Immunotoxicity and hepatic function evaluation in Nile tilapia (Oreochromis niloticus) exposed to diazinon

The LC(50) of the organophosphorus pesticides (OPs) diazinon to Nile tilapia (Oreochromis niloticus) was determined, thereafter, hepatic activity, phagocytic index, percentages of active cells, relative spleen weight, total IgM concentration and lymphoproliferation rates were compared between diazinon exposed groups (LC(50) and (1/2)LC(50)) and non-exposed control group. Experimental data show that diazinon is highly toxic for juvenile Nile tilapia (LC(50)=7.830 ppm) and presents immunotoxic properties which affect both the innate and cellular adaptive immune responses of this fish, as revealed by the fact that splenocyte proliferation and phagocytic indices were significantly decreased after acute exposure to the pesticide. However, the hepatic biochemical parameters and the total circulating IgM concentrations were not affected in this experimental model.     

The activation of diazinon by ganglia of the cockroach Periplaneta americana L. and its action on nerve conduction and cholinesterase activity

When sixth abdominal ganglia of the cockroach Periplaneta americana were irrigated continuously with diazinon solution in situ, its effects on nerve conduction and cholinesterase activity closely resembled those of diazoxon; spontaneous activity and after-discharge increased until conduction was blocked, which happened while some cholinesterase was still uninhibited. The symptoms were only slightly relieved by irrigating ganglia with saline. Though the LD50's of diazinon and diazoxon applied topically to adult male P. americana were similar (2.5 ± 0.33 and 4.5 ± 0.38 μig. per insect), diazoxon was about 300 times more active than diazinon against nerve function and cholinesterase activity in the sixth abdominal ganglion. This is probably because in the nerve preparations contact between the insecticide and the tissues surrounding the nerve cord, which in whole insects convert diazinon, a thionophosphate, into its phosphate analogue diazoxon, a more active anticholinesterase, was minimized. Indeed, taking into account the evidence of workers who previously compared in vitro the anticholinesterase activities of several thionophosphates with those of their phosphate analogues and found the phosphates much more active, the effect of diazinon on cholinesterase activity and nerve function in our experiments was unexpectedly great. By applying diazinon to nerve cords with SKF 525-A, a compound likely to prevent oxidation of diazinon to diazoxon, an attempt was therefore made to decide whether diazinon directly affected nerve conduction or whether the effect resulted either from its conversion to diazoxon within the nerve tissue or from impurities in the diazinon used. Results were inconclusive, for SKF 525-A (p-diethylaminoethyl diphenylpropylacetate hydrochloride) not only failed to prevent the inhibition of cholinesterase, but interfered with the action of both diazinon and diazoxon on nerve conduction, and itself affected nerve conduction when applied alone. The possibility that diazinon is itself a mild anticholinesterase was not excluded. SKF 525-A applied to sixth abdominal ganglia at 2 × 10^-4M blocked conduction from cereal nerves to giant fibres in 50–97 min. and at 4 × 10^-5M decreased the post-synaptic response; applied to giant fibres at 2 × 10^-4M it blocked conduction in 90–208 min. The effects of the larger concentration were not completely reversible. Although SKF 525-A has been widely used to study the metabolism of drugs, its direct effects on conduction in nerve axons seem not to have been noted previously.     

Factors affecting the toxicity of diazinon to Musca domestica L

Processes affecting the toxicity of diazinon to a susceptible and a resistant strain of houseflies were examined. More evidence was obtained to show that slower penetration of diazinon through the integument of resistant flies is a cause of resistance. Small amounts of two decomposition products were found in both strains. The decomposition mechanisms, in these strains were differently distributed and, although detoxication of diazinon in the two strains is quantitatively similar and small, it may contribute to resistance. Traces of diazoxon were detected when diazinon was incubated with tissue extracts of either strain. Tissue extracts of resistant, but not of susceptible, flies decomposed significant amounts of diazinon in 1 hr. and the ability to decompose diazoxon seems to be an important cause of resistance. Tissues of both strains sorbed diazinon from aqueous solution similarly; the quantities sorbed were large and suggest that sorption may increase the amount of poison needed inside the insects to kill, by between five and forty times.     

Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver

The metabolism of diazinon, an organophosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague–Dawley (SD) rats or SD rats treated with different P450‐specific inducers (phenobarbital, dexamethasone, β‐napthoflavone, and pyrazole). Results obtained indicate an involvement of CYP2C11, CYP3A2, and CYP2B1/2, whereas CYP2E1 and CYP1A1 do not contribute to the pesticide oxidative metabolism. Indeed, diazinon was metabolized by microsomes from control rats; among the inducers, phenobarbital and dexamethasone only increased the production of either metabolites, although to different extents. The production of the two metabolites is self‐limiting, due to P450 inactivation; therefore, the inhibition of CYP‐specific monooxygenase activities after diazinon preincubation has been used to selectively identify the competent CYPs in diazinon metabolism. Results indicate that, after diazinon preincubation, CYP3A2‐catalyzed reactions (2β‐ and 6β‐testosterone hydroxylation) are very efficiently inhibited; CYP2C11‐ and CYP2B1/2‐catalyzed reactions (2α‐ and 16β‐testosterone hydroxylation, respectively) are weakly inhibited, while CYP2E1‐, CYP2A1/2‐, and CYP1A1/2‐related activities were unaffected. Results obtained by using chemical inhibitors or antibodies selectively active against specific CYPs provide a direct evidence for the involvement of CYP2C11, CYP3A2, and CYP2B1/2, indicating that each of them contributed about 40–50% of the diazinon metabolism, in hepatic microsomes from untreated, phenobarbital‐, and dexamethasone‐treated rats, respectively. The higher diazoxon/pyrimidinol ratio observed after phenobarbital‐treatment together with the significantly more effective inhibition toward diazoxon production exerted by metyrapone in microsomes from phenobarbital‐treated rats supports the conclusion that CYP2B1/2 catalyze preferentially the production of diazoxon. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 53–61, 1999     

In situ evaluation of the genotoxic potential of the river Nile: II. Detection of DNA strand-breakage and apoptosis in Oreochromis niloticus niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822)

This work is part of a wider eco-toxicological study proposed to evaluate the biological impact of contaminants along the whole course of the river Nile, Egypt. Here we present data on the presence of DNA strand-breaks and apoptotic cells assessed by use of comet and diffusion assays in erythrocytes of Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus). The results showed high degrees of DNA damage and increased frequencies of apoptotic nuclei in blood of fish collected from downstream compared with those sampled from upstream river Nile. Qualitative analysis revealed a shift in the frequency of DNA-damage classes towards higher damage levels correlating with the increasing pollution gradient. The degree of DNA damage measured by use of comet assay and diffusion assay exhibited seasonal variations. Both fish species showed significant increases in DNA damage during the summer. The results of our study indicated that the alkaline comet assay seems to be a useful technique for in situ genotoxic monitoring. At the same time the diffusion assay is sensitive enough to detect low frequencies of apoptotic nuclei. The results reveal species-specific differences in sensitivities, suggesting that Nile tilapia may serve as a more sensitive test species compared with the African catfish. Based on the outcome of the comet and diffusion assays, it can be concluded that the water quality of the river Nile with respect to the presence of genotoxic compounds needs to be improved, especially in its estuaries. As far as we know this is the first time that the comet and diffusion assays are used for genotoxic monitoring of the river Nile.     

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.     

四个罗非鱼选育品种抗链球菌病能力差异研究

为筛选出抗病力优良的罗非鱼品种, 以奥利亚罗非鱼“夏奥1号”、尼罗罗非鱼“99”埃及品系、吉富罗非鱼“中威1号”和奥尼罗非鱼为研究对象, 33℃水温暂养7d后分别进行无乳链球菌人工感染实验, 连续7d统计累计死亡率, 并于人工感染后0、24h、48h和72h采集血液和组织样本, 研究这4个罗非鱼选育品种抗链球菌病能力的差异。结果显示: 感染7d后奥尼罗非鱼的累计死亡率最低; 奥尼罗非鱼的谷草转氨酶(AST)感染前后始终都低于其余3个品种罗非鱼(P<0.05); 埃及尼罗和奥尼在感染72h后球蛋白(GLO)分别显著升高1.13倍和1.41倍; 奥尼罗非鱼白蛋白/球蛋白(A/G)在感染前后没有显著性变化(P>0.05), 而其余3个品种罗非鱼A/G比值在感染后都显著性降低(P<0.05); 埃及尼罗的碱性磷酸酶(AKP)在感染72h后显著降低(P<0.05), 奥利亚和吉富的AKP表现为先上升后下降, 奥尼的AKP感染前后没有显著性变化(P>0.05); 各品种罗非鱼血清中的乳酸脱氢酶(LDH)感染后都显著升高(P<0.05); 奥利亚、吉富和奥尼罗非鱼的超氧化歧化酶(SOD)感染48h时都显著升高(P<0.05); 奥尼罗非鱼在感染前后溶菌酶(LZM)活性都显著高于其余3个品种罗非鱼(P<0.05)。组织病理学结果显示:吉富和奥尼肝细胞水肿变性, 而奥利亚和埃及尼罗出现大面积肝细胞脂肪变性; 每个品种罗非鱼均呈现严重的脾炎, 奥利亚、埃及尼罗和吉富的脾脏中大量铁血黄素沉积; 每种罗非鱼呈现不同程度的肾小球萎缩, 肾小管上皮细胞变性、坏死。研究表明奥尼罗非鱼抗链球菌病能力最强, 感染后血清中AST水平与肝受损程度呈一定的正相关, LZM水平和罗非鱼抗链球菌病能力呈一定的正相关。     

Analysis of apoptosis of lymphoid cells in fish exposed to immunotoxic compounds

BACKGROUND: Chemical induction of apoptosis in cells is believed to contribute to toxicity. Techniques for measuring apoptosis have increased in both sensitivity and number and in many cases can be readily extended to nontraditional research species. A comparison of established assays for measuring apoptosis of lymphoid cells has thus far not been performed in the fish and thus would be efficacious in assessing immunotoxicity. METHODS: The present study evaluated chemical-induced immune cell apoptosis in fish (tilapia, Oreochromis niloticus) exposed to two known immunotoxic chemicals, azathioprine and T-2 toxin. Cytocentrifugation and light microscopy of leukocyte-enriched cell samples from the pronephros (i.e., the fish primary hematopoietic compartment) demonstrated chemical-related increases in apoptotic bodies. This observation was examined further with the ApoAlert Annexin V Apoptosis kit and two DNA-binding dyes employed for detecting apoptosis, 7-aminoactinomycin D (7-AAD) and propidium iodide (PI). RESULTS: The apoptotic probes confirmed the microscopic observations of increased apoptosis in the chemical-exposed fish. The ApoAlerttrade mark annexin V and 7-AAD assays, which discriminate early and late apoptosis/necrosis, compared well in identifying apoptotic populations. PI staining in Vindelov's solution was unable to detect early apoptosis. CONCLUSIONS: The present data suggest that apoptotic immune cells may be a useful marker for certain immunotoxicant exposures in fish. These findings agree with those of previous reports that fish may respond immunologically in a manner similar to mammals after immunotoxicant challenge.     

Comparative age and growth of Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis> L.) in lakes Nabugabo and Wamala,Uganda

Age and growth of Nile tilapia (Oreochromis niloticus) from Lake Nabugabo and Lake Wamala, Uganda, were determined using cross-sectioned sagittal otoliths. Marginal-increment and edge analyses of Nile tilapia otoliths from Lake Nabugabo indicated formation of two annuli per 12-month period. Opaque zones associated with faster growth were observed between April and June and between September and December, coincident with the two rainy seasons of the year. Within both lakes, males were larger at age than females. Nile tilapia from Lake Nabugabo, however, had faster growth rates than Nile tilapia from Lake Wamala, and fish >3 years old from Lake Nabugabo were larger at age than those from Lake Wamala. Ages ranged from 0 to 8.0 years for Nile tilapia from Lake Nabugabo, and from 0.5 to 6.5 years for tilapia from Lake Wamala. Differences in the patterns of growth in Nile tilapia between lakes may reflect, at least in part, the relatively energy-rich omnivorous diet of Nile tilapia in Lake Nabugabo versus a phytoplanktivorous diet in Lake Wamala. Diet differences of Nile tilapia between the two lakes are ascribed to trophic changes in the lakes due to the introduction of Nile perch (Lates niloticus) into Lake Nabugabo but not Lake Wamala. Alternatively, the greater exploitation of Nile tilapia in Lake Nabugabo may have resulted in increased growth rates, whereas Nile tilapia in Lake Wamala may be subject to slower, density-dependent growth. Handling editor: J. Cambray     

尼罗罗非鱼早期发育形态及其在珠江水系的空间分布

【背景】罗非鱼作为联合国粮农组织（FAO）向全世界推广养殖的优良品种之一,有多个品系,其养殖范围已遍布85个国家和地区。1956年我国从越南引进莫桑比克罗非鱼,经过养殖及推广,2006年我国罗非鱼产量达到100万t。但是,该外来物种在给我国带来良好经济价值的同时,对土著种类及水环境造成了极大的影响。【方法】对近几年珠江水系渔获物的调查数据进行整理,并观察尼罗罗非鱼早期发育形态,统计尼罗罗非鱼苗对其他鱼苗的最大捕食量及捕食规格,以分析罗非鱼生物学特性及其在珠江水系的入侵现状。【结果】尼罗罗非鱼早期发育快,卵黄营养非常丰富,比珠江水系土著种类更有竞争性;罗非鱼苗呈现很强的攻击性与捕食性;尼罗罗非鱼已经扩散到珠江水系各主要河流,并在部分江段形成优势种群。【结论与意义】尼罗罗非鱼种群快速扩张,对土著种已构成严重威胁,有必要将其列为珠江水系高危入侵种。     

The impacts of invasive Nile tilapia (Oreochromis niloticus) on the fisheries in the main rivers of Guangdong Province,China

Nile tilapia (Oreochromis niloticus) is one of the most widespread invasive fish species, and this species has successfully established populations in the major rivers of Guangdong Province, China. Field surveys and manipulative experiments were conducted to assess the impacts of Nile tilapia on fisheries. We determined that the increase of Nile tilapia in these rivers not only affects the CPUE (catch-per-unit-per-effort) of the fish community and native fish species but also reduces the income of fishermen. In the manipulative experiments, we observed that the growth of native mud carp decreased in the presence of Nile tilapia. Our results suggest that the invasion of Nile tilapia negatively affected the fishery economy and native fish species, and suitable control measurements should be taken.     

Sertoli cell proliferation in the adult testis--evidence from two fish species belonging to different orders

Germ cell survival and development critically depend on the cells' contact with Sertoli cells in the vertebrate testis. Fish and amphibians are different from mammals in that they show a cystic type of spermatogenesis in which a single germ cell clone is enclosed by and accompanied through the different stages of spermatogenesis by an accompanying group of Sertoli cells. We show that in maturing and adult testes from African catfish and Nile tilapia, Sertoli cell proliferation occurs primarily during spermatogonial proliferation, allowing the cyst-forming Sertoli cells to provide the increasing space required by the growing germ cell clone. In this regard, coincident with a dramatic increase in cyst volume and number of germ cells per cyst, in Nile tilapia, the number of Sertoli cells per cyst was strikingly increased from primary spermatogonia to spermatocyte cysts. In both African catfish and Nile tilapia, Sertoli cell proliferation is strongly reduced when germ cells have proceeded into meiosis, and stops in postmeiotic cysts. We conclude that Sertoli cell proliferation is the primary factor responsible for the increase in testis size and sperm production observed in teleost fish. In mammals, Sertoli cell proliferation in the adult testis is not observed under natural conditions. However, on the level of the individual spermatogenic cyst--similar to mammals--Sertoli cell proliferation ceases when germ cells have entered meiosis and when tight junctions are established between Sertoli cells. This suggests that fish are valid vertebrate models for studying Sertoli cell physiology.     

Exposure of rats to a 50-Hz, 100 muTesla magnetic field does not affect the ex vivo production of interleukins by activated T or B lymphocytes.

Two separate, independent experiments were conducted to evaluate the effects of exposure of rats to a 50-Hz linearly polarized, 100 microT magnetic field (MF) on the ex vivo production of interleukins (ILs) by mitogen-stimulated splenic lymphocytes. IL-1 and IL-2 were determined by proliferation assays, using IL-dependent murine T cell lines. In the first experiment, female Sprague-Dawley rats were treated with 7,12-dimethylbenz[a]anthracene (DMBA] at a dose of 20 mg per rat (four weekly gavage doses of 5 mg), and were either MF-exposed or sham-exposed for 14 weeks. This experimental protocol has previously been shown to result in a significant increase in breast cancer growth in response to MF exposure. Furthermore, MF exposure at 50-100 microT for 3 months was recently found to induce a suppressed ex vivo proliferation of splenic T cells in response to mitogen stimulation, which could be a result of reduced IL production of spleen lymphocytes. However, the present experiments failed to demonstrate any significant difference between MF- and sham-exposed groups in production of IL-1 by mitogen-activated splenic B cells. In a second experiment, shorter MF exposure periods were studied with respect to IL production from mitogen-stimulated B and T cells. Groups of rats were MF- or sham-exposed for 1 day, 1 week, or 2 weeks, followed by preparation and activation of spleen lymphocytes. No significant difference in IL-1 or IL-2 production from stimulated B or T cells was seen. The data indicate that in vivo MF exposure of rats does not affect the ex vivo IL production of B or T lymphocytes, suggesting that the recently reported changes in T cell proliferation in response to MF exposure may not be mediated via alterations in B or T cell IL production.     

Major histocompatibility complex class IIA and IIB genes of Nile tilapia Oreochromis niloticus: Genomic structure,molecular polymorphism and expression patterns

Major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism, and it plays an important role in the immune response of vertebrates. In the present study, we isolated MHC class II genes from Nile tilapia in order to investigate the immune mechanism in tilapia and develop better strategies for disease prevention. Moreover, we cloned the full-length cDNA sequences of MHC IIA and IIB from Nile tilapia by the RACE approach. In addition, the genomic structure, molecular polymorphism and expression patterns of MHC II genes in Nile tilapia were also examined. Compared with that of other teleosts, Nile tilapia MHC class IIA contained four exons and three introns. The deduced amino acid sequence of the MHC IIA molecule shared 25.4–64.5% similarity with those of other teleosts and mammals. Six exons and five introns were identified from Nile tilapia MHC IIB, and the deduced amino acid sequence shared 26.9–74.7% similarity with those of other teleosts and mammals. All the characteristic features of MHC class II chain structure could be identified in the deduced sequences of MHC IIA and IIB molecules, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. A total of 12 MHC IIA alleles were identified from six individuals. Four alleles originating from a single individual suggested that at least four MHC IIA loci existed. Moreover, 10 MHC IIB alleles were identified, among which four were detected in a single individual, suggesting that at least four MHC IIB loci existed. The expression of MHC IIA and IIB at the mRNA level in 10 types of normal tissues was determined using quantitative real-time PCR analysis. The highest expression level was detected in stomach and gill, whereas the lowest expression was detected in muscle and brain. Furthermore, MHC IIA and IIB were probably two candidate immune molecules involved in the resistance against streptococcosis, because their expression was significantly up-regulated in gill, kidney, intestine and spleen after the intraperitoneal injection of Streptococcus agalactiae.     

Effects of serum on phagocytic activity and proteomic analysis of tilapia (Oreochromis mossambicus) serum after acute osmotic stress

The objective of the study was to analyze the effect of serum from freshwater (FW) exposed tilapia or from 25 ppt seawater (SW) exposed tilapia on the ability to mediate the phagocytic activity of tilapia phagocytes. To analyze the phagocytic activity, head kidney (HK) and spleen leukocytes were tested in 300 or 500 mOsm medium using three different treatment groups (a) control, (b) addition of 25% serum from freshwater (FW) exposed tilapia, and (c) addition of 25% of serum from 25 ppt seawater (SW) exposed tilapia. HK leukocytes cultured in 300 and 500 mOsm media for 4 h showed an increase of phagocytic ability in the control group as compared to the addition of serum from either FW or SW exposed tilapia. HK leukocytes exposed to 500 mOsm medium showed a higher phagocytic ability than those leukocytes exposed to 300 mOsm medium in each corresponding group. Concurrently, spleen leukocytes in the control group showed a higher phagocytic ability than those leukocytes with the addition of serum from FW or SW exposed tilapia. As compared to spleen leukocytes cultured in 300 mOsm medium, leukocytes cultured in 500 mOsm medium showed an increase of phagocytic ability within their respective group. To further investigate the observed phenomenon, 2D-gel electrophoresis was performed for analyzing the differentially expressed proteins in serum that was thought to influence the phagocytic ability. Up-regulated serum proteins in SW exposed tilapia contained complement C3 protein, NADH dehydrogenase (Ubiquinone) Fe–S protein 3, Mg²⁺-dependent neutral sphingomyelinase, Semaphorins, and Caspase 3. Taken together these results suggest that addition of serum decreased the phagocytic activity in HK and spleen leukocytes in vitro, furthermore, induced proteins semaphorin, complement C3, Mg²⁺-dependent neutral sphingomyelinase, and Caspase 3 are up-regulated in the serum, which might have decreased the phagocytic activity upon exposure to hyperosmotic solutions.     

Effects of low concentration of endosulfan on proliferation, ERK1/2 pathway, apoptosis and senescence in Nile tilapia (Oreochromis niloticus) splenocytes

Endosulfan is a potent organochlorinated pesticide that is known to induce side effects in aquatic organisms, including Oreochromis niloticus (Nile tilapia). It has been previously shown that endosulfan induces oxidative stress and non-specific activation of splenic macrophages and exacerbated serum interleukin-2 synthesis in Nile tilapia. Endosulfan may promote proliferation of T cells through MAP kinase (MAPK) activated signal transductions. The ERK family of MAPKs includes ERK1 and ERK2. Phosphorylated ERK1/2 (pERK1/2) molecules are involved in many aspects of cellular survival, and are important for apoptosis or oxidative stress-induced senescence. In order to study the mechanisms by which endosulfan affects fish health, the present study was aimed at evaluating the in vitro effects of this insecticide on proliferation, the ERK1/2 pathway, apoptosis and cell senescence in splenocytes from Nile tilapia. Lymphoproliferation was evaluated by colorimetric method using the WST-1 assay. Flow cytometry was used to assess pERK1/2, apoptosis and senescence, using Annexin V-FITC and β-galactosidase respectively. Experimental data showed that exposure to 7 μg mL(-1) of endosulfan per se increased cellular proliferation, but decreased the lymphoproliferative response to mitogenic stimulus with PMA + ionomycin. Splenocytes exposed to endosulfan for 15-180 min showed significantly higher levels of pERK1/2 than the non-exposed control. Endosulfan mediated a decrease in etoposide-induced apoptosis and provoked cell senescence. In conclusion, exposure of immune cells to a low concentration of endosulfan deregulates their function and may facilitate the development of multiple diseases.     

Effect of Selenomethionine Supplementation in Food on the Excretion and Toxicity of Arsenic Exposure in Female Mice

Selenium (Se) is an essential component of several major metabolic pathways and controls immune function. Arsenic (As) is a human carcinogen with immunotoxic and genotoxic activities, functioning mainly by producing oxidative stress. Due to the ability of Se to interact with As and to possibly block its toxic effects, we investigated the impact of dietary Se-methionine (Se-Met) supplementation on the toxicity of As exposure in vivo in a mouse model. Sufficient and excess levels of Se-Met (0.2 and 2 ppm, respectively) were fed to C57BL/6N female mice exposed to sodium arsenite (3, 6 and 10 mg/kg) in tap water for 9 days. We observed that As exposure increased Se-Met excretion in the urine. Se-Met supplementation increased the relative liver weight and decreased the concentration of total liver proteins in animals exposed to 10 mg/kg of As. Se-Met supplementation maintained a normal pool of glutathione in the liver and increased glutathione peroxidase concentration, although the lipoperoxidation level was increased by Se-Met even without As exposure. Se-Met supplementation helped to maintain the CD4/CD8 ratio of lymphocytes in the spleen, although it increased the proportion of B cells. Se-Met supplementation prior to As exposure increased the secretion of interleukin-4, IL-12 and interferon-γ and the stimulation index of the spleen cells in in vitro assays. Se-Met intake improved the basal immunological parameters but did not reduce the damage caused by oxidative stress after low-dose As exposure.     

Effects of in vivo exposure to GSM-modulated 900 MHz radiation on mouse peripheral lymphocytes

The aim of this study was to evaluate whether daily whole-body exposure to 900 MHz GSM-modulated radiation could affect spleen lymphocytes. C57BL/6 mice were exposed 2 h/day for 1, 2 or 4 weeks in a TEM cell to an SAR of 1 or 2 W/kg. Untreated and sham-exposed groups were also examined. At the end of the exposure, mice were killed humanely and spleen cells were collected. The number of spleen cells, the percentages of B and T cells, and the distribution of T-cell subpopulations (CD4 and CD8) were not altered by the exposure. T and B cells were also stimulated ex vivo using specific monoclonal antibodies or LPS to induce cell proliferation, cytokine production and expression of activation markers. The results did not show relevant differences in either T or B lymphocytes from mice exposed to an SAR of 1 or 2 W/kg and sham-exposed mice with few exceptions. After 1 week of exposure to 1 or 2 W/kg, an increase in IFN-gamma (Ifng) production was observed that was not evident when the exposure was prolonged to 2 or 4 weeks. This suggests that the immune system might have adapted to RF radiation as it does with other stressing agents. All together, our in vivo data indicate that the T- and B-cell compartments were not substantially affected by exposure to RF radiation and that a clinically relevant effect of RF radiation on the immune system is unlikely to occur.