Effect Of Microcystin-LR On Metabolic Regulation Of Thyroid Hormones In Fish

With the development of human civilization, much more sanitary sewage and agriculture fertilizers poured into water, resulting the increasingly serious eutrophic of lakes, and trigger the widespread occurrence of cyanobacterial blooms. This has been regarded as a serious environmental issue and received increasing attention. A potentially hazardous consequence of cyanobacterial blooms is the production of several kinds of cyanotoxins. The microcystins(MCs) are the most commonly identified toxins in freshwater blooms, and microcystin-LR(MC-LR) is recognized as being the most toxic and is distributed worldwide. Fish are easily exposed to MCs either by ingestion or absorption through the gills.MC-LR is thought to a hepatotoxin, for it preferentially accumulates in the liver, and result in the serious liver damage. Some studies revealed that MCs also have adverse impacts on development and growth of fish, and induced the oxidative damage in fish. In recent years, a few studies have shown that MCs could alter thyroid hormone(TH) levels, indicates that MCs could disrupt the thyroid endocrine systems in fish. Although MCs can disturb the normal physiological processes of TH metabolism in fish, the mechanism underlying the changes in TH levels is still unclear.In order to study the effects of MCs on metabolism regulation of THs in fish. We chose the hepatic cell line of the grass carp(L8824), the juvenile and adult zebrafish as the experimental model. Firstly, we evaluated the effects of MC-LR on the gene transcription and activities of iodothyronine deiodinases(IDs) in L8824 cells. Then, the juvenile and adult zebrafish were used to assess the disruption of thyroid endocrine system caused by acute and subacute MC-LR exposure respectively. Studies were under taken with the following contents: cell culture and cell viability test, the measurement of TH levels, the observation and analysis of the changes of morphology in thyroid follicle epithelial cells, the measurement of the activities of IDs, and the gene transcription involved in TH synthesis and metabolism. The analysis of this combination of factors involved in TH metabolism allows for a more complete assessment of the mechanisms of thyroid endocrine disruption in fish exposed to MCs.Results and conclusions:1. The L8824 cells viabilities were not significantly affected by 1 μg/L-1000 μg/L MC-LR after exposed for either 24 or 48 h compared with the control group. The m RNA expression of type I reduced after exposure to 100 μg/L and 1000 μg/L MC-LR. And type II iodothyronine deiodinase(ID2) reduced after exposure to 1、10、100 and 1000 μg/L MC-LR. However, MC-LR exposure led to the increase in the m RNA expression of type III iodothyronine deiodinase(ID3). Moreover, significant ID1 and ID2 activities decline were also observed in the L8824 cells exposed to MC-LR, and the activity of ID3 increased significantly in the MC-LR treated group. Therefore, The MC-LR exposure significantly alters the gene transcription and activities of IDs in the L8824 cells. Since the IDs play a crucial role in the metabolism of THs, the alterations of the IDs activities caused by MC-LR may disturb the normal THs metabolism processes.2. Acute MC-LR exposure(50 μg/L-400 μg/L) significantly decreased both T4 and T3 levels in juvenile zebrafish. The gene transcription of corticotropin-releasing hormone(crh), thyroid-stimulating hormone(tsh), thyroid peroxidase(tpo), and transthyretin(ttr) were present a rising trend after lower first following the MC-LR exposure. The gene transcriptions of thyroid receptors(tr-α and tr-β) were down-regulation all the time during the exposure period. Moreover, the acitivities of ID1 and ID2 were also significantly decline firstly, and then present a rising trend following the MC-LR exposure. The activity of ID3 decreased significantly during the exposure time. The results indicate that acute MC-LR exposure disturbs the THs homostasis and result in a hypothyroidism state in juvenile zebrafish. The changes in the gene transcriptions and IDs revealed that MC-LR significantly disturbs the normal physiological function of thyroid endocrine system, leading to a decline in TH production.3. No significant difference in T4 content was observed in male zebrafish. However, the T4 content was significantly decrased in female zebrafish after exposed to 100、200 and 400 μg/L MC-LR, and the T4 content gradually returned to control level after 96 h of exposure. In adition, acute MC-LR exposure(100 、 200 and 400 μg/L) significantly decreased T3 contents in both male and female zebrafish. Moreover, acute exposure to MC-LR caused serious changes in gene transcription involved in TH synthesis and metabolism in brain or liver of adult zebrafish, and also significantly alters the activities of IDs in liver. As the extension of exposure, the results present the dynamic fluctuations. These results illustrate that acute MC-LR exposure also significantly alters the activities of IDs and the gene transcription involved in TH sunthesis and metabolism in adult zebrafish. Due to the well developed of all the tissues and organs in adult zebrafish, they can make more effective self-regulation mechanism than in juvenile zebrafish. As a result, MC-LR exposure did not caused the serious changes in TH levels in adult zebrafish.4. Subacute MC-LR exposure at environmentally relevant concentrations(1、5 and 25 μg/L) significantly increased T4 content in juvenile zebrafish. However, the T3 content was decreased after exposed to 5 or 25 μg/L MC-LR in juvenile zebrafish. We also observed hypertrophy and hyperplasia of the thyroid follicle epithelial cells, as well as up-regulation of crh, tsh, tpo and ttr genes expression. The decreases in ID1 and ID2 activities were observed in 5 and 25μg/L MC-LR treatment groups. An increase in ID3 activity was observed in 1、5 and 25 μg/L MC-LR treatment groups. Thus, subacute MC-LR exposure at environmentally relevant concentrations significantly alters the activities of IDs and causes a drop in T3 contents, led to a hypothyroidism state in juvenile zebrafish. In addition, the hyper-stimulation of TH synthesis and secretion, including the hypertrophy and hyperplasia of the thyroid follicle epithelial cells, the elevations in T4 contents as well as the up-regulated genes involved in TH synthesis, can be mainly regard as the negative feedback from the hypothalamus and pituitary due to the decreased contents of T3.5. No differences in histopathology of thyroid follicle epithelial cells, as well as unchanged T4 contents were observed in adult zebrafish. The significant decline in T3 or FT3 contentls associated with the decreased in ID2 activity in male zebrafish were observed after exposed to 1、5 and 25 μg/L MC-LR treatment groups for 21 days. Moreover, MC-LR exposure significantly increases the ID1 or ID2 activity and decreases the ID3 activity respectively with the continuous exposure and the T3 content gradually returned to control disruption in TH levels in adult zebrafish. The MC-LR also alters the gene transcription involved in TH synthesis and metabolism. Thus, although the subacute MC-LR exposure at environmentally relevant concentrations could alters the activities of IDs and the gene transcription of crh, tsh, ttr and tr, the adult zebrafish can trigger an effective compensatory mechanism to maintain the homeostasis of TH.