Akira Kubota

Hisato Iwata
email: iwatah@agr.ehime-u.ac.jp

Eun-Young Kim

Catalytic function of avian cytochrome P450 1A4 and 1A5 (CYP1A4 and CYP1A5) enzymes heterologously expressed using in vitro yeast system

Keywords: CYP1A4, CYP1A5, Birds, Heterologous expression, EROD, Biomarker, 2,3,7,8-tetrachlorodibenzofuran

The present study clarifies the enzymatic properties of two avian cytochrome P4501A (CYP1A) paralogs, CYP1A4 and 1A5, using a yeast-based vector system.

Recombinant CYP1A4 and 1A5 proteins from common cormorant (Phalacrocorax carbo) were expressed in yeast cells, and showed typical reduced CO-difference spectra with a peak at 446 nm. Kinetic analysis of O-dealkylase of methoxy-, ethoxy-, pentoxy-and benzyloxyresorufin catalyzed by the CYP1A enzymes revealed that V max value for ethoxyresorufin O-deethylase (EROD) activity was much higher than that for the other three O-dealkylase activities for both isozymes. Interestingly, remarkable substrate specificity of the CYP1As was observed for O-dealkylation of benzyloxyresorufin and methoxyresorufin; CYP1A4 was highly specific for catalyzing benzyloxyresorufin O-debenzylase activity, whereas CYP1A5 was more efficient in catalyzing methoxyresorufin O-demethylase activity.

The present study also measured CYP1A-dependent EROD activity in the presence of 2,3,7,8-tetrachlorodibenzofuran (TCDF) to evaluate the ability of this dioxin-like congener to inhibit the EROD activity. One hundred nM TCDF noncompetitively inhibited CYP1A5-dependent EROD activity, although no inhibitory effect was detected for CYP1A4-dependent EROD activity. These results indicate that the avian CYP1A paralogs have different affinities for substrate and inhibitor, thus suggesting their distinct physiological and toxicological roles.

known that the induced CYP1A is involved in the disruption of the signaling pathway regulated by endogenous substrates and the production of reactive oxygen species, thus indicating that CYP1A induction by xenobiotics is linked with the toxic effects of environmental contaminants.

Our recent study (Kubota et al., 2006) revealed that the common cormorant (Phalacrocorax carbo) possesses at least two paralogous CYP1A genes that are orthologous to chicken (Gallus gallus) CYP1A4 and 1A5. Furthermore, we have clarified that cormorants collected from Lake Biwa in Japan accumulate high levels of DRCs in the liver and induce CYP1A4 and 1A5 at transcriptional and translational levels (Kubota et al., 2004(Kubota et al., , 2005(Kubota et al., , 2006)).

While extensive data are available on mammalian CYP1A1 and 1A2, enzymatic properties of CYP1A4 and 1A5 isozymes in avian species have not been clarified until now. Therefore, the present study investigated enzymatic properties of avian CYP1A4 and 1A5 that were heterologously expressed in yeast cells.

The full-length cDNAs of common cormorant CYP1A4 and 1A5 were isolated from the hepatic cDNA library (Kubota et al., 2006). The CYP1A4 and 1A5 were expressed with plasmids containing cDNA of each isozyme in an in vitro yeast expression system following the method of Chung et al. (2004). Expression of recombinant CYP1A proteins in yeast microsomes was confirmed by the reduced CO-difference spectrum and detected by western blotting. Kinetics and specificity of the CYP1A enzymes toward O-dealkylation of methoxy-, ethoxy-, pentoxy-and benzyloxyresorufin (MROD, EROD, PROD and BROD, respectively) were investigated as described by Kubota et al. (2005) with some modifications.

Yeast microsomes containing each common cormorant CYP1A enzyme showed quantifiable Soret maximum at 446 nm. The concentrations of CYP1A4 and 1A5 proteins were estimated to be 351 and 275 pmol/mg protein, respectively. By immunoblotting using an anti-rat CYP1A1 polyclonal antibody, a single cross-reactive band was detected in the yeast microsomes containing CYP1A4 or 1A5. Kinetic analysis of AROD activity catalyzed by the recombinant CYP1A enzymes revealed that the AROD reaction follows typical Michaelis Menten kinetics. Among all the AROD activities, V max value of EROD activity was the highest for both isozymes (V max = 1.0 and 1.5 nmol/min/nmol CYP for CYP1A4 and 1A5, respectively). Regarding BROD activity, CYP1A4 (V max = 0.98 nmol/min/nmol CYP) was more specific than CYP1A5 (V max = 0.028 nmol/min/nmol CYP). As for MROD activity, CYP1A5 (0.89 nmol/min/nmol CYP) exhibited a greater capacity than CYP1A4 (0.095 nmol/min/nmol CYP). On the other hand, both CYP1A4 and 1A5 showed relatively weak V max values for PROD activity with 0.10 and 0.15 nmol/min/nmol CYP, respectively. Although the activities reported for the two recombinant CYP isozymes under the conditions present in a yeast background may not reflect enzyme activity in vivo, the clear difference in AROD profiles between the avian CYP1A paralogs implies that the two CYP1As have distinct potential for substrate recognition.

CYP1A4-or 1A5-dependent EROD activity was measured in the presence and absence of TCDF to examine the inhibitory effect of this DRC congener. The results showed that CYP1A5-dependent EROD activity was specifically inhibited by TCDF, whereas no inhibition was observed for CYP1A4-dependent EROD activity (Fig. 1). Considering that treatment with TCDF lowered the maximum CYP1A5 activity (V max ), but less affected the K m (0.152 M and 0.169 M with or without addition of TCDF, respectively), the mode of inhibition by TCDF is regarded to be noncompetitive [inhibitor constant (K i ) = 74 nM]. The noncompetitive inhibition indicates that TCDF does not affect binding of ethoxyresorufin to CYP1A5, and binds CYP1A5 at a site distinct from binding site of ethoxyresorufin. Several previous investigations have reported that the catalytic function of rodent CYP1A2 was inhibited by certain DRCs (Staskal, et al., 2005). These results clearly indicate that TCDF also inhibits the enzymatic activity of avian CYP1A5, which supports the previous hypothesis that the avian CYP1A5 is orthologous to mammalian CYP1A2 (Goldstone and Stegeman, 2006;Kubota et al., 2006).

Further study is necessary to understand physiological and toxicological roles of avian CYP1A4 and 1A5. The approach using recombinant CYP proteins may provide a novel insight into risk assessment of environmental contaminants in avian species. [S] (ethoxyresorufin, µ µM)

Fig. 1 .Fig

 1 Figure caption:

Acknowledgments

The authors thank Prof. A. Subramanian, Ehime University, for critical reading of this manuscript. This study was supported by Grants-in-Aid for Scientific Research (A) (No. 17208030) and (C) (No. 18510059) from Japan Society for the Promotion of Science. Financial assistance was also provided by "21 st Century COE Program" and "Global COE Program" from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by Feasibility Studies for Basic Research in ExTEND2005 (Enhanced Tack on Endocrine Disruption) from the Ministry of the Environment, Japan. This research was also supported in part by Hayashi Memorial Foundation for Female Natural Scientists.


Richard F Lee

Felipe H Luis

Niencheski

Karrie Brinkley

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

Keywords: DNA strand breaks, Comet assay, Toxicity, Coal ash, Sediments, Embryo

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.

Grass shrimp are an important component of the estuarine food web in the southeast United States. Grass shrimp embryos, enclosed in egg sacs, go through 10 stages while attached to the female pleopods. After stage 10 a free swimming zoea emerges from the egg sac. Several DNA lesions (e.g. strand breaks, modified bases, DNA-DNA crosslinks, DNA-protein crosslinks) are produced after exposure to genotoxicants, e.g. metals and PAHs. Reduced embryo production observed when grass shrimp are exposed to metals and PAHs (Lee et al., 2004) may be linked to damaged DNA. In the present study comparisons were made between reproduction processes in grass shrimp exposed to sediments containing coal combustion residues or highway runoff and hatching rates/ DNA strand breaks (comet assay) in embryos exposed to pore water from these sediments.

Sediments were collected from three stations in an estuary in Hilton Head, South Carolina (USA): station A -next to a highway storm drain; station B -100 m away from the drain; station C -500m away from the drain. For coal fly ash exposure experiments, 400 g of coal fly ash from power plants in Augusta, GA (USA) or from Candiota, Rio Grande do Sul (Brazil) was blended with 2000 g of reference sediment. Coal ash is one of the residues from coal combustion and is composed primarily of metal oxides. Reference sediment was from the Skidaway River estuary (Savannah, GA) Pore water was collected by centrifugation of sediments at 1000 X g for 30 min. Sediments to be analyzed for PAHs in sediment were freeze dried, extracted with methylene chloride and extracts passed through a silica gel column. The PAH fractions from the silica gel column were analyzed with a Hewlett Packard 6890 Series Plus Gas Chromatography coupled to a 5973 mass spectrometer. Twenty three individual PAH analytes were quantified with a detection limit of 10 ng/g for each analyte. Metals (V, Cr, Mn, Ni, Cu, Zn, As, Se, Ag, Cd, Pb) in sediment pore water samples were determined by ICP-MS (Fison Plasma Quad II Plus) equipped with a nebulizer. To determine reproductive parameters, 20 juvenile grass shrimp were added to aquaria contain 700 g of test sediment and 40 l of seawater. Three aquaria were used for each station or experiment (n=3). Salinity of the water was 28 ppt and temperature was maintained at 27 o C. Shrimp were fed Artemia and kept under 12 h light/12 h dark regime. Every 5 days the following parameters were determined: (a) mortality; (b) number of females with mature ovaries; (c) number of females with attached embryos. Grass shrimp were exposed to test sediments for 60 days with no change of seawater. Embryo hatching rates were determined after transferring 25 stage 8 embryos to polystyrene plates with each well containing 1.2 ml of estuarine water and 1 embryo. For pore water bioassays, 0.1 ml of sediment pore water was added to 1.1ml of filtered estuarine water in a polystyrene plate with each well containing one stage 8 embryo (25 embryos per test) and hatching rates determined. For assessment of DNA strand breaks, the comet assay was carried out on stage 7 embryos exposed to sediment pore water (diluted 1:10 with filtered estuarine water) for 24 h. The comet assay followed the procedures described by Singh et al., (1988) with modifications described by Lee and Kim (2002). A computerized image-analysis system (Komet Version 4.01, Kinetic Imaging Ltd.) was used to determine % DNA in tail of fifty randomly cells per slide.

At different distances from the highway storm drain, total PAH concentrations (sum of 24 analytes) were 29, 1.5 and 0.1 µg/g sediment for stations A, B, and C, respectively (Table 1). Concentrations of the different PAH analytes in these stations were previously reported (Lee et al., 2004). In the coal fly ash experiment, the concentrations of vanadium and selenium in sediment pore water are reported (Table 1) since the concentrations of these metals in coal fly ash sediment pore water (V: 129-278 µg/l; Se: 115-256 µg/l) was much higher than their concentrations in reference sediment pore water (V: 1-2 µg/l; Se: 0.04-0.1 µg/l). High selenium concentrations were found in sediment pore water near a coal-fired power station (Jung and Batley, 2004). Grass shrimp exposed to coal combustion resides has increased DNA strand breaks and had accumulated selenium (Kuzmick et al., 2007). Embryos exposed to pore water from sediment close to a highway storm drain (station A) showed significant (p< 0.05) difference with respect DNA strand breaks (38% DNA in tail) and reduction in embryo hatching (35%) when compared with embryos exposed to sediments distant from the storm drain (Stations B and C) (Table 1). Embryos exposed to sediment with coal fly ash had reduced hatching (25-35%) and increased DNA strand breaks (38-41% DNA in tail) compared to controls. Zoea that emerged died within a few hours. Female grass shrimp exposed to highway runoff sediment and coal fly ash sediment, showed significant reduction in embryo production and embryo hatching (Table 2) compared to females exposed to reference sediment. Reference sediment 71 ± 14 88 ± 4 1.5 a 400g of coal ash added to 2000 g of reference sediment *Denotes significant difference from reference sediment at P< 0.05 (ANOVA, F-test)

Table 1

 1 Bioassays with grass shrimp embryos exposed to sediment pore water from reference station,

	highway runoff stations and sediment with added
	coal fly ash
	__________________________________________
	____________________________________
	Station or	DNA Strand Breaks-Embryo Hatching	Vanadium
	Selenium
	Experiment	Comet Assay	(% hatching into	(µg/L in	(µg/L
	in
		(% DNA in tail)	zoea)	pore water) pore
	water)
		Mean ± SE; n=3)	Mean ± SD; n=3
	__________________________________________
	____________________________________
	I. Sediments at Different Distances from Highway Storm Drain
	Station A	38 ± 10*	35 ± 5*	2	0.1
	Station B	15 ± 7	70 ± 7	2	0.1
	Station C	9 ± 6	87 ± 12	1	0.04
	II. Reference Sediment with Coal Fly Ash from Power Plants in Brazil and United States
	Brazil ash	38 ± 8*	35 ± 10*	278	115
	USA ash	41 ± 11*	25 ± 12*	129	256
	III. Reference	11 ± 7	92 ± 11	3	0.05
	__________________________________________
	____________________________________
	*Denotes significant difference from reference sediment at P<0.05 (ANOVA, F-test)

Table 2 .

 2 Reproduction parameters in female grass shrimp exposed to sediments from highway runoff stations or sediments with coal fly ash Coal fly ash from power plants in Brazil and United States added to reference sediments a

	________________________________________________________________________
	Station or	Embryo production	Embryo hatching	Total PAHs
	Experiment	by gravid females	(% hatching into	of sediment
		(% gravid females	zoea stage)	(µg/g sediment)
		producing embryos)	Mean ± SD (n=3)
		Mean ± SD (n=3)
	________________________________________________________________________
	Sediments at different distances(in parenthesis) from highway storm drain
	Station A (0 m)	42 ± 11*	35 ± 5*	28.8
	Station B (100 m)	53 ± 6	46 ± 13*	12.3
	Station C (500 m)	65 ± 12	85 ± 6	1.0
	Brazil ash	52 ± 7*	24 ± 8*	3.4
	USA ash	30 ± 12*	7 ± 8*	2.9


Juliano Zanette

Flores Fabrício

Igor Dias Nunes

Marília Nardelli Medeiros

Siebert

Joaquim Jacó

Karim Hahn Mattos

Lüchmann

Manoel Cláudio

Afonso Rodrigues De Melo

Dias Celso

Bainy
email: bainy@mbox1.ufsc.br

Fabrício Flores Nunes

Igor Dias Medeiros

Marília Nardelli Siebert

Joaquim Mattos

Karim Hahn Lüchmann

Manoel Rodrigues De Melo

Afonso Celso

Dias Bainy

Comparison of the antioxidant defense system in C and C exposed to domestic sewage discharges

Keywords: Pollution, Antioxidant defenses, Biomarkers, Oyster, Domestic sewage

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche

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catalase (CAT), glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR), as previously described (Carlberg and Mannervik, 1985;Silva et al., 2005). One-way ANOVA, HSD for unequal sample size, P<0.05, was used to compare the data of stations from each species and tissue, considering the animals from site 4 as the reference group. Fecal coliforms (FC), used as indicator of the presence of domestic sewage discharges were analyzed by multipletube fermentation technique according to Standard Methods (1999). The FC average values recorded during the experiment (1, 7 and 14 days) at stations 1, 2, 3 and 4 were 14,100; 1,083; 103; and 46 MPN per 100 ml, evidencing the presence of a gradient of the sewage discharge in the study area. Likewise, the average turbidity and dissolved phosphate levels at the station 1 was about 4 to 10-fold higher than the other sites (data not shown). Correlations between the FC and enzymatic activities of the organisms from each station were performed by Pearson's coefficient (P<0.05).

Higher CAT activity was observed in both tissues of C. rhizophorae kept at station 1, when compared to the other stations (Figure 1A). Oysters from the station 2 showed higher CAT activity in gills, when compared to the reference site organisms (Figure 1A). Positive correlation between water FC levels and CAT activity was observed in both tissues of C. rhizophorae, suggesting a possible association between this enzyme and chemicals discharged in the untreated domestic sewage (Table 1).

Elevated CAT activity has been associated with the presence of peroxisome proliferators (PP) in mussel digestive gland (Cancio et al., 1999). CAT activity copes the increase in hydrogen peroxide produced by peroxisomal enzymes (eg.: acyl-CoA oxidases, poly amine oxidase and xanthine oxidase) [START_REF] Schrader | [END_REF]. In the present study, we could hypothesize that compounds present in the domestic sewage Higher G6PDH activity in the digestive gland of C. gigas from station 1 was observed (Figure 1C). The G6PDH is the main regulatory enzyme in the pentose phosphate shunt which produces NAPDH, required for the maintenance of the cellular antioxidant capacity, fatty acid biosynthesis, and xenobiotic biotransformation (Silva et al., 2005). NADPH donates electrons to reduce oxidized glutathione (GSSG) through the enzyme glutathione reductase (GR), recycling GSH. Higher GR activity was observed in gills of C. gigas from station 1 (Fig. 1D), possibly to compensate a decrease in GSH levels. Lower levels of GSH were found in gills of C. gigas from station 1 (data not show). These results suggest that the increase of G6PDH and GR activity in C. gigas exposed to domestic sewage occurs in a tissue distinct manner to cope the increase in cellular pro-oxidants and/or depletion of NADPH/GSH employed as substrate by antioxidant and biotransformation enzymes (eg.: GPx and CYP, respectively).

In conclusion, C. rhizophorae and C. gigas showed different responses in the activity of antioxidant and ancillary enzymes after in situ exposure to domestic sewage.

However, the native Atlantic southwestern oyster C. rhizophorae showed more significant changes in CAT suggesting that this organism is more adequate monitor in this kind of effluent. Further studies need to be carried out to investigate the presence of peroxisome proliferator-inducing compounds in these discharges.

Figure captions

  Domestic sewage discharges are frequently observed in Brazilian coast, where only 20 % of the cities possess wastewater treatment plant (IBGE, 2000). In the present study, a suite of antioxidant and ancillary enzymes were evaluated in two potential monitor species, the oysters Crassostrea rhizophorae and Crassostrea gigas. C. rhizophorae is distributed from Southern Brazil to the Caribbean and the Pacific oyster C. gigas is worldwide distributed being the most cultivated oyster in the world. Specimens of C. rhizophorae and C. gigas, six-months old (2-3 cm and 4-6 cm, respectively) were transplanted from a farming area, in September 2006, to four sites in São José, SC, Brazil, where they were kept for fourteen days. These stations were chosen according to a contamination gradient starting at 0.01, 0.4, 1.5 and 4.5 Km far from the mouth of Bücheler River, the most contaminated site (stations 1, 2, 3 and 4, respectively). Gill and digestive gland from each individual were excised, homogenized, centrifuged and the obtained S9 fraction was prepared to quantify the activity of

  might have induced peroxisome proliferation, with associated increase in the CAT ACCEPTED MANUSCRIPT activity. Zanette et al. (2006) also reported increases in CAT activity in the gills of C. rhizophorae sampled at sites contaminated by sewage discharges. Further studies are required to investigate which chemical could be responsible for the increase in CAT activity observed in the present report.

Fig. 1 .

 1 Fig. 1. Catalase (CAT), glutathione S-transferase (GST), glucose-6-phosphate

  Figure 1.

Table 1 .

 1 Correlations of faecal coliforms in water (FC) with catalase (CAT), glutathione Stransferase (GST), glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activities in gills and digestive glands (dg) of C. gigas and C. rhizophorae oysters transplanted to field stations (Pearson correlation coefficients). * Indicates significant correlations. Values of FC and enzyme activities were Log transformed.

	Oyster	Tissue	Enzyme	fecal coliform	P -value of
				correlation	correlation
				coefficient
	C. gigas	gill	CAT	0.557	0.443
			GST	-0.678	0.322
			G6PDH	0.825	0.175
			GR	-0.860	0.351
		dg	CAT	0.457	0.542
			GST	-0.222	0.778
			G6PDH	-0.056	0.943
			GR	0.759	0.434
	C. rhizophorae gill	CAT	0.981	0.019 *
			GST	-0.436	0.563
			G6PDH	-0.475	0.524
			GR	0.649	0.140
		dg	CAT	0.976	0.023 *
			GST	-0.879	0.120
			G6PDH	-0.475	0.524
			GR	0.565	0.240

Acknowledgements

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Supported by CNPq-CT-Hidro-CT Agro to ACDB. ACDB is recipient of CNPq productivity fellowship.


Gro Harlaug Olsen

Jolynn Carroll

Eirin Sva

Lionel Camus

Cellular energy allocation in the Arctic sea ice amphipod Gammarus wilkitzkii exposed to Water Soluble Fraction of oil

Keywords: Arctic, Amphipods, Oil, Cellular energy allocation, Gammarus wilkitzkii

Increasing offshore oil and gas activities in the European Arctic has raised concerns of the potential anthropogenic impact of oil-related compounds on the polar marine ecosystem.

We measured cellular energy allocation (CEA) in the sea ice amphipod Gammarus wilkitzkii after exposure for one month to the water soluble fraction (WSF) of oil. The CEA biomarker measures the energy budget of organisms by biochemically assessing changes in carbohydrates, protein and lipid content as well as the electron transport system activity. A significantly higher protein content was observed in the medium dose compared to controls, while the total energy budget was not affected in G. wilkitzkii. This indicates that parts of the energy budget of G. wilkitzkii, which is a key species in the Arctic ecosystem, is affected by a WSF of oil.

*Corresponding Author: Tel: +47 77 75 03 13; fax: +47 77 75 03 01 E-mail address: lionel@akvaplan.niva.no (L. Camus) Oil and gas activity in the Barents Sea is increasing as is the risk of accidental discharges, which underscores the need for more information on the effects of oil on Arctic organisms. PAHs have been shown to be some of the most toxic components of crude oil, bioaccumulating in organisms and disrupt cell and tissue function [START_REF] French-Mccay | Oil spill impact modeling: development and validation[END_REF]). To our knowledge no studies have focused on effects of oil-related compounds on sea ice species.

In sea ice, amphipods are the dominant organisms and they represent a direct link between lower and higher trophic levels (Lønne and Gulliksen, 1991).

Exposure to pollutants may increase the energy requirement of animals and hence result in reduced energy available for growth and reproduction (Calow and Sibly, 1990). The cellular energy allocation methodology measures energy available (Ea) (protein, lipid and carbohydrate content) and energy consumed (Ec) (electron transport system activity, ETS). Ea and Ec can be combined into a value expressing the net energy budget (De Coen and Janssen, 1997).

In this study, we exposed ovigerous females of the sea ice amphipod G. wilkitzkii to the water soluble fraction of crude oil to investigate whether the energy budget of G. wilkitzkii The protein levels were significantly higher in the medium dose compared to controls (Fisher's LSD: p= 0.02) (Fig. 1), indicating that WSF altered the energy budget related to protein metabolism in G. wilkitzkii. There were no significant differences in any of the other Ea parameters, Ec and CEA (Fig. 2) among treatments (ANOVA p>0.05), indicating that WSF did not affect the other energy parameters.

The higher protein level observed in organisms from the medium dose demonstrates that the WSF affected parts of the energy budget in G. wilkitzkii. A higher protein value can reflect sub lethal stress that induced compensatory changes in the organisms' energy metabolism. This is in accordance with a study from Verslycke et al. (2004) showing protein induction in Neomysis integer from a polluted area. Increased protein level could be caused by an increased lysosomal activity as lysosomes have shown to be a target of toxic stress induced by PAHs and PCBs (Gagnaire et al., 2006). Lysosomal enzymes are considered to play an important role in invertebrate defense reaction systems. Exposure to WSF may therefore have triggered increased production of lysosomal activities, explaining higher protein level in exposed group compared to controls.

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Higher protein levels were observed in the medium dose only. Explanations for this may be that high levels of PAHs may have reduced the physiological response systems (gene and protein expression) in the amphipods receiving high dose.

Negative CEA values were observed in all treatments, including the controls. This indicates that animals used more energy than they were able to allocate to their reserves. It is likely that a reduced food uptake and stress caused these negative values. In all cases, a higher Ec value compared to Ea were measured. This can be explained by the fact that it is assumed that ETS activity is an overestimation of the actual respiration (Båmstedt, 1980).

In the Arctic Ocean, the sea ice amphipod G. wilkitzkii is a key species and provide a link between primary production, detritus and higher trophic levels. Our results show that parts of the energy metabolism of the ice-amphipod were affected by WSF of oil. More time points are needed to definitively link the responses of energy budget parameters to petroleum hydrocarbons, but the results show evidence for disturbed protein metabolisms in G. wilkitzkii exposed to WSF of oil.

Figure caption

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  was altered by sub-lethal concentration of oil. This study is the first application of CEA technique on an Arctic sea ice species. Ovigerous female specimens of G. wilkitzkii were sampled in August 2006 in the Arctic Ocean (ca. N81 o 43' E15 o 46'). To simulate oil spill conditions, an exposure system was set-up according to Carls et al. (1999). After one month exposure Ea, Ec and Cea analyses and ACCEPTED MANUSCRIPT calculations were performed on each individual (De Coen, 1997). Dissolved PAH concentrations were determined by the modified standard US Environmental Protection Agency (EPA) procedure 3510 C. Total PAH concentrations in the water were as follows at day 0 and day 30: high dose, 55-8 ppm; medium dose, 10-2ppm; and low dose, 5-1ppm. The concentrations of PAHs decreased with time and also the relative PAH composition changed, a higher proportion of soluble compounds were found in the beginning and less soluble compounds in the end of the experiment. Ten females per treatment were used, and no replicates were done owing to a limited number of animals. Embryos developmental stages were different between females.

Fig. 1 .

 1 Fig.1. Protein content (mg/g wwt) in Gammarus wilkitzkii in control (C) , low dose (LD),

Fig. 2 .

 2 Fig. 2. Cellular energy allocation (CEA) in Gammarus wilkitzkii in control (C) , low dose

  Figure 1

Acknowledgements

This study is financed by the Norwegian Research Council under the program PROOF N o 164407, N o 159016/S40 and Statoil 'Ice Edge Project'.


Natàlia Garcia-Reyero

Ira Adelman

Li Liu

Nancy Denslow
email: ndenslow@ufl.edu

Gene expression profiles of Fathead minnows exposed to surface waters above and below a sewage treatment plant in Minnesota

Keywords: Gene expression profiling, Sewage treatment plants, Pharmaceuticals

Water treatment plants are often not effective in removing pharmaceuticals, personal care products or natural hormones from their effluents. To test the effects of these effluents on fish, we exposed male fathead minnows (Pimephales promelas) for 48 hours to effluents at two sites, one up-stream and the other down-stream from a water treatment plant. Gene expression profiling showed that significant changes occurred in the gonad of fish exposed below, compared to above the treatment plant and to laboratory control fish. Among the biological processes affected were the innate immune response, response to stress, control of homeostasis, control of transcription, metabolism, and cell communication. This work suggests that fish are impacted by

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exposures to the sewage treatment effluents and effects can be detected rapidly by gene expression profiling.

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Pharmaceuticals, natural hormones and personal care products in municipal effluents and surface waters around the world may cause hazards, which have not yet been characterized (Huggett et al., 2002;Daughton and Ternes, 1999). These waterways are complex soups of compounds, ranging from antibiotics to acetaminophen to soaps and other personal care products, many of which may be affecting aquatic life. Many of these pollutants are endocrine active compounds that disrupt reproduction at different levels along the hypothalamus-pituitarygonadal axis. In this work, we use expression profiling on fathead minnows to determine changes in gene expression in fish exposed to river waters up-stream and down-stream from a municipal sewage treatment facility located in Minnesota.

Fathead minnows (FHM) were placed in wire mesh minnow traps that were anchored to the bottom in the stream current. Fish were removed from the traps after 48 hours and transported back to the laboratory in stream water. Gonads were removed from four males for total RNA isolation, using Stat-60 reagent (Tel-test, Friendswood, TX), as previously described (Sabo Atwood et al., 2004). Total RNA was treated with an RNase inhibitor (RNASecure, Ambion) and with DNase (DNA-free, Ambion) to remove traces of DNA. RNA integrity and quantity were measured on the Agilent bioanalyzer and the nanodrop spectrophotometer, respectively.

Microarray analyses were performed on the individual samples using competitive hybridization on the 4 X 44K FHM microarray (EcoArray, Alachua, FL). These microarrays are oligonucleotide based and contain 22,000 genes. A reference design was used, where the reference sample, consisting of equal aliquots of control and treated samples, was labeled with the Cy3 dye, while experimental samples were labeled with the Cy5 dye, following Agilent protocols.

Microarrays were analyzed by ANOVA followed by Tukey's Honestly Significantly Difference post-hoc test to determine the set of differentially regulated genes at site #12 compared to site #11. Hierarchical clustering was performed for genes with a P value < 0.05.

To determine biological processes that were specifically changed in fish at site 12, we analyzed for Gene Ontology (GO) categories that are differentially enriched at site #12 compared to site #11, using Fisher's exact test and also calculated the false discovery rate.

The two sites in our study are located on the Des Moines River, one several miles upstream (site #11) and the other, immediately downstream (site #12) from the Windom sewage treatment plant. The upstream site # 11 drains extensive agricultural land, although few animal feeding operations are within 5 miles of the site. These sites were characterized by [START_REF] Lee | Waterresources Investigations report 00-4202[END_REF] who found significant effects in resident male carp at site #12, including the presence of plasma vitellogenin (< 10 ug/ml), decreased levels of 11-keto testosterone, and higher amounts of ceroid/lipofuscin deposits in the gonads, which correlate with cellular abnormalities.

In our study of the caged FHM, we analyzed differentially expressed genes by hierarchical clustering for the gonad (Fig. 1). The individual gonad samples clustered together for the two sites and the expression patterns were quite distinct from each other. If we add laboratory controls to the hierarchical clustering, they cluster with site #11, suggesting that fish at site # 11 are more closely related to controls (data not shown). In the gonad, 119 genes were up-regulated and 128 genes were down-regulated at P < 0.05.

We determined the molecular pathways most affected in the gonad by the downstream effluent by calculating P values for each GO category for Biological Process. GO categories are plotted as a function of increasing P value (Fig. 2) and identified in Fig. 2B, along with their P values. Interestingly, there are more pathways represented in the list of down-regulated genes (30 categories) than in the list of up-regulated genes (3 categories) (Fig 2B). The main pathways up-regulated by the exposure include cell communication, signal transduction, and the protein kinase cascade. Among the pathways that are down-regulated we note at the top of the list innate immune response and response to stress. Other pathways include control of homeostasis, control of transcription, metabolism, and embryonic development.

These results suggest that exposure of fish to the downstream effluent results in changes in gene transcription that may adversely affect reproduction, growth and susceptibility to disease.

The documented changes in gonad structure in carp collected at this site by [START_REF] Lee | Waterresources Investigations report 00-4202[END_REF] are consonant with the changes in the pathways we have measured. To date there are no analytical measurements of contaminants in the downstream waters, but these findings, together with the phenotypic changes seen in carp collected at the site, strongly indicate that such measurements should be carried out and that this site should be evaluated more thoroughly by traditional assessments to determine the risk to fish.

The use of gene expression changes to evaluate exposure of fish to toxicants is still in its infancy. We have shown that a 48-hour exposure of caged fish to the effluent is sufficiently long to change gene expression in a robust and specific way. As more controlled laboratory experiments are performed with individual chemicals, gene expression signatures will be identified that relate back to individual chemicals.

Fig. 1 .

 1 Figure captions:

Fig. 2 .

 2 Fig. 2. (A) Differential expression of biological function pathways in fish exposed to site 12

  Fig. 2A

Acknowledgements

This work is the result of research sponsored by the Minnesota Sea Grant College Program supported by the NOAA Office of Sea Grant, United States Department of Commerce, under grant No. NA03OAR4170048. The U.S. Government is authorized to reproduce and ACCEPTED MANUSCRIPT distribute reprints for government purposes, not withstanding any copyright notation that may appear hereon. This paper is journal reprint No. JR 538 of the Minnesota Sea Grant College Program. Also, by a fellowship from the Spanish Ministry of Sciences and Technology (EX-2004-0986) to NGR.


Lionel Camus

Gro Harlaug Olsen

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

Keywords: Arctic, Sea ice, Amphipod, Crude oil, Embryo, Malformation

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 Gammarus wilkitzkii 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.

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As the oil industry expands in the Barents Sea, there are increasing risks of accidental oil spill with potential drift and incorporation into sea ice for several years. Animals in the sea ice, notably amphipods (Lønne and Gulliksen, 1991), may be exposed chronically to polycyclic aromatic hydrocarbons (PAH) and other oil-related compounds. More data are needed on the toxicity the toxicity of PAH on Arctic amphipods (Chapman and Riddle, 2005).

In addition, the amphipod population (Ampelisca sp) has yet to fully recover studies of the 'Amoco Cadiz' oil spill in Northern coast of France (>20 years ago) (Poggiale and Dauvin, 2001). A potential explanation is that amphipods are brooders (female amphipod carries her young in the brood pouch until they hatch as fully developed immature specimens); hence, pollutants affecting adults will also affect offspring since they are released into the same local environment. Furthermore re-colonisation is much slower since amphipods do not have pelagic larvae. In this study, we exposed ovigerous females of the sea ice amphipod The principle of a rock column was used to simulate oil spill (Carls et al 1999). The WSF of crude oil was extracted by passing continuously cold seawater through a column filled up with glass beads (3mm) coated with crude oil. Four doses (including control) were made with the extract to expose the ovigerous females for 30 days using a continuous flow system. Concentration of PAH decreased with time, in addition, relative composition changes, with a higher proportion of soluble compounds at the beginning and less soluble in the end (Carls et al., 1999). Ten females per treatment were used, and no replicates were done owing to a limited number of animals. Embryo developmental stages were different between females.

Embryo were scored separately for abnormalities by 2 scientists using stereo microscopes (see Sundelin and Eriksson, 1998). The frequency of malformations/aberrations per brood per female was counted, and no attempt was made to distinguish between the different types of aberrations due to small sample sizes.

The females showed a frequency of 2 to 6% malformed embryos, 0 to 5% undifferentiated and 0 to 6% dead eggs. A high proportion of enlarged embryos had a relatively wide gap between the membrane and the fetus. This may be a direct impact of the PAH on the embryo cell membrane, allowing water to enter. No differences in developmental stages were seen among treatments, indicating that WSF did not alter the period of embryogenesis as observed by (Lawrence and Polter, 2001) in the amhipod Chaetogammarus marinus exposed to benzo (a) pyrene. Additionally, a higher proportion of smaller embryos, indicating a reduced development, seem to correlate the smaller hatched juveniles from exposed group versus control observed by Lawrence and Polter (2001). Finally, the lack of a membrane delimiting the pouch in G. wilkitzkii, contributes to enhanced effects of water borne contaminants on the embryos. To conclude, embryo development of the sea ice amphipod can be impaired by WSF. In addition, it would be interesting to expose adult amphipods prior and during mating and additionally to investigate the survival and fitness of the released offsprings.

Figure captions

  Embryo aberrations in sea ice amphipod Gammarus wilkitzkii exposed to water soluble fraction Please cite this article as: Camus, L., Olsen, G.H., Embryo aberrations in sea ice amphipod Gammarus wilkitzkii exposed to water soluble fraction of oil, Marine Environmental Research (2008), doi: 10.1016/j.marenvres. 2008.02.074

  Gammarus wilkitzkii to the water soluble fraction (WSF) of crude oil to investigate the effects on the developmental stages and malformations of G. wilkitzkii. Divers collected female individuals of G. wilkitzkii (3cm length) in late August 2006 in the Arctic Ocean (ca. N81 o 43' E15 o 46') from the research vessel of the University of Tromsø (RV JanMayen).

  PAH concentrations in water were analyzed according to the modified standard US Environmental Protection Agency (EPA) procedure 3510 C. PAH analyses were performed at UNILAB, Tromsø, Norway, accredited laboratory. Total PAH concentrations in the water were as follow at day 0 and day 30: high dose, 55-8 ppm; medium dose, 10-2ppm; and low dose, 5-1ppm.The reproductive stages of G. wilkitzkii were similar to the stages described in the Baltic Sea amphipod Monoporeia affinis(Sundelin and Eriksson, 1998). Across treatments, the females in this study ranged from stage three to nine. No females had embryos in the first development stages (stage 1 and 2). Three of the females were empty (no embryos or juveniles already released) and four females died during the course of the experiment.Average number of eggs/juveniles per female was 84 (Min: 1, Max 201). The different observed aberrations were also the same as described inSundelin and Eriksson (1998); an example of malformed embryos is shown in Fig.1. There were no significant differences in reproductive stage, eggs per female or weight among the different treatments (Unequal N post-hoc p 0.64), indicating that the females in the different treatments were in similar stages, had similar weight and similar amounts of embryos. The frequency of embryo aberrations was significantly higher in the high dose compared to controls (Unequal N post hoc test; p= 0.025; Fig.2). The significant higher number of aberrations observed in the high dose demonstrated that 30 days exposure to the WSF of crude oil cause severe damages to the embryo of G. wilkitzkii. This finding is in agreement withSundelin and Eriksson (1998) that reported elevated frequencies of malformed embryos among females collected near an aluminum smelter where high concentrations of PAHs were measured. In this study, no attempt was made to distinguish between the different types of malformations due to small sample size. The percentage of total embryo aberrations was 10.4 % in control, 4% in low dose, 12.2 % in medium and 29 % in high dose. In a four-year study in the Baltic proper and Bothnian Sea with a large sample size,Sundelin and Eriksson (1998) reported that gravid

Fig. 1 .

 1 Fig. 1. Example of healthy (A) and malformed embryos (B) at stage 7 observed after 30 days

Fig. 2 .

 2 Fig. 2. Aberration frequencies (total number of malformations/total number of eggs per

  Figure 1

Acknowledgements

The authors are grateful to the crew of the research vessel R/V Jan Mayen from the University of Tromsø. We thank Jeep Rice and Mark Carls (Auke Bay, Juneau, NOAA) and


Daniela Volcan Almeida

Gilma Santos Trindade

Laura A Geracitano

Daniela Martí Barros

José Maria Monserrat

Luis Fernando Marins

Initial results in the development of a reporter cell line for toxicology studies at gene expression level: activation of the electrophile-responsive element

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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electrophile responsive element fused to the mouse metallothionein (mt1) minimal promoter. The rat HTC hepatoma cells were transfected with the EpREmt-GFP construct and successfully selected with G418 antibiotic. EpREmt-GFP HTC cells were treated with 0.002mg.L -1 , 0.02 mg.L -1 , 0.2 mg.L -1 and 2 mg.L -1 copper sulfate and 0.001 mg.L -1 , 0.01 mg.L -1 , 0.1 mg.L -1 and 1 mg.L -1 methyl parathion for 48h. GFP expression was directly quantified in living cells using a microplate fluorimeter. GFP expression was significantly increased in higher concentrations of both pollutants, showing a 1.80-and 2.58-fold induction of GFP at 2 mg copper.L -1 and 1 mg methyl parathion.L -1 , respectively (p<0.01).

The results obtained in the present study demonstrate that the EpREmt-GFP HTC cell line can be an interesting model for further development for the study of the cellular response to aquatic pollutants as well as a new tool for environmental monitoring at the molecular level.

Keywords: Electrophile response element; Green fluorescent protein; Hepatoma cells;

Copper; Methyl parathion.

Corresponding author: E-mail address: dqmluf@furg.br (L.F. Marins)

Electrophile responsive elements (EpRE) are within the regulatory region of a number of genes involved with xenobiotic metabolism and antioxidant defense. These include NAD(P)H:quinone oxidoreductase (NQO), glutathione S-transferase Ya subunit (GST Ya), heme oxygenase 1 (HO-1), and -glutamylcysteine synthetase (GCL) (for review see Jaiswal, 2004). EpRE core sequence (5'-TMAnnRTGAYnnnGCR-3') was characterized by defining regions of the sequence required for basal and xenobiotic inducible expression (Rushmore et al., 1991). Copper and methyl parathion are pollutants capable of generating oxidative stress in zebrafish cells and initiating the transcription of an EpRE-regulated reporter gene (Carvan III et al., 2001). The objective of the present study DNA extraction [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF] and PCR analysis, which confirmed the presence of the transgene. For validation of the model, the stable cell lines were exposed to the pollutants at the concentrations cited above and the GFP expression visualized by epifluorescence microscopy (Fig. 1). The results of GFP fluorimeter quantification showed a 1.8-fold induction of GFP at 2 mg copper.L -1 (Fig. 2A, p<0.01) and 2.58 times at 1 mg methyl parathion.L -1 over controls (Fig. 2B, p<0.01). These results indicate an EpRE induction only at concentrations that exceed 100 times the value permitted by the Brazilian legislation. However, copper concentration up to 216 mg.L -1 has been found in aquatic environment at Southern Brazil (Niencheski and Baumgarten, 2000), which makes this model suitable for this level of contamination. In terms of comparison between the pollutants studied here, Carvan III et al. (2001) also verified a larger induction capacity for methyl parathion when compared to copper, corroborating our results. Profiles showing induction of GFP reporter gene expression were described by [START_REF] Zhu | [END_REF]. These authors have considered that EpRE-mediated gene activation is related to cellular detoxification processes. Even though we have demonstrated that EpRE can be induced in HTC cells by high concentrations of copper and methyl parathion, a high level of basal EpRE-GFP induction was also observed in this cellular type (Fig. 1). In fact, it is known that genes involved with xenobiotic metabolism and antioxidant defense are highly expressed in tumoral cells (for review see Nair et al., 2007). This could explain the lack of significant differences at lower pollutant concentrations (Fig. 2). In addition, both pollutants used here could be chelated or complexed with other components present in the

Figure captions

Fig. 1. Green fluorescence protein (GFP) expression visualized using an epifluorescence microscope (×400 magnification). Cells were treated with 0.002 mg.L -1 (C2), 0.02 mg.L - 1 (C3), 0.2 mg.L -1 (C4) and 2 mg.L -1 (C5) copper sulfate and 0.001 mg.L -1 (C2), 0.01 mg.L - 1 (C3), 0.1 mg.L -1 (C4) and 1 mg.L -1 (C5) methyl parathion for 48h. Controls (C1) were treated with solvents (water for copper and acetone for methyl parathion). a. phase-contrast; b. fluorescence.

  Initial results in the development of a reporter cell line for toxicology studies at gene expression level: activation of the electrophile-responsive element by copper and methyl parathion Daniela Volcan Almeida, Gilma Santos Trindade, Laura A. Geracitano, Daniela Martí Barros, José Maria Monserrat, Luis Fernando Please cite this article as: Almeida, D.V., Trindade, G.S., Geracitano, L.A., Barros, D.M., Monserrat, J.M., Marins, L.F., Initial results in the development of a reporter cell line for toxicology studies at gene expression level: activation of the electrophile-responsive element by copper and methyl parathion, Marine Environmental Research (2008), doi: 10.1016/j.marenvres.2008.02.075

  was to create and validate a stable reporter cell line using green fluorescent protein (GFP) expression as a reporter of EpRE-mediated transcriptional activation. A stable cell line was produced which carries a transgene comprised of the GFP reporter gene under transcriptional control of the mouse glutathione-S-transferase (gst1) electrophile-responsive element fused to the mouse metallothionein (mt1) minimal promoter. The cells used in the present study were a rat hepatoma line (HTC) obtained from the Rio de Janeiro Cell Bank (Brazil) and grown in RPMI 1640 (Gibco) medium supplemented with sodium bicarbonate (0.2 g/L), L-glutamine (0.3 g/L), HEPES (25 mM) and -mercaptoethanol (5 × 10 -5 M), with 10% fetal bovine serum (Gibco), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco), in cell culture flasks, at 37 °C. In order to create the stably transfected EpRE-GFP reporter cell lines, 5x10 5 cells were transfected with 5 µg of the EpREmt-GFP construct and 25 µg DOTAP cationic lipid (Acros) in 2 mL serum-free RPMI medium and incubated at 37 °C for 24 h. The medium was then replaced with 2 mL RPMI medium and geneticin (500 µg/mL; Gibco) was added after 48 h. Presence of the transgene in the cells ). For model validation, the EpREmt-GFP HTC cells were plated at a concentration of 1x10 6 cells/well in a 24-well plate and were treated with 0.002mg.L -1 , 0.02 mg.L -1 , 0.2 mg.L -1 and 2 mg.L -1 copper sulfate and 0.001 mg.L -1 , 0.01 mg.L -1 , 0.1 mg.L -1 or 1 mg.L -1 methyl parathion for 48h. The compounds were dissolved in water (copper) or acetone (methyl parathion) and added in a final concentration of 0.01% (v/v) in the cell culture medium. The concentrations of 0.02 mg.L -1 and 0.01 mg.L -1 indicated maximum values permitted by the Brazilian legislation in freshwater for copper and methyl parathion, respectively. Controls were treated with solvents (water for copper and acetone for methyl parathion). GFP expression was directly quantified in living cells using a microplate fluorimeter (Victor 2, Perkin Elmer) with excitation/emission at 485 nm/530 nm. GFP expression values were normalized to total cell number. Fluorescence data were subjected to an analysis of variance (ANOVA). Post-hoc comparisons were made by Tukey's test and all statistical tests used fixed type I error of 5% ( =0.05). After selection with G418 antibiotic, the cells were subjected to genomic

  medium and thus unavailable to the cells. An interesting perspective of the present study is the development of new reporter cell lines of non-tumoral origin, which are currently underway in our laboratory. The employment of such cell lines as environmental monitoring models through utilization of transgenes constituted by pollutant responsive elements directing reporter gene expression could compose a sensitive, low-cost and practical method for biological monitoring of aquatic pollution. In summary, the initial results obtained in the present study are promising and have stimulated further development of in vitro models as tools for the study of cellular response to aquatic pollutants.

Fig. 2 .

 2 Fig. 2. Green fluorescence protein (GFP) expression in EpREmt-GFP HTC cell line. Cells

Figure 1

Acknowledgments

The authors would like to thank to Maira Proietti for valuable corrections of the manuscript. This work was supported by FAPERGS (Proc. N. 0412405). D.V. Almeida and L.A. Geracitano, a graduate student and post-doctoral fellow, respectively, were financed by CAPES. J.M. Monserrat and D. Martí Barros are research fellows from CNPq.