1-Nitropyrene (1-NP) is a neurodevelopmental toxicant. This study was to evaluate the impact of exposure to 1-NP after weaning on anxiety-like behavior. Five-week-old mice were administered with 1-NP (0.1 or 1 mg/kg) daily for 4 weeks. Anxiety-like behaviour was measured using elevated-plus maze (EPM) and open field test (OFT). In EPM test, time spending in open arm and times entering open arm were reduced in 1-NP-treated mice. In OFT test, time spent in the center region and times entering the center region were diminished in 1-NP-treated mice. Prefrontal dendritic length and number of dendrite branches were decreased in 1-NP-treated mice. Prefrontal PSD95, an excitatory postsynaptic membrane protein, and gephyrin, an inhibitory postsynaptic membrane protein, were downregulated in 1-NP-treated mice. Further analysis showed that peripheral steroid hormones, including serum testosterone (T) and estradiol (E2), testicular T, and ovarian E2, were decreased in 1-NP-treated mice. Interestingly, T and E2 were diminished in 1-NP-treated prefrontal cortex. Prefrontal T and E2 synthases were diminished in 1-NP-treated mice. Mechanistically, GCN2-eIF2α, a critical pathway that regulates ribosomal protein translation, was activated in 1-NP-treated prefrontal cortex. These results indicate that exposure to 1-NP after weaning induces anxiety-like behaviour partially by inhibiting steroid hormone synthesis in prefrontal cortex.

OBJECTIVE: To observe the expression of the retinoic acid(RA) pathway in hypothalamus and pituitary damage induced by combined exposure of low-level lead and 1-nitropyrene in mice, and to explore the relationship between the changes of RA pathway and hypothalamus and pituitary damage.
METHODS: A total of 84 4-week-old ICR mice were randomly divided into the control group, Pb~(2+) tainted group(0.008 mg/L), 1-NP tainted group(0.1 mg/kg), low(0.008 mg/L Pb~(2+)+0.004 mg/kg 1-NP), medium(0.008 mg/L Pb~(2+)+0.02 mg/kg 1-NP), and high-dose co-toxicity group(0.008 mg/L Pb~(2+)+0.1 mg/kg 1-NP) according to body weight, with 14 mice in each group. Among them, Pb~(2+) was provided by lead acetate, added to deionized water and ingested by mice drinking freely, 1-NP was given by intraperitoneal injection, 1-NP was administered by intraperitoneal injection. Record daily water intake and food intake. After 21 consecutive days of exposure, body mass was measured, histological changes in the hypothalamus and pituitary were observed under an optical microscope, and lead content in brain tissue was measured by atomic absorption spectrometry. The real-time fluorescence quantitative PCR was used to detect the abundance of retinoic acid pathway members and c-Jun N-terminal kinases genes(Jnks), and the western blot method was used to detect expression levels of acetaldehyde dehydrogenase 2(ALDH2), cytochrome P450 family member 26A1(CYP26a1) proteins.
RESULTS: There was no difference in the mean weekly water intake and food intake of the mice in each group. The body weight of the high-dose co-toxicity group mice((27.4±1.9)g) was lower than that of the control group((29.8±2.3)g)(P<0.05). The level of serum follicle-stimulating hormone(FSH) in the middle and high dose co-toxicity groups((265.01±2.99), (260.42±3.61)pg/mL, respectively) was lower than that in the control group((279.00±1.30)pg/mL, P<0.05). The content of Pb~(2+) in the brain of each group containing Pb~(2+) was higher than that of the control group. In the hypothalamic and pituitary tissues, the abundance of Adh1, Adh2, Rar and Rxr, and ALDH2 levels in the medium and high dose co-toxicity groups were higher than those in the control group(P<0.05). Cyp26a1 gene abundance and protein levels were lower in the medium and high dose co-toxicity groups than in the control group(P<0.05). The abundance of Jnks in the high-dose co-toxicity group was higher than that in the control group(P<0.05).
CONCLUSIONS: Continuous exposure to 0.008 mg/L Pb~(2+)+0.1 mg/kg 1-NP for 21 days can cause damage to the hypothalamus and pituitary of mice, and activate the RA signaling pathway.

BACKGROUND: Diesel exhaust (DE) exposures pose concerns for serious health effects, including asthma and lung cancer, in California communities burdened by multiple stressors.
OBJECTIVE: To evaluate DE exposures in disproportionately impacted communities using biomonitoring and compare results for adults and children within and between families.
METHODS: We recruited 40 families in the San Francisco East Bay area. Two metabolites of 1-nitropyrene (1-NP), a marker for DE exposures, were measured in urine samples from parent-child pairs. For 25 families, we collected single-day spot urine samples during two sampling rounds separated by an average of four months. For the 15 other families, we collected daily spot urine samples over four consecutive days during the two sampling rounds. We also measured 1-NP in household dust and indoor air. Associations between urinary metabolite levels and participant demographics, season, and 1-NP levels in dust and air were evaluated.
RESULTS: At least one 1-NP metabolite was present in 96.6% of the urine samples. Detection frequencies for 1-NP in dust and indoor air were 97% and 74%, respectively. Results from random effect models indicated that levels of the 1-NP metabolite 6-hydroxy-1-nitropyrene (6-OHNP) were significantly higher in parents compared with their children (p-value = 0.005). Urinary 1-NP metabolite levels were generally higher during the fall and winter months. Within-subject variability was higher than between-subject variability (~60% of total variance versus ~40%, respectively), indicating high short-term temporal variability.
CONCLUSIONS: Biomonitoring, coupled with air monitoring, improves understanding of hyperlocal air pollution impacts. Results from these studies will inform the design of effective exposure mitigation strategies in disproportionately affected communities.

Fetal growth restriction (FGR) is a prevalent complication in obstetrics, yet its exact aetiology remains unknown. Numerous studies suggest that the degradation of the living environment is a significant risk factor for FGR. 1-Nitropyrene (1-NP) is a widespread environmental pollutant as a representative substance of nitro-polycyclic aromatic hydrocarbons. In this study, we revealed that 1-NP induced FGR in fetal mice by constructing 1-NP exposed pregnant mice models. Intriguingly, we found that placental trophoblasts of 1-NP exposed mice exhibited significant ferroptosis, which was similarly detected in placental trophoblasts from human FGR patients. In this regard, we established a 1-NP exposed cell model in vitro using two human trophoblast cell lines, HTR8/SVneo and JEG-3. We found that 1-NP not only impaired the proliferation, migration, invasion and angiogenesis of trophoblasts, but also induced severe cellular ferroptosis. Meanwhile, the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively rescued 1-NP-induced trophoblast biological function impairment. Mechanistically, we revealed that 1-NP regulated ferroptosis by activating the ERK signaling pathway. Moreover, we innovatively revealed that CYP1B1 was essential for the activation of ERK signaling pathway induced by 1-NP. Overall, our study innovatively identified ferroptosis as a significant contributor to 1-NP induced trophoblastic functional impairment leading to FGR and clarified the specific mechanism by which 1-NP induced ferroptosis via the CYP1B1/ERK signaling pathway. Our study provided novel insights into the aetiology of FGR and revealed new mechanisms of reproductive toxicity of environmental pollutants.

Airborne polycyclic aromatic hydrocarbon (PAH) exposure can adversely affect human health by generating reactive oxygen species (ROS) and increasing oxidative stress, which causes changes in mitochondrial DNA copy number (mtDNAcn), a key indicator of mitochondrial damage and dysfunction. This study aimed to determine the effects of atmospheric benzo[a]pyrene (BaP) and 1-nitropyrene (1-NP) exposure on mtDNAcn in humans. One hundred and eight adults living in Cheongju, South Korea, were included in this study. Atmospheric BaP and 1-NP concentrations and urinary 6-hydroxy-1-nitropyrene (6-OHNP), N-acetyl-1-aminopyrene (1-NAAP), and 1-hydroxypyrene concentrations were measured. Blood samples were also collected to assess mtDNAcn. The mean mtDNAcn was 9.74 (SD 4.46). mtDNAcn decreased significantly with age but was not significantly associated with sex, sampling season, or smoking habit. While there was a borderline significant increase in mtDNAcn with increasing ambient total PAH levels, ambient PAH or urinary 1-hydroxypyrene concentrations showed no significant association with mtDNAcn. However, urinary 6-OHNP or 1-NAAP concentrations, 1-NP metabolites, were significantly associated with mtDNAcn. These results suggest that the metabolism of absorbed NPs generates excess ROS, which damages mitochondrial DNA, resulting in increased mtDNAcn.

Intrauterine growth retardation (IUGR) is a major cause of perinatal morbidity and mortality. Previous studies showed that 1-nitropyrene (1-NP), an atmospheric pollutant, induces placental dysfunction and IUGR, but the exact mechanisms remain uncertain. In this research, we aimed to explore the role of mitophagy on 1-NP-evoked placental progesterone (P4) synthesis inhibition and IUGR in a mouse model. As expected, P4 levels were decreased in 1-NP-exposed mouse placentas and maternal sera. Progesterone synthases, CYP11A1 and 3βHSD1, were correspondingly declined in 1-NP-exposed mouse placentas and JEG-3 cells. Mitophagy, as determined by LC3B-II elevation and TOM20 reduction, was evoked in 1-NP-exposed JEG-3 cells. Mdivi-1, a specific mitophagy inhibitor, relieved 1-NP-evoked downregulation of progesterone synthases in JEG-3 cells. Additional experiments showed that ULK1/FUNDC1 signaling was activated in 1-NP-exposed JEG-3 cells. ULK1 inhibitor or FUNDC1-targeted siRNA blocked 1-NP-induced mitophagy and progesterone synthase downregulation in JEG-3 cells. Further analysis found that mitochondrial reactive oxygen species (ROS) were increased and GCN2 was activated in 1-NP-exposed JEG-3 cells. GCN2iB, a selective GCN2 inhibitor, and MitoQ, a mitochondria-targeted antioxidant, attenuated GCN2 activation, FUNDC1-mediated mitophagy, and downregulation of progesterone synthases in JEG-3 cells. In vivo, gestational MitoQ supplement alleviated 1-NP-evoked reduction of placental P4 synthesis and IUGR. These results suggest that FUNDC1-mediated mitophagy triggered by mitochondrial ROS may contribute partially to 1-NP-induced placental P4 synthesis inhibition and IUGR.

Polycyclic aromatic hydrocarbons (PAHs) have been reported to cause oxidative stress in metabolic processes. This study aimed to evaluate the relationship between exposure to PAHs, including benzo[a]pyrene (BaP) and 1-nitropyrene (1-NP), in the atmosphere and oxidative stress levels in the human body. This study included 44 Korean adults who lived in Cheongju, Republic of Korea. Atmospheric BaP and 1-NP concentrations and urinary 6-hydroxy-1-nitropyrene (6-OHNP), N-acetyl-1-aminopyrene (1-NAAP), and 1-hydroxypyrene (1-OHP) concentrations were measured. The oxidative stress level was assessed by measuring urinary thiobarbituric acid-reactive substances (TBARS) and 8-hydroxydeoxyguanosine (8-OHdG) concentrations. Urinary TBARS and 6-OHNP concentrations significantly differed between winter and summer. BaP exposure was significantly associated with urinary 8-OHdG concentrations in summer. However, atmospheric 1-NP did not show a significant correlation with oxidative stress marker concentrations. Urinary 1-NAAP concentration was a significant determinant for urinary 8-OHdG concentration in summer. Oxidative stress in the body increases in proportion to inhalation exposure to BaP, and more 8-OHdG is produced in the body as the amount of 1-NP, which is metabolized to 1-AP or 1-NAAP, increases.

1-Nitropyrene (1-NP), a typical nitro-polycyclic aromatic hydrocarbon, is a developmental toxicant. This study was to evaluate gestational 1-NP-induced anxiety-like behavior in male adult offspring. Pregnant mice were orally administered to 1-NP daily throughout pregnancy. Anxiety-like behaviors, as determined by Elevated Plus-Maze (EPM) and Open-Field Test (OFT), were showed in male adult offspring whose mothers were exposed to 1-NP. Gestational 1-NP exposure reduced dendritic arborization, dendritic length and dendritic spine density in ventral hippocampus of male adult offspring. Additional experiments showed that gephyrin, an inhibitory synaptic marker, was reduced in fetal forebrain and hippocampus in male adult offspring. Nrg1 and Erbb4, two gephyrin-related genes, were reduced in 1-NP-exposed fetuses. Accordingly, 5hmC contents in two CpG sites (32008909 and 32009239) of Nrg1 gene and three CpG sites (69107743, 69107866 and 69107899) of Erbb4 gene were decreased in 1-NP-exposed fetuses. Mechanistically, ten-eleven translocation (TET) activity and alpha-ketoglutarate (α-KG) content were decreased in 1-NP-exposed fetal forebrain. Supplementation with α-KG alleviated 1-NP-induced downregulation of gephyrin-related genes, prevented hippocampal synaptic damage, and improved anxiety-like behavior in male adult offspring. These results indicate that early-life 1-NP exposure causes anxiety-like behavior in male adulthood partially by altering hippocampal epigenetic reprogramming of synaptic plasticity.

1-nitropyrene (1-NP) is representative nitropolycyclic aromatic hydrocarbon pollutant widely present in exhaust particles of internal combustion engine, which is known for its carcinogenicity and mutagenicity. Previous studies have demonstrated that 1-NP has reproductive toxicity, but the specific mechanism is unknown. In this study, Human decidual stromal cells (HDSCs) were treated by 1-NP, exosomes were extracted from the conditioned medium of HDSCs, which were then used to treat human chorionic trophoblast cells (HTR8/SVneo) for 24 h. The findings showed that human decidual stromal cell-derived exosomes (HDSC-EXOs) can promote the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT; Vimentin and N-cadherin) of HTR8/SVneo by about 64%, 17%, 23%, 81% and 13%. The process of regulating the biological behaviors of embryonic trophoblast cells by maternal decidual stromal cells during pregnancy was simulated. Further investigations showed that HDSC-EXOs treatment activated the Wnt/β-catenin signaling pathway in HTR8/SVneo. Co-treatment by dickkopf-1 (DKK-1) significantly suppressed the activation of Wnt/β-catenin signaling pathway in HTR8/SVneo, and inhibited the proliferation, migration, invasion and EMT (N-cadherin and E-cadherin) of HTR8/SVneo by about 60%, 22%, 42%, 25%, 55% and 21%. These findings indicated that 1-NP exposure could induce the secretion of HDSC-EXOs from HDSCs, which in turn activate the Wnt/β-catenin signaling pathway and enhance the proliferation, migration, invasion and EMT of HTR8/SVneo.

Nitropyrene (1-NP) is a specific indicator of exposure to diesel exhaust and is partly metabolized to 1-aminopyrene (1-AP) and N-acetyl-1-aminopyrene (1-NAAP), which are excreted in urine. This study was conducted to evaluate the effects of gene polymorphisms of metabolic enzymes for 1-NP on the urinary concentrations of 1-AP and 1-NAAP. The study participants were 70 South Koreans who were occupationally or environmentally exposed to diesel exhaust. To evaluate 1-NP exposure levels, we sampled airborne particulate matters with a personal air sampler and measured urinary 1-AP and 1-NAAP concentrations. The genetic polymorphisms of the 1-NP metabolic enzymes (CYP1A1, CYP1B1, EPHX1, NQO1, and NAT2) were determined by direct sequencing. The mean 1-NP exposure level was 20.40 pg/m3, and the mean urinary concentrations of 1-AP and 1-NAAP were 0.074 nM and 0.213 nM, respectively. The correlation coefficient between the 1-NP exposure level and urinary 1-AP concentrations was 0.0138 and that between the 1-NP exposure level and urinary 1-NAAP concentrations was 0.1493, and neither correlation coefficient was statistically significant. The correlation coefficient between the 1-NP exposure level and urinary 1-AP concentrations showed statistically significant differences according to the CYP1A1 and CYP1B1 genotypes, and that between the 1-NP exposure level and urinary 1-NAAP concentrations was significantly different according to the CYP1A1, CYP1B1, and NAT2 genotypes. The urinary concentration of 1-NAAP is a better biomarker for exposure to 1-NP or DEPs because the former is higher, easier to measure, and more strongly correlated with 1-NP exposure levels than that of 1-AP. The relationship between 1-NP exposure and urinary 1-AP or 1-NAAP concentration depends on the single nucleotide polymorphism types of CYP1A1, CYP1B1, NQO1, and NAT2.