACC
and FAS (Pradhan et al., 2018; Srinivasan et al., 2008). The correlation
between ACA and ACS (and also CPT and ACC) and between ATGL and GPAT
demonstrated that DEHTP significantly promoted the overall lipid
metabolism. Especially, such promotion occurred at environmental levels
of DEHTP (e.g., 0.008 mg/L), showing the potential of DEHTP as an
environmental obesogen. Combining the apical and biochemical effects,
the positive correlations supported the connection between energy supply
and reproduction. Meanwhile, the negative correlations indicated that
the energy supply was balanced and even allocated with preference
towards reproduction instead of locomotion behavior.

Notably, there were significant mismatches between lipid metabolism and
the apical responses (i.e., locomotion). Such mismatch can be resulted
from other energy consuming responses. It was reported that oxidative
stress and antioxidant investment are known to cost energy and may
directly involve trade-offs with other life traits (e.g., growth) (Yu et
al., 2018). The influences of DEHTP on oxidative stress and antioxidant
responses had been demonstrated in the human body (Huang et al., 2020).
The mismatch urged further studies on the energy budget and also
multi-omic analysis (e.g., lipidomic and transcriptomic) to reveal the
essential connections among various responses.

\textbf{4.3. Neural regulation underlying the multi-generational
effects}

The opposite effects between AChE and ACH supported the function of AChE
in hydrolyzing ACH (Behl et al., 2023). The up-regulated expressions
of\emph{ser-1} (encoding G-protein coupled receptors family 1 profile
domain-containing protein), \emph{ser-6} , \emph{mod-1} (encoding
serotonin-gated chloride channel) and \emph{dop-3} (encoding dopamine
receptor 3) are involved in the increases of 5-HT and GABA transductions
(McDonald et al., 2006; Srinivasan et al., 2008). However, the effects
of DEHTP on their expressions were not consistent with those on 5-HT or
GABA. The lack of such consistence suggests that more regulations were
involved in impacts on the neural regulation other than \emph{ser-1}
,\emph{ser-6} , \emph{mod-1} and \emph{dop-3} . The neuro-toxicities of
DEHTP showed some similarities to those of DEHP on nematodes and fishes
(Huang et al., 2022a; Huang et al., 2022b), which raises more concerns
about the toxicities of the plasticizer substitutes.

It is well known that neural signals regulate behavior. Both 5-HT and DA
support the nematode locomotion and feeding (Cermak et al., 2020;
Srinivasan et al., 2008), and DA is essential in avoidance responses of
nematodes (Chou et al., 2022). However, the lack of correlations between
the effects on neural regulations and those on locomotion demonstrated
that there was more complicated regulation between the biochemical
neural signals and the apical locomotion.

\textbf{4.4. Combined analysis on the multi-generational influences of
DEHTP}

Neural signals not only regulate behavior, but also connect with lipid
metabolism and reproduction (Luo et al., 2022; Magnan et al., 2015). The
positive correlations between biochemicals in the neural regulation and
those in lipid metabolism supports the neural regulation on lipid
metabolism (Ali and Mirza, 2021; Crane et al., 2015). The oscillatory
changes in the HCA results demonstrated the active responses of
organisms to the multi-generational signals. Such multigenerational
responses can involve epigenetic regulations (e.g., DNA methylations),
which were observed in the disturbances of DEHP on liver in mice over
three generations (Wen et al., 2020). Notably, the 5-HT results in HCA
demonstrated an oscillatory pattern over generations, which could be
related with the oscillation between stimulatory and inhibitory apical
effects of DEHTP over generations. Future studies should consider both
transcriptomic and epigenomic analysis to further explore mechanism.

\textbf{5. Conclusion}

In summary, DEHTP showed a regular oscillation pattern in the
multigenerational reproductive toxicities, with stimulation in F1 and F3
generations and inhibition in F2 and F4 ones. The influences of DEHTP on
reproduction and lifespan showed trade-off relationships. Further
analysis demonstrated the involvement of lipid metabolism and neural
regulation underlying the multi-generational effects. HCA results
pointed out the oscillatory connection between 5-HT and other neural
regulation in F1 and F3 and between 5-HT and other indicators in F2 and
F4. The overall oscillatory effects of DEHTP over generations need
further transcriptomic and epigenomic analysis to explore the underlying
mechanisms.

\textbf{Acknowledgement}

This work was supported by National Key Research and Development Program
of China (2021YFC3200803) and National Natural Science Foundation of
China (21407061).

\textbf{Conflict of interest}

The authors declare no conflict of interest associated with this
publication to disclose.

\textbf{Reference}

Agathokleous, E., Guedes, R.N.C., Calabrese, E.J., Fotopoulos, V.,
Azevedo, R.A., 2022. Transgenerational hormesis: What do parents
sacrifice for their offspring? Current Opinion in Environmental Science
\& Health 29, 100380.

Ali, M., Mirza, L., 2021. Morbid obesity due to prolactinoma and
significant weight loss after dopamine agonist treatment. AACE Clinical
Case Reports 7, 204-206.

Behl, T., Kaur, I., Sehgal, A., Singh, S., Sharma, N., Gupta, S.,
Albratty, M., Najmi, A., Alhazmi, H.A., Bungau, S., 2023. AChE as a
spark in the Alzheimer's blaze - Antagonizing effect of a cyclized
variant. Ageing Research Reviews 83, 101787.

Bernard, L., Masse, M., Boeuf, B., Chennell, P., Decaudin, B., Durand,
N., Genay, S., Lambert, C., Basle, Y.L., Moreau, E., Pinguet, J.,
Ponsonnaille, V., Richard, D., Saturnin, N., Storme, L., Sautou, V.,
ARMED, 2023. Medical devices used in NICU: The main source of
plasticisers' exposure of newborns. Science of the Total Environment
858, 159994.

Bulbul, M., Bhattacharya, S., Ankit, Y., Yadav, P., Anoop, A., 2022.
Occurrence, distribution and sources of phthalates and petroleum
hydrocarbons in tropical estuarine sediments (Mandovi and Ashtamudi) of
western Peninsular India. Environmental Research 214, 113679.

Cermak, N., Yu, S.K., Clark, R., Huang, Y.C., Baskoylu, S.N., Flavell,
S.W., 2020. Whole-organism behavioral profiling reveals a role for
dopamine in state-dependent motor program coupling in \emph{C. elegans}
. Elife 9, e57093.

Chen, M.Y., Liu, H.P., Cheng, J., Chiang, S.Y., Liao, W.P., Lin, W.Y.,
2019. Transgenerational impact of DEHP on body weight
of\emph{Drosophila} . Chemosphere 221, 493-499.

Chou, S.-H., Chen, Y.-J., Liao, C.-P., Pan, C.-L., 2022. A role for
dopamine in \emph{C. elegans} avoidance behavior induced by
mitochondrial stress. Neuroscience Research 178, 87-92.

Costantini, D., 2022. For better or worse: benefits and costs of
transgenerational plasticity and the transhormesis hypothesis. In
Costantini and Marasco (eds), Development strategies and biodiversity:
Darwinian fitness and evolution in the Anthropocene, Springer, 37-50.

Costantini, D., Metcalfe, N.B., Monaghan, P., 2010. Ecological processes
in a hormetic framework. Ecology Letters 13, 1435-47.

Costantini, D., Monaghan, P., Metcalfe, N.B., 2014. Prior hormetic
priming is costly under environmental mismatch. Biology Letters 10,
20131010.

Crane, J.D., Palanivel, R., Mottillo, E.P., Bujak, A.L., Wang, H., Ford,
R.J., Collins, A., Bl\selectlanguage{ngerman}ümer, R.M., Fullerton, M.D., Yabut, J.M., Kim,
J.J., Ghia, J.E., Hamza, S.M., Morrison, K.M., Schertzer, J.D., Dyck,
J.R.B., Khan, W.I., Steinberg, G.R., 2015. Inhibiting peripheral
serotonin synthesis reduces obesity and metabolic dysfunction by
promoting brown adipose tissue thermogenesis. Nature Medicine 21,
166-172.

Dhawan, G., Kapoor, R., Dhawan, R., Singh, R., Monga, B., Giordano, J.,
Calabrese, E.J., 2020. Low dose radiation therapy as a potential life
saving treatment for COVID-19-induced acute respiratory distress
syndrome (ARDS). Radiotherapy and Oncology 147, 212-16.

Dziewi\selectlanguage{polish}ę\selectlanguage{english}cka, M., Flasz, B., Roszkowska, M.R., K\selectlanguage{polish}ę\selectlanguage{english}dziorski, A.,
Kochanowicz, A., Augustyniak, M., 2020. Graphene oxide as a new
anthropogenic stress factor - multigenerational study at the molecular,
cellular, individual and population level of \emph{Acheta domesticus} .
Journal of Hazardous Materials 396, 122775.

Hamilton, L.K., Dufresne, M., Jopp\selectlanguage{ngerman}é, S.E., Parent, M., Chaurand, P.,
Fernandes, K.J.L., 2015. Aberrant lipid metabolism in the forebrain
niche suppresses adult neural stem cell proliferation in an animal model
of Alzheimer's disease. Sell Stem Cell 17, 397-411.

Hansen, M., Flatt, T., Aguilaniu, H., 2013. Reproduction, fat
metabolism, and life span: what is the connection? Cell Metabolism 17,
11-19.

Huang, M.L., Yen, P.L., Chang, C.H., Liao, V.H.C., 2022a. Chronic
di(2-ethylhexyl) phthalate exposure leads to dopaminergic neuron
degeneration through mitochondrial dysfunction in \emph{C. elegans} .
Environmental Pollution 307, 119574.

Huang, P.-C., Waits, A., Chen, H.-C., Chang, W.-T., Jaakkola, J.J.K.,
Huang, H.-B., 2020. Mediating role of oxidative/nitrosative stress
biomarkers in the associations between phthalate exposure and thyroid
function in Taiwanese adults. Environment International 140, 105751.

Huang, W.L., Xiao, J.F., Shi, X.L., Zheng, S.K., Li, H.L., Liu, C.X.,
Wu, K.S., 2022b. Effects of di-(2-ethylhexyl) phthalate (DEHP) on
behavior and dopamine signaling in zebrafish (\emph{Danio rerio} ).
Enviormental Toxicology and Pharmacology 93, 103885.

Lemieux, G.A., Ashrafi, K., 2016. Investigating connections between
metabolism, longevity, and behavior in \emph{Caenorhabditis elegans} .
Trends in Endocrinology \& Metabolism 27, 586-596.

Lessmann, F., Kolossa-Gehring, M., Apel, P., Rüther, M., Pälmke, C.,
Harth, V., Brüning, T., Koch, H.M., 2019. German Environmental Specimen
Bank: 24-hour urine samples from 1999 to 2017 reveal rapid increase in
exposure to the para-phthalate plasticizer di(2-ethylhexyl)
terephthalate (DEHTP). Environment International 132, 105102.

Li, Z., Ai, F., Zhang, J., Yu, Z., Yin, D., 2019.
Using\emph{Caenorhabditis elegans} for studying trans- and
multi-generational effects of toxicants. Journal of Visualized
Experiments 149, e59367.

Li, Z., Yu, Z.Y., Gao, P., Yin, D.Q., 2020. Multigenerational effects of
perfluorooctanoic acid on lipid metabolism of \emph{Caenorhabditis
elegans} and its potential mechanism. Science of the Total Environment
703, 134762.

Liu, H., Mo, L.Y., Hou, M.F., Zhang, J., 2020. Life stage-dependent
toxicities of 1-ethyl-3-methylimidazolium bromide on\emph{Caenorhabditis
elegans} . Environmental Pollution 265, 114848.

Liu, S.S., Li, A.Y., Witt, C.M., Leon, A.A.P.d., 2013. Effects of
reserpine on reproduction and serotonin immunoreactivity in the stable
fly Stomoxys calcitrans. Journal of Insect Physiology 59, 974-82.

Luo, Z.L., Yu, Z.Y., Yin, D.Q., 2022. Obesogenic effect of erythromycin
on \emph{Caenorhabditis elegans} through over-eating and lipid
metabolism disturbances. Environmental Pollution 294, 118615.

Ma, S., Hu, X., Tang, J., Cui, J., Lin, M., Wang, F., Yang, Y., Yu, Y.,
2022. Urinary metabolites and handwipe phthalate levels among adults and
children in southern China: Implication for dermal exposure. Journal of
Hazardous Materials 439, 129639.

Magnan, C., Levin, B.E., Luquet, S., 2015. Brain lipid sensing and the
neural control of energy balance. Molecular and Cellular Endocrinology
418, 3-8.

Matyja, K., Rybak, J., Hanus-Lorenz, B., Wróbel, M., Rutkowski, R.,
2020. Effects of polystyrene diet on \emph{Tenebrio molitor} larval
growth, development and survival: Dynamic Energy Budget (DEB) model
analysis. Environmental Pollution 264, 114740.

McDonald, P.W., Jessen, T., Field, J.R., Blakely~, R.D., 2006. Dopamine
signaling architecture in \emph{Caenorhabditis elegans} . Cellular and
Molecular Neurobiology 26, 591-616.

Nagorka, R., Conrad, A., Scheller, C., Süßenbach, B., Moriske, H.-J.,
2011. Diisononyl 1,2-cyclohexanedicarboxylic acid (DINCH) and
Di(2-ethylhexyl) terephthalate (DEHT) in indoor dust samples:
Concentration and analytical problems. International Journal of Hygiene
and Environmental Health 214, 26-35.

Pacyga, D.C., Patti, M.A., Papandonatos, G.D., Haggerty, D.K., Calafat,
A.M., Gardiner, J.C., Braun, J.M., Schantz, S.L., Strakovsky, R.S.,
2023. Associations of individual and cumulative urinary phthalate and
replacement biomarkers with gestational weight gain through late
pregnancy. Science of the Total Environment 855, 158788.

Pradhan, A., Olsson, P.E., Jass, J., 2018. Di(2-ethylhexyl) phthalate
and diethyl phthalate disrupt lipid metabolism, reduce fecundity and
shortens lifespan of \emph{Caenorhabditis elegans} . Chemosphere 190,
375-82.

Quinnies, K.M., Doyle, T.J., Kim, K.H., Rissman, E.F., 2015.
Transgenerational effects of di-(2-ethylhexyl) phthalate (DEHP) on
stress hormones and behavior. Endocrinology 156, 3077--83.

Rattan, S., Brehm, E., Gao, L., Flaws, J.A., 2018. Di (2-ethylhexyl)
phthalate exposure during prenatal development causes adverse
transgenerational effects on female fertility in mice. Toxicological
Sciences 163, 420-29.

Schwedler, G., Rucic, E., Koch, H.M., Lessmann, F., Bruning, T., Conrad,
A., Schmied-Tobies, M.I.H., Kolossa-Gehring, M., 2020. Metabolites of
the substitute plasticiser Di-(2-ethylhexyl) terephthalate (DEHTP) in
urine of children and adolescents investigated in the German
Environmental Survey GerES V, 2014-2017. International Journal of
Hygiene and Environmental Health 230, 113589.

Sedha, S., Lee, H., Singh, S., Kumar, S., Jain, S., Ahmad, A., Jardan,
Y.A.B., Sonwal, S., Shukla, S., Simal-Gandara, J., Xiao, J., Huh, Y.S.,
Han, Y.-K., Bajpai, V.K., 2021. Reproductive toxic potential of
phthalate compounds -- State of art review. Pharmacological Research
167, 105536.

Seyourm, A., Pradhan, A., 2019. Effect of phthalates on development,
reproduction, fat metabolism and lifespan in \emph{Daphnia magna} .
Science of the Total Environment 654, 969-77.

Shi, Y., Meng, X.Z., Zhang, J., 2021. Multi- and trans-generational
effects of N-butylpyridium chloride on reproduction, lifespan, and
pro/antioxidant status in \emph{Caenorhabditis elegans} . Science of the
Total Environment 778, 146371.

Speakman, J.R., 2020. Why does caloric restriction increase life and
healthspan? The `clean cupboards' hypothesis. National Science Review 7,
1153-56.

Sree, C.G., Buddolla, V., Lakshmi, B.A., Kim, Y.-J., 2023. Phthalate
toxicity mechanisms: An update. Comparative Biochemistry and Physiology
Part C: Toxicology \& Pharmacology 263, 109498.

Srinivasan, S., Sadegh, L., Elle, I.C., Christensen, A.G.L., Faergeman,
N.J., Ashrafi, K., 2008. Serotonin regulates \emph{C. elegans} fat and
feeding through independent molecular mechanisms. Cell Metabolism 7,
533-44.

Wang, L., Wu, D., Yu, Z., Huang, S., Zhang, J., 2023. Hormone-mediated
multi- and trans-generational reproductive toxicities of
1-ethyl-3-methylimidazolium hexafluorophosphate on \emph{Caenorhabditis
elegans} . Science of the Total Environment 863, 160958.

Wang, M.-C., Furukawa, F., Wang, C.-W., Peng, H.-W., Lin, C.-C., Lin,
T.-H., Tseng, Y.-C., 2022a. Multigenerational inspections of
environmental thermal perturbations promote metabolic trade-offs in
developmental stages of tropical fish. Environmental Pollution 308,
119605.

Wang, X., Okoffo, E.D., Banks, A.P.W., Li, Y., Thomas, K.V., Rauert, C.,
Aylward, L.L., Mueller, J.F., 2022b. Phthalate esters in face masks and
associated inhalation exposure risk. Journal of Hazardous Materials 423,
127001.

Watts, J.L., 2009. Fat synthesis and adiposity regulation
in\emph{Caenorhabditis elegans} . Trends in Endocrinology and Metabolism
20, 58-65.

Wen, Y., Rattan, S., Flaws, J.A., Irudayaraj, J., 2020. Multi and
transgenerational epigenetic effects of di-(2-ethylhexyl) phthalate
(DEHP) in liver. Toxicology and Applied Pharmacology 402, 115123.

Xie, Z., Zhang, X., Xie, Y., Wu, J., Wu, Y., 2023. Occurrences and
potential lipid-disrupting effects of phthalate metabolites in humpback
dolphins from the South China Sea. Journal of Hazardous Materials 441,
129939.

Yu, Y., Hua, X., Chen, H., Wang, Z., Han, Y., Chen, X., Yang, Y., Xiang,
M., 2022. Glutamatergic transmission associated with locomotion-related
neurotoxicity to lindane over generations in \emph{Caenorhabditis
elegans} . Chemosphere 290, 133360.

Yu, Z.Y., Jiang, L., Yin, D.Q., 2011. Behavior toxicity
to\emph{Caenorhabditis elegans} transferred to the progeny after
exposure to sulfamethoxazole at environmentally relevant concentrations.
Journal of Environmental Science 23, 294-300.

Yu, Z.Y., Sun, G.H., Liu, Y.J., Yin, D.Q., Zhang, J., 2017.
Trans-generational influences of sulfamethoxazole on lifespan,
reproduction and population growth of \emph{Caenorhabditis elegans} .
Ecotoxicology and Environmental Safety 135, 312-318.

Yu, Z.Y., Yin, D.Q., Hou, M.F., Zhang, J., 2018. Effects of food
availability on the trade-off between growth and antioxidant responses
in \emph{Caenorhabditis elegans} exposed to sulfonamide antibiotics.
Chemosphere 211, 278-85.

Yue, W.Y., Mo, L.Y., Zhang, J., 2021. Reproductive toxicities of
1-ethyl-3-methylimidazolium bromide on \emph{Caenorhabditis elegans}with
oscillation between inhibition and stimulation over generations. Science
of the Total Environment 765, 144334.

Zhang, J., Chen, R., Yu, Z., Xue, L., 2017. Superoxide dismutase (SOD)
and catalase (CAT) activity assay protocols for \emph{Caenorhabditis
elegans} . Bio-protocol 7, e2505.

Zhang, J., Feng, L., 2022. Intermittent multi-generational effects of
1-hexyl-3-methylimidazolium nitrate on \emph{Caenorhabditis
elegans}mediated by lipid metabolism. Science of The Total Environment
812, 152479.

Zhang, J., Shi, Y., Yu, Z., 2022. Balances among reproduction,
antioxidant responses and lipid metabolism underlying the
multi-generational effects of N-butylpyridinium bromide
on\emph{Caenorhabditis elegans} . Science of The Total Environment 846,
157468.

Zhao, Y., Li, X.-N., Zhang, H., Cui, J.-G., Wang, J.-X., Chen, M.-S.,
Li, J.-L., 2022. Phthalate-induced testosterone/androgen receptor
pathway disorder on spermatogenesis and antagonism of lycopene. Journal
of Hazardous Materials 439, 129689.

Zheng, Y.G., Yu, Z.Y., Zhang, J., 2022. Multi-generational effects of
enrofloxacin on lifespan and reproduction of \emph{Caenorhabditis
elegans} with SKN-1-mediated antioxidant responses and lipid metabolism
disturbances. Science of The Total Environment 804, 150250.

\textbf{Hosted file}

\verb`2023-08-02-Fig. 1-5.docx`  available at \url{https://authorea.com/users/650036/articles/658712-impacts-of-di-2-ethylhexyl-terephthalate-on-multi-generational-fitness-of-caenorhabditis-elegans-via-lipid-metabolism-and-neural-regulation}

\selectlanguage{english}
\FloatBarrier
\end{document}

