Long-Term Pyrene Exposure of Grass Shrimp, Palaemonetes pugio, Affects Molting and Reproduction of Exposed Males and Offspring of Exposed Females.

The objective of this study was to investigate the impact of long-term pyrene exposure on molting and reproduction in the model estuarine invertebrate, the grass shrimp (Palaemonetes pugio). Grass shrimp were exposed to measured concentrations of 5.1, 15.0, and 63.4 ppb ([micro]g/L) pyrene for 6 weeks, during which time we determined molting and survivorship. At the end of the exposure, we immediately sacrificed some of the shrimp for biomarker (CYP1A and vitellin) analyses. The remaining shrimp were used to analyze fecundity and embryo survivorship during an additional 6 weeks after termination of pyrene exposure. Male shrimp at the highest pyrene dose (63 ppb) experienced a significant delay in molting and in time until reproduction, and showed elevated ethoxycoumarin o-deethylase (ECOD) activity immediately after the 6-week exposure period. In contrast, 63 ppb pyrene did not affect these parameters in female shrimp. Females produced the same number of eggs per body weight, with high egg viability (98-100%) at all exposure levels, but with decreased survival for the offspring of the 63-ppb pyrene-exposed females. In addition, vitellin levels were elevated only in females at 63 ppb pyrene after the 6-week exposure. We hypothesize that the elevated vitellin binds pyrene and keeps it biologically unavailable to adult females, resulting in maternal transfer of pyrene to the embryos. This would account for the lack of effect of pyrene exposure on ECOD activity, molting, and reproduction in the adult females, and for reduced survival of their offspring. Key words: cytochrome P450, molting, pyrene, reproduction, shrimp, vitellin. Environ Health Perspect 108:641-646 (2000). [Online 2 June 2000]

http://ehpnet1.niehs.nih.gov/docs/2000 /108p641-646oberdorster/abstract.html

Grass shrimp, Palaemonetes pugio, are a key link in the estuarine detritus food chain. Grass shrimp life history is well studied (1), and shrimp can be easily maintained in the laboratory, making them ideal and important model estuarine invertebrates. Molting and reproduction in grass shrimp are controlled by the molting hormone, ecdysone (2,3). A spike in ecdysone stimulates molting (4), and as with all steroids, ecdysone regulates gene and protein expression by interacting with its receptor, the ecdysone receptor (5). This includes the expression of ovarian egg yolk protein, vitellin (Vt). (6-8). Female grass shrimp molt, and must be mated within 7 hr of molting (1). Eggs are fertilized and extruded, then held on the pleopods of the female's abdomen until larvae are released 12-15 days later. The female molts again within a few days after spawning, and produces an additional brood. The breeding season varies with climate, but can be several months long in the estuaries of the southeastern United States. Larvae develop through a series of metamorphic molt stages and reach maturity within a few months to 1 year, depending on climate.

Estuaries often receive large amounts of anthropogenic contaminants, including polycyclic aromatic hydrocarbons (PAHs). In some areas, sediment concentrations [is greater than] 4 mg/g have been found, although most highly contaminated areas are in the range of 1-2 [micro]g/g (9-11). Water concentrations have been recorded in the nanogram-per-liter to microgram-per-liter range (10,12). In a controlled sediment-exposure study, pyrene water concentrations reached up to 18 [micro]g/L in the filtered water from sediments containing 2.25 [micro]g/g pyrene (12). In a separate study, grass shrimp were exposed to PAH-contaminated sediments including up to 2.1 [micro]g/g pyrene, and water levels of pyrene reached up to 165 [micro]g/L (10). However, in this study, water was not filtered and pyrene was most likely associated with suspended solids. Considering that pyrene levels can reach milligram-per-gram sediment levels in contaminated estuaries, it is reasonable to assume that pyrene concentrations in the field are in the microgram-per-liter range in the water column.

Biomarker induction, especially cytochrome P4501A (CYP1A) induction, has been well documented after the exposure of fish to PAHs (13). The induction of CYP1A by PAHs involves binding to the aryl-hydrocarbon receptor (AhR), which is translocated to the nucleus by the accessory protein, ARNT. The complex binds to xenobiotic response element in the promoter/enhancer region of the CYP1A gene, resulting in gene transcription and ultimately CYP1A protein induction (14). The ligand that best fits into the AhR is a 10 [Angstrom] x 3 [Angstrom] planar ring (15), such as found in the PAHs and polychlorinated biphenyls.

PAHs are able to induce CYP1A-like protein activity in crustaceans as well (16-23). However, the specificity of this inducibility is slightly different from that in vertebrates. For example, 3-methylcholanthrene (3MC) does not induce ethoxyresorufin o-deethylase activity or benzo[a]pyrene hydroxylase in lobster (17), whereas in vertebrates (reptiles, birds, fish, and mammals) 3MC induces a wide variety of P450s, specifically CYP1A (24-29).

In addition to interacting with AhR, PAHs can interact with the vertebrate steroid hormone systems by acting as antiestrogens in reporter gene assays and MCF-7 breast cancer cell proliferation assays, and as antiandrogens in whole animals (30-32). PAHs are also able to interact with the invertebrate ecdysteroid hormone system (33). Benzo[a]pyrene, pyrene, chrysene, and benzo[b]fluoranthene enhance ecdysone-dependent reporter gene expression and cell differentiation.

Several studies have been done on whole animals to investigate physiologic responses to PAH exposure in crustaceans (34-36). These studies showed that molting patterns are altered in crabs exposed to crude oil (35). Tanner crabs exposed to 0.56 mL crude oil per liter seawater autotomized (spontaneously lost) several limbs. Autotomy of limbs stimulates ecdysis (molt), and it is difficult to assess whether changes in molt are due to this physiologic response or to PAH effects. At 0.32 mL/L, molting success was increased, and crabs closest to molt were resistant to acute toxicity. Crabs exposed to PAHs during molt were less able to metabolize PAHs, presumably because PAHs are competitive substrates for some of the P450s that also metabolize 20-OH ecdysone to its inactive form (34,37,38). It could be argued that PAHs in molting crabs would compete with and interfere with normal molting due to interaction with the metabolism of ecdysone.

In a study using sediments from the Elizabeth River in Virginia, a PAH mixture was used that included pyrene levels up to 2 [micro]g/g sediment and over 160 [micro]g/L (10). At these levels, there was a slight increase in mortality from 5% in controls to 12% in exposed shrimp after 96 hr. Also, there was a slight decrease in respiration rates in shrimp exposed to the contaminated sediment.

In blue crab, PAHs are taken up primarily into the hepatopancreas, where they are metabolized and eliminated (20,39). During PAH metabolism, reactive intermediates are often produced, which lead to toxicity. Organic contaminants (dinitrochlorobenzene, hexachlorbiphenyl, and organotins) are not only distributed to the hepatopancreas for metabolism, but are also bound by Vt (the protein that serves the nutritional needs of the embryo before feeding begins). Up to 6% of the hepatopancreas burden of these contaminants was ultimately transported into the ovaries and oocytes after 6 days (40). Considering that PAHs are extremely genotoxic, this could have serious implications for the developing embryo. In fish, Vt can bind both dioxin and benzo[a]pyrene (41). This may be one mechanism by which organic contaminants can become biologically unavailable for metabolism to reactive intermediates, thereby mitigating toxicity to the adult animal, but promoting toxicity in their offspring.

The objective of this study was to investigate the impact of long-term exposure of pyrene on molting and reproduction in the model estuarine invertebrate, the grass shrimp. Because several PAHs, including pyrene, are able to interact with ecdysone-dependent gene transcription and cell proliferation in vitro (33), we hypothesized that pyrene will interfere with normal growth, reproduction, and Vt production of grass shrimp in vivo.

Materials and Methods

We collected grass shrimp, Palaemonetes pugio, near Ocean Springs, Mississippi, and held them at the Institute of Marine Science aquatic facility (Ocean Springs, MS) for 6 months before use in the exposures. Shrimp for all treatments were maintained in flowthrough condition at 27 [degrees] C at 15 ppt well-aerated seawater and fed ad libitum with brine shrimp once daily and commercial flake food once daily. Seawater was transported from the U.S. Environmental Protection Agency Environmental Research Laboratory in Gulf Breeze, Florida, adjusted to 15 ppt salinity with nonchlorinated well water, and filtered before use. Female shrimp were separated when identified (gravid), and held separately until use.

The 6-week exposure of shrimp to pyrene was done using the methods of Walker et al. (42). We used 12 test aquaria with duplicates of three exposure levels of pyrene (10, 25, and 100 ppb) plus 2 aquaria for seawater control and 4 aquaria for solvent control [triethylene glycol (TEG)]. Four aquaria of the solvent control were used because the controlled reproductive studies required a large number of TEG (solvent)-control shrimp. Aquaria were housed within a central water bath to maintain tank temperature at 27 [degrees] C. TEG concentrations in solvent controls and all treatments were kept below 0.1 mL/L seawater. We chose these levels of pyrene from preliminary static exposure 48-hr range-find assays, median lethal concentration studies from PAH sediment-exposure studies in grass shrimp, and from water concentrations reported in the literature (9,10,12). In the range-find assay, larval shrimp ([is less than] 1 month of age) were exposed to nominal concentrations of 1, 10, 100, or 1,000 ppb pyrene for 48 hr. After 48 hr, there was 60% mortality at 1 ppm and 20% mortality at 100 ppb. From the literature, a mixture of PAHs including pyrene levels of up to 165 [micro]g/L led to 5-12% mortality after 96 hr in adult grass shrimp (10).

Before the start of the exposures, the flowthrough system was allowed to operate at the selected pyrene concentrations for approximately 1 week. We measured the pyrene concentrations in the aquaria daily by HPLC [Beckman Gold System (Beckman Coulter, Inc., Fullerton, CA) with 4.6 mm x 25 cm C18 reverse-phase column eluted with 90% acetonitrile at 0.8 mL/min] with fluorometric detector [Jasco FP-920 fluorescence detector (Jasco, Inc., Easton, MD) with excitation at 235 nm and emission at 390 nm]. Pyrene concentrations (mean [+ or -] SE) were 5.1 [+ or -] 0.5, 15.0 [+ or -] 0.7, and 63.4 [+ or -] 2.5 ppb.

We exposed 50 shrimp, 25 in each of two replicate aquaria per treatment; each shrimp was housed in an individual mesh netting cage within the aquaria. This compartmentalization of test organisms precluded cannibalism, isolated males from females to avoid premature mating, and enabled us to enumerate individual molt data. We used a 16-hr light/8-hr dark photoperiod. Shrimp were checked daily for molting and survivorship. Water quality (pH, salinity, temperature, and dissolved oxygen) and pyrene levels were checked twice weekly. The average pH was 8.25 [+ or -] 0.2; salinity was 15 [+ or -] 0.4 ppt; temperature was 27.1 [+ or -] 0.3 [degrees] C; and dissolved oxygen was 7.2 [+ or -] 1.8 mg/L (ppm; 100% saturation at these conditions is 7.2 ppm). There were no differences between any of the tanks for any of these parameters.

After the 42-day exposure, a reproductive phase was initiated for an additional 6 weeks (42 days) by setting up controlled matings of treatment and control shrimp. This phase of the study was carried out in clean seawater using the following scenario: exposed males x exposed females; TEG males x exposed females; exposed males x TEG females; and seawater control males x seawater control females. Because of mortalities in the 15- and 63-ppb exposure group and 11 instances of misidentifications of sexes (e.g., female-female pairs), between 4 and 10 male-female pairs were ultimately used in the mating studies (Table 1). Pairs were checked daily for egg production, and were sacrificed after the female was gravid for a minimum of 2 days. The thorax, including hepatopancreas and gonads, was frozen at -80 [degrees] C in 200 [micro]L storage buffer (100 mM [K.sub.2][HPO.sub.4], pH 7.4, 1 mM DTT, 1 mM EDTA, 20% glycerol and aprotinin at 0.67 trypsin inhibitor units/mL). We collected, counted, and determined the viability of the eggs. A subset of 20 viable eggs/female was incubated individually in 4 mL sterile seawater/plate in 24-well polystyrene culture plates. Plates were shaken on an orbital shaker at 60 rpm at 27 [+ or -] 1 [degrees] C. Percent embryo survival was determined by successful hatch by day 10 postisolation. Animals not used in the mating studies were sacrificed immediately after the 6-week exposure period, and hepatopancreas and abdomens were collected as described previously.

Table 1. Numbers of pairs used in the controlled mating study.

                            Male

                                    Pyrene

Female     Seawater   TEG   5 ppb   15 ppb   63 ppb

Seawater      8
TEG           -        6      8       9        5
Pyrene
  5 ppb       -        10     6       -        -
  15 ppb      -        8      -       8        -
  63 ppb      -        5      -       -        4

As a measure of CYP1A-like protein induction, we measured ethoxycoumarin O-deethylase (ECOD) activity in crude hepatopancreas extract. Tissues from the three collection times were used: immediately after the exposure phase; as soon as eggs were produced; and, if no eggs were produced, immediately at the end of the reproductive phase. Hepatopancreas/thorax was homogenized with a Teflon homogenizer in storage buffer and centrifuged at 14,000 rpm at 4 [degrees] C in a microcentrifuge for 15 min. We used the supernatant in the ECOD assay. Protein was measured via the Bradford method (43). A 96-well plate assay was developed for the ECOD assay using the Spectramax Gemini plate reader (Molecular Devices Corp., Sunnyvale, CA) set at 380 nm excitation and 448 nm emission. Preliminary studies showed that metabolism was linear for at least 30 min at 29 [degrees] C. A standard curve of hydroxycoumarin from 0 to 500 pM was used in incubation buffer (50 mM Tris, pH 7.6, 2 mM 7-ethoxycoumarin). We incubated 140 [micro]L incubation buffer and 10 [micro]L supernatant proteins for 5 min at 29 [degrees] C. We added 3 [micro]L NADPH (5 mM) to each well and measured fluorescence immediately for 30 min at 45 sec intervals. [V.sub.max] values were calculated and relative fluorescence units were converted to picomoles coumarin. Final numbers are reported as picomoles coumarin per minute per milligram protein. Each sample was measured in triplicate.

Dot-blots for Vt were done using the crude hepatopancreas/thorax homogenates that also contained gonads. The polyvinylidene difluoride (PVDF) (Millipore, Inc., Bedford, MA) membrane was wetted in methanol, rinsed in water for 5 min, and used in the BioRad dot-blot apparatus (BioRad Laboratories, Hercules, CA). Two rinses of 100 [micro]L phosphate-buffered saline (PBS) were done under mild vacuum, and samples were applied to each well afterward. A standard curve using partially purified Vt from grass shrimp eggs (10-160 [micro]g protein) was used to quantify Vt from samples from all phases of the study. Ten microliters of homogenates in triplicate were applied to each well of the BioRad dot-blot after heating in 2.5 [micro]L sample buffer [0.8 M Tris-HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 5% [Beta]-mercaptoethanol, and 0.05% w/v bromophenol blue] at 95 [degrees] C for 5 min. We added an additional 50 [micro]L PBS to each well, and samples were allowed to gravity filter through the unit for 2 hr at room temperature. The membrane was blocked overnight in PBS plus 3% bovine serum albumin at 4 [degrees] C, rinsed 4 times with PBS plus 0.05% Tween 20 (PBST), incubated for 2 hr at room temperature with a 1:3 dilution of S-15-2 monoclonal anti-Vt antibody (44), rinsed 4 more times with PBST, and incubated 1 hr at room temperature with 1:50,000 dilution of goat-antimouse IgG coupled to horseradish peroxidase secondary antibody. Dots were visualized using the enhanced chemiluminescence kit from Amersham (Pharmacia Biotech, Inc., Piscataway, NJ), and multiple exposures of X-ray film were taken. The films were developed using a Mini-Med film processor (AFP Imaging Corporation, Elmsford, NY), and were analyzed using the BioRad gel documentation system. Western blot analysis was used to ascertain that increased density of the dot blots was indeed due to elevated levels of Vt. To this end, a subset of homogenates (5 [micro]g each) was subjected to separation on a 6% SDS-PAGE gel and transferred to PVDF membrane for 6 hr at 60V and blotted as described previously.

Statistical analysis was done using SYSTAT version 8.0 (SPSS, Inc., Chicago, IL) for the personal computer. Because some ECOD and embryo mortality data were non-normal, the data were square-root transformed (ECOD) or arcsine square-root transformed (percent embryo mortality) using the equation

[x.sup.1] = [square root of (x + 0.5)] or [p.sup.1] = arcsin([square root of p])

(45). If outliers still remained (Durbin-Watson t-statistic), they were removed from the data set. Analysis of variance (ANOVA) was run on data from all assays, and if p [is less than] 0.05, the post hoc Tukey test was used to determine which groups were different. If data had to be transformed to meet normality requirements, ANOVA was done on transformed data. Linear regression analyses of embryo mortality were done on percentages, not on transformed data.

Results

Molting was affected only in males at the highest exposure level (Table 2). Males exposed to 63 ppb pyrene had fewer molts during the 6-week exposure period than males at other exposure levels. There was no effect of pyrene on molting in females. At 63 ppb pyrene, higher mortality occurred in both males and females (Table 2). An interesting observation was that deaths of the 63-ppb exposure group occurred at the time of, or in close proximity to, molt--shrimp were found dead while only partially or newly molted. The average day of death ([+ or -] SE) for the males in the 63-ppb group was day 25.6 [+ or -] 3.5 (range 7-42); for females it was 29.5 [+ or -] 1.7 (range 23-41) during the exposure phase of the study. Deaths occurred more frequently during the second half of the exposure study. The average number of molts before death for males was 2.4 [+ or-] 0.5 and for females was 3.3 [+ or -] 0.4. These animals were not used to calculate the number of molts per exposure group in Table 2. An additional two females in the 10-ppb and one female in the 63-ppb dose groups died during the following 42-day reproductive study in clean seawater.

Table 2. Number of molts and percent survivorship of grass shrimp during 42 days of pyrene exposure.

Sex,                 Survivorship       Molts per
treatment    No.         (%)            shrimp(a)

Male
  Control    24          96         4.29 [+ or -] 0.25
  TEG        49          98         3.77 [+ or -] 0.21
  Pyrene
    5 ppb    23(b)       96         3.70 [+ or -] 0.24
    15 ppb   25         100         3.60 [+ or -] 0.22
    63 ppb   14          56(*)      2.86 [+ or -] 0.46(*)
Female
  Control    25         100         4.16 [+ or -] 0.24
  TEG        50         100         4.24 [+ or -] 0.20
  Pyrene
    5 ppb    25         100         3.96 [+ or -] 0.25
    15 ppb   24          96         3.58 [+ or -] 0.21
    63 ppb   15          60(*)      4.00 [+ or -] 0.29

(a) Molts per shrimp are calculated using only those shrimp surviving to day 43.

(b) Started with 24 shrimp.

(*) p < 0.05.

Time until successful mating (as evidenced by gravid females) was significantly delayed in males, but not in females (Figure 1). In both groups where 63-ppb pyrene-exposed males were mated with either control females or exposed females, the time until the first brood was delayed until 24 or 14 days, respectively. Control and low-dose males were able to produce a brood after 4-7 days. By the end of the 42-day reproductive period in clean seawater, the males had recovered and there was no difference in the percent of pairs producing a brood.

[Figure 1 ILLUSTRATION OMITTED]

Females were able to reproduce at normal levels even after exposure to the highest dose (63 ppb) pyrene (Figure 1). At all exposure levels in all groups, when females produced eggs, the number of eggs/female body weight did not differ between any exposure groups, and the percent viable eggs was not different, ranging from 98 to 100% viability (Table 3). However, there was a significant increase in embryo mortality in pyrene-exposed females at 63 ppb when mated with both control and exposed males (Figure 2). In addition, linear regression analysis showed a significant increase in mortality with increasing pyrene concentrations for both mating groups (p [is less than] 0.01 for exposed males x exposed females, slope = 0.24; p [is less than] 0.002 for TEG males x exposed females; slope = 0.66).

[Figure 2 ILLUSTRATION OMITTED]

Table 3. Number of eggs per milligram female weight and percent viability.

                             Male

Female          Seawater                TEG

Seawater   0.53 [+ or -] 0.12           -
                 99.1%
TEG                -            0.70 [+ or -] 0.07
                                       100%
Pyrene
  5 ppb            -            0.62 [+ or -] 0.05
                                       100%
  15 ppb           -            0.68 [+ or -] 0.07
                                        99.9%
  63 ppb           -            0.71 [+ or -] 0.09
                                       100%

                             Male

                             Pyrene

Female            5 ppb               15 ppb

Seawater           -                    -

TEG        0.66 [+ or -] 0.06   0.59 [+ or -] 0.08
                  100%                99.5%
Pyrene
  5 ppb    0.63 [+ or -] 0.06           -
                   99.6%
  15 ppb           -            0.71 [+ or -] 0.11
                                      99.5%
  63 ppb           -                    -

                  Male

                 Pyrene

Female           63 ppb

Seawater           -

TEG        0.63 [+ or -] 0.07
                   98.3%
Pyrene
  5 ppb            -

  15 ppb           -

  63 ppb   0.74 [+ or -] 0.14
                  100%

ECOD activity was highly variable within exposure groups (Table 4). At the end of the exposure phase, there was significant elevation of ECOD activity for males in the 63-ppb exposure group when compared to all other exposures. There was no significant induction in females after the exposure phase, and there was no significant induction of ECOD at any time during the reproductive phase of the study in either males or females (Table 4).

Table 4. ECOD activity (pmol/min/100 [micro]g protein [+ or -] SE) in grass shrimp hepatopancrei.

ECOD activity              Males, (n)

Postexposure
  Control             3.39 [+ or -] 1.86 (5)
  TEG control         5.46 [+ or -] 2.57 (5)
  Pyrene
    5 ppb            13.38 [+ or -] 4.81 (5)
    15 ppb            4.35 [+ or -] 1.78 (5)
    63 ppb           42.25 [+ or -]8.91(*,**)(4)
Reproductive
  Control             8.00 [+ or -] 3.13 (6)
  TEG control         7.93 [+ or -] 2.24 (22)
  Pyrene
    5 ppb            12.64 [+ or -] 3.50 (11)
    15 ppb            2.33 [+ or -] 1.28 (10)
    63 ppb            9.73 [+ or -] 3.46 (7)
Nonreproductive(a)
  Control             3.84 [+ or -] 2.89 (4)
  TEG control         8.02 [+ or -] 2.40 (14)
  Pyrene
    5 ppb             7.86 [+ or -] 5.52 (5)
    15 ppb            3.84 [+ or -] 0.72 (9)
    63 ppb           12.99 [+ or -] 6.98 (3)

ECOD activity              Females, (n)

Postexposure
  Control            27.51 [+ or -] 12.66 (5)
  TEG control         3.56 [+ or -] 1.45 (5)
  Pyrene
    5 ppb            10.08 [+ or -] 5.42 (5)
    15 ppb            7.22 [+ or -] 3.98 (4)
    63 ppb           13.16 [+ or -] 4.27 (5)
Reproductive
  Control            13.63 [+ or -] 3.58 (5)
  TEG control        11.87 [+ or -] 2.67 (21)
  Pyrene
    5 ppb            17.46 [+ or -] 3.14 (10)
    15 ppb           11.58 [+ or -] 3.26 (12)
    63 ppb           13.12 [+ or -] 2.90 (7)
Nonreproductive(a)
  Control             3.43 [+ or -] 0.61 (4)
  TEG control         5.45 [+ or -] 1.71 (11)
  Pyrene
    5 ppb             5.04 [+ or -] 1.54 (8)
    15 ppb            6.81 [+ or -] 3.22 (7)
    63 ppb           13.72 [+ or -] 5.32 (2)

(a) Animals that did not reproduce during the reproductive phase.

(*) p < 0.001 from control, TEG, and 15 ppb.

(**) p < 0.03 from 5 ppb.

The percent of protein as Vt identified by dot blots was significantly elevated in females after exposure to 63 ppb pyrene for 6 weeks (Figure 3). Western blot analysis of a subset of four females confirmed these results. Vt levels were not different from control or solvent control levels at any time in males or in all females that had just reproduced or that had not reproduced after 6 weeks. There was a high variability in Vt levels in both males and females. To further examine the somewhat unexpected presence of Vt in males, we used Western blotting to analyze a subset of six males that showed Vt via dot blots. In three of these we found a single band that corresponded to the molecular weight of the Vt standard, whereas the other three did not contain Vt. There appears to be material in homogenates from some of the males that cross-react with the anti-Vt antibodies. The nature of this cross-reactivity is not clear, but is under further investigation. Similar Vt cross-reactivity has been shown in juvenile and male prawn (46). It is clear, however, that consistently low levels of apparent Vt in males do not change with pyrene exposure.

[Figure 3 ILLUSTRATION OMITTED]

Discussion

Grass shrimp are a key link in the estuarine detritus food chain. However, many of our estuaries are impacted by anthropogenic contaminants that can adversely impact this ecologically important resource. Because grass shrimp life history is well studied (1), shrimp can be easily maintained in the laboratory, making them an ideal and important model estuarine invertebrate.

In our study, grass shrimp were exposed to 5, 15, and 63 ppb pyrene, which is similar to what has been measured in the water column in both the field and in laboratory sediment toxicity testing (10,12). Mortality was high (56% male and 60% female) at the 63-ppb dose, with shrimp dying just before or during the molt. In a study with blue crabs, animals were unable to metabolize and eliminate PAHs the closer they were to molt, resulting in a higher body burden (34). Mothershead and Hale (34) suggested that this was due to competition for substrates by P450s necessary to metabolize ecdysone for the molt. Extrapolating to this current study with a chronic pyrene exposure to shrimp, this increased PAH uptake could lead to increasingly higher body burdens of pyrene with each successive molt, eventually leading to increased mortality. In support of this hypothesis, mortality occurred primarily in the second half of the exposure study after an average of two to three molts.

The exposure of grass shrimp to pyrene resulted in significant effects on male shrimp. At the highest pyrene dose (63 ppb), males had a significantly decreased number of molts, elevated ECOD activity, and delay in mating success. Male shrimp sacrificed immediately after they mated showed that ECOD activity had returned to control levels. ECOD activity is an indicator of pyrene exposure, and it appears that once males were able to depurate enough pyrene, they were able to successfully mate.

In contrast to males, female molting, ECOD activity, and reproduction were not affected by any of these doses of pyrene. However, there is a significant induction of Vt levels at 63 ppb pyrene exposure in surviving females. Pyrene is able to increase ecdysone-dependent gene expression in vitro (33). Because the active ecdysteroid, 20-hydroxyecdysone (20HE), regulates protein expression via ecdysteroid-dependent gene expression and controls vitellogenesis (4,8), this may be the mechanism by which Vt levels are elevated in pyrene-treated shrimp. It is possible that Vt binds pyrene, resulting in reduced production of toxic reactive intermediates of PAH metabolism. This may explain the lack of adverse effects of pyrene on adult females.

Our hypothesis further predicts that Vt may serve as a vehicle for the transport of pyrene from the mother to the oocytes of embryos. Studies in blue crab and fish showed that egg yolk proteins are able to bind several classes of organic contaminants and transfer them to the oocytes (40,41). This mechanism is supported by our observed pyrene-dependent decrease in embryo survival. Interestingly, exposed females mated with solvent-control males had a steeper slope in the regression analysis of embryo mortality than exposed females mated with exposed males. Successful mating of exposed males was significantly delayed, which may explain the lower slope for this group. The delay would allow for females to depurate pyrene for a longer period of time, leading to decreased pyrene in the oocytes, and less severe effects on the developing embryo, as compared to exposed females who immediately produced a brood after mating with solvent-control males.

Conclusions

This study shows that there may be a link between molting, reproduction, Vt levels, and P450 activity in shrimp. Males, but not females, are significantly delayed in molting and reproduction after exposure to environmentally relevant levels of pyrene. In the field, where exposure is continuous, this would lead to the impairment of reproductive function of male shrimp. In males, ECOD activity was significantly elevated immediately after the 6-week exposure, but in females ECOD activity was not elevated. There was no change in molting pattern or reproduction in females, but high-dose females had increased Vt levels. Our study suggests that embryo mortality would be higher in contaminated areas than in clean areas possibly became of Vt-mediated transport of PAHs from the adult to the embryos. Finally, our study shows that high pyrene concentrations can be lethal to adult shrimp after a series of molts. Taken together, these effects of PAHs may result in a reduction of the grass shrimp population, and hence decreased food availability for animals dependent on grass shrimp for prey, ultimately resulting in effects at the community and ecosystem level.

REFERENCES AND NOTES

(1.) Anderson G. Species profiles: life histories and environmental requirements of coastal fishes and invertebrates (Gulf of Mexico): grass shrimp. In: U.S. Fish and Wildlife Service Biological Report, Vol. 82(11.35). Lafayette, LA:National Wetlands Research Center, 1985.

(2.) Freeman JA, Bartell CK. Some effects of the molt-inhibiting hormone and 20-hydroxyecdysone upon molting in the grass shrimp, Palaemonetes pugio. Gen Comp Endocrinol 28:131-142 (1976).

(3.) Chang ES. Comparative endocrinology of molting and reproduction: insects and crustaceans. Annu Rev Entomol 38:161-180 (1993).

(4.) Chan SM. Possible roles of 20-hydroxyecdysone in the control of ovary maturation in the white shrimp Penaeus vannamei (Crustacea, Decapoda). Comp Biochem Physiol 112C:51-59 (1995).

(5.) Yao T, Forman MB, Jlang Z, Cherbas L, Chen JD, McKeown M, Cherbas P, Evans M. Functional ecdysone receptor is the product of EcR and ultraspiracle genes. Nature 366:476-479 (1993).

(6.) Wilder MN, Okumura T, Aida K. Accumulation of ovarian ecdysteroids in synchronization with gonadal development in the giant freshwater prawn, Macrobrachium rosenbergii. Zool Sci 8:919-927 (1991).

(7.) Young NJ, Webster SG, Rees HH. Ovarian and hemolymph ecdysteroid titers during vitellogenesis in Macrobranchium rosenbergii. Gen Comp Endocrinol 90:183-191 (1993).

(8.) Young NJ, Webster SG, Rees HH. Ecdysteroid profiles and vitellogenesis in Penaeus monodon (Crustacea: Decapoda). Invertebr Reprod Dev 24:107-118 (1993).

(9.) Oberdorster E, Martin M, Ide CF, McLachlan JA. Benthic community structure and biomarker induction in grass shrimp in an estuarine system. Arch Environ Contam Toxicol 157:152-158 (1999).

(10.) Alden RW III, Butt AJ. Statistical classification of the toxicity and polynuclear aromatic hydrocarbon contamination of sediments from a highly industrialized seaport. Environ Toxicol Chem 6:673-684 (1987).

(11.) Long ER, Macdonald DD, Smith SL, Calder FD. Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments. Environ Manag 19:81-97 (1995).

(12.) Djomo JE, Garrigues P, Narbonne JF. Uptake and depuration of polycyclic aromatic hydrocarbons from sediment by the zebrafish (Brachydanio rerio). Environ Toxicol Chem 15:1177-1181 (1996).

(13.) Stegeman JJ, Brouwer M, DiGiulio RT, Forlin L, Fowler BA, Sanders BM, VanVeld PA. Molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure and effect. In: Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress (Huggett RJ, Kimerle RA, Mehrle PMJ, Bergman HL, eds). Ann Arbor, MI:Lewis Publishers, 1992;235-335.

(14.) Whitlock JP Jr, Denison MS. Induction of cytochrome P450 enzymes that metabolize xenobiotics. In: Cytochrome P450: Structure, Mechanism, and Biochemistry (deMontellano PO, ed). New York:Plenum Press, 1995;367-389.

(15.) Poland A, Knutson JC. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22:517-554 (1982).

(16.) James MO. Cytochrome P450 monooxygenases in crustaceans. Xenobiotica 19:1063-1076 (1989).

(17.) James MO, Little PJ. 3-Methylcholanthrene does not induce in vitro xenobiotic metabolism in spiny lobster hepatopancreas, or affect in vivo disposition of benzo[a]pyrene. Comp Biochem Physiol 78C:241-245 (1984).

(18.) James MO. Catalytic properties of cytochrome p-450 in hepatopancreas of the spiny lobster, Panulirus argus. Mar Environ Res 14:1-11 (1984).

(19.) James MO, Boyle SM. Cytochromes P450 in crustacea. Comp Biochem Physiol 121C:157-172 (1998).

(20.) Lee RF. Metabolism and accumulation of xenobiotics within hepatopancreas cells of the blue crab, Callinectes sapidus. Mar Environ Res 28:93-97 (1989).

(21.) O'Hara SCM, Corner EDS, Forsberg TEV. Studies on benzo[a]pyrene mono-oxygenase in the shore crab Carcinus maenas. J Mar Biol Assoc UK 62:339-357 (1982).

(22.) Sparagano O, Sage T, Fossi MC, Lari L, Child P, Savva D, Depledge N, Bamber S, Walker C. Induction of a putative monooxygenase of crabs (Carcinus spp.) by polycyclic aromatic hydrocarbons. Biomarkers 4:203-213 (1999).

(23.) Singer SC, March PE Jr, Gonsoulin F, Lee RF. Mixed function oxygenase activity in the blue crab, Callinectes sapidus: characterization of enzyme activity from stomach tissue. Comp Biochem Physiol 65C:129-134 (1980).

(24.) Greenlee WF, Poland A. An improved assay of 7-ethoxycoumarin O-deethylase activity: induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Pharmacol Exper Ther 205:596-605 (1978).

(25.) Ertl RP, Stegeman JJ, Winston GW. Induction time course of cytochromes P450 by phenobarbital and 3-methylcholanthrene pretreatment in liver microsomes of Alligator mississippiensis. Biochem Pharmacol 55:1513-1521 (1998).

(26.) Bani MH, Fukuhara M, Kimura M, Ushio F. Modulation of snake hepatic cytochrome P450 by 2-methylcholanthrene and phenobarbital. Comp Biochem Physiol 119C:143-148 (1998).

(27.) Leaver MJ, Pirrit L, George SG. Cytochrome P450 1A1 cDNA from plaice (Pleuronectes platessa) and induction of P450 1A1 mRNA in various tissues by 3-methylcholanthrene and isosafrole. Mol Mar Biol Biotechnol 2:338-345 (1993).

(28.) Ueng YF, Ueng TH. Induction and purification of cytochrome P450 1A1 from 3-methylcholanthrene-treated tilapia, Orechromis niloticus x Orechromis aureus. Arch Biochem Biophys 322:347-356 (1995).

(29.) Lin S, Bullock PL, Addison RF, Bandiera SM. Detection of cytochrome P4501A in several species using antibody against a synthetic peptide derived from rainbow trout cytochrome P4501A1. Environ Toxicol Chem 17:439-445 (1998).

(30.) Dorfman RI, Dorfman AS. A test for anti-androgens. Acta Endocrinol 33:308-316 (1960).

(31.) Arcaro KF, O'Keefe PW, Yang Y, Clayton W, Gierthy JF. Antiestrogenicity of environmental polycyclic aromatic hydrocarbons in human breast cancer cells. Toxicology 133:115-127 (1999).

(32.) Tran DQ, Ide CF, McLachlan JA, Arnold SF. The antiestrogenic activity of selected polynuclear aromatic hydrocarbons in yeast expressing human estrogen receptor. Biochem Biophys Res Commun 229:102-108 (1998).

(33.) Oberdorster E, Wilmot FA, Cottam DM, Milner MJ, McLachlan JA. Interaction of PAHs and PCBs with ecdysone-dependent gene expression and cell proliferation. Toxicol Appl Pharmacol 180:101-108 (1999).

(34.) Mothershead RF, Hale RC. Influence of ecdysis on the accumulation of polycyclic aromatic hydrocarbons in field exposed blue crabs (Callinectes sapidus). Mar Environ Res 33:145-156 (1992).

(35.) Karinen JF, Rice SD. Effects of Purdhoe Bay crude oil on molting Tanner crabs, Chionecetes bairdi. Mar Fish Rev 36:31-37 (1974).

(36.) Flowers GC, Suhayda JN, Clymire JW, McPherson GL, Koplitz LC, Poirrier MA. Impact of industrial effluent diversion on Bayou Trepagnier, Louisiana. In: Quarterly Report for the U.S. DOE. New Orleans, LA:Tulane University, 1997;1-52.

(37.) Singer SC, Lee RF. Mixed function oxygenase activity in blue crab, Callinectes sapidus: tissue distribution and correlation with changes during molting and development. Biol Bull 153:377-386 (1977).

(38.) 0'Hara SCM, Neal AC, Corner EDS, Pulsford AL. interrelationships of cholesterol and hydrocarbon metabolism in the shore crab, Carcinus. J Mar Biol Assoc UK 65:113-131 (1985).

(39.) Lee RF, Neuhauser ML. Fate of petroleum hydrocarbons taken up from food and water by the blue crab, Callinectes sapidus. Mar Biol 37:363-370 (1976).

(40.) Lee RF. Passage of xenobiotics and their metabolites from hepatopancreas into ovary and oocytes of blue crabs, Callinectes sapidus: possible implications for vitellogenesis. Mar Environ Res 35:1-2 (1993).

(41.) Monteverdi G, DiGiulio RT. In vitro and in vivo association of 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(a)pyrene with the yolk-precursor protein vitellogenin. Environ Toxicol Chem (in press).

(42.) Walker WW, Overstreet RM, Manning CS, Hawkins WE. Development of aquarium fish models for environmental carcinogenesis: an intermittent-flow exposure system for volatile, hydrophobic chemicals. J Appl Toxicol 5:255-260 (1985).

(43.) Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 77:248-254 (1976).

(44.) Oberdorster E, Rice CD, Irwin LK. Unpublished data.

(45.) Zar JH. Data transformation. In: Biostatistical Analysis. 3rd ed. Upper Saddle River, NJ:Prentice Hall, 1996;279-284.

(46.) Wilder MN, Okumura T, Suzuki Y, Fusetani N, Aida K. Vitellogenin production induced by eyestalk ablation in juvenile giant freshwater prawn Macrobrachium rosenbergii and trial methyl farnesoate administration. Zool Sci 11:45-53 (1994).

Eva Oberdorster,(1) Marius Brouwer,(2) Thea Hoexum-Brouwer,(2) Steve Manning,(2) and John A. McLachlan(3,4)

(1) Department of Environmental Toxicology, Clemson University, Pendleton, South Carolina, USA; (2) Institute of Marine Sciences, University of Southern Mississippi, Ocean Springs, Mississippi, USA; (3) Tulane/Xavier Center for Bioenvironmental Research, New Orleans, Louisiana, USA; (4) Department of Pharmacology, Tulane University, New Orleans, Louisiana, USA

Address correspondence to E. Oberdorster, Department of Environmental Toxicology, 509 Westinghouse Road, Box 709, Pendleton, SC 29670 USA. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

This work was supported by a Department of Energy grant, Environmental Management Science Program.

Received 22 December 1999; accepted 8 March 2000.