Oxidative damage to mitochondrial DNA in maternal zebrafish (Danio rerio) exposed to dibutyl phthalate at environmentally relevant level.

Dibutyl phthalate (DBP) is a widely-used plasticizer that is dispersed in various environments, causing significant pollution and health risks. The toxic mechanism of DBP has been discussed in recent years, while the susceptibility of mitochondrial DNA (mtDNA) to DBP exposure and the resulting damage remain unclear. In this study, maternal zebrafish were exposed to environmentally relevant concentration of DBP for 0, 2, 4, and 6 weeks. Results showed that DBP exposure impaired health status, leading to the reduced body length and weight, condition factor, hepatosomatic index, and gonadosomatic index. Furthermore, DBP exposure induced oxidative stress and ATP deficiency in the gill and liver in a time-dependent manner. The oxidized mtDNA (ox-mtDNA) levels in the D-loop and ND1 regions were assessed in different tissues, showing distinct response patterns. The high energy-consuming tissues such as heart, brain, gill, and liver exhibited elevated susceptibility to mitochondrial damage, with a rapid increase in ox-mtDNA levels in the short term. Conversely, in muscle, ovary, eggs, and offspring, ox-mtDNA gradually accumulated over the exposure period. Notably, the ox-mtDNA levels in the D-loop region of blood showed a prompt response to DBP exposure, making it convenient for evaluation. Additionally, decreased hatching rates, increased mortality, lipoperoxidation, and depressed swimming performance were observed in offspring following maternal DBP exposure, suggesting the inherited impairments of maternal mtDNA. These findings highlight the potential for ox-mtDNA to serve as a convenient biomarker for environmental contamination, aiding in ecological risk assessment and forewarning systems in aquatic environment.

Analysis of morphology, histology characteristics, and circadian clock gene expression of Onychostoma macrolepis at the overwintering period and the breeding period.

Fish typically adapt to their environment through evolutionary traits, and this adaptive strategy plays a critical role in promoting species diversity. Onychostoma macrolepis is a rare and endangered wild species that exhibits a life history of overwintering in caves and breeding in mountain streams. We analyzed the morphological characteristics, histological structure, and expression of circadian clock genes in O. macrolepis to elucidate its adaptive strategies to environmental changes in this study. The results showed that the relative values of O. macrolepis eye diameter, body height, and caudal peduncle height enlarged significantly during the breeding period. The outer layer of the heart was dense; the ventricular myocardial wall was thickened; the fat was accumulated in the liver cells; the red and white pulp structures of the spleen, renal tubules, and glomeruli were increased; and the goblet cells of the intestine were decreased in the breeding period. In addition, the spermatogenic cyst contained mature sperm, and the ovaries were filled with eggs at various stages of development. Throughout the overwintering period, the melano-macrophage center is located between the spleen and kidney, and the melano-macrophage center in the cytoplasm has the ability to synthesize melanin, and is arranged in clusters to form cell clusters or white pulp scattered in it. Circadian clock genes were identified in all organs, exhibiting significant differences between the before/after overwintering period and the breeding period. These findings indicate that the environment plays an important role in shaping the behavior of O. macrolepis, helping the animals to build self-defense mechanisms during cyclical habitat changes. Studying the morphological, histological structure and circadian clock gene expression of O. macrolepis during the overwintering and breeding periods is beneficial for understanding its unique hibernation behavior in caves. Additionally, it provides an excellent biological sample for investigating the environmental adaptability of atypical cavefish species.

Mitochondrial DNA Stress-Mediated Health Risk to Dibutyl Phthalate Contamination on Zebrafish (Danio rerio) at Early Life Stage.

Plastics contaminations are found globally and fit the exposure profile of the planetary boundary threat. The plasticizer of dibutyl phthalate (DBP) leaching has occurred and poses a great threat to human health and the ecosystem for decades, and its toxic mechanism needs further comprehensive elucidation. In this study, environmentally relevant levels of DBP were used for exposure, and the developmental process, oxidative stress, mitochondrial ultrastructure and function, mitochondrial DNA (mtDNA) instability and release, and mtDNA-cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway with inflammatory responses were measured in zebrafish at early life stage. Results showed that DBP exposure caused developmental impairments of heart rate, hatching rate, body length, and mortality in zebrafish embryo. Additionally, the elevated oxidative stress damaged mitochondrial ultrastructure and function and induced oxidative damage to the mtDNA with mutations and instability of replication, transcription, and DNA methylation. The stressed mtDNA leaked into the cytosol and activated the cGAS-STING signaling pathway and inflammation, which were ameliorated by co-treatment with DBP and mitochondrial reactive oxygen species (ROS) scavenger, inhibitors of cGAS or STING. Furthermore, the larval results suggest that DBP-induced mitochondrial toxicity of energy disorder and inflammation were involved in the developmental defects of impaired swimming capability. These results enhance the interpretation of mtDNA stress-mediated health risk to environmental contaminants and contribute to the scrutiny of mitochondrial toxicants.

Species distribution modeling combined with environmental DNA analysis to explore distribution of invasive alien mosquitofish (Gambusia affinis) in China.

China has become one of the most serious countries suffering from biological invasions in the world. In the context of global climate change, invasive alien species (IAS) are likely to invade a wider area, posing greater ecological and economic threats in China. Western mosquitofish (Gambusia affinis), which is known as one of the 100 most invasive alien species, has distributed widely in southern China and is gradually spreading to the north, causing serious ecological damage and economic losses. However, its distribution in China is still unclear. Hence, there is an urgent need for a more convenient way to detect and monitor the distribution of G. affinis to put forward specific management. Therefore, we detected the distribution of G. affinis in China under current and future climate change by combing Maxent modeling prediction and eDNA verification, which is a more time-saving and reliable method to estimate the distribution of species. The Maxent modeling showed that G. affinis has a broad habitat suitability in China (especially in southern China) and would continue to spread in the future with ongoing climate change. However, eDNA monitoring showed that occurrences can already be detected in regions that Maxent still categorized as unsuitable. Besides temperature, precipitation and human influence were the most important environmental factors affecting the distribution of G. affinis in China. In addition, by environmental DNA analysis, we verified the presence of G. affinis predicted by Maxent in the Qinling Mountains where the presence of G. affinis had not been previously recorded.

Dibutyl phthalate exposure induced mitochondria-dependent ferroptosis by enhancing VDAC2 in zebrafish ZF4 cells.

Dibutyl phthalate (DBP) contamination has raised global concern for decades, while its health risk with toxic mechanisms requires further elaboration. This study used zebrafish ZF4 cells to investigate the toxicity of ferroptosis with underlying mechanisms in response to DBP exposure. Results showed that DBP induced ferroptosis, characterized by accumulation of ferrous iron, lipid peroxidation, and decrease of glutathione peroxidase 4 levels in a time-dependent manner, subsequently reduced cell viability. Transcriptome analysis revealed that voltage-dependent anion-selective channel (VDAC) in mitochondrial outer membrane was upregulated in ferroptosis signaling pathways. Protecting mitochondria with a VDAC2 inhibitor or siRNAs attenuated the accumulation of mitochondrial superoxide and lipid peroxides, the opening of mitochondrial permeability transition pore (mPTP), and the overload of iron levels, suggesting VDAC2 oligomerization mediated the influx of iron into mitochondria that is predominant and responsible for mitochondria-dependent ferroptosis under DBP exposure. Furthermore, the pivotal role of activating transcription factor 4 (ATF4) was identified in the transcriptional regulation of vdac2 by ChIP assay. And the intervention of atf4b inhibited DBP-induced VDAC2 upregulation and oligomerization. Taken together, this study reveals that ATF4-VDAC2 signaling pathway is involved in the DBP-induced ferroptosis in zebrafish ZF4 cells, contributing to the in-depth understanding of biotoxicity and the ecological risk assessment of phthalates.

Exploring the toxicity of biodegradable microplastics and imidacloprid to earthworms (Eisenia fetida) from morphological and gut microbial perspectives.

Biodegradable microplastics (BMPs) pose serious environmental problems to soil organisms, and their adsorption capacity might make pesticides more dangerous for soil organisms. Therefore, in this study, polylactic acid (PLA) BMPs and imidacloprid (IMI) were used as a representative of BMPs and pesticides, respectively. Eisenia fetida was used as a test animal to investigate the effects of environmentally relevant concentrations of single and compound contaminated PLA BMPs and IMI on mortality, growth, number of offspring, tissue damage, and gut microorganisms of E.fetida. Exposure to PLA BMPs treatment and PLA BMPs + IMI treatment resulted in a sustained increase in E.fetida mortality, reaching 16.7% and 26.7%, respectively. The growth inhibition rate of single treatments was significantly increased. The compound contamination had the greatest effect on E.fetida offspring compared to the control. PLA BMPs and IMI cause histological damage to E.fetida, with the compound treatment causing the most severe damage. Based on the results of 16S sequencing, the bacterial communities in E.fetida gut and soil treated to PLA BMPs and IMI were significantly different. PLA BMPs + IMI treatment suppresses the abundance and diversity of E.fetida gut microorganisms, disrupting the homeostasis of bacterial communities and causing immune and metabolic dysfunction. These findings highlight the more severe damage of combined PLA BMPs and IMI pollution to E.fetida, and help to assess the risk of earthworm exposure to environmentally relevant concentrations of PLA BMPs and IMI.

Insight into the health risk implicated in mitochondrial toxicity of dibutyl phthalate exposure on zebrafish (Danio rerio) cells.

Dibutyl phthalate (DBP) is commonly applied plasticizer in plastic products such as face masks, easily leaches or migrates into environment and its widespread contamination posed profound health risks. Further concerns rise regarding to the toxicity of DBP at subcellular level, while little is known about the ranging effects on mitochondrial susceptibility. Present study investigated the mitochondrial impairments with implicated cell death upon DBP exposure on zebrafish cells. Elevated mitochondrial oxidative stress reduced its membrane potential and count, enhanced fragmentation, and impaired ultrastructure that showed smaller size and cristae rupture. Afterwards, the critical function of ATP synthesis was damaged and the stabilized binding capacity between DBP with mitochondrial respiratory complexes was simulated by the molecular docking. And the top pathways enrichment of mitochondrion and metabolism by transcriptome analyses verified the mitochondrial dysfunction that indicated the human diseases risks. The mitochondrial DNA (mtDNA) replication and transcription with DNA methylation modifications were also disrupted, reflecting the genotoxicity on mtDNA. Moreover, the activated autophagy and apoptosis underlying mitochondrial susceptibility integrated into cellular homeostasis changes. These findings provide the first systemic evidence broadening and illustrating the mitochondrial toxicity of DBP exposure on zebrafish model that raise concern on phthalates contamination and ecotoxicological evaluation.

Oxidative stress and gene expression induced by biodegradable microplastics and imidacloprid in earthworms (Eisenia fetida) at environmentally relevant concentrations.

The environmental issues caused by biodegradable microplastics (BMPs) from polylactic acid (PLA) as well as pesticides are of increasing concern nowadays. In this study, the toxicological effects of the single and combined exposure of PLA BMPs and imidacloprid (IMI), a neonicotinoid insecticide, on earthworms (Eisenia fetida) were investigated in terms of oxidative stress, DNA damage, and gene expression, respectively. The results showed that compared with the control, SOD, CAT and AChE activities in the single and combined treatments decreased significantly, and POD activity showed an "inhibition-activation" trend. SOD and CAT activities of combined treatments on day 28 and AChE activity of combined treatment on day 21 were significantly higher than those of the single treatments. For the rest of the exposure period, SOD, CAT and AChE activities in the combined treatments were lower than those in the single treatments. POD activity in the combined treatment was significantly lower than those of single treatments at day 7 and higher than that of single treatments at day 28. MDA content showed an "inhibition-activation-inhibition" trend, and the ROS level and 8-OHdG content increased significantly in both the single and combined treatments. This shows that both single and combined treatments led to oxidative stress and DNA damage. ANN and HSP70 were expressed abnormally, while the SOD and CAT mRNA expression changes were generally consistent with the corresponding enzyme activities. The integrated biomarker response (IBR) values were higher under combined exposures than single exposures at both biochemical and molecular levels, indicating that combined treatment exacerbated the toxicity. However, the IBR value of the combined treatment decreased consistently at the time axis. Overall, our results suggest that PLA BMPs and IMI induce oxidative stress and gene expression in earthworms at environmentally relevant concentrations, thereby increasing the risk of earthworms.

Ecological risk assessment of environmentally relevant concentrations of propofol on zebrafish (Danio rerio) at early life stage: Insight into physiological, biochemical, and molecular aspects.

Propofol is an intravenous anesthetic injection extensively used in clinic, which has been proved to be neurotoxic in humans. Improper use and disposal of propofol may lead to its release into the aquatic environment, but the potential ecological risk of propofol to aquatic organisms remains poorly understood. For this study, we comprehensively explored the ecotoxicological effects and potential mechanisms of propofol (0.04, 0.2 and 2 mg L-1) on 120 hpf zebrafish (Danio rerio) embryos from physiological, biochemical, and molecular perspectives. The results showed that propofol has moderate toxicity on zebrafish embryos (96 h LC50 = 4.260 mg L-1), which could significantly reduce the hatchability and delay the development. Propofol can trigger reactive oxygen species (ROS) generation, lipid peroxidation (Malondialdehyde, MDA) and DNA damage (8-hydroxy-2-deoxyguanosine, 8-OHdG). The glutathione peroxidase (GPX) activity of zebrafish embryos in 0.04 and 0.2 mg L-1 propofol treatment group was activated in response to oxidative damage, while activities of superoxide dismutase (SOD), catalase (CAT) and GPX in zebrafish treated with 2 mg L-1 was significant inhibited compared with the control group (p＜0.05). Moreover, the expression of antioxidant genes and related pathways was inhibited. Apoptosis was investigated at genes level and histochemistry. Molecular docking confirmed that propofol could change in the secondary structure of acetylcholinesterase (AChE) and competitively inhibited acetylcholine (ACh) binding to AChE, which may disturb the nervous system. These results described toxic response and molecular mechanism in zebrafish embryos, providing multiple aspects about ecological risk assessment of propofol in water environment.

Mitochondrial changes in fish cells in vitro in response to serum deprivation.

Mitochondria are critical to cellular activity that implicated in expansive networks to maintain organismal homeostasis under external stimuli of nutrient variability, a common and severe stress to fish performance during the intensive culture conditions. In the present study, zebrafish embryonic fibroblast cells were used to investigate the fish mitochondrial changes upon serum deprivation. Results showed that mitochondrial content and membrane potential were significantly reduced with increased intracellular ROS level in the serum deprivation treated fish cells. And the impaired mitochondria were characterized by rough and fracted outer membrane, and more fused mitochondria were frequently observed with the upregulated mRNA expressions of mitochondrial fusion genes (mfn1b, mfn2, and opa1). Besides, the mitochondrial DNA (mtDNA) copy numbers of mtatp6, mtcox1, mtcytb, mtnd4, and mtnd6 were overall showing the highly significant reduction, together with the mRNA expressions of these genes significantly increased, exhibiting the compensatory effects in mitochondria. Furthermore, the methyl-cytosine of whole mtDNA was compared and the methyl-reads numbers were distinctly increased in the treatment group, reflecting the instability of fish mtDNA with mitochondrial dysfunction under nutrient fluctuations. Collectively, current findings could facilitate the integrated research between fish mitochondrial response and external variables that indicates the potentially profound and durative deficits in fish health during the aquaculture processes.

Anthropogenic impacts on antibiotic resistance genes and their hosts from pristine to urban river using metagenomic and binning approaches.

Driven by anthropogenic pressure, Antibiotic resistance genes (ARGs) could transfer from the environmental resistome into human commensals or even pathogens. The transport of ARGs through aquatic ecosystems is crucial and has attracted attention. Here, we employed metagenomic and binning to compare ARGs profiles, their co-occurrence with metal resistance genes (MRGs) and mobile genetic elements (MGEs), and their hosts between pristine and anthropogenic influenced rivers and explore the ecological mechanisms underlying the dissemination of ARGs induced by anthropogenic activities. The significantly increased relative abundance of macrolide-lincosamide-streptogramins, vancomycin, ß-lactam and sulfonamide resistance genes along the environmental gradient from pristine to polluted sediments implied that anthropogenic impact aided the emergence and dissemination of certain ARGs. At the lower reach of the Ba River, the higher ratios for contigs carrying more than one ARG suggested that anthropogenic pollution favored the co-occurrence of multiple ARGs. Anthropogenic pressures also increased the relative abundance of advantaged hosts, including Chloroflexi, Firmicutes and Euryarchaeota. At the lower reach of Ba River, Romboutsia timonensis carrying multiple ARGs and ICEs were successfully recovered, posing a serious threat to human health by affecting the metabolism of gut microbiomes. And Methanothrix soehngenii affiliated to archaea carrying multiple ARGs, MRGs and ICEs were also recovered from the lower Ba River. The partial least squares path modeling revealed that MGEs were the most predominant factors inducing the ARG profiles, and the antibiotic resistance could be enriched by co-transfer with MRGs. Furthermore, environmental factors could impact the ARG profiles indirectly by first influencing the ARGs' hosts.

Biofilm characteristics and transcriptomic profiling of Acinetobacter johnsonii defines signatures for planktonic and biofilm cells.

Most bacteria in the natural environment have a biofilm mode of life, which is intrinsically tolerant to antibiotics. While until now, the knowledge of biofilm formation by Acinetobacter johnsonii is not well understood. In this study, the characteristics and the effect of a sub-inhibitory concentration of antibiotic on A. johnsonii biofilm and planktonic cells were determined. We discovered a positive relationship between biofilm formation and tetracycline resistance, and biofilms rapidly evolve resistance to tetracycline they are treated with. Persister cells commonly exist in both planktonic and biofilm cells, with a higher frequency in the latter. Further transcriptomic analysis speculates that the overexpression of multidrug resistance genes and stress genes were mainly answered to sub lethal concentration of tetracycline in planktonic cells, and the lower metabolic levels after biofilm formation result in high resistance level of biofilm cells to tetracycline. Altogether, these data suggest that A. johnsonii can adjust its phenotype when grown as biofilm and change its metabolism under antibiotic stress, and provide implications for subsequent biofilm control.

Chronic exposure to Bisphenol A resulted in alterations of reproductive functions via immune defense, oxidative damage and disruption DNA/histone methylation in male rare minnow Gobiocypris rarus.

Bisphenol A (BPA) is a widely used chemical that represents a reproductive hazard in fish. However, the molecular pathways mediating reproductive toxicity under chronic BPA exposure remain unclear. To study the reproductive hazards associated with chronic BPA exposure, adult male rare minnows (Gobiocypris rarus) were treated with 15 µg L - 1 and 225 µg L - 1 BPA for 90 days. Results showed that chronic BPA treatment induced reproductive impairments with decreased fertilization capacity and movement time of sperm. Transcriptome analysis indicated 1421 transcripts that were differentially expressed in response to BPA exposure, which are involved in the biological process of oxidative stress, immune responses and DNA/histone methylation. BPA caused the oxidative stress via significantly increasing hydrogen peroxide (H2O2) levels and inhibiting the activities of antioxidant-related enzymes (Catalase, CAT). BPA caused an inflammatory response in the testes by significantly increasing IL-1ß levels and inducing infiltration of inflammatory cells. Moreover, exposure to 15 µg L - 1 BPA significantly decreased the genomic DNA methylation level. These data revealed that chronic BPA exposure had adverse effects on male reproduction. Oxidative stress, inflammatory response and DNA/histone methylation might account for the decreased sperm quality.

Phenotype profiles and adaptive preference of Acinetobacter johnsonii isolated from Ba River with different environmental backgrounds.

Acinetobacter johnsonii is a potentially opportunistic pathogen widely distributed in nosocomial and natural environments, but little attention has been paid to this bacillus. Here A. johnsonii strains from Ba River with different pollution levels were isolated. In this study, we found that the increasing anthropogenic contaminants accounted for the emergence of multidrug-resistant (MDR) A. johnsonii strains. Correlation analysis results showed that the resistance phenotype of strains could be generated by co-selection of heavy metals or non-corresponding antibiotics. The whole genome sequence analysis showed that the relative heavy pollution of water selects strains containing more survival-relevant genes. We found that only some genes like blaOXA-24 were responsible for its corresponding resistance profile. Additionally, the tolerance profiles toward heavy metals also attribute to the expression of efflux pumps rather than corresponding resistance genes. In summary, our finding revealed that the resistance profiles of A. johnsonii could be generated by cross or co-selection of anthropogenic contaminants and mediated by efflux pumps instead of corresponding resistance determinants. Our study also has deep-sight into the adaptive preference of bacteria in natural environments, and contributes to surveillance studies and MDR- A. johnsonii monitoring worldwide.

Stress responses of testicular development, inflammatory and apoptotic activities in male zebrafish (Danio rerio) under starvation.

Food deprivation is a severe stress across multiple fields and challenged to organismal development and immune system. Here, adult male zebrafish were used to investigate the starvation stress on organismal development, spermatogenesis, testicular inflammation and apoptosis. Results showed that the biological indexes, blood parameters, and RNA/DNA ratio in testis dramatically decreased after 1-3 weeks of starvation. The testicular architecture was impaired and the spermatogenesis was retarded with increased proportions of spermatogonia and spermatocytes, and decreased proportion of spermatozoa in the starved fish. The mRNA expressions of amh and sycp3 were downregulated, the retinoic acid content increased at later stage of starvation through the transcriptional regulation of aldh1a2 and cyp26a1. Besides, the immune response was elevated with upregulated mRNA and protein expressions of TNF-α, IL-6, and IL-1ß, which indicated the inflammation of opportunistic risk in testis. The apoptotic activity was stimulated, accompanied by differentially upregulated expressions of baxa, casp9, casp3, casp2, and decreased ratio of Bcl-2/Bax in the attenuate testis. Taken together, our findings revealed that the stress responses of testicular development, inflammatory and apoptotic activities in male zebrafish under starvation and pointed out the susceptibility of fish gonad to food fluctuation.

Resveratrol protects boar sperm in vitro via its antioxidant capacity.

The objective of the present study was to elucidate whether resveratrol could facilitate the survival of boar sperm during liquid preservation and fast cooling processes. Boar semen were diluted with Modena extender containing different concentrations of resveratrol. Sperm motility was evaluated by visual estimation. Membrane integrity, acrosome integrity and mitochondrial membrane potentials were measured by SYBR-14/PI, FITC-PNA and JC-1 staining, respectively. Moreover, the levels of reactive oxygen species (ROS), malonaldehyde (MDA) and total antioxidant capacity (T-AOC) were measured using commercial assay kits. B-cell lymphoma protein-2 (BCL2) content was determined by western blotting. During liquid preservation at 17oC, the addition of 50 µM resveratrol to the Modena extender significantly improved sperm motility, membrane integrity, acrosome integrity, and sperm mitochondrial membrane potentials. Similar results were also observed in the 150 µM resveratrol group during the fast cooling process. Furthermore, addition of resveratrol led to a decrease of ROS and MDA, and an increase in the content of T-AOC and BCL2. These observations suggest that addition of resveratrol to Modena extender protects boar sperm against oxidative stress. The optimal concentrations of resveratrol are 50 µM and 150 µM during liquid preservation and fast cooling process, respectively.

Genomic responses of DNA methylation and transcript profiles in zebrafish cells upon nutrient deprivation stress.

Environmental stress such as nutrient deprivation across multiple fields in nature causes physiological and biochemical changes in organism. Understanding the potential epigenetic modulations to phenotypic variation upon nutrient deprivation stress is crucial for environmental assessments. Here, the methyl-cytosine at single-base resolution was mapped across the whole genome and the methylation patterns and methylation levels coordinated with transcript analysis were systemically elaborated in zebrafish embryonic fibroblast cells under serum starvation stress. The down-regulated genes mainly annotated to the pathways of DNA replication and cell cycle that were consistent with cell physiological changes. Vast differentially methylated regions were identified in genomic chromosome and showed enrichment in the intron and intergenic regions. In an integrated transcriptome and DNA methylation analyses, 135 negatively correlated genes were determined, wherein the hub genes of gins2, cdca5, fbxo5, slc29a2, suv39h1b, and zgc:174160 were predominant responsive to the nutrient condition changes. Besides, nutrient recovery and DNA methyltransferases inhibitor supplements partly rescued cell proliferation with decrease of DNA methylation and reactivation of several depressed genes, implying the possible intrinsic relationships among cell physiological state, mRNA expression, and DNA methylation. Collectively, current study proved the broad role of DNA methylation in governing cellular responses to nutrient deprivation and revealed the epigenetic risk of starvation stress in zebrafish.

Discrepant dose responses of bisphenol A on oxidative stress and DNA methylation in grass carp ovary cells.

Bisphenol A (BPA), is a common contaminant in diverse environmental compartments and its endocrine disruptive effect on living organisms has been widely reported. Further works are still required to facilitate the research on cytotoxicity and genotoxicity. In the present study, grass carp ovary (GCO) cells were used to investigate cellular oxidative stress and genomic DNA methylation under BPA exposure. Results showed that BPA exposure for 48 h arrested cell proliferation and viability. The oxidative stress was distinctly enhanced with increased reactive oxygen species (ROS), malondialdehyde level, and oxidation of reduced glutathione (GSH) in 30 µM BPA group. Furthermore, the global 5-methylcytosine (5 mC) level was elevated and showed inverted U-shaped responses to the BPA doses. Besides, one-carbon metabolism and de novo GSH synthesis were disrupted at 30 µM BPA. Current data suggested that low dose of BPA exposure could exhibit hormesis in recycling circular biosynthesis of GSH and scavenging ROS to create a relatively reductive intracellular environment, and up-regulate transcripts of methyltransferases that increased the 5 mC level in GCO cells. While high dose of BPA distinctly induced oxidative stress, elevated de novo GSH synthesis, and then attenuated transmethylation activity and decreased 5 mC level. Current study highlighted the discrepant dose responses of BPA in fish ovary cells that facilitated the understanding of pleiotropic consequences in organisms.

The cellular responses of autophagy, apoptosis, and 5-methylcytosine level in zebrafish cells upon nutrient deprivation stress.

Here we reported the stress responses of nutrient deprivation and extended observation of autophagy, apoptosis, and DNA methylation in zebrafish embryonic fibroblast (ZF4) cells. Our results showed that serum deprivation resulted in the changes of cell shape and adherent ability, the suppressed cell growth and viability, and the inhibited proliferation and cell cycle. Besides, the appearance of lysosome and autophagosome/autolysosome with significantly increased expression of mRNAs (ulk1a, becn1, atg12, sqstm1, maplc3, and lamp1) and proteins (Atg12, Becn1, Sqstm1, and Lamp1) indicate the autophagic activity was boosted at initial stage but relatively weakened at 48 h of serum starvation. When autophagy no longer mitigate for the stress, cell apoptosis detected by the mRNA expression of caspases, Bcl-2/Bax expression, and Annexin V/PI was gradually enhanced to execute the death plan upon prolonged starvation process. Furthermore, the methyl group metabolism was increased in accordance with autophagic activity and was suppressed by enhanced apoptotic activity. These data suggested that the recycle activity induced by autophagy could compensate the substrates and reactions of DNA transmethylation, which obviously increased 5-methylcytosine (5 mC) level in ZF4 cells. In summary, our results discovered the cellular responses under prolonged serum starvation stress and elaborated the switch from autophagy to apoptosis and corresponding correlation with 5 mC level changes in teleost fish in vitro.

Resveratrol Improves Boar Sperm Quality via 5'AMP-Activated Protein Kinase Activation during Cryopreservation.

Mammalian sperm is highly susceptible to the reactive oxygen species (ROS) stress caused by biochemical and physical modifications during the cryopreservation process. 5'AMP-activated protein kinase (AMPK) is involved in regulating both cell metabolism and cellular redox status. The aim of the present study was to investigate whether the resveratrol protects boar sperm against ROS stress via activation of AMPK during cryopreservation. Boar sperm was diluted with the freezing medium supplemented with resveratrol at different concentrations (0, 25, 50, 75, 100, and 125 µM). It was observed that the addition of 50 µM resveratrol significantly improved the postthaw sperm progressive motility, membrane integrity, acrosome integrity, mitochondrial activity, glutathione (GSH) level, activities of enzymatic antioxidants (glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase), and the phosphorylation of AMPK. Meanwhile, the lipid peroxidation, ROS levels, and apoptosis of postthaw sperm were reduced in the presence of 50 µM resveratrol. Furthermore, when fresh boar sperm was incubated with the medium in the presence of 50 µM resveratrol and 30 µM Compound C (an AMPK inhibitor), the effects of the resveratrol were partly counteracted by the Compound C. These observations suggest that the resveratrol protects boar sperm via promoting AMPK phosphorylation. In conclusion, the addition of resveratrol to the freezing extenders protects boar sperm against ROS damage via promoting AMPK phosphorylation for decreasing the ROS production and improving the antioxidative defense system of postthaw sperm. These findings provide novel insights into understanding the mechanisms of resveratrol on how to protect boar sperm quality contrary to the ROS production during cryopreservation.