Retinoid X receptor (RXR), estrogen receptor (ER) and other nuclear receptors in tissues of the mussel Mytilus galloprovincialis: cloning and transcription pattern.

Bivalve molluscs accumulate chemical compounds from the environment that could cause alterations in lipid homeostasis and endocrine system. In vertebrates such cell processes are modulated by transcription factors belonging to the superfamily of nuclear receptors (NRs). The goal of this study was to clone fragments of mussel Mytilus galloprovincialis NR genes that could mediate cell responses such as peroxisome proliferation and endocrine disruption. PCR-based screening of mussel digestive gland cDNA using degenerate primers provided cDNA fragments or whole ORFs of retinoid X receptor (RXR), estrogen receptor (ER) and 5 proteins belonging to the NR1 subfamily highly similar to the arthropod ecdysone inducible protein E75. NR1G, whose whole ORF was cloned, is related to the nematode and trematode G group of NR1 receptors; NR1DEF is related to the D, E and F groups, and NR1Dv1, NR1Dv2 and NR1DΔ belong to the D group. mRNA transcripts for all these receptors were detected in gill, mantle and digestive gland. In all cases, except ER, transcript levels were lower in June than in January. NR1Dv1 and NR1DΔ did not show identical transcription levels, although both were at their lowest in digestive gland in June. On the contrary, NR1Dv2 and NR1DΔ transcription profiles were similar. Further studies are needed to determine the function(s) of mussel RXR, ER and novel NR1 subfamily receptors and their possible role in the regulation of physiological cell responses and/or adaptive response to xenobiotic exposures.

Distribution of organic microcontaminants, butyltins, and metals in mussels from the Estuary of Bilbao.

Mussels are used as bioindicators of chemical pollution in coastal and estuarine waters. We measured the concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters (PEs), butyltins, and metals (Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, V, and Zn) in mussel tissues collected from the lower Bilbao estuary (Arriluze, north of Spain) every 2 months from November 2002 to March 2004. The concentration (microg g(-1) dry weight) of PAHs, PCBs, and PEs ranged from 5.1 to 18.3, from 0.04 to 0.2, and from 1.5 to 27.6, respectively. Temporal pattern variations, including maximum and minimum values, were determined for metals and BTs from their concentration profiles during a period of 1 year. The main feature of organic microcontaminants was relatively high concentration values, reflecting the overall industrial and harbour activities of the site. Moreover, the ratios of methylated species and certain other diagnostic ratios suggested a petrogenic origin for PAHs. Finally, the relations among the concentrations found in mussel tissues and the levels of several cell biomarkers were established by a partial least squares model.

Cloning and expression pattern of peroxisome proliferator-activated receptors, estrogen receptor alpha and retinoid X receptor alpha in the thicklip grey mullet Chelon labrosus.

Aquatic organisms are exposed to diverse xenobiotics that cause peroxisome proliferation and/or endocrine disruption, both modulated in vertebrates by transcription factors of the nuclear receptor (NR) superfamily. Peroxisome proliferators are agonists of peroxisome proliferator-activated receptors (PPARs) that heterodimerize with the retinoid X receptor (RXR). Many xenoestrogens activate the estrogen receptor (ER). Here, 1090 bp of PPARalpha, 1255 bp of PPARgamma, 278 bp of RXRalpha, and 578 bp of ERalpha of thicklip grey mullet Chelon labrosus were cloned. Sequences were highly conserved, although relevant changes with respect to mammalian homologs were identified in PPARgamma and ERalpha. Semi-quantitative RT-PCR was used to determine if these NRs were expressed in different tissues of male, female and undifferentiated mullets captured in January and June. Expression of PPARs was highest in liver and lowest in muscle. RXRalpha expression was homogeneous excepting a low expression in male and female gill in January and brain and heart of undifferentiated fish in January and June. ERalpha expression predominated in liver and female gonad in June. The expression level of PPARs and ERalpha was significantly higher in liver in January than in gills in January or June. The present results show tissue-dependent modulation of expression of NRs in mullets.

Cloning and transcription of nuclear receptors and other toxicologically relevant genes, and exposure biomarkers in European hake (Merluccius merluccius) after the Prestige oil spill.

In November 2002 the tanker Prestige released more than 60,000t of a heavy fuel oil which spread over Galician waters and the Biscay Bay, affecting coastal ecosystems. Polycyclic aromatic hydrocarbons are the main components of the Prestige fuel oil and induce biotransformation metabolism and peroxisome proliferation in marine organisms. In vertebrates, this later response involves peroxisome proliferator-activated receptors (PPARs), transcription factors belonging to the nuclear receptor superfamily, that act upon heterodimerization with the retinoid X receptor (RXR). In order to assess the possible biological effects of the Prestige oil spill in the Biscay Bay, male and female juvenile and adult European hakes Merluccius merluccius were sampled in June and December 2004 and 2005. PCR screening of hake liver cDNA with degenerate primers resulted in cloning and sequencing of cDNA fragments of PPARα (1011bp), PPARγ (812bp), RXR (270bp) and of the PPARα target gene palmitoyl-CoA oxidase (AOX1, 792bp). Fragments of another 9 toxicologically relevant genes were also cloned and sequenced. PPARα mRNA expression was not significantly different among groups. In juvenile females transcription of PPARγ, RXR and AOX1 significantly increased in June 2005 when compared to June 2004. In adult males levels of AOX1 decreased in the same period. AOX1 and 7-ethoxyresorufin O-deethylase (EROD) activities, measured as exposure biomarkers, differed between years only in males sampled in June. EROD activity was higher in 2004 than in 2005 in adults, whereas both juvenile and adults showed higher AOX1 activity in 2005. The lack of historical data previous to the accident or in areas not affected by the accident did not allow to relate observed variations in gene transcription levels and enzyme activities to the Prestige oil spill. Reported data could be useful for comparison purposes for future studies in European hake and contributes gene sequence information relevant for future toxicological studies.

Cloning and expression pattern of peroxisome proliferator-activated receptor alpha in the thicklip grey mullet Chelon labrosus.

Aquatic organisms living in coastal and estuarine areas are exposed to diverse contaminants which can cause peroxisome proliferation. Peroxisome proliferators are agonists of peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor superfamily. We have recently demonstrated expression of the three PPAR isoforms in liver of mullet Chelon labrosus and other fish species by immunohistochemistry. The goal of the present study was first to clone PPARalpha and second to investigate its expression pattern in various tissues of mullet. PCR-based screening of mullet cDNA with PPARalpha specific degenerate primers resulted in amplification, subcloning and sequencing of a 1090 bp cDNA fragment (AY618315) that encodes mullet PPARalpha and exhibits highest amino acid identity to fish Sparus aurata PPARalpha (90%). Semi-quantitative RT-PCR was used to characterize the expression of PPARalpha in brain, muscle, liver, spleen, gill, heart and female gonad of juvenile and adult male and female mullet. For this, mullet 18S-rRNA (AY825252), beta-actin (AY836368) and elongation factor alpha (AY836369) were cloned and used as internal reference for RT-PCR. Expression of PPARalpha was detected in all tissues, was highest in liver and lowest in adult male and female muscle.