journal of environmental sciences 137 (2024) 615–625

Available online at www.sciencedirect.com

www.elsevier.com/locate/jes

Tributyltin causes generational

neurodevelopmental toxicity and the protective
effect of folic acid in zebrafish

Haoxing Cai, Naying Zheng, Chen Tang, Yuxuan Zhang, Zhenghong Zuo∗,
Chengyong He∗
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences,
Xiang’an Hospital of Xiamen University, Xiamen University, Xiamen 361005, China

a r t i c l e i n f o a b s t r a c t

Article history: Tributyltin (TBT), a common organotin environmental pollutant, may pose a threat to hu-
Received 29 November 2022 man development during critical early-life periods. We aimed to assess the neurodevel-
Revised 8 March 2023 opmental intergenerational toxicity of early-life exposure to TBT and the protective effect
Accepted 8 March 2023 of DNA methyl donor folic acid (FA). Specifically, after early-life exposure (1–21 days post-
Available online 21 March 2023 fertilization, dpf) to TBT (0, 1, 10 and 100 ng/L), zebrafish (Danio rerio) were cultured in clean
medium until sexual maturity. The exposed females were mated with unexposed males to
Keywords: produce embryos (F1). The F1 generation were cultured (4–120 hours post-fertilization, hpf)
Tributyltin with and without 1 mmol/L FA. The neurotoxic effects of early-life TBT exposure for ze-
Neurodevelopment brafish and their offspring (F1) were significantly enhanced anxiety and reduced aggression,
Intergeneration decreased gene expression of DNA methyltransferase in the brain and increased serotonin
DNA methylation levels in the body. Moreover, the intergenerational neurodevelopmental toxicity, as mani-
Folic acid fested in the F1 generation, was attenuated by FA supplementation. In summary, early-life
Zebrafish TBT exposure led to intergenerational neurodevelopmental deficits in zebrafish, and DNA
methyl donors had a protective effect on F1 neurodevelopment, which can inform the pre-
vention and treatment of intergenerational neurotoxicity due to organotin pollutants.
© 2023 The Research Center for Eco-Environmental Sciences, Chinese Academy of
Sciences. Published by Elsevier B.V.

of the Mucuripe Bay (Moreira et al., 2021) and Vitoria estuary

Introduction (Abreu et al., 2021), respectively, after dredging activities.
Worryingly, TBT levels in sediments are as high as 500 ng/g in
Tributyltin (TBT), an antifungal preservative and antifouling
heavily polluted marine protected areas (Castro et al., 2021).
agent, is widely used in industry and agriculture and thus has
In Europe, the sedimental TBT concentrations range from < 5
contaminated the environment. In Asia, it has been reported
to 850 ng/g with a mean of 260 ng/g in the Thames (Vane et al.,
that TBT levels range from (25 ± 4.2) to (303 ± 7.4) ng/L in
2022). Of particular note, a mean TBT level of 0.32 ng/g fresh
coastal water, from (17 ± 1.4) to (107 ± 4.1) ng/kg in sediment
weight was detected in the placenta of new-borns in a Finland
and from (4 ± 1.2) to (42 ± 2.2) ng/kg in the wet weight of Perna
and Denmark cohort study (Rantakokko et al., 2013a, 2014b).
viridis in Sri Lanka (Bandara et al., 2021). In South America, TBT
Increasing evidence has linked early-life adversity to later
levels reach 52.6 and 113 ng Sn/g dry weight in the sediment
neurotransmitter levels and neurobehavioural outcomes

∗
Corresponding authors.
E-mails: zuozhenghong@xmu.edu.cn (Z. Zuo), hecy@xmu.edu.cn (C. He).

https://doi.org/10.1016/j.jes.2023.03.015
1001-0742/© 2023 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
616 journal of environmental sciences 137 (2024) 615–625
(Doherty et al., 2019, Li et al., 2020, Weber-Stadlbauer et al.,
2017a, 2021b). Exposure to environmental levels of TBT has
significant adverse effects on the nervous system of various
experimental models. In juvenile common carp (Cyprinus
carpio), 60 days of exposure to 75 ng/L TBT induced brain func-
tional damage (Li et al., 2015). In female Sebastiscus marmora-
tus, exposure to 1, 10, and 100 ng/L TBT causes cell apoptosis,
reactive oxygen species production and elevated nitric oxide
levels in the brain, which might influence shoaling, sensory
function and movement (Zhang et al., 2008). After 50 days
of 10, 100 and 1000 ng/L TBT exposure, dopamine (DA) levels
increased and serotonin (5-HT) and norepinephrine levels de-
creased in S. marmoratus (Yu et al., 2013). These studies show
that early-life exposure to TBT causes neurodevelopmental
abnormalities in fishes. However, the intragenerational effects
of organotin exposure, especially TBT, remain unclear.
Epigenetic regulation mediates the adverse intergenera-
tional or transgenerational transcriptional effects caused by
environmental toxicants; of these mechanisms, DNA methy-
lation is the most studied (Gruzieva et al., 2019; Martin and
Fry, 2018; Rider and Carlsten, 2019; Virani et al., 2016). DNA
methylation is a key regulatory mechanism during embryonic
development. Methylation typically occurs on CpG dinu-
cleotides and regulates transcriptional cascades, genomic
imprinting, genomic stability and chromatin structure. DNA
methylation is catalysed by DNA methyltransferases (Dnmt)
of the Dnmt1 and Dnmt3 families. DNMT3 is a paralogue
of DNMT, and more than one duplication event has been
found in zebrafish (Danio rerio). Chemical toxicants have been
reported to affect DNA methylation-related gene expression,
such as nicotine, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and
oxygenated polycyclic aromatic hydrocarbons (Faillace and
Bernabeu, 2022; Volz et al., 2016; Wirbisky-Hershberger et al.,
2017; Yun et al., 2019).
An excellent model organism, the zebrafish lays a large
number of eggs, has transparent embryos, and has clear
developmental cycles. Therefore, our study assessed the
neurodevelopmental effects and intergenerational effects of
early-life exposure to ambient TBT levels (0, 1, 10, and 100
ng/L). Folate metabolism is closely related to the synthesis
of S-adenosylmethionine, which is a direct methyl donor for
DNA methylation (Reynolds, 2006). The effect of folic acid (FA)
supplementation in TBT-induced intergenerational neurotox-
icity was further studied. This study is of great importance
for the prevention and treatment of the intergenerational
neurotoxicity due to organotin pollutants.
1. Methods and materials
1.1. Reagents and assay kits
TBT (CAS no.:1461-22-9; 96.0% purity) was purchased from
Acmec (Shanghai, China). Real-time PCR kits were purchased
from Abclonal (Wuhan, China). 5-HT and DA levels were
determined using a competitive enzyme immunoassay tech-
nique, and commercial kits were purchased from Chunshi
(Shanghai, China) and Meibiao (Jiangsu, China), respectively.
Dimethyl sulfoxide (DMSO) and all other reagents used for
RNA isolation and in the standard water (NaCl, KCl, NaHCO3 ,
and CaCl2 ) were of analytical grade.
1.2. Protocol for exposure, delivery, and sampling
Our protocol was approved by the Animal Ethics Committee
of Xiamen University. Wild-type (WT) zebrafish were raised
in a previously described manner (Tang et al., 2021). Briefly,
fertilized eggs were dispersed at random into 20 glass petri
dishes, holding 80 embryos each, and provided with 10 mL
of ordinary water containing various TBT concentrations.
TBT exposure concentrations were 0 (control), 1, 10, and 100
ng/L. Previously reported ambient values were equivalent to
the maximum concentration (100 ng/L) (Chen et al., 2020).
The DMSO concentration was the same in all control and
TBT-exposed groups (0.01%, V/V).
As early as the first day post-fertilization (dpf) in the
embryonic stage, abundant primordial germ cells (PGCs) are
formed to ensure later ovary differentiation and development
in zebrafish (Ye et al., 2019). Zebrafish germlines undergo
four processes, including PGC specification, migration, mi-
gration to the genital ridge, and proliferation, from 1 to 21
dpf (Wang et al., 2021). Exposure of neonatal rats to 100
ng/kg bw TBT leads to dysfunction of adult oocyte-granulosa
cell communication (Chen et al., 2022), which suggests that
exposure to TBT during primordial germ cell development
from postnatal days 1 to 21 poses health risks for later life.
From 1 to 21 dpf, experimental zebrafish were treated with
TBT in order to evaluate the impact of TBT exposure on the
neurodevelopment of both the F0 and F1 generation during
the development of PGCs. Twice daily, the exposure solution
was changed to maintain optimal water quality for the ze-
brafish. The larvae were moved to aquaria at the end of the ex-
posure (21 dpf), and 50 adolescents were reared to sexual ma-
turity in each aquarium. Every day, approximately two-thirds
of the water was replaced. Eight F0 zebrafish from each repli-
cation (n = 3) were chosen at random for neurobehavioural
tests between 35 and 60 dpf. Six F0 zebrafish were quick-
frozen in liquid nitrogen at 60 days of gestation and kept at -
80°C until their 5-HT and DA levels were measured. The brains
of six more F0 zebrafish were separated, quickly quick-frozen
in liquid nitrogen, and kept at -80°C until transcriptional al-
terations in genes associated with DNA were detected. At ap-
proximately 140 dpf, unexposed male and control or 100 ng/L
TBT exposed female F0 zebrafish were placed in a mating tank
to collect fertilized eggs for the F1 generation. The F1 zebrafish
embryos were cultivated during crucial stages of nervous sys-
tem development (4–120 hours post-fertilization, hpf) with
and without FA (1 mmol/L, pH 8.5). The time window of 4–120
hours post-fertilization was chosen based on the developmen-
tal stages described in established atlases of normal zebrafish
development (Kimmel et al., 1995). The dose levels of FA (1
mmol/L) were chosen based on published research (Ma et al.,
2012). The intervention solutions were kept in a dark place and
renewed twice daily. Analogously, at 35–60 dpf, five F1 larvae
were randomly chosen (n = 3) from each replicate for neurobe-
havioural tests. At 60 dpf, six F1 zebrafish were euthanized us-
ing ice water. Then, their brains were removed, quickly frozen
in liquid nitrogen, and kept at -80°C until transcriptional alter-
ations in the genes involved in DNA methyltransferase were
 journal of environmental sciences 137 (2024) 615–625
observed. Six more F1 zebrafish were quick-frozen in liquid ni-
trogen and kept at -80°C until their 5-HT and DA levels were
measured. The remaining F0 and F1 zebrafish were narcotized
and euthanized using ice water until their operculum move-
ment ceased. There was an approximately 5:1 ratio of ice to
water (Thurman et al., 2019). A dissecting stereomicroscope
was used to describe morphological anomalies at 72 hpf, in-
cluding spinal curvature and pericardial and yolk-sac oedema.
1.3. Behavioural test
At 35 dpf, F0 and F1 zebrafish were randomly selected for
assessment in the novel tank diving test, mirror attack
test, and novel object (marble) avoidance test. In each neu-
robehavioural test, after a two-minute acclimatization, a
five-minute video was recorded for each fish using S5KHMX
(SAMSUNG Inc., Korea). The video data were acquired and
analysed using Ethovision XT 12.0 software (Noldus Informa-
tion Technology, the Netherlands). In the analysis software
setting, a cumulative delay duration of more than or equal to
10 sec is needed before the start of tracking to avoid interfer-
ence factors such as water waves and shadows in the video.
1.3.1. Novel tank diving test
The duration in the top of the tank can be used as a measure
of anxiety, as zebrafish naturally prefer to dive to the bottom
of a novel tank before adapting to the environment. A de-
creased duration in the top of the tank is a sign of heightened
anxiety (Kysil et al., 2017). To calculate the total distance
travelled, velocity, number of entries into and duration in the
top of the novel tank, a camera was placed on the side of the
aquarium to track the swimming trajectory of each zebrafish
that was individually netted and placed into the tank.
1.3.2. Mirror attack test
When a zebrafish approaches the mirror during a mirror at-
tack test, the fish’s reflection seems to slowly approach them,
which leads to aggressive behaviour. We have described this
methodology previously (Zhou et al., 2021). To calculate the
frequency and cumulative duration in close proximity to
the mirror, a camera was placed on the side of the tank to
track the swimming trajectories of each zebrafish that was
individually netted and placed into the tank.
1.4. Neurotransmitter assays
5-HT and DA levels were determined in 60-dpf whole fish
(n = 6). The zebrafish were anaesthetized and euthanized
using ice water until operculum movement ceased. Zebrafish
were blotted from the surface on filter paper and then
weighed and recorded. Precooled PBS (pH 7.2) was added, and
the samples were homogenized by means of a tissue lyser
(JingXin, Shanghai, China). The samples were homogenized
at a low temperature (4°C, 3000 × g) and then centrifuged for
20 min. The supernatant was collected. Both 5-HT and DA
levels were measured in accordance with the instructions
provided with commercial ELISA kits (CS-00E196321, Chunshi,
Shanghai and MB-10128A, Meibiao biology, Jiangsu, China,
respectively). The manufacturer reported a limit of detection
(LOD) of 2 pg/mL for 5-HT and 50 pg/mL for DA. Absorbance
was measured at 450 nm.
617
1.5. Expression levels of genes related to DNA
methyltransferase
Total RNA was extracted from brain samples in two gener-
ations of zebrafish (one brain was considered one sample,
n = 6) using RNAex Pro Reagent (Accurate Biotechnology,
Hunan, China). The quality of total RNA and the validity of
primers were verified by electrophoresis. First-strand cDNA
was synthesized using a thermal cycler (Bio-Rad, Hercules,
CA, USA) and an ABScript III Reverse Transcriptase kit (Ab-
clonal). The relative expression levels of the reference and
target genes were measured with an automatic medical
PCR analysis system (Tianlong Technology, Xi’an, China)
and SYBR Green PCR Kit (Abclonal). In the supplementary
material, the primer sequences are displayed (Appendix A
Table S1). As an internal standard, the gapdh gene, which
codes for glyceraldehyde-3-phosphate dehydrogenase, was
chosen.
1.6. Statistical analysis
The mean and standard error of the mean (SEM) are dis-
played for every data point. The Shapiro-Wilk test was used
to determine if the model’s residuals were normally dis-
tributed. Levene’s test was used to analyse the homogeneity
of variances before analysis of variance (ANOVA). To exam-
ine the variations in neurotransmitter levels between groups,
an independent-sample t test was utilized. Additionally, using
IBM SPSS version 22.0, all additional significant group differ-
ences were assessed by one-way ANOVA, followed by Duncan’s
post hoc analysis or Dunnett’s T3 analysis (IBM Inc., Chicago, IL,
USA). P values less than 0.05 were regarded as significant for
all comparisons.
2. Results
2.1. Development indicators in embryos
The results of early-life TBT exposure on zebrafish develop-
mental indicators are shown in Appendix A Table S2. Although
there was no significant difference, the survival rates of em-
bryos exposed to TBT (1 ng/L: 99.67% ± 0.82%, 10 ng/L: 99.83%
± 0.41%, and 100 ng/L: 99.67% ± 0.52%) were progressively re-
duced compared with the control group (control: 100.0% ±
0.00%) at 36 hpf. Similarly, the frequency of embryo movement
in embryos exposed to TBT (1 ng/L: 1.51 ± 2.86, 10 ng/L: 1.12 ±
1.22, and 100 ng/L: 1.06 ± 1.16 times/min) were also decreased
gradually compared to the control (2.80 ± 1.87 times/min).
These results suggest that early-life exposure to 1, 10, and 100
ng/L TBT had little embryotoxicity.
2.2. Behavioural tests in juvenile zebrafish
Zebrafish behavioural performance in the novel tank diving
test is shown in Fig. 1. Compared to the control group, early-
life exposure to 100 ng/L TBT significantly shortened the time
in the top of the tank (control: 185.68 ± 8.63 sec, n = 22 vs.
100 ng/L: 138.15 ± 12.09 sec, n = 21) and decreased the num-
ber of entries into the top of the tank (control: 100.32 ± 18.12,
618 journal of environmental sciences 137 (2024) 615–625

Fig. 1 – Early–life tributyltin (TBT) exposure increased anxiety–like behavior in juvenile in novel tank diving test. (a)
Locomotor traces; (b) Total distance traveled in the top; (c) Velocity traveled in the top; (d) The number of entries to the top;
(e) The time spent in the top. The data are expressed as the mean ± standard error of the mean (SEM). Different letters
above the error bars represent significant between groups (P < 0.05).

n = 22 vs. 100 ng/L: 87.62 ± 14.93, n = 21). There was no
significant difference in locomotor behaviour, including to-
tal distance travelled and velocity in the top of the tank be-
tween the control fish and fish exposed in early life to 100
ng/L TBT. These results show that early-life exposure to 100
ng/L TBT leads to more anxiety-like behaviours in juvenile
zebrafish.
The performance of zebrafish in the mirror attack test is
shown in Fig. 2. Compared to the control group, a signifi-
cant reduction in the cumulative duration in close proxim-
ity to the mirror was observed in juveniles with early-life
exposure to TBT (control: 48.75 ± 7.56 sec, n = 21 vs. 100
ng/L: 29.36 ± 5.46 sec, n = 21), and a significant decrease
in the frequency of moving in close proximity to the mir-
ror (control: 53.10 ± 5.18; n = 21 vs. 100 ng/L: 35.30 ± 3.86;
n = 20). These results indicate that aggression is sup-
pressed in juvenile zebrafish exposed to 100 ng/L TBT in
early life.
2.3. Neurotransmitter content in juvenile zebrafish
The 5-HT and DA are closely associated with abnormal
anxiety and aggressive behaviour (de la Mora et al., 2010;
Zangrossi and Graeff, 2014). Thus, we examined the 5-HT and
 journal of environmental sciences 137 (2024) 615–625 619

Fig. 2 – Early–life TBT exposure reduced aggression and cognitive ability in juvenile in the mirror exposure test. (a) The
heatmaps of cumulative locomotor traces, from dark blue to dark red, the colors at both ends indicate the minimum and
maximum values of the per-pixel frequency; (b) The frequency close to the mirror; (c) The cumulative duration close to the
mirror. The data are expressed as the mean ± SEM. Different letters above the error bars represent significant between
groups (P < 0.05).

Fig. 3 – Early–life TBT exposure increased neurotransmitter levels in juvenile. (a) Zebrafish serotonin (5-HT) levels; (b)
Zebrafish dopamine (DA) levels. The data are expressed as the mean ± SEM. Different letters above the error bars represent
significant between groups (P < 0.05).

DA levels in whole juvenile fish. In the 60-dpf juveniles, sig-
nificant increases in 5-HT (control: 21.93 ± 3.24 vs. 100 ng/L:
42.45 ± 1.46 pg/mL each fish) (Fig. 3a) and DA levels (control:
1560.14 ± 141.03 vs. 100 ng/L: 1927.00 ± 68.87 pg/mL each fish)
were observed (Fig. 3b).
2.4. Dnmt expression in zebrafish juvenile brain
DNA methylation plays a role in controlling important cellu-
lar processes in neuronal development and brain plasticity
(Bayraktar and Kreutz, 2018). Therefore, we measured dnmt
620 journal of environmental sciences 137 (2024) 615–625
Fig. 4 – Zebrafish early exposure to TBT reduced
methyltransferase–related gene expression levels in the
brain of juvenile. The data are expressed as the
mean ± SEM relative to β-actin.
expression in the juvenile (60 dpf) zebrafish brain by RT-
qPCR. The expression of dnmt1, dnmt3aa, dnmt3ab, dnmt3ba,
dnmt3bb.2, and dnmt3bb.3 in the brain was decreased (0.80-,
0.44-, 0.16-, 0.66-, 0.73-, and 0.30- fold, respectively) in the
100 ng/L group (Fig. 4). These results show that exposure to 100
ng/L TBT in early life alters DNA methyltransferase expression
in the brain.
2.5. Development indicators in F1 embryos
Considering that any alterations in the PGCs can have trans-
generational impacts (Leitch et al., 2013), the findings in the F0
zebrafish motivated us to explore the transgenerational neu-
rodevelopmental toxicity of early-life TBT exposure and the
potential protective effect of the DNA methyl donor, FA, on the
F1 generation of zebrafish. To understand the developmental
effects of early-life exposure to 100 ng/L TBT and 1 mmol/L
FA supplementation on offspring, we explored the effects of
early-life TBT exposure of zebrafish on the development of
their F1 embryos, as shown in Appendix A Table S3. There was
no significant difference in survival rates, embryo movement,
or normal rate of F1 embryos of zebrafish exposed to 100 ng/L
TBT compared with those of the control group.
2.6. FA rescues TBT-induced neurobehavioural
abnormalities in the F1 generation
To observe the neurotoxic effects of early-life exposure to 100
ng/L TBT on offspring and the protective effects of FA supple-
mentation against TBT-induced intergenerational neurotoxic-
ity, we performed the same behavioural tests in the F1 genera-
tion. The effects of early-life TBT exposure on anxiety-like be-
haviours in the F1 generation in the novel tank diving test are
shown in Fig. 5. The number of entries into the top of the tank
of the F1 offspring of zebrafish exposed to 100 ng/L TBT (100
ng/L-F1 juveniles, 19.83 ± 4.39) were significantly lower than
those of zebrafish in the control group (control-F1 juveniles,
38.08 ± 5.25) and those that received FA supplementation (100
ng/L+FA-F1 juveniles, 36.25 ± 5.50).
The effects of early-life TBT exposure on cognition and ag-
gressive behaviour in F1 offspring in the mirror attack test
are shown in Fig. 6. The cumulative duration in close prox-
imity to the mirror of F1 offspring from zebrafish exposed to
100 ng/L TBT (100 ng/L-F1 juveniles, 28.76 ± 4.22 sec) was sig-
nificantly lower than that of F1 offspring from zebrafish in
the control group (control-F1 juveniles, 64.27 ± 9.63 sec). It
is worth noting that the time spent in the top of the tank
was significantly lower in 100 ng/L-F1 juveniles than that in
100 ng/L+FA-F1 juveniles (54.51 ± 37.32 sec). The frequency of
moving in close proximity to the mirror of F1 offspring from
zebrafish exposed to 100 ng/L TBT (100 ng/L-F1 juveniles, 21.60
± 2.70) was significantly lower than that of F1 offspring from
zebrafish in the control group (control-F1 juveniles, 33.80 ±
4.55) and that of F1 offspring that received FA supplementa-
tion (100 ng/L+FA-F1 juveniles, 34.86 ± 4.49). Parental early-
life exposure to 100 ng/L TBT increased anxiety and decreased
aggression in offspring. Moreover, FA supplementation pro-
tected offspring from the TBT-induced intergenerational
neurotoxicity.
2.7. FA rescued TBT-induced neurotransmitter
abnormalities in the F1 generation
To understand the relationship between TBT-induced in-
creases in anxiety and decreases in aggression as well as neu-
rotransmitter levels and the role of FA supplementation in the
F1 generation, we measured 5-HT and DA levels in whole ju-
veniles from the F1 generation. The 5-HT level of F1 offspring
from zebrafish exposed to 100 ng/L TBT (100 ng/L-F1 juve-
niles, 67.47 ± 7.07 pg/mL each fish) were significantly higher
than that of F1 offspring from zebrafish in the control group
(control-F1 juveniles, 53.16 ± 2.53 pg/mL each fish) and those
in the FA intervention group (100 ng/L+FA-F1 juveniles, 49.68
± 3.54 pg/mL each fish) (Fig. 7a).
The DA levels of F1 offspring from zebrafish exposed to
100 ng/L TBT (100 ng/L-F1 juveniles) were significantly lower
than that of F1 offspring from zebrafish in the control group
(control-F1 juveniles) and those in the FA intervention group
(100 ng/L+FA-F1 juveniles) (Fig. 7b). The results indicated that
FA reduced the TBT-induced elevation of 5-HT and DA levels
in the F1 generation to a certain extent.
2.8. Dnmt expression in the brain of juvenile F1 zebrafish
In the brain of juvenile F1 zebrafish, the expression of
dnmt1, dnmt3aa, dnmt3ab, dnmt3ba, and dnmt3bb.2 was de-
creased (by 0.84-, 0.49-, 0.49-, 0.53- and 0.37-fold, respec-
tively) (Fig. 8). Early-life TBT exposure decreased the expres-
sion levels of genes involved in the regulation of DNA methy-
lation (dnmt1, dnmt3aa, dnmt3ab, and dnmt3bb.2) in offspring
that were attenuated by FA, although the changes were not
significant.
 journal of environmental sciences 137 (2024) 615–625 621

Fig. 5 – Folic acid (FA) rescues zebrafish early–life TBT exposure induced increase anxiety behavior in their F1 offspring in a
novel tank diving test. (a) Locomotor traces; (b) Total distance traveled in the top; (c) Velocity traveled in the top; (d) The
number of entries to the top; (e) The time spent in the top zone. The data are expressed as the mean ± SEM. Different letters
above the error bars represent significant between groups (P < 0.05).

age to these cells can be passed on to future generations

3. Discussion (Leitch et al., 2013). It is crucial to study the effects of expo-
sure to environmental pollutants during this critical develop-
TBT, a marine coating widely used in ships, vessels and
mental period. Therefore, we focused on the effects of expo-
tankers, is released into the marine environment and is found
sure on later life and offspring in zebrafish at 1–21 dpf. In a
in harbours, coastal waters and rivers (Abreu et al., 2021;
recent study, prolonged co-exposure to 100 ng/L TBT and cad-
Bandara et al., 2021; Castro et al., 2021; Moreira et al., 2021;
mium in both parental and offspring zebrafish induced devel-
Vane et al., 2022). This pollutant is also used as a molluscicide
opmental neurotoxicity in three generations (Li and Li, 2020).
in agriculture and is released into soils. It may be enriched
Specifically, both F1 and F2 generations were continuously ex-
in organisms through the food chain. In this study, the neu-
posed to TBT and cadmium from the start as germ cells, which
rotoxicity of early-life TBT exposure in zebrafish and their F1
induced developmental neurotoxicity in the F1 and F2 gen-
offspring led to significantly enhanced anxiety and reduced
erations, including embryotoxicity and growth inhibition in
aggression, decreased gene expression levels of DNA methyl-
fish larvae, decreased DA and 5-HT levels and inhibited acetyl-
transferases in the brain and increased 5-HT levels. More-
cholinesterase (AChE) activity. Their study emphasized that
over, the intergenerational neurodevelopmental toxicity man-
the effects of co-exposure are complex and require environ-
ifested in the F1 generation was attenuated by supplementa-
mental risk assessments that consider the actual situation,
tion with a DNA methyl donor. This study can inform the pre-
which is different from our research purpose. Our study sub-
vention and treatment of intergenerational neurotoxicity due
stantially narrows the exposure window for inducing these in-
to organotin pollutants.
tergenerational neurobehavioural effects.
We investigated the neurodevelopmental and intergener-
In the present study, early-life exposure to TBT caused in-
ational effects of TBT exposure during the development of
tergenerational neurodevelopmental toxicity. These changes
PGCs. The occurrence and development of PGCs is a critical
were characterized by increased anxiety and fear and de-
period in early life. Exposure to environmental pollutants dur-
creased aggression in juveniles. In another study, acute ex-
ing PGCs development can have significant impacts on hu-
posure to TBT (100 and 500 ng/L for 28 days) in zebrafish re-
man health and fertility. This is because the PGCs are the pre-
sulted in neurobehavioural changes such as anxiety and fear
cursors to sperm and egg cells, and any mutations or dam-
in adult males (Tu et al., 2020). In other fish, such as the rare
622 journal of environmental sciences 137 (2024) 615–625

Fig. 6 – FA rescues zebrafish early–life TBT exposure induced reduced aggression and cognitive ability of their F1 offspring
in the mirror exposure test. (a) The heatmaps of cumulative locomotor traces, from dark blue to dark red, the colors at both
ends indicate the minimum and maximum values of the per-pixel frequency; (b) Distance moved close to the mirror; (c) The
frequency close to the mirror; (d, e) The cumulative duration close to the mirror. The data are expressed as the mean ± SEM.
Different letters above the error bars represent significant between groups (P < 0.05).

Fig. 7 – FA rescues zebrafish early–life TBT exposure induced increased neurotransmitter levels of their F1 offspring. (a)
Zebrafish 5–HT levels; (b) Zebrafish DA levels. The data are expressed as the mean ± SEM, P < 0.05 is considered significant
between groups.
 journal of environmental sciences 137 (2024) 615–625
Fig. 8 – FA rescues zebrafish early–life TBT exposure
induced reduced methyltransferase–related gene
expression levels in the brain of their F1 offspring. The data
are expressed as the mean ± SEM relative to β-actin.
gudgeon (Gobiocypris rarus), environmental concentrations of
1, 10 and 100 ng/L TBT were also found to have alter female
anxiety behaviour, manifested as fewer entries into the up-
per half of the tank (Zhang et al., 2016). These studies sug-
gest that there may be similar target organs and biological
mechanisms. In mammalian studies, DA plays a critical role
in the regulation of fear and anxiety. Dopaminergic neurons
in the ventral tegmental area are involved in the regulation
of both fear and anxiety in mammals (de la Mora et al., 2010).
Serotonergic projections from the dorsal raphe nucleus inner-
vate the basolateral amygdala, activating 5-HT2C receptors
to regulate inhibitory avoidance and evasion (Zangrossi and
Graeff, 2014). In the current study, early-life TBT exposure re-
sulted in increased 5-HT and DA levels in zebrafish later in
life. Increased DA levels have also been reported in previ-
ous studies of zebrafish and mice exposed to perfluorodode-
canoic acid and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(Guo et al., 2018; Lohr et al., 2014). The increased 5-HT levels
enhance activity in the amygdala, a brain region involved in
processing emotions including fear and anxiety, which could
lead to increased anxiety (Bocchio et al., 2016). This same in-
crease in 5-HT levels can also lead to the decreased activity
in the prefrontal cortex, a brain region involved in regulating
emotions and controlling behaviour, resulting in reduced in-
hibitory control over aggressive behaviour and decreased ag-
gression (Seo et al., 2008). The increased 5-HT levels can mod-
ulate the activity of other neurotransmitter systems, such as
the noradrenergic and dopaminergic systems, which are also
involved in regulating anxiety and aggression (Seo et al., 2008).
In our study, FA supplementation was found to signifi-
cantly rescue TBT-induced changes in offspring behaviour
(increased anxiety-like behaviour and reduced aggressive
behaviour) and cognition. The present study partially demon-
623
strated that the intergenerational neurotoxic effects induced
by TBT in the early life of parents are related to DNA methyla-
tion by replenishing an important component methyl donor.
Similarly, a previous study showed that FA protects zebrafish
heart development from PM2.5 -extractable organic matter by
rescuing DNA methylation levels (Jiang et al., 2019). In our
study, the expression levels of the brain methyltransferase-
related genes dnmt1, dnmt3aa, dnmt3ab, dnmt3ba and dnmt3bb.2
were decreased in both early-life TBT exposure parents and
their offspring. An earlier study showed that TBT induced
reduced DNA methylation levels in the liver tissue of S. mar-
moratus (Wang et al., 2009). Chamorro-Garcia et al. (2013) also
reported prenatal TBT-induced transgenerational inheritance
of metabolic phenotypes in mice. In a subsequent study, the
TBT-induced transgenerational thrifty phenotype in mice
was verified to be related to DNA methylation (Chamorro-
Garcia et al., 2017). DNMT3A and 3B are the major de novo
DNA methyltransferases (DNMTs) in the brain, which intro-
duce new methylation markers into unmethylated regions in
postmitotic neurons. FA is an exogenous folate supplement
commonly used during pregnancy to prevent neural tube
defects in the foetus. A previous study showed that 1 mmol/L
FA supplementation ameliorated cardiac and neurological
deficits in selenate-treated embryos (Ma et al., 2012). In
the present study, the FA-induced rescue effects may be
partly through upregulation of the expression levels of DNA
methyltransferase-related genes (dnmt1, dnmt3aa, dnmt3ab
and dnmt3bb.2). Our study can inform for the prevention and
treatment of TBT-induced intergenerational neurotoxicity.
This study has two major limitations that can be ad-
dressed in future studies. First, this study focused on the
intergenerational effects of early-life TBT exposure on neu-
rodevelopment. Although FA supplementation rescued some
of the neurobehavioural changes, it is still unclear which as-
sociated target-gene promoter sites had altered methylation
levels. Second, PGC development may be a critical period
for TBT exposure leading to intergenerational neurotoxicity.
More experimental techniques, such as a combination of in
vitro cell culture and in vivo transgenerational studies, should
be utilized in the future to better elucidate this biological
mechanism.
4. Conclusions
The current study found that early-life TBT exposure resulted
in neurobehavioural changes in juveniles, including increased
anxiety and fear and decreased aggression, with such changes
observed even in the next generation (F1). It is worth noting
that FA supplementation was protective against intergenera-
tional toxicity from early-life TBT exposure, which can inform
the prevention and treatment of intergenerational neurotoxi-
city due to organotin pollutants.
Declaration of competing interest
The authors declare that they have no known competing fi-
nancial interests or personal relationships that could have ap-
peared to influence the work reported in this article.
624 journal of environmental sciences 137 (2024) 615–625
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Nos. 32071301, 31971234 and 42177411)
and the Natural Science Foundation of Fujian Province, China
(No. 2020J01027).
Appendix A Supplementary data
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jes.2023.03.015.
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