DNA strand breaks (comet assay) and embryo development effects in grass shrimp (Palaemonetes pugio) embryos after exposure to genotoxicants.

Grass shrimp embryos develop in egg sacs (stages 1-10) attached to the female for 14-20 days after which they 'hatch' from the egg sacs into a swimming zoea stage (stage 11). Until they emerge from the egg sacs, embryos depend on lipids and lipovitellin stored within the egg. The percent of embryos which hatch after exposure to toxicants relative to controls was the basis of an embryo development assay. Exposure of embryos to chromium(III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) resulted in a reduced hatching rate. In addition to effects on embryo development, DNA strand damage tests were carried out on contaminant-exposed embryos, using the single-cell electrophoresis method often referred to as comet assay. Development of stage 4 embryos was more affected by MNQ exposure than stage 7 embryos. The hatching rates of stages 4 and 7 embryos exposed to MNQ (172 micrograms/l) were 0 and 90%, respectively. DNA strand damage, measured as DNA tail moments, were 3.4 and 4.4, respectively. Thus, exposure of an early embryo stage to MNQ prevented full embryo development while development of later embryo stages was not affected. It may be that the DNA repair systems are more efficient in later embryo stages than in early stages and thus DNA damaged in the early stages affects development.     

Comet assays to assess DNA damage and repair in grass shrimp embryos exposed to phototoxicants

Exposure of grass shrimp (Palaemonetes pugio) embryos to four compounds (anthracene, pyrene, alpha-terthienyl, methylene blue) along with solar exposure resulted in extensive DNA strand damage using the comet assay. DNA tail moments of embryos exposed to these chemicals in the dark ranged from 1.8 to 4.3, while exposure to chemicals and solar resulted in tail moments of 14.3-15.3. Reduction of DNA tail moments when solar exposed embryos were transferred to the dark, suggested DNA repair systems were active. The comet assay can be used to follow both DNA damage and repair following exposure to phototoxic chemicals.     

DNA strand breaks in grass shrimp embryos exposed to highway runoff sediments and sediments with coal fly ash

Embryo production was reduced in female grass shrimp exposed to sediments with added coal fly ash and to sediments collected from an estuarine station containing high PAH concentrations due to its proximity to a highway storm drain. Grass shrimp embryos exposed to pore water from the high PAH and high metal sediments showed both reduced hatching and increases in DNA strand breaks (comet assay). Sediments with added coal fly ash had high concentrations of vanadium and selenium which may have contributed to effects similar to those observed with sediments with high PAH. The embryo pore water bioassay (hatching/DNA strand breaks) gave results comparable to those observed for reproduction effects (reduced embryo production/embryo hatching) with female grass shrimp exposed to whole sediment.     

Interactive effects of UV, benzo[alpha] pyrene, and cadmium on DNA damage and repair in embryos of the grass shrimp Paleomonetes pugio

We examined the link between DNA strand breaks and hatching rates in grass shrimp, (Paleomonetes pugio), embryos exposed to 0.2 microM benzo[alpha] pyrene (BP), 5 microM cadmium (Cd) and 330 kJ/m(2) UV light, either alone or together. After exposure, embryos were transferred to clean seawater with or without 5 microM Cd. Hatching rates and DNA strand breaks (Comet Assay) were determined. DNA lesions caused by exposure to BP, UV light, or BP/cadmium were rapidly repaired and were not associated with any effects on hatching. Exposure to Cd after exposure to BP or UV did not affect embryological development or DNA repair. Exposure to BP/UV resulted in a high level of DNA lesions which were slowly repaired. Exposure to cadmium following BP/UV exposure inhibited hatching and DNA repair. Adducts formed during exposure to BP/UV exposure may be difficult to excise or may saturate the nucleotide excision repair system.     

Exposure of grass shrimp to sediments receiving highway runoff: Effects on reproduction and DNA

A grass shrimp bioassay was carried out on sediments from three estuarine stations which were different distances from a highway storm drain. Total polycyclic aromatic hydrocarbon (PAH) concentrations were 29, 1.5 and 0.1 μg/g sediment at stations A (next to drain), B (100 m from drain) and C (500 m from drain), respectively. Lower embryo production and embryo hatching rates and a higher level of DNA strand breaks (comet assay) were observed in grass shrimp exposed to stations A and B sediments. There appeared to be an association between reproduction abnormalities and increased DNA strand breaks as a result of grass shrimp exposure to estuarine sediments receiving highway runoff.     

Stress-induced apoptosis in sea urchin embryogenesis

We find that early sea urchin embryos have the capability to induce programmed cell death, or apoptosis, in response to chemical and physical stress. Strongylocentrotus purpuratus embryos (fertilized, 4 cell, 16 cell, 64 cell, and early blastula) were exposed to known cytotoxins, in order to determine when apoptosis occurs naturally and in response to stress. Using cell permeability as an indicator of early stage apoptosis, caspase activation as a mid-stage indicator, and DNA fragmentation as a late stage indicator, we find that during the cleavage stage of embryogenesis apoptosis is almost completely absent. However, a statistically significant (p<0.001) rise in apoptosis in stressed embryos is evident around 24 h after fertilization, during the early blastula stage and shortly after hatching. Before this stage, exposed embryos show no statistically significant increases in apoptosis in comparison to the controls. This pattern of apoptosis in development is similar to that seen in lower vertebrate models in which stress-induced apoptosis occurs only around the mid-blastula transition. We conclude that apoptosis may be used to rid embryos of aberrant or damaged cells in early development, but this response is stage-dependant. Repair, rather than apoptosis, may be utilized during earlier stages, or alternatively, embryos exposed to such stressors may continue development with damaged cells and perhaps damaged DNA. Our continued studies will focus on these alternative hypotheses.     

Effects of salinity on embryonic and larval development of Chinese mitten crab Eriocheir sinensis(Decapoda: Brachyura) and salinity-induced physiological changes

To investigate the effects of salinity on early development of Chinese mitten crab ( Eriocheir sinensis ), and the salinity tolerance mechanism of embryos, different developmental stages of embryos (gastrula, eyespot and pre-hatching stage), and hatched stage I zoea and megalopa, were exposed to a range of salinities (1, 5, 10, 15 (control), 20, 25, 30, 35 and 40). Hatching, survival and molting were monitored. Effects of 24-hour hypersaline (35) and hyposaline (1) stress on egg diameter, water content, Na +/K +-ATPase (NKA) activity, and crustacean hyperglycemic hormone (CHH) gene mRNA expression in embryos and megalopa, are reported. Embryos are more tolerant of low (≤ 5) than high (≥25) salinities, with optimum ranges for gastrula and pre-hatching stage embryos being 5-20, and for eyespot embryo and stage I zoea, 10-20. Most megalopa can molt to the first juvenile instar by day 5 at salinities between 1 and 40, whereas molting of megalopa stages was delayed at 40. Hypersaline conditions resulted in a loss of moisture, reduction of egg volume, and a signifi cant increase in NKA activity and CHH mRNA expression at some developmental stages. Hyposaline conditions did not affect moisture content or egg volume, but resulted in decreased NKA activity and CHH mRNA expression in embryos. For megalopa stages, NKA activity was significantly upregulated following both hypo- and hypersaline stress. Our results suggest high salinity will inhibit development and hatching of E. sinensis embryos, and low salinity will affect the survival of their stage I zoea. Increased NKA activity in embryos and megalopa stages might indicate a hyporegulation response under hypersaline conditions. These findings provide useful information for spawning ground protection of indigenous E . sinensis and enrich the knowledge of embryonic tolerance mechanisms of hyperregulating crustaceans following osmotic stress.     

The toxicity of one-hour silver exposures to early-life stages of the surf Spisula solidissima (Dillwyn)

Early life stages of Spisula solidissima (1-, 4-, 8- and 12-h post-fertilization) were exposed to various concentrations of silver in solution (0, 4, 8, 24, 44, 124 and 278 μg liter^?1) for 1 h. An additional group of 1-h post-fertilized eggs was treated with 0, 4, 11 and 24 μg liter^?1 silver continuously for 48h. Results indicate that younger embryos are more susceptible to Ag. Exposure of one-hour-old embryos to silver for only 1 h gives results similar to a continuous 48-h exposure to the same concentration of Ag. The calculated EC₅₀ for 48-h continuously exposed embryos was similar to the EC₅₀ for 1-h post-fertilized embryos that were exposed to silver for only 1 h.     

DNA damage and apoptosis in the mussel Mytilus galloprovincialis

The effects of known genotoxic substances (4-nitroquinoline-N-oxide, benzo[a]pyrene, teniposide, etoposide, cycloheximide, tributyltin) on human cells (FLC, HL-60) and on mussels were investigated. The correlations between formation of DNA strand breaks and DNA fragmentation characteristic for the process of apoptosis were estimated. Strand breaks induced by 4-nitroquinoline-N-oxide and benzo[a]pyrene did not correlate with DNA fragmentation detected in the process of apoptosis. Induction of internucleosomal DNA fragmentation in HL-60 cells was initiated by teniposide, etoposide and tributyltin, while in the gills of mussels this was detected only with tributyltin. Levels of DNA strand breaks in natural mussel populations, living at locations under the influence of urban and industrial wastes, do not mirror the apoptotic processes.     

Measurement of DNA single-strand breaks in gill and hemolymph cells of mussels, Mytilus sp., collected on the French Atlantic Coast

DNA single-strand breaks were measured by the comet assay in both gill and hemolymph cells of mussels collected in 3 sampling areas of the French coast (Pointe du Castelli, Pen Bron and Saint-Nazaire Harbour). Whole mussel tissue samples were also collected for the chemical determination of PAH, PCB and heavy metal concentrations. In mussel, a higher level of DNA strand breaks was measured in gill than in hemolymph cells (p < 0.01). Despite a factor of contamination from 2 to 3 between sites, no difference in the extent of mussel DNA strand breaks was shown between sampling locations (p > 0.05), questioning the sensitivity of the assays used in biomonitoring studies.     

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.     

Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay

The single-cell microgel electrophoresis assay or the comet assay was used to evaluate DNA damage of dispersed crude oil on sea urchins (Strongylocentrotus droebachiensis) and mussels (Mytilus edulis L.). Sea urchins were exposed to 0.06 and 0.25 mg/L dispersed crude oil in a continuous flow system, while the mussels were exposed to 0.015, 0.06 and 0.25 mg/L dispersed crude oil. Sea urchin coelomocytes and mussel haemocytes were sampled after 4 and 5 weeks exposure, respectively. In the sea urchin coelomocytes, there was a significant concentration-related increase in the percentage of DNA in comet tail. In mussel haemocytes, there was a significantly higher percentage of DNA in comet tail for all treatments compared to the control. The responses were concentration-related up to 0.06 mg/L oil. The two highest exposure concentrations of mussels were not significantly different from each other. These results indicate that the comet assay can be used for biomonitoring of DNA damage in marine invertebrates following oil contamination.     

Embryo aberrations in sea ice amphipod Gammarus wilkitzkii exposed to water soluble fraction of oil

Offshore oil and gas activities have gained momentum in the European Arctic, raising concerns of the potential impact of oil-related chemicals on the polar marine ecosystem, notably on sea ice communities. Herein, malformations on embryos of the Arctic sea ice amphipod Gammaruswilkitzkii exposed to the water soluble fraction of oil were studied. The females ranged from development stage three to nine. No differences in reproductive stage were observed among the different treatments after 30 days of exposure. Frequency of embryo aberrations was significantly higher in the high-dose compared to controls, indicating that the embryos of G. wilkitzkiii were affected by oil.     

Monitoring of the chemical pollution of the river Rhône through measurement of DNA damage and cytochrome P4501a induction in chub (Leuciscus cephalus)

The in vivo response of freshwater fish exposed to pollutants was assessed using two biomarkers, 7-ethoxyresorufin-O-deethylase (EROD) induction and DNA single strand breaks. Chub (Leuciscus cephalus) were caught in spring and in fall at various locations in the river Rhône watershed. EROD activity was measured in the liver while DNA damage was evaluated in chub erythrocytes using the recently developed Comet assay. Chemical contamination was evaluated both in fish muscle (PCBs) and in sediment (PCBs, PAHs, heavy metals) collected at each sampling station. Sex of individuals was shown to influence the level of EROD activity but not the level of DNA damage. The EROD activity as well as the DNA damage were found to be higher in the mostly contaminated stations compared to the reference one. This study shows that multibiomarker-based approach provides complementary informations about early effects in feral fish exposed to complex chemical pollution and highlights the interest of the Comet assay in genotoxicity assessment.     

Effect of parental exposure to trenbolone and the brominated flame retardant BDE-47 on fertility in rainbow trout (Oncorhynchus mykiss)

We exposed sexually maturing male rainbow trout (Oncorhynchus mykiss) to BDE-47 (a polybrominated diphenyl ether) and female rainbow trout to trenbolone (an anabolic steroid). Male trout were orally exposed for 17 days to 55 microg/kg/day BDE-47 and female trout continuously exposed for 60-77 days to a measured trenbolone water concentration of 35 ng/L. After the exposure, eggs and semen were collected and in vitro fertilization trials performed using a sperm:egg ratio of 300,000:1. In the BDE-47 study, eggs from control females were fertilized with semen from exposed males, while in the trenbolone study, eggs from exposed females were fertilized with semen from control males. All treatments were evaluated at two-three early developmental time-points representing first cleavage (0.5 day), embryonic keel (9 days), and eyed stages (19 days), respectively. The results indicated that BDE-47 exposure did not alter fertility as embryonic survival was similar between control and exposed groups. Trenbolone exposure also did not alter embryo survival. However, in the embryos fertilized with eggs from trenbolone exposed females, a noticeable delay in developmental progress was observed. On day 19 when eye development is normally complete, the majority of the embryos either lacked eyes or displayed under-developed eyes, in contrast to control embryos. This finding suggests steroidal androgen exposure in sexually maturing female rainbow trout can impact developmental timing of F1 offspring.     

双锯鱼(Amphiprion)胚胎不同发育阶段和成鱼不同组织中内参基因的筛选及应用

双锯鱼(Amphiprion)亦称为海葵鱼,又称为小丑鱼,是一类经济价值较高的海水观赏鱼。目前国内在双锯鱼的胚胎发育、人工饲养以及形态学观察等方面已经取得了有效成果,但在分子生物学水平上对其基因表达的研究较少。为了筛选出适用于双锯鱼胚胎不同发育阶段以及成鱼组织的内参基因,分析酪氨酸酶(TYR)基因的表达情况,以眼斑双锯鱼(A.ocellaris)和白条双锯鱼(A.frenatus)为材料,利用实时定量聚合酶链式反应(qPCR)对18SrRNA、GAPDH(甘油醛-3-磷酸脱氢酶基因)、Ef-1α(转录延伸因子基因)和β-actin(β-肌动蛋白基因)这4个候选内参基因的表达水平进行检测,同时通过geNorm和Norm Finder软件对其稳定性进行评估,最后以合适的内参基因作为参考,研究TYR mRNA的表达水平。结果表明,在双锯鱼胚胎的不同发育阶段,18S和β-actin的表达量相对于其他基因较为稳定;在双锯鱼的不同组织中,稳定性依次为18S>β-actin>Ef-1α>GAPDH。以18S作为内参基因时分析发现TYR基因的表达在双锯鱼胚胎发育过程中呈先上升后下降的趋势,在体节期表达量最高;在双锯鱼各组织中均有表达,且在眼、尾和红皮中表达量最高。     

Influence of sample manipulation on contaminant flux and toxicity at the sediment-water interface

Toxicities of sediments from San Diego and San Francisco Bays were compared in laboratory experiments using sea urchin (Strongylocentrotus purpuratus) embryos exposed to pore water and at the sediment-water interface (SWI). Toxicity was consistently greater to embryos exposed at the SWI to intact (unhomogenized) sediment samples relative to homogenized samples. Measurement of selected trace metals indicated considerably greater fluxes of copper, zinc, and cadmium into overlying waters of intact sediment samples. Inhibition of sea urchin embryo development was generally greater in sediment pore waters relative to SWI exposures. Pore water toxicity may have been due to elevated unionized ammonia concentrations in some samples. The results indicate that invertebrate embryos are amenable to SWI exposures, a more ecologically relevant exposure system, and that sediment homogenization may create artifacts in laboratory toxicity experiments.     

Effects of the duration of exposure to mercuric chloride on the embryogenesis of the estuarine teleost, Fundulus heteroclitus

Embryos of Fundulus heteroclitus in the 4–8 cell stage were exposed nine Hg⁺⁺ (as mercuric chloride) concentrations from 0 to 100 μg/litre (ppb) for 32 days, 5 days, 2 days and 1 day. All but the chronically (32-day) exposed embryos were allowed to continue development in mercury-free water (20% S) after the exposure period to determine the effect of duration of exposure on three parameters of development. Ninety-six hour survival, total hatching success and per cent incidence of lateral spinal curvature were measured. Only embryos exposed to mercury for a single day showed a significant increase in survival in comparison with embryos exposed for a longer period of time after four days of development. Under all conditions of exposure, survival was reduced at concentrations greater than 40 ppb Hg⁺⁺. Hatching success of chronically exposed embryos was significantly reduced at concentrations greater than 10 ppb Hg⁺⁺. Significant increases in total hatchability were effected by reducing the duration of exposure to five days and one day. Of eleutheroembryos emerging following chronic exposure to mercury, increases in the incidence of lateral spinal curvature were noted at all concentrations above 20 ppb Hg⁺⁺. Significant reductions of this disorder were observed among eleutheroembryos exposed to mercury for 5 days, 2 days or 1 day. Of the three parameters measured, the incidence of lateral spinal curvature and the resulting VH₅₀ value gave a more sensitive evaluation of the embryotoxic effects of Hg⁺⁺ as mercuric chloride on the estuarine teleost, Fundulus heteroclitus.     

Selenium-induced teratogenicity in Sacramento splittail (Pogonichthys macrolepidotus)

Selenium is a potent reproductive and teratogenic environmental contaminant and there are concerns over possible reproductive effects of selenium on the Sacramento splittail (Pogonichthys macrolepidotus) population, a threatened species, in California, USA. In this study, the teratogenic effects of selenium were examined in splittail embryos exposed to 0.0, 5.0, and 15.0 mg l(-1) sodium selenite for 48-h at 18.0 degrees C under static conditions, with renewal every 12 h. Embryo development was evaluated daily for abnormalities from initiation of exposure (stage 27) to initiation of exogenous feeding. At the end of evaluation, prelarvae were preserved for histological analysis. There were no significant differences in mortality or hatching success between control and exposed embryos. Exposed fish had pericardial edema and deformities of skeletal tissues (loss of tail, lordosis, scoliosis, and kyphosis). Other histological alterations were limited to dysplasia, hyperplasia and metaplasia of skeletal tissues in the deformed fish. This study showed that a short exposure of embryos during somite development has significant effects on the musculoskeletal development.     

DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis during anoxic stress and aerobic recovery

Anoxia-induced DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis was assessed with the alkaline comet assay (single-cell gel electrophoresis). The alkaline comet assay method for detecting DNA strand breaks and alkali labile sites in individual cells. DNA damage was determened in the scallops (M. yessoensis) gill cells. The scallops were exposed to air for 8 h showing a clear increase in the levels of DNA damage. After the air exposure, M. yessoensis were re-submersed for a period of 12 h, leading values to return to a pre-aerial exposure level. Control animals were kept immersed during the whole period. The resulting data demonstrate that natural influences, such as oxygen depletion (anoxia) in seawater, can be responsible for the induction of DNA damage. If the scallops were re-immersed in oxic conditions, the anoxically induced breaks were repaired. The main mechanisms influencing the integrity of the DNA structure are discussed in this paper.