Effects of benzo(a)pyrene exposure on the antioxidant enzyme activity of scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

Scallop Chlamys farreri was exposed to different concentrations of benzo(a)pyrene (BaP) (0.5 μg/L, 1.0 μg/L, 10.0 μg/L and 50.0 μg/L) for 30 days in seawater. The 7-ethoxyresorufin O-deethylase (EROD) activity was significantly induced, and increased with the increasing BaP concentration. The glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx) activities increased in short time at low concentration of BaP, and was significantly depressed at high concentrations. Scallop gill was more sensitive to BaP than the digestive gland, and the digestive gland was the main tissue to deal with oxyradicals. The contents of malondialdehyde (MDA) increased with the exposure time and there was a positive correlation (concentration-effect) between the MDA content and the concentration of BaP. The biomarkers determined in this experiment had important roles in detoxification, and showed great potential as biomarkers for oxidative stress. Controlled laboratory experiments designed to simulate field exposure scenarios are particularly useful in ascertaining biomarkers suitable for use with complex contaminant mixtures in the marine environment. Supported by the Technology Development Program of Shandong (No. 2008GG1005010) and the Program of Introducing Talents of Discipline of Universities (111 Project, No. B08049)     

Effects of benzo（a）pyrene exposure on the antioxidant enzyme activity of scallop Chlamysfarreri

Scallop Chlamys farreri was exposed to different concentrations of benzo(a)pyrene (BaP) (0.5 μg/L, 1.0 μg/L, 10.0 μg/L and 50.0 μg/L) for 30 days in seawater. The 7-ethoxyresorufin O-deethylase (EROD) activity was significantly induced, and increased with the increasing BaP concentration. The glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx) activities increased in short time at low concentration of BaP, and was significantly depressed at high concentr...     

Effect of benzo[a]pyrene on detoxification and the activity of antioxidant enzymes of marine microalgae

The objective of this study was to examine the effect of benzo[a]pyrene(Ba P) on the detoxification and antioxidant systems of two microalgae,Isochrysis zhanjiangensis and Platymonas subcordiformis.In our study,these two algae were exposed to Ba P for 4 days at three different concentrations including 0.5 μg~(L-1)(low),3 μg~(L-1)(mid) and 18 μg~(L-1)(high).The activity of detoxification enzymes,ethoxyresorufin O-deethylase(EROD) and glutathione S-transferase(GST) increased in P.subcordiformis in all Ba P-treated groups.In I.zhanjiangensis,the activity of these two enzymes increased at the beginning of exposure,and then decreased in the groups treated with mid-and high Ba P.The activity of antioxidant enzyme superoxide dismutase(SOD) increased in I.zhanjiangensis in all Ba P-treated groups,and then decreased in high Ba P-treated group,while no significant change was observed in P.subcordiformis.The activity of antioxidant enzyme catalase(CAT) increased in I.zhanjiangensis and P.subcordiformis in all Ba Ptreated groups.The content of malondialdehyde(MDA) in Isochrysis zhanjiangensis increased first,and then decreased in high Ba P-treated group,while no change occurred in P.subcordiformis.These results demonstrated that Ba P significantly influenced the activity of detoxifying and antioxidant enzymes in microalgae.The metabolic related enzymes(EROD,GST and CAT) may serve as sensitive biomarkers of measuring the contamination level of Ba P in marine water.     

Molecular cloning and differential expression patterns of sigma and omega glutathione S-transferases from <Emphasis Type="Italic">Venerupis philippinarum</Emphasis> to heavy metals and benzo[a]pyrene exposure

Glutathione S-transferases (GSTs) are a class of enzymes that facilitate the detoxification of xenobiotics, and also play important roles in antioxidant defense. We identified two glutathione S-transferase isoforms (VpGSTS, sigma GST; VpGSTO, omega GST) from Venerupis philippinarum by RACE approaches. The open reading frames of VpGSTS and VpGSTO were of 612 bp and 729 bp, encoding 203 and 242 amino acids with an estimated molecular mass of 22.88 and 27.94 kDa, respectively. The expression profiles of VpGSTS and VpGSTO responded to heavy metals and benzo[a]pyrene (B[a]P) exposure were investigated by quantitative real-time RT-PCR. The expression of VpGSTS and VpGSTO were both rapidly up-regulated, however, they showed differential expression patterns to different toxicants. Cd displayed stronger induction of VpGSTS expression with an approximately 12-fold increase than that of VpGSTO with a maximum 6.4-fold rise. Cu exposure resulted in similar expression patterns for both VpGSTS and VpGSTO. For B[a]P exposure, the maximum induction of VpGSTO was approximately two times higher than that of VpGSTS. Altogether, these findings implied the involvement of VpGSTS and VpGSTO in host antioxidant responses, and highlighted their potential as a biomarker to Cd and B[a]P exposure.     

Effects of heavy metal ions (Cu2+, Pb2+ and Cd2+) on DNA damage of the gills, hemocytes and hepatopancreas of marine crab, Charybdis japonica

There are rising concerns about the hazardous effects of heavy metals on the environment. In this study, comet assay and DNA alkaline unwinding assay were conducted on the tissues (gills, hepatopancreas, and hemocytes) of Charybdis japonica in order to illustrate genotoxicity of three heavy metal ions (Cu2+, Pb2+, and Cd2+) on the marine crabs C. japonica. The crabs were exposed to Cu2+ (10, 50, and 100 ?g.L?1), Pb2+ (50, 250, and 500 ?g L?1) and Cd2+ (5, 25, and 50 ?g L?1), and the tissues were sampled at days 0.5, 1, 3, 6, 9, and 15. DNA alkaline unwinding assay was used for testing the DNA single strand break in gills and hepatopancreas and comet assay was employed for testing the DNA damage in hemocytes. The results showed that the DNA damage (F-value) of gills in the crabs exposed to the three heavy metals was decreased gradually during the exposure periods and there was a dose-time response relationship in certain time, suggesting that the levels of DNA single strand break in all the experimental groups increased significantly compared to the controls. Changes of F-value in hepatopancreas of the crabs exposed to the three heavy metals were similar to those in gills except that the peak values were found in the 500 ?g L?1 Pb2+ treatment group at day 3 and the 50 ?g L?1 Cd2+ treatment group at day 9. The ranks of DNA damage in gills and hepatopancreas induced by the three heavy metal ions (50 ?g L?1, day 15) were Cd2+ >Pb2+ >Cu2+ and Pb2+ >Cu2+ >Cd2+. The levels of DNA damage in gills were higher than those in hepatopancreas in the same experimental group. It can be concluded that indices of DNA damage can be used as the potential biomarkers of heavy metal pollution in marine environment.     

Effect of hypoxia on the blood of large yellow croaker (Pseudosciaena crocea)

Large areas of hypoxic water have recently been reported in the East China Sea. It is hypothesized that hypoxia may be partially responsible for the decline of some fish stocks. We evaluated the effect of hypoxia on large yellow croaker (Pseudosciaena crocea). The fish were exposed to three concentrations of dissolved oxygen (DO; 1.5 mg/L and 2.0 mg/L, and 6.5 mg/L control). We collected blood after 6, 12, 24, 48, and 96 h exposure. There was a significant increase in red blood count, hematocrit, hemoglobin concentration, and mean corpuscular hemoglobin in the group exposed to 1.5 mg/L DO after 6 h or 12 h, and a delayed increase (only elevated after 48 h and 96 h) in these indices in the group exposed to 2.0 mg/L DO. Plasma glucose concentrations increased significantly in both hypoxic groups after 24 h. Furthermore, plasma lactate and lactate dehydrogenase activity increased significantly after the first 6 h exposure in both hypoxic groups. Our results suggest that large yellow croakers could not maintain the aerobic pathway and instead use anaerobic metabolism for survival when DO levels fall below 2.0 mg/L. We conclude that the occurrence of hypoxia (<2 mg/L DO) in the East China Sea could cause metabolic stress for large yellow croakers and may be partially responsible for the population decline of this species.     

Genotoxic potential of copper oxide nanoparticles in the bivalve mollusk <Emphasis Type="Italic">Mytilus trossulus</Emphasis>

Copper oxide nanoparticles (CuO-NPs) are among the most widely used metal oxide nanoparticles, which increases the chance of their being released into the marine environment. As the applications of these particles have increased in recent years, their potential impact on the health of marine biota has also increased. However, the toxicological effects of these NPs in the marine environment are poorly known. In the present study, the DNA damaging potential of CuO-NPs in the marine eastern mussel Mytilus trossulus was evaluated and compared to that of dissolved copper exposures. Genotoxicity was assessed by the single cell gel electrophoresis (comet) assay in mussel gill and digestive gland cells. The results showed that copper in both forms (CuO-NPs and dissolved copper) was accumulated to different extents in mussel tissues. The mussel exposed to the dissolved copper attained higher concentrations of copper in the gills than in the digestive gland. In contrast to these results, it was found that CuO-NPs could induce much higher copper accumulation in the digestive gland than in the gills. A clear and statistically significant increase in DNA damage was found in both tissues of the Cu-exposed group compared to the control mussels. Our results indicated that the CuO-NP exposure produced remarkable effects and increased DNA damage significantly in mussel gill cells only. It should be noted that the digestive gland cells were prone to accumulation following CuO-NPs when compared to the gill cells, while the gill cells were more sensitive to the genotoxic effects of CuO-NPs. These results also suggested the need for a complete risk assessment of engineered particles before its arrival in the consumer market.     

MONITORING POLYNUCLEAR AROMATIC HYDROCARBONS IN SEDIMENT POREWATER BY SPMD

A new mimic biological Semi-permeable Membrane Device (SPMD) introduced for sampling organic pollutants yielded satisfactory results when it was frrst used as a passive sampler to concentrate and determine 16 kinds of polynuclear aromatic hydrocarbons (PAHs) by means of capillary GC on an HP 5890 GC-FID in coastal sediment perewater. The concentration of PAHs in sediment porewater for naphthalene(N), acenaphthlene(AL), acenaphthene (AE), fluorene (F), phenaphthene(P), anthracene(A), fluoranthene(FA), pyrene(Py), benzo[a]anthracene(B[a]A), chrysene(Chr), benzo[b] fluor- anthene(B[b]F), benzo[k]fluoranthene(B[k]F), benzo[a]pyrene(B[a]P),indeno[1,2,3,-cd]-Pyrene(I[123]P), dibenz[a,h]anthracene(D[ab]A) and benzo[g,h,i] perylene(B[ghi]P) were:50.36, under detection limits(UD), 18.19, 8.41, 8.40, 1.44, UD, 8.01, 524.15, 168.47, 50.13,123.66, 63.48, 27.40, 82.04 and 58,81 ng/L, respectively.     

A multi-generation Schmakeria poplesia culturing system for use in ecotoxicological study

Crustacean zooplankton form the keystone link between primary producers and fish stocks in marine and estuary ecosystems. We have established a multi-generation cultivation system for zooplankton with which future experiments on the biological effects of pollutants in marine and estuary environments can be better performed. A population of calanoid copepod, Schmakeria poplesia, was collected in December 2003 and maintained in a static system through all stages (eggs to adults). The population exhibited an average developmental time of 13.6 d in conditions corresponding to the natural environment (water temperature 20°C, salinity 15). A series of experiments were performed to examine copepod egg production and hatching success as functions of food type and feeding concentration. Results in our study showed that Isochrysis galbana was more favored for the reproduction of copepods than Phaeodactylum tricornutum, and 10×10⁴cells mL⁻¹ was the most practical algae concentration. We have demonstrated that the Schmakeria poplesia population can be maintained in the laboratory through multiple generations. In addition, methods to control egg production through changes in food concentration have been established, making it feasible to control the start date of exposure experiments or the timing of the collection of offspring to initiate a new generation.     

Genotoxic Potential of Copper Oxide Nanoparticles in the Bivalve Mollusk Mytilus trossulus

Copper oxide nanoparticles(CuO-NPs) are among the most widely used metal oxide nanoparticles,which increases the chance of their being released into the marine environment.As the applications of these particles have increased in recent years,their potential impact on the health of marine biota has also increased.However,the toxicological effects of these NPs in the marine environment are poorly known.In the present study,the DNA damaging potential of CuO-NPs in the marine eastern mussel Mytilus trossulus was evaluated and compared to that of dissolved copper exposures.Genotoxicity was assessed by the single cell gel electrophoresis(comet) assay in mussel gill and digestive gland cells.The results showed that copper in both forms(CuO-NPs and dissolved copper) was accumulated to different extents in mussel tissues.The mussel exposed to the dissolved copper attained higher concentrations of copper in the gills than in the digestive gland.In contrast to these results,it was found that CuO-NPs could induce much higher copper accumulation in the digestive gland than in the gills.A clear and statistically significant increase in DNA damage was found in both tissues of the Cu-exposed group compared to the control mussels.Our results indicated that the CuO-NP exposure produced remarkable effects and increased DNA damage significantly in mussel gill cells only.It should be noted that the digestive gland cells were prone to accumulation following CuO-NPs when compared to the gill cells,while the gill cells were more sensitive to the genotoxic effects of CuO-NPs.These results also suggested the need for a complete risk assessment of engineered particles before its arrival in the consumer market.     

Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.     

Effects of tributyltin （TBT） on enzyme activity and oxidative stress in hepatopancreas and hemolymph of small abalone, Haliotis diversicolor supertexta

We investigated the effect of tributyltin (TBT) exposure on the concentration of malondialdehyde (MDA) and the activity levels of the superoxide dismutase (SOD), catalase (CAT), and acid and alkaline phosphatase (ACP and AKP) enzymes in the small abalone, Haliotis diversicolor supertexta. We collected samples of the hepatopancreas and hemolymph 2, 6, 24, 48, 96, and 192 h after exposure to 0.35 μg (Sn)/L TBT. In the hepatopancreas, ACP activity was significantly higher in animals exposed to TBT 2, 24, and 96 h post-exposure compared with the control animals. AKP activity was also higher after 2 h, but SOD and CAT activity was unchanged. The concentration of MDA in the hemolymph was significantly higher than the control animals 2 and 6 h post-exposure. In the hemolymph of animals exposed to TBT, ACP activity was significantly lower than in the control animals 192 h post-exposure, whereas AKP activity was significantly lower 2 and 192 h post-exposure. Hemolymph SOD activity and levels of MDA were significantly lower than in the control animals 24 h after exposure but significantly higher after 96 h. Our results demonstrate that exposure to TBT cause rapid changes in ACP and AKP activity as well as altering the concentration of MDA in the hepatopancreas and hemolymph. SOD and CAT do not appear to be involved in the detoxification of TBT in the hepatopancreas of small abalone.     

Growth inhibition and oxidative stress in two species of marine diatoms exposed to 1-phenylethanol

1-phenylethanol(1-PEA)is a flavor extensively used in the production of co smetics,beverages,and food.The release of 1-PEA into coastal environments has aroused great concern.However,its potential effects on marine organisms are still unknown.In order to provide a better understanding of the ecological risks of 1-PEA in marine environments,this study determined the toxic effects of 1-PEA on two marine diatoms(Phaeodactylum tricornutum and Skeletonema costatum).The diatoms were grown in culture medium containing different concentrations of 1-PEA for 96 h.The contents of chlorophyll a,chlorophyll c,glutathione(GSH),malondialdehyde(MDA),and the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx),were measured at the end of the exposure period.1-PEA was shown to significantly inhibit the growth of diatoms,with 96-h EC_(50) values of 257.14 mg/L and 126.46 mg/L in P.tricornutum and S.costatum,respectively.In P.tricornutum,the levels of SOD,CAT,GPx,GSH,and MDA were stimulated only when 1-PEA concentrations were close to or greater than the 96-h EC_(50)value.However,in S.costatum,the activities of SOD and CAT,and the syntheses of two chlorophylls were inhibited even at an exposure concentration below the 96-h EC_(50) value.Taken together,these findings indicate a potential ecological risk by discharging 1-PEA into coastal areas and its species-specific toxic effects on marine organisms.     

Laboratory study on the ecological impact of sophorolipid used for harmful algae elimination

We studied the role of sophorolipid in inhibiting harmful algae bloom (HAB). Different sophorolipid concentrations were tested on marine microalgae, zooplankton, fish, and bivalve (Mytilus edulis) in laboratory. The result shows that sophorolipid could inhibit the growth of algal species selectively. Among three algae species selected, Platymonas helgolandica var. tsingtaoensis was promoted with increasing sophorolipid concentration; Isochrysis galbana was inhibited seven days later in sophorolipid concentration below 40 mg/L; and Nitzschia closterium f. minutissima was inhibited obviously in only a high sophorolipid concentration over 20 mg/L. Therefore, sophorolipid in a low concentration at <20 mg/L could remove certain harmful algae species effectively without harming other non-harmful microalgae. For other animals, sophorolipid could inhibit the growth of ciliate Strombidium sp. by 50% at 20 mg/L sophorolipid concentration after 96 h. The concentration in 96-h LC₅₀ for Calanus sinicus, Neomysis awatschensis, Lateolabrax japonicus, and Paralichthys olivaceus was 15, 150, 60, and 110 mg/L, respectively. The 24 h LC₅₀ value for Artemia salina was 600 mg/L. The relative clearance rate of mussel Mytilus edulis decreased to 80%, 40%, and 20% of the control group after being exposed to 20, 50, and 100 mg/L sophorolipid for 24 h. Therefore, the toxicity for mitigation of harmful algae bloom at previously recommended concentration of 5–20 mg/L sophorolipid is low for most tested organisms in this reaserch.     

CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM (Pagrosomus major) FIN CELL LINE RSBF

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Acute Toxicity of Formalin to the Red-tide Dinoflageilate Prorocentrum minimum Schiller （Protozoa, Mastigophora）

The acute toxic levels of formalin to a marine red-tide dinoflagellate Prorocentrum minimum Schiller (Mastigophora, Dinoflagellida) were determined by linear regression analysis. The data obtained revealed that there is a close regression relationship between logarithmic concentrations of formalin and mortality probit scales of the organism when exposure times are 1, 2, 6, 12, 24 h. The median lethal concentrations (LC50 values) obtained from correlation analysis for these exposure times (with 95% confidence intervals), were 11.83, 6.76, 4.37, 4.27 and 3.98mgL^-1 respectively. A toxicity curve was obtained to connect the exposure time and LC50 value from the correlation between them. The results indicated that P. minimum could be killed or fixed by formalin in the concentration range of 4-12mgL^-1 within 1-24h.     

Immunotoxicity Effect of Benzo[α]Pyrene on Scallop Chlamys farreri

The toxic effects of benzo[α]pyrene (B[α]P) at different concentrations (0.1, 0.5, 1, 2.5 and 7.5 ugL-1) on scallop (Chla-mys farreri) immune system were studied. The results showed that B[a]P had significant toxic effects on the haemocyte counts, neu-tral red uptake, phagocytosis, bacteriolytic and antibacterial activity (P<0.05), while the seawater control and acetone control had no significant differences. The haemocyte counts, neutral red uptake, phagocytosis and bacteriolvtic activity in all B[α]P treatment groups as well as antibacterial activity, in groups of 0.5, 1, 2.5 and 7.5 ugL-1 B[α]P decreased significantly (P<0.05). Some of these indices tended to be stable on the sixth day and others on the ninth day, and the indices showed clear time- and concentra-tion-response to B[α]P. Bacteriolytic activity in 0.1ugL-1 B[α]P treatment group and antibacterial activity in 0.1 ugL-1 and 0.5 ugL-1 B[α]P treatment groups increased at the beginning of exposure and reached their peaks on day 1 and day 6, respectively. Following that, both activities decreased gradually and became stable after day 9. When all the indices reached stability, they were significantly lower than those in control group (P<0.05), except for antibacterial activity in 0.1 ugL-1 B[α]P treatment group (P<0.05). Thus, B[α]P has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immu-nomodulation in aquatic organisms.     

CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM（Pagrosomus major）FIN CELL LINE RSBF

A continuous marine fish cell line RSBF(i.e.Red Sea Bream Fin)was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine(PEI)and nickel cholride(NiCl2)in this study on the deleterious effects of aquatic genotoxins on fish.At the 0.01 to 1μgml concentration tested,PEI had acute toxicity to the treated RSBF cells(IC50=1.12,0.92,0.88 and 0.64μg/ml PEI for time 0 h,24 h,48 h and 72 h after treatment,respectively)and markedly inhibited their proliferation in a dose-dependent manner.At the 0.001 to 5 μmol/L concentration tested,NiCl2 posed no acute toxicity but significantly stimulated their growth(107?214?of control).Random amplified polymorphic DNA(RAPD)technique was used to detect the genotoxic effects of PEI and NiCl2 by comparing the RAPD banding patterns of the control and treated cells.RAPD analysis indicated that at the concentrations tested,PEI was more genotoxic than NiCl2 to RSBF cells;that there was a slight dose-dependent response in the genotoxic effect of PEI bue not NiCl2;and that RAPD technique might provide a sensitive,non-specif-ic gentoxic endpoint.And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene vector in fish gene transfer and human gene therapy.     

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared 100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6–50 days post-vaccination. These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for the development and application of this promising DNA vaccine.