Un-Ki Hwang

Microplastic Size-Dependent Toxicity, Oxidative Stress Induction, and p-JNK and p-p38 Activation in the Monogonont Rotifer (Brachionus koreanus)

In this study, we evaluated accumulation and adverse effects of ingestion of microplastics in the monogonont rotifer (Brachionus koreanus). The dependence of microplastic toxicity on particle size was investigated by measuring several in vivo end points and studying the ingestion and egestion using 0.05-, 0.5-, and 6-μm nonfunctionalized polystyrene microbeads. To identify the defense mechanisms activated in response to microplastic exposure, the activities of several antioxidant-related enzymes and the phosphorylation status of mitogen-activated protein kinases (MAPKs) were determined. Exposure to polystyrene microbeads of all sizes led to significant size-dependent effects, including reduced growth rate, reduced fecundity, decreased lifespan and longer reproduction time. Rotifers exposed to 6-μm fluorescently labeled microbeads exhibited almost no fluorescence after 24 h, while rotifers exposed to 0.05- and 0.5-μm fluorescently labeled microbeads displayed fluorescence until 48 h, suggesting that 6-μm microbeads are more effectively egested from B. koreanus than 0.05- or 0.5-μm microbeads. This observation provides a potential explanation for our findings that microbead toxicity was size-dependent and smaller microbeads were more toxic. In vitro tests revealed that antioxidant-related enzymes and MAPK signaling pathways were significantly activated in response to microplastic exposure in a size-dependent manner.

Gamma rays induce DNA damage and oxidative stress associated with impaired growth and reproduction in the copepod Tigriopus japonicus

Nuclear radioisotope accidents are potentially ecologically devastating due to their impact on marine organisms. To examine the effects of exposure of a marine organism to radioisotopes, we irradiated the intertidal copepod Tigriopus japonicus with several doses of gamma radiation and analyzed the effects on mortality, fecundity, and molting by assessing antioxidant enzyme activities and gene expression patterns. No mortality was observed at 96h, even in response to exposure to a high dose (800Gy) of radiation, but mortality rate was significantly increased 120h (5 days) after exposure to 600 or 800Gy gamma ray radiation. We observed a dose-dependent reduction in fecundity of ovigerous females; even the group irradiated with 50Gy showed a significant reduction in fecundity, suggesting that gamma rays are likely to have a population level effect. In addition, we observed growth retardation, particularly at the nauplius stage, in individuals after gamma irradiation. In fact, nauplii irradiated with more than 200Gy, though able to molt to copepodite stage 1, did not develop into adults. Upon gamma radiation, T. japonicus showed a dose-dependent increase in reactive oxygen species (ROS) levels, the activities of several antioxidant enzymes, and expression of double-stranded DNA break damage genes (e.g. DNA-PK, Ku70, Ku80). At a low level (sub-lethal dose) of gamma irradiation, we found dose-dependent upregulation of p53, implying cellular damage in T. japonicus in response to sub-lethal doses of gamma irradiation, suggesting that T. japonicus is not susceptible to sub-lethal doses of gamma irradiation. Additionally, antioxidant genes, phase II enzyme (e.g. GSTs), and cellular chaperone genes (e.g. Hsps) that are involved in cellular defense mechanisms also showed the same expression patterns for sublethal doses of gamma irradiation (50-200Gy). These findings indicate that sublethal doses of gamma radiation can induce oxidative stress-mediated DNA damage and increase the expression of antioxidant enzymes and proteins with chaperone-related functions, thereby significantly affecting life history parameters such as fecundity and molting in the copepod T. japonicus.

Effects of UV radiation on hatching, lipid peroxidation, and fatty acid composition in the copepod Paracyclopina nana

To evaluate the effects of UV radiation on the reproductive physiology and macromolecules in marine zooplankton, several doses of UV radiation were used to treat the copepod Paracyclopina nana, and we analyzed in vivo endpoints of their life cycle such as mortality and reproductive parameters with in vitro biochemical biomarkers such as reactive oxygen species (ROS), the modulated enzyme activity of glutathione S-transferase (GST) and superoxide dismutase (SOD), and the production of a byproduct of peroxidation (e.g. malonedialdehyde, MDA). After UV radiation, the survival rate of P. nana was significantly reduced. Also, egg sac damage and a reduction in the hatching rate of offspring were observed in UV-irradiated ovigerous females. According to the assessed biochemical parameters, we found dose-dependent increases in ROS levels and high levels of the lipid peroxidation decomposition product by 2 kJ m(-2), implying that P. nana was under off-balanced status by oxidative stress-mediated cellular damage. Antioxidant enzyme activities of GST and SOD increased over different doses of UV radiation. To measure UV-induced lipid peroxidation, we found a slight reduction in the composition of essential fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These findings indicate that UV radiation can induce oxidative stress-triggered lipid peroxidation with modulation of antioxidant enzyme activity, leading to a significant effect on mortality and reproductive physiology (e.g. fecundity). These results demonstrate the involvement of UV radiation on essential fatty acids and its susceptibility to UV radiation in the copepod P. nana compared to other species.

Triclosan (TCS) and Triclocarban (TCC) cause lifespan reduction and reproductive impairment through oxidative stress-mediated expression of the defensome in the monogonont rotifer (Brachionus koreanus)

Triclosan (TCS) and Triclocarban (TCC) are used as antimicrobial agents and have been widely dispersed and detected in the marine environment. However, the toxicities of TCS and TCC have been poorly investigated in marine invertebrates. In this study, the effects of TCS and TCC on mortality, population growth, lifespan, and fecundity were examined in the monogonont rotifer (Brachionus koreanus) using cellular ROS levels, GST enzymatic activity, and gene expression of defensomes. The median lethal concentration (LC50) of TCS (393.1μg/L) and TCC (388.1μg/L) was also determined in the same species. In TCS- and TCC-exposed B. koreanus, growth retardation and reduced fecundity were observed and were shown to have a potentially deleterious effect on the life cycle of B. koreanus. In addition, time-dependent increases in ROS content (%) and GST enzymatic activity were shown in response to TCS and TCC exposure. Additionally, transcript levels of detoxification proteins (e.g., CYPs), antioxidant proteins (e.g., GST-sigma, Cu/ZnSOD, CAT), and heat shock proteins (Hsps) were modulated in response to TCS and TCC exposure over a 24h period. Our results indicate that TCS and TCC induce oxidative stress and transcriptional regulation of detoxification, antioxidant, and heat shock proteins, resulting in changes in lifespan and fecundity.

Adverse Effects, Expression of the Bk-CYP3045C1 Gene, and Activation of the ERK Signaling Pathway in the Water Accommodated Fraction-Exposed Rotifer

To examine the deleterious effects of the water accommodated fraction (WAF) of crude oil, the growth curve, fecundity, and lifespan of the monogonont rotifer (Brachionus koreanus) were measured for 24 h in response to three different doses (0.2×, 0.4×, and 0.8×) of WAFs. A higher dose of WAFs significantly reduced the fecundity and lifespan. A rotifer 32K microarray chip showed that the Bk-CYP3045C1 gene had the highest expression. Of the 25 entire CYP genes, the Bk-CYP3045C1 gene showed a significant expression for different doses and times in response to WAFs and chemical components of WAFs (naphthalene and phenanthrene); also, glutathione S-transferase genes, ABC transporter, and other genes showed dose responses upon exposure to 80% WAF over time. Different doses of WAFs increased the oxidative stress with an induction of reactive oxygen species (ROS) and a depletion of glutathione (GSH). Exposure to WAFs did not show toxic effects on survivability in B. koreanus; however, toxicity to WAFs was shown when piperonyl butoxide, a potent inhibitor of cytochrome P450 (CYP) enzymes, was added. This toxicity was dose-dependent. After WAFs exposure, p-ERK was activated over time in response to WAFs, which suggests that WAFs can be activated by the p-ERK signaling pathway.

Dose- and time-dependent expression of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) in PCB-, B[a]P-, and TBT-exposed intertidal copepod Tigriopus japonicus.

The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon nuclear translocator (ARNT) genes from the copepod Tigriopus japonicus (Tj) were cloned to examine their potential functions in the invertebrate putative AhR-CYP signaling pathway. The amino acid sequences encoded by the Tj-AhR and Tj-ARNT genes showed high similarity to homologs of Daphnia and Drosophila, ranging from 68% and 70% similarity for the AhR genes to 56% for the ARNT genes. To determine whether Tj-AhR and Tj-ARNT are modulated by environmental pollutants, transcriptional expression of Tj-AhR and Tj-ARNT was analyzed in response to exposure to five concentrations of polychlorinated biphenyl (PCB 126) (control, 10, 50, 100, 500 μg L(-1)), benzo[a]pyrene (B[a]P) (control, 5, 10, 50, 100 μg L(-1)), and tributyltin (TBT) (control, 1, 5, 10, 20 μg L(-1)) 24h after exposure. A time-course experiment (0, 3, 6, 12, 24h) was performed to analyze mRNA expression patterns after exposure to PCB, B[a]P, and TBT. T. japonicus exhibited dose-dependent and time-dependent upregulation of Tj-AhR and Tj-ARNT in response to pollutant exposure, and the degree of expression was dependent on the pollutant, suggesting that pollutants such as PCB, B[a]P, and TBT modulate expression of Tj-AhR and Tj-ARNT genes in the putative AhR-CYP signaling pathway.

Cloning of circadian rhythmic pathway genes and perturbation of oscillation patterns in endocrine disrupting chemicals (EDCs)-exposed mangrove killifish Kryptolebias marmoratus

To investigate the effect of endocrine disrupting chemicals (EDCs) on the circadian rhythm pathway, we cloned clock and circadian rhythmic pathway-associated genes (e.g. Per2, Cry1, Cry2, and BMAL1) in the self-fertilizing mangrove killifish Kryptolebias marmoratus. The promoter region of Km-clock had 1 aryl hydrocarbon receptor element (AhRE, GTGCGTGACA) and 8 estrogen receptor (ER) half-sites, indicating that the AhRE and ER half sites would likely be associated with regulation of clock protein activity during EDCs-induced cellular stress. The Km-clock protein domains (bHLH, PAS1, PAS2) were highly conserved in five additional fish species (zebrafish, Japanese medaka, Southern platyfish, Nile tilapia, and spotted green pufferfish), suggesting that the fish clock protein may play an important role in controlling endogenous circadian rhythms. The promoter regions of Km-BMAL1, -Cry1, -Cry2, and -Per2 were found to contain several xenobiotic response elements (XREs), indicating that EDCs may be able to alter the expression of these genes. To analyze the endogenous circadian rhythm in K. marmoratus, we measured expression of Km-clock and other circadian rhythmic genes (e.g. Per2, Cry1, Cry2, and BMAL1) in different tissues, and found ubiquitous expression, although there were different patterns of transcript amplification during different developmental stages. In an estrogen (E2)-exposed group, Km-clock expression was down-regulated, however, a hydroxytamoxifen (TMX, nonsteroid estrogen antagonist)-exposed group showed an upregulated pattern of Km-clock expression, suggesting that the expression of Km-clock is closely associated with exposure to EDCs. In response to the exposure of bisphenol A (BPA) and 4-tert-octyphenol (OP), Km-clock expression was down-regulated in the pituitary/brain, muscle, and skin in both gender types (hermaphrodite and secondary male). In juvenile K. marmoratus liver tissue, expression of Km-clock and other circadian rhythmic pathway-associated genes showed a regular oscillation pattern over a period of approximately 24h during a 12L:12D cycle. However, the circadian rhythm of BPA-exposed juvenile K. marmoratus liver tissue was perturbed over a 12L:12D period. This study will aid in our understanding of how EDCs perturb endogenous circadian rhythms, particularly in BPA-exposed fish liver tissue.

Effects of water accommodated fractions (WAFs) of crude oil in two congeneric copepods Tigriopus sp.

Oil pollution has deleterious effects on marine ecosystems. However, the toxicity of crude oil towards Antarctic marine organisms has not been well studied. We compared the deleterious effects of water accommodated fractions (WAFs) of crude oil on reproduction, intracellular reactive oxygen species (ROS) levels, and antioxidant enzymatic activity in Antarctic (Tigriopus kingsejongensis) and temperate (Tigriopus japonicus) copepods. Reproductive rates of T. kingsejongensis and T. japonicus were significantly reduced (P < 0.05) in response to WAFs. Furthermore, T. kingsejongensis showed elevated levels of ROS and higher antioxidant enzyme (glutathione peroxidase [GPx]) activity than T. japonicus in response to WAFs. CYP genes from congeneric copepods were identified and annotated to better understand molecular detoxification mechanisms. We observed significant up-regulation (P < 0.05) of Tk-CYP3024A3 and Tj-CYP3024A2 in response to WAFs, suggesting that CYP genes may contribute to the detoxification mechanism in response to WAF exposure. These finding also suggest that WAFs may induce oxidative stress, leading to reproductive impairment in copepods. Furthermore, Tk-CYP3024A3 and Tj-CYP3024A2 genes can be considered as potential biomarkers of WAF toxicity in the congeneric copepods T. kingsejongensis and T. japonicus. This study will be helpful for enhancing our knowledge on the harmful effects of WAFs in Antarctic and temperate copepods and provides insight into the underlying molecular mechanisms.

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.

Nine co-localized cytochrome P450 genes of the CYP2N, CYP2AD, and CYP2P gene families in the mangrove killifish Kryptolebias marmoratus genome: Identification and expression in response to B[α]P, BPA, OP, and NP

The CYP2 genes are the largest and most diverse cytochrome P450 (CYP) subfamily in vertebrates. We have identified nine co-localized CYP2 genes (∼55kb) in a new cluster in the genome of the highly resilient ecotoxicological fish model Kryptolebias marmoratus. Molecular characterization, temporal and tissue-specific expression pattern, and response to xenobiotics of these genes were examined. The CYP2 gene clusters were characterized and designated CYP2N22-23, CYP2AD12, and CYP2P16-20. Gene synteny analysis confirmed that the cluster in K. marmoratus is similar to that found in other teleost fishes, including zebrafish. A gene duplication event with diverged catalytic function was observed in CYP2AD12. Moreover, a high level of divergence in expression was observed among the co-localized genes. Phylogeny of the cluster suggested an orthologous relationship with similar genes in zebrafish and Japanese medaka. Gene expression analysis showed that CYP2P19 and CYP2N20 were consecutively expressed throughout embryonic development, whereas CYP2P18 was expressed in all adult tissues, suggesting that members of each CYP2 gene family have different physiological roles even though they are located in the same cluster. Among endocrine-disrupting chemicals (EDCs), benzo[α]pyrene (B[α]P) induced expression of CYP2N23, bisphenol A (BPA) induced CYP2P18 and CYP2P19, and 4-octylphenol (OP) induced CYP2AD12, but there was no significant response to 4-nonylphenol (NP), implying differential catalytic roles of the enzyme. In this paper, we identify and characterize a CYP2 gene cluster in the mangrove killifish K. marmoratus with differing catalytic roles toward EDCs. Our findings provide insights on the roles of nine co-localized CYP2 genes and their catalytic functions for better understanding of chemical-biological interactions in fish.