Bo-Mi Kim

Copper induces apoptotic cell death through reactive oxygen species-triggered oxidative stress in the intertidal copepod Tigriopus japonicus

The copepod, Tigriopus japonicus is an important model for toxicity testing. However, no attempt has been made in analyzing the effect of toxicants at the level of the ROS-mediated signal transduction pathway. To understand copper-induced cytotoxicity at the molecular level, we employed several cellular and biochemical assays after exposure to copper, and found a significant induction of enzyme activities of antioxidant proteins with increased intracellular reactive oxygen species (ROS) as well as an increase of TUNEL-positive cells, but a decrease of BrdU-positive cells. In addition, several important genes such as p38 MAPK, antioxidant-related genes, Hsps, and apoptosis-related genes were significantly modulated by copper exposure. Taken together, we suggest that copper-induced cytotoxicity is mediated by the formation of intracellular ROS and oxidative stress in T. japonicus. Whole body biochemical assays such as TUNEL- and BrdU-assay will provide a better understanding of cellular responses such as apoptosis and cell death upon cytotoxic exposure of copper in T. japonicus.

Heavy metals induce oxidative stress and trigger oxidative stress-mediated heat shock protein (hsp) modulation in the intertidal copepod Tigriopus japonicus.

Heat shock proteins (hsps) are induced by a wide range of environmental stressors including heavy metals in aquatic organisms. However, the effect of heavy metals on zooplankton at the molecular level remains still unclear. In this study, we measured the intracellular reactive oxygen species (ROS) level and the antioxidant enzyme activities for 96 h after exposure to five heavy metals: arsenic (As), cadmium (Cd), copper (Cu), silver (Ag), and zinc (Zn) in the intertidal copepod Tigriopus japonicus. Activities of the antioxidant enzymes were highly elevated in metal-exposed copepods, indicating that heavy metals can induce oxidative stress by generating ROS, and stimulate the involvement of antioxidant enzymes as cellular defense mechanisms. Subsequently, transcriptional changes in hsp gene families were further investigated in the metal-exposed groups for 96 h. The ROS level and glutathione (GSH) content were significantly increased in Ag-, As-, and Cu-exposed copepods, while they were only slightly elevated in Cd- and Zn-exposed groups. Based on the numbers of significantly modulated hsp genes and their expression levels for 96 h, we measured the effect of heavy metals to stress genes of T. japonicus in the following order: Cu > Zn > Ag > As > Cd, implying that Cu acts as a stronger oxidative stress inducer than other heavy metals. Of them, the expression of hsp20 and hsp70 genes was substantially modulated by exposure to heavy metals, indicating that these genes would provide a sensitive molecular biomarker for aquatic monitoring of heavy metal pollution.

Bisphenol A modulates expression of sex differentiation genes in the self-fertilizing fish, Kryptolebias marmoratus.

Endocrine disrupting chemicals (EDCs) have been a major concern in the normal reproduction and development of aquatic organisms. In the teleost, steroid hormones are synthesized via the steroidogenesis pathway, and play a key physiological role in the regulation of gonadal sex differentiation. The protogynous hermaphroditic fish, Kryptolebias marmoratus is the only vertebrate capable of reproducing through internal self-fertilization. To uncover the effect of bisphenol A (BPA) on sex differentiation genes on transcription, we investigated the expression patterns of several sex differentiation-related genes such as dax1, dmrt1, mis, sf1, figlα, StAR and wt1 after BPA exposure with controls (E2 and TMX). In response to 17β-estradiol (E2) exposure, a testis-specific gene, dmrt1 mRNA was down-regulated in the gonad of the secondary male but the expression of the female-specific gene, dax1 mRNA was significantly elevated in the brain and gonad. A high level of StAR mRNA was detected in the brain and gonad of both hermaphrodite and secondary males, suggesting that the elevated expression of dax1 and StAR genes would be involved in E2 exposure. As expected, upon BPA exposure, the dmrt1 and MIS mRNA level decreased in both hermaphrodite and secondary males, while the female-specific gene, figlα mRNA level increased in the gonad of both genders. BPA showed an opposite mode of action on the expression of dax1 (induction, P>0.05) and sf1 mRNA (inhibition, P>0.05) in the brain and gonad against both genders. The sensitivity of dax1 to BPA on expression was relatively high in the secondary male. The wt1 mRNA was up-regulated in most tissues except in the liver of BPA-exposed secondary males. Regarding the time course study, the figlα mRNA level increased at 6 h after BPA exposure. In addition, BPA elevated the expression of StAR, dax1, and wt1 mRNA but repressed sf1 mRNA. In this paper, we demonstrated that BPA may modulate the expression of sex differentiation and steroidogenesis pathway genes, and this finding would provide a better understanding on the modulation of transcription upon BPA exposure in steroidogenesis and sex differentiation in the hermaphroditic fish, K. marmoratus.

Evaluation of biomarker potential of cytochrome P450 1A (CYP1A) gene in the marine medaka, Oryzias melastigma exposed to water-accommodated fractions (WAFs) of Iranian crude oil

CYP1A is involved in the metabolism of diverse chemicals, including polycyclic aromatic hydrocarbons and alkylated-PAHs, as a first line of detoxification mechanism. First, we identified and characterized the CYP1A gene from the marine medaka, Oryzias melastigma. O. melastigma CYP1A (Om-CYP1A) showed a high similarity of motifs/domains compared to those of vertebrates in their amino acid sequences. To check whether the Om-CYP1A would be inducible, we tested two strong CYP1A inducers, β-naphthoflavone (β-NF) and benzo[α]pyrene (B[α]P), and observed concentration-dependent transient expression on transcripts of Om-CYP1A for 96 h over a wide range of concentrations. Om-CYP1A mRNA level was significantly increased in exposure to different concentrations of β-NF and B[α]P, and its expression was highly transcribed within 12 h upon the exposure to low concentrations of both chemicals. Inducible transcript profiles revealed that Om-CYP1A would be associated with the toxicant metabolism via AhREs/DREs/XREs in its promoter region. To uncover the effects of the water-accommodated fraction (WAF) of crude oil on transcripts of Om-CYP1A, we measured mRNA expression of Om-CYP1A towards different concentrations of WAF for 24h. As a result, WAF exposure significantly increased Om-CYP1A transcripts at all concentrations as well as during time-course experiments for 96 h. In this paper, we demonstrated that WAF would trigger up-regulation of the CYP1A gene that would be associated with the initiation of the cellular defense systems. This finding provides a better understanding of the molecular mechanism of cellular protection particularly that involved in the WAF-mediated cellular response in O. melastigma.

Effect of pharmaceuticals exposure on acetylcholinesterase (AchE) activity and on the expression of AchE gene in the monogonont rotifer, Brachionus koreanus

Pharmaceuticals are widely used in human and veterinary medicine. However, they are emerging as a significant contaminant in aquatic environments through wastewater. Due to the persistent and accumulated properties of pharmaceuticals via the food web, their potential harmful effects on aquatic animals are a great concern. In this study, we investigated the effects of six pharmaceuticals: acetaminophen, ATP; atenolol, ATN; carbamazepine, CBZ; oxytetracycline, OTC; sulfamethoxazole, SMX; and trimethoprim, TMP on acetylcholinesterase (AChE; EC 3.1.1.7) activity and its transcript expression with chlorpyrifos (as a positive control) in the monogonont rotifer, Brachionus koreanus. ATP, CBZ, and TMP exposure also remarkably inhibited Bk-AChE activity at 100 μg/L (24 h) and 1000 μg/L (12 h and 24 h). ATP, CBZ, and TMP exposure showed a significant decrease in the Bk-AChE mRNA level in a concentration-dependent manner. However, in the case of OTC and SMX, a slight decrease in Bk-AChE mRNA expression was found but only at the highest concentration. The time-course experiments showed that ATP positively induced Bk-AChE mRNA 12 h after exposure at both 100 and 1000 μg/L, while the Bk-AChE mRNA expression was significantly downregulated over 6 to 24 h after exposure to 1000 μg/L of CBZ, OTC, SMX, and TMP. Our findings suggest that Bk-AChE would be a useful biomarker for risk assessment of pharmaceutical compounds as an early signal of their toxicity in aquatic environments. Particularly, ATP, CBZ, and TMP may have a toxic cholinergic effect on rotifer B. koreanus by inhibiting AChE activity.

Effects of benzo[a]pyrene on whole cytochrome P450-involved molecular responses in the marine medaka Oryzias melastigma.

Despite being a strong toxicant for aquatic ecosystems, the effect of benzo[a]pyrene (B[a]P) on whole cytochrome P450 (CYP) biotransformation mechanisms has not been deeply investigated in aquatic organisms. To understand the mode of action of B[a]P on CYP molecular responses in fish, we analyzed the full spectrum of cyp genes and the activities of enzymes that are involved in detoxification and antioxidant defense systems after exposure to different concentrations of B[a]P over different time courses in the marine medaka, Oryzias melastigma. Upon B[a]P exposure, we found significant downregulation of cyp genes associated with steroidogenesis with decreased concentrations of actual hormones including estradiol (E2) and testosterone (11-KT), indicating that B[a]P-treated groups were closely associated with the dysfunction of hormone synthesis in a dose-dependent manner. In addition, B[a]P exposure strongly influenced transcriptional levels of antioxidant-related genes and their enzyme activities. Based on these results, we suggest that B[a]P induced the CYPs-involved systematic biotransformation mechanism with oxidative stress in the juvenile marine medaka, resulting in changes of endogenous hormonal levels and transcriptional levels of several steroidogenic metabolism-related CYPs.

Genome-wide identification of whole ATP-binding cassette (ABC) transporters in the intertidal copepod Tigriopus japonicus

BackgroundsThe ATP-binding cassette (ABC) transporter superfamily is one of the largest transporter gene families and is observed in all animal taxa. Although a large set of transcriptomic data was recently assembled for several species of crustaceans, identification and annotation of the large ABC transporter gene family have been very challenging.ResultsIn the intertidal copepod Tigriopus japonicus, 46 putative ABC transporters were identified using in silico analysis, and their full-length cDNA sequences were characterized. Phylogenetic analysis revealed that the 46 T. japonicus ABC transporters are classified into eight subfamilies (A-H) that include all the members of all ABC subfamilies, consisting of five ABCA, five ABCB, 17 ABCC, three ABCD, one ABCE, three ABCF, seven ABCG, and five ABCH subfamilies. Of them, unique isotypic expansion of two clades of ABCC1 proteins was observed. Real-time RT-PCR-based heatmap analysis revealed that most T. japonicus ABC genes showed temporal transcriptional expression during copepod development. The overall transcriptional profile demonstrated that half of all T. japonicus ABC genes were strongly associated with at least one developmental stage. Of them, transcripts TJ-ABCH_88708 and TJ-ABCE1 were highly expressed during all developmental stages.ConclusionsThe whole set of T. japonicus ABC genes and their phylogenetic relationships will provide a better understanding of the comparative evolution of essential gene family resources in arthropods, including the crustacean copepods.

Gamma irradiation‐induced oxidative stress and developmental impairment in the hermaphroditic fish, Kryptolebias marmoratus embryo

This study investigated the effects of gamma radiation on the early developmental stages in hermaphroditic fish embryos of Kryptolebias marmoratus. The authors measured reactive oxygen species (ROS) level and antioxidant enzyme activities with the endpoint hatching rate after gamma irradiation of different embryonic stages. Then, the transcriptional changes of antioxidant enzyme-coding genes were evaluated by quantitative real-time reverse transcription polymerase chain reaction in response to gamma radiation on embryonic stages. Gamma radiation inhibited hatching rate and caused developmental impairment in a dose-dependent manner. Embryos showed tolerances in a developmental stage-dependent manner, indicating that early embryonic stages were more sensitive to the negative effects of gamma radiation than were later stages. After 5 Gy rate of radiation, the ROS level increased significantly at embryonic stages 2, 3, and 4 with a significant induction of all antioxidant enzyme activities. The expressions of glutathione S-transferase isoforms, catalase, superoxide dismutase (Mn-SOD, Cu/Zn-SOD), glutathione reductase, and glutathione peroxidase mRNA were upregulated in a dose-and-developmental stage-dependent manner. This finding indicates that gamma radiation can induce oxidative stress and subsequently modulates the expression of antioxidant enzyme-coding genes as one of the defense mechanisms. Interestingly, embryonic stage 1 exposed to gamma radiation showed a decreased expression in most antioxidant enzyme-coding genes, suggesting that this is also related to a lower hatching rate and developmental impairment. The results of this study provide a better understanding of the molecular mode of action of gamma radiation in aquatic organisms.

UV-B radiation-induced oxidative stress and p38 signaling pathway involvement in the benthic copepod Tigriopus japonicus

Ultraviolet B (UV-B) radiation presents an environmental hazard to aquatic organisms. To understand the molecular responses of the intertidal copepod Tigriopus japonicus to UV-B radiation, we measured the acute toxicity response to 96 h of UV-B radiation, and we also assessed the intracellular reactive oxygen species (ROS) levels, glutathione (GSH) content, and antioxidant enzyme (GST, GR, GPx, and SOD) activities after 24 h of exposure to UV-B with LD50 and half LD50 values. Also, expression patterns of p53 and hsp gene families with phosphorylation of p38 MAPK were investigated in UV-B-exposed copepods. We found that the ROS level, GSH content, and antioxidant enzyme activity levels were increased with the transcriptional upregulation of antioxidant-related genes, indicating that UV-B induces oxidative stress by generating ROS and stimulating antioxidant enzymatic activity as a defense mechanism. Additionally, we found that p53 expression was significantly increased after UV-B irradiation due to increases in the phosphorylation of the stress-responsive p38 MAPK, indicating that UV-B may be responsible for inducing DNA damage in T. japonicus. Of the hsp family genes, transcriptional levels of hsp20, hsp20.7, hsp70, and hsp90 were elevated in response to a low dose of UV-B radiation (9 kJ m(-2)), suggesting that these hsp genes may be involved in cellular protection against UV-B radiation. In this paper, we performed a pathway-oriented mechanistic analysis in response to UV-B radiation, and this analysis provides a better understanding of the effects of UV-B in the intertidal benthic copepod T. japonicus.

Expression pattern analysis of DNA repair-related and DNA damage response genes revealed by 55K oligomicroarray upon UV-B irradiation in the intertidal copepod, Tigriopus japonicus.

Ultraviolet-B (UV-B) radiation affects the genome stability of aquatic organisms by absorption of certain wavelength at the molecular level. Recently, extensive gene information has been identified from the intertidal copepod, Tigriopus japonicus. Here, we developed a 55K (54,254 genes) oligomicroarray and tested its usefulness to identify the effect of single dose of UV-B irradiation (12 kJ/m(2)) on transcriptomes of the copepod T. japonicus. A total of 35,361 spots were identified to be significantly modulated on the 55K oligomicroarray by hierarchical clustering after exposure to UV-B irradiation over 48 h (6, 12, 24, and 48 h). Of them, 1300 and 588 genes were observed to be up-regulated and down-regulated at all time points, respectively. Particularly, it was observed that several genes involved in DNA repair mechanism were significantly modulated in the UV-B-exposed T. japonicus by microarray and quantitative real-time RT-PCR analysis. In detail, UV-B irradiation specifically up-regulated some genes in non-homologous end-joining (NHEJ), homologous recombination (HR), base excision repair (BER), and mismatch repair (MMR) pathways. On the other hand, a majority of down-regulated genes were representatives for the nucleotide excision repair (NER) mechanism. These results demonstrated that DNA damage would be induced by UV-B irradiation in this species, resulting in reliable induction or repression of various DNA repair mechanism on UV-B-induced DNA damage. In this report, we suggest that a high density microarray-based approach for risk assessment of UV-B irradiation would be useful to elucidate the mechanistic analysis in a non-model organism. This study could also provide a better understanding of molecular mechanisms of cellular protection against UV-B-induced stress.