Ecotoxicology and Environmental Safety 220 (2021) 112416

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Chlorinated disinfection byproducts of diazepam perturb cell metabolism

and induce behavioral toxicity in zebrafish larvae
Xiaoyong Huang a, b, c, Xiaole Zhao c, Xin Zhang a, Peng Wang b, Kui Zhu c, Bing Shao a, *
a
Beijing Center for Disease Prevention and Control, Beijing Research Center for Preventive Medicine, Beijing 100013, China
b
College of Science, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China
c
College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China

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

Edited by: Dr G Liu Numerous byproducts resulting from chlorinated disinfection are constantly being generated during water
treatment processes. The potential risks of these new emerging pollutions remain largely unknown. Here, we
Keywords: determined the risks of chlorinated disinfection byproducts of diazepam (DZP) in the cellular and zebrafish
Diazepam exposure experiments. The cytotoxicity of disinfection byproducts (MACB and MBCC) was greater than DZP in
Chlorinated disinfection byproducts
macrophage raw 264.7 cells at 10 mg/L. We further found that the effects of MBCC on the metabolism of glycine,
Cytotoxicity
serine, threonine and riboflavin were far greater than DZP by the targeted metabolomics methods. Moreover,
Behavioral toxicity
Zebrafish MBCC significantly decreased the peak amplitude of neuronal action potential in primary embryonic rat (Spragu-
Dawley SD) hippocampal neurons. We finally determined behavioral toxicity of DZP and byproducts in zebrafish
larvae. MBCC significantly decreased the maximal swim-activity and peak duration of zebrafish after 72 h
exposure. Altogether, these findings indicate the MBCC pose serious pressures on public health.

1. Introduction
Diazepam (DZP) is a classical benzodiazepine drug most frequently
prescribed to treat anxiety, insomnia, muscle spasms, seizures and
alcohol withdrawal (Calcaterra and Barrow, 2014). Due to its efficacy,
the usage of DZP has exceeded 2.3 billion doses in the United States in
1978 (Sternbach, 1979) and is still increasing year by year worldwide
(INCB, 2019). However，the high incidences of DZP misuse or overdose
(Yamamoto et al., 2019) give rise to serious environmental pollution
(Patel et al., 2019). Nowadays, DZP have been widely detected in
aquatic systems including surface water (lakes, rivers and streams),
groundwater, wastewater treatment plants (WWTP) influent and
effluent and drinking water with the highest concentration up to mi­
crograms per liter levels (Kosjek et al., 2012; Lei et al., 2021; Subedi
et al., 2017; Wu et al., 2015; Zhang et al., 2019). What’s more, many
elimination methods including photolysis, chlorination oxidation and
biodegradation cannot completely remove DZP from aqueous systems
(Patel et al., 2019) and the removal rate was below 50% (Wang et al.,
2017). These residues pose a potential risk for environmental health.
A number of studies have reported the environmental toxicity of DZP
in wildlife. For example, DZP at ranges from 0.25 µM to 20 µM (71 μg/L
to 5.68 mg/L) decreased zebrafish motor activity (Bruni et al., 2016;
McCarroll et al., 2019) (Zahid et al., 2018). The metabolite of DZP,
oxazepam, was reported to increase behavior of wild European perch
(Perca fluviatilis) and Atlantic salmon smolt at 1.8 or 1.9 µg/L (Brodin
et al., 2013; Hellström et al., 2016). DZP was also predicted to pose an
environmental risk (Risk Quotient = 2.0) to surface water according to
European Medicines Agency (EMA) Guideline (Cunha et al., 2019). Even
worse, some transformation products generated during water treatment
processes were more toxic than the precursor compound (Bedner and
MacCrehan, 2006b; Carpinteiro et al., 2017; X et al., 2019). For
example, the chlorination products of bisphenol F, acetaminophen or
azithromycin exhibited stronger toxicity than the original compounds
(Bedner and MacCrehan, 2006a; Guo et al., 2018; Zheng et al., 2016).
The increased toxicity may be due to the formation of ’supramolecular
ligands’, making it easier to combine with nuclear receptors (Delfosse
et al., 2015). In our previous study, we have determined the chlorinated
disinfection byproducts of DZP in drinking water (Zhang et al., 2019).
However, the risks of these new emerging chemicals remain virtually
unknown.
Here, we performed the limited toxicity estimations with the T.E.S.T.
tool and deduced that the toxicities of 2-Methylamino-5-chlorobenzo­
phenone (MACB) and methyl-(2-benzoyl-4-chlorophenyl)(methyl)
carbamate (MBCC) were greater than DZP (Table 1). The cytotoxicity of

* Corresponding author.
E-mail address: shaobingch@sina.com (B. Shao).

https://doi.org/10.1016/j.ecoenv.2021.112416
Received 20 March 2021; Received in revised form 3 June 2021; Accepted 8 June 2021
Available online 11 June 2021
0147-6513/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Huang et al. Ecotoxicology and Environmental Safety 220 (2021) 112416

MACB and MBCC was associated with one-carbon metabolism. We 2.5. Metabolites measurement
further determined MBCC significantly disturbed action potential
propagation in hippocampal neurons. Finally, we confirmed the After 24 h incubation with DZP, MACB or MBCC at 1 µg/L or 1 mg/L,
behavioral toxicity of MBCC in zebrafish larvae by the established the content of target nutrients in cell cultural medium was were
behavioral profiling assay. These findings indicate that the chlorinated extracted with ACN. Each group contained four independent biological
disinfection byproducts of DZP pose serious pressures on public health. replicates. Samples were centrifuged at 12000 rpm for 15 min. Super­
natants were collected and analyzed by a liquid chromatograph mass
2. Materials and methods spectrometer (LC-MS 8045, Shimadzu Corporation) through a cell-
cultural-profiling program according to previous study (Huang et al.,
2.1. Chemical reagents 2021). The targeted metabolomics includes sugars (5), nucleic acid
associated compounds (18), amino acid and derivatives (38), vitamins
DZP (CAS: 439–14–5, purity > 99%) was purchased from National (17) and others (16). Chromatographic separations were carried out on a
Institutes for Food and Drug Control (Beijing, China). Analytical stan­ Discovery® HS F5 HPLC column (Sigma, 567503-U) at 40 ◦ C. The mo­
dards of MACB (purity > 95%) and MBCC (purity > 99%) were isolated bile phase consisted of 0.1% FA in water and ACN. The flow rate was set
and synthesized in the laboratory. The purities were calculated by HPLC at 0.35 mL/min, the injection volume was 1 µL. Information for full
chromatogram (Waters 2695, Milford, USA). Stock solution of each details of the characterization of related compounds was listed in
chemical (10 mg/mL) was prepared using Dimethyl sulfoxide (DMSO, Table 2. The height intensity of 2-isopropylmalic acid was determined as
Sigma-Aldrich, D4540). Acetonitrile (ACN, Thermo Scientific, A955–4) the internal standard.
and formic acid (FA, Sigma-Aldrich, 56302) were LC-MS grade.
2.6. Electrophysiology
2.2. Zebrafish cultivation and embryo collection
Primary embryonic rat (Spragu-Dawley SD) hippocampal neurons
were obtained through enzymatic dissociation. Cells were re-suspended
Adult zebrafish (wild type, AB strain) were purchased from China
in plating medium consisted of neurobasal medium (Gibco, 21103049)
Zebrafish Resource Center (Wuhan, Hubei, China). Zebrafish were
with 2% w/v B-27 supplement (Gibco,17504044), 0.5 mM glutamine
maintained at 28 ± 0.5 ◦ C in a five-shelf standalone recirculating system
(Sigma, 59202 C), and 1% penicilline/streptomycin (Hyclone,sv30010).
(ESEN-AW-S1, Beijing, China) under 14 h light/10 h dark cycles. The
Patch pipettes were prepared from borosilicate glass (Sutter,
fertilized eggs were collected according to previously described method
BF150–86–10) with a Sutter P97 pipette puller. The tight GΩ-seal was
(Huang et al., 2019). Naturally fertilized eggs were staged by hours post
obtained in microelectrode system (Sutter, MP285) under inverted mi­
fertilization (hpf). All fish were treated with regard for the alleviation of
croscope (Olympus IX71). Rupture the membrane with negative pres­
suffering in accordance with the approved Institutional Animal Care and
sure to obtain the whole cell configuration. Cells were continuously
Use Committee protocols of China Agriculture University.
perfused with extracellular solution (140 mM NaCl, 4 mM KCl, 1 mM
MgCl2, 2 mM CaCl2, 5 mM D-Glucose monohydrate, 10 mM HEPES; with
2.3. Cell lines pH adjusted with NaOH to pH=7.4) at room temperature. Pipettes were
filled with intracellular solution (5 mM NaCl, 140 mM K-Gluconate, 0.1
Macrophage RAW 264.7 cell and microglial BV-2 cell were main­ mM CaCl2, 1 mM MgCl2, 2 mM Mg-ATP, 1 mM EGTA, 10 mM HEPES;
tained in our laboratory. The cardiomyocyte cell line H9C2 was pur­ with pH adjusted with KOH to pH = 7.2). When the background current
chased from China Infrastructure of Cell Line Resource. BV-2 cells were was stabilized, the action potentials were recorded in current-clamp
cultured in RPMI 1640 Medium (Gibco, 61870036) with 10% fetal model on an EPC-10 amplifier (EPC-10 USB; Heka) using PatchMaster
bovine serum (FBS, CellMax, SA212.02) and penicillin/streptomycin software (Heka). Depolarizing currents of − 60–250 pA (500 ms
(pen/strep, ThermoFisher Scientific, 15140122). Both RAW 264.7 cells
and H9C2 cells were cultured in DMEM (Gibco, C11995500CP) with Table 2
10% FBS and 1% pen/strep. All the cells were incubated in 5% CO2 The information of related compounds in LC-MS method.
humidified atmosphere at 37 ◦ C.
Compound Name Polarity Target Ion Confirmation CE
Ion
2.4. Cell viability assay 2-Isopropylmalic MRM(-) 175.15 > 175.15 > 17.0 16.0
acid 115.05 113.05
RAW 264.7 cells were seeded into 96-well plates at a density of 5 × Serine MRM 105.90 > -12.0
(+) 60.10
103 cells per well and incubated for six hours in 100 µL DMEM with 10% Glycine MRM 75.90 > -11.0
FBS. Before exposure, the culture medium was replaced with 100 µL (+) 30.15
fresh DMEM with 1% FBS. RAW 264.7 cells were then incubated with Threonine MRM 120.10 > 120.10 > 56.05 -13.0–17.0
DZP, MACB or MBCC solutions at concentrations ranging from 100 ng/L (+) 74.15
Folic acid MRM 442.00 > 442.00 > -15.0–41.0
to 10 mg/L with four replicates for 24 h. The cell viability assay was
(+) 295.15 176.05
examined by CCK-8 assay (Beyotime, China, C0042) according to the Riboflavin MRM 377.00 > 377.00 > -23.0–40.0
manufacturers instructions. Optical densities (OD) were determined at (+) 243.05 172.00
450 nm by a multiskan-FC microplate reader (Thermo). Cell viability
MRM: multiple reaction monitoring.
was normalized to that treated with DMSO.

Table 1
The predicted toxicity of compounds by T.E.S.T.
Chemicals Fathead minnow LC50 (96 hr) mg/L Daphnia magna LC50 (48 hr) mg/L Oral rat LD50 mg/kg Developmental Toxicity value Bioaccumulation factor value

DZP 0.48 3.84 1772.09 0.83 26.64

MACB 2.72 1.28 2712.57 0.68 73.43
MBCC 0.54 3.09 681.34 1.07 89.56

T.E.S.T: The Toxicity Estimation Software Tool.

2
X. Huang et al. Ecotoxicology and Environmental Safety 220 (2021) 112416

duration) were delivered in increments of 10 pA until an AP was evoked. independent variable or two independent variables were evaluated by
For quality control, only patch clamps with series resistance below 20 one-way or two-way analysis of variance (ANOVA) test respectively
MΩ and a seal resistance above 1 GΩ were accepted for further analysis. followed with Tukey’s multiple comparisons test.

2.7. Automated rest/wake behavioral assay 3. Results

For the sedative effect of DZP, we prolonged the dark duration time
after lights out according to the described rest/wake behavioral assay
(Rihel et al., 2010; Xue et al., 2013) to compare the interference effect
between DZP and MACB or MBCC. Briefly, the hatched zebrafish larvae
at 48 hpf was collected and randomly incubated in DZP, MACB or MBCC
solutions at 1 mg/L or 100 ng/L in 60 mm glass dish at 28 ◦ C for 72 h.
Individual zebrafish larvae were placed into a single well of a clear
flat-bottomed 96-well plate with 300-µL medium at 120 hpf. All ex­
periments contained at least 8 zebrafish larvae per group. The 96-well
plates were then placed inside the DanioVision® tracking system (Nol­
dus, EthoVision XT 13, Holland), which included a high-resolution
digital video camera. The larvae acclimated to the system for 30 min
before the start of data acquisition. The light intensity of the observation
chamber was set to 4000 lux. Spontaneous free-swimming trajectories
were recorded using the EthoVision XT-13 software, including the speed
of each minute, peak value (the extent of the burst of activity) and peak
duration (the duration time of peak bout), the timing of the first rest
bout after a light transition (rest latency).
2.8. Statistical analysis
All data was represented as means ± SEM and performed by
Graphpad Prism 7. Differences between two groups were evaluated by
two-tailed, unpaired Student’s t-test. More than three groups with one
3.1. MBCC reduced cell viability
To determine the effects of DZP and chlorine disinfection byproducts
of DZP on cell viability, we incubated cells with chemicals at various
concentrations for 24 h. The results showed that DZP did not affect cell
viability up to 10 mg/L (Fig. 1A). However, the viability of the cells
decreased dramatically with the increase of the concentrations of MACB
and MBCC. The viability values were higher than 80% for cells treated
with MACB and MBCC at 1 mg/L. However, MACB and MBCC at 10 mg/
L reduced the cell viability to 30.7% and 39.9%, respectively. Thus,
chlorine disinfection byproducts of DZP were more cytotoxic than DZP.
3.2. MBCC perturbed cell metabolism
For further evaluation of the effects of MACB and MBCC on cellular
metabolic activity, we determined the changes in the composition of cell
culture medium after 24 h incubation at 1 mg/L. The contents of serine
in MBCC treated groups were significantly (P < 0.001) higher than
DMSO treated group (Fig. 1B). Moreover, MBCC significantly
(P < 0.001) increased the level of glycine. Interestingly, we observed
similar results in H9C2 cells exposure experiments (Fig. 1C). The effects
of MBCC on serine and glycine metabolism were stronger than MACB.
Additionally, we also confirmed that MBCC interfered with the meta­
bolism in BV-2 cell, especially for the metabolism of threonine and

Fig. 1. MBCC reduced cell viability and perturbed cell metabolism. A, The dose dependent inhibitory effect of DZP, MACB and MBCC on cellular viability. B, C, D,
Metabolic changes in exposure of macrophage RAW 264.7 cells, cardiomyocyte H9C2 cells and brain microglial BV-2 cells to DZP, MACB and MBCC at 1 mg/L for
24 h. Data were analyzed by one-way analysis of variance (ANOVA) test within each target. All the data are expressed as mean ± SEM, (n = 4). * , P < 0.05; and * *,
P < 0.01; * ** , P < 0.001; and * ** *, P < 0.0001; treated groups versus DMSO group.

3
X. Huang et al.
riboflavin (Fig. 1D). These results suggested that MBCC was prone to
induce metabolic dysfunction.
3.3. The time-dependent effects of MBCC on metabolism
For the interconversion among serine, glycine and threonine
occurred in metabolic processes (Fig. 2A), we next determined the
changes of nutrients in cell culture medium at different time points. Cells
were incubated with MACB or MBCC medium at 1 µg/L. The content of
serine decreased over time and no differences were observed among
treated groups (Fig. 2B). The levels of glycine reached to the highest
values after 36 h incubation. Both DZP and byproducts promoted the
biosynthesis of glycine (Fig. 2C). The level of threonine reached to the
lowest value after 36 h incubation (Fig. 2D). MBCC inhibited the uptake
of threonine in comparison with control groups. For the interconversion
between serine and glycine occur through folate metabolism, we further
determined the changes of folic acid and riboflavin in cell culture me­
dium. The level of folic acid increased over time in control groups
(Fig. 2E). However, DZP and byproducts inhibited the biosynthesis of
folic acid. Notably, MBCC, promoted the uptake of riboflavin during the
Fig. 2. The time dependent effects of MBCC on metabolism. A, Diagram depicting nutrients interconversion in one-carbon metabolism. The level of serine (B),
glycine (C), threonine (D), folic acid (E) and riboflavin (F) in cell cultural medium over time with DZP and byproducts at 1 µg/L. All the data are expressed as mean
± SEM, (n = 4).
4
Ecotoxicology and Environmental Safety 220 (2021) 112416
early 36 h incubation (Fig. 2F). These results suggested that MBCC
deeply interfere with the finely controlled metabolic regulation.
3.4. MBCC decreased peak amplitude of action potentials
For metabolic energy was required for action potentials in neurons,
we determined the effects of MACB and MBCC on action potentials in
current-clamp model after a single dose at 10 mg/L. From the re­
cordings, we derived the action potential (AP), rheobase (the minimum
current required to elicit action potential), threshold, peak amplitude,
half-width and number of AP. DZP did not alter electrophysiological
characteristics including AP, rheobase, threshold (data not shown), peak
amplitude (Fig. 3A) and number of AP during electrical stimulation
(Fig. 3B). Although peak amplitude remained unchanged in MACB
treated group (Fig. 3C), the number of AP decreased at high current
stimulation compared with control groups (Fig. 3D). However, MBCC
significantly (P < 0.05) decreased the peak amplitude (Fig. 3E) and the
number of AP in the high-current region (Fig. 3F). Altogether, these
results suggested that MBCC disturbed neurotransmission that might
induce behavioral abnormalities.
X. Huang et al.
Fig. 3. MBCC decreased peak amplitude of action potentials. The effect of DZP (A and B), MACB (C and D) and MBCC (E and F) on the peak amplitude and number of
action potentials. All the data are expressed as mean ± SEM, (n = 3). The statistical significance of the differences was determined by the unpaired Student’s t-test.
3.5. MBCC decreased peak duration of swimming burst activity in
zebrafish
We then established the automated rest/wake behavioral assay in
zebrafish (Fig. 4A) to determine the actual behavioral toxicity of DZP,
MACB and MBCC. Under white light illumination, zebrafish exhibited no
or very little swim activity. The transition from light to dark can result in
a burst of swim activity in zebrafish. Normally, zebrafish spend about
13 min with the burst of activity after the transition. The time–velocity
curve forms a behavioral fingerprint for each compound. To avoid the
interference of chorions, we collected the hatched zebrafish larvae at 48
hpf and exposed to each compound for 72 h. There were no deaths
observed during the exposure. DMSO (0.01%) treated zebrafish
exhibited similar phenotype with water group (Fig. 4B). MACB and
MBCC at 1 mg/L induced similar behavioral phenotypes in zebrafish
with DZP (Fig. 4B), decreasing peak duration from 13 min to less than
5
Ecotoxicology and Environmental Safety 220 (2021) 112416
9 min after a transition (Fig. 4C). Notably, the reduction level was more
prominent for MBCC treatment than for DZP treatment. Meanwhile,
MACB and MBCC significantly (P < 0.001) decreased the velocity of
zebrafish at the corresponding time points (Fig. 4D). MACB and MBCC
had not effect on the peak duration at low concentration (100 ng/L)
(Fig. 5A and B). However, MBCC still significantly (P < 0.05) decreased
the velocity of zebrafish at the burst of activity at 100 ng/L (Fig. 5C).
These results suggested that the behavioral toxicity of MBCC was greater
than DZP.
4. Discussion
Disinfection byproducts in drinking water has become an emerging
concern. In this study, we compared the cytotoxicity and behavioral
toxicity of DZP with its disinfection byproducts. Byproducts have pro­
found effects on cell viability and metabolism. Both DZP and byproducts
X. Huang et al.
decrease locomotor activity of zebrafish at high concentration. The ef­
fects of byproduct MBCC were more substantial than DZP at low con­
centration. These results suggested that disinfection byproducts of DZP
pose serious risks for wildlife.
DZP is a sedative, anxiolytic and anticonvulsant muscle relaxant.
This study found that DZP decreased locomotor activity, which in
accordance with previous study demonstrating the reduced activity
induced by DZP at 0.63 μM (179 μg/L) in zebrafish behavioral profiling
(Bruni et al., 2016). MACB and MBCC exhibited similar behavioral
phenotypes at the high concentration might be associated with the
target of DZP. In addition, MBCC at the low concentration still could
decrease the extent of the burst of activity in zebrafish. This may due to
MBCC have the stronger hydrophilic property and the higher bio­
accumulation factor than DZP. Moreover, the increased anxiety in
response to the current coronavirus disease 2019 (COVID-19) pandemic
(He et al., 2021) would further increase the use of DZP. It was reported
that the usages of DZP were 121 mg/d/1000 people in Guangzhou,
China (Lei et al., 2021). Unfortunately, DZP can enhance intracellular
acidosis of macrophages (Sanders et al., 2013) and induce immuno­
toxicity (Luebke et al., 2006) increasing the incidence of pneumonia by
6
Ecotoxicology and Environmental Safety 220 (2021) 112416
Fig. 4. MBCC decreased the peak duration of zebrafish. A,
Automated rest/wake behavioral assay in zebrafish and the
chemical structures of DZP, MACB and MBCC. The bio-
activation of chemicals by N-Dealkylation was assessed
computationally according the structural formulas (Dang
et al., 2018). B, C, MBCC decreased Peak duration DZP and
byproducts reduced locomotor activity of zebrafish at high
concentration. A, The behavioral fingerprints of each
compound after the transition from light to dark. deter­
mined in zebrafish individuals by the lasting time for the
speed over 0.5 mm/s (n > = 8 larvae/group). Data were
analyzed by one-way analysis of variance (ANOVA) test (F
(4, 46) = 7.75, P < 0.0001). C, The swimming speed of
zebrafish at the corresponding time (n > = 8 lar­
vae/group). Data were analyzed by two-way analysis of
variance (ANOVA) test (Interaction, F (12, 183) = 2.871,
P = 0.0012. Row factor, F (3, 183) = 37.99, P < 0.0001.
Column factor, F (4, 183) = 10.43, P < 0.0001). All the
data are expressed as mean ± SEM. * , P < 0.05; and * *,
P < 0.01; * ** , P < 0.001; and * ** *, P < 0.0001; treated
groups versus DMSO group.
49% and mortality by 27% in the community population (Obiora et al.,
2013). If measures were not taken to decrease the residues of DZP and
disinfection byproducts of DZP, the burden of disease would grow in the
coming years.
Metabolic modulation is associated with changes in host behavior.
The altered metabolome associated with behavior can be derived from
astrocytes (Chao et al., 2019), microglia (García-Cáceres et al., 2019),
peripheral CD4+ T cells (Fan et al., 2019) and even gut microbiota
(Hsiao et al., 2013; Lynch and Hsiao, 2019). In this study, we deter­
mined the metabolite changes in different cell lineages after incubation
with DZP or disinfection byproducts of DZP. The increased level of
glycine, which is an important inhibitory neurotransmitter (Harvey and
Yee, 2013), probably the main reason for the reduced activity in DZP
and byproducts treated zebrafish. Serine can be metabolized to glycine
by SHMT2 (serine hydroxymethyltransferase) supporting folate meta­
bolism in mitochondrial one-carbon cycle (Kory et al., 2018). This may
explain the reason that DZP and byproducts promoted the biosynthesis
of folic acid. Serine synthesis is also constrained in cellular redox state
(Diehl et al., 2019) due to the behavioral changes in developing zebra­
fish (Paganotto Leandro et al., 2020) and attenuates temporal lobe
X. Huang et al.
epilepsy (Beesley et al., 2020). In addition, threonine can also be
catabolized to glycine by threonine dehydrogenase in mitochondrial
further facilitating one-carbon metabolism (Wang et al., 2009). The high
level of threonine in microglial BV-2 cell cultural medium might due to
the mitochondrial dysfunction induced by DZP and byproducts. Notably,
MBCC promoted the uptake of riboflavin (7,8-dimethyl-10-ribityl-i­
soalloxazine; vitamin B2) at low concentration might through enhancing
riboflavin transporter, which associated with mitochondrial electron
transport chain (ETC) (Carreau et al., 2020; Manole et al., 2017).
Therefore, the cytotoxicity of MABC or MBCC might induce by mito­
chondrial metabolism dysfunction. However, the mechanisms through
which metabolic processes need to be further studied.
5. Conclusions
The chlorination disinfection byproducts in drinking water pose
serious risks for human health. In this study, we determined the cyto­
toxicity of DZP and chlorinated disinfection byproducts of DZP.
Byproducts have profound effects on cellular metabolism and exhibit
higher behavioral toxicity than DZP. Chemicals with the variation in
structures increase environmental health risks. Therefore, future studies
should explore the intervention strategies to restrict the usage and
discharge of such psychoactive drugs and combined methods including
filtration or bio-adsorption to remove the parent contaminants and by-
products.
CRediT authorship contribution statement
B.S. P.W. and K.Z. conceived the project. X.H., X.Z. X.Z. performed
the experiments. X.H. did data analysis and wrote the manuscript. All
authors read and approved the manuscript. The authors have declared
that no competing interests exist.
Declaration of Competing Interest
The authors declare that they have no known competing financial
7
Ecotoxicology and Environmental Safety 220 (2021) 112416
Fig. 5. Chlorinated disinfection byproducts decrease the
extent of the burst of activity in zebrafish at low concen­
tration. A, The behavioral fingerprints of each compound
after the transition from light to dark. B, Peak duration
determined in zebrafish individuals by the lasting time for
the speed over 0.05 mm/s (n > = 18 larvae/group). Data
were analyzed by one-way analysis of variance (ANOVA)
test (F (4, 94) = 1.096, P = 0.3634). C, The swimming
speed of zebrafish at the corresponding time (n > = 18
larvae/group). The speeds at each minute were analyzed by
two-way analysis of variance (ANOVA) test (Interaction, F
(12, 375) = 1.977; P = 0.0252. Row factor, F (3, 375)
= 135.7, P < 0.0001. Column factor, F (4, 375) = 6.788,
P < 0.0001). All the data are expressed as mean ± SEM. * ,
P < 0.05; treated groups versus DMSO group.
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This study was supported by National Natural Science Foundation of
China (21677019), Capital Foundation of Medicine Research and
Development (2018-1-302) and Beijing Postdoctoral Research Founda­
tion (2020-ZZ-114).
References
Bedner, M., MacCrehan, W.A., 2006a. Transformation of acetaminophen by chlorination
produces the toxicants 1,4-benzoquinone and N-acetyl-p-benzoquinone imine.
Environ. Sci. Technol. 40, 516–522.
Bedner, M., MacCrehan, W.A., 2006b. Transformation of acetaminophen by chlorination
produces the toxicants 1,4-benzoquinone and N-acetyl-p-benzoquinone imine.
Environ. Sci. Technol. 40, 516–522.
Beesley, S., Sullenberger, T., Crotty, K., Ailani, R., D’Orio, C., Evans, K., Ogunkunle, E.O.,
Roper, M.G., Kumar, S.S., 2020. D-serine mitigates cell loss associated with temporal
lobe epilepsy. Nat. Commun. 11, 4966.
Brodin, T., Fick, J., Jonsson, M., Klaminder, J., 2013. Dilute concentrations of a
psychiatric drug alter behavior of fish from natural populations. Science 339,
814–815.
Bruni, G., Rennekamp, A.J., Velenich, A., McCarroll, M., Gendelev, L., Fertsch, E.,
Taylor, J., Lakhani, P., Lensen, D., Evron, T., Lorello, P.J., Huang, X.P.,
Kolczewski, S., Carey, G., Caldarone, B.J., Prinssen, E., Roth, B.L., Keiser, M.J.,
Peterson, R.T., Kokel, D., 2016. Zebrafish behavioral profiling identifies multitarget
antipsychotic-like compounds. Nat. Chem. Biol. 12, 559–566.
Calcaterra, N.E., Barrow, J.C., 2014. Classics in chemical neuroscience: diazepam
(valium). ACS Chem. Neurosci. 5, 253–260.
Carpinteiro, I., Rodil, R., Quintana, J.B., Cela, R., 2017. Reaction of diazepam and related
benzodiazepines with chlorine. Kinetics, transformation products and in-silico
toxicological assessment. Water Res. 120, 280–289.
Carreau, C., Benoit, C., Ahle, G., Cauquil, C., Roubertie, A., Lenglet, T., Cosgrove, J.,
Meunier, I., Veauville-Merllié, A., Acquaviva-Bourdain, C., Nadjar, Y., 2020. Late-
onset riboflavin transporter deficiency: a treatable mimic of various motor
neuropathy aetiologies. J. Neurol. Neurosurg. Psychiatry.
Chao, C.C., Gutiérrez-Vázquez, C., Rothhammer, V., Mayo, L., Wheeler, M.A., Tjon, E.C.,
Zandee, S.E.J., Blain, M., de Lima, K.A., Takenaka, M.C., Avila-Pacheco, J.,
Hewson, P., Liu, L., Sanmarco, L.M., Borucki, D.M., Lipof, G.Z., Trauger, S.A.,
Clish, C.B., Antel, J.P., Prat, A., Quintana, F.J., 2019. Metabolic control of astrocyte
pathogenic activity via cPLA2-MAVS. Cell 179, 1483–1498.e1422.
X. Huang et al.
Cunha, D.L., Mendes, M.P., Marques, M., 2019. Environmental risk assessment of
psychoactive drugs in the aquatic environment. Environ. Sci. Pollut. Res. Int. 26,
78–90.
Dang, N.L., Hughes, T.B., Miller, G.P., Swamidass, S.J., 2018. Computationally assessing
the bioactivation of drugs by N-dealkylation. Chem. Res Toxicol. 31, 68–80.
Delfosse, V., Dendele, B., Huet, T., Grimaldi, M., Boulahtouf, A., Gerbal-Chaloin, S.,
Beucher, B., Roecklin, D., Muller, C., Rahmani, R., Cavaillès, V., Daujat-
Chavanieu, M., Vivat, V., Pascussi, J.M., Balaguer, P., Bourguet, W., 2015.
Synergistic activation of human pregnane X receptor by binary cocktails of
pharmaceutical and environmental compounds. Nat. Commun. 6, 8089.
Diehl, F.F., Lewis, C.A., Fiske, B.P., Vander Heiden, M.G., 2019. Cellular redox state
constrains serine synthesis and nucleotide production to impact cell proliferation.
Nat. Metab. 1, 861–867.
Fan, K.Q., Li, Y.Y., Wang, H.L., Mao, X.T., Guo, J.X., Wang, F., Huang, L.J., Li, Y.N.,
Ma, X.Y., Gao, Z.J., Chen, W., Qian, D.D., Xue, W.J., Cao, Q., Zhang, L., Shen, L.,
Zhang, L., Tong, C., Zhong, J.Y., Lu, W., Lu, L., Ren, K.M., Zhong, G., Wang, Y.,
Tang, M., Feng, X.H., Chai, R.J., Jin, J., 2019. Stress-induced metabolic disorder in
peripheral CD4(+) T cells leads to anxiety-like behavior. Cell 179, 864–879.e819.
García-Cáceres, C., Balland, E., Prevot, V., Luquet, S., Woods, S.C., Koch, M., Horvath, T.
L., Yi, C.X., Chowen, J.A., Verkhratsky, A., Araque, A., Bechmann, I., Tschöp, M.H.,
2019. Role of astrocytes, microglia, and tanycytes in brain control of systemic
metabolism. Nat. Neurosci. 22, 7–14.
Guo, Q., Du, Z., Shao, B., 2018. Simulation and experimental study on the mechanism of
the chlorination of azithromycin. J. Hazard. Mater. 359, 31–39.
Harvey, R.J., Yee, B.K., 2013. Glycine transporters as novel therapeutic targets in
schizophrenia, alcohol dependence and pain. Nat. Rev. Drug Discov. 12, 866–885.
He, L., Wei, D., Yang, F., Zhang, J., Cheng, W., Feng, J., Yang, W., Zhuang, K., Chen, Q.,
Ren, Z., Li, Y., Wang, X., Mao, Y., Chen, Z., Liao, M., Cui, H., Li, C., He, Q., Lei, X.,
Feng, T., Chen, H., Xie, P., Rolls, E.T., Su, L., Li, L., Qiu, J., 2021. Functional
Connectome Prediction of Anxiety Related to the COVID-19 Pandemic. Am. J.
Psychiatry, 202020070979 appiajp202020070979.
Hellström, G., Klaminder, J., Finn, F., Persson, L., Alanärä, A., Jonsson, M., Fick, J.,
Brodin, T., 2016. GABAergic anxiolytic drug in water increases migration behaviour
in salmon. Nat. Commun. 7, 13460.
Hsiao, E.Y., McBride, S.W., Hsien, S., Sharon, G., Hyde, E.R., McCue, T., Codelli, J.A.,
Chow, J., Reisman, S.E., Petrosino, J.F., Patterson, P.H., Mazmanian, S.K., 2013.
Microbiota modulate behavioral and physiological abnormalities associated with
neurodevelopmental disorders. Cell 155, 1451–1463.
Huang, X., Yan, Z., Zhu, K., Ding, S., 2019. Ca(2+) protect zebrafish embryos from water
acidification. Ecotoxicol. Environ. Saf. 172, 65–71.
INCB, 2019. Technical Report on Psychotropic Substances, in: Board, I.N.C. (Ed.), htt
ps://www.incb.org/.
Kory, N., Wyant, G.A., Prakash, G., Uit de Bos, J., Bottanelli, F., Pacold, M.E., Chan, S.H.,
Lewis, C.A., Wang, T., Keys, H.R., Guo, Y.E., Sabatini, D.M., 2018. SFXN1 is a
mitochondrial serine transporter required for one-carbon metabolism. In: Science,
362.
Kosjek, T., Perko, S., Zupanc, M., Zanoski Hren, M., Landeka Dragicevic, T., Zigon, D.,
Kompare, B., Heath, E., 2012. Environmental occurrence, fate and transformation of
benzodiazepines in water treatment. In: Water Res., 46, pp. 355–368.
Lei, H.J., Yang, B., Ye, P., Yang, Y.Y., Zhao, J.L., Liu, Y.S., Xie, L., Ying, G.G., 2021.
Occurrence, fate and mass loading of benzodiazepines and their transformation
products in eleven wastewater treatment plants in Guangdong province, China. Sci.
Total Environ. 755, 142648.
Luebke, R.W., Chen, D.H., Dietert, R., Yang, Y., King, M., Luster, M.I.,
Immunotoxicology, W., 2006. The comparative immunotoxicity of five selected
compounds following developmental or adult exposure. J. Toxicol. Environ. Health B
Crit. Rev. 9, 1–26.
Lynch, J.B., Hsiao, E.Y., 2019. Microbiomes as sources of emergent host phenotypes.
Science 365, 1405–1409.
Manole, A., Jaunmuktane, Z., Hargreaves, I., Ludtmann, M.H.R., Salpietro, V., Bello, O.
D., Pope, S., Pandraud, A., Horga, A., Scalco, R.S., Li, A., Ashokkumar, B.,
Lourenço, C.M., Heales, S., Horvath, R., Chinnery, P.F., Toro, C., Singleton, A.B.,
Ecotoxicology and Environmental Safety 220 (2021) 112416
Jacques, T.S., Abramov, A.Y., Muntoni, F., Hanna, M.G., Reilly, M.M., Revesz, T.,
Kullmann, D.M., Jepson, J.E.C., Houlden, H., 2017. Clinical, pathological and
functional characterization of riboflavin-responsive neuropathy. Brain J. Neurol.
2820–2837.
McCarroll, M.N., Gendelev, L., Kinser, R., Taylor, J., Bruni, G., Myers-Turnbull, D.,
Helsell, C., Carbajal, A., Rinaldi, C., Kang, H.J., Gong, J.H., Sello, J.K., Tomita, S.,
Peterson, R.T., Keiser, M.J., Kokel, D., 2019. Zebrafish behavioural profiling
identifies GABA and serotonin receptor ligands related to sedation and paradoxical
excitation. Nat. Commun. 10, 4078.
Obiora, E., Hubbard, R., Sanders, R.D., Myles, P.R., 2013. The impact of benzodiazepines
on occurrence of pneumonia and mortality from pneumonia: a nested case-control
and survival analysis in a population-based cohort. Thorax 68, 163–170.
Paganotto Leandro, L., Siqueira de Mello, R., da Costa-Silva, D.G., Medina Nunes, M.E.,
Rubin Lopes, A., Kemmerich Martins, I., Posser, T., Franco, J.L., 2021. Behavioral
changes occur earlier than redox alterations in developing zebrafish exposed to
Mancozeb. Environ. Pollut. 268, 115783.
Patel, M., Kumar, R., Kishor, K., Mlsna, T., Pittman Jr., C.U., Mohan, D., 2019.
Pharmaceuticals of emerging concern in aquatic systems: chemistry, occurrence,
effects, and removal methods. Chem. Rev. 119, 3510–3673.
Rihel, J., Prober, D.A., Arvanites, A., Lam, K., Zimmerman, S., Jang, S., Haggarty, S.J.,
Kokel, D., Rubin, L.L., Peterson, R.T., Schier, A.F., 2010. Zebrafish behavioral
profiling links drugs to biological targets and rest/wake regulation. Science 327,
348–351.
Sanders, R.D., Godlee, A., Fujimori, T., Goulding, J., Xin, G., Salek-Ardakani, S.,
Snelgrove, R.J., Ma, D., Maze, M., Hussell, T., 2013. Benzodiazepine augmented
γ-amino-butyric acid signaling increases mortality from pneumonia in mice. Crit.
Care Med. 41, 1627–1636.
Sternbach, L.H., 1979. The benzodiazepine story. J. Med. Chem. 22, 1–7.
Subedi, B., Balakrishna, K., Joshua, D.I., Kannan, K., 2017. Mass loading and removal of
pharmaceuticals and personal care products including psychoactives,
antihypertensives, and antibiotics in two sewage treatment plants in southern India.
Chemosphere 167, 429–437.
Wang, C., Hou, L., Li, J., Xu, Z., Gao, T., Yang, J., Zhang, H., Li, X., Du, P., 2017.
Occurrence of diazepam and its metabolites in wastewater and surface waters in
Beijing. Environ. Sci. Pollut. Res. Int. 24, 15379–15389.
Wang, J., Alexander, P., Wu, L., Hammer, R., Cleaver, O., McKnight, S.L., 2009.
Dependence of mouse embryonic stem cells on threonine catabolism. Science 325,
435–439.
Wu, M., Xiang, J., Que, C., Chen, F., Xu, G., 2015. Occurrence and fate of psychiatric
pharmaceuticals in the urban water system of Shanghai, China. Chemosphere 138,
486–493.
X, L., J, Z., Q, W., D, W., H, X., J, M., W, Q., T, H., 2019. Sonolytic degradation of
bisphenol S: effect of dissolved oxygen and peroxydisulfate, oxidation products and
acute toxicity. Water Res. 165, 114969.
Xue, J.Y., Li, X., Sun, M.Z., Wang, Y.P., Wu, M., Zhang, C.Y., Wang, Y.N., Liu, B.,
Zhang, Y.S., Zhao, X., Feng, X.Z., 2013. An assessment of the impact of SiO2
nanoparticles of different sizes on the rest/wake behavior and the developmental
profile of zebrafish larvae. Small 9, 3161–3168.
Yamamoto, T., Dargan, P.I., Dines, A., Yates, C., Heyerdahl, F., Hovda, K.E., Giraudon, I.,
Sedefov, R., Wood, D.M., 2019. Concurrent use of benzodiazepine by heroin users-
what are the prevalence and the risks associated with this pattern of use? J. Med.
Toxicol. 15, 4–11.
Zahid, H., Tsang, B., Ahmed, H., Lee, R.C.Y., Tran, S., Gerlai, R., 2018. Diazepam fails to
alter anxiety-like responses but affects motor function in a white-black test paradigm
in larval zebrafish (Danio rerio). Prog. Neuropsychopharmacol. Biol. Psychiatry 83,
127–136.
Zhang, X., Yang, Y., Zhang, J., Yang, Y., Shen, F., Shen, J., Shao, B., 2019. Determination
of emerging chlorinated byproducts of diazepam in drinking water. Chemosphere
218, 223–231.
Zheng, S., Shi, J.C., Hu, J.Y., Hu, W.X., Zhang, J., Shao, B., 2016. Chlorination of
bisphenol F and the estrogenic and peroxisome proliferator-activated receptor
gamma effects of its disinfection byproducts. Water Res. 107, 1–10.