Jun Chul Park

Effects of triclosan (TCS) on fecundity, the antioxidant system, and oxidative stress-mediated gene expression in the copepod Tigriopus japonicus

Triclosan (TCS) is an antimicrobial agent that has been widely dispersed and detected in the marine environment. However, the effects of TCS in marine invertebrates are poorly understood. In this study, the effects of TCS on life cycle history (e.g. mortality and fecundity) along with cellular reactive oxygen species (ROS) levels, GSH content, antioxidant enzymatic activities, and mRNA expression levels of oxidative stress-mediated genes were measured in the copepod Tigriopus japonicus. The no observed effect concentration (NOEC) and median lethal concentration (LC50) of TCS in the adult stage were determined to be 300μg/L and 437.476μg/L, respectively, while in the nauplius stages the corresponding values were 20μg/L, and 51.76μg/L, respectively. Fecundity was significantly reduced (P<0.05) in response to TCS at 100μg/L. Concentration- and time-dependent analysis of ROS, GSH content (%), and antioxidant enzymatic activities (e.g. GST, GPx, and SOD) were significantly increased (P<0.05) in response to TCS exposure. Additionally, mRNA expression of detoxification (e.g., CYPs) and antioxidant (e.g., glutathione S-transferase-sigma isoforms, Cu/Zn superoxide dismutase, catalase) genes was modulated in response to TCS exposure at different concentrations over a 24h period. Our results revealed that TCS can induce reduced fecundity and oxidative stress with transcriptional regulation of oxidative stress-mediated genes with activation of the antioxidant system in the copepod T. japonicus.

Ultraviolet B radiation induces impaired lifecycle traits and modulates expression of cytochrome P450 (CYP) genes in the copepod Tigriopus japonicus

To evaluate the effects of ultraviolet B (UV-B) radiation at the developmental, reproductive, and molecular levels in aquatic invertebrates, we measured UV-B-induced acute toxicity, impairments in developmental and reproductive traits, and UV-B interaction with the entire family of cytochrome P450 (CYP) genes in the intertidal benthic copepod Tigriopus japonicus. We found a significant, dose-dependent reduction (P<0.05) in the survival of T. japonicus that began as a developmental delay and decreased fecundity. The 48h LD10 and LD50 were 1.35 and 1.84kJ/m2, and the CYP inhibitor (PBO) elevated mortality, confirming the involvement of CYP genes in UV-B induced toxicity. Low-dose UV-B (1.5kJ/m2) induced developmental delays, and higher doses (6-18kJ/m2) caused reproductive impairments in ovigerous females. The significant up-regulation of CYP genes belonging to clans 2/3/MT/4/20 in T. japonicus exposed to UV-B (12kJ/m2) confirmed molecular interaction between UV-B and CYP genes. Moreover, orphan CYPs, such as CYP20A1, provide good insight on the deorphanization of invertebrate CYPs. Overall, these results demonstrate the involvement of UV-B radiation in the expression of all the CYP genes in T. japonicus and their susceptibility to UV-B radiation. This will provide a better understanding of the mechanistic effects of UV-B in copepods through the predicted AhR-mediated up-regulation of CYP genes.

Interrelationship of salinity shift with oxidative stress and lipid metabolism in the monogonont rotifer Brachionus koreanus

Salinity is a critical key abiotic factor affecting biological processes such as lipid metabolism, yet the relationship between salinity and lipid metabolism has not been studied in the rotifer. To understand the effects of salinity on the monogonont rotifer B. koreanus, we examined high saline (25 and 35psu) conditions compared to the control (15psu). In vivo life cycle parameters (e.g. cumulative offspring and life span) were observed in response to 25 and 35psu compared to 15psu. In addition, to investigate whether high salinity induces oxidative stress, the level of reactive oxygen species (ROS) and glutathione S-transferase activity (GST) were measured in a salinity- (15, 25, and 35psu; 24h) and time-dependent manner (3, 6, 12, 24h; 35psu). Furthermore composition of fatty acid (FA) and lipid metabolism-related genes (e.g. elongases and desaturases) were examined in response to different salinity conditions. As a result, retardation in cumulative offspring and significant increase in life span were demonstrated in the 35psu treatment group compared to the control (15psu). Furthermore, ROS level and GST activity have both demonstrated a significant increase (P<0.05) in the 35psu treatment. In general, the quantity of FA and mRNA expression of the lipid metabolism-related genes was significantly decreased (P<0.05) in response to high saline condition with exceptions for both GST-S4 and S5 demonstrated a significant increase in their mRNA expression. This study demonstrates that high salinity induces oxidative stress, leading to a negative impact on lipid metabolism in the monogonont rotifer, B. koreanus.

De novo assembly and annotation of the marine mysid (Neomysis awatschensis) transcriptome.

We sequenced the whole transcriptome of the marine mysid Neomysis awatschensis (Crustacea: Mysidacea) using Illumina RNA-seq. De novo assembly was performed with 14,018,702 raw reads using Trinity, resulting in 82,434 contigs. Transdecoder found 22,141 candidate coding contigs with homology to other species by BLAST analysis. Functional gene annotation was performed by Gene Ontology, InterProScan, and KEGG pathway analyses. We generated an expressed gene catalog for the mysid N. awatschensis to serve as a resource for marine environmental genomic and ecotoxicogenomic studies focused on uncovering the molecular mechanisms underlying the responses of N. awatschensis to environmental stressors and chemicals.

Aging extension and modifications of lipid metabolism in the monogonont rotifer Brachionus koreanus under chronic caloric restriction

To examine the interrelationship of aging extension and modification of lipid metabolism under chronic caloric restriction (CCR; reduced concentration of the green algae Tetraselmis suecica) in the monogonont rotifer Brachionus koreanus, we assessed life cycle parameters, fatty acid composition, and expression of sirtuin and genes related to lipid metabolism. B. koreanus in the 5% T. suecica group showed an increased life span but decreased reproduction. Based on this finding, we chose 5% T. suecica for further experiments and compared the data with those for 100% T. suecica. Upregulation of sirtuin gene expression was observed under CCR. In addition, despite the reduction in the amount of total fatty acid (FA) and the area of triacylglycerol, increases in the ratios of saturated fatty acid and monounsaturated fatty acid (MUFA) to total FA in 5%-exposed B. koreanus were observed. Furthermore, mRNA expression analysis confirmed that CCR promoted the synthesis of MUFA through Δ9 desaturase. Moreover, expression of the docosahexaenoic acid (DHA) synthesizing gene Δ4 desaturase was also upregulated, together with DHA content. These data suggest that CCR modified protein acetylation and lipid metabolism, leading to a decrease in reproduction and consequently resulting in life span extension.

Effects of bisphenol A and its analogs bisphenol F and S on life parameters, antioxidant system, and response of defensome in the marine rotifer Brachionus koreanus

To understand the adverse outcome in response to bisphenol A and its analogs bisphenol F and S (BPA, BPF, and BPS), we examined acute toxicity, life parameter, and defensome in the marine rotifer Brachionus koreanus. Among the bisphenol analogs, BPA showed the highest acute toxicity and then BPF and BPS, accordingly in the view of descending magnitude of toxicity. In life parameters including life span and reproduction, BPA, BPF, and BPS were found to cause adverse effect. Both intracellular ROS level and GST activity were significantly increased (P < 0.05) in response to each dosage of bisphenol analogs exposures. In response to bisphenol analogs, defensomes of phase I, II, and III detoxification mechanism demonstrated inverse relationship between the lipophilicity of bisphenol analogs and the expression patterns of defensomes. BPA and BPF were found to have significant modulation (P < 0.05) in the expression of cytochrome P450 (CYP) and GST genes. In phase III, BPS with comparatively lower lipophilicity demonstrated highly diversified expressional pattern, suggesting that BPS is likely caused less toxicity compared to BPA and BPF. In this study, via phase I, II, and III detoxification mechanism, bisphenol A and its analogs F and S demonstrated specific detoxification mechanism in rotifer.

Genome-wide identification of the entire 90 glutathione S-transferase (GST) subfamily genes in four rotifer Brachionus species and transcriptional modulation in response to endocrine disrupting chemicals

Genome-wide identification of glutathione S-transferase (GST), a major phase II detoxification enzyme, was investigated in four different aquatic model rotifer species Brachionus koreanus, B. plicatilis, B. rotundiformis, and B. calyciflorus. GSTs are ubiquitous antioxidant enzymes that play versatile function including cellular detoxification, stress alleviation, and production of the radical conjugates. Among the four rotifers, B. rotundiformis was found with the least number of GST genes (total 19 GST genes), whereas the other three species shared 23 to 24 GST genes. Among the identified GST genes, belonging to the cytosolic GST superfamily, the expansion of GST sigma classes mainly occurs through tandem duplication, resulting in tandem-arrayed gene clusters on the chromosomes. Overall, the number of genes discovered in this study was highest in the sigma class, zeta, alpha, and omega in descending order. With integration of phylogenetic analysis and xenobiotic-mediated GST mRNA expression patterns along with previous enzymatic activities, the functional divergence among species-specific GST genes was clearly observed. This study covers full identification of GST classes in three marine rotifer and one fresh-water rotifer species and their important role in marine environmental ecotoxicology.

Adverse effects of two pharmaceuticals acetaminophen and oxytetracycline on life cycle parameters, oxidative stress, and defensome system in the marine rotifer Brachionus rotundiformis

To investigate the adverse effect of two widely used pharmaceuticals, paracetamol (acetaminophen [APAP]) and oxytetracycline (OTC) on the marine rotifer Brachionus rotundiformis (B. rotundiformis), the animals were exposed to various environmentally-relevant concentrations. Up to date, acetaminophen and oxytetracycline have been considered as toxic, if used above threshold concentration, i.e. overdosed. However, this study demonstrated these two pharmaceuticals even at low concentration (i.e., μg/L scale) elicited oxidative stress through the generation of reactive oxygen species (ROS) along with the increased glutathione S-transferase activity, despite no-observed effect in in-vivo population growth. To validate the adverse effects of the two pharmaceuticals at relatively low concentrations, mRNA expression analysis was performed of the entire set of genes encoding 26 cytochrome P450s (CYPs) of phase I and 19 glutathione S-transferases (GSTs) of phase II of the rotifer B. rotundiformis. The mRNA expression analysis suggested specific genes CYP3045A2 and GSTσ1, GSTσ4, and GSTω1 take part in detoxification of APAP and OTC, resulting in no significant changes in the population growth and undetermined no observed effect concentration (NOEC) in the marine rotifer B. rotundiformis.

Effects of environmental stressors on lipid metabolism in aquatic invertebrates

Lipid metabolism is crucial for the survival and propagation of the species, since lipids are an essential cellular component across animal taxa for maintaining homeostasis in the presence of environmental stressors. This review aims to summarize information on the lipid metabolism under environmental stressors in aquatic invertebrates. Fatty acid synthesis from glucose via de novo lipogenesis (DNL) pathway is mostly well-conserved across animal taxa. The structure of free fatty acid (FFA) from both dietary and DNL pathway could be transformed by elongase and desaturase. In addition, FFA can be stored in lipid droplet as triacylglycerol, upon attachment to glycerol. However, due to the limited information on both gene and lipid composition, in-depth studies on the structural modification of FFA and their storage conformation are required. Despite previously validated evidences on the disturbance of the normal life cycle and lipid homeostasis by the environmental stressors (e.g., obesogens, salinity, temperature, pCO2, and nutrients) in the aquatic invertebrates, the mechanism behind these effects are still poorly understood. To overcome this limitation, omics approaches such as transcriptomic and proteomic analyses have been used, but there are still gaps in our knowledge on aquatic invertebrates as well as the lipidome. This paper provides a deeper understanding of lipid metabolism in aquatic invertebrates.

Adverse effects of the insecticides chlordecone and fipronil on population growth and expression of the entire cytochrome P450 (CYP) genes in the freshwater rotifer Brachionus calyciflorus and the marine rotifer Brachionus plicatilis

Chlordecone and fipronil are used as an insecticide and have been widely detected in the aquatic environments. However, their toxicity is still poorly investigated in aquatic invertebrates. In this study, we examined effects of chlordecone and fipronil on population growth and transcriptional regulation of the entire cytochrome P450 (CYP) genes in the freshwater rotifer Brachionus calyciflorus and the marine rotifer B. plicatilis. In B. calyciflorus, a 24 h-no observed effect concentration (NOEC-24 h) and a 24 h-median lethal concentration (LC50-24 h) of chlordecone were determined as 100 μg/L and 193.8 μg/L, respectively, while NOEC-24 h and LC50-24 h of fipronil were determined as 1000 μg/L and 2033.0 μg/L, respectively. In B. plicatilis, NOEC-24 h and LC50-24 h of chlordecone were 100 μg/L and 291.0 μg/L, respectively, while NOEC-24 h and LC50-24 h of fipronil were determined as 1000 μg/L and 5735.0 μg/L, respectively. Moreover, retardation in the population growth were observed in response to chlordecone and fipronil in both rotifer species, suggesting that chlordecone and fipronil have a potential adverse effects on life cycle parameters of two rotifer species. Additionally, modulation in the expressions of the entire CYP genes were demonstrated in response to chlordecone and fipronil at 24 h period. These results provide the better understanding on how chlordecone and fipronil can affect in population growth of two rotifers and CYP gene expressions in chlordecone- and fipronil-exposed rotifers.