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높은 구리 농도가 홍해 잉어 Pagrus major의 독성 및 산화 스트레스 반응에 미치는 영향. 이 연구는 다양한 농도의 구리에 노출된 후 홍해 잉어(Pagrus major)에서 독성과 산화 스트레스를 유발하는 구리의 농도 범위를 결정하기 위해 수행되었습니다. 구리가 참돔의 독성 스트레스와 세포사멸에 미치는 영향.

또한, TUNEL 분석 결과, 구리 농도가 30 μg/L인 실험군에서 가장 많은 세포사멸이 관찰되었다. 따라서 참돔의 경우 구리농도가 30μg/L 이상이면 독성이 있어 스트레스 및 세포사멸을 유발하는 것으로 판단된다. 구리가 참돔의 산화스트레스와 면역반응에 미치는 영향.

Effects of Waterborne Copper on Toxicity and Oxidative Stress Responses in Red Seabream, Pagrus major

Effects of waterborne copper on toxicity and oxidative stress responses in red sea bream, Pagrus major. ACTH), cortisol and glucose)] and toxic stress indices [(metallothionein (MT) and Na+/K+-ATPase (NKA)] in red sea bream exposed to different concentrations of Cu. As a result, the concentration of CRH, ACTH, cortisol , MT, H2O2 and caspase-3 concentrations increased significantly after Cu exposure times were observed in the experimental groups exposed to 30 and 40 μg/L. In conclusion, when exposed to Cu concentrations of 30 μg/L or more, toxic in the fish body, indicating stress and apoptosis.

Effects of aqueous copper on oxidative stress and immune responses in red sea carp, Pagrus major.

Effects of waterborne copper on oxidative stress and immune responses in red seabream, Pagrus major

ACTH), cortisol and glucose)] and toxic stress indices [(metallothionein (MT) and Na+/K+-ATPase (NKA)] in red sea bream exposed to various concentrations of Cu. The comet assay also showed that the highest level of nuclear DNA damage occurred experiment group, which was exposed to 30 μg/L for 120 h. In red sea bream, Cu concentration above 30 μg/L was considered to have a negative effect on the physiological function of red sea bream such as induction of secretion of antioxidant activity hormone and reduces the expression of substance-related immune index.

Chapter 1

Effects of waterborne copper on toxicity stress and apoptosis responses in red seabream, Pagrus major

Introduction
Materials and methods

Experimental fish
Cu treatment and sampling
Plasma parameter analysis
TUNEL assay
Statistical analysis

Results

Changes in plasma levels of CRH and ATCH
Changes in plasma levels of cortisol and glucose
Changes in plasma levels of MT
Changes in plasma levels of NKA concentrations
Change in plasma caspase-3 and H 2 O 2 levels
TUNEL assay

Discussion
Chapter 2

To this end, I analyzed changes in the physiological stress response, monitored by the HPI axis (CRH, ACTH, cortisol and glucose) and toxic stress indices (MT and NKA) in sea bream exposed to different Cu concentrations. Furthermore, I measured the activity of caspase-3 and H2O2 in the blood to confirm the apoptotic response of sea bream exposed to Cu, and performed a terminal transferase dUTP nick end labeling (TUNEL) assay to determine the cell death rate in gill tissue. The changes in plasma levels of CRH and ACTH in sea bream exposed to different concentrations of Cu and 40 μg/L are shown in Fig.

In this study, I exposed sea bream to different concentrations of copper to examine the nature and degree of the stress response and apoptosis. In addition, as a defense mechanism against stress, the HPI axis was activated in sea bream, as indicated by increased blood levels of CRH and ACTH. I analyzed changes in plasma cortisol and glucose concentrations secreted in response to CRH and ACTH.

Plasma cortisol and glucose concentrations were significantly increased in the experimental group exposed to 40 μg/L Cu, and the concentrations tended to increase with an increase in exposure time. Increases in MT levels were observed in red sea bream exposed to 30 and 40 μg/L Cu, and with an increase in the time exposed to Cu. Therefore, in the present study, exposure to high concentrations of Cu (30 and 40 μg/L) acted as a toxic stress in the fish body, leading to increased MT concentration as a defense mechanism against metal exposure.

In this study, we also analyzed the plasma concentrations of NKA, which plays a role in ion regulation, to investigate the effect of Cu concentration-induced stress on gill function. A similar study by Wu et al. 2015) reported that NKA activity was significantly reduced when tilapia Oreochromis mossambicus was exposed to environmental Cu (0.2, 1, and 2 mg/L) and that the level of injury was generally higher in the 2 mg/L group. In the present study, I also analyzed the effects of exposure to different concentrations of copper on changes in plasma H2O2 and caspase-3 in sea bream.

I observed that apoptosis was induced to the greatest extent in fish gill cells exposed to a Cu concentration of 30 μg/L for 120 h compared to Cont. Therefore, in the present study, I suspect that high concentrations of Cu induced apoptosis in the gill tissue of red mushrooms. In conclusion, when red carp were exposed to Cu concentrations of at least 30 μg/L, stress was induced in the fish and to cope with this stress, CRH, ACTH, cortisol activity and glucose levels related to the axis HPI. hormones have increased significantly.

Fig. 1. Changes in plasma corticotrophin-releasing hormone (CRH) (A) and adrenocorticotropic hormone (ACTH) (B) levels during exposure to Cu [0 (Cont.), 10, 20, 30, and 40 μg/L]

Effects of waterborne copper on oxidative stress and immune responses in red seabream, Pagrus major

Experimental fish and treatment

The fish were reared with automatic temperature control systems (JS-WBP-170RP; Johnsam Co., Seoul, Korea) and allowed to acclimatize for 1 week in thirty 300-liter circulating filter tanks (with one tank raised per experimental group to minimize density stress, n= 5) in the laboratory. We provided a commercial pellet diet twice daily (at 9:00 AM and 4:00 PM) until the day before sampling, but not during the experiment. During the experimental period, salinity and photoperiod were maintained at 35 psu and a period of 12 h (h) light (L): 12 h dark (D), respectively.

Sampling

Total RNA extraction, cDNA synthesis

Quantitative real-time PCR (qPCR)

Western blot analysis

180 V and the gels were immediately transferred to a 0.2-µm polyvinylidene difluoride membrane (Bio-Rad) at 85 V for 3 min using Trans-Blot® Turbo™. Membrane images were scanned with a high-resolution scanner, and band density was estimated with a computer program (Image LabTM Software, version 3.0; Bio-Rad).

SOD and CAT activities analysis

Plasma lysozyme, IgM, and melatonin levels

Comet assay

Statistical analysis

The expressions and activities of anti-oxidant enzymes SOD and CAT in the liver and plasma

The lowercase letters indicate significant (P < 0.05) differences between the different exposure times at the same Cu concentrations. The numbers above the bars indicate significant differences between the fish exposed to different Cu concentrations within an exposure period. The numbers above the bars indicate significant differences between the fish exposed to different Cu concentrations within an exposure period.

Fig. 7. Expression (A) and activity (B) of superoxide dismutase (SOD) in the liver and plasma of red seabream exposed to Cu [0 (Cont.), 10, 20, 30, and 40 μg/L]

Plasma lysozyme and IgM levels

Plasma Melatonin levels

Western blot of immune expression [lysozyme (B, 32 kDa), IgM (D, 32 kDa)] in abdominal liver and β-tubulin (55 kDa) was used as an internal control. Western blots of the expression of melatonin (A, 45 kDa) in the liver of stonefish and β-tubulin (55 kDa) were used as an internal control.

Fig. 10. Changes in plasma of lysozyme (A) and IgM (C) levels during exposure to Cu [0 (Cont.), 10, 20, 30, and 40 μg/L] in red seabream, as measured using a microplate reader

Comet assay

First, the analysis of mRNA expressions and activities of SOD and CAT, which are antioxidant enzymes in red sea carp, showed that mRNA expression and activity were significantly increased according to Cu concentration and exposure time in the groups of exposure 30 and 40 μg/L Cu. . This study confirms the previous study (Jiang et al., 2014) which reported that when Jian carp, Cyprinus carpio, was exposed to a high concentration of Cu, 0.6 mg/L (= 600 μg/L), for 4 days, ROS was redundant. produced in the brain and antioxidant enzyme mRNA expressions (SOD, CAT and GPX) were increased to increase antioxidant activity in the body. Furthermore, plasma H2O2 and LPO are widely used as indicators of the degree of oxidative stress in the body (Halliwell and Gutteridge, 1984; Woo et al., 2006).

Therefore, in the results of the current study, the concentration of toxic H2O2 in the muscle caused the toxicity to exceed the antioxidant capacity of the body and the initiation of lipid oxidation in the muscle (Jin et al., who examined the H2O2 and LPO factors measurements, reported that when goldfish were exposed to different concentrations of selenium, the levels of H2O2 and LPO were significantly increased at high concentrations of 3 and 4 mg/l, indicating that selenium was toxic at these concentrations. Cu concentration and exposure time at 10 and 20 μg/L Cu, but in the 30 and 40 μg/L Cu experimental groups, the levels of immune-related hormones decreased as Cu concentration and exposure time increased. They found that lysozyme and IgM, which are indices of immunity, were significantly reduced in the experimental group (diet with Cu).

Recent studies have also shown that in the case of rock bream, when the concentration of bisphenol A was more than 10 ng/L, ROS formation was induced and these ROS lowered plasma melatonin levels (Choi et al., 2016b). Therefore, similar to previous studies on organisms exposed to heavy metals such as Cu, ROS were induced in the body of red sea bream exposed to high Cu concentrations, and therefore ROS were considered to significantly reduce the immunity of red sea bream . Finally, I performed a comet assay to measure the degree of nuclear DNA damage in the liver cells of red sea bream exposed to high concentrations of Cu.

Nuclear DNA in bream hepatocytes exposed to cont., 10 and 20 μg/L Cu was normal, but in bream hepatocytes exposed to 30 and 40 μg/L Cu, tail length and % DNA in the tail were significantly increased. In summary, our results indicate that copper concentrations above 30 μg/L induce ROS in the body of sea bream and act as a factor to increase oxidative stress. In the future, studies on the improvement of antioxidant reactions and immunity of sea bream should be carried out.

Differential diurnal rhythms of melatonin in the pineal gland and gut of the goldfish, Carassius auratus in response to light. Administration of melatonin increases the activity of antioxidant enzymes and reduces lipid peroxidation in erythrocytes of rainbow trout (Oncorhynchus mykiss, Walbaum). Gill cell changes induced by copper exposure in the South American tropical freshwater fish Prochilodus scrofa.