Molecular effects of diethanolamine exposure on Calanus finmarchicus (Crustacea: Copepoda).

Alkanolamines are surface-active chemicals used in a wide range of industrial, agricultural and pharmaceutical applications and products. Of particular interest is the use of alkanolamines such as diethanolamine (DEA) in the removal of CO(2) from natural gas and for CO(2) capture following fossil fuel combustion. Despite this widespread use, relatively little is known about the ecotoxicological impacts of these compounds. In an attempt to assess the potential effects of alkanolamines in the marine environment, a key species in the North Atlantic, the planktonic copepod Calanus finmarchicus, was studied for molecular effects following sublethal exposure to DEA. DEA-induced alterations in transcriptome and metabolome profiling were assessed using a suppression subtractive hybridization (SSH) gene library method and high resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR), respectively. Effects were observed on transcription of genes reportedly involved in lipid metabolism, antioxidant systems, metal binding, and amino acid and protein catabolism. These effects were accompanied by altered expression of fatty acid derivates, amino acids (threonine, methionine, glutamine, arginine, alanine and leucine) and cholines (choline, phosphocholine and glycerophosphocholine). Together, SSH and HR-MAS NMR offer complementary screening tools for the assessment of molecular responses of C. finmarchicus to DEA and can be used in the study of other chemicals and organisms. Concentration-response and time-response relationships between DEA exposure and single gene transcription were investigated using quantitative PCR. Specific relationships were found between DEA exposure and the transcription of genes involved in protein catabolism (ubiquitin-specific protease-7), metal ion homeostasis (ferritin) and defence against oxidative stress (gamma-glutamylcysteine synthase, glutathione synthase and Cu/Zn-superoxide dismutase). At the lowest alkanolamine concentration used in these experiments, which corresponded to 0.5% of the LC(50) concentration, no transcriptional effects were observed, giving information regarding the lower molecular effect level. Finally, similar transcription patterns were observed for a number of different genes following exposure to DEA, which indicates analogous mechanisms of toxicity and response.

Modulation of salmon ovarian steroidogenesis and growth factor responses by the xenoestrogen, 4-nonylphenol.

Endocrine-disrupting chemicals are known to influence organismal reproductive processes, including the production and regulation of gonadal steroids. This study evaluated the effects of a xenoestrogen (nonylphenol: NP) on salmon ovarian steroidogenesis and growth factors using an in vitro organ culture system. Ovarian tissues were cultivated for 3 and 7d with different concentrations of NP (0 (control), 1, 10 and 50 microM) dissolved in ethanol (0.1%). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450-mediated cholesterol side-chain cleavage (P450scc), aromatase isoforms, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), Cyp11beta-, Cyp17 and 21-hydroxylase, and insulin-like growth factors (IGF-1 and IGF-2) and IGF1-receptor (IGF1-R) were quantified by real-time PCR. Tissue levels of estradiol-17beta (E2), testosterone (T) and 11-ketotestosterone (11-KT) were quantified using enzyme immunoassays. Our data show that nominal NP levels produced time- and concentration-specific effects on the expression of steroidogenesis- and IGF-related transcripts in salmon ovarian tissues. Tissue levels of ovarian E2, T and 11-KT were significantly modulated after NP exposure. Interestingly, elevated ovarian E2 levels after 10 microM NP exposure at day 3 paralleled P450Arom isoforms mRNA expression at the same time interval. The expression patterns of other steroidogenic protein and enzyme genes, such as StAR, P450scc, 3beta-HSD and Cyp17 inversely paralleled this pattern, displaying consistent decreased transcript levels. These findings show that NP (an ubiquitous environmental pollutant) can produce variations in gonadal steroidogenesis and growth regulating responses with potential consequences for overt fecundity in teleosts.

Effects of naphthalene on gene transcription in Calanus finmarchicus (Crustacea: Copepoda).

The planktonic copepod Calanus finmarchicus is a key species in the Northern Atlantic food web; an oceanic area with extensive oil production. Naphthalene is one of the major constituents of produced water and water soluble fractions of petrogenic oils. This study investigates the effects on gene transcription of a short term exposure to naphthalene at levels well below LC(50) concentrations. This was done in order to establish a molecular basis of naphthalene toxicity in a species which has previously been subject only to very limited studies at the molecular level. Naphthalene exposure to C. finmarchicus was found to cause glutathione S-transferase (GST) induction, indicating lipid peroxidation as the major mode of naphthalene toxicity. There is no clear evidence that the putative cytochrome P450 enzymes CYP1A2 and CYP330A1 mRNAs are parts of a detoxification enzyme system. Instead, an observed decrease in CYP330A1 mRNA levels at the highest naphthalene exposure concentration may indicate an effect on ecdysteroidogenesis. Only the lowest naphthalene concentration lead to increased mRNA levels of antioxidants SOD and CAT, indicating no clear evidence for general cellular oxidative stress following exposure. Small and insignificant changes in the HSP-70, HSP-90 and ubiquitin mRNA levels indicate a small degree of protein damage owing to naphthalene exposure. The established culture of C. finmarchicus at the SINTEF/NTNU Sealab, and the use of gene transcription analyses provide excellent tools for improving the understanding of biochemical mechanisms involved in the defense against environmental impacts and the molecular modes of toxicity in this species.

Steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage (P450scc) as molecular and cellular targets for 17alpha-ethynylestradiol in salmon previtellogenic oocytes.

Gonadal steroids are known to modulate both the synthesis and the release of gonadotropins by the pituitary and influence several brain functions that are apparently responsible for gender-specific differences in the regulation of the hypothalamus-pituitary-gonadal (HPG) axis. It is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and subsequent conversion to pregnenolone by P450-mediated cholesterol side chain cleavage (P450 scc). In the present study, we have evaluated the effects of 17alpha-ethynylestradiol (EE2) on salmon previtellogenic oocytes using an in vitro culture system and molecular, histological, and physiological methods. The in vitro culture technique was based on an agarose floating method recently validated for xenoestrogens in our laboratory. Tissue was cultured in a humidified incubator at 10 degrees C for 3, 7, and 14 days with different concentrations of EE2 [0 (control), 0.01, 0.1, and 1 microM] dissolved in ethanol (0.1%). The StAR, P450 scc, P450 arom isoforms, and insulin-like growth factor 2 (IGF-2) mRNA expressions were performed using validated real-time polymerase chain reaction (PCR) with specific primers, and immunohistochemistry of the StAR and P450 scc proteins was performed using antisera prepared against synthetic peptide for both proteins and estradiol-17beta (E2); testosterone (T) and 11-ketotestosterone (11-KT) tissue levels were performed using enzyme immunoassay (EIA). Our data show that EE2 produced time- and concentration-specific effects on the StAR protein, P450 scc, P450 arom isoforms, and IGF-2 gene expressions in salmon gonadal tissues. Cellular expression of the StAR and P450 scc proteins was mainly demonstrated in follicular cells of the oocyte membrane, showing time- and EE2 concentration-dependent differences in staining intensities. Tissue levels of E2, T, and 11-KT in salmon were differentially modulated by EE2 in a time- and concentration-specific manner. Although an apparent negative relationship between E2 and T that reflected aromatization of T to E2 was observed at day 3 postexposure, T and 11-KT showed an apparent concentration-dependent effect after EE2 exposure at day 14. The consistencies between our data at day 14 postexposure suggest that the EE2 modulates steroidogenesis by targeting the initial and rate-limiting step that involves the StAR protein. In general, these findings show that the synthetic pharmaceutical endocrine disruptor and ubiquitous environmental pollutant also produce variations in key gonadal steroidogenic and growth-regulating pathways. These effects and the hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.