Multidimensional occurrence and diet risk of emerging contaminants in freshwater with urban agglomerations.

Freshwater systems near highly urbanized areas are extremely susceptible to emerging contaminants (ECs), yet their stereoscopic persistence in aquatic ecosystems and related risks remain largely unknown. Herein, we characterized the multi-mediums distribution of 63 ECs in Baiyangdian Lake, the biggest urban lake in the North of China. We identified variations in the seasonal patterns of aquatic EC levels, which decreased in water and increased in sediment from wet to dry seasons. Surprisingly, higher concentrations and a greater variety of ECs were detected in reeds than in aquatic animals, indicating that plants may contribute to the transferring of ECs. Source analysis indicated that human activity considerably affected the distribution and risk of ECs. The dietary risk of ECs is most pronounced among children following the intake of aquatic products, especially with a relatively higher risk associated with fish consumption. Besides, a comprehensive scoring ranking method was proposed, and 9 ECs, including BPS and macrolide antibiotics, are identified as prioritized control pollutants. These findings highlight the risks associated with aquatic ECs and can facilitate the development of effective management strategies.

Characterization and removal mechanism of fluoroquinolone-bioremediation by fungus Cladosporium cladosporioides 11 isolated from aquacultural sediments.

Antibiotics have been widely detected in aquatic environments, and fungal biotransformation receives considerable attention for antibiotic bioremediation. Here, a fungus designated Cladosporium cladosporioides 11 (CC11) with effective capacity to biotransform fluoroquinolones was isolated from aquaculture pond sediments. Enrofloxacin (ENR), ciprofloxacin (CIP) and ofloxacin (OFL) were considerably abated by CC11, and the antibacterial activities of the fluoroquinolones reduced significantly after CC11 treatment. Transcriptome analysis showed the removal of ENR, CIP and OFL by CC11 is a process of enzymatic degradation and biosorption which consists well with ligninolytic enzyme activities and sorption experiments under the same conditions. Additionally, CC11 significantly removed ENR in zebrafish culture water and reduced the residue of ENR in zebrafish. All these results evidenced the potential of CC11 as a novel environmentally friendly process for the removal of fluoroquinolones from aqueous systems and reduce fluoroquinolone residues in aquatic organisms.

Bisphenol A Analogues Induce Neuroendocrine Disruption via Gut-Brain Regulation in Zebrafish.

There is epidemiological evidence in humans that exposure to endocrine-disrupting chemicals such as bisphenol A (BPA) is tied to abnormal neuroendocrine function with both behavioral and intestinal symptoms. However, the underlying mechanism of this effect, particularly the role of gut-brain regulation, is poorly understood. We exposed zebrafish embryos to a concentration series (including environmentally relevant levels) of BPA and its analogues. The analogue bisphenol G (BPG) yielded the strongest behavioral impact on zebrafish larvae and inhibited the largest number of neurotransmitters, with an effective concentration of 0.5 µg/L, followed by bisphenol AF (BPAF) and BPA. In neurod1:EGFP transgenic zebrafish, BPG and BPAF inhibited the distribution of enteroendocrine cells (EECs), which is associated with decreased neurotransmitters level and behavioral activity. Immune staining of ace-α-tubulin suggested that BPAF inhibited vagal neural development at 50 and 500 µg/L. Single-cell RNA-Seq demonstrated that BPG disrupted the neuroendocrine system by inducing inflammatory responses in intestinal epithelial cells via TNFα-trypsin-EEC signaling. BPAF exposure activated apoptosis and inhibited neural developmental pathways in vagal neurons, consistent with immunofluorescence imaging studies. These findings show that both BPG and BPAF affect the neuroendocrine system through the gut-brain axis but by different mechanisms, revealing new insights into the modes of bisphenol-mediated neuroendocrine disruption.

Significant decline of water pollution associated with inland fishery across China.

Water pollution seriously threatens the sustainable development of fisheries in China. To inform effective pollution control policies, a comprehensive understanding of the fishery environment status is needed. However, nationwide data on the temporal changes of major pollutants in the fishery waters of China are scarce. This study collected data on the major water pollutants, including total nitrogen, total phosphorus, heavy metals, and total petroleum hydrocarbons (TPHs), from 2003 to 2017 to evaluate dynamic changes in the inland fishery water environment across China. We discovered that the levels of four heavy metals (Cu, Zn, Pb, and Cd) and TPH decreased during the 15-year period, corresponding to the reduced national discharge of pollution sources from 2003 to 2015. However, nitrogen and phosphorus levels in the inland fishery waters showed no significant changes during this period. A comparative analysis of water quality in different periods indicated that these improvements were highly associated with effective measures for water pollution control in China. In addition, the decline in pollution was consistent among the three regions of China (north, west, and southeast) from 2003 to 2017, while southeast China exhibited the weakest pollution mitigation among the three regions. These findings suggest that the inland fishery water quality improved during 2003-2017, but still faced eutrophication risk.

Bisphenol F induces liver-gut alteration in zebrafish.

The unease of consumers with bisphenol A has led to the increased industrial usage of bisphenol F (BPF), which is a new hazard to environmental health. Here, zebrafish were exposed to three BPF concentrations (0.5, 5, and 50 µg/L) from the embryonic stage for 180 days. Results showed that zebrafish body length and weight decreased and hepatosomatic index values increased, even at environmentally relevant concentration. Histological analysis identified the occurrence of hepatic fibrosis and steatosis in 5 and 50 µg/L groups, which indicated the liver injury caused by BPF. Based on the untargeted metabolomics results, a dose-dependent variation in the effects of BPF on liver metabolism was found, and amino acids, purines and one carbon metabolism were the main affected processes in the 0.5, 5, and 50 µg/L treatments, respectively. At the same time, BPF induced a shift in intestinal microbiome composition, including decreased abundance of Erysipelotrichaceae, Rhodobacteraceae and Gemmobacter. In addition, the correlation analysis suggested an association between gut microbiome changes and affected hepatic metabolites after BPF exposure. These findings indicate that a liver-gut alteration is induced by long-term BPF exposure.

Integrated toxicity assessment of DEHP and DBP toward aquatic ecosystem based on multiple trophic model assays.

To comprehensively understand the toxic risks of phthalates to aquatic ecosystems, we examined the acute toxicity of di-(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) on multiple trophic models, including algae (Chlorella vulgaris), Daphnia magna and fish (Danio rerio, Pseudorasbora parva). Thus, a 15-day zebrafish exposure was conducted to trace the dynamic changes of phthalate-induced toxic effects. Among the four species, D. magna exhibited the strongest sensitivity to both DEHP and DBP, followed by D. rerio and P. parva. C. vulgaris exhibited the lowest sensitivity to phthalates. The sub-chronic zebrafish assay demonstrated that 1000 µg/L DBP induced significant mortality at 15 days post-exposure (dpe), and DEHP exhibited no lethality at the tested concentrations (10-5000 µg/L). Zebrafish hepatic SOD activity and sod transcription levels were inhibited by DBP from 3 dpe, which was accompanied by increased malondialdehyde level, while zebrafish exposed to DEHP exhibited less oxidative damage. Both DEHP and DBP induced time-dependent alterations on Ache activity in zebrafish brains, thus indicating the potential neurotoxicity toward aquatic organisms. Additionally, 1000 µg/L and higher concentration of DBP caused hepatic DNA damage in zebrafish from 7 dpe. These results provide a better understanding of the health risks of phthalate to water environment.

Bisphenol F Impaired Zebrafish Cognitive Ability through Inducing Neural Cell Heterogeneous Responses.

The central nervous system (CNS) is a sensitive target for endocrine-disrupting chemicals, such as bisphenol analogues. Bisphenol A (BPA) usage is associated with the occurrence of many neurological diseases. With the restricted use of BPA, bisphenol F (BPF) has been greatly introduced for industrial manufacture and brings new hazards to public CNS health. To understand how BPF affects the neural system, we performed a cognitive test for zebrafish that are continuously exposed to environmentally relevant concentrations (0.5 and 5.0 µg/L) of BPF since embryonic stage and identified suppressed cognitive ability in adulthood. Single-cell RNA sequencing of neural cells revealed a cell composition shift in zebrafish brain post BPF exposure, including increase in microglia and decrease in neurons; these changes were further validated by immune staining. At the same time, a significant inflammatory response and increased phagocytic activity were detected in zebrafish brain post BPF exposure, which were consistent with the activation of microglia. Cell-specific transcriptomic profiles showed that abnormal phagocytosis, activated brain cell death, and apoptosis occurred in microglia post BPF exposure, which are responsible for the neuron loss. In addition, certain neurological diseases were affected by BPF in both excitatory and inhibitory neurons, such as the movement disorder and neural muscular disease, however, with distinctly involved genes. These findings indicate that BPF exposure could lead to an abnormal cognitive behavior of zebrafish through inducing heterogeneous changes of neural cells in brain and revealed the dominating role of microglia in mediating this effect.

Bisphenol analogues induced metabolic effects through eliciting intestinal cell heterogeneous response.

The metabolic effects of endocrine-disrupting chemicals, such as bisphenol analogues, have drawn increasing attention. Bisphenol A (BPA) usage is associated with the occurrence of many metabolic diseases. With the restricted use of BPA, alternatives like bisphenol F (BPF) and bisphenol AF (BPAF) have been greatly introduced for industrial manufacture, and brings new hazard to public health. To understand how bisphenol analogues induced metabolic effects, zebrafish are continuous exposed to environmental level (0.5 µg/L) of BPA, BPF and BPAF since embryonic stage, and identified hepatic steatosis and insulin resistance at 60-day post fertilization. Hepatic transcriptional profile indicated that pancreatic disease pathways were activated by BPA, but were inhibited by BPF. At the same time, increased lipid secretion and gluconeogenesis pathways in zebrafish liver was found post BPAF exposure. Significant inflammatory response, histological injury and increased mucus secretion was detected in zebrafish intestine post exposure of three bisphenol analogues. Single-cell RNA sequencing of zebrafish intestinal cells revealed activation of lipid uptake and absorption pathways in enterocyte lineages, which well explained the hepatic steatosis induced by BPA and BPF. Besides, genes related to carbohydrate metabolism, diabetes and insulin resistance were activated in intestinal immune cell types by three bisphenol analogues. These findings indicated that BPA and its alternatives could lead to abnormal lipid and carbohydrate metabolism of zebrafish through inducing cell heterogeneous changes in gut, and revealed both molecular and cellular mechanism in mediating this effect.

Environmental level of bisphenol F induced reproductive toxicity toward zebrafish.

Bisphenol F (BPF), as an important bisphenol A substitute, is being increasingly used for industrial production. Here we performed large scale fecundity test for zebrafish that are continuous exposed to environmental levels of BPF (0.5, 5 and 50 µg/L) from embryonic stage, and identified suppressed spawning capacity of females and reduced fertility rate of males in adulthood. Although pathological change is only observed in female gonads, the transcriptional change in the hypothalamic-pituitary-gonad axis genes occurred in the gonads of both female and male fish at 150 days post-exposure. F1 generation embryos showed abnormal developmental outcomes including decreased heart rate, reduced body length, and inhibition of spontaneous movement after parental exposure to BPF. RNA-sequencing showed that the genes involved in skeletal/cardiac muscle development were significantly altered in F1 embryos spawned by BPF-treated zebrafish. The advanced pathway analysis showed that cancer and tumour formation were the most enriched pathways in the offspring of 0.5 and 5.0 µg/L groups; organismal development and cardiovascular system development were mainly affected after parental exposure to 50 µg/L of BPF; these changes were mediated by several involved regulators such as GATA4, MYF6, and MEF2C. These findings confirmed that long-term exposure to BPF at environment relevant concentration would result in reproductive toxicity among zebrafish indicating the urgent demand for the control of BPA substitutes.

A multi-omics approach reveals molecular mechanisms by which phthalates induce cardiac defects in zebrafish (Danio rerio).

The potential risks of phthalates affecting human and animal health as well as the environment are emerging as serious concerns worldwide. However, the mechanism by which phthalates induce developmental effects is under debate. Herein, we found that embryonic exposure of zebrafish to di-(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) increased the rate of heart defects including abnormal heart rate and pericardial edema. Changes in the transcriptional profile demonstrated that genes involved in the development of the heart, such as tbx5b, nppa, ctnt, my17, cmlc1, were significantly altered by DEHP and DBP at 50 µg/L, which agreed with the abnormal cardiac outcomes. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) further showed that significant hypomethylation of nppa and ctnt was identified after DEHP and DBP exposure, which was consistent with the up-regulation of these genes. Notably, hypermethylation on the promoter region (<1 kb) of tbx5b was found after DEHP and DBP exposure, which might be responsible for its decrease in transcription. In conclusion, phthalates have the potential to induce cardiac birth defects, which might be associated with the transcriptional regulation of the involved developmental factors such as tbx5b. These findings would contribute to understand the molecular pathways that mediated the cardiac defects caused by phthalates.

The pigmentation interference of bisphenol F and bisphenol A.

Bisphenol A (BPA) and bisphenol F (BPF) are widely distributed in the environment and daily consumptions, leading to exposure toward human and environmental animals. The potential risk of bisphenol analogs on pigment and skin health is not well documented. In this study, we found that 0.05 mg/L BPF (tolerated daily intake (TDI) value of BPA) affected the particle size and color density of zebrafish melanin. While BPA caused less depigmentation effect toward zebrafish with effective concentration of 5.0 mg/L. The downregulation of melanin synthases induced by BPF is associated with the reduction in melanin. Molecular dynamics indicated that both BPF and BPA could act as ligands of zebrafish and human Tyr family proteins; however, these compounds have completely different energetics and spatial steric effects, potentially explaining their varying depigmentation effects. Additionally, an in vitro assay using A375 melanoma cells demonstrated that the inhibitory effect of BPF on human melanin production was primarily attributed to Tyr inhibition. These findings provide an important basis for understanding the molecular mechanisms of BPF and BPA in melanin inhibition, and the results reflect the skin pigmentation interference risk of these compounds, which are ubiquitous in everyday personal products.

Evaluation of the spinal effects of phthalates in a zebrafish embryo assay.

Phthalates (phthalate esters, PAEs) are commonly used as plasticizers and are emerging concerns worldwide for their potential influence on the environment and general public health. Thus, identification of the negative effects and involved mechanisms of PAEs is necessary. Herein, we found that embryonic exposure of zebrafish to di-(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) significantly induced spinal defects, such as inhibited spontaneous movement at 24 h post-fertilization (hpf), spine curvature and body length decrease at 96 hpf. The transcriptional level of the genes that are related to the development of the notochord (col8a1a and ngs), muscle (stac3, klhl41a and smyd2b) and skeleton (bmp2, spp1) were significantly altered by DEHP and DBP at 50 and 250 µg/L, which might be associated with the observed morphological changes. Notably, DBP and DEHP altered the locomotor activity of zebrafish larvae at 144 hpf, which might be due to the abnormal development of the spine and skeletal system. In conclusion, phthalates caused spinal birth defects in zebrafish embryos, induced transcriptional alterations of the spinal developmental genes, and led to abnormal behavior.

Bisphenol F-Induced Neurotoxicity toward Zebrafish Embryos.

In this study, the influence of bisphenol F (BPF) toward central nervous system (CNS) was assessed using zebrafish embryos. We found that BPF could induce significant neurotoxicity toward zebrafish embryos, including inhibited locomotion, reduced moving distance, and CNS cell apoptosis at an effective concentration of 0.0005 mg/L. Immunofluorescence assay showed that both microglia and astrocyte in zebrafish brain were significantly activated by BPF, indicating the existence of neuroinflammatory response. Peripheral motor neuron development was significantly inhibited by BPF at 72 hpf. RNA-seq data indicated that neuronal developmental processes and cell apoptosis pathways were significantly affected by BPF exposure, which was consistent with the phenotypic results. Chip-seq assay implied that the transcriptional changes were not mediated by ERα. Additionally, no significant change was found in neurotransmitter levels (5-hydroxytryptamine, dopamine, and acetylcholine) or acetylcholinesterase (Ache) enzyme activity after BPF exposure, indicating that BPF may not affect neurotransmission. In conclusion, BPF could lead to abnormal neural outcomes during zebrafish early life stage through inducing neuroinflammation and CNS cell apoptosis even at environmentally relevant concentration.

The mechanisms underlying the developmental effects of bisphenol F on zebrafish.

With the increasing use of bisphenol F (BPF) as BPA alternative, BPF are widely distributed in multiple environment media. Our previous study demonstrated that BPF possess equivalent toxicity towards zebrafish as BPA, while its toxic mechanism remains largely unknown. To investigate the mechanisms mediating the developmental effects of BPF, zebrafish embryos were exposed to 0.0005, 0.5, and 5.0 mg/L BPF. Morphological examination indicated that BPF exposure led to depigmentation, decreased heart rate, inhibited spontaneous movement, hatch inhibition, and spinal deformation. Motor neuron-green fluorescence zebrafish assay indicated that exposure to 0.5 or 5.0 mg/L BPF affected embryonic motor neuron development, which is consistent with the spinal defect and spontaneous movement inhibition. Transcriptomic analysis showed that genes associated with the observed symptoms, including neuron development (ngln2a, socs3a, fosb), cardiac development (klf2a), and spinal deformation (ngs, col8a1a, egr2a), were down-regulated after exposure to either 0.0005 (environmental relevant concentration) or 0.5 mg/L BPF. This partially explained the mechanisms underlying the effects of BPF. In conclusion, BPF had the potential to affect zebrafish development even at environmental level through down-regulating associated genes.

Global microRNA and isomiR expression associated with liver metabolism is induced by organophosphorus flame retardant exposure in male Chinese rare minnow (Gobiocypris rarus).

To reveal the adverse effects of organophosphorus flame retardants (OPFRs) on aquatic organisms at the epigenetic level, male Chinese rare minnows were exposed to 0.24 mg/L tris(2­butoxyethyl) phosphate (TBOEP), 0.04 mg/L tris(1,3­dichloro­2­propyl) phosphate (TDCIPP), or 0.012 mg/L triphenyl phosphate (TPHP) for 14 days. The effects of sub-acute OPFR exposure on liver miRNA and the 3' isomiR expression profiles of Chinese rare minnows were investigated. Through small RNA sequencing and bioinformatics analysis, a total of 32, 84, and 19 differentially expressed miRNAs were detected for TBOEP, TDCIPP, and TPHP exposure, respectively (p < 0.05). Target prediction of the differentially expressed miRNAs and pathway enrichment analysis indicated that predicted altered mRNAs for all three OPFRs were associated with metabolic pathways, whereas base excision repair was only predicted to be perturbed by the TPHP treatment. In addition, 3' isomiR-Us were unexpectedly abundant in all groups (e.g., miR-143), and TDCIPP strongly increased the ratio of 3' isomiR-U expression. Finally, histological examination and metabolic enzyme activity analyses werein agreement with the predicted metabolic pathways. As such, our study indicates that the investigation of epigenetics changes in miRNA gene transcription is a considerable method for the assessment of aquatic toxicity.

Triphenyl Phosphate (TPHP)-Induced Neurotoxicity in Adult Male Chinese Rare Minnows ( Gobiocypris rarus).

The neurotoxicity of triphenyl phosphate (TPHP) in exposed humans and laboratory animals is under debate. The rapid crossing of the blood-brain barrier (BBB) and high distribution of TPHP in fish brains have raised widespread concerns about potential neurotoxicity. Adult male Chinese rare minnows ( Gobiocypris rarus) were used as a model and exposed to 0, 20, or 100 µg/L TPHP for 28 days. We evaluated the BBB permeability, neuroinflammatory response, cell proliferation and apoptosis, synaptic plasticity and synapse loss in fish brains via the learning/memory performance of fish following 28 days of TPHP exposure. TPHP significantly increased the BBB permeability, activated the neuroinflammatory response, and decreased the tight junction-related mRNA levels of claudin-5α and occludin in the fish brain. In addition, cell proliferation was inhibited by treatment with 100 µg/L TPHP, but no significant apoptosis was observed in the brain. Fish exposed to 100 µg/L TPHP exhibited significantly decreased dendritic arborization in pyramidal neurons in the cerebellum (Ce), and the maze test indicated impaired learning/memory performance. Taken together, these findings provide scientific evidence that TPHP is neurotoxic to fish and further suggest that TPHP may not be a safe alternative for aquatic organisms.

The neuropeptides of Asian freshwater clam (Corbicula fluminea) as new molecular biomarker basing on the responses of organophosphate chemicals exposure.

In the present study, to discover new biomarker of Asian freshwater clam (Corbicula fluminea) to assess impact of environmental pollutions, cholecystokinin (CCK), conopressin, and Neuropeptide FF (FFamide) in C. fluminea were selected as potent biomarkers. Therefore, their full-length cDNAs were cloned and characterized to investigate the molecular characteristics and expression patterns of neuropeptides in C. fluminea. According to the sequence analysis, CCK, conopressin, and FFamide encoded proteins of 173, 152, and 90 amino acids, respectively. Moreover, the multiple sequence alignment revealed that the bioactive regions of these neuropeptides were well conserved among different invertebrates. In addition, under basal conditions, CCK, conopressin and FFamide mRNA were mainly expressed in the visceral mass, whereas the FFamide mRNA was rarely detected in the foot and mantle. Exposure to 20 and 200 µg/L Tris (2-butoxyethyl) phosphate (TBOEP) and tri-butyl-phosphate (TBP) exposure significantly up-regulated the expression of the CCK and FFamide mRNAs in the visceral mass (p < 0.05), whereas no significant changes in conopressin mRNA levels were observed in response to any treatment. Therefore, CCK and FFamide of C. fluminea neuropeptides are feasible new biomarkers for screening and assessing responses to organophosphate chemicals.

New cytokines and TLR pathway signaling molecules in Chinese rare minnow (Gobiocypris rarus): Molecular characterization, basal expression, and their response to chlorpyrifos.

In this study, the cDNA fragments of cytokines (il-8) and toll-like receptor (TLR) pathway signaling molecules (myd88, irak-1, irf5, and irf7) in the Chinese rare minnow were cloned and exhibited a high amino-acid sequence identity compared to other cyprinid fish orthologs. The mRNA expressions of these genes in the different tissues (liver, brain, spleen, kidney, and skin) were observed. The highest expression levels of myd88, irak-1, and irf5 were detected in the spleen, whereas il-8 and irf7 were detected in the kidney and liver respectively. The mRNA expression of irak-1, irf5, and irf7 in the liver from 0.1 µg/L and 0.5 µg/L CPF treatments were significantly increased on day 7 (p < 0.05), whereas the levels of only irak-1 and irf7 were markedly increased on day 28 (p < 0.05). Moreover, the mRNA expression of il-8 in the spleen following 0.5 µg/L CPF treatments was significantly decreased on day 7 (p < 0.05), whereas significantly decrease were observed in the levels of irf7 in the spleen at 2.5 µg/L CPF on days 7 and 28 (p < 0.05). The 0.1 µg/L and 0.5 µg/L of CPF significantly induced the levels of irak-1 and myd88 in the spleen after 28 d exposure (p < 0.05). Therefore, the high induction of cytokines and TLR pathway signaling molecules demonstrated that Chinese rare minnow was immune-compromised exposed to CPF. Moreover, our finding indicated that these immune-related genes could be feasible to screen for substances hazardous to the immune system of fish.

Phenanthrene-Induced Apoptosis and Its Underlying Mechanism.

Phenanthrene (Phe) is one of the most abundant low-molecular-weight polycyclic aromatic hydrocarbons (PAHs). Widespread human and aquatic organism exposure to Phe has been reported, but the toxic effects of Phe and potential mechanisms are unclear. We focused on the chronic hepatotoxicity of Phe in adult Chinese rare minnows (Gobiocypris rarus) and the underlying mechanisms. The chronic effects of exposing Chinese rare minnows to 8.9, 82.3, or 510.0 µg/L Phe for 30 days were examined by histopathological observation, TUNEL assays, caspase activity assays, and gene expression profiles. The liver lesion frequency and hepatocyte apoptosis were increased in Phe-exposed groups. Caspase 9 and caspase 3 enzyme activity in liver tissues was markedly increased. The expression of miR-17/92 cluster members was significantly increased in the 82.3 and 510.0 µg/L groups. Moreover, the response of primary hepatocytes indicated a significant decrease in the mitochondrial membrane potential (MMP) after a 48 h exposure to Phe. Interestingly, miR-18a was significantly decreased in primary hepatocytes in all treatments. Moreover, molecular docking indicated that Phe might have the same binding domain as pri-miR-18a, forming pi-pi and pi-σ interactions with heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Given the above, Phe caused liver lesions and induced hepatocyte apoptosis through the intrinsic apoptosis pathway, and the interaction of Phe with hnRNP A1 contributes to the suppression of miR-18a expression and hepatocyte apoptosis.

Benzo(a)pyrene-induced a mitochondria-independent apoptosis of liver in juvenile Chinese rare minnows (Gobiocypris rarus).

To examine the effects of BaP on tissue apoptosis, laboratory studies were conducted using juvenile Chinese rare minnows (Gobiocypris rarus) exposed to 1, 5, 20, and 80 µg/L of BaP for 28 days. The post-treatment pathological findings in the liver were associated with hepatocyte swelling, karyopyknosis, and karyorrhexis. Moreover, an increase in the goblet cells in the intestine, epithelial hyperplasia of the gills and fusion of gill lamellae were observed. Significant increases in hepatocyte apoptosis using the TUNEL stain were observed in the liver tissue but not in the intestine and gills. In addition, BaP exposure significantly up-regulated the mRNA levels of cyp1a1, p53, bax, bcl-2, and caspase-9 in the liver following the 5, 20, and 80 µg/L treatments, whereas the apaf-1 was significantly down-regulated following all treatments. Moreover, the activities of caspase 3 and caspase 8 were markedly elevated, whereas the protein expression levels of Apaf-1 were down-regulated following the 20 and 80 µg/L treatments. Taken together, our results suggested that BaP strongly induces tissue-specific apoptosis in vivo, leading to significant pathological changes. The responsiveness of apoptotic-related genes demonstrates that BaP induced apoptosis in the liver may be through a mitochondria-independent pathway.