Effects of polynuclear aromatic hydrocarbons on hemocyte characteristics of the Pacific oyster, Crassostrea gigas.

ABSTRACT To investigate the effect of varying concentrations of PAHs and laboratory holding on hemocyte characteristics, Pacific oysters, Crassostrea gigas, were exposed to varying concentrations of polycyclic aromatic hydrocarbons for 40 days. The circulating hemocyte count (HC) was significantly decreased over the experimental day for all treatments including controls, by day 20; after 10 days, there were no differences among treatments. Similar pattern of decrease was observed over the experimental day in phagocytic index (PI) with relatively constant PI after 20 days. Statistical significance observed only at 10 days. Percent of mobile hemocyte (PMH) also exhibited significant decrease with an association of PAH concentrations without effect of laboratory holding. Lysosomal membrane destabilization (LD) increased continuously over the experimental day with exposed level-dependent differences. Our result indicate that PMH and LD are useful tools for biomarker with sensitivity to toxicant regardless of the effect of laboratory holding, however, HC and PI are significantly influenced by laboratory holding and exposure effect is negligible at the end of the experiment.

KEY WORDS: Crassostrea gigas, hemocyte, polycyclic aromatic hydrocarbon

INTRODUCTION

The growing frequency of oil spillages in coastal waters and the industrialization and urbanization of the coastal area have increased the opportunities for marine organisms to be exposed to various xenobiotics. This may be one cause for the decreased productivity in coastal bivalve farming.

Polycyclic aromatic hydrocarbons (PAHs) are one class of toxic environmental pollutants that are accumulated by coastal organisms. Although PAHs can originate from natural sources (Cerniglia & Yang 1984, Guerin & Jones 1988), a major portion can be attributed to anthropogenic sources, particularly from fuel combustion, pyrolytic processes, spillage of petroleum products, waste incinerators and domestic heaters (Frreman & Cattell 1990, Wakeham et al. 1980). Because of a stable chemical structure and low solubility, PAHs have been detected in a variety of environmental samples and may have accumulated in marine organisms (Baumard et al. 1999, Juhasz & Naidu 2000). As they accumulate in marine organisms, the xenobiotics may cause sublethal changes that arise from chemical insult as well as physiologic mechanisms elicited to avert chemical damage (Oliver et al. 2001).

Bivalve mollusks are often used as sentinel organisms to detect xenobiotics in the marine environment. The functions of bivalve hemocytes are often studied as an end point for xenobiotic stresses (Auffret & Oubella 1997, Oliver et al. 2001, Dyrynda et al. 1998, Sauve et al. 2002, Baier-Anderson & Anderson 2000).

PAHs threaten hemocyte function by causing malfunctions and malformations of the hemocyte. Highly reactive oxygen species (ROS) such as superoxide anion generated during the metabolism of PAHs can cause protein oxidation, enzyme inactivation, DNA damage and cytoskeletal protein damage of hemocytes (Bellomo et al. 1990, Grundy et al. 1998). This can lead to a loss in defense function of hemocyte such as suppressed adhesion, aggregation, motility and phagocytosis (Fires & Tripp 1980, Grundy et al. 1996), decreased circulating hemocyte numbers (Sami et al. 1992) and enhanced disease expression (Chu et al, 2002). Field observation for hemocyte function, however, includes various disagreements depending on the studies.

In this study, we investigated alteration in hemocyte functions of the Pacific oyster, Crassostrea gigas, exposed to PAHs for 40 days. Time course alteration and exposed level dependent impact were observed to elucidate the PAH effects on hemocytic characteristics of the Pacific oyster, C. gigas.

MATERIALS AND METHODS

Experimental Animals

Pacific oysters, Crassostrea gigas, were collected from an oyster farm and transported to the laboratory in December 2002. After 3 days acclimation, the oysters were allocated to polypropylene tanks containing 15 L of filtered seawater (25 oysters per tank), which exchanged 3 times a day. The water temperature was maintained at 10[degrees]C and gently aerated with a commercial air stone using air blower.

PAH Exposure

The seawater was changed every day and the tank dosed with PAH cocktails at final concentration of 50, 100 and 200 ppb respectively (3 replicates) but only the same amount of solvent was administrated in control. The stock solution was prepared from equivalent amounts of 10 species of PAHs with acetone. Acenaphthene, acenaphthylene, benz(a)phenanthrene, benz(a)anthracene, benzo(a)pyrene, chrysene, dibenz(a,h)anthracene, fluoranthene, fluorene, phenanthrene and pyrene were purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan).

The oysters were fully fed marine microalgae: Chaetoceros simplex, C. gracilis, Isochrysis galbana and Tetraselmis tetrathele mixed in equal proportions when seawater was exchanged. Five or six animals per tank were used for experimental measurements every 10 days.

Hemolymph Collection and Circulating Hemocyte Count

Approximately 0.5 mL hemolymph per oyster was extracted from the pericardial cavity with a syringe and a 22 G needle under aseptic conditions. The cells from 2 to 3 individuals were pooled in each tube (duplicate pools per tank) and stored on ice during manipulations. The hemolymph was gently mixed to ensure homogeneity. A drop of hemolymph collected from each individual was introduced into an improved Neubauer hemocytometer and the number of hemocytes determined microscopically.

Hemocyte Mobility

A hemocyte monolayer was obtained by allocating hemolymph (50 [micro]L) and 0.2 [micro]m filtered seawater (FSW) (150 [micro]L) onto a Laboratory-Tek 8 chamber slide and incubating for 30 min in a 27[degrees]C incubator. After discarding the hemolymph, the movements of monolayered hemocytes were recorded for 15 min with a computer controlled camera under a differential interference contrast microscope (BX-5, Olympus). Selected hemocytes (12-15 cells) were traced for 3 min using an image analyzer program (ImagePro., Media Cybernetics, Inc.). The PMH was expressed as a percentage of the number of mobile hemocytes against the total number of hemocytes (Fisher et al. 1996) and the rate of hemocyte locomotion (RHL) as a mean distance of hemocyte movement per minute ([micro]m/min).

Phagocytic Index

Pre-counted hemocytes were introduced into Laboratory-Tek 8 chamber slides (~10,000 cells). Micrococcus lysocleikticus (Sigma-Aldrich Co., St Louis, Missouri) hydrated in FSW at room temperature for 30 min and then the yeast concentration was determined using an improved Neubauer hemocytometer. Approximately 10,000 cells were introduced to the chamber (1:1) and incubated for 60 min at 27[degrees]C. After removing the chamber from the slide, excessive suspension and yeast were discarded by FSW. The slides were microscopically examined to determine the number of hemocytes bound or ingested yeast. A minimum of 100 hemocytes per well were examined to determine the PI, which was expressed as the function (Oliver & Fisher 1995, Fisher et al. 1996):

PI = [(Number of haemocytes displaying bound or ingested yeast)/ Total number of haemocytes observed] x 100

Lysosomal Destabilization

A modified neutral red retention (NRR) time assay was applied (Lowe et al. 1995a, Lowe et al. 1995b). Approximately 10,000 hemocytes of each sample were allocated onto a [poly-.sub.L]-lysine (Sigma Diagnostics Inc., St. Louis, Missouri) coated glass slide and incubated in a lightproof moisture chamber for 30 min to allow the cells to attach on the slide. Excess solution was gently removed and 40 [micro]L of neutral red (Sigma Chemical Co. St. Louis, Missouri) working solution was added. The cells were incubated in the chamber at room temperature for 15 min and examined under the light microscope (total magnification x400). Following an additional hour incubation, the percentage of small granular hemocytes that exhibited movement of dye from lysosomes into the cytosol was determined using a microscope (BX-5, Olympus).

Statistics

All data were analyzed with the SPSS statistics package (SPSS Inc.). The homogeneities of the means were tested with 1-way analysis of variance (ANOVA) (P < 0.05). Tukey HSD test was used to identify the differences between means of treatments.

RESULTS

Circulating Hemocyte Count

Hemocite Count at 10 days in controls (134.0 x [10.sup.4] cells/mL) was not significantly different from HC at the beginning of the experiment (135.6 x [10.sup.4] cells/mL). As the experiment proceeded, however, HC in controls significantly decreased ranging from 65.0-72.2 x [10.sup.4] cells/mL (Fig. 1) without significant difference except at 30 days (P < 0.05).

[FIGURE 1 OMITTED]

For exposed groups, HC after 10 days (94.0-100.4 x [10.sup.4] cells/ mL) was higher than any other days (range 48.5-65.0 x [10.sup.4] cells/ mL) HC after 20 days in 200 ppb was lowest during the experiment (P < 0.05). Although not being consistent with exposed concentration, mean of HC in exposed groups was always lower than in control while statistical significance was not always observed (P < 0.05) (Fig. 1).

Hemocyte Mobility

PMH ranged 38.6% to 70.0% during the experiment. Although no significant change of PMH was observed in control remaining around 66.2% to 70.0% (P < 0.05), PMH in exposed groups significantly varied during the experiment in the range of 54.8% to 68.2% for 50 ppb, 49.8% to 56.0% for 100 ppb and 38.6% to 53.4% for 200 ppb. PMH in 50 ppb was continuously decreased during the experiment whereas those in 100 and 200 ppb slightly increased after 20 days with lowest at 10 days (P < 0.05) (Fig. 2).

[FIGURE 2 OMITTED]

Although being slightly decreased from 16.4 to 14.6 [micro]m/min, RHL in control did not significantly change during the experiment. For exposed groups, although continuous decrease of RHLs in 50 ppb observed during experiment, those in 100 and 200 ppb were increased in comparison to that after 10 days. During the experimental day, RHLs in control were always higher than those in exposed groups whereas statistical significance was not always observed (P < 0.05) (Fig. 3).

[FIGURE 3 OMITTED]

Phagocytic index

Phagocytic Index ranged from 48.8% to 14.0% with significant change during the experiment (P < 0.05). Although PI in the control was significantly decreased from 48.8% to 23.0% (P < 0.05), PI in the control remained around 21.6% to 25.1% after significant decrease between 10 and 20 days. For exposed groups, after 20 days, PI decreased to 18.3% for 50 ppb, 18.1% for 100 ppb and 14.0% for 200 ppb at 20 days and then increased to 21.0% for 50 ppb, 20.4% for 100 ppb and 19.1% for 200 ppb (P < 0.05). Exposure dependent PI was only observed at 10 day (P < 0.05). Although not being significant statistically, mean of phagocytosis was decreased as exposed level increased (Fig. 4).

[FIGURE 4 OMITTED]

Lysosomal Destabilization

Lysosomal destabilization ranged from 38.8% to 62.8% with significant changes during the experiment (P < 0.05). LD of each groups continuously increased during the experiment: 38.8% to 62.8% for control, 46.5% to 66.0% for 50 ppb, 44.0% to 66.0% for 100 ppb and 56.6% to 77.0% for 200 ppb. Although not being consistent with exposed level, LD increased as exposed level increased the experiment. In control, LD was steadily increased over the experimental day with significant difference at 40 days (P < 0.05). At every experimental day, LD in 200 ppb was significantly higher than those in the control (P < 0.05) (Fig. 5).

[FIGURE 5 OMITTED]

DISCUSSION

Organic contamination is a major threat to marine bivalve farms. A cultured bivalve is often affected by organic compounds through acute or chronic exposure. When exposed to PAHs, an organic contaminant, marine bivalves tend to accumulate and metabolize the compound, which results in toxicity to the host animal and stimulates the internal defense system.

Hemocytes play an essential role in the internal defense of bivalves (Stanber 1950, Cheng 1985, Cheng 1986). It has been reported that the immune system of a bivalve can be adversely affected by PAHs (Grundy et al. 1996). Hemocyte functions have been applied as a useful biomarker of the coastal environment (Dyrynda et al. 1998, Fisher et al. 2000). We investigated changes in hemocyte function of cultured oysters exposed to varying concentration of PAHs for 40 days. The PAH levels in this study were similar to concentrations found in coastal waters (Table 1).

The impact of xenobiotics on bivalve HC is still controversial. Sami et al. (1992) reported decreased numbers of hemocytes, which contrasted with Coles et al. (1994). The number of hemocytes is highly affected by various stresses (i.e., anthropogenic stress) (Coles et al. 1994, Coles et al. 1995), natural stress (Renwrantz 1990), mechanical stress (Lacoste et al. 2002) and seasonal change (Fisher et al. 1996), and it is reversible upon withdrawal of the stressor (Lacoste et al. 2002).

In this study, however, the control at 10 days did not differ from the initial number, but then decreased and remained constant. HC in control was significantly affected by laboratory holding with significant decrease at 20 days. Similar decreasing patterns were observed in the exposed group. At 40 days, no significant difference was observed between HC in the control and the exposed groups. The differences of HC between the control and the exposed groups, however, became negligible as the experiment proceeded. This implied that the exposure impact was high at the beginning of the exposure, and then might remain at a certain level as the physiologic adaptation progressed.

PMH and RHL were also significantly affected by PAH exposure, but did not exhibit a dose-response. The contaminant effect on hemocytes activities can still be disputed. Some field works have reported that hemocyte number and activities were positively associated with contaminated sites (metals and PAHs), but a few negative associations were also found (Oliver et al. 2001, Fisher et al. 2000). The elevated hemocyte activities might, therefore, require increased energy demand, but the retarded physiologic response and depletion of cellular ATP might not meet the energy demand. PAH interruption in ATP-related homeostasis might lead to suppression of hemocyte activities. It is known for oyster to represent different physiologic response depending on contaminant levels; in low level, bivalves tend to increase intake metabolism such as food and oxygen to facilitate the metabolism or elimination mechanism of contaminant (Gillfillan et al. 1976, Widdows et al. 1982, Hartwell et al. 1991, Toro et al. 2003). On the other hand, at high levels, they tend to reduce their metabolism to avoid an intrusion of the xenobiotics (Takayanagi et al. 2000). PAHs can also affect the physiologic parameters that are directly related to the nutrient status. Eertman et al. (1995) reported that PAHs significantly reduced the feeding rate of mussels in agreement with Toro et al. (2003) who worked with field samples contaminated with organochlorines and PAHs. They reported that the clearance rate, a major component of energy intake, is affected by toxic compounds (Howell et al. 1984, Bourdelin 1996). Further, they reported a significant negative correlation between contaminant and SFG (scope for growth). In another experiment, significant energy loss was found at 100 and 200 ppb with significantly decreased intake metabolisms and decreased absorption efficiency (Jeong et al. 2004). The decreased energy reservoir may influence hemocyte mobility, which is more apparent in PHM than in RHL because of greater variability in RHL. Since no oyster mortalities were observed and the oysters were fully fed 3 times a day with a same amount to each group, short of food supplies might be a negligible cause for decreased hemocyte functions. However, results from contaminated oysters by PAHs were different case by case, and there has been disagreement between field and laboratory results. This might be associated with tolerability of the animal.

In this study, although not clearly dose-dependent, PI was suppressed when oysters were exposed to PAHs (except at 40 days). Suppressed phagocytosis has been known to be associated with chemical exposure (Fries & Tripp 1980, Cheng & Sullivan 1984, Coles et al. 1995, Fisher et al. 2000). On the other hand, stimulated phagocytosis has also been noted after exposure to phenolic compounds and low doses of some heavy metals (Cheng & Sullivan 1984, Cheng 1988, Coles et al. 1995, Brousseau et al. 2000).

Lysosomal destabilization was highly associated with PAH levels and exposure day. Unlike previous parameters, lysosomal destabilization continuously increased with the duration of exposure. Intake of PAHs in bivalves is directly related to the level in the surrounding waters (Nott & Moore 1987) but, although some of them are metabolized, tend to be accumulated in organelles such as lysosomes. Lysosomal destabilization, therefore, is a useful biomarker for environmental monitoring and has been widely used in toxicity tests (Lowe et al. 1995a, Lowe et al. 1995b; Ringwood et al. 1999, Grundy et al. 1996).

Some controversial disagreements are still disputed about the effect of contaminants on hemocyte functions. Some field works have suggested that stimulated immuncompetence and hemocyte activities were highly associated with contaminated sites. In controlled experiments, however, those decreased with chemical exposure (Fries & Tripp 1980, Sami et al. 1992, Pipe & Coles 1995, Grundy et al. 1996) in agreement with our study, whereas some studies indicate stimulated activities at low contaminant concentrations (Fisher et al. 1990, Pipe et al. 1999). In the this study, hemocyte functions were initially suppressed and then re-elevated, which might be a sign of acclimation to the contaminated environment. PAHs have been also known to affect cellular-calcium homeostasis that have an influence on cellular signal pathway and deplete the cellular ATP content (Seagrave & Burchiel 2000). During phagocytosis, ATP falls dramatically (Densen & Mandell 1978).

When toxicant stress is applied, increased hemocyte function at the beginning may deplete the ATP reserves sufficiently to cause the declines in hemocyte function. As adaptation proceeded, hemocyte functions would be restored at a certain level, which might be significantly lower than those in unexposed.

In conclusion, hemocyte characteristics were significantly affected by PAH exposure and laboratory holding. Our result suggests that PMH and LD are useful biomarkers with sensitivity to this toxicant regardless of the effect of laboratory holding. However, HC and PI were significantly influenced by laboratory holding, and exposure effect was negligible at the end of the experiment.

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WOO-GEON JEONG AND SANG-MAN CHO *

Institute of Marine Industry & Department of Marine biology and Aquaculture, College of Marine Science, Gyeongsang National University, 445 Inpyeong, Tongyeong, 650-160, Korea

* Corresponding author: E-mail: [e-mail]

TABLE 1.
The summary of the PAH level in estuary and coast water.

Location                          N *   Range (ppb)

Chesapeake Bay, USA                17    20-65.7
Coast around England
  and Wales UK                     15     1-24,821
Rhode Island, USA                  26    ND-115,000
Seine River and Estuary, France    11     4-36
Winyah Bay, USA                    14    ND-49
Western Xiamen Sea, China          16   106-945

Location                                  Reference

Chesapeake Bay, USA               Gustafson & Dickhut (1997)
Coast around England
  and Wales UK                    Law et al. (1997)
Rhode Island, USA                 Reddy et al. (2001)
Seine River and Estuary, France   Fernandes et al. (1997)
Winyah Bay, USA                   Kucklick & Bidleman (1994)
Western Xiamen Sea, China         Zhou et al. (2000)

* Species number of PAHs analyzed.