P450 1A Research Articles

Identification, functional characterization and expression profiling of cytochrome p450 1A (CYP1A) gene in Labeo rohita against emamectin benzoate

The cytochrome p450 1A (CYP1A) plays vital role in detoxification of xenobiotic compounds in living organisms. In the present study, full-length CYP1A gene was sequenced from liver of Labeo rohita and mRNA expression analysis were carried out at 0, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h (h) time points after emamectin benzoate treatment. The full-length cDNA sequence of CYP1A was 1741 bp which consist of open reading frame (ORF) of 1618 bp, 5′-untranslated region (UTR) 48 bp and 75 bp 3′-UTR respectively. ORF encodes 526 amino acids with a molecular mass a 59.05 kDa and an isoelectric point of 8.74. The subcellular localization confirmed presence of the CYP1A protein was higher in plasma membrane (45.8%), followed by the mitochondrial region (13.9%) and nuclear region (9.2%). The CYP1A protein interaction was found to intermingle more with other CYP family proteins. Analysis of tissue distribution revealed that CYP1A gene was predominantly expressed in the liver compared to other tissues kidney, gills, muscle and intestine. Furthermore, present study reveals that CYP1A mRNA level in emamectin benzoate treated group @ 20 mgkg−1 body was significantly (p < 0.05) higher compared with the control. The CYP1A mRNA expression levels were found upregulating with time and highest expression levels at 24 h. Histological examination found that emamectin benzoate treated liver revealed vacuolisation, hepatocyte infiltrations, cytoplasmic degeneration of hepatocytes compared to control. Overall, present results lay a strong basis for CYP1A is important biomarker for drug detoxification in aquatic animals.

Correlation between cytochrome P450 1A (cyp1a) mRNA expression and ambient phenanthrene and pyrene concentration in Javanese Medaka Oryzias javanicus

Cytochrome P450 1A (Cyp1a) catabolizes polycyclic organic compounds. In this study, we sequenced the cyp1a gene from two Javanese medaka Oryzias javanicus strains and examined gene expression in response to two organic pollutants, phenanthrene and pyrene. Sequence comparisons among the two strains identified several single-nucleotide polymorphisms (SNPs), two of which resulted in amino acid substitutions. Reverse transcription PCR analysis of major organs revealed that the cyp1a mRNA level is highest in the liver. After determining LC50s against phenanthrene and pyrene for 96 h (3.9 and 2.5 µM, respectively), cyp1a mRNA expression in the liver against sublethal concentrations of phenanthrene and pyrene was examined. Using quantitative real-time PCR, a positive correlation was discovered between the cyp1a mRNA levels and pyrene concentration, and similar tendency was observed for phenanthrene. cyp1a may serve as a quantitative indicator of a broad range of pollutants.

Gene expression and histopathological changes of Nile tilapia (Oreochromis niloticus) infected with Aeromonas hydrophila and Pseudomonas fluorescens

Hepcidin, interleukin 1-β (IL-1β), and cytochrome P450 1A (CYP1A) genes play an important role in the adaptive immune system in fish. To our knowledge, this is the first observation among the expression level of these genes in response to infection with Aeromonas hydrophila and Pseudomonas fluorescens in Nile tilapia (Oreochromis niloticus). Also, the effect of these experimentally induced infections was investigated regarding the mortality rate, clinical signs, and histopathological changes in the liver and kidney tissues. Three experimental fish groups were injected intraperitoneally (IP) by 0.1 ml of phosphate-buffered saline, two of these groups were injected by 0.1 ml of 1 × 105 CFU/ml of the suspension of either A. hydrophila or P. fluorescens. The infected groups showed extensive skin hemorrhages, cloudiness of eyes, protrusion of the scales, and severe swelling of the abdomen with inflammation of the vent and the mortality rate (MR%) was 42.75 and 37.5% in A. hydrophila and P. fluorescens groups, respectively. The histological changes of the infected O. niloticus with A. hydrophila and P. fluorescens showed fibrosis, karyolitic necrosis, the disappearance of hepatocytes wall and deposits of hemosiderin in the liver, while kidney showed dilation in Bowman's spaces, disconnection of renal tubules and hemorrhage and necrotic areas between them besides the accumulation of hemosiderin. The relative expression levels of hepcidin were higher after A. hydrophila than P. fluorescens infection in most studied organs while other studied genes expression levels were varied between infected fish groups. The relative expression of hepcidin was increased in the liver, blood, pituitary, kidney, and ovary while IL-1β transcripts were increased in the liver, kidney, muscle, gill, brain, and blood. CYP1A transcripts were increased in the liver, kidney, gill, and testis. The magnitude of the increase in IL-1β levels differed from that observed for the hepcidin and CYP1A genes. The relative expression levels of studied genes were significantly increased in the liver followed by the kidney. No significant differences were observed in the relative expression of studied genes among the gill, blood, brain, and pituitary and among the ovary, testis, brain, and pituitary during the bacterial challenge (p < .05). These findings in response to pathogen challenge could improve our understanding of the defense mechanism of adaptive immunity genes in O. niloticus organs.

Effects of short-term exposure to environmentally-relevant concentrations of benzo(a)pyrene-sorbed polystyrene to White seabass (Atractoscion nobilis)☆

Plastic marine debris hyper-concentrates hydrophobic contaminants such as polycyclic aromatic hydrocarbons (PAHs) and can transfer these sorbed contaminants to biota following ingestion. PAHs are known to induce cardiotoxicity and visual toxicity at sublethal doses. Juvenile White seabass (Atractoscion nobilis) fish were fed environmentally relevant concentrations of either virgin polystyrene or benzo(a)pyrene (BaP)-sorbed polystyrene for 5 days and were monitored for changes in phototactic response, swimming behavior, and hepatic cytochrome p450 1A (CYP1A) enzyme activity. No significant differences in the monitored endpoints were recorded in fish that ingested either polystyrene or BaP-sorbed polystyrene relative to control fish following the short-term exposure. However, fish exposed to 252 μg/L BaP alone as a positive control had significantly elevated CYP1A enzyme activity (p = 0.046) and impaired phototactic response (p = 0.020), though no altered swimming behavior was observed (p = 0.843) relative to control fish. These results demonstrate that pelagic fish ingesting environmentally relevant concentrations of BaP-sorbed polystyrene for a short, 5-day duration do not demonstrate measurable changes in vision, swimming activity, nor CYP1A activity. High variability within enzyme activity and behavioral responses suggest that lack of significant effects may be due to low sample size.

Molecular characterization and expression of arginine vasotocin V1a2 receptor in Atlantic croaker brain: Potential mechanisms of its downregulation by PCB77.

The arginine vasotocin (AVT)-V1a receptor mediates critical reproductive behaviors of the nonapeptide vasotocin in the teleost brain. In this study, we report the molecular characterization of the AVT-V1a2 receptor and its messenger RNA (mRNA) and protein expressions in the Atlantic croaker brain after exposure to the planar polychlorinated biphenyl congener 3,3',4,4'-tetrachlorobiphenyl (PCB77). The full-length sequence of croaker AVT-V1a2 receptor complementary DNA (cDNA) is highly homologous to other teleost AVT-V1a2 receptor cDNAs. Double-labeled immunohistochemistry showed coexpression of AVT-V1a2 receptor and gonadotropin-releasing hormone-I (GnRH-I, a neuropeptide that regulates gonadotropin secretion) in hypothalamic neurons, thereby providing the anatomical basis for possible AVT modulation of croaker reproduction through alterations in GnRH-I secretion. AVT-V1a2 receptor mRNA and protein levels as well as GnRH-I mRNA levels were markedly decreased in hypothalamic tissues of croaker exposed to PCB77 (dose: 2 and 8 µg/g body weight for 4 weeks) compared with levels in untreated (control) fish. In contrast, hypothalamic cytochrome P450 1A (CYP1A, a monooxygenase enzyme) and interleukin-1β (IL-1β, a cytokine indicator of inflammation and response to neuronal damage) mRNA levels, and plasma protein carbonyl (PC, an indicator of reactive oxygen species) contents, important biomarkers of neural stress, were increased in PCB77-exposed fish compared with controls. Collectively, these results suggest that the downregulation of hypothalamic AVT-V1a2 receptor and GnRH-I transcripts due to PCB77 exposure is associated with induction of CYP1A, cellular inflammation and oxidative stress in Atlantic croaker, a marine teleost that inhabits estuaries along the US Atlantic coast and the Gulf of Mexico that are often contaminated with persistent organic pollutants such as PCBs.

Dual immunological and oxidative responses in Oreochromis niloticus fish exposed to lambda cyhalothrin and concurrently fed with Thyme powder (Thymus vulgaris L.): Stress and immune encoding gene expression

The present study was performed to explore the immunotoxicological effects of the lambda cyhalothrin (LCH) insecticide and evaluate the efficiency of Thyme powder (TP) as a fish supplement in attenuation of LCH impact on Oreochromis niloticus (O. niloticus) fish. Fish was sampled following 30-days exposure to LCH (1/6 LC50: 0.48 μg/L) and TP (2%) supplementation, individually or in combination. The growth performance, immune status, biochemical indices, and mRNA expression pattern changes of stress and immune-encoding genes in the liver and spleen tissues, respectively, through real-time polymerase chain reaction (RT-PCR) analysis, were evaluated. The findings showed that LCH exposure caused a significant lowering in most of the estimated variables including growth performance, hematological and immunological indices. Moreover, LCH disrupted the oxidant/antioxidant status and dysregulated the expression of stress and immune-related genes, downregulating the mRNA transcript level of Immunoglobulin M heavy chain (IgM), Interferon (IFN-γ), CXC-chemokine, and Toll-like receptors (TLR-7) in the spleen. However, mRNA expression of Myxovirus resistance (Mx) gene remained unaffected. In liver tissue, the heat shock protein (HSP-70) expression was upregulated, while that of cytochrome P450 1A (CYP 1A) was downregulated. TP (2%) supplementation elicited a significant modulation in aforementioned indices; however, their levels did not attain that of the control values. Our findings concluded that LCH affects the O. niloticus immune response through the negative transcriptional influence on genes linked to immunity and induction of oxidative injury of the immune organs. Besides, dietary TP (2%) could be a proper candidate to modulate the compromised immunity in response to LCH exposure in O. niloticus aquaculture.

Effects of metal contamination on liver in two fish species from a highly impacted neotropical river: A case study of the Fundão dam, Brazil.

Environmental disasters such as the rupturing of mine tailings dams are a major concern worldwide. In the present study, we assess the effects of the release of mine waste due to the rupture of the Fundão dam on two native fish species (Hoplias intermedius and Hypostomus affinis) from the Doce River basin. Two sampling sites were chosen: S1, a reference site, and S2, contaminated by mining waste. Water and sediment were collected to evaluate metals concentration. Adult fish were caught to analyse biological parameters, hepatic histopathology, and biomarkers of metal contamination. Compared to site S1, the concentration of manganese was statistically higher in water while lead, nickel, and arsenic were statistically higher in the sediment from site S2, and iron had no significant difference between sites. At site S1, fish of both species presented hepatic tissue with normal architecture. At site S2, hepatic alterations, such as cytoplasmic vacuolization and necrosis were frequently found in both species. Regarding the histopathological index, higher values were found in both species from site S2. The positive antibody reactions for cytochrome P450 1A (CYP1A) and metallothionein (MT) were statistically greater in site S2 for both species. The oxidative stress biomarkers, superoxide dismutase (SOD) and catalase (CAT) were statistically higher in H. intermedius from site S2, but only CAT was statistically greater in H. affinis at site S2. These results demonstrate that the release of mineral residues from the rupture of the Samarco mine dam is provoking hepatic damage in the fish from the Doce River besides inducing the expression of proteins and enzymes related to metal contamination.

Exacerbation of gefitinib-induced liver injury by glutathione reduction in mice.

Gefitinib (GEF) is the first selective tyrosine kinase inhibitor of epidermal growth factor receptor. It is associated with the occurrence of clinical drug-induced liver injury. Although GEF is metabolized to chemically reactive metabolites by cytochrome P450 3A and 1A enzymes and then conjugated to glutathione (GSH), whether these reactive metabolites contribute to GEF-induced toxicity remains unknown. In this study, we investigated whether GSH depletion can sensitize mice to liver injury caused by GEF. Male C57BL/6J mice were intraperitoneally pretreated with L-buthionine (S,R)-sulfoximine (BSO) at 700 mg/kg to inhibit GSH synthesis and then orally administered GEF at 500 mg/kg every 24 hr for 4 consecutive days. The coadministration of BSO and GEF increased plasma alanine aminotransferase (ALT) levels to approximately 700 U/L and 1600 U/L at 72 and 96 hr after the first administration, respectively, whereas the increase in plasma ALT levels in mice receiving GEF at 500 mg/kg alone was limited, suggesting that GSH plays a protective role in GEF-induced liver injury. Histological examination showed nuclear karyorrhexis and sporadic single hepatocyte death in the livers of BSO+GEF coadministered mice. In these mice, the hepatic expression levels of heme oxygenase 1 (Hmox1) and metallothionein 2 (Mt2) mRNA, caspase 3/7 enzymatic activity, and the amounts of 2-thiobarbiuric acid reactive substances were significantly increased, suggesting the presence of oxidative stress, which may be associated with hepatocellular death. Together, these results show that oxidative stress as well as the reactive metabolites of GEF are involved in GEF-induced liver injury in GSH-depleted mice.

Inhibitory Effects of Several Fluoroquinolones on Feline CYP1A and 3A in Hepatic Microsomes

In this study, the effects of several fluoroquinolones (FQs), such as Ciprofloxacin (CPFX); Orbifloxacin (OBFX); Norfloxacin (NFX); Ofloxacin (OFX); and Enerofloxacin (EFX) on activities of both Cytochrome P450 1A (CYP1A) and Cytochrome P450 3A (CYP3A) of feline microsomes by in vitro tests were studied. Ethoxyresorufin O-deethylation (EROD) and Midazolam 1' hydroxylation and 4-hydroxylation (MDZ1'H and MDZ4H) were analyzed by High Performance Liquid Chromatography (HPLC). All the FQs inhibited the reactions by a competitive or noncompetitive and irreversible manner. The inhibitory constants (Ki) were as followings: CYP1A; ranged from 0.12 to 1.23 mM for NFX, OBFX, EFX, CPFX, OFX and CYP3A, for MDZ1'H; ranged from 5.8 to 35 and MDZ4H; 9 to 29 mM, respectively. As these values are higher by 24 to 200-times of given single clinical dose of serum levels after application of FQs. It indicates that if co-administrated with these FQs by reversible inhibitory manner, the inhibition of CYP1A and CYP3A effect on CYP1A and 3A actions is not very significant to cause drug interaction with above mentioned enzyme substrates. Out of the FQs tested, CPFX and NFX for CYP1A, and CPFX for CYP3A showed irreversible inhibitory effects (time-dependent), so it has been concluded that these drugs may cause drug-drug interaction by accumulation, when they are repeatedly administrated. Since EFX is biotransformed to CPFX by the liver, it could have the identical risk too.

Phylogenetic Analysis and In Silico Characterization of Cytochrome P450 1A (Cyp1A) Protein from the African Catfish, Clarias gariepinus (Burchell, 1822)

The CYP family enzymes are broadly used as biomarkers because of their pattern of expression. This study describes the application of in silico tools to predict the physico-chemical characters of CYP1A protein from the catfish, Clarias gariepinus. The nucleotide sequence analysis of C. gariepinus CYP1A gene showed higher similarity with C. batrachus and reflected in the phylogenetic tree. The comparative modelling results showed this CYP1A protein was highly similar with the 3-D crystal structure of human Cytochrome p450 1A1 (PDB: 1BE3). The prediction results depicted that most of the amino acids formed alpha helix. The predicted pI was 9.10, hydropathycity was -0.226, exposed and buried residues were 61.67, 38.33% respectively. Ramachandran plot analysis showed that most of the amino acids falling on the favoured region and exhibited right- handed alpha helices as the most stable secondary structure. Some amino acids were also found to form loops to interconnect different helices. The CYP1A protein was predicted to be localized in the mitochondrion of the eukaryotic cell.

Functional cytochrome P450 1A enzymes are induced in mouse and human islets following pollutant exposure

Aims/hypothesisExposure to environmental pollution has been consistently linked to diabetes incidence in humans, but the potential causative mechanisms remain unclear. Given the critical role of regulated insulin secretion in maintaining glucose homeostasis, environmental chemicals that reach the endocrine pancreas and cause beta cell injury are of particular concern. We propose that cytochrome P450 (CYP) enzymes, which are involved in metabolising xenobiotics, could serve as a useful biomarker for direct exposure of islets to pollutants. Moreover, functional CYP enzymes in islets could also impact beta cell physiology. The aim of this study was to determine whether CYP1A enzymes are activated in islets following direct or systemic exposure to environmental pollutants.MethodsImmortalised liver (HepG2) and rodent pancreatic endocrine cell lines (MIN6, βTC-6, INS1, α-TC1, α-TC3), as well as human islets, were treated in vitro with known CYP1A inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3-MC). In addition, mice were injected with either a single high dose of TCDD or multiple low doses of TCDD in vivo, and islets were isolated 1, 7 or 14 days later.ResultsCYP1A enzymes were not activated in any of the immortalised beta or alpha cell lines tested. However, both 3-MC and TCDD potently induced CYP1A1 gene expression and modestly increased CYP1A1 enzyme activity in human islets after 48 h. The induction of CYP1A1 in human islets by TCDD was prevented by cotreatment with a cytokine mixture. After a systemic single high-dose TCDD injection, CYP1A1 enzyme activity was induced in mouse islets ~2-fold, ~40-fold and ~80-fold compared with controls after 1, 7 and 14 days, respectively, in vivo. Multiple low-dose TCDD exposure in vivo also caused significant upregulation of Cyp1a1 in mouse islets. Direct TCDD exposure to human and mouse islets in vitro resulted in suppressed glucose-induced insulin secretion. A single high-dose TCDD injection resulted in lower plasma insulin levels, as well as a pronounced increase in beta cell death.Conclusions/interpretationTransient exposure to TCDD results in long-term upregulation of CYP1A1 enzyme activity in islets. This provides evidence for direct exposure of islets to lipophilic pollutants in vivo and may have implications for islet physiology.

Nonlethal Biomarkers of Oxidative Stress in Oiled Sediment Exposed Southern Flounder (Paralichthys lethostigma): Utility for Field-Base Monitoring Exposure and Potential Recovery.

The Deepwater Horizon (DWH) blowout resulted in the deposition of toxic polycyclic aromatic hydrocarbons (PAHs), in the coastal sediments of the Gulf of Mexico. The immediate effects on an ecosystem from an oil spill are clearly recognizable, however the long-term chronic effects and recovery after a spill are still not well understood. Current methodologies for biomonitoring wild populations are invasive and mostly lethal. Here, two potential nonlethal biomonitoring tools for the assessment of PAH toxicity and induced biological alterations in the field, were identified using laboratory-validated methods. In this study, subadult southern flounder (Paralichthys lethostigma) were chronically exposed to DWH surrogate oiled sediments for 35 days; a subset of these exposed flounder were then provided a clean nonexposure period to ascertain the utility of selected biomarkers to monitor recovery post exposure. After chronic exposure, there was an increase in gene expression of cytochrome P450 1A but not glutathione S-transferase. There was also a notable imbalance of oxidants to antioxidants, measured as reduced glutathione, oxidized glutathione, and their ratio in the blood. Evidence of subsequent oxidative damage due to chronic exposure was found through lipid peroxidation and DNA damage assessments of liver, gill, and blood.

Biological responses of shoal flounder (Syacium gunteri) to toxic environmental pollutants from the southern Gulf of Mexico

The Gulf of Mexico (GoM) is exposed to a diversity of contaminants, such as hydrocarbons and heavy metal(oid)s, either from natural sources or as a result of uncontrolled coastal urbanisation and industrialisation. To determine the effect of these contaminants on the marine biota along the Mexican GoM, the biological responses of the shoal flounder Syacium gunteri, naturally exposed, were studied. The study area included all the Mexican GoM, which was divided into three areas: West-southwest (WSW), South-southwest (SSW) and South-southeast (SSE). The biological responses included the global DNA methylation levels, the expression of biomarker genes related to contaminants (cytochrome P450 1A, glutathione S-transferase, glutathione reductase, glutathione peroxidase, catalase, and vitellogenin), histopathological lesions and PAH metabolites in bile (hydroxynaphthalene, hydroxyphenanthrene, hydroxypyrene and Benzo[a]pyrene). The correlation between the biological responses and the concentration of contaminants (hydrocarbons and metal(oid)s), present in both sediments and organisms, were studied. The shoal flounders in WSW and SSW areas presented higher DNA hypomethylation, less antioxidative response and biotransformation gene expression and a higher concentration of PAH metabolites in bile than SSE area; those responses were associated with total hydrocarbons and metals such as chromium (Cr). SSE biological responses were mainly associated with the presence of metals, such as cadmium (Cd) and copper (Cu), in the tissue of shoal flounders. The results obtained on the physiological response of the shoal flounder can be used as part of a permanent active environmental surveillance program to watch the ecosystem health of the Mexican GoM.

Assessment of Gene Expression Biomarkers in the Chilean Pencil Catfish, Trichomycterus areolatus, from the Choapa River Basin, Coquimbo Chile.

The objective of this study was to describe changes in the gene expression in the Chilean catfish, Trichomycterus areolatus, based on their geographic location within the Choapa River. Genes of choice included those that are biomarkers of exposure to metals, oxidative stress, and endocrine disruption. Male and female T. areolatus were sampled from four sites in January 2015 differently impacted by human activities. In males, but not females, hepatic gene expression of heat shock protein (HSP70) and cytochrome P450 1A (CYP1A) were significantly elevated at the site adjacent to the small city of Salamanca, relative to the other sites. In females, hepatic HSP70, the aryl hydrocarbon receptor (AHR), and the estrogen responsive genes, vitellogenin (VTG) and estrogen receptor alpha (ERα), were significantly lower at the site located furthest downstream. A similar downstream pattern of lower expression levels also was found in ovarian tissue for the genes, HSP70 and ERα. Gill gene expression showed a unique pattern in females as levels of metallothionein were elevated at the site furthest downstream. While analytical chemistry of water samples provided limited evidence of agrichemical contamination, the gene expression data are consistent with an exposure to agrichemicals and metals. T. areolatus may be a valuable sentinel organism and its use as a bioindicator species in some rivers within Chile can provide considerable insight, particularly in situations analytical chemistry is limited by environmental constraints.

Self-protective transcriptional alterations in ZF4 cells exposed to Pb(NO3 )2 and AgNO3.

In this study, gene expression alterations of phase I to III enzymes, transcription factors, and microRNA (miRNA) in embryonic zebrafish fibroblasts (ZF4) cells after the treatment of Pb(NO3 )2 and AgNO3 were investigated, to illustrate the possible detoxification pathway of heavy metal ions. It was observed that both metals caused concentration-dependent death and moderate elevation of oxidative stress in ZF4 cells. In response to such toxicity, upregulation of multidrug resistance protein (mdr)4 and multiresistance-associated protein (mrp)1 were found. However, enhanced expression of glutathione S-transferase (gst) and cytochrome P450 (cyp)1a could only be detected during the exposure of Pb2+ . In addition, both metals induced extensive upregulation of pregnane X receptor (pxr), but only moderate elevation of E2-related factor (nrf2), while they suppressed the expression of miR-122 and miR-126. In conclusion, Pb2+ and Ag+ shared the same detoxification mechanism including ABC transporters, Pxr, and miRNA in ZF4 cells, which needs further investigation.

Oxylipin concentrations in bovine corpora lutea during maternal recognition of pregnancy

The objective was to determine the effects of pregnancy status on oxylipin profiles and eicosanoid metabolizing enzymes and in corpora lutea (CL) or endometrial (caruncle; CAR and intercaruncle; IC) tissues. Angus crossed cattle were synchronized with the CO-Synch protocol and artificially inseminated (AI). Sixteen days after AI, cattle were euthanized, and reproductive tracts collected from 6 non-pregnant and 6 pregnant cows. Oxylipin profiles and concentrations of progesterone (P4) were obtained from CL tissues. The activity of cytochrome P450 1A (CYP1A) and UDP-glucuronosyltransferase (UGT) enzymes were determined using specific luminogenic substrates. Data were analyzed using the MIXED procedure of SAS, and the model included pregnancy status. Corpora lutea of pregnant cattle contained greater (P < 0.05) concentrations of 9,10-DiHODE, 15,16-DiHODE, and 9,10-DiHOME. These oxylipins have been observed to increase cellular proliferation and vasodilation. Activity of CYP1A in the CL and UGT in CAR and IC was not different (P > 0.05) between pregnant and non-pregnant cattle. In the CL, activity of UGT was decreased (P < 0.05) in pregnant vs. non-pregnant cattle. The decrease in CL UGT activity during pregnancy indicates alterations in local hormone metabolism, while no differences in CL weight nor amount of P4 in CL were different between pregnant and non-pregnant cattle. Moreover, the increase in specific concentrations of oxylipins in the CL may indicate a novel pathway of steroid and eicosanoid metabolism during maternal recognition of pregnancy.

208 The Aryl hydrocarbon receptor (AhR) induces the expression of aldo-keto reductase (AKR) 1C3 in keratinocytes in a ligand-specific manner

The aldo-keto reductase 1C3 (AKR1C3) belongs to a superfamily of NAD(P)H-dependent oxidoreductases, which metabolize prostaglandins and steroid hormones. It is involved in the pathogenesis of various skin malignancies, such as atopic dermatitis (AD) and squamous cell carcinoma, which are triggered by polycyclic aromatic hydrocarbons and related environmental pollutants. These chemicals are able to bind to the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that, upon activation, rapidly translocates to the nucleus to induce the expression of target genes, for instance encoding for cytochrome P450 (CYP) 1A monooxygenases. In this study, we identified AKR1C3 as a target gene of the AhR in immortalized keratinocytes. Interestingly, activation of AhR by benzo(a)pyrene (BaP), but not by PCB126, a dioxin-like polychlorinated biphenyl, induced AKR1C3 expression. Chemical and genetic inhibition of AhR reversed the BaP-mediated expression of AKR1C3 and CYP1A1, providing evidence for an AhR-dependent effect. As the expression of CYP1A1 was induced by both ligands, we hypothesize that AKR1C3 is regulated by a non-canonical AhR signaling pathway. Upon ligand-binding, AhR undergoes conformational changes and c-Src, as an integral component of the cytosolic multiprotein complex, is released. This tyrosine kinase, in turn, activates EGFR-MEK-ERK signal transduction. Accordingly, chemical inhibition of either c-Src, EGFR and MEK abrogated the BaP-triggered induction of AKR1C3. SDS-PAGE/western blot analyses confirmed the AhR-dependent activation of this cascade in response to BaP treatment. Taken together, these data provide a new mechanism through which pollutants may regulate AKR1C3 expression and thereby contribute to the progression of inflammatory and malignant skin diseases.

Interspecies Variation in NCMN-O-Demethylation in Liver Microsomes from Various Species.

NCMN (N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide), a newly developed ratiometric two-photon fluorescent probe for human Cytochrome P450 1A (CYP1A), shows the best combination of specificity and reactivity for real-time detection of the enzymatic activities of CYP1A in complex biological systems. This study aimed to investigate the interspecies variation in NCMN-O-demethylation in commercially available liver microsomes from human, mouse, rat, beagle dog, minipig and cynomolgus monkey. Metabolite profiling demonstrated that NCMN could be O-demethylated in liver microsomes from all species but the reaction rate varied considerably. CYP1A was the major isoform involved in NCMN-O-demethylation in all examined liver microsomes based on the chemical inhibition assays. Furafylline, a specific inhibitor of mammalian CYP1A, displayed differential inhibitory effects on NCMN-O-demethylation in all tested species. Kinetic analyses demonstrated that NCMN-O-demethylation in liver microsomes form rat, minipig and cynomolgus monkey followed biphasic kinetics, while in liver microsomes form human, mouse and beagle dog obeyed Michaelis-Menten kinetics, the kinetic parameters from various species are much varied, while NCMN-O-demethylation in MLM exhibited the highest similarity of specificity, kinetic behavior and intrinsic clearance as that in HLM. These findings will be very helpful for the rational use of NCMN as a practical tool to decipher the functions of mammalian CYP1A or to study CYP1A associated drug-drug interactions in vivo.

Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

Effects of stocking density on stress response, innate immune parameters, and welfare of turbot (Scophthalmus maximus)

This experiment was conducted to investigate the growth performance, stress and immune responses, and welfare of juvenile turbot (Scophthalmus maximus) under three different densities (initial density 9.3, 13.6, and 19.1 kg m−2) for 120 days in a recirculating aquaculture system. Turbot were measured every 20 days to evaluate growth biometrically and sampled every month to measure biochemical parameters and mRNA levels of some stress-related genes. No significant differences were detected in the parameters and gene expression among density groups until the final sampling, except Fulton’s condition factor, lysozyme, immunoglobulin M, and complement C3. At the end of the experiment (final density 26.11, 38.22, and 52.25 kg m−2), turbot reared in the high-density (HD) group had lower body mass increase, specific growth rate, and Fulton’s condition factor, as well as higher feed conversion ratio and coefficient of variation for weight than those reared in the low-density (LD) group (P < 0.05). Fish in the HD group had higher serum cortisol, glucose, lactate, and cholesterol levels than fish in the other groups, whereas they had lower lysozyme, immunoglobulin M, and complement C3 and C4 contents (P < 0.05). Fish reared in the HD group also had higher serum chloride and osmolality levels and higher sodium-potassium adenosine triphosphatase (Na+,K+-ATPase) activity and higher Na+,K+-ATPase gene expression levels in gills compared to the other groups (P < 0.05). The mRNA levels of cytochrome P450 1A (CYP1A) and heat-shock proteins 70 and 90 (HSP70 and HSP90) were significantly upregulated, whereas glutathione S-transferase (GST) mRNA levels were significantly downregulated in the head kidney of fish in the HD group relative to fish in the other groups at the end of this trial (P < 0.05). These results indicated that overly high stocking (~50 kg m−2) density can negatively affect the growth performance, serum biochemical parameters, osmolality levels, stress-related gene expression, and overall welfare of turbot.