Immigrant reproductive dysfunction facilitates ecological speciation.

The distributions of species are not only determined by where they can survive - they must also be able to reproduce. Although immigrant inviability is a well-established concept, the fact that immigrants also need to be able to effectively reproduce in foreign environments has not been fully appreciated in the study of adaptive divergence and speciation. Fertilization and reproduction are sensitive life-history stages that could be detrimentally affected for immigrants in non-native habitats. We propose that "immigrant reproductive dysfunction" is a hitherto overlooked aspect of reproductive isolation caused by natural selection on immigrants. This idea is supported by results from experiments on an externally fertilizing fish (sand goby, Pomatoschistus minutus). Growth and condition of adults were not affected by non-native salinity whereas males spawning as immigrants had lower sperm motility and hatching success than residents. We interpret these results as evidence for local adaptation or acclimation of sperm, and possibly also components of paternal care. The resulting loss in fitness, which we call "immigrant reproductive dysfunction," has the potential to reduce gene flow between populations with locally adapted reproduction, and it may play a role in species distributions and speciation.

Mixture effects between different azoles and ß-naphthoflavone on the CYP1A biomarker in a fish cell line.

The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) - CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist ß-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2-4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48h, whereas exposure to the omeprazole for 48h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50=1.3-7.7µM), whereas omeprazole had no effect on this activity (IC50=72µM). Exposure to 10µM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10µM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24h at concentrations that are known to disassemble microtubules at 24h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules.

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor.

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.

Interactions of pharmaceuticals and other xenobiotics on key detoxification mechanisms and cytoskeleton in Poeciliopsis lucida hepatocellular carcinoma, PLHC-1 cell line.

Fish are exposed to chemicals, including pharmaceuticals, in their natural habitat. This study focuses on effects of chemicals, including nine classes of pharmaceuticals, on key detoxification mechanisms in a fish liver cell-line (PLHC-1). Chemical interactions were investigated on efflux pumps, P-glycoprotein (Pgp) and multidrug resistance associated proteins (MRP1/MRP2), and on biotransformation enzymes, cytochrome P450 (CYP1A/CYP3A). Diclofenac and troleandomycin inhibited efflux activities, whereas ethinylestradiol activated efflux function. Exposure to troleandomycin and ß-naphthoflavone induced MRP2 mRNA levels, but no effects were seen on MRP1 or Pgp expressions. Inhibition of CYP1A activities were seen in cells exposed to α-naphthoflavone, ß-naphthoflavone, clotrimazole, nocodazole, ketoconazole, omeprazole, ethinylestradiol, lithocholic acid, rifampicin and troleandomycin. Exposure to fulvestrant, clotrimazole and nocodazole resulted in induction of CYP1A mRNA levels. Although, exposure to nocodazole resulted in disassembled microtubules. A CYP3A-like cDNA sequence was isolated from PLHC-1, but basal expression and activities were low and the gene was not responsive to prototypical CYP3A inducers. Exposure to ibuprofen, lithocholic acid and omeprazole resulted in fragmentation of microtubules. This study revealed multiple interactions on key detoxification systems, which illustrates the importance of study effects on regulation combined with functional studies to provide a better picture of the dynamics of the chemical defense system.

One-way inhibiting cross-talk between arylhydrocarbon receptor (AhR) and estrogen receptor (ER) signaling in primary cultures of rainbow trout hepatocytes.

The aryl hydrocarbon receptor (AhR) and the estrogen receptor (ER) are ligand-activated transcription factors, both of which can be activated by environmental pollutants. The AhR regulates cytochrome P450 1A (CYP1A) expression and can be induced by aromatic hydrocarbons. The ER regulates vitellogenin (VTG) expression and can be induced by estrogenic substances. Both receptor responses are established biomarkers used to assess the effects of pollutants in the aquatic environment. The receptors can also be affected in situations of mixed exposure. Cross-talk between these receptor pathways has been suggested, although there are conflicting data in the literature. We investigated cross-talk between ER-VTG and AhR-CYP1A signaling pathways in primary cultures of rainbow trout hepatocytes, using quantitative PCR (qPCR) for mRNA analyses and studies of CYP1A catalytic function and protein expression. The model agonists ß-naphthoflavone (BNF) and 17α-ethinylestradiol (EE(2)) were used for AhR and ER activation, respectively. Combined exposure to BNF and EE(2) reduced the EE(2)-mediated induction of VTG mRNA levels by about 40%, but had no effect on the BNF-mediated CYP1A mRNA levels, indicative of a one-way inhibiting AhR-ER cross-talk. However, basal levels of CYP1A mRNA were reduced 40% upon exposure to EE(2) alone, implying different cross-talk mechanism between basal and induced CYP1A mRNA levels. The mammalian ER antagonist fulvestrant (ICI) is commonly described as an absolute ER antagonist. However, ICI failed to reverse the ER activation caused by EE(2) in the present study. The CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activity was reduced by 80% in cells co-treated with BNF and EE(2), compared to cells exposed to BNF alone. In vitro inhibiting studies suggests that this reduction was a result of inhibition of the CYP1A catalyst by EE(2) since EE(2) acted as a potent inhibitor (IC(50): 4.6µM) of the EROD activity. In addition, ICI also acted as a potent inhibitor of the EROD enzyme (IC(50): 0.6µM). Taken together, our data supports a one-way inhibiting AhR-ER cross-talk in rainbow trout hepatocytes exposed to a mixture of BNF and EE(2).

Interactions of pharmaceuticals and other xenobiotics on hepatic pregnane X receptor and cytochrome P450 3A signaling pathway in rainbow trout (Oncorhynchus mykiss).

The pregnane X receptor (PXR) belongs to the nuclear hormone receptor (NR) superfamily and is commonly described as a xenophore or a pharmacophore, as it can be activated by a wide array of xenobiotics, including numerous pharmaceuticals and other environmental pollutants. The PXR regulates expression of e.g. cytochrome P450 3A (CYP3A) and the P-glycoprotein (P-gp) that are involved in excretion of lipophilic xenobiotics and endobiotics. A full length PXR cDNA was isolated from rainbow trout liver and it was expressed in a descending order of magnitude in liver>intestine>kidney>heart. A rainbow trout PXR reporter assay was developed and a suite of pharmaceuticals and other xenobiotics were screened. However, no specific activation of rainbow trout PXR was observed with the substances tested. Interactions of prototypical PXR agonists on PXR signaling in rainbow trout were further investigated in cells of hepatic origin exposed in vitro and in juvenile rainbow trout exposed in vivo. The rainbow trout hepatoma cell line (RTH-149), displayed 600 times lower expression of CYP3A mRNA compared to primary cultures of hepatocytes, and did not respond to treatment with either pregnenolone 16α-carbonitrile (PCN), ketoconazole (KCZ) or rifampicin (RIF), which implies a non-functional PXR in this cell line. Exposure of hepatocytes to PCN and lithocholic acid (LA), resulted in a weak concentration-dependent induction of CYP3A and P-gp mRNA levels, though, exposure to the higher concentration of LA (50 µM) decreased PXR mRNA levels. Exposure to dexamethasone (DEX) resulted in a decrease in PXR mRNA, without affecting CYP3A mRNA levels in hepatocytes in vitro. Injections of rainbow trout in vivo with 1 mg LA/kg fish resulted in a slight (albeit not significant) increase in CYP3A mRNA levels without affecting PXR mRNA levels. Although, injection with 10mg omeprazole (OME)/kg fish had no effect on PXR and CYP3A mRNA levels, a 60% inhibition of CYP3A enzyme activities was evident. An in vitro screening of the chemicals used showed that OME and RIF acted as weak CYP3A inhibitors whereas LA and DEX did not affect the CYP3A activity. In contrast, PCN acted as an activator of the CYP3A enzyme activity in vitro. Taken together, these data show that some prototypical PXR agonists weakly affect PXR activation in rainbow trout. Besides, some of these agonists have a stronger effect on the CYP3A catalyst. This study demonstrates the importance of investigation effects of pharmaceuticals on the PXR signaling pathway in non-target animals such as fish.