P450 1A Research Articles

Beta-naphthoflavone-inducedCYP1A expression in the guppy Jenynsia multidentata: Time-dependent response, anesthetic MS-222 effect and fin analysis

Cytochrome P450 1A (CYP1A) expression in fish is used as a biomarker of exposure to organic contaminants, such PAHs, PCBs and dioxins, in the aquatic environment. South American guppy fish Jenynsia multidentata were exposed to the prototypical aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF; 1μM) and the fins were biopsied to characterize different aspects of CYP1A induction. RTq-PCR was used to quantify CYP1A mRNA levels in fish tissues. CYP1A induction in the gill, liver and anal fin (gonopodium) occurred within the first hour of waterborne exposure to BNF and persisted throughout 2, 4, 8, 24, 48 and 96h compared to controls (DMSO vehicle; p<0.05). The organ-specific temporal pattern of induction was marked by mRNA levels consistently augment as duration of exposure increases and tend to a sustained induction from 24h to 96h for gill and liver (∼15-fold and ∼50-fold over control, respectively). In gonopodium, there was a maximum CYP1A mRNA level at 4h (∼34-fold over control). Basal CYP1A mRNA levels and its induction following BNF exposure were not affected by administration of a chemical anesthetic (fish immersion in 100mgl−1 MS-222 for 2–5min) in the gill, liver, gonopodium, dorsal or tail fin (p<0.05). In an ex vivo assay, in which small pieces of biopsied fins were exposed to BNF for 4h, high CYP1A induction was observed in the tail and gonopodium (∼49-fold and ∼69-fold, respectively) but not in the dorsal fin compared to controls. To our knowledge, this is the first study to show that a 1h waterborne exposure to an AHR agonist is sufficient to cause CYP1A induction in fish organs and fins. The present study added new information to the field regarding the use of MS-222 as an anesthetic on fish and the analysis of biopsied fins as an alternative non-lethalex vivo assay for evaluating the CYP1A biomarker in fish. This observation could be useful for planning fish toxicological bioassays and biomonitoring studies on the aquatic environments in South America.

Differential expression of HO-1 and CYP1A2 during up-regulation of ERK in stressed fish hepatocytes.

Induction of heme oxygenase-1 (HO-1) and extracellular signal-regulated kinase (ERK) is a crucial step in the cellular response to oxidative stress. Altered expression of such proteins in response to stress conditions is a key factor for the maintenance of cellular integrity and survival. Hypoxia-inducible factor-1 (HIF-1) and cytochrome P450 1A (CYP1A) are critical indicators of environmental exposure to hypoxia. The current study was aimed to analyse the expression pattern of HO-1, p-ERK, HIF-1α and CYP1A2 in hepatocytes of Mugil cephalus inhabiting Kovalam (unpolluted site) and Ennore (polluted site) estuaries. The impact of stress on hepatocytes was assessed by measuring the level of lipid hydroperoxides (LHP) and reduced glutathione (GSH) in hepatocytes of M. cephalus inhabiting these estuaries. The expression of HO-1, p-ERK, HIF-1α and CYP1A2 was analysed by ELISA and immunoblot analysis, respectively. There was significant decrease of GSH (19%) and CYP1A2 (34%) along with significant increase of LHP (31%), HO-1 (96%), p-ERK (p < 0.01) and HIF-1α (41%) in hepatocytes of M. cephalus inhabiting Ennore estuary than Kovalam estuary. The present study shows that increased synthesis of HO-1 mainly regulated by p-ERK and HIF-1α may significantly contribute to the environmental adaptation processes of grey mullet, by protecting the cells from oxidative stress and stress-induced degenerative changes.

Hypoxia and the pharmaceutical diclofenac influence the circadian responses of three-spined stickleback

Pollution with low concentrations of pharmaceuticals, especially when combined with low-oxygen conditions (hypoxia), is a threat to aquatic ecosystems worldwide. The non-steroidal anti-inflammatory drug diclofenac is commonly detected in wastewater effluents, and has potential to accumulate in the bile of fish. Diclofenac has been shown to activate aryl hydrocarbon receptor (AHR), which induces transcription in the metabolic enzyme cytochrome P450 1a (cyp1a). Previously, crosstalk has been shown to occur between AHR and hypoxia inducible factor 1 (HIF-1). In addition, both of these transcription factors interact with the proteins regulating circadian (24-h) rhythms in vertebrates. Yet little is known about the significance of these interactions during simultaneous exposure to chemicals and hypoxia in fish in vivo. We exposed wild-caught three-spined sticklebacks (Gasterosteus aculeatus) to diclofenac (1μg/L, 14 days), hypoxia (2.0mg/L, up to 24h) and the combination of both. We then analyzed markers of chemical biotransformation (EROD activity, cyp1a and ahr mRNA levels), glycolysis (lactate dehydrogenase (LDH) enzyme activity, ldh and enolase 1a mRNA levels), and the transcription of core circadian clock genes clock and period 1 in liver tissue. Samples were taken at three time points during the light period in order to address disturbances in the circadian variation of metabolic processes. The results show that mRNA levels and LDH activity tended to be lowest before the dark period, but this pattern was disturbed by hypoxia and diclofenac. Diclofenac and hypoxia co-exposure induced EROD activity more strongly than diclofenac exposure alone, while cyp1a mRNA level was increased also by hypoxia and diclofenac alone. LDH activity and mRNA expression showed a clear time-dependent response during hypoxia, which is consistent with the previously suggested decreased accumulation of HIF-1 during the dark period. Furthermore, LDH activity and transcription was disturbed by diclofenac, indicating important effects of environmental pollutants in disturbing natural acclimation. This study demonstrates the need for more studies to understand the potential disturbances in endogenous rhythms caused by environmental pollution in natural populations.

Effects of aflatoxin B1, fumonisin B1 and their mixture on the aryl hydrocarbon receptor and cytochrome P450 1A induction

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are mycotoxins widely found as cereal contaminants and their co-occurrence in corn has been associated with a high incidence of liver cancer. Both toxins are immunotoxic, with AFB1 being a procarcinogen, and its bioactivation through specific cytochrome P450 (Cyp) enzymes, such as Cyp1A, being a requirement for hepatocarcinogenic and toxic activities. This study evaluated the effects of these mycotoxins, alone or combined, on activation and expression of Cyp1A and its transcription factor aryl hydrocarbon receptor (Ahr) in hepatoma cell line H4IIE and spleen mononuclear cells of rats. The results demonstrate that in H4IIE cells, AFB1 induced an increase in Cyp1A activity and cyp1A transcription, associated with an enhanced Ahr activity, which suggests that this toxin can act as an Ahr agonist. Moreover, FB1 caused a small rise in Cyp1A activity and cyp1A expression. Similarly in spleen cells, AFB1 and FB1 induced overexpression of cyp1A and ahr genes. This work shows that the response potency was significantly higher for the mixture, indicating the existence of an interaction between both toxins. This study proposes the Ahr pathway activation as a toxicity mechanism of AFB1 and FB1, and highlights that FB1 may increase AFB1 bioactivation.

Effect of 2 corpora lutea on blood perfusion, peripheral progesterone, and hepatic steroid-inactivating enzymes in dairy cattle

The luteal structure that develops postovulation is critical to the facilitation and maintenance of pregnancy in dairy cattle. The objectives of this experiment were to determine if the induction of an accessory corpus luteum (CL), via human chorionic gonadotropin, altered blood perfusion of CL, peripheral concentrations of progesterone, or hepatic steroid-inactivating enzymes. Twenty-eight late-lactation Holstein cows were synchronized using the Ovsynch protocol and randomly assigned to 1 of 2 treatment groups. Cows received either an injection of human chorionic gonadotropin (1,000IU, i.m.) to induce an accessory CL (cows had exactly 2CL in 1 ovary) or no treatment (cows had exactly 1CL). Corpora lutea were examined daily from d 10 to 18 (d 0 was induced ovulation) via Doppler ultrasonography and a blood sample was collected. Volume of the CL was recorded, as well as images and videos of each CL, which were analyzed for blood perfusion. On d 13, a liver biopsy was performed to analyze hepatic steroid-inactivating enzymes. Cows with 1 or 2CL had similar peripheral concentrations of progesterone. Cows with 2CL had similar luteal volumes to cows with 1CL but cows with 2CL had greater total luteal blood perfusion. Hepatic enzyme [cytochrome P450 (CYP) 1A, 3A, and 2C, aldo-keto reductase 1C, and uridine diphosphate glucuronosyltransferase] activities did not differ between cows with 1 and 2CL. Overall, the observed increase in total luteal blood perfusion in cows with 2CL did not correspond to differences in peripheral concentrations of progesterone or clearance of progesterone measured by the hepatic enzyme activity. This could indicate that induction of an accessory CL would not affect concentrations of progesterone necessary to maintain pregnancy.

Mutagenic and antimutagenic effects of Heterotheca inuloides.

The antioxidant and hepatoprotective effects of Heterotheca inuloides have been reported before, nevertheless its use as a possible chemopreventive agent has not been documented. The aim of this study was to evaluate the mutagenic and antimutagenic activities of H. inuloides extracts using the Ames test. Both, the methanolic and acetonic extracts, were mutagenic in the TA98 but not in TA100 or TA102 strains. On the other hand, the methanolic extract reduced the mutagenicity of norfloxacin, benzo[a]pyrene and 2-aminoanthracene. Quercetin, one of the main components in the methanolic extract, also presented a mutagenic/antimutagenic dual effect and is an inhibitor of Cytochrome P450 (CYP) 1A. The antigenotoxic properties of H. inuloides could be due to the antioxidant properties previously reported and to its CYP inhibitory effect mediated by quercetin. Further studies with in vivo systems will afford information about H. inuloides beneficial and detrimental properties.

Abstract 1587: Mechanistic role of cytochrome P4501B1 in DNA adduct formation by 3-methylcholanthrene in mice: Implications for lung cancer in humans

Abstract Humans are frequently exposed to polycyclic aromatic hydrocarbons (PAHs). Cytochrome P450 (CYP)1A and 1B1 enzymes are known to play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to DNA-binding metabolites, which in turn mediate carcinogenesis in target organs such as lung, breast, and skin. In this investigation, we tested the hypothesis that mice lacking the gene for Cyp1b1 will show attenuation of MC-DNA adduct formation, compared to WT mice. Eight week-old female wild type (WT) (C57BL/6J) mice or mice lacking the gene for Cyp1b1 were treated with MC (100 µmol/kg), once daily for 2 days, and animals sacrificed 1 day after the last treatment. DNA adducts from liver and lung genomic DNA were analyzed by the 32P-postabeling assay. Control animals received only the vehicle corn oil (CO). In WT mice, CO-treated group did not show any adducts, whereas MC-treated animals showed 11 adduct spots in the lung. In Cyp1b1-null mice, although there were no qualitative differences in the pulmonary adduct pattern, the levels of each adduct was markedly suppressed (25-90%), with total levels of adducts being about 15% of those formed in WT mice. In liver of WT as well as Cyp1b1-null mice, MC induced formation of 11 adducts, and the adduct pattern was similar to that of lung, but the total levels of adducts in WT mice were about a third of those seen in the lungs of the corresponding animals. In Cyp1b1-null mice, MC adducts were significantly decreased compared to WT mice, but the extent of attenuation in liver was lesser than that in lung. These results suggest that CYP1B1 plays a major role in formation of DNA adducts from MC. Because DNA adducts are pre-mutagenic lesions, our results are highly relevant to PAH-mediated pulmonary carcinogenesis. Our data suggest that CYP1B1 could be a molecular target for developing preventive and therapeutic strategies against lung cancer in humans. Citation Format: Bhagavatula Moorthy, Guodong Zhou, Lihua Wang, Sudha R. Kondraganti, Weiwu Jiang. Mechanistic role of cytochrome P4501B1 in DNA adduct formation by 3-methylcholanthrene in mice: Implications for lung cancer in humans. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1587. doi:10.1158/1538-7445.AM2014-1587

Defining molecular sensors to assess long-term effects of pesticides on carcinogenesis.

The abundance of dioxins and dioxin-like pollutants has massively increased in the environment due to human activity. These chemicals are particularly persistent and accumulate in the food chain, which raises major concerns regarding long-term exposure to human health. Most dioxin-like pollutants activate the aryl hydrocarbon receptor (AhR) transcription factor, which regulates xenobiotic metabolism enzymes that belong to the cytochrome P450 1A family (that includes CYP1A1 and CYP1B1). Importantly, a crosstalk exists between estrogen receptor α (ERα) and AhR. More specifically, ERα represses the expression of the CYP1A1 gene, which encodes an enzyme that converts 17β-estradiol into 2-hydroxyestradiol. However, (ERα) does not repress the CYP1B1 gene, which encodes an enzyme that converts 17β-estradiol into 4-hydroxyestradiol, one of the most genotoxic estrogen metabolites. In this review, we discuss how chronic exposure to xenobiotic chemicals, such as pesticides, might affect the expression of genes regulated by the AhR–ERα crosstalk. Here, we focus on recent advances in the understanding of molecular mechanisms that mediate this crosstalk repression, and particularly on how ERα represses the AhR target gene CYP1A1, and could subsequently promote breast cancer. Finally, we propose that genes implicated in this crosstalk could constitute important biomarkers to assess long-term effects of pesticides on human health.

Individual and joint toxic effects of cadmium sulfate and α-naphthoflavone on the development of zebrafish embryo.

This paper aims to evaluate the individual and joint toxicities of cadmium sulfate (CdSO4) and α-naphthoflavone (ANF) in zebrafish embryos. As a result, CdSO4 caused both lethal and sub-lethal effects, such as 24 h post-fertilization (hpf) death and 72 hpf delayed hatching. However, ANF only caused sub-lethal effects, including 48 hpf cardiac edema and 72 hpf delayed hatching. Taking 24 hpf death and 48 hpf cardiac edema as endpoints, the toxicities of CdSO4 and ANF were significantly enhanced by each other. Consistently, both CdSO4 and ANF caused significant oxidative stress, including decreases in the reduced glutathione (GSH) level, inhibition of superoxide dismutase (SOD) activity, as well as increases in malondialdehyde (MDA) content in zebrafish embryos, but these mixtures produced much more significant alterations on the biomarkers. Co-treatment of CdSO4 and ANF significantly down-regulated the mRNA level of multidrug resistance-associated protein (mrp) 1 and cytochrome P450 (cyp) 1a, which constituted the protective mechanisms for zebrafish embryos to chemical toxins. In conclusion, co-treatment of CdSO4 and ANF exhibited a much more severe damage in zebrafish embryos than individual treatment. Meanwhile, production of oxidative stress and altered expression of mrp1 and cyp1a could be important components of such joint toxicity.

Emamectin benzoate treatment alters the expression and activity of CYP1A, FMO and GST in different tissues of rainbow trout (Oncorhynchus mykiss)

Abstract Emamectin benzoate (EMB) is one of the drugs used for the treatment and control of infections by Caligus rogercresseyi on salmonids. EMB is orally delivered to the salmon in food, which could be affecting the activity of biotransformation markers in these fish. The aim of this study was to determine the effect of EMB treatment on the mRNA and protein expression levels and enzymatic activity of cytochrome P450 1A (CYP1A), flavin-monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues of rainbow trout (Oncorhynchus mykiss). Rainbow trout were collected between 10 and 25 days after completion of medication with EMB by a standard seven-day treatment in different salmon farms from southern Chile. The CYP1A, FMO and GST mRNA, protein levels and activity in the liver, muscle, gill, kidney and intestine tissues from rainbow trout were measured. Most mRNA and protein levels were significantly affected by treatment with EMB. The highest mRNA transcriptional up-regulations were observed with CYP1A in the liver and FMO in the liver, muscle and kidney and were very marked with regard to the increases of CYP1A and FMO in the gill and GST in the gill and intestine. These effects only correlated slightly with FMO protein levels in the liver muscle and gill. The highest up-regulations for protein levels were observed for GST in the kidney. While, the down-regulation measured in CYP1A was similar between protein levels in the liver, gill and intestine, and also in CYP1A activity levels in all tissues examined. On the other hand, GST mRNA levels were essentially without great changes, except slight increases in the gill and intestine, while GST protein and activity levels increased in all tissues measured. These results show that treatment with EMB induces changes of the expression of detoxification enzymes in most trout tissues, and this effect was consistent with increases of activity for FMO in the intestine and was comparable to the increases of activity for GST observed in all trout tissues analyzed. By contrast, EMB decreased the CYP1A activity levels in all tissues examined. These results show that treatment with EMB in trout alters the expression and activity of CYP1A, FMO and GST, which could be affecting detoxification processes and immuno-modulatory mechanisms. Moreover, the data obtained will allow insight into the mechanisms involved in the biotransformation of EMB in salmonids and its putative relation with drug resistance observed in EMB treatments.

Glucosinolate hydrolysis products of cruciferous vegetables inhibit cytochrome P450 1A activity and mediate oxidative stress

Glucosinolate hydrolysis products of cruciferous vegetables inhibit cytochrome P450 1A activity and mediate oxidative stress

Hepatic steroid metabolizing enzyme activity during early, mid, and late bovine pregnancy

The objective was to examine hepatic steroid inactivating enzymes throughout gestation and determine the effect of early to mid-gestation maternal nutrient restriction followed by realimentation on the activity of these enzymes. On day 30 of gestation, cows were assigned to dietary treatments: control (CON; 100% National Research Council; n = 18) and restricted (RES; 60% National Research Council; n = 30). On day 85, cows were slaughtered (CON, n = 6 and RES, n = 6), remained on control (CC, n = 12) and restricted (RR, n = 12), or were realimented to control (RC, n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5), or were realimented to control (RRC, n = 6). On day 254, all remaining cows were slaughtered. Jugular blood samples were collected before the slaughter for steroid analysis. At slaughter, maternal liver samples were collected for hepatic enzyme activity analysis. Activity of cytochrome P450 3A decreased (P = 0.05) from mid- to late-gestation. Peroxisome proliferator-activated receptor alpha DNA binding activity was increased (P < 0.01) on day 140 and 254 of gestation vs day 85. Concentrations of estradiol-17β (E2) increased (P < 0.01) as gestation proceeded, whereas progesterone concentrations (P4) tended to increase (P = 0.06) from mid- to late-gestation. Activity of cytochrome P450 1A and 2C were decreased (P < 0.05) in nutrient restricted cows vs control, whereas concentrations of E2 were increased (P < 0.05) in nutrient restricted cows vs control. A longer period of nutrient realimentation from mid- to late-gestation increased (P < 0.05) aldo-keto reductase 1C activity and decreased (P < 0.05) P4 concentrations compared with the shorter period of nutrient realimentation. In addition, significant negative correlations were observed for cytochrome P450 3A activity vs E2 (r2 = −0.30; P < 0.05) and aldo-keto reductase 1C activity vs P4 (r2 = −0.29; P < 0.05). The present study implicates hepatic steroid inactivation in the partial modulation of peripheral concentrations of E2 and P4 during gestation.

Effects of water accommodated fractions of crude oils and diesel on a suite of biomarkers in Atlantic cod (Gadus morhua)

The aim of this study was to characterize concentration- and time-dependent responses in juvenile Atlantic cod (Gadus morhua) following exposure for one and three weeks to the water-soluble fraction (WAF) of three weathered oils: Arabian Light crude oil (ALC), North Sea crude oil (NSC) and ship-diesel. The sum of polycyclic aromatic hydrocarbons (PAH) in water was highest after one week of exposure and within environmentally relevant concentrations. PAH metabolites in bile confirmed exposure to and uptake of PAHs. Hepatic cytochrome P450 1A (CYP1A) gene expression (mRNA quantification) increased dramatically following exposure to all three oil types (fold-change up to 165) and there was a time lag between gene and protein expression. Hepatic CYP1A protein concentration and ethoxyresorufin-O-deethylase (EROD) activity were more variable among individuals and treatments than gene expression. EROD activity in liver and gills increased in fish exposed to WAF from the two crude oils, but not in fish exposed to WAF from diesel. Exposure to diesel appeared to induce oxidative stress to a greater extent than exposure to crude oils. Other biomarkers (glutathione S-transferases, acetylcholine esterase, vitellogenin) did not appear to respond to the exposure and hence did not discriminate among oils. Biomarker responses in cod after exposure to weathered crude oils and diesel suggested that the CYP1A system and oxidative stress markers have the highest potential for discriminating among different oil types and to monitor the environmental consequences of spills.

Transient suppression of AHR activity in early red seabream embryos does not prevent the disruption of peripheral nerve projection by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The toxicity of dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is mainly mediated by an aryl hydrocarbon receptor (AHR), which regulates the transcription of multiple target genes including cytochrome P450 1A (CYP1A). Our pervious study identified the presence of TCDD-induced defects of peripheral nerve projection in red seabream (Pagrus major) embryos. However, it remains unclear whether the TCDD-induced peripheral neurotoxicity is mediated by the AHR. To assess the contribution of the red seabream AHR (rsAHR) signaling pathway to the neuronal toxicity, red seabream embryos at 10h post-fertilization (hpf) were treated for 80min with TCDD (0, 0.3, 5.3, and 37nM in seawater) alone or in combination with CH223191 (500nM in seawater), which is an AHR antagonist. A preliminary in vitro reporter gene assay confirmed that TCDD-induced transcriptional activity via rsAHR1 and rsAHR2 was suppressed by CH223191 treatment in a dose-dependent manner. CYP1A mRNA expression in embryos was determined by 2-step real time quantitative-polymerase chain reaction at 24 and 120hpf and in situ hybridization at 48, 72, 96 and 120hpf. The morphology of the peripheral nerve system (PNS) was also microscopically observed by fluorescent staining using an anti-acetylated tubulin antibody at 120hpf. CYP1A mRNA expression was dose-dependently induced by TCDD at all of the examined developing stages. The suppression of TCDD-induced CYP1A expression by CH223191 treatment was observed in embryos at 24 and 48hpf, while the effect of the rsAHR antagonist disappeared at 96 and 120hpf. This phenomenon indicated the transient suppression of TCDD-induced rsAHR activation by CH223191 treatment. The immunostaining of peripheral nerves at 120hpf demonstrated that the projections of the craniofacial nerve were altered in TCDD-treated embryos, and the frequency of TCDD-induced abnormal projections was not prevented by co-treatment with CH223191. These results indicate that the transient suppression of TCDD-induced rsAHR activation during the early developing stages of the red seabream does not influence the abnormal projection of peripheral nerves. In conclusion, transient rsAHR activation in the early stages of development is not involved in the neurotoxicity.

Expression patterns of metallothionein, cytochrome P450 1A and vitellogenin genes in western mosquitofish (Gambusia affinis) in response to heavy metals

The aim of this study was to evaluate the effects of three metals (Zn, Cd and Pb) on hepatic metallothionein (MT), cytochrome P450 1A (CYP1A) and vitellogenin (Vtg) mRNA expression in the liver of adult female mosquitofish (Gambusia affinis) after 1, 3 or 8d. Both concentration-response and time-course effects of hepatic MT, CYP1A and Vtg at the transcription level were determined by quantitative real-time PCR. The results from this study showed that Zn, Cd and Pb could significantly induced MT, CYP1A and Vtg mRNA expression levels in mosquitofish. In general, this study demonstrated that heavy metals modulate MT, CYP1A and Vtg mRNA expression levels in a metal-, concentration- or time-dependent manner.

Endocrine and AhR-CYP1A Pathway Responses to the Water-Soluble Fraction of Oil in Zebrafish (Danio rerio Hamilton)

Crude oil is a complex mixture of compounds of which the water-soluble fraction (WSF) is considered to be bioavailable and potentially toxic to aquatic biota. Containing numerous compounds, WSF becomes a source of multiple chemical stressors to wildlife when introduced into the environment. To study the combined effects of WSF components on aquatic biota, the model species zebrafish (Danio rerio Hamilton) was exposed for 24 or 72 h to 10 or 50% WSF solution of known composition, generated from artificially weathered North Sea crude oil. Hepatic expression of genes involved in the aryl hydrocarbon receptor-cytochrome P-450 1A (AhR-CYP1A) pathway (AhR2, AhRR1, CYP1A1) and steroidogenesis (StAR, CYP11A, 3β-HSD, CYP19A, CYP19B) was measured, as well as estrogen receptors ERα and ERβ1. Induction of CYP1A and particularly of AhRR1 was observed while ERα and steroidogenic enzymes CYP11A and 3β-HSD were downregulated. Regression analysis demonstrated a negative relationship between AhR-CYP1A pathway and endocrine transcript levels, although causality remains to be established. These findings indicate that exposure to WSF of oil disrupts steroidogenesis and may therefore constitute a potential risk for reproductive ability of aquatic organisms. In addition, it is proposed that hepatic gene expression of AhRR1 may serve as a novel biomarker of WSF exposure.

Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1

Graphene and its derivatives are an emerging class of carbon nanomaterial with great potential for a broad range of industrial and consumer applications. However, their increasing production and use is expected to result in release of nano-sized graphene platelets into the environment, where they may interact with chemical pollutants modifying their fate and toxic potential. The objective of this study was to assess whether graphene nanoplatelets can act as vector for aromatic environmental pollutants increasing their cellular uptake and associated hazardous effects in vitro. For this purpose, cell cultures of the topminnow fish (Poeciliopsis lucida) hepatoma cell line PLHC-1 were simultaneously (and successively) exposed to graphene nanoplatelets (graphene oxide (GO) or carboxyl graphene (CXYG)) and an aryl hydrocarbon receptor (AhR) agonist (β-naphthoflavone (β-NF), benzo(k)fluoranthene (BkF) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169)). Following exposure cytochrome P450 1A (Cyp1A) induction was assessed by measuring cyp1A mRNA expression levels using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Cyp1A-dependent ethoxyresorufin-O-deethylase (EROD) activity. It was observed that pre- and co-exposure of cells to GO and CXYG nanoplatelets had a potentiating effect on β-NF, BkF, and PCB169-dependent Cyp1A induction suggesting that graphene nanoplatelets increase the effective concentration of AhR agonists by facilitating their passive diffusion into the cells by damaging the cells' plasma membrane and/or by transporting them over the plasma membrane via a Trojan horse-like mechanism. The results demonstrate the existence of combination effects between nanomaterials and environmental pollutants and stress the importance of considering these effects when evaluating their respective hazard.

Integrated biomarkers in wild crucian carp for early warning of water quality in Hun River, North China

Metabolizing enzymes play important roles in the detoxification of various pollutants in aquatic organisms, thereby they can also be used to provide early-warning signals of environmental risks. Real-time quantitative reverse-transcription polymerase chain reaction assays were developed to quantify cytochrome P450 1A (CYP1A), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione-S-transferase (GST) in crucian carp (Carassius auratus). The methods were then used to detect the respective mRNA expression levels in liver tissue in wild crucian carp from the Hun River, North China. CYP1A mRNA expression was significantly up-regulated in fish from stations S5, S6, and S8 (p < 0.05). SOD mRNA expression was significantly down-regulated in downstream areas relative to fish from upstream sites (p < 0.05); GPx and CAT mRNA expression levels were also down-regulated at S9 (p < 0.05). In contrast, GST mRNA expression showed no obvious change between fish collected from up- or downstream areas of the river. Finally, an integrated biomarker response was used to evaluate the integrated impact of pollutants in the Hun River and allow better comprehension of the real toxicological risk of these investigated sites.

Increased susceptibility to hyperoxic lung injury and alveolar simplification in newborn rats by prenatal administration of benzo[a]pyrene

Maternal smoking is one of the risk factors for preterm birth and for the development of bronchopulmonary dysplasia (BPD). In this study, we tested the hypothesis that prenatal exposure of rats to benzo[a]pyrene (BP), a component of cigarette smoke, will result in increased susceptibility of newborns to oxygen-mediated lung injury and alveolar simplification, and that cytochrome P450 (CYP)1A and 1B1 enzymes and oxidative stress mechanistically contribute to this phenomenon. Timed pregnant Fisher 344 rats were administered BP (25mg/kg) or the vehicle corn oil (CO) on gestational days 18, 19 and 20, and newborn rats were either maintained in room air or exposed to hyperoxia (85% O2) for 7 or 14 days. Hyperoxic newborn rats prenatally exposed to the vehicle CO showed lung injury and alveolar simplification, and inflammation, and these effects were potentiated in rats that were prenatally exposed to BP. Prenatal exposure to BP, followed by hyperoxia, also resulted in significant modulation of hepatic and pulmonary cytochrome P450 (CYP)1A and 1B1 enzymes at PND 7–14. These rats displayed significant oxidative stress in lungs at postnatal day (PND) 14, as evidenced by increased levels of the F2-isoprostane 8-iso-PGF2α. Furthermore, these animals showed BP-derived DNA adducts and oxidative DNA adducts in the lung. In conclusion, our results show increased susceptibility of newborns to oxygen-mediated lung injury and alveolar simplification following maternal exposure to BP, and our results suggest that modulation of CYP1A/1B1 enzymes, increases in oxidative stress, and BP-DNA adducts contributed to this phenomenon.

The influence of dicoumarol on the bioactivation of the carcinogen aristolochic acid I in rats

Aristolochic acid I (AAI) is the major toxic component of the plant extract AA, which leads to the development of nephropathy and urothelial cancer in human. Individual susceptibility to AAI-induced disease might reflect variability in enzymes that metabolise AAI. In vitro quinone oxidoreductase (NQO1) is the most potent enzyme that activates AAI by catalyzing formation of AAI-DNA adducts, which are found in kidneys of patients exposed to AAI. Inhibition of renal NQO1 activity by dicoumarol has been shown in mice. Here, we studied the influence of dicoumarol on metabolic activation of AAI in Wistar rats in vivo. In contrast to previous in vitro findings, dicoumarol did not inhibit AAI-DNA adduct formation in rats. Compared with rats treated with AAI alone, 11- and 5.4-fold higher AAI-DNA adduct levels were detected in liver and kidney, respectively, of rats pretreated with dicoumarol prior to exposure to AAI. Cytosols and microsomes isolated from liver and kidney of these rats were analysed for activity and protein levels of enzymes known to be involved in AAI metabolism. The combination of dicoumarol with AAI induced NQO1 protein level and activity in both organs. This was paralleled by an increase in AAI-DNA adduct levels found in ex vivo incubations with cytosols from rats pretreated with dicoumarol compared to cytosols from untreated rats. Microsomal ex vivo incubations showed a lower AAI detoxication to its oxidative metabolite, 8-hydroxyaristolochic acid (AAIa), although cytochrome P450 (CYP) 1A was practically unchanged. Because of these unexpected results, we examined CYP2C activity in microsomes and found that treatment of rats with dicoumarol alone and in combination with AAI inhibited CYP2C6/11 in liver. Therefore, these results indicate that CYP2C enzymes might contribute to AAI detoxication.