武秋立,楊鵬,王大勇

東南大學(xué)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)系、發(fā)育與疾病相關(guān)基因教育部重點(diǎn)實(shí)驗(yàn)室,江蘇南京 210009)

Mercury(Hg)is a hazardous heavy m eta,l which could be released into the environment from both natural and anthropogenic sources.Natural sources ofHg include volcanic emissions,volatilization from the ocean,and degassing from soi.l Use of Hg in industrial such as the manufacture of plastic,chlorine, caustic soda, caustic potash and antifouling pain,tis 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 environments[1]. In addition, there is a high potential for Hg bioaccu mulation 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 mammals[2].H igh levels of Hg content were also found in commercial pelagic fish in the W estern Indian Ocean,and large fish can naturally bioaccumulateHg[1].Especially,in the su mmer of 2 000,Hg spillswere discovered in the basements of some Chicago-area homes a-f ter removal of gas regulators by gas company contractors[3].The risk of residentialHg conta m ination after gas regulator removal ranged from 0.9/1 000 to 4.3/1 000 homes[3].So far,Hg has been considered as one of the most serious environmental conta m ination threats to human,fish andw ildlife of the globa.l

Mice,rodents,fish,birds and some other mamm als have ever been used asmodels to evaluate the Hg toxicity and related regu lation mechanisms.E le m entalHg is a si-l very metal that is liquid at room temperature.Human absorption of elemental Hg occurs primarily through inhalation of Hg vapor[3].The pri mary targets of acute exposure to Hg are liver,kidneys and central nervous system in fish,birds andmamm als[4-5].Inhaled Hg vapor results in accumulation with highest concentrations in the cerebe-l lum and brainstem nuclei of rats and mice[6].Hg can cause toxic effects at concentrations even below 1 ppb in water and the effects include loss of appetite,brain lesions,cataracts,abnor malmotor coordination and abnormal behavioral changes[4-5,7].Aspects affected by Hg exposure also contain the reproduction,growth,metabolism,blood chemistry, immunity, and oxygen exchange[8-11].Several theories about the mechanism of Hg toxicity have already been raised and these theories suggest that the Hg exposure can causemulti-biological toxicities by affecting specific signaling pathways and lipid peroxidation[12-13].However,for the possible lim it of the experi mental organisms of m ice,fish,birds and other mammals,whether the multi-biological toxicities caused by Hg exposure can be transferred from exposed animals to their progeny remains still unclear.

Caenorhabditis elegans,a free-living soil nematode,has been found favor as a biomarker organism,because it is one of the best-characterized animals at the genetic,physiologica,l molecular, and developmental levels[14].C.elegans has the properties of short life cycle,sm all size,ease of cultivation,a simple cell lineage that has been completed characterized,and behavior easily mon-i tored under the m icroscope.Moreover,its great potential for for ward and reverse genetic analysismake 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 soil[15-16]andwater[17-19].M oreover, transgenic hsp-16-GFP-lacZ and hsp-16-GFP nematodeshave been constructed for the study of environmental monitoring and toxicology[20-25]. 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)GuideE2172-01 in 2002[26].

In the present study,we selected the C.elegans organism to examine whether the multi-biological toxic-i ties induced by Hg exposure can be transferred from exposed animals to their progeny.Our results suggest that most of thesemulti-biological toxicities induced by Hg exposure can be considered to be transferable from parental generations to their progeny,and some specific defects in progeny appeared evenmore severe than in their parental generations.

1 Materials and methods

1.1 Chemicals

The Hg concentrations used in this report were selected aspreviously described[20,27].Three concentrations of HgC l2solutionwere used in the currentwork,and they were 2.5 μ mol·L-1,75 μ mol·L-1and 200 μmol·L-1,respectively.All the chemicalswere obtained from Sigm a-A ldrich(S.t Louis,MO,USA).

1.2 Strains

All nematodesused were wild-typeN2,originally obtained from the Caenorhabditis Genetics Center(CGC).They were maintained on ne matode grow th medium(NGM) plates seeded with E scherichia coli OP50 at 20℃as described[28].Gravid nematodes were washed off the plates into centrifuge tubes and were lysed with a bleaching mixture(0.45 mol·L-1NaOH,2%HOCl).Age synchronous populations of N2(L4-larvae stage)were obtained by the collection as described[29].The L4-larvae stage ne matodeswere washed with double-distilled water t w ice,followed by washing with K mediu monce(50mmol·L-1NaC,l 30 mmol·L-1KC,l 10 mmol·L-1NaOAc,p H 5.5).Exposureswere perfor med in 12-well sterile tissue culture plates.A ll exposureswere 48 h,and were carried out in 20℃incubator in the absence of food.To evaluate the Hg tox ic in progeny,eggswere obtained from ne matodes subjecting to the Hg exposure with the bleaching mixture,and then transferred to a nor mal NGM plates with out 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 size

The methods were perfor med as previously described[30-32].For life span assay,the exposed and progeny animals were picked onto the assay plates and the ti me 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 per-i od.The numbers of survivorswere scored every day.Animals that failed to respond to repeated touch stimulation were considered asdead.Life span graphs are representative of at least three trials.Body size was deter mined by measuring the flat surface area of nematodesusing the I mage-Pro Express software.For each tes,t at least 15 an-i malswere picked for assay.

1.4 Head thrash frequency

The thrashes were assayed aspreviously described[33-35].To assay the head thrash frequency,nematodeswere washed with the double-distilled water,fo-l lowed by washing with K medium.Every animal was transferred into a microtiter well containing 60 μl of K medium on the top of agar.After a 1min recovery per-iod,the head thrashes were counted for 1 m in.A thrash was defined as a change in the direction of bending at the m id body.Fifteen nematodes were examined per trea-t men.t

1.5 Body bend frequency

The method wasperformed as previously described[33-35].To assay the body bend frequency,ne matodeswere picked onto a second plate and scored for the nu mber 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 treatmen.t

1.6 Chemotaxis assay and conditioning procedure

Chemotaxis assays and conditioning procedure were perfor med as previously described[22,36].Approxi mately 100 nematodeswere used for each tria.l An agar plug excised from the plate with additional 100 mmol·L-1NaCl was placed on the surface of assay plate containing 5 mmol·L-1potassium phosphate,p H 6.0,1 mmol·L-1CaCl2,1mmol·L-1MgSO4and 20 g·L-1agar for at least 14 h.Shortly before analysis,the plug was removed and 1 μl 0.5mol·L-1Na N3was spotted at the centre of plug to anaesthetize the nematodes.NaN3was also spotted 4 c m away from the centre of theNaC l gradient as a contro.l The chemotaxis index CI was calculated as CI=(the number with in NaCl gradient-the numberwithin control)/the total number of nematodes on the plate.

To analyze the learning, the treated nematodes(young adults)were washed three ti m es w ith washing buffer containing 5 mmol·L-1potassium phosphate,p H 6.0,1 mmol·L-1CaCl2,1 mmol·L-1MgSO4and 0.5 g·L-1gelatin.Nematodes were starved for 3 h at NaC l-E.coil plates(NaCl-free and E.coli-free plates)or+NaCl-E.coil plates.And then,they were collected w ith washing bufferand placed equidistant(about 3.5 cm)from those two spots mentioned above on the as-say plate to let them move freely for 45m in at 20℃.The nematodes with in 1.5 cm of these two spotswere counted.

1.7 Statistical analysis

A ll data in this article were expressed as means±S.D.Graphs were generated using Microsoft Excel(M-i crosoft Corp.,Red mond,WA).An overall ANOVA was used for comparison between control and the metal treated groups,followed by pairw ise comparison tests.The probability levels of 0.05 and 0.01 were considered statist-i cally significan.t

2 Results

2.1 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 ne matodes[19,22,31].Because C.elegans has a very short life cycle,it is more convenient to investigate the aging and to elucidate the mechanism of ani m als’lifespan[30]. In other organisms,H g was found to be able to accelerate the aging process possibly by affecting the neurotoxicity and oxidative injury[13]. In C. elegans, as shown in Fig 1,high concentrations(75 μ mol· L-1and 200 μmol·L-1)ofHg exposure caused more severe lifespan defects compared to low concentration(2.5 μ mol· L-1)of Hg exposure and control(0 μmol· L-1).W hen nematodes were exposed to 75 μmol·L-1and 200 μ mol·L-1concentrations of Hg,their maxi mum lifespans were reduced by nearly 4 days compared to contro.l The mean lifespans of nematodes exposed to 200 μmol· L-1H g was nearly half of those in control nematodes.

Fig 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 m axi mu m 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

W e next exam ined the effects of Hg exposure on nematode develop ment by observing the body size and morphology of animals.As shown in Fig 2,the body sizes of nematodeswere significantly(P＜0.01)reduced after Hg exposure,and the toxicity on develop ment was also concentration-dependen.t 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 μmol·L-1Hg, the body sizes were also more severely(P＜0.01)suppressed than in their parents.

Fig 2 Body sizes of nematodes exposed to different concentrations of Hg and their progeny

In 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 for m ed in progeny of nematodes exposed to Hg.

2.3 Loco motion behavior defects in Hg exposed nematodes and their progeny

Hg exposure can not only influence the lifespan and the developmen,tit may also affect the development and function of nervous system[34].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 μmol·L-1Hg.More severe body bend defectswere observedwhen nematodeswere exposed to high concentrations(75 μ mol·L-1and 200 μ mol·L-1)of Hg,whereas no distinct differences were found for the head thrash defects in nematodes exposed to 2.5 μmol· L-1of Hg from those in nematodes exposed to 75 μ mol·L-1and 200 μmol·L-1of Hg.Investigation on their progeny ind-i cates 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 μmol·L-1of Hg,and the head thrash frequencies in progeny of nematodes exposed to 75 μmol·L-1and 200 μmol·L-1of Hg could be recovered approxi mately 21% and 14%,respectively.

Fig 3 Loco motion behaviors of nematodes exposed to different concentrations of Hg and their progeny

2.4 Che motaxis plasticity defects in Hg exposed nematodes and their progeny

The chemotaxis plasticity is one of the simple forms for behavioral plasticity,which m ight be able to reflect a for m of associative learning[36].Lastly,we examined the possible toxic effects ofH g 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 culturem ed-i um containing the NaCl can make the chemotaxis of an-i mals towards to NaCl fall dramatically[36].As shown in Fig 4,nematodes exposed to 75 μ mol·L-1and 200 μmol·L-1concentrations of Hg displayed severe che motaxis plasticity defects(P＜0.01)compared to control and those exposed to 2.5 μ mol·L-1Hg.In genera,l nematodes starved on-NaC l-E.coil plates show chemotaxis toward NaC,lbut starved on+NaC l-E.coil plates lose most of this property[36].Consequently, the association bet ween NaCl and the food might be blocked in nematodes exposed to 75 μmol· L-1and 200 μmol· L-1Hg,which then made the nematodes stillmove to the spots ofNaC.l The chemotaxis plasticity in nematodes exposed to 2.5 μ mol· L-1ofHg only exhibited amoderate defec.t Further more,we observed that the chemotaxis plasticity defects could be transferred from exposed nematodes to their progeny.A-l though 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.

Fig 4 Che motaxis plasticity of nematodes exposed to different concentrations of Hg and their progeny

3 Discussion

Hg exposure in the environment is one of the most increasing health concerns so far. Its ability to form mono-methylmercury throughm icrobe bio-transformation leads to accumulation in the food chains.In C.elegans,early in 1982,Popham and Webster ever analyzed the u-l trastructural changes of animals exposed to Hg[37].The stress response,mortality,reproduction,and structures and functions of sensory neuronswere also examined previously in Hg exposed nematodes[20,23,38-40].However,the systematical multi-biological toxicities have not been investigated ye.t In this repor,t endpoints of lifespan,body size,body bend,head thrash,and chemotaxisplasticity were used for the acute toxicity testing to examine the multi-biological tox icities from Hg exposure.Our results indicate that the Hg exposure could causemulti-biological defectsw ith a concentration-dependent manner in C.elegans,which are largely consistent with the conclusions drawn from other organisms[1-13].

Moreover,among thesemulti-biological toxicities,we found that the develop mental defects are very specific for the Hg toxicity.Hg exposure specially caused the appearance of slimanimals at high concentrations.Metallothionein gene expression in the larvae of C.elegans has been raised as a potential bio marker for Hg toxicity[41].However,the metallothionein gene expression can also indicate the toxicity from cad m iu m exposure.Therefore,the morphological defects will be valuable to be used as a key monitor to evaluate the specific Hg toxicity from environments.Especially,a co mbination of this phenotype with the transgenic reporter for metallothionein gene would be more valuable for the assessment of the Hg toxicity.

According to the results and analysis in this projec,t we can summarize the defects caused byHg exposure into four groups based on the transferable properties from exposed nematodes to their progeny.F irs,t the defects caused by Hg exposure could be largely recovered in progeny,such as the locomotion behaviors in progeny of nematodes exposed to low concentration of Hg.Second,the defects caused by Hg exposure could be only partially recovered in progeny,such as the chemotaxis plasticity.Third,no rescue phenomena could be observed for the defects caused by Hg toxicity,such as the body sizes in progeny of nematodes exposed to low concentration of Hg.Fourth,the defects caused by Hg toxicity became more severe in progeny than in their parents,such as the body sizes in progeny of nematodes exposed to high concentrations of Hg.Thus,our data suggest that the multi-biological defects of phenotypes and behaviors caused by Hg exposure could largely be considered as transferable in C.elegans.

However,the transferable properties of Hg exposure could not be considered as a kind of heredity in genetics,since some of the defects caused by Hg exposure could still be partially recovered in progeny nematodes.Therefore,we suppose that gain of the transferable properties for nematodes exposed to Hg might be largely due to the deposition of Hg toxicity in their eggs.Organic and inorganic Hg has been found to be able to be all transferred to the fetal rat via placenta and milk[42-43].Residual Hg levels in egg yolk were also found to greatly surpass the level found in the egg white[44].But at the same ti me,we also noticed that some of the defects in progeny nematodes appeared even more severe phenotypes than in their parents,which is still an interesting question needed to be deeply elucidated.

In conclusion,our results showed that the Hg exposure can result in multi-toxicity,and most of these multibiological defects can be transferred to progeny from Hg exposed nematodes.

Ac know ledge ments Strain used in this work was provided by the Caenorhabd its Genetics Center(funded by the NI H,National Center for Research Resource).This work was supported by the grants from the National Natural Science Foundation of China(No.30870810)and the Progra m for New Century Excellent Talents in University.

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