汞暴露导致秀丽线虫后代中出现可传递的表型和行为缺陷.doc

汞暴露导致秀丽线虫后代中出现可传递的表型和行为缺陷【摘要】 目的：研究汞暴露秀丽线虫表型和行为缺陷在后代中的可传递特性。方法：寿命、身体尺寸、身体弯曲频率、头部摆动频率和化学趋向可塑性用于汞急性毒害评价,并进一步分析表型和行为缺陷从汞暴露的当代到后代的可传递特性。结果：汞暴露能够引起包括寿命、身体尺寸、身体弯曲频率、头部摆动频率和化学趋向可塑性等参数在内的多重生物学缺陷,且其毒害与所暴露汞浓度之间密切相关。大部分毒害能够从暴露线虫当代传递给后代。汞暴露对线虫后代寿命的毒害不能够明显恢复。汞暴露线虫后代身体尺寸的缺陷甚至比当代更为严重。此外,高浓度汞暴露通常导致非常纤细的线虫动物出现,且在后代中可观察到更多具有这种表型的线虫。汞暴露导致的线虫身体弯曲和头部摆动频率缺陷在后代中可以大部分或者完全恢复。尽管化学趋向可塑性在汞暴露线虫后代中能够部分恢复,但是化学趋向可塑性仍受到严重的破坏。结论：汞暴露引起的多重生物学毒害可以从暴露当代传递给它们的后代,并且在后代中出现的一些缺陷甚至比当代更为严重。 【关键词】 汞暴露; 多重毒性; 可传递性; 表型; 行为; 秀丽线虫Mercury(Hg) is a hazardous heavy metal, which could be released into the environment from both natural and anthropogenic sources. Natural sources of Hg include volcanic emissions, volatilization from the ocean, and degassing from soil. Use of Hg in industrial such as the manufacture of plastic, chlorine, caustic soda, caustic potash and antifouling paint, is the major anthropogenic sources. Use of Hg in agriculture such as fossil fuel burning, base metal smelting, waste incinerators and Hg based fungicides are other important input sources of Hg in the environments1. In addition, there is a high potential for Hg bioaccumulation and biomagnification in different organisms. The levels of Hg in some commercial fish in New Jersey were detected in the range known to cause some sublethal effects in sensitive predatory birds and mammals2. High levels of Hg content were also found in commercial pelagic fish in the Western Indian Ocean, and large fish can naturally bioaccumulate Hg1. Especially, in the summer of 2 000, Hg spills were discovered in the basements of some Chicagoarea homes after removal of gas regulators by gas company contractors3. The risk of residential Hg contamination after gas regulator removal ranged from 0.9/1 000 to 4.3/1 000 homes3. So far, Hg has been considered as one of the most serious environmental contamination threats to human, fish and wildlife of the global.Mice, rodents, fish, birds and some other mammals have ever been used as models to evaluate the Hg toxicity and related regulation mechanisms. Elemental Hg is a silvery metal that is liquid at room temperature. Human absorption of elemental Hg occurs primarily through inhalation of Hg vapor3. The primary targets of acute exposure to Hg are liver, kidneys and central nervous system in fish, birds and mammals4-5. Inhaled Hg vapor results in accumulation with highest concentrations in the cerebellum and brainstem nuclei of rats and mice6. Hg can cause toxic effects at concentrations even below 1 ppb in water and the effects include loss of appetite, brain lesions, cataracts, abnormal motor coordination and abnormal behavioral changes4-5,7. Aspects affected by Hg exposure also contain the reproduction, growth, metabolism, blood chemistry, immunity, and oxygen exchange8-11. Several theories about the mechanism of Hg toxicity have already been raised and these theories suggest that the Hg exposure can cause multibiological toxicities by affecting specific signaling pathways and lipid peroxidation12-13. However, for the possible limit of the experimental organisms of mice, fish, birds and other mammals, whether the multibiological toxicities caused by Hg exposure can be transferred from exposed animals to their progeny remains still unclear.Caenorhabditis elegans, a freeliving soil nematode, has been found favor as a biomarker organism, because it is one of the bestcharacterized animals at the genetic, physiological, molecular, and developmental levels14. C. elegans has the properties of short life cycle, small size, ease of cultivation, a simple cell lineage that has been completed characterized, and behavior easily monitored under the microscope. Moreover, its great potential for forward and reverse genetic analysis make it very powerful for deeply elucidating the mechanisms of metal toxicity. By virtue of these properties, several toxicity tests using C. elegans have been developed for ecological risk assessment in soil15-16 and water17-19. Moreover, transgenic hsp16GFPlacZ and hsp16GFP nematodes have been constructed for the study of environmental monitoring and toxicology20-25. In addition, a standardized method for conducting laboratory soil toxicity tests using C. elegans was published in the America Society for Testing and Materials(ASTM) Guide E217201 in 200226.In the present study, we selected the C. elegans organism to examine whether the multibiological toxicities induced by Hg exposure can be transferred from exposed animals to their progeny. Our results suggest that most of these multibiological toxicities induced by Hg exposure can be considered to be transferable from parental generations to their progeny, and some specific defects in progeny appeared even more severe than in their parental generations.1 Materials and methods1.1 ChemicalsThe Hg concentrations used in this report were selected as previously described20,27. Three concentrations of HgCl2 solution were used in the current work, and they were 2.5 molL-1, 75 molL-1 and 200 molL-1, respectively. All the chemicals were obtained from SigmaAldrich(St. Louis, MO, USA).1.2 StrainsAll nematodes used were wildtype N2, originally obtained from the Caenorhabditis Genetics Center(CGC). They were maintained on nematode growth medium(NGM) plates seeded with Escherichia coli OP50 at 20 as described28. Gravid nematodes were washed off the plates into centrifuge tubes and were lysed with a bleaching mixture(0.45 molL-1 NaOH, 2% HOCl). Age synchronous populations of N2(L4larvae stage) were obtained by the collection as described29. The L4larvae stage nematodes were washed with doubledistilled water twice, followed by washing with K medium once(50 mmolL-1 NaCl, 30 mmolL-1 KCl, 10 mmolL-1 NaOAc, pH 5.5). Exposures were performed in 12well sterile tissue culture plates. All exposures were 48h, and were carried out in 20 incubator in the absence of food. To evaluate the Hg toxic in progeny, eggs were obtained from nematodes subjecting to the Hg exposure with the bleaching mixture, and then transferred to a normal NGM plates without addition of Hg solution. Endpoints of lifespan, body size, body bend, head thrash, and chemotaxis plasticity were used for the acute toxicity testing in C. elegans.1.3 Lifespan and body sizeThe methods were performed as previously described30-32. For life span assay, the exposed and progeny animals were picked onto the assay plates and the time was recorded as t=0. About twenty animals were placed onto a single plate and adult animals were transferred every 2 days to fresh plates during the brood period. The numbers of survivors were scored every day. Animals that failed to respond to repeated touch stimulation were considered as dead. Life span graphs are representative of at least three trials. Body size was determined by measuring the flat surface area of nematodes using the ImagePro Express software. For each test, at least 15 animals were picked for assay.1.4 Head thrash frequencyThe thrashes were assayed as previously described33-35. To assay the head thrash frequency, nematodes were washed with the doubledistilled water, followed by washing with K medium. Every animal was transferred into a microtiter well containing 60 l of K medium on the top of agar. After a 1 min recovery period, the head thrashes were counted for 1 min. A thrash was defined as a change in the direction of bending at the mid body. Fifteen nematodes were examined per treatment.1.5 Body bend frequencyThe method was performed as previously described33-35. To assay the body bend frequency, nematodes were picked onto a second plate and scored for the number of body bends in an interval of 20 s. A body bend was counted as a change in the direction of the part of the animals corresponding to the posterior bulb of the pharynx along the y axis, assuming that the nematodes were traveling along the x axis. Fifteen nematodes were examined per treatment.1.6 Chemotaxis assay and conditioning procedureChemotaxis assays and conditioning procedure were performed as previously described22,36. Approximately 100 nematodes were used for each trial. An agar plug excised from the plate with additional 100 mmolL-1 NaCl was placed on the surface of assay plate containing 5 mmolL-1 potassium phosphate, pH 6.0, 1 mmolL-1 CaCl2, 1 mmolL-1 MgSO4 and 20 gL-1 agar for at least 14 h. Shortly before analysis, the plug was removed and 1 l 0.5 molL-1 NaN3 was spotted at the centre of plug to anaesthetize the nematodes. NaN3 was also spotted 4 cm away from the centre of the NaCl gradient as a control. The chemotaxis index CI was calculated as CI=(the number within NaCl gradientthe number within control) / the total number of nematodes on the plate.To analyze the learning, the treated nematodes(young adults) were washed three times with washing buffer containing 5 mmolL-1 potassium phosphate, pH 6.0, 1 mmolL-1 CaCl2, 1 mmolL-1 MgSO4 and 0.5 gL-1 gelatin. Nematodes were starved for 3 h at NaClE. coil plates(NaClfree and E. colifree plates) or+NaClE. coil plates. And then, they were collected with washing buffer and placed equidistant(about 3.5 cm) from those two spots mentioned above on the assay plate to let them move freely for 45 min at 20 . The nematodes within 1.5 cm of these two spots were counted.1.7 Statistical analysisAll data in this article were expressed as means S.D. Graphs were generated using Microsoft Excel(Microsoft Corp., Redmond, WA). An overall ANOVA was used for comparison between control and the metal treated groups, followed by pairwise comparison tests. The probability levels of 0.05 and 0.01 were considered statistically significant.2 Results2.1 Lifespan defects in Hg exposed nematodes and their progeny Lifespan is often used as a main parameter to evaluate the toxicity of a specific metal or compound in nematodes19,22,31. Because C. elegans has a very short life cycle, it is more convenient to investigate the aging and to elucidate the mechanism of animals lifespan30. In other organisms, Hg was found to be able to accelerate the aging process possibly by affecting the neurotoxicity and oxidative injury13. In C. elegans, as shown in Fig 1, high concentrations(75 molL-1 and 200 molL-1) of Hg exposure caused more severe lifespan defects compared to low concentration(2.5 molL-1) of Hg exposure and control(0 molL-1). When nematodes were exposed to 75 molL-1 and 200 molL-1 concentrations of Hg, their maximum lifespans were reduced by nearly 4 days compared to control. The mean lifespans of nematodes exposed to 200 molL-1 Hg was nearly half of those in control nematodes.A. Lifespans of nematodes exposed to 2.5 molL-1 Hg. B. Lifespans of nematodes exposed to 75 molL-1 Hg. C. Lifespans of nematodes exposed to 200 molL-1 Hg. D. Lifespans of progeny from nematodes exposed to 2.5 molL-1 Hg. E. Lifespans of progeny from nematodes exposed to 75 molL-1 Hg. F. Lifespans of progeny from nematodes exposed to 200 molL-1 Hg.G. Comparison of the mean lifespan for nematodes exposed to 2.5 molL-1, 75 molL-1 and 200 molL-1 Hg, respectively.H. Comparison of the mean lifespan for progeny from nematodes exposed to 2.5 molL-1, 75 molL-1 and 200 molL-1 Hg, respectively. Bars represent mean S.D. a. P<0.01 vs 0 molL-1Fig 1 Lifespans of nematodes exposed to different concentrations of Hg and their progeny To investigate whether the Hg toxicity on lifespan could be transferred from exposed nematodes to their progeny, we analyzed the changes of lifespan in progeny of nematodes exposed to Hg. Surprisingly, the toxicity on lifespan from Hg exposure could not be obviously recovered in progeny nematodes. Severe defects could still be observed for both the maximum lifespan and the mean lifespan in progeny nematodes. Therefore, the toxicity on lifespan from Hg exposure can be transferred from exposed nematodes to their progeny, and Hg exposure can exert severely adverse effects on the lifespan of progeny nematodes.2.2 Developmental defects in Hg exposed nematodes and their progeny We next examined the effects of Hg exposure on nematode development by observing the body size and morphology of animals. As shown in Fig 2, the body sizes of nematodes were significantly(P<0.01) reduced after Hg exposure, and the toxicity on development was also concentrationdependent. Moreover, the body size defects in progeny of nematodes exposed to different concentrations of Hg appeared even more severe than in their parents. Even in progeny of nematodes exposed to 2.5 molL-1 Hg, the body sizes were also more severely(P<0.01) suppressed than in their parents.0 molL-12.5 molL-175 molL-1200 molL-1Hg exposedProgenyA. Morphological comparison of nematodes exposed to different concentrations of Hg and their progeny. All images are representative of threeday post hatch nematodes.B. Comparison of body sizes of nematodes exposed to different concentrations of Hg. C. Comparison of body size of progeny from nematodes exposed to different concentrations of Hg. Bars represent mean S.D. a. P<0.01 vs 0 molL-1Fig 2 Body sizes of nematodes exposed to different concentrations of Hg and their progenyIn addition, high concentrations of Hg exposure usually caused the appearance of very slim nematodes, and more nematodes with this phenotype were found in progeny population. Thus, more severe development defects can be formed in progeny of nematodes exposed to Hg.2.3 Locomotion behavior defects in Hg exposed nematodes and their progeny Hg exposure can not only influence the lifespan and the development, it may also affect the development and function of nervous system34. To test the influences of Hg exposure on locomotion behaviors, the body bend and the head thrash were assayed. As shown in Fig 3, both the body bends and the head thrashes in nematodes were dramatically impaired even exposed to a very low concentration of 2.5 molL-1 Hg. More severe body bend defects were observed when nematodes were exposed to high concentrations(75 molL-1 and 200 molL-1) of Hg, whereas no distinct differences were found for the head thrash defects in nematodes exposed to 2.5 molL-1 of Hg from those in nematodes exposed to 75 molL-1 and 200 molL-1 of Hg. Investigation on their progeny indicates that the defects of body bends could be largely or completely recovered. The defects of head thrashes could be largely recovered in nematodes exposed to 2.5 molL-1 of Hg, and the head thrash frequencies in progeny of nematodes exposed to 75 molL-1 and 200 molL-1 of Hg could be recovered approximately 21% and 14%, respectively.A. Body bend frequencies of nematodes exposed to Hg. B. Body bend frequencies in progeny of nematodes exposed to Hg.C. Head thrash frequencies of nematodes exposed to Hg.D. Head thrash frequencies in progeny of nematodes exposed to Hg. Bars represent mean S.D. a. P<0.05 vs 0 molL-1; b. P<0.01 vs 0 molL-1Fig 3 Locomotion behaviors of nematodes exposed to different concentrations of Hg and their progeny2.4 Chemotaxis plasticity defects in Hg exposed nematodes and their progeny The chemotaxis plasticity is one of the simple forms for behavioral plasticity, which might be able to reflect a form of associative learning36. Lastly, we examined the possible toxic effects of Hg exposure on nematodes chemotaxis plasticity. In this research system, the conditioning requires both the presence of NaCl and the absence of a bacterial food source, because starvation on culture medium containing the NaCl can make the chemotaxis of animals towards to NaCl fall dramatically36. As shown in Fig 4, nematodes exposed to 75 molL-1 and 200 molL-1 concentrations of Hg displayed severe chemotaxis plasticity defects(P<0.01) compared to control and those exposed to 2.5 molL-1 Hg. In general, nematodes starved onNaClE. coil plates show chemotaxis toward NaCl, but starved on+NaClE. coil plates lose most of this property36. Consequently, the association between NaCl and the food might be blocked in nematodes exposed to 75 molL-1 and 200 molL-1 Hg, which then made the nematodes still move to the spots of NaCl. The chemotaxis plasticity in nematodes exposed to 2.5 molL-1 of Hg only exhibited a moderate defect. Furthermore, we observed that the chemotaxis plasticity defects could be transferred from exposed nematodes to their progeny. Although the defects of chemotaxis plasticity in progeny of Hg exposed nematodes could be partially recovered, the chemotaxis plasticity was still seriously impaired.Taken together, our data suggest that Hg exposure can result in the transferable toxicities or defects for both the locomotion behaviors and the behavioral plasticity from exposed nematodes to their progeny in C. elegans.A. Chemotaxis performance of nematodes exposed to Hg. B. Chemotaxis performance in progeny of nematodes exposed to Hg. About 100 nematodes were put on each plate. CI=(the number within NaCl gradientthe number within control) / the total number of nematodes in plate. Bars represent mean S.D. a. P<0.01 vs 0 molL-1Fig 4 Chemotaxis plasticity of nematodes exposed to different concentrations of Hg and their progeny3 DiscussionHg exposure in the environment is one of the most increasing health concerns so far. Its ability to form monomethyl mercury through microbe biotransformation leads to accumulation in the food chains. In C. elegans, early in 1982, Popham and Webster ever analyzed the ultrastructural changes of animals exposed to Hg37. The stress response, mortality, reproduction, and structures and functions of sensory neurons were also examined previously in Hg exposed nematodes20,23,38-40. However, the systematical mult