Abstract

   Pyridaben (PY) is a widely used organochlorine acaricide, which can be
   detected in the peripheral blood of pregnant women. Available evidence
   suggests that PY has reproductive toxicity. However, it remains
   uncertain whether prenatal PY exposure impacts neurobehavioral
   development in offspring. Here, we administered PY to pregnant mice at
   a dose of 0.5 and 5 mg kg^−1 day^−1 via gavage and observed
   anxiety-like behaviors in PY offspring aged five weeks. We then
   integrated the metabolome and transcriptome of the offspring's brain to
   explore the underlying mechanism. Metabolome data indicated that the
   vitamin B6 metabolism pathway was significantly affected, and the
   pyridoxal 5′-phosphate (PLP) concentration and the active form of
   vitamin B6 was significantly reduced. Moreover, the transcriptome data
   showed that both PLP generation-related Pdxk and anxiety-related Gad1
   were significantly down-regulated. Meanwhile, there was a decreasing
   trend in the concentration of GABA in the hippocampal DG region. Next,
   we supplemented PLP at a dose of 20 mg kg^−1 day^−1 to the PY offspring
   via intraperitoneal injection at three weeks. We found up-regulated
   expression of Pdxk and Gad1 and restored anxiety-like behaviors. This
   study suggests that prenatal exposure to PY can disrupt vitamin B6
   metabolism, reduce the concentration of PLP, down-regulate the
   expression levels of Pdxk and Gad1, inhibit the production of GABA, and
   ultimately lead to anxiety-like behaviors in offspring.

   Keywords: Pyridaben, Prenatal exposure, Pyridoxal 5′-phosphate,
   Anxiety-like behaviors

Graphical abstract

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Highlights

     * •
       Prenatal pyridaben (PY) exposure induces anxiety-like behaviors in
       offspring.
     * •
       Five mRNA and two metabolites are involved in vitamin B6
       metabolism.
     * •
       Prenatal PY exposure disturbs vitamin B6 metabolism and reduces PLP
       production.
     * •
       PLP supplementation restores the anxiety-like behaviors in
       offspring caused by PY.

1. Introduction

   Pyridaben (PY), a type of organochlorine pesticide (OCPs), has become
   the fastest-developing and most widely used acaricide due to its high
   activity and rapid effects. PY is used to protect farmland, fruit, and
   vegetable crops. Furthermore, its residues have been detected in
   various foods [[39]1,[40]2], indicating a broad source of exposure in
   daily life. Structurally, PY is similar to rotenone, with a cellular
   inhibitory effect on mitochondrial complexes and NADH dehydrogenase
   [[41]3]. Despite the adverse impacts observed in animal experiments,
   such as growth inhibition of uterine epithelial cells [[42]4] and
   mitochondrial dysfunction of testicular cells [[43]5], the usage of PY
   is still increasing. In our previous study, it has even been detected
   in the peripheral blood of pregnant women. Given the chance of exposure
   in the fetus, the potential health risk of early-life PY exposure could
   never be neglected.

   Anxiety has become one of the most common mental illnesses worldwide
   [[44]6]. The lifetime prevalence of “any anxiety disorder” in studies
   with children or adolescents is approximately 15%–20% [[45]7]. The
   occurrence and development of anxiety are closely related to
   environmental and genetic factors [[46][8], [47][9], [48][10]].
   Additionally, early environmental exposure may increase anxiety risk
   later in life [[49]11]. Mounting epidemiological evidence indicates a
   positive correlation between exposure to OCPs during pregnancy and
   neurodevelopmental disorders, including anxiety, impaired motor
   development, and memory loss [[50][12], [51][13], [52][14], [53][15]].
   Similarly, in vivo studies also show increased anxiety-like behavior
   levels in mice offspring exposed to OCPs during lactation [[54]16]. PY
   is an organochlorine pesticide (OCPs), similar to rotenone in
   structure. Although PY has been found to impair mitochondrial function
   in dopaminergic neurons [[55]17], whether its prenatal exposure affects
   the neurological behavior of offspring remains unknown.

   The metabolic process of the body is quite complex, and its homeostasis
   is essential to maintain the health of the human body [[56]18].
   Moreover, amino acid metabolism is found to be dysregulated under OCPs
   exposure during pregnancy [[57]19], which is closely associated with
   various adverse pregnancy outcomes such as abortion [[58]20], birth
   weight loss [[59]21], and cognitive birth deficits [[60]22] in
   children. B vitamins boost glutamate's metabolism and ensure the
   release of neurotransmitters such as GABA and serotonin, which are
   essential for nervous system signaling. Because 80% of PY is
   metabolized and binds glucuronic acid and remains in the body, we
   hypothesized that prenatal PY exposure might affect metabolism and
   induce neurobehavior toxicity in the offspring.

   In this study, we built a prenatal PY exposure model, evaluated the
   neurobehavior of mice offspring, identified differential metabolites of
   the brain, and investigated PYs metabolites in mice offspring to
   explore the effects and underlying mechanisms of prenatal PY exposure
   on neurobehavior development.

   In this study, we demonstrated that prenatal PY exposure inhibited GABA
   generation by down-regulating the levels of PLP and GAD1 in the brain,
   resulting in anxiety-like behaviors of mice offspring, which could be
   restored by supplementation with PLP.

2. Methods and materials

2.1. Animals and treatment

   Six-week-old C57BL/6J mice were purchased from the Animal Experimental
   Center of Nanjing Medical University. The animals were kept under
   light/dark conditions for 12 h with a temperature of 21 ± 3 °C and a
   humidity of 52 ± 8%. Food and water are provided ad libitum. All mice
   were adaptively fed for seven days. Then, the female mice were exposed
   to daily PY gavage. After seven days, mice were mated in cages with a
   2:1 ratio of female to male. When the vaginal plug was detected, it was
   recorded as 0.5 days of gestation (GD 0.5). The mice continued to be
   gavaged from GD 0.5 to delivery. It has been reported that PY exerts
   reproductive toxicity [[61]4]. The mice were extremely aggressive
   during lactation and ate the offspring mice in the study [[62]23]. To
   obtain adequate offspring for the subsequent experiments, we tried to
   keep the maternal mouse with 3–4 males and female offspring in the same
   cage. Afterward, we randomly selected an equal number of mice offspring
   from each cage in each group for subsequent behavioral tests. Mice
   offspring on PND1–PND21 are not suitable for behavioral experiments
   such as OFT and EPM, while five-week-old mice are in a stage that is
   less affected by the estrous cycle and sex hormones [[63]24] and can
   stably reflect the neurodevelopmental indicators [[64]25]. Therefore,
   behavior tests were performed on offspring aged five weeks.

   In the National Food Safety Standard–Maximum Residue Limits for
   Pesticides in Food in China (Chinese National Food Safety Standard,
   2019), the ADIs of PY is set to be 0.01 mg kg^−1 bw^−1. In the exposure
   model, taking the ADI and the NOAEL dose of rat as references, we