Effects of benzophenone-3 and its metabolites on the marine diatom Chaetoceros neogracilis: Underlying mechanisms and environmental implications.

The wide application of benzophenones (BPs), such as benzophenone-3 (BP3), as an ingredient in sunscreens, cosmetics, coatings, and plastics, has led to their global contamination in aquatic environments. Using the marine diatom Chaetoceros neogracilis as a model, this study assessed the toxic effects and mechanisms of BP3 and its two major metabolites (BP8 and BP1). The results showed that BP3 exhibited higher toxicity on C. neogracilis than BP8 and BP1, with their 72-h median effective concentrations being 0.4, 0.8 and 4 mg/L, respectively. Photosynthesis efficiencies were significantly reduced after exposure to environmentally relevant concentrations of the three benzophenones, while cell viability, membrane integrity, membrane potential, and metabolic activities could be further impaired at their higher concentrations. Comparative transcriptomic analysis, followed by gene ontology and KEGG pathway enrichment analyses unraveled that all the three tested benzophenones disrupted photosynthesis and nitrogen metabolism of the diatom through alteration of similar pathways. The toxic effect of BP3 was also attributable to its unique inhibitory effects on eukaryotic ribosome biosynthesis and DNA replication. Taken together, our findings underscore that benzophenones may pose a significant threat to photosynthesis, oxygen production, primary productivity, carbon fixation, and the nitrogen cycle of diatom in coastal waters worldwide.

Bioaccumulation of pollutants in the green-lipped mussel Perna viridis: Assessing pollution abatement in Victoria Harbour and its adjacent aquaculture area, Hong Kong, and the minimal human health risks from mussel consumption.

The green-lipped mussel Perna viridis was utilised for pollution biomonitoring in Victoria Harbour and its adjacent aquaculture area in Hong Kong. P. viridis was collected from a reference site and redeployed at five study sites for five weeks during the dry and wet seasons of 2019. Our study found various polycyclic aromatic hydrocarbons (PAHs) and heavy metals in the mussel tissue, while polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) were not detected. P. viridis at the reference site generally displayed lower levels of pollutants. Comparing with previous research in the 1980s and 2000s, we observed substantial reduction in the tissue levels of PAHs, PCBs, OCPs and heavy metals in P. viridis. The human health risks associated with consuming these mussels were determined to be insignificant. Our findings imply that the Harbour Area Treatment Scheme has been effective in improving the water quality in Victoria Harbour and its adjacent aquaculture area.

Hypoxia in aquatic invertebrates: Occurrence and phenotypic and molecular responses.

Global deoxygenation in aquatic systems is an increasing environmental problem, and substantial oxygen loss has been reported. Aquatic animals have been continuously exposed to hypoxic environments, so-called "dead zones," in which severe die-offs among organisms are driven by low-oxygen events. Multiple studies of hypoxia exposure have focused on in vivo endpoints, metabolism, oxidative stress, and immune responses in aquatic invertebrates such as molluscs, crustaceans, echinoderms, and cnidarians. They have shown that acute and chronic exposure to hypoxia induces significant decreases in locomotion, respiration, feeding, growth, and reproduction rates. Also, several studies have examined the molecular responses of aquatic invertebrates, such as anaerobic metabolism, reactive oxygen species induction, increased antioxidant enzymes, immune response mechanisms, regulation of hypoxia-inducible factor 1-alpha (HIF-1α) genes, and differently expressed hemoglobin/hemocyanin. The genetic basis of those molecular responses involves HIF-1α pathway genes, which are highly expressed in hypoxic conditions. However, the identification of HIF-1α-related genes and understanding of their applications in some aquatic invertebrates remain inadequate. Also, some species of crustaceans, rotifers, sponges, and ctenophores that lack HIF-1α are thought to have alternative defense mechanisms to cope with hypoxia, but those factors are still unclear. This review covers the formation of hypoxia in aquatic environments and the various adverse effects of hypoxia on aquatic invertebrates. The limitations of current hypoxia research and genetic information about the HIF-1α pathway are also discussed. Finally, this paper explains the underlying processes of the hypoxia response and presents an integrated program for research about the molecular mechanisms of hypoxic stresses in aquatic invertebrates.

Chromatin modifiers: A new class of pollutants with potential epigenetic effects revealed by in vitro assays and transcriptomic analyses.

A great variety of endocrine-disrupting chemicals (EDCs) have been used extensively and become widespread in the environment nowadays. Limited mammalian studies have shown that certain EDCs may target chromosome and epigenome of the germline, leading to adverse effects in subsequent generations, despite these progenies having never been exposed to the EDC before. However, the underlying mechanisms of chromosomal changes induced by these pollutants remain poorly known. Using the human ovarian granulosa tumor cell line COV434 as a model, we investigated and compared the transcriptomic changes induced by nine EDCs with diverse chemical structures (i.e. BDE-47, BPA, BP-3, DEHP, DHP, EE2, TCS, TDCPP and NP), to inquire if there is any common epigenetic modification associated with reproductive functions induced by these EDCs. Our results showed that COV434 cells were more responsive to BP-3, NP, DEHP and EE2, and more importantly, these four EDCs altered the expression of gene clusters related to DNA damage response, cell cycle, proliferation, and chromatin remodeling, which can potentially lead to epigenetic modifications and transgenerational inheritance. Furthermore, dysregulation of similar gene clusters was common in DEHP and NP treatments. Bioinformatics analysis further revealed that BP-3 disturbed signaling pathways associated with reproductive functions, whereas alterations in telomere-related pathways were highlighted upon EE2 exposure. Overall, this study highlighted chromatin modifications caused by a class of chemicals which that may potentially lead to epigenetic changes and transgenerational reproductive impairments.

Embryo developmental toxicity in marine medaka (Oryzias melastigma) due to parental and embryonic 17α-ethinylestradiol exposure.

The synthetic estrogen 17α-ethinylestradiol (EE2) is a common component of hormone therapy and oral contraceptives and has been widely used for nearly 60 years. Numerous studies have shown that exposure to EE2 can affect embryonic development in a number of fish species. The effects of parental and embryonic EE2 exposure on embryo developmental toxicity and the underlying molecular mechanisms, however, have rarely been examined. In this study, embryos collected from parental EE2-exposed adult fish were examined to assess EE2-induecd toxicity during embryo development. The rate of embryo development including heart rate, hatching rate, and larval locomotion were measured to assess embryo developmental toxicity. The embryonic transcriptome was used to delineate the related developmental toxicity pathways. Our results suggest that parental and embryonic EE2 exposure resulted in growth retardation including a reduction in embryo heart rate, a delay in the appearance eye pigmentation, decreased hatching rate and impaired larval locomotion. In addition, gene ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Ingenuity Pathway Analysis (IPA) of transcriptome revealed that these impairments are controlled by estrogen receptor and related to eye structure, neuronal and synaptic structure, and behaviour. The key factors identified, including PRKAA2, APOB, EPHB2, OXTR, NR2E3, and POU4F2, could serve as biomarkers for assessing EE2-induced embryo developmental toxicity. For the first time, our results show that eye pigmentation is a potentially sensitive marker of EE2-induced embryo developmental toxicity.

Continuous 17α-ethinylestradiol exposure impairs the sperm quality of marine medaka (Oryzias melastigma).

17α-ethinylestradiol (EE2) is an anthropogenic estrogen that is widely used for hormone therapy and oral contraceptives. It was reported that EE2 exposure induced reproductive impairments through processes affecting reproduction behavior and inducing ovotestis. However, the effects of continuous EE2 exposure on the reproductive performance remain largely unknown. In this study, adult marine medaka fish (Oryzias melastigma) were exposed to EE2 (85 ng/L) for one (F0) and two (F1) generations. Our results indicate that continuous EE2 exposure reduced fecundity and sperm motility. The testicular transcriptome, followed by bioinformatic analysis revealed the dysregulation of pathways related to steroidogenesis, sperm motility, and reproductive system development. Collectively, our findings indicate that continuous EE2 exposure directly affected sperm quality via the alteration of steroidogenesis and dysregulation of reproductive system development. The identified key factors including DNM1, PINK1, PDE7B, and SLC12A7 can serve as biomarkers to assess EE2-reduced sperm motility.

Corrigendum: Low-Dose Radiation Can Cause Epigenetic Alterations Associated With Impairments in Both Male and Female Reproductive Cell.

[This corrects the article DOI: 10.3389/fgene.2021.710143.].

Low-Dose Radiation Can Cause Epigenetic Alterations Associated With Impairments in Both Male and Female Reproductive Cells.

Humans are regularly and continuously exposed to ionizing radiation from both natural and artificial sources. Cumulating evidence shows adverse effects of ionizing radiation on both male and female reproductive systems, including reduction of testis weight and sperm count and reduction of female germ cells and premature ovarian failure. While most of the observed effects were caused by DNA damage and disturbance of DNA repairment, ionizing radiation may also alter DNA methylation, histone, and chromatin modification, leading to epigenetic changes and transgenerational effects. However, the molecular mechanisms underlying the epigenetic changes and transgenerational reproductive impairment induced by low-dose radiation remain largely unknown. In this study, two different types of human ovarian cells and two different types of testicular cells were exposed to low dose of ionizing radiation, followed by bioinformatics analysis (including gene ontology functional analysis and Ingenuity Pathway Analysis), to unravel and compare epigenetic effects and pathway changes in male and female reproductive cells induced by ionizing radiation. Our findings showed that the radiation could alter the expression of gene cluster related to DNA damage responses through the control of MYC. Furthermore, ionizing radiation could lead to gender-specific reproductive impairment through deregulation of different gene networks. More importantly, the observed epigenetic modifications induced by ionizing radiation are mediated through the alteration of chromatin remodeling and telomere function. This study, for the first time, demonstrated that ionizing radiation may alter the epigenome of germ cells, leading to transgenerational reproductive impairments, and correspondingly call for research in this new emerging area which remains almost unknown.

Hypoxia causes sex-specific hepatic toxicity at the transcriptome level in marine medaka (Oryzias melastigma).

Hypoxia, a low environmental oxygen level, is a common problem in the ocean globally. Hypoxia has been known to cause disruption to the endocrine system of marine organisms in both laboratory and field studies. Our previous studies have demonstrated the sex-specific response to hypoxia in the neural and reproductive systems of marine fish. In the current report, we aim to study the sex-specific hepatic response of fish at the transcriptome level to hypoxic stress. By using a comparative transcriptome analysis, followed by a systematic bioinformatics analysis including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA), we found that hypoxia altered expression of genes related to cell proliferation and apoptosis of hepatocytes, which are associated with human pathologies, such as liver inflammation hepatic steatosis and steatohepatitis. Furthermore, we observed sex-specific responses in the livers of fish through different cell signaling pathways. In female fish, hypoxia causes dysregulation of expression of genes related to impairment in endoplasmic reticulum structure and liver metabolism. In male fish, genes associated with redox homeostasis and fatty acid metabolism were altered by hypoxic stress. The findings of this study support the notion that hypoxia could cause sex-specific changes (hepatic toxicity and changes) in marine fish.

Ultra violet filters in the urine of preschool children and drinking water.

Benzophenones (BPs) and other ultra violet (UV) filters (UV-filters) are widely used in sunblock and other personal care products, raising concerns about their adverse health risks to human, especially for children. In the present study, BP-type UV-filters and other four widely used UV-filters were evaluated in the child urinary samples (4-6 years, n = 53), tap water and commercial distilled water in Hong Kong. The results suggested that the target chemicals are ubiquitous in the subject. BP1, BP2, BP3 and BP4 in children urine samples contributed closely to the overall children exposure of UV filters, with detection rates above 58% and geometric means ranging from 44.2 to 76.7 ng/mL. As a contrast, BP3 was the major substance found in the tap water and distilled bottle water, with detection rates of 100% and geometric means of 9.64 and 14.5 ng/L, respectively. There were some significant relationships between urinary UV filters and personal characteristics (BMI values, sex, income level, hand washing frequency, and body location usage), but the health risks associated with UV-filters in Hong Kong children might not be concerning. Only two children applied sun creams in this research, indicating that there were other sources to exposure these chemicals.

Construction of High-Resolution RAD-Seq Based Linkage Map, Anchoring Reference Genome, and QTL Mapping of the Sex Chromosome in the Marine Medaka Oryzias melastigma.

Medaka (Oryzias sp.) is an important fish species in ecotoxicology and considered as a model species due to its biological features including small body size and short generation time. Since Japanese medaka Oryzias latipes is a freshwater species with access to an excellent genome resource, the marine medaka Oryzias melastigma is also applicable for the marine ecotoxicology. In genome era, a high-density genetic linkage map is a very useful resource in genomic research, providing a means for comparative genomic analysis and verification of de novo genome assembly. In this study, we developed a high-density genetic linkage map for O. melastigma using restriction-site associated DNA sequencing (RAD-seq). The genetic map consisted of 24 linkage groups with 2,481 single nucleotide polymorphism (SNP) markers. The total map length was 1,784 cM with an average marker space of 0.72 cM. The genetic map was integrated with the reference-assisted chromosome assembly (RACA) of O. melastigma, which anchored 90.7% of the assembled sequence onto the linkage map. The values of complete Benchmarking Universal Single-Copy Orthologs were similar to RACA assembly but N50 (23.74 Mb; total genome length 779.4 Mb; gap 5.29%) increased to 29.99 Mb (total genome length 778.7 Mb; gap 5.2%). Using MapQTL analysis with SNP markers, we identified a major quantitative trait locus for sex traits on the Om10. The integration of the genetic map with the reference genome of marine medaka will serve as a good resource for studies in molecular toxicology, genomics, CRISPR/Cas9, and epigenetics.

Hypoxia Causes Transgenerational Impairment of Ovarian Development and Hatching Success in Fish.

Hypoxia is a pressing environmental problem in both marine and freshwater ecosystems globally, and this problem will be further exacerbated by global warming in the coming decades. Recently, we reported that hypoxia can cause transgenerational impairment of sperm quality and quantity in fish (in F0, F1, and F2 generations) through DNA methylome modifications. Here, we provide evidence that female fish ( Oryzias melastigma) exposed to hypoxia exhibit reproductive impairments (follicle atresia and retarded oocyte development), leading to a drastic reduction in hatching success in the F2 generation of the transgenerational group, although they have never been exposed to hypoxia. Further analyses show that the observed transgenerational impairments in ovarian functions are related to changes in the DNA methylation and expression pattern of two gene clusters that are closely associated with stress-induced cell cycle arrest and cell apoptosis. The observed epigenetic and transgenerational alterations suggest that hypoxia may pose a significant threat to the sustainability of natural fish populations.

Transcriptomic analysis reveals transgenerational effect of hypoxia on the neural control of testicular functions.

There are over 400 hypoxic zones in the ocean worldwide. Both laboratory and field studies have shown that hypoxia causes endocrine disruption and reproductive impairments in vertebrates. More importantly, our recent study discovered that parental (F0) hypoxia exposure resulted in the transgenerational impairment of sperm quality in the F2 generation through the epigenetic regulation of germ cells. In the present study, we aim to test the hypothesis that the brain, as the major regulator of the brain-pituitary-gonad (BPG) axis, is also involved in the observed transgenerational effect. Using comparative transcriptomic analysis on brain tissues of marine medaka Oryzias melastigma, 45 common differentially expressed genes caused by parental hypoxia exposure were found in the hypoxic group of the F0 and F2 generations, and the transgenerational groups of the F2 generation. The bioinformatic analysis on this deregulated gene cluster further highlighted the possible involvement of the brain in the transgenerational effect of hypoxia on testicular structure, including abnormal morphologies of the epididymis and the seminal vesicle, and degeneration of the seminiferous tubule. This finding is concordant to the result of hematoxylin and eosin staining, which showed the reduction of testicular lobular diameter in the F0 and F2 generations. Our study demonstrated for the first time the involvement of the brain in the transgenerational effect of hypoxia.

Overexpression and Knockdown of Hypoxia-Inducible Factor 1 Disrupt the Expression of Steroidogenic Enzyme Genes and Early Embryonic Development in Zebrafish.

Hypoxia is an important environmental stressor leading to endocrine disruption and reproductive impairment in fish. Although the hypoxia-inducible factor 1 (HIF-1) is known to regulate the transcription of various genes mediating oxygen homeostasis, its role in modulating steroidogenesis-related gene expression remains poorly understood. In this study, the regulatory effect of HIF-1 on the expression of 9 steroidogenic enzyme genes was investigated in zebrafish embryos using a "gain-of-function and loss-of-function" approach. Eight of the genes, CYP11a, CYP11b2, 3ß-HSD, HMGCR, CYP17a1, 17ß-HSD2, CYP19a, and CYP19b, were found to be differentially upregulated at 24 and 48 hpf following zHIF-1α-ΔODD overexpression (a mutant zebrafish HIF-1α protein with proline-414 and proline-557 deleted). Knockdown of zHIF-1α also affected the expression pattern of the steroidogenic enzyme genes. Overexpression of zHIF-1α and hypoxia exposure resulted in downregulated StAR expression but upregulated CYP11a and 3ß-HSD expression in zebrafish embryos. Conversely, the expression patterns of these 3 genes were reversed in embryos in which zHIF-1α was knocked down under normoxia, suggesting that these 3 genes are regulated by HIF-1. Overall, the findings from this study indicate that HIF-1-mediated mechanisms are likely involved in the regulation of specific steroidogenic genes.

Hypoxia alters testicular functions of marine medaka through microRNAs regulation.

Hypoxia is a global environmental concern and poses a significant threat to aquatic ecosystems, including the sustainability of natural fish populations. The deleterious effects of hypoxia on fish reproductive fitness, as mediated by disruption of sex hormones and gene expression along the Brain-Pituitary-Gonad axis, have been well documented. Recently, we further demonstrated that the observed disruption of steroidogenesis in the ovary of marine medaka Oryzias melastigma is mediated through microRNAs (miRNAs). More importantly, we reported the transgenerational epigenetic effect of hypoxia on the male reproductive impairment of marine medaka. This study attempts to elucidate the function of miRNAs and its potential role in the transgenerational effect of hypoxia in the male medaka testis, using small RNA sequencing. A total of 558 miRNAs were found in the testis, of which 9 were significant upregulated and 5 were downregulated by hypoxia. Bioinformatics analysis further revealed that among the 2885 genes targeted by the hypoxia-responsive miRNAs, many are closely related to stress response, cell cycle, epigenetic modification, sugar metabolism and cell motion. Furthermore, the integrated analysis of transcriptome data and the result of target gene prediction demonstrated 108 genes and 65 genes were concordantly upregulated and downregulated, respectively. In which, euchromatic histone-lysine N-methyltransferase 2, the epigenetic regulator of transgenerational reproductive impairment caused by hypoxia, is found to be targeted by miR-125-5p. The present findings not only reveal that miRNAs are crucial downstream mediators of hypoxic stress in fish male gonad, but also shed light on the underlying epigenetic mechanism for the reproductive impairments of hypoxia on male fish, including the observed transgenerational effects.

Hypoxia causes transgenerational impairments in reproduction of fish.

Hypoxia is amongst the most widespread and pressing problems in aquatic environments. Here we demonstrate that fish (Oryzias melastigma) exposed to hypoxia show reproductive impairments (retarded gonad development, decrease in sperm count and sperm motility) in F1 and F2 generations despite these progenies (and their germ cells) having never been exposed to hypoxia. We further show that the observed transgenerational reproductive impairments are associated with a differential methylation pattern of specific genes in sperm of both F0 and F2 coupled with relevant transcriptomic and proteomic alterations, which may impair spermatogenesis. The discovered transgenerational and epigenetic effects suggest that hypoxia might pose a dramatic and long-lasting threat to the sustainability of fish populations. Because the genes regulating spermatogenesis and epigenetic modifications are highly conserved among vertebrates, these results may also shed light on the potential transgenerational effects of hypoxia on other vertebrates, including humans.

Transcriptomic responses of marine medaka's ovary to hypoxia.

Hypoxia, an endocrine disruptor, is pressing global problem affecting marine organisms in over 400 "Dead Zones" worldwide. There is growing evident demonstrated the disruptive effect of hypoxia on reproductive systems of marine fish through the impairments of steroidogenic gene expression, leading to the alteration of sex hormone production in gonads. But the detailed molecular mechanism underlying the responses of female reproductive systems to hypoxic stress remains largely unknown. In the present report, we used marine medaka Oryzias melastigma as a model, together with high-throughput transcriptome sequencing and bioinformatics analysis, aiming to determine the changes in transcriptional signature in the ovary of marine fish under hypoxic stress. Our result discovered over two hundred differential expressed genes in ovary in response to hypoxia. The bioinformatics analysis together with quantitative RT-PCR validation on the deregulated genes highlighted the dysregulations of a number of female reproductive functions including interruptions of ovarian follicle development, gonad development and steroid metabolic process. Additionally, we revealed that these deregulations are through the modulation of leukemia inhibitory factor (LIF), insulin-like growth factor 1 receptor (IGF1R) and follicle stimulating hormone (FSH). The result of this work complements previous studies and provides additional insights into the underlying molecular mechanism of hypoxia-induced impairment of female reproductive system.

Discovery and functional characterization of novel miRNAs in the marine medaka Oryzias melastigma.

The marine medaka Oryzias melastigma has often been used as a marine fish model to investigate the biological responses to environmental stresses and pollutants in marine environments. miRNAs are post-transcriptional regulators of many biological processes in a variety of organisms, and have been shown to be affected by environmental stresses, but the novel miRNA profile of marine medaka has not been reported. Using both genome and small RNA sequencings coupled with different bioinformatics analyses, we have discovered 58, 82, 234, and 201 unannotated miRNAs in the brain, liver, ovary and testis tissues of marine medaka, respectively. Furthermore, these novel miRNAs were found to target genes with tissue-specific roles such as neuron development and synaptic transmission in the brain, glucose and fat metabolism in the liver and steroidogenesis in the gonads. We here report, for the first time, novel miRNA profile of marine medaka, which will provide a foundation for future biomarkers and transgenerational studies for the assessment of environmental stresses and pollutions in the marine environments. In a boarder context, our data will provide novel insight into our knowledge of miRNome and miR research.

Hypoxia alters steroidogenesis in female marine medaka through miRNAs regulation.

Hypoxia is a worldwide environmental problem in marine ecosystems, leading to serious declines in fishery production over large areas. Our previous studies demonstrated that hypoxia is an endocrine disruptor which can cause reproductive impairment through the regulation of miRNAs, suggesting the functional role of miRNAs in reproductive systems in response to hypoxia. In this study, we used small RNA sequencing to determine the change in miRNA profile in ovary of marine medaka Oryzias melastigma under hypoxic stress. A total of 509 miRNAs were found in the ovary of marine medaka, in which, 33 and 10 miRNAs were found to be statistically significant upregulated and downregulated under hypoxia, respectively. Bioinformatics analysis highlighted that a large number of hypoxia-suppressed miRNAs that target a variety of steroidogenic enzymes including steroidogenic acute regulatory protein, aromatase, and 17-alpha-monooxygenase. Also, estrogen receptor 2 and androgen receptor were found to be targeted by hypoxia-responsive miRNAs. For the first time, our results showed that hypoxia may upregulate specific steroidogenic enzymes and hormone receptors through actions of miRNA, and hence provide a novel mechanism for the observed female reproductive impairment caused by hypoxia.

Differential responses of female and male brains to hypoxia in the marine medaka Oryzias melastigma.

Hypoxia, an endocrine disruptor, affects synthesis and balance of sex steroid hormones, leading to reproductive impairment in both female and male fish. Cumulating reports demonstrated the alternation of hypothalamus-pituitary-gonad axis (HPG-axis) by hypoxia. However, the detail mechanism underlying how hypoxia may alter other brain functions remains largely unknown. In this report, we used marine medaka as a model and conducted a high-throughput RNA sequencing followed by bioinformatics analysis on hypoxia-exposed brain tissues, aiming to determine the change of transcriptional signature and to unravel the pathways that are induced by hypoxia. We found that hypoxia lead to dysregulation of brain functions (including synaptic transmission, axon guidance, potassium ion transport, neuron differentiation, and development of brain and pituitary gland), and also signaling pathways (e.g., gap junction, calcium signaling pathway, and GnRH signaling pathway). Our results further demonstrate gender-specific responses to hypoxia in female and male fish's brains, which provides novel insights into the mechanism underlying the hypoxia induced sex specific brain functions impairments.