Carp transferrin can protect spermatozoa against toxic effects of cadmium ions

Cadmium is a widespread heavy metal that enters the aquatic environment and affects many processes involved in fish reproduction such as sperm motility. Fish seminal plasma proteins can protect spermatozoa against toxic effects of heavy metals. The objective of this study was to demonstrate the ability of a major carp seminal plasma protein-transferrin (TF) to bind cadmium ions and to neutralize the toxic effect of cadmium on carp sperm motility. To obtain a high quantity of carp seminal plasma TF necessary for the experiment, immunoaffinity chromatography as a one-step isolation procedure was established. The titration of TF with cadmium ions spectrophotometrically at 247nm revealed that TF binds cadmium ions at only one spectrophotometrically-sensitive binding site, which suggests that TF is capable of neutralizing the cadmium toxic effect. Indeed, the addition of carp TF to carp semen incubated with 50ppm cadmium for 48h led to about a four-times higher percentage of sperm motility (30.3±1.1%) in comparison to samples incubated with only 50ppm cadmium (8.2±5.2%). Similarly, higher values of other parameters of sperm movement measured by a computer-assisted sperm motility analysis system (VSL, VCL and ALH) were observed at the presence of transferrin. In conclusion, our study provides the first evidence that transferrin from carp seminal plasma can protect sperm motility from cadmium toxicity.     

Complex physiological traits as biomarkers of the sub-lethal toxicological effects of pollutant exposure in fishes

Complex physiological traits, such as routine aerobic metabolic rate or exercise performance, are indicators of the functional integrity of fish that can reveal sub-lethal toxicological effects of aquatic pollutants. These traits have proved valuable in laboratory investigations of the sub-lethal effects of heavy metals, ammonia and various xenobiotics. It is not known, however, whether they can also function as biomarkers of the complex potential range of effects upon overall functional integrity caused by exposure to mixtures of chemicals in polluted natural environments. The current study used portable swimming respirometers to compare exercise performance and respiratory metabolism of fish exposed in cages for three weeks to either clean or polluted sites on three urban European river systems: the river Lambro, Milan, Italy; the rivers Blythe, Cole and Tame, Birmingham, UK; and the river Amstel, Amsterdam, The Netherlands. The UK and Italian rivers were variously polluted with high levels of both bioavailable heavy metals and organics, and the Amstel by mixtures of bioavailable organics at high concentrations. In both the UK and Italy, indigenous chub (Leuciscus cephalus) exposed to clean or polluted sites swam equally well in an initial performance test, but the chub from polluted sites could not repeat this performance after a brief recovery interval. These animals were unable to raise the metabolic rate and allocate oxygen towards exercise in the second trial, an effect confirmed in successive campaigns in Italy. Swimming performance was therefore a biomarker indicator of pollutant exposure in chub exposed at these sites. Exposure to polluted sites on the river Amstel did not affect the repeat swimming performance of cultured cloned carp (Cyprinus carpio), indicating either a species-specific tolerance or relative absence of heavy metals. However, measurements of oxygen uptake during swimming revealed increased rates of routine aerobic metabolism in both chub and carp at polluted sites in all of the rivers studied, indicating a sub-lethal metabolic loading effect. Therefore, the physiological traits of exercise performance and metabolic rate have potential as biomarkers of the overall sub-lethal toxic effects of exposure to complex mixtures of pollutants in rivers, and may also provide insight into why fish do not colonize some polluted environments.     

三峡库区水样中重金属含量及其对斑马鱼胚胎发育的毒性评价

三峡库区水体污染对库区鱼类生存的影响尚不明晰, 因此测试库区水体中污染物含量及其对鱼类早期发育的影响对于库区生态渔业的安全发展具重要意义。研究于三峡库区坝前段典型区域设置3 个采样点,用ICP-MS分析了原水中13 种重金属的含量, 并将斑马鱼胚胎暴露于纤维膜过滤后的水样中, 测试其内分泌及发育毒性效应。结果表明, 三峡库区水样中13 种重金属的浓度均低于地表水环境标准规定的限值, 且3 号采样点重金属浓度普遍比1 号和2 号采样点高。经7d 暴露后, 处理组和对照组中斑马鱼胚胎的孵化率、相对成活率和畸形率(畸形指标包括原肠胚终止、心跳/心率、脊柱畸形、心包囊水肿)均无明显差异;但幼鱼体内生殖相关基因(如 型雌激素受体, 雄激素受体和芳香化酶)、甲状腺相关基因(如甲状腺激素受体、甲状腺激素释放激素、Ⅱ型脱碘酶)和神经发育相关基因(如1 微管蛋白、突触蛋白、生长相关蛋白)的转录水平较之对照组(曝气24h 的自来水)均显著下降。上述结果表明, 三峡库区水体中13 种重金属的含量仍处于较低水平, 且在此污染水平下不会对斑马鱼胚胎发育过程产生显著影响。此外, 污染水体导致的分子水平上的变化是否会引起鱼类生长发育过程中的长期影响尚有待进一步研究。       

Some aspects of hepatic function in feral brown trout, Salmo trutta, living in metal contaminated water

Brown trout, Salmo trutta, exposed to heavy metals (mainly Cd and Zn) for at least 2 years in the Eagle River, Colorado, were examined for liver size and activity of the growth-promoting enzyme, ornithine decarboxylase (ODC) and compared to trout living in an uncontaminated site. Liver-somatic index (LSI) was greater for trout living in the uncontaminated site with the LSI of females being significantly greater than that of males. The LSI for females at the uncontaminated site was greater than that of females at the contaminated site, but males were not different statistically. ODC activity in the livers of both males and females was lower at the contaminated site. However, males and females did not differ with respect to ODC activity. These data suggest that chronic exposure to heavy metals may have important implications for growth and reproduction and possibly survival. The activity of ODC in liver might serve as a useful biomarker when assessing chronic toxicity of metals to naturally reproducing fish populations.     

Reproductive effects of prenatal exposure to nonylphenol on zebrafish (Danio rerio)

Mature female and male zebrafish were separated and exposed to nonylphenol (NP) at 0.1, 1, 10, 50, 100 and 500 microg/L, respectively, for 3 weeks. Gonadosomatic index (GSI) in both sexes and vitellogenin (VTG) induction in males was measured as the bioindicators for the impairment to the parents. The results indicated that 50 microg/L of NP was the non-observed effect concentration (NOEC) for GSI and VTG induction. Afterwards, the 50 microg/L NP exposed females and males, and the control females and males were cross-wise pair-bred in the control water for one week to examine the reproductive effects. The embryonic cathepsin D (CAT D) activity, eggshell thickness, fecundity, hatching rate and malformation (vertebral column flexure) rate of offspring were determined in the four pair-bred groups. While endpoints remained unchanged in the groups with exposed males, prenatal exposure of females to 50 microg/L of NP resulted in the impairment of reproduction in groups with exposed females including inhibition of CAT D activity (P < 0.05), decrease of eggshell thickness (by 23.6%) and elevation of malformation rate (P < 0.001). These results suggested NP could induce reproductive damage to zebrafish at NOEC for parents. The results also imply that alterations of CAT D activity and eggshell thickness may be more sensitive biomarkers to indicate the reproductive effects caused by endocrine disrupting chemicals.     

鱼类对重金属胁迫的分子反应机理

水体中浓度超标的重金属对鱼类的正常生命活动产生了严重影响,甚至导致鱼类的死亡.鱼体受重金属离子胁迫时体内会产生一系列相关基因的表达变化,并合成相关的蛋白和酶,如热激蛋白、金属硫蛋白、转运铁蛋白、谷胱甘肽转移酶、过氧化氢酶、超氧化物歧化酶等以应对重金属对机体产生的危害.这是生物体对环境的一种应激保护性反应,是生物体对周围环境中过量重金属的一种防御性机制.通过鱼类在重金属胁迫下组织中相关基因的表达可以从分子水平上来阐述有毒物质对鱼体产生的影响.综述了重金属离子胁迫下鱼类机体的分子反应机理.     

The effects of vinclozolin,an anti-androgenic fungicide,on male guppy secondary sex characters and reproductive success

Despite the enormous volume of research concerning the various effects of chemicals with endocrine-disrupting properties in fish, there is still very little evidence that endocrine disruption can adversely affect individual fertility and, hence, pose problems for the population. In the present study, guppies (Poecilia reticulata) were fed with the anti-androgenic fungicide vinclozolin at concentrations ranging from 1.8 to 180 mg/kg from 8-14 wk of age. Male sperm count and the intensity of his sexual display behavior were significantly reduced in treatment groups, which was in line with the results of previous studies. Here, we show further that these impairments translate into reduced fertility, measured as the size of the female's first clutch. Also, this reduced fertility was correlated to the male sperm count, but not to the intensity of the male sexual display. Finally, by crossing exposed with unexposed animals, we show that the adverse effect of vinclozolin on reproduction is mediated through the male alone.     

Counteracting effects of heavy metals and antioxidants on male fertility

Infertility is regarded as a global health problem affecting 8–12% of couples. Male factors are regarded as the main cause of infertility in 40% of infertile couples and contribute to this condition in combination with female factors in another 20% of cases. Abnormal sperm parameters such as oligospermia, asthenospermia, and teratozoospermia result in male factor infertility. Several studies have shown the deteriorative impact of heavy metals on sperm parameters and fertility in human subjects or animal models. Other studies have pointed to the role of antioxidants in counteracting the detrimental effects of heavy metals. In the currents study, we summarize the main outcomes of studies that assessed the counteracting impacts of heavy metal and antioxidants on male fertility. Based on the provided data from animal studies, it seems rational to administrate appropriate antioxidants in persons who suffer from abnormal sperm parameters and infertility due to exposure to toxic elements. Yet, further human studies are needed to approve the beneficial effects of these antioxidants.

     

Environmental chemical mediated male reproductive toxicity: Drosophila melanogaster as an alternate animal model

Industrialization and indiscriminate use of agrochemicals have increased the human health risk. Recent epidemiological studies raised a concern for male reproduction given their observations of reduced sperm counts and altered semen quality. Interestingly, environmental factors that include various metals, pesticides and their metabolites have been causally linked to such adversities by their presence in the semen at levels that correlate to infertility. The epidemiological observations were further supported by studies in animal models involving various chemicals. Therefore, in this review, we focused on male reproductive toxicity and the adverse effects of different environmental chemicals on male reproduction. However, it is beyond the scope of this review to provide a detailed appraisal of all of the environmental chemicals that have been associated with reproductive toxicity in animals. Here, we provided the evidence for reproductive adversities of some commonly encountered chemicals (pesticides/metals) in the environment. In view of the recent thrust for an alternate to animal models in research, we subsequently discussed the contributions of Drosophila melanogaster as an alternate animal model for quick screening of toxicants for their reproductive toxicity potential. Finally, we emphasized the genetic and molecular tools offered by Drosophila for understanding the mechanisms underlying the male reproductive toxicity.     

Radiation-induced bystander effects in the Atlantic salmon (salmo salar L.) following mixed exposure to copper and aluminum combined with low-dose gamma radiation

Very little is known about the combined effects of low doses of heavy metals and radiation. However, such “multiple stressor” exposure is the reality in the environment. In the work reported in this paper, fish were exposed to cobalt 60 gamma irradiation with or without copper or aluminum in the water. Doses of radiation ranged from 4 to 75 mGy delivered over 48 or 6 h. Copper doses ranged from 10 to 80 μg/L for the same time period. The aluminum dose was 250 μg/L. Gills and skin were removed from the fish after exposure and explanted in tissue culture flasks for investigation of bystander effects of the exposures using a stress signal reporter assay, which has been demonstrated to be a sensitive indicator of homeostatic perturbations in cells. The results show complex synergistic interactions of radiation and copper. Gills on the whole produce more toxic bystander signals than skin, but the additivity scores show highly variable results which depend on dose and time of exposure. The impacts of low doses of copper and low doses of radiation are greater than additive, medium levels of copper alone have a similar level of effect of bystander signal toxicity to the low dose. The addition of radiation stress, however, produces clear protective effects in the reporters treated with skin-derived medium. Gill-derived medium from the same fish did not show protective effects. Radiation exposure in the presence of 80 μg/L led to highly variable results, which due to animal variation were not significantly different from the effect of copper alone. The results are stressor type, stressor concentration and time dependent. Clearly co-exposure to radiation and heavy metals does not always lead to simple additive effects.     

Gene delivery to pancreatic exocrine cells in vivo and in vitro

Background

Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein).

Results

The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1?C2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17?-oestradiol (E₂), the synthetic oestrogen 17??- ethinyloestradiol (EE₂), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE₂; 10?ng/L, E₂; 100?ng/L, after 72?h exposures). For the NP exposures, GFP expression was observed at 10???g NP/L after 72?h (100???g NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally.

Conclusion

Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.     

Nonylphenol modulates expression of androgen receptor and estrogen receptor genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERalpha and ERbeta) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERalpha, and ERbeta from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERalpha, and ERbeta genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ERalpha and Rm-ERbeta cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERbeta mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERalpha mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERalpha mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERbeta mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 microg/l) and BisA (600 microg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERalpha, and Rm-ERbeta mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERalpha, and Rm-ERbeta genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.     

邻苯二甲酸二乙酯和壬基酚联合暴露对杜氏盐藻生长的影响

为了探讨环境激素类物质邻苯二甲酸二乙酯(DEP)和壬基酚(NP)对海洋微藻的联合毒性效应,选取杜氏盐藻(Dunaliella salina)为受试生物,以环境激素对杜氏盐藻单一暴露的96h EC₅₀的毒性效应作为一个毒性单位(IU),采用毒性单位法比较研究了DEP和NP单一暴露以及两者以三种不同混合比例(毒性单位比:1:1、1:4和4:1)暴露对杜氏盐藻的细胞生长、叶绿体色素含量、可溶性蛋白含量、SOD活性以及最大光能转化效率(Fv/Fm)的影响.实验结果表明:DEP和NP单一暴露对杜氏盐藻的96h EC₅₀分别为69.54 mg/L和1.47 mg/L,两种环境激素对杜氏盐藻均有抑制作用,且NP较DEP对杜氏盐藻的毒性更强.DEP和NP联合暴露较单一暴露对杜氏盐藻的细胞生长、叶绿体色素和可溶性蛋白的合成有较强的抑制作用,两种环境激素在毒性单位比为1:1、1:4、4:1三个比例水平上的联合毒性效应均表现为协同效应,其中比例为1:1的协同效应最强.     

Combined action of X-rays and nonylphenol on mouse sperm

The aim of this study was to assess the effects of 2-weeks’ X-ray and/or nonylphenol (NP) exposure on male mice’s sperm count and quality. Pzh:SFIS mice were exposed to X-rays (0.05 Gy, 0.10 Gy, 0.20 Gy) or to nonylphenol (25 mg/kg bw, 50 mg/kg bw, 100 mg/kg bw) or to both agents (0.05 Gy + 25 mg/kg bw NP, 0.10 Gy + 50 mg/kg bw NP). At 24 h and 5 weeks after the end of exposure the sperm count, morphology and frequency of DNA damage in the male germ cells were estimated. Each agent alone diminished sperm count and morphology. The dose of 0.05 Gy of X-rays decreased the frequency of DNA damage. Combined exposure to lower doses of both agents significantly improved sperm morphology and decreased the level of DNA damage compared to one agent alone. Combined exposure to higher doses reduced the frequency of DNA damage compared to the effect of the appropriate dose of NP. Results of combined exposure to low doses of both agents suggest that 0.05 Gy of X-rays stimulate the DNA damagecontrol system and in consequence repair of DNA caused by X-rays and NP. It may be correlated with increased antioxidant capacity.     

Amino acid cues emanating from Pacific salmon eggs and ovarian fluid

The eggs of salmonid fishes are an important food source for many aquatic predators that detect eggs using olfaction. Moreover, chemicals from eggs and ovarian fluid aid sperm cells in detecting and locating eggs for fertilization, and ovarian fluid is attractive to conspecific males. Thus chemicals from eggs and ovarian fluid may facilitate reproduction but may also attract egg predators. The authors sampled mature females of three Pacific salmon species – Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch) and sockeye (Oncorhynchus nerka) – and determined the proportional representation of amino acids, potent fish odorants, from their eggs and ovarian fluid (Chinook and coho salmon only). They then tested juvenile coho salmon, an egg predator, for responses to ovarian fluid and egg odours using the electro-olfactogram (EOG) recording technique. The amino acid compositions of the salmon species were significantly and positively correlated with each other, and the interspecific differences were comparable to those between individuals of the same species. The egg water samples were, on average, dominated by lysine, alanine and glutamine (12.6%, 12.4% and 10.9%, respectively). The ovarian fluid samples were dominated by lysine (20.5%), followed by threonine (9.7%), glycine (9.2%) and arginine (8.8%). EOG recordings demonstrated the ability of juvenile coho salmon to detect the chemical traces of eggs and ovarian fluid. It is concluded that salmon eggs are a potent source of odours for potential predators but likely not highly differentiated among salmon species.     

Biochemical changes and adaptive strategies of plants under heavy metal stress

Heavy metal contamination of soil, aqueous waste stream and ground water causes major environmental and human health problems. Heavy metals are major environmental pollutants when they are present in high concentration in soil and show potential toxic effects on growth and development in plants. Due to unabated, indiscriminate and uncontrolled discharge of hazardous chemicals including heavy metals into the environment, plant continuously have to face various environmental constraints. In plants, seed germination is the first exchange interface with the surrounding medium and has been considered as highly sensitive to environmental changes. One of the crucial events during seed germination entails mobilization of seed reserves which is indispensable for the growth of embryonic axis. But, metabolic alterations by heavy metal exposure are known to depress the mobilization and utilization of reserve food by affecting the activity of hydrolytic enzymes. Some plants possess a range of potential mechanisms that may be involved in the detoxification of heavy metals by which they manage to survive under metal stress. High tolerance to heavy metal toxicity could rely either on reduced uptake or increase planned internal sequestration which is manifested by an interaction between a genotype and its environment. Such mechanism involves the binding of heavy metals to cell wall, immobilization, exclusion of the plasma membrane, efflux of these toxic metal ions, reduction of heavy metal transport, compartmentalization and metal chelation by tonoplast located transporters and expression of more general stress response mechanisms such as stress proteins. It is important to understand the toxicity response of plant to heavy metals so that we can utilize appropriate plant species in the rehabilitation of contaminated areas. Therefore, in the present review attempts have been made to evaluate the effects of increasing level of heavy metal in soils on the key behavior of hydrolytic and nitrogen assimilation enzymes. Additionally, it also provides a broad overview of the strategies adopted by plants against heavy metal stress.     

Heavy metals toxicity in plants: An overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants

Plants experience oxidative stress upon exposure to heavy metals that leads to cellular damage. In addition, plants accumulate metal ions that disturb cellular ionic homeostasis. To minimize the detrimental effects of heavy metal exposure and their accumulation, plants have evolved detoxification mechanisms. Such mechanisms are mainly based on chelation and subcellular compartmentalization. Chelation of heavy metals is a ubiquitous detoxification strategy described in wide variety of plants. A principal class of heavy metal chelator known in plants is phytochelatins (PCs), a family of Cys-rich peptides. PCs are synthesized non-translationally from reduced glutathione (GSH) in a transpeptidation reaction catalyzed by the enzyme phytochelatin synthase (PCS). Therefore, availability of glutathione is very essential for PCs synthesis in plants at least during their exposure to heavy metals. Here, I reviewed on effect of heavy metals exposure to plants and role of GSH and PCs in heavy metal stress tolerance. Further, genetic manipulations of GSH and PCs levels that help plants to ameliorate toxic effects of heavy metals have been presented.     

Cadmium and copper inhibit both DNA repair activities of polynucleotide kinase

Human exposure to heavy metals is of increasing concern due to their well-documented toxicological and carcinogenic effects and rising environmental levels through industrial processes and pollution. It has been widely reported that such metals can be genotoxic by several modes of action including generation of reactive oxygen species and inhibition of DNA repair. However, although it has been observed that certain heavy metals can inhibit single strand break (SSB) rejoining, the effects of these metals on SSB end-processing enzymes has not previously been investigated. Accordingly, we have investigated the potential inhibition of polynucleotide kinase (PNK)-dependent single strand break repair by six metals: cadmium, cobalt, copper, nickel, lead and zinc. It was found that micromolar concentrations of cadmium and copper are able to inhibit the phosphatase and kinase activities of PNK in both human cell extracts and purified recombinant protein, while the other metals had no effect at the concentrations tested. The inhibition of PNK by environmentally and physiologically relevant concentrations of cadmium and copper suggests a novel means by which these toxic heavy metals may exert their carcinogenic and neurotoxic effects.