Biphasic modulation of neuro- and interrenal steroidogenesis in juvenile African sharptooth catfish (Clarias gariepinus) exposed to waterborne di-(2-ethylhexyl) phthalate.

Receptor (i.e. genomic) and non-receptor (or non-genomic) effects of endocrine toxicology have received limited or almost non-existent attention for tropical species and regions. In the present study, we have evaluated the effects of di-(2-ethylhexyl) phthalate (DEHP) on neuro- and interrenal steroidogenesis of the African catfish (Clarias gariepinus) using molecular, immunochemical and physiological approaches. Juvenile fish (mean weight and length: 5.6±0.6g and 8.2±1.2cm, respectively), were randomly distributed into ten 120L rectangular glass tanks containing 60L of dechlorinated tap water, at 50 fish per exposure group. The fish were exposed to environmentally relevant concentrations of DEHP, consisting of 0 (ethanol solvent control), 10, 100, 200, and 400µg DEHP/L water and performed in two replicates. Brain, liver and head kidney samples were collected at day 3, 7 and 14 after exposure, and analysed for star, p450scc, cyp19a1, cyp17, cyp11ß-, 3ß-, 17ß- and 20ß-hsd, and 17ß-ohase mRNA expression using real-time PCR. The StAR, P450scc and CYP19 proteins were measured using immunoblotting method, while estradiol-17ß (E2) and testosterone (T) were measured in liver homogenate using enzyme immunoassay (EIA). Our data showed a consistent and unique pattern of biphasic effect on star and steroidogenic enzyme genes with increases at low concentration (10µg/L) and thereafter, a concentration-dependent decrease in both the brain and head kidney, that paralleled the expression of StAR, P450scc and CYP19 proteins. Cellular E2 and T levels showed an apparent DEHP concentration-dependent increase at day 14 of exposure. The observed consistency in the current findings and in view of previous reports on contaminants-induced alterations in neuro- and interrenal steroidogenesis, the broader toxicological and endocrine disruptor implication of our data indicate potentials for overt reproductive, metabolic, physiological and general health consequences for the exposed organisms.

Hydrolyzed fish proteins modulates both inflammatory and antioxidant gene expression as well as protein expression in a co culture model of liver and head kidney cells isolated from Atlantic salmon (Salmo salar).

Hydrolyzed fish proteins (H-pro) contain high concentrations of free amino acids and low molecular peptides that potentially may benefit fish health. The following study aimed to test whether the water-soluble phase of H-pro could attenuate lipopolysaccharide (LPS) provoked inflammation in liver cells and head kidney cells isolated from Atlantic salmon. Cells were grown as mono cultures or co cultures to assess possible crosstalk between immune cells and metabolic cells during treatments. Cells were added media with or without H-pro for 2 days before LPS exposure and harvested 24 h post LPS exposure. Respective cells without H-pro and LPS were used as controls. H-pro alone could affect expression of proteins directly as H-pro increased catalase protein expression in head kidney- and liver cells, regardless of culturing methods and LPS treatment. Leukotriene B4 (LTB4) production was also increased by H-pro in head kidney cells co cultured with liver cells. H-pro increased LPS induced interleukin 1ß (IL-1ß) transcription in liver cells co cultured with head kidney cells. All cultures of head kidney cells showed a significant increase in IL-1ß transcription when treated with H-pro + LPS. H-pro decreased caspase-3 transcription in liver cells cultured co cultured with head kidney cells. Peroxisome proliferator activated receptor α (PPAR α) was upregulated, regardless of treatment, in liver cells co cultured with head kidney cells clearly showing that culturing method alone affected gene transcription. H-pro alone and together with LPS as an inflammation inducer, affect both antioxidant and inflammatory responses.

A co culture approach show that polyamine turnover is affected during inflammation in Atlantic salmon immune and liver cells and that arginine and LPS exerts opposite effects on p38MAPK signaling.

This study assess which pathways and molecular processes are affected by exposing salmon head kidney cells or liver cells to arginine supplementation above the established requirements for growth support. In addition to the conventional mono cultures of liver and head kidney cells, co cultures of the two cell types were included in the experimental set up. Responses due to elevated levels of arginine were measured during inflammatory (lipopolysaccharide/LPS) and non -inflammatory conditions. LPS up regulated the genes involved in polyamine turnover; ODC (ornithine decarboxylase), SSAT (spermidine/spermine-N1-acetyltransferase) and SAMdc (S-adenosyl methionine decarboxylase) in head kidney cells when co cultured with liver cells. Regardless of treatment, liver cells in co culture up regulated ODC and down regulated SSAT when compared to liver mono cultures. This suggests that polyamines have anti-inflammatory properties and that both salmon liver cells and immune cells seem to be involved in this process. The transcription of C/EBP ß/CCAAT, increased during inflammation in all cultures except for liver mono cultures. The observed up regulation of this gene may be linked to glucose transport due to the highly variable glucose concentrations found in the cell media. PPARα transcription was also increased in liver cells when receiving signals from head kidney cells. Gene transcription of Interleukin 1ß (IL-1ß), Interleukin-8 (IL-8), cyclooxygenase 2 (COX2) and CD83 were elevated during LPS treatment in all the head kidney cell cultures while arginine supplementation reduced IL-1ß and IL-8 transcription in liver cells co cultured with head kidney cells. This is probably connected to p38MAPK signaling as arginine seem to affect p38MAPK signaling contrary to the LPS induced p38MAPK signaling, suggesting anti-inflammatory effects of arginine/arginine metabolites. This paper shows that co culturing these two cell types reveals the connection between metabolism and inflammation, suggesting different pathways and candidate biomarkers to be further explored.

Isolation and characterisation of two cDNAs encoding transglutaminase from Atlantic cod (Gadus morhua).

Two cDNAs encoding transglutaminase (TG) were identified in a subtractive cDNA library prepared from the head kidney of poly I:C stimulated Atlantic cod (Gadus morhua). Full-length TG-1 and TG-2 cDNA were cloned from the head kidney by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The deduced amino acid (aa) sequence for TG-1 was 695 aa with an estimated molecular mass of 78.3 kDa, while TG-2 was a 698 aa protein with an estimated molecular mass of 78.8 kDa. The two proteins were named TG-1 and TG-2 and both possess transglutaminase/protease-like homologous domains (TGc) and full conservation of amino acids cysteine, histidine, and aspartate residues that form the catalytic triad. Sequence analysis showed high similarity (93.1%) with Alaska pollock TG, and the TGs were grouped together with TGs from chum salmon, Japanese flounder, Nile tilapia, and red sea bream in addition to Alaska pollock in phylogenetic analysis. Interestingly, they showed different tissue distribution with highest constitutive expression in reproductive and immunological organs, indicating important roles in these organs. Furthermore, the up-regulation of TG-1 and TG-2 in head kidney after stimulating Atlantic cod with poly I:C suggested a role of TGs in immune response in Atlantic cod.

In situ forming microparticle implants for delivery of sex steroids in fish: Modulation of the immune response of gilthead seabream by testosterone.

Current knowledge on the sensitivity of marine fish to androgenic environmental chemicals is limited, despite the growing interest in the effects of endocrine disrupting chemicals. To study in vivo the effects of testosterone (T) on the fish immune response, we used a microencapsulation implant technique, the in situ forming microparticle system, containing 1 mg T/kg body weight (T-ISM), in adult specimens of gilthead seabream (Sparus aurata L.), a species of great economic interest. We demonstrated that implants themselves (without T) have no significant effect on most of the parameters measured. In T-ISM implanted fish, T serum levels reached supraphysiological concentrations accompanied by a slight increase in 11-ketotestosterone and 17ß-estradiol levels 21 days post-implantation (dpi). Liver and head-kidney samples were processed 7 and 21 dpi to assess T-ISM effect on (i) the mRNA expression of genes involved in the metabolism of steroid hormones and in the immune response, and (ii) phagocyte activities. The expression profile of cytokines, chemokines and immune receptors was altered in T-ISM implanted animals that showed an early pro-inflammatory tendency, and then, a mixed pro-/anti-inflammatory activation during longer exposure. Furthermore, the enhancement of phagocytic activity and the production of reactive oxygen species by leukocytes 21 dpi in T-ISM implanted specimens suggest fine modulation of the innate immune response by T. Taken together, these data demonstrate for the first time the feasibility of using ISM implants in an aquatic species, and provide new data on the role played by T on the immune response in fish.

Evidence for pituitary adenylate cyclase-activating peptide as a direct immunoregulator in teleost head kidney.

In mammals, pituitary adenylate cyclase activating polypeptide (PACAP) is a potent anti-inflammatory factor, showing that it inhibits the expression and release of proinflammatory cytokines and enhances the production of anti-inflammatory factors. However, whether fish PACAP plays similar regulatory roles as seen in mammals remains unclear. In the present study, expression of PACAP-specific receptor PAC1-R was shown in grass carp head kidney and spleen, supporting that PACAP may have a direct effect on fish immune cells. To test this hypothesis, the immunoregulatory role of grass carp PACAP (gcPACAP) was examined in head kidney leucocytes (HKLs). Results showed that gcPACAP inhibited basal and further attenuated lipopolysaccharide (LPS)-stimulated cell viability of HKLs, indicating that gcPACAP may possess similar inhibitory property at cellular level as seen in mammals. Curiously, in vitro and in vivo studies revealed that gcPACAP stimulated proinflammatory factors (IL-1ß and TNF-α) but not IL-10 mRNA expression in HKLs and head kidney. Moreover, bacterial infection and LPS enhanced IL-1ß, TNF-α and IL-10 mRNA expression in grass carp head kidney and HKLs, respectively, and these stimulatory effects were not influenced by gcPACAP. These findings suggest that PACAP plays distinct roles, at least does not function as an anti-inflammatory factor, in fish compared with that in mammals.

Cadmium and copper reduce hematopoietic potential in common carp (Cyprinus carpio L.) head kidney.

The effects of cadmium and copper on activity of common carp head kidney hematopoietic tissue were evaluated. The fish were subjected to short-term (3 h, Cd-s and Cu-s) or long-term (4 weeks, Cd-l and Cu-l) exposures to 100% 96hLC50 or 10% 96hLC50, respectively. Head kidneys were isolated weekly from 5 fish of each group for 4 weeks (post-short-term exposure and during long-term exposure). Percentage of early blast cells among the hematopoietic precursors was calculated. Proliferative and apoptotic activity were evaluated using immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and caspase 3, respectively. Hematopoietic activity was calculated as the ratio of proliferating to apoptotic cells. All metal exposures induced an increase in frequency of early blast cells. The frequency of proliferating (PCNA-positive) cells also significantly increased. A considerable and significant increase in the frequency of apoptotic cells was the most pronounced effect of metal exposures. Both short-term and long-term treatments caused similar effects, but in case of Cd exposures, the reaction was more pronounced. All metal exposures reduced hematopoietic potential of fish measured as the ratio of proliferating to apoptotic precursor cell frequency. However, in all cases, hematopoietic activity was higher than 1 showing that the rate of repair of hematopoietic tissue prevailed over destruction.

Matrix metalloproteinase 2 of grass carp Ctenopharyngodon idella (CiMMP2) is involved in the immune response against bacterial infection.

The gene encoding matrix metalloproteinase 2 (MMP2) was cloned from grass carp (Ctenopharyngodon idella), and its expression levels during Aeromonas hydrophila infection and embryonic development stages were evaluated. The complete open reading frame of CiMMP2 was 1974 bp in length, encoding a 658-amino acid polypeptide. The deduced MMP2 protein contained four conserved domain structures, including an N-terminal signal sequence, a propeptide domain, three repeats of fibronectin-type II domain inserted in the catalytic domain and a C-terminal hemopexin-like domain. Phylogenetic analysis of MMP2s grouped grass carp with other teleosts. Detected in all fish tissues examined, CiMMP2 expression increased in the spleen and head kidney at 4 h and was significantly downregulated at 1 d after A. hydrophila infection. CiMMP2 transcripts were present in unfertilized eggs, suggesting its maternal origin. These findings implicate an important role for CiMMP2 in A. hydrophila-related diseases and early embryonic developmental stages of grass carp.